New strategies to optimize the secretion capacity
for heterologous proteins in Bacillus subtilis
Dissertation
zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie an der Internationalen Graduiertenschule
Biowissenschaften der Ruhr-Universität Bochum
angefertigt am Institut für Molekulare Enzymtechnologie
vorgelegt von Ulf Brockmeier
aus Münster
Bochum 2006
Neue Strategien zur Optimierung der Sekretionsleistung für heterologe Proteine in Bacillus subtilis
Dissertation
zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie an der Internationalen Graduiertenschule
Biowissenschaften der Ruhr-Universität Bochum
angefertigt am Institut für Molekulare Enzymtechnologie
vorgelegt von Ulf Brockmeier
aus Münster
Bochum 2006
Die vorliegende Arbeit wurde im Rahmen des Europäischen Graduiertenkollegs der
Ruhr-Universität Bochum (EGC 795): Regulatory Circuits in Cellular Systems:
Fundamentals and Biotechnological Applications angefertigt.
Referent: Prof. Dr. K.-E. Jäger Korreferent: Prof. Dr. A. J. M. Driessen Tag der mündlichen Prüfung:
Erklärung
Hiermit erkläre ich, dass ich die Arbeit selbständig verfasst und bei keiner anderen
Fakultät eingereicht und dass ich keine anderen als die angegebenen Hilfsmittel
verwendet habe. Es handelt sich bei der heute von mir eingereichten Dissertation um
fünf in Wort und Bild völlig übereinstimmende Exemplare.
Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten
und in keinem Fall inhaltsverändernde Bildbearbeitung vorgenommen wurde.
Bochum, den
_____________________________________
(Unterschrift)
Danksagungen Herrn Prof. Dr. K.-E. Jäger danke ich für die Überlassung des interessanten wissenschaftlichen Themas, für das Interesse am Fortschritt meiner Arbeit, für zahlreiche konstruktive Diskussionen und die Gelegenheit, diese Arbeit frei und selbständig zu gestalten. I would like to thank Prof. Dr. A. J. M. Driessen for agreeing to co-supervise this thesis. Ich möchte mich für die Finanzierung meiner Forschungsarbeit im Rahmen des Europäischen Graduiertenkollegs EGC 795 und insbesondere bei den Organisatoren Herrn Prof. Dr. M. Rögner und Frau Dr. C. Wüllner für viele interessante Tagungen und Kurse bedanken. Vielen Dank auch an alle Mitglieder des Kollegs für nette Gesprächsrunden und die freundliche Atmosphäre während unserer Treffen. Bei Herrn Prof. Dr. R. Freudl sowie bei Herrn M. Caspers (IBT1, FZ Jülich) möchte ich mich für die erfolgreiche Zusammenarbeit und für die durchgeführten Pulse-Chase Experimente bedanken. Desweiteren danke ich Herrn Prof. Dr. T. Noll und Herrn Dr. A. Jockwer (ehemals IBT2, FZ Jülich) für die Herstellung der polyklonalen Antikörper gegen die Cutinase. Frau E. Knieps-Grünhagen (IBT2, FZ-Jülich) möchte ich für die Aufreinigung der Cutinase danken. Herrn Prof. Dr. J. Büchs und Herrn F. Kensy (Lehrstuhl für Bioverfahrenstechnik, RWTH Aachen) danke ich für die gute Zusammenarbeit und die durchgeführten Wuchs- und Expressionsversuche von B. subtilis in Mikrotiterplatten. Herrn Dr. T. Eggert gilt mein besonderer Dank für die exzellente wissenschaftliche und auch persönliche Unterstützung während meiner gesamten Promotionszeit. Vielen Dank auch für die aufmerksame Durchsicht dieses Manuskriptes. Für ihre große Hilfsbereitschaft sowie für die kritische Durchsicht des Manuskriptes möchte ich mich besonders herzlich bei Herrn Dr. C. Leggewie sowie Herrn M. Puls bedanken. Ich hoffe darauf, dass Freundschaften mit der Entfernung wachsen werden... Meiner langjährigen Arbeitskollegin und „Leidensgenossin“ Frau M. Wendorff möchte ich herzlich danken für die vielen netten Gespräche und aufmunternden Worte, die einen in stressigen Zeiten am Leben halten. Vielen Dank an alle hier nicht namentlich erwähnten Mitglieder meiner Arbeitsgruppe. Ich wünsche Euch für die Zukunft weiterhin ein so gutes Arbeitsklima, wie ich es während meiner Zeit am Forschungszentrum erleben durfte. Ich möchte mich darüber hinaus sehr herzlich bei allen Mitarbeitern und Kollegen des Instituts für Molekulare Enzymtechnologie für die freundschaftliche Atmosphäre bedanken. Ein ganz persönliches Dankeschön geht an meine Familie und meine Freunde. Ihr Rückhalt bedeutet mir sehr viel.
Für Ulla
..wenn der Nahanni fällt, hast Du gelebt.....
Publikationen Brockmeier, U., M. Caspers, R. Freudl, A. Jockwer, T. Noll and T. Eggert. Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. Submitted for publication. Brockmeier, U., M. Wendorff and T. Eggert. 2006. Versatile expression and secretion vectors for Bacillus subtilis. Curr. Microbiol. 52:143-148.
Eggert, T., U. Brockmeier, M. J. Dröge, W. J. Quax, and K. E. Jaeger. 2003. Extracellular lipases from Bacillus subtilis: regulation of gene expression and enzyme activity by amino acid supply and external pH. FEMS Microbiol. Lett. 225:319-324.
Posterpräsentationen Brockmeier, U., K.-E. Jaeger and T. Eggert. 2004. Evolution of signal peptides to optimize heterologous secretion in Bacillus subtilis. International Congress on Biocatalysis- Biocat 2004, Hamburg, Germany. Brockmeier, U., K.-E. Jaeger and T. Eggert. 2004. Improvement of the Bacillus
secretion machinery using Directed Evolution. Biospektrum special issue of VAAM Annual conference, Braunschweig, Germany, p. 116. Brockmeier, U., K.-E. Jaeger and T. Eggert. 2003. Secretion of two lipolytic enzymes in Bacillus subtilis. Biospektrum special issue of VAAM Annual conference, Berlin, Germany, p. 77. Brockmeier, U., K.-E. Jaeger and T. Eggert. 2003. Improvement of the Bacillus
secretion machinery using Directed Evolution, 1st combined GBB-EGC Symposium, 11.-12.09. 2003, Groningen, The Netherlands. Vorträge Brockmeier, U., T. Eggert and K.-E. Jaeger. 2002. Secretion pathways for biocatalysts in Bacillus subtilis. EGC-GBB Symposium, 22.11. 2002, Bochum, Germany. Brockmeier, U., K.-E. Jaeger and T. Eggert. 2005. Heterologous protein expression in Bacillus subtilis: Successful manipulation of the Sec pathway by directed evolution. Third combined GBB-EGC Symposium, 15.-16.09. 2005, Groningen, The Netherlands.
Contents
I
Contents
List of Figures……………………………………………………………………………….. III List of Tables………………………………………………………………………………… IV Abbreviations………………………………………………………………………………… V 1. Introduction………………………………………………………………………... 1 1.1 The necessity of protein translocation in cells…………………………………………… 1 1.2 Protein sorting in cellular traffic- The crucial role of signal peptides………….……….. 2 1.3 Signal peptide classification in B. subtillis ……………………………………………….. 4 1.4 The Sec pathway as the major export route……………………………….…………….. 6 1.5 Industrial use of Bacillus species…………………………………………….……………. 8 1.6 Export bottlenecks for foreign recombinant proteins in B. subtilis…………………….. 9 1.7 Strategies to bypass the limitations in protein production…………………….………… 11 1.8 Scope of this thesis…………………………………………………………………………. 14 2. Materials and Methods…………………………………………………………... 16 2.1 Chemicals and enzymes………………………………………………………….………... 16 2.2 Bacterial strains and plasmids…………………………………………………………….. 16 2.3 Oligonucleotides and PCR-primers………………………………………………………. 17 2.4 Culture media and growth conditions…………………………………………………….. 19 2.5 Storage of bacteria…………………………………………………………………………. 20 2.6 Isolation of nucleic acid…………………………………………………………………….. 20 2.7 Agarose gel electrophoresis………………………………………………………………. 20 2.8 In vitro recombination of DNA……………………………………………………………… 20 2.9 Transformations of bacteria………………………………………………………………... 21 2.9.1 Chemical transformation of E. coli………………………………………………………… 21 2.9.2 Electroporation of E.coli …………………………………………………………………… 21 2.9.3 Transformation of B. subtillis………………………………………………………………. 21 2.10 Polymerase chain reaction (PCR)……………………………………………….………... 22
2.10.1 Standard PCR................................................................................................………... 22 2.10.2 Site directed mutagenesis…………………………………………………………………. 22 2.10.3 Saturation mutagenesis……………………………………………………………………. 23 2.10.4 Random mutagenesis…………………………………………………………….………… 23 2.10.5 Cassette mutagenesis of the PPX-domain of secA…………………………………….. 23
2.11 Construction of integration plasmid pMCut………………………………………………. 24 2.12 DNA sequencing……………………………………………………………………………. 24 2.13 Enzyme assays……………………………………………………………………………… 25
2.13.1 Lipolytic plate assay………………………………………………………………………… 25 2.13.2 α-amylase activity plate assay…………………………………………………………….. 25 2.13.3 Lipolytic activity assay……………………………………………………………………… 25 2.13.4 Esterase activity assay……………………………………………………………………... 26 2.13.5 High throughput screening (HTS) assay for cutinase/EstCL1
secretion using a pipetting robot…………………………………………………………...
26 2.14 Determination of protein concentration…………………………………………………… 27 2.15 TCA precipitation of proteins………………………………………………………………. 27 2.16 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)………………………………… 27 2.17 Cell culture and production of cutinase-specific antibodies by hybridoma cells……… 28 2.18 Immunodetection of proteins………………………………………………………………. 28 2.19 Purification of overexpressed cutinase in E. coli………………………………………… 29 2.20 Pulse-chase protein labelling……………………………………………………………… 30 2.21 Cell fractioning of B. subtilis……………………………………………………………….. 31 2.22 Computational methods……………………………………………………………………. 31 3. Results……………………………………………………………………………… 32
3.1 New expression and secretion vectors for Bacillus subtilis……………………………. 32 3.1.1 Choice of expression host: B. subtillis versus E. coli……………………………………. 32 3.1.2 Construction of vector pBSMuL1 and pBSMuL2………………………………………… 33
Contents
II
3.1.3 Overexpression, secretion and purification of His-tagged cutinase in pBSMuL vectors………………………………………………………………………………………...
34
3.1.4 Stability of pBSMuL vectors in B. subtilis under non selective conditions……………. 36 3.2 In search of the most functional signal peptide for heterologous protein
secretion in B. subtillis………………………………………………………………………
38 3.2.1 Establishment of a high-throughput screening (HTS-) system to evaluate
secretion efficiency for heterologous proteins in B. subtillis…………………………….
38 3.2.1.1 Construction of a Bacillus vector system using cutinase as a secretion reporter……. 39 3.2.1.2 Analysis of growth differences in microtiter plates………………………………………. 41 3.2.1.3 Development of a HTS-process for detection of export efficiency using a
pipetting robot………………………………………………………………………………..
42 3.2.1.4 Validation of the automated HTS process in B. subtillis………………………………… 44
3.2.2 Library construction and high-throughput screening of all Sec-type signal sequences fused to cutinase……………………………………………………………….
45
3.2.3 The highest D-score does not necessarily predict the optimal signal peptide for secretion of the heterologous cutinase………………………………………………..
47
3.2.4 Translocation efficiency is not solely responsible for the varying export amounts of cutinase……………………………………………………………………………………
53
3.2.5 High level production of cutinase in B. subtilis using the best identified signal peptide………………………………………………………………………………………..
55
3.3 One-step optimization of the heterologous esterase EstCL1 for secretion using the SP-library………………………………………………………………………….
57
3.4 Directed Evolution of SP-AmyE to achieve high yield of cutinase export…………….. 59 3.4.1 Random mutagenesis of SP-AmyE did not lead to increased amount of exported
cutinase ………………………………………………………………………………………
60 3.4.2 Saturation mutagenesis of SP-AmyE revealed improved variants…………………….. 61
3.5 Coexpression and mutagenesis of B. subtilis secA to increase cutinase export…….. 67 3.5.1 Construction and characterization of marker strain B. subtilis Marc1…………………. 67 3.5.2 Co-overexpression of SecA improves cutinase secretion……………………………… 69 3.5.3 The precursor form of cutinase fused to SP-LipA is processed rapidly under
condition of SecA-overexpression…………………………………………………………
70 3.5.4 A SecA variant leading to eight-fold enhanced protein export………………………… 71 3.5.5 The improved export effect of SecA variant A23T, K809D was neutralised
by a further amino acid exchange H289Y in the PPX-domain………………………….
72 4. Discussion…………………………………………………………………………. 73 4.1 A new Bacillus vector series……………………………………………………………….. 74 4.2 A new secretion marker system for HTS processes in B. subtillis…………………….. 76 4.3 Improvement of heterologous protein secretion in B. subtilis by signal sequence
screening……………………………………………………………………………………..
77 4.4 Saturation mutagenesis as a useful tool for the characterization and optimization
of a signal peptide…………………………………………………………………………...
81 4.5 Improvement of heterologous protein secretion in B. subtilis by
co-overexpression of SecA…………………………………………………………………
85 4.6 High level secretion for any foreign protein in B. subtilis- prospects for a
general strategy……………………………………………………………………………...
88 5. Summary…………………………………………………………………………… 92 6. Zusammenfassung……………………………………………………………….. 94 7. References…………………………………………………………………………. 97 8. Appendix…………………………………………………………………………… 111
Contents
III
List of Figures
Fig. 1 Simplified model for the interaction of a signal peptide with a cell
membrane……………………………………………………………………………….
3 Fig 2 Classification of cleavable N-terminal signal peptides in B. subtillis……………… 5 Fig. 3 Schematic overview of the targeting of secretory proteins containing
a Sec-type signal peptide in B. subtilis……………………………………………….
7 Fig. 4 Plasmid map of the pBSMuL series………………………………………………….. 34 Fig. 5 Purification of His-Tag cutinase from B. subtillis culture supernatant…………….. 35 Fig. 6 Expression and secretion efficiency of cutinase in B. subtillis using
vectors pBSMuL1,2……………………………………………………………………..
36 Fig. 7 Stability of pBSMuL vectors in B. subtillis …………………………………………... 37 Fig. 8 Schematic overview about the hydrolysis-reaction which the assay
is based on…………………………………………...................................................
39 Fig. 9 Plasmid map of shuttle vector pBSMuL3……………………………………………. 40 Fig. 10 Growth of B. subtillis in DeepWell microtiter plates………………………………… 41 Fig. 11 The pipetting robot “TECAN workstation Genesis 200 Freedom”………………… 42 Fig. 12 Two different Software scripts providing the automated pipetting process………. 43 Fig. 13 Reproducibility and stability study of cutinase activity of B. subtillis
supernatant in the HTS …………………………………………………....................
44 Fig. 14 Fluctuations in cutinase activity due to transformation events…………………….. 45 Fig. 15 Schematic overview of amplification, cloning and screening of all
Sec-type signal peptides of B. subtillis ………………………………………………
46 Fig. 16 Overall composition of signal peptides fused to mature cutinase
and its computer analysis exercising SignalP 3.0…………………………………..
48 Fig. 17 Comparison of all 148 screened signal peptides used for export
of the heterologous cutinase in B. subtillis ………………………………………….. 49
Fig. 18 Immunodetection of cutinase in B. subtillis expression strain……………………... 50 Fig. 19 Analysis of the processing kinetics of cutinase precursor proteins
with different SPs in B. subtillis via pulse-chase experiment………………………
54 Fig. 20 Cutinase production by B. subtillis via secretion into the medium… 56 Fig. 21 Identification of the most efficient signal peptide in secretion
of the heterologous esterase EstCL1 in B. subtillis ………………………………...
58 Fig. 22 Composition of hybrid protein SP-AmyE-cutinase………………………………….. 60 Fig. 23 Screening results after saturation mutagenesis generated
in the C-region and +1 position of SP-AmyE fused to cutinase…………………..
63 Fig. 24 Processing kinetics of cutinase precursor protein with SP-AmyE
in B. subtillis …………………………………………………………………………….
64 Fig. 25 Screening results of saturation mutagenesis libraries generated in
the N-region of the SP-AmyE fused to cutinase……………………………………..
65 Fig. 26 Comparative analysis of export efficiency and calculated D-score for
chosen variants of SP-AmyE with amino acid exchanges in the N-region……….
66 Fig. 27 Construction and characterization of B. subtillis secretion
reporter strain MArc1…………………………………………………………………...
68 Fig. 28 The effect of overexpressed SecA for cutinase export in B.subtillis
marker strain Marc1…………………………………………………………………….
69 Fig. 29 Processing of precursor protein cutinase fused to SP-LipA in different
B. subtilis Marc1 strains………………………………………………………………..
70 Fig. 30 The effect of two B. subtilis SecA variants for the export level of cutinase………. 71 Fig. 31 Localisation of amino acid substitutions in the protein structure of
B. subtilis SecA that effected cutinase secretion …………………………………...
87 Fig. 32 Comparison of cutinase expressing B. subtillis strains in LB- and TB-medium
using the on-line light scattering measurement technique for shaken MTPs…….
89 Fig. 33 Idealized model of a general strategy to optimize secretion yields of
heterologous proteins…………………………………………………………………..
91 Fig. 34 Cassette mutagenesis to generate a library of the PPX-domain of SecA……….. 116 Fig. 35 Overview of the complete Gemini pipetting
Script Ulf_PnPP1_10_tiptouch_bis_10PL……………………………………………
116
Contents
IV
List of Tables Tab. 1 Various destinations of exoproteins and their export pathways
in B. subtilis……………………………………………………………………………...
4 Tab. 2 Protein production of commercial interest in different Bacillus species………….. 9 Tab. 3 Performed or planned strategies to overcome export bottlenecks
in B. subtilis……………………………………………………………………………...
14 Tab. 4 Bacterial strains and plasmids used in this study…………………………………… 16 Tab. 5 Oligonucleotides used in this study…………………………………………………... 18 Tab. 6 Relevant antibiotics used for growth selection……………………………………… 19 Tab. 7 Comparison of chosen screened signal peptides used for cutinase export……… 51 Tab. 8 Signal peptides not tolerated in B. subtillis when constitutively
expressed in front of heterologous cutinase………………………………………....
52 Tab. 9 Positions of SP-AmyE chosen for saturation mutagenesis………………………... 61 Tab. 10 Base composition in oligonucleotides mix to saturate the C-region
of SP-AmyE……………………………………………………………………………...
62 Tab. 11 Properties and applications of the pBSMuL vector series…………………………. 74 Tab. 12 Oligonucleotides used for PCR-amplification of all Sec-dependent
signal sequences from genomic DNA of B.subtilis 168……………………………
111 Tab. 13 Comparison of all screened signal sequences used for export of
heterologous cutinase in B. subtilis…………………………………………………...
113
Contents
V
Abbreviations
A Ampere aa Amino acid A. dest Distilled water Amp Ampicillin ATP Adenosine 5`-triphosphate bp Base pair BSA Bovine serum albumin °C Degree Celsius Cm Chloramphenicol CM Cell membrane Da Dalton DMSO Dimethyl sulfoxide DNA Desoxyribonucleic acid dATP Desoxyadenosine-5`-
triphosphate dCTP Desoxycytosin-5`-
triphosphate dGTP Desoxyguanosin-5`-
triphosphate dTTP Desoxythymidin-5`-triphosphate dNTP Desoxyribonucleoside
triphosphate DW-MTP Deepwell microtiter plate EDTA Ethylendiamine tetra acetic acid eP-PCR Error-prone PCR EtOH Ethanol Ffh Fifty four homologoue Fig Figure g Gram GRAS Generally recognized as safe h hour HTS High-throughput screening IPTG Isopropyl-β-D-thiogalactoside k Kilo kb Kilobases kDa Kilodalton Km Kanamycin L Litre M Molarity (mol/L) m Mili µ Micro MCS Multiple cloning site min Minutes n Nano NCBI National Center for
Biotechnology Information OD Optical density ON Over night orf Open reading frame ori Origin of replication p.A. Per Analyse
PAGE Polyacrylamid gel electrophoresis PCR Polymerase chain reaction PDB Protein database bank pNPC para-nitrophenyl-caproat pNPP para-nitrophenyl-palmitate pI Isolelectric points of proteins rbs Ribosome binding site
RNA Ribonucleic acid rpm Rounds per minute RT Room temperature scRNA Small cytoplasmic RNA sec Seconds SDS Sodium dodecyl sulfate SP(s) Signal peptide(s) SRP Signal recognition particle ss Signal sequence t time Tab. Table TBE Tris-Borat-EDTA TCA Trichlor acetic acid Tm Melting temperature TM Transmembrane segment Tris Tris(hydroxyl methyl) amino
methane U Units V Volt v/v Volume per volume w/v Weight per volume
Introduction
1
1. Introduction
1.1 The necessity of protein translocation in cells
The permanent adaptation is obligatory for the survival of living cells in a
continuously changing environment. Therefore the whole cell metabolism must be
strictly regulated to avoid a waste of energy resources. Cell membranes (CM) serve
as protective barriers to maintain composition and concentration of cytoplasmic
molecules, on the other hand they must allow and control the transfer of molecules
and proteins from and to the environment to enable essential events like energy
supply (e.g. chemical gradients or nutrition mobilization and uptake), cell division, cell
communication, enemy defence and DNA uptake. This dilemma is solved by a
membrane composition of amphiphilic lipids and transmembranel proteins. The lipids
form a flexible but nearly impermeable structure whereas proteins can build
membrane channels for regulated molecule and enzyme transport. While eukaryotic
cells contain a complex network of membranes separating different organelles,
bacteria belong to the prokaryotic organisms that show only a single cell
compartment surrounded by one or two membranes.
Nevertheless, protein transfer from the site of synthesis, the cytoplasm, to other cell
compartments or the exterior is a quite complex process even in bacteria (151, 178).
Due to the extended use of microorganisms in commercial production processes
many efforts have been undertaken to decipher their secretome. But despite the
increasing knowledge of bacterial protein export at molecular level continuously
documented by numerous publications over the last decades (33, 47, 62, 142, 144,
151, 167, 178, 188, 200, 220) there are still many secrets of the bacterial secretome
left behind building a platform for extensive future research studies.
Introduction
2
1.2 Protein sorting in cellular traffic- The crucial role of signal peptides
Proteins destined for the transport and secretion across the cytoplasmic membrane
in pro- and eukaryotes are synthesized as preproteins containing a similar N-terminal
amino acid extension, the so-called signal peptide (SP). The domain organization of
signal sequences shows a common pattern (Fig. 2). They consist of a positively
charged amino terminus (N-region) showing high affinity for phospolipids of CMs, a
central hydrophobic core (H-region) of certain length (10-12 residues) enough to
span a hydrophobic region of 35 Å in a β structure, and a more polar carboxyl-
terminal end (23, 58, 59, 173, 202) recognized by a signal peptidase. Each region
was shown to be essential for a functioning SP.
However, based on the primary structure of a SP its functionality is hardly predictable
except the change of essential parts or residues like the hydrophobic core or the
cleavage recognition site (59, 225). Several studies focussed on the conformation
and orientation of isolated SPs interacting with monophospolipids (22, 26, 34, 59,
123). In summary, a conformational change of a SP was found depending on the
environmental conditions: Most SPs form a random structure in aqueous solution,
adopt a β structure after interaction with a lipid phase without insertion, but prefer an
α-helical conformation after insertion into a lipid phase. The structural change of a SP
into an α-helix was proposed to result in the further insertion of 10 to 15 residues of
the mature exoprotein into the membrane as shown in Fig. 1.
Introduction
3
These findings demonstrate that the conformational flexibility is an important feature
of a SP to support the insertion of a protein into a CM. Furthermore, the SP and its
secondary structure influences the folding and therefore the targeting of a secretory
protein on the whole way to the CM (52, 145, 154).
During the targeting process along the general secretion pathway (Sec pathway) of
bacteria (151) or into the lumen of the endoplasmatic reticulum (ER) in most
eukaryotes (97) SPs interact with several components of the cell: (i) Binding to the
signal recognition particle (SRP) (127) or other cytoplasmic factors (see Fig. 3) acting
as intracellular chaperones. (ii) Binding to the membrane. (iii) Initiation and facilitation
of translocation. (iv) Recognition and cleavage by a signal peptidase (SPase) at the
trans-site of the membrane that are highly conserved processes as shown for
archaeal and eukaryotes SPs fully functional in bacteria (59, 149, 183). Due to the
Fig. 1: Simplified model for the interaction of a signal peptide with a cell membrane. A SP contains a positively charged N-region (N, highlighted in blue), a central hydrophobic region (H, marked in black) and the cleavage site recognized by a signal peptidase I (C, highlighted in red). The N-terminal SP is fused to the target protein (T) forming the precursor protein. (A) The SP is unstructured in the aqueous cytoplasm. As the initial step the positively charged N-region of a SP interacts with the negatively charged phospholipids of the membrane. The high tendency of the SP to insert into the membrane is indicated by black arrows. (B) While the N-region stays “fixed” at the cytoplasmic site of the membrane, the hydrophobic part of the SP continuously inserts into the membrane thereby forming an α-helical structure. (C) Due to this “threading” process the N-terminal part of the mature target protein is pulled through the membrane. The exposed C-region of the SP at the trans-site of the membrane is recognized and cleaved by a signal peptidase I (coloured in green). For clarity cytoplasmic targeting factors (e.g. SRP, SecA, SecB) or other components essential for protein secretion are not considered in this model.
A B
T
++N
CH
C
cytoplasm
extracellular
cell membrane
++N
T
T
++N
C
H
SPase I
H
C
Introduction
4
involvement in all stages of protein export from synthesis until membrane crossing,
SPs can be regarded as zip codes essential for the selective protein targeting.
Stimulated by the relevance of SPs for protein export in microorganisms, prediction
tools have been developed (58) with SignalP being the most popular and user-
friendly program, which is freely accessible via the world wide web
(http://www.cbs.dtu.dk/service/SignalP/) (9, 135). The SignalP 3.0 server is able to
predict the likelihood of a particular amino acid sequence to act as a signal peptide or
not by calculating a discrimination score termed D-score. Sequences leading to
values above 0.5 are classified as signal peptides. Sequences showing calculated D-
scores of > 0.7 are signal peptides with the highest probability.
1.3 Signal peptide classification in B. subtilis
B. subtilis, a Gram-positive endospore forming soil bacterium, is ubiquitous and
developed to a paradigm for Gram-positive bacteria due to intensive research over
the last decades. Even if B. subtilis shows a relatively simple cell organisation, non-
cytoplasmic proteins can be addressed to various destinations via different pathways
depending on the presence or absence of a SP and/or retention signals (Table 1).
Table 1: Various destinations of exoproteins and their export pathways in B. subtilis. Abbreviations are TM, transmembran segments; CWB, cell wall binding repeats.
destination of
exoprotein signal peptide retention signal export pathway
Sec-type - Sec pathway
Tat-dependent - Tat pathway (90) extracellular
medium leader peptide - ABC transporters (119)
pseudopilin-SP TM Com pathway (30, 31)
Sec-type TM trans-site of
membrane Sec-type lipobox
cell wall Sec-type CWB
Sec pathway
Introduction
5
In contrast to Gram-negative bacteria secretory proteins have to overcome only one
CM in B. subtilis before being released into the extracellular medium. By using the
SignalP prediction tool, Tjalsma et al. were able to predict essentially all secreted
proteins in the Gram-positive bacterium Bacillus subtilis (188). The classification of
Fig. 2: Classification of cleavable N-terminal signal peptides in B. subtilis modified according to Tjalsma et al. (187, 188). The predicted SPs were divided into five distinct classes on the basis of SPase cleavage site and the export pathways via which the preproteins are exported. Amino acids (aa) are shown in the one-letter code. “X” is defined as any aa. The N-, the H- and the C-regions (if known) of a SP are distinguished by blue, white and red coloured boxes, respectively. The N-terminal part of the mature protein is indicated as a grey coloured open box. SP cleavage sites are indicated by a black arrow. (A) Sec-type SPs, precursors are targeted across the CM by the general secretion pathway. The cleavage site is recognized by one of the five type I SPase. (B) Sec-type lipoprotein SPs contain a so-called lipobox with the consensus motif L-X-X-C. After transport across the CM via the Sec pathway, the Cys of the lipobox is modified by lipids, before the type II SPase cleaves the peptide bond in front of the Cys which is afterwards attached to an additional acyl group serving as a membrane anchor in the CM. (C) The proteins ComGC, ComGE, ComGD and ComGG are required for DNA binding and uptake during competence and contain prepilin-like SPs. These are targeted by the Com pathway completely bypassing the Sec pathway. Since the C-region is located next to the N-region, the H-region stays fused to the mature protein after cleavage by ComC, a Com specific SPase. (D) N-terminal SP of bacteriocins and pheromones are often called leader peptides and lack a H-region. So far three secretory proteins, the bacteriocins sublancin 168, subtilosin and the pheromone ComX were identified. The leader peptides prevent premature activity and provide secretion via ABC transporters. (E) Several exoproteins were identified containing a Sec-type SP with the consensus motif R-R-X-#-# indicating the potential to be secreted by the Tat pathway. So far, only two proteins, PhoD and YwbN, showed a Sec-independent export by the Tat machinery.
Sec-type signal peptidesA
N H
++ AXA A
SPase I
C +1
C +1
Sec-type lipoprotein signal peptidesB
++ LXX C
SPase II
HN
++ R-R-X-X- # - # AXA
C +1
SPase I
N H
Twin-arginine signal peptidesE
Prepilin-like signal peptidesC
EF
ComC
H N C +1
KG
D Bacteriocin and pheromone signal peptide
SunT, AlbE/F
N C +1
Sec pathway
Com pathway
ABC transporter
Tat pathway
Introduction
6
SPs into five distinct groups is based on the export pathway and the SPase cleavage
sites as shown in Fig. 2. Regarding the relevance of each SP class for the complete
secretome in the following this study will focus only on the Sec-type SPs recognized
and targeted by the general secretion pathway which is described in detail in the next
chapter.
1.4 The Sec pathway as the major export route
Next to secretion by ABC transporters or via the Tat pathway, the majority of the
predicted ~300 potential secretory proteins in B. subtilis use the Sec pathway,
therefore called the general secretion pathway (188, 195), even if a proteomic
analysis of B. subtilis culture supernatant showed a discrepancy between genome-
based prediction and the experimentally detected secretome: For several proteins no
secretion was predicted due to a lack of a SP or cell retention signals. Surprisingly,
these proteins were found in the growth medium instead of at the predicted sites in
the cytoplasm or bound to the CM. These results imply the presence of additional
mechanisms for protein release into the extracellular medium like protein shedding
(proteolysis or inefficient retention) for membrane proteins and lipoproteins (5). The
occurrence of cell wall-bound proteins in the extracellular medium might be a result
of cell wall turnover, cell lysis or even a so far unknown secretion pathway as
discussed for protein release lacking a SP (187).
However, this study focusses on the Sec pathway in B. subtilis, the main road for
export proteins, which is most likely co-translational, since no clear evidence of a
SecB homologon or analogon to E. coli was described (187, 220). SecA, the
preprotein translocase ATPase, is the central motor protein of the Sec pathway,
interacting with most of the contributing components like acidic phospolipids of the
CM, the SecYEG proteins which form the membrane pore, intracellular chaperones,
the signal peptide as well as the mature protein of a precursor (44, 126, 144, 205).
The translocation process is driven by the SecA protein through multiple rounds of
ATP binding and hydrolysis. SecA alternates between “the inserted” and “deinserted”
state, resulting in processive translocation along the precursor substrate and export
of the translocating protein across the membrane in steps of 20-30 amino acids (175,
192).
Introduction
7
Whether SecA functions as a monomer or a dimer during the translocation process
has been the subject of considerable controversy (40, 42, 76, 89). Depending on
--- ---
----
- -
scRNA
HBsu
Ffh
SP
ribosome
mRNA
cytoplasm
cell membrane
cell wall
extracellular
FtsY
Sec
A
FtsY
SecA
Sip
SecYEG
SecD
F
Sp
pA
TepA
PrsA
BdbB/C
Yq
jG
Sp
oIIIJ
proteases
mature exoprotein
--- ---
----
- - --- ---
----
- ---- ---
----
- -
--- ---
----
- ---- ---
----
- -WprACa2+ Ca2+
Fe3+ Fe3+
Fe3+
Ca2+
1 2
3
4
Fig 3: Schematic overview of the targeting of secretory proteins containing a Sec-type signal peptide in B. subtilis . The presented model summarizes findings reported in various publications (131, 136, 181, 187-189, 195, 220, 221, 227) and does not claims for completeness. (1) Ribosomally synthesized exoproteins are directly bound at the exposed N-terminal SP by the Ffh protein to guarantee a translocation-competent state in an (partially) unfolded conformation. Ffh is next to HBsu and scRNA part of the signal recognition particle (SRP)-complex providing co-translational translocation. (2) The SRP-precursor complex interacts with the SecA protein via binding to the SP. (3) The SRP-precursor-SecA complex is targeted to the membrane embedded translocase consisting of the pore forming proteins SECY, SecE, SecG and accessory proteins SecDF, SpoIIIJ and YqjG. After cleavage of the SP at the trans-site of the membrane by one of the SPases I (SipS-W), the precursor protein is pushed step by step through the Sec-pore by SecA energized mainly by ATP and supported by the proton motif force. The FtsY protein can bind to Ffh and is required for release of the SRP from the membrane to ensure the next targeting cycle. (4) Whereas the SP is degraded by one of the two SPPases SppA and TepA, in the late stage of secretion the processed exoprotein must pass through cell wall thereby undergoing quality control by proteases like the cell wall associated WprA and folding to its native conformation supported by the extracellular folding catalyst PrsA, a lipoprotein anchored into the CM. Extracytoplasmic thiol-disulfide oxidoreductases like BdbB and BdbC provide disulfide bond formation if required. The negatively charged cell wall environment harbours several proteases and huge amounts of Ca2+- and Fe3+-ions, which are necessary to stabilize some exoproteins.
Introduction
8
temperature and salt concentration, the occurrence of both, SecA monomers and -
dimers in solution, was reported (212).
Interestingly, even if the common assumption is that SecA is active as a dimer, a
recent study reported the full functionality of only monomeric SecA for the cell (138).
The complete process can be subdivided into the early stage of secretion comprising
the targeting to the membrane by several intracellular chaperones including SecA (1-
2), the actual translocation process across the membrane (3) and the late stage of
secretion including extracellular folding, degradation and release of the target protein
into the environment (4). A more detailed description is given in figure 3.
1.5 Industrial use of Bacillus species
Long before useful genetic techniques for strain improvement were developed,
B. subtilis and its relatives were used in traditional fermentation processes (63). An
important feature of this diverse group of organisms is a tremendous protein export
capacity up to 25 g/L for homologous proteins (81, 82) allowing a direct enzyme
transport into the cell-surrounding environment avoiding intracellular accumulation.
The phenomenon of protein accumulation occurs during protein overexpression
especially in Gram-negative bacteria in the cytoplasm as well as in the periplasm and
can lead to toxicity problems to the cells depending on the kind of product (8). Yet, as
another positive effect of the protein export, the genus Bacillus bypasses the
reducing conditions in the cytoplasm that might support formation of misfolded
proteins and inclusion bodies (124). The secretion is a natural separation step
simplifying further purification processes of the product. In contrast to Gram negative
bacteria, B. subtilis and other representatives like Bacillus licheniformis or Bacillus
amyloliquefaciens are classified as endotoxin free microorganisms by the Food and
Drug Administration and therefore generally recognized as save (GRAS) what makes
them preferred candidates for commercial applications especially in the food industry.
Additionally, Bacillus species show easy culturing conditions and high cell growth
during fermentation processes. In consequence since the early 60es several Bacillus
spp. were used and further improved for the fermentative production of primary
homologous hydrolytic enzymes. In 2004, the world market for industrial enzymes
was estimated to be 1,6 billion $US with increasing tendency. Between 50% and
Introduction
9
60% of the commercially available enzymes are produced by Bacillus species (172).
Two of the most important bulk enzymes are alkaline proteases (subtilisins) used in
household detergents and amylases used in industrial applications (143). Table 2
gives an overview of homologous and heterologous protein production in various
Bacillus strains with respect to industrial applications.
Table 2: Protein production of commercial interest in different Bacillus species. In the case of B. subtilis as the production host the corresponding column is shaded in grey.
1.6 Export bottlenecks for foreign recombinant proteins in B. subtilis
Despite this dominant position in industrial enzyme production it is remarkable that
E. coli is still preferred over Bacillus species for the commercial therapeutic
production (160). Although a variety of heterologous proteins could be successfully
produced in Bacillus species on laboratory scale (see also Table 2), there still exists
product application origin production
host reference
alkaline protease detergents B. subtilis/licheniformis B. subtilis/
licheniformis (155)
alkaline amylase detergents B. licheniformis B. licheniformis (172) α-amylase starch degradation B. licheniformis B. licheniformis (172) pullulanase starch degradation B. halodurans B. halodurans (129) glucose isomerase starch processing B. coagulans B. coagulans (172)
CGTase starch conversion to cyclodextrins
B. firmus B. firmus (57)
β-glucanase glucan modification B. subtilis B. subtilis (17) xylanase food processing B. subtilis B. subtilis (103) cellulase cellulose degradation B. subtilis B. subtilis (75) chitinase chitin degradation B. thuringiensis B. thuringiensis (185)
levansucrase Hydrolase/transferase activity for
B. circulans B. circulans (140)
esterase lipolytic degradation B. circulans B. circulans (91, 92) growth hormon medicin human B. subtilis (64) interleukin-1beta medicin human B. subtilis (172) proinsulin medicin human B. subtilis (172) penicillin G acylase medicin B. megaterium B. subtilis (222) cyclic oligopeptides antibiotic B. licheniformis B. licheniformis (95) basic peptides antibiotic Brevibacillus brevis Brevibacillus brevis (150) endotoxin biodegradable insecticide B. thuringiensis B. thuringiensis (172) purine nucleotides medicin, flavour enhancer B. subtilis B. subtilis (172) riboflavin vitamin ingredient B. subtilis B. subtilis (37)
D-ribose flavor enhancer B. subtilis, B. pumilus B. subtilis,
B. pumilus (39)
2-acetyl-1-pyrroline popcorn flavour B. cereus B. cereus (162) polyhydroxybutyrate biodegradable plastics bacterial B. megaterium (74) streptavidin biotin-binding protein Streptomyces species B. subtilis (214) antigen displaying spore oral vaccination tetanus toxin fragment C B. subtilis (45)
Introduction
10
a clear reservation to use Bacillus in their industrial applications. Even for the model
organism of Gram positive bacteria B. subtilis, whose complete genome is available
since 1997 (104) and diverse DNA recombination methods led to improved
production strains over the last years, several bottlenecks still limit the universal
application of B. subtilis for heterologous protein production. An overview is given
in Table 3.
(i) Since the phenomenon of plasmid instability for replicating vectors was
reported for Bacillus species, much more integration vectors have been
constructed which show stable integration in the chromosome (25). But the
clear disadvantage of these one-copy plasmids is the lower expression
rate in contrast to replicating plasmids (~50 copies per cell).
(ii) The problem of plasmid instability can partly explain the lack of versatile,
stable replicating expression vectors for B. subtilis (208) comparable to the
various available, user-friendly vector systems in E. coli (e.g. pUC-Vectors,
or the pET-vectors series commercialised by Novagen, Madison, USA).
(iii) The Signal peptides, labelling proteins to be secreted, accompany them all
the way to the CM thereby getting in contact to most of the Sec
components (see Fig. 3). Thus SPs play one of the most important roles in
the efficient translocation across the membrane. Numerous secretion
studies demonstrated a key role of SPs for efficient production of
homologous and heterologous proteins in B. subtilis (13, 32, 55, 58, 70,
111, 112, 130, 137, 164, 210, 225).
(iv) Interestingly, even for the secretion of foreign proteins the formation of
inclusion bodies can be a limiting factor as reported for the antidigoxin
single-chain antibody (SCA) production in B. subtilis (215, 217), although
the intracellular concentration should continuously be reduced by the Sec
machinery.
Introduction
11
(v) Another critical factor for protein export is the sufficient processing of
precursors at the outer site of the membrane by one of the five type I signal
peptidases (5, 187, 188).
(vi) At least 8 extracellular proteases are expressed by B. subtilis resulting in
proteolytic degradation of particularly heterologous products (208). These
foreign proteins are more accessible for extracellular proteases than
homologous enzymes due to their very slowly folding process into the
mature conformation.
(vii) A frequent problem is the misfolding of foreign exoproteins after the
translocation across the CM as a consequence of missing or insufficient
chaperone activity or their misfolding after translocation. (11, 20, 83, 101,
102). The misfolding of heterologous proteins, that contain structural
relevant disulfide bridges, represent a potential bottleneck as shown for the
secretion of a staphylokinase fusion with the fibrin-targeting Kringle domain
of human plasminogen, for the secretion of human pancreatic α-amylase
(HPA) as well as for secretion of human serum albumin (12, 171, 213).
(viii) Before processed proteins finally reach the culture medium, they must
pass through the 10-50 nm thick cell wall. It is composed of peptidoglycan
and anionic polymers forming a negatively charged environment thereby
expose affinity to especially positively charged secretory proteins. These
can interact with the cell wall leading to low yields in the culture
supernatant like shown for the human serum albumin, an α-amylase from
B. licheniformis and a recombinant B. anthracis protective antigen (171,
180, 186).
1.7 Strategies to bypass the limitations in protein production
For all listed reasons (1.6) the well studied E. coli expression systems are still mostly
used in large scale production of heterologous proteins. Meanwhile various projects
have been started to enhance the productivity of B. subtilis for foreign proteins:
Introduction
12
(i) One possibility to overcome the problem of plasmid instability might be the
deletion of the responsible BsuM restriction-modification system in
B. subtilis as done in a previous study (207). This system was found to be
responsible for the limitation of using plasmids for high level production
(60).
(ii) Over the last years, some novel plasmids were constructed with different
and improved properties and applications to extend the range of available
Bacillus expression systems (106, 108, 147).
(iii) The export inefficiency for foreign products could be improved
conspicuously by various modifications of the SPs: For instance a study of
Hemilä et al. showed that a modified junction between SP and a
heterologous pectinase from Erwinia carotovora yielded in a high level
production of 0,8 g/L protein (68). Furthermore, Saunders et al. changed
the C-region of a SP from a B. amyloliquefaciens neutral protease in a way
to increase export of a 34 aa fragment of human parathyroid hormone
(170). A structural modified SP α-amylase was the reason for a
significantly enhanced secretion of the β-lactamase (132).
(iv) However, another approach to enhance the secretion yield in B. subtilis
was the additional overexpression of intracellular chaperones GroE and
DnaK. It decreased the insoluble amount and increased the secreted
amount of the antidigoxin single-chain antibody (scFv) in B. subtilis
significantly (215).
(v) Limitations in the crucial processing step can be decreased by an
overexpressed SPase I like shown in a previous study (193). But enhanced
processing does not inevitably result in enhanced amount of secreted
protein like observed in a study by Vitikainen et al. (198).
(vi) Several B. subtilis strains were constructed showing low proteolytic activity
by elimination of extracellular proteases and could be used in successful
production of some heterologous proteins, which were sensitive to
Introduction
13
proteolytic degradation (96, 211, 214, 216, 217, 223). On the other hand,
different studies reported undesirable side effects of the inactivation of
proteases like increased cell lysis and growth reduction in protein-rich
industrial media what might lower the production yield (161, 179). A
change of growth temperature or pH variation of the medium can also help
to reduce proteolytic activity (208).
(vii) The occurrence of misfolded proteins often results in massive degradation.
Yet the single overexpression of extracellular chaperon PrsA led to an
increase of export yield of a protease and an α-amylase (102, 198). A
simple overexpression strategy applied for the thiol-disulfide
oxidoreductases BdbB and BdbC to reach a better disulfide bond formation
might be a successful strategy, but needs to be tested in the future.
(viii) To overcome the problem of protein retention in the cell wall due to a high
pI (> 9) protein variants can be engineered bearing equal enzymatic
properties but a lowered net charge resulting in a better release into the
culture medium as described in a previous study (180).
(ix) Since the whole genome sequence of B. subtilis is available, a totally
different approach to overcome production limits in B. subtilis is the
minimization of the chromosome as reported for E. coli (100). However, of
the total amount of ∼ 4100 protein encoding genes in B. subtilis only 270
genes were revealed to be indispensable for growth in rich medium at
37°C (99). The idea is the elimination of all unnecessary gene products to
avoid useless gene expression what might prevent a maximal production
yield for the protein of interest or lead to enhanced cost due to additional
contaminations of the product.
Table 3 summarizes the known secretion problems for B. subtilis and shows potential
solutions.
Introduction
14
Table 3 : Performed or planned strategies to overcome export bottlenecks in B. subtilis. Improved/not improved production yields of correctly folded protein are indicated by a “+” or a”-“. A”?” indicates a strategy that still needs to be tested. “+/-“ indicate both positive and negative effects of an applied strategy in different studies.
1.8 Scope of this thesis
Because several bottlenecks still limit the production for heterologous proteins in the
Gram-positive host B. subtilis, this work focusses on the optimization of secretion
capacity by different strategies:
(i) In the broad field of biotechnology the availability of useful Bacillus expression
plasmids is limited. Especially high throughput screening processes require
user-friendly cloning and expression strategies. Therefore a clear preference
exist for the Gram-negative bacterium E. coli as expression host. To overcome
this dilemma, several useful Bacillus plasmids should be constructed and
bottleneck strategy resulting
production yield stage of
secretion
(i) plasmid instability deletion of BsuM-rstriction-modification system ?
(ii) lack of expression vectors construction of new plasmids +
(iii) “wrong” signal peptide
change or modification of the signal peptide +/-
lowering the growth Tm (iv) inclusion bodies overexpression of intracellular
chaperones GroE and DnaK +
early
(v) inefficient processing
overexpression of Sip proteins +/-
construction of protease deficient strains +
(vi) extracellular proteases adaptation of Tm or pH to
reduce protease activity +/-
overexpression of extracellular chaperone PrsA +
(vii)
misfolding of exoproteins/ inefficient disulfide bond formation
overexpression of extracellular Bdb proteins ?
(viii) retention to cell wall engineering of protein variant with a lower pI +
late
(ix) unwanted site products
deletion of relevant genes- minimizing the genome ?
Introduction
15
tested with respect to (a) non inducible, optional intracellular expression, (b)
secretion, (c) easy purification and (d) application for HTS processes.
(ii) Since highest secretion efficiencies up to 20 g/L are obtained only with
homologous proteins from Bacillus species fused to the original signal peptide
(SP), one major limitation in secretion of foreign proteins is caused by using
the wrong SP resulting in inefficient and unsatisfying low yields.
Hence the main goal of this work was to solve this secretion handicap by a so
far new strategy. The idea was a systematic approach to identify the optimal
SP in a pool of all natural B. subtilis SPs with respect to its heterologous
secretion partner. The first step to realize this project was the generation of a
functional secretion reporter system in B. subtilis using a heterologous
enzyme of biotechnological relevance. Consequently, this system must allow
testing of different SPs fused to a heterologous marker enzyme for secretion
efficiency. Next, this system should be adapted to a high throughput screening
(HTS) format in B. subtilis enabling a general fast SP-optimization for export of
any target protein. Once established, this HTS system can form the basis to
use various mutagenesis techniques as an alternative way to improve the
functionality of a SP.
(iii) Another target for optimization was SecA, the central motor protein of the
general secretion pathway. Since translocation across the CM strongly
depends on the efficient formation of a complex of SecA and the precursor
proteins, variants of SecA should be constructed by Directed Evolution and
screened in the generated HTS system for improved export of a secretion
reporter.
Materials and Methods
16
2. Materials and Methods
2.1 Chemicals and enzymes
All used chemicals and enzymes were obtained in p.a. quality from the following
companies:
Antibiotics: Serva (Heidelberg), Sigma-Aldrich (Taufkirchen)
Chemicals: Fluka (Sternheim), Merck (Darmstadt), Roth (Karsruhe), Sigma-Aldrich
(Taufkirchen), DIFCO (Detroit, USA), Roche (Grenzach, Germany)
Enzymes: Lysozyme (Sigma-Aldrich), Pfu-DNA Polymerase (Stratagene,
Heidelberg), TurboPfu-polymerase (Stratagene, Heidelberg, Germany), Taq-DNA
polymerase (MGI Fermentas (St. Leon-Roth, Germany), Taq-Goldstar Polymerase
(Stratagene, Heidelberg, Germany), restriction enzymes (MGI Fermentas (St. Leon-
Roth), T4 DNA Ligase (MGI Fermentas, St. Leon-Roth), T7 DNA Polymerase (MGI
Fermentas, St. Leon-Roth),
2.2 Bacterial strains and plasmids
The strains and plasmids used in this study are listed in Table 4.
Table 4: Bacterial strains and plasmids used in this study.
Strains or plasmids Genotype/ Characteristics Source or reference
Strains
E. coli JM109
e14-(McrA-) recA1 endA1 gyrA96 thi-1 hsdR17(rK- mK+)
supE44 relA1 D(lac-proAB) [F’ traD36 proAB lacIqZDM15]
Stratagene, Heidelberg
B. subtilis 168 trpC2 (104)
B. subtilis DB430 his nprE aprE bpf ispI (41)
B. subtilis TEB1030 B. subtilis DB430 lipA lipB (48)
B. subtilis Marc0 B. subtilis TEB1030 amyE::pMCut This study
B. subtilis Marc1 B. subtilis TEB1030 amyE::pMCut1 This study
Plasmids
pMA5 ColE1 repB Kmr Ampr P HpaII (228)
pDG268 spoVG-lacZ Ampr Cmr (6)
pDG268-∆lacZ
pDG268 plasmid EcoRI / BpU1102I restricted and religated
after blunting; thereby, deleting the spoVG-lacZ fragment
This study
Materials and Methods
17
Plasmids Characteristics Source or reference
pMCut pDG268-XY containing a PHpaII-cutinase-fusion This study
pMCut1
pMCut containing a HindIII / EcoRI fragment coding the
B. subtilis lipase A signal sequence (sslipA), thereby
generating an in-frame fusion of sslipA and cutinase under
control of PHpaII
This study
pBSMuL1
B. subtilis – E. coli shuttle vector for overexpression
(ribosome binding site, PHpaII ) , secretion (sslipA) and
purification (C-terminal 6x-His-Tag); ColE1 repB Kmr Amp
r
This study
pBSMuL1-Cut pBSMuL1 containing a 662 bp EcoRI-XhoI fragment of
Fusarium solani pisi cutinase in frame with H6-Tag
This study
pBSMul2 pBSMuL1 vector with a second constitutive promoter P59 This study
pBSMuL2-Cut pBSMuL2 containing a 662 bp EcoRI-XhoI fragment of
Fusarium solani pisi cutinase in frame with H6-Tag
This study
pBSMuL3
pBSMuL1 derivative without ribosome binding site and
without sslipA; inverted MCS
This study
pBSMuL3-Cut
pBSMuL3 containing a 655 bp EcoRI-BamHI fragment of
Fusarium solani pisi cutinase without signal sequence
This study
pBSMuL3-Cut-ss
pBSMuL3-Cut containing an in-frame fusion of all Sec-
dependent signal sequences of B. subtilis ; ss is an
abbreviation for the paticular signal sequence; according to
the 174 different signal sequences of B. subtilis, 174
different pBSMul3-Cut-ss plasmids were constructed
This study
pBSMuL3-Cutintra pBSMuL3 containing a 655 bp HindIII-BamHI fragment of
Fusarium solani pisi cutinase without signal sequence This study
pBSMuL3-EstCL1
pBSMuL3 containing a 964 bp EcoRI-BamHI fragment of a
metagenome lipase (EstCL1) without signal sequence
This study
pBSMuL3-EstCL1-ss
pBSMuL3-Cut containing an in-frame fusion of all Sec-
dependent signal sequences of B. subtilis comparable to
pBSMuL3-Cut-ss
This study
pBSMuL3secA
pBSMuL3 containing a 2500 bp XhoI / BamHI fragment
encoding secA∆SacI of B. subtilis
This study
pBSMuL3secA-ep
pBSMuL3 containing 2500 bp XhoI / BamHI fragments
encoding ep-PCR-mutated secA of B. subtilis
This study
2.3 Oligonucleotides and PCR-primers
All listed oligonucleotides were ordered online from Thermo Electron Corporation at
website “http://www.thermohybaid.de/cgi-bin/oligos-new/order.py” and were obtained
Materials and Methods
18
as high purified salt free and lyophilized. The primers were dissolved in A. dest
resulting in a concentration of 50 pmol/µl and stored at –20°C.
Table 5: Oligonucleotides used in this study*. Restriction sites are highlighted in bold letters, start or stop codons are underlined. Wobbled bases (N = equimolar mix of all dNTPs; S = equimolar mix of dCTP and dGTP) are marked in italics.
Primer Sequence 5´ →→→→ 3´ Modifications
Cut1 ATATGAATTCGCGCCTACTAGTAACCCTGCT EcoRI
Cut2 ATATCTCGAGAGCAGAACCACGGACAGCCCG XhoI
Cut_low ATATGGATCCTCAAGCAGAACCACGGACAGC BamHI,
Cut_up ATATAAGCTTATTTGAATTCGCGCCTACTAGTAACCCTGCT HindIII, EcoRI
cutintra-up ATATAAGCTTAAGGAGGATATTATGGCGCCTACTAGTAACCCTGCT HindIII
Cut_down ATATGCATGCATATGGATCCTCAAGCAGAACCACGGACAGC PaeI, BamHI,
hpaII_up ATATGCATGCTTAAAGGTGGAGATTTTTTGA PaeI,
hpaII_down ATATAAGCTTCCCTGATTTCACTTTTTGCATTCT HindIII
EstCL1_up ATATGAATTCATGACCGATCCCTATGTGCGC EcoRI
EstCL1_low ATATGGATCCTCATGCTTCTGCCATAACCCC BamHI,
PPX_up ATTTCTGGACAAGCTGCAAAA -
PPX_down CAACGTCATGCTTTCGTTTTG -
secA_up ATATCTCGAGCTCTAAAATAGGCGTGTGATGATAGAGGAGCGTTATAA
ATG
XhoI,
secA_low ATATGGATCCATGGTACCTCAGTAAACTTGCCGGGGCGAACTA BamHI,
secA∆SacI-up GATGAAATGCTTGAACTCATTATGGATCGC
secA∆SacI-low GCGATCCATAATGAGTTCAAGCATTTCATC
silent base exchange to delete internal SacI restriction site in the secA gene
U1 TAATATATTAATAAGGAGGACATATGAAATTTGTAAAAAGAAGGATCAT
TGCACTTGTAACAATTTTGAT
VspI
U2 GCTGTCTGTTACATCGCTGTTTGCGTTGCAGCCGTCAGCAAAAGCCGC
CGAATTCGGTACCGTCGACCCC
-
U3 GGGAAGCTTGCGGCCGCGATATCTCTCGAGCACCACCACCACCACCA
CTGAATTAATATAT
VspI
L1 TGCAATGATCCTTCTTTTTACAAATTTCATATGTCCTCCTTATTAATATA
T VspI
L2 GGCTGCAACGCAAACAGCGATGTAACAGACAGCATCAAAATTGTTACA
AG -
L3 CCGCAAGCTTCCCGGGGTCGACGGTACCGAATTCGGCGGCTTTTGCT
GAC -
L4 TAATATATTAATTCAGTGGTGGTGGTGGTGGTGCTCGAGAGATATCGC
GG
VspI
AmyE-up ATATAAGCTTAAGGAGGATATTATGTTTGCAAAACGATTCAAA HindIII
AmyE-2NNS-up ATATAAGCTTAAGGAGGATATTATGNNSGCAAAACGATTCAAA HindIII, Phe2 saturated
AmyE-3NNS-up ATATAAGCTTAAGGAGGATATTATGTTTNNSAAACGATTCAAAACC HindIII, Ala3 saturated
AmyE-4NNS-up ATATAAGCTTAAGGAGGATATTATGTTTGCANNSCGATTCAAAACCTCT HindIII, Lys4 saturated
AmyE-5NNS-up ATATAAGCTTAAGGAGGATATTATGTTTGCAAAANNSTTCAAAACCTCT
TTA HindIII, Arg5 saturated
AmyE-6NNS-up ATATAAGCTTAAGGAGGATATTATGTTTGCAAAACGANNSAAAACCTCT
TTACTG
HindIII, Phe6 saturated
AmyE-7NNS-up ATATAAGCTTAAGGAGGATATTATGTTTGCAAAACGATTCNNSACCTCT
TTACTGCCG
HindIII, Lys7 saturated
Materials and Methods
19
Primer Sequence 5´ →→→→ 3´ Modifications
ssamyE-low ATATGAATTCAGCAGCACTCGCAGCCGCCGGTCC EcoRI
ssamyE
(+1NNS)– low ATATGAATTCSNNAGCACTCGCAGCCGCCGGTCC EcoRI, +1 position
saturated
P59-up ATGGCTTGACAGGGAGAGATAGGTTTGATAGAATATAATAGTTGTCGC
GGAAGCCATCCATGAAG BstXI
P59-low CTTCTACCGAACTGTCCCTCTCTATCCAAACTATCTTATATTATCAACA
GCGCCTTCGGTAGGTA BstXI
*All oligonucleotides used for PCR-amplification of all Sec-dependent signal sequences from genomic DNA of B. subtilis 168 are listed in the Appendix.
2.4 Culture media and growth conditions
LB-medium: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl
LB-agar: LB-medium,15 g/L agar
TB-medium: 5g/L glycerol, 12 g/L tryptone, 24 g/L yeast extract, 12,54 g/L K2HPO4,
2,31 g/L KH2PO4 (pH7)
If not stated elsewhere the following growth conditions were applied:
E. coli cells were grown at least for 16 hours in selective LB-medium at 37°C.
B. subtilis precultures were incubated overnight in selective LB-medium at 37°C and
diluted 1:1000 to inoculate expression cultures. The expression B. subtilis cultures
were grown for 20 hours in selective LB-medium at 37°C.
Tab. 6: Relevant antibiotics used for growth selection
substrate final concentration [µg/mL] organism
ampicillin 100 E. coli
kanamycin 50 B. subtilis
chloramphenicol 5 B. subtilis
Materials and Methods
20
2.5 Storage of bacteria
E.coli and B. subtilis strains grown on agar plates were stored at 4°C and RT,
respectively. For longer storage 1,3 mL of a liquid culture was mixed with DMSO
(8 % v/v end concentration) and stored at -80°C.
2.6 Isolation of nucleic acid
Isolation of plasmid DNA was prepared by using the plasmid purification kits from
QIAGEN (Hilden, Germany). Genomic DNA of bacteria was prepared by using the
Genomic-tip 20/G-Kit from QIAGEN (Hilden, Germany) under conditions
recommended by the manufacturer.
2.7 Agarose gel electrophoresis
Agarose gel electrophoresis for DNA analysis and DNA isolation was carried out in a
i-Mupid mini gel system (Eurogentec, Köln, Germany) as described by Sambrook et
al. (165). For DNA electrophoresis 0,5x TBE buffer was used (89 mM Tris, 89 mM
Boris acid, 2,5 mM Na2-EDTA). An Eagleeye II (Stratagene, Heidelberg, Germany)
was used for visualisation and documentation of DNA. For purification of DNA
fragments out of agarose gels, the QIAEX II kit (Qiagen, Hilden, Germany) was
applied according to the protocol provided. The “Gene Ruler 1 kb DNA Ladder” from
Fermentas (St. Leon-Rot, Germany) was used as a molecular weight standard.
2.8 In vitro recombination of DNA Restriction reactions, blunting ends and ligation reactions were performed using
enzymes from Fermentas (St. Leon-Rot, Germany) under conditions recommended
by the manufacturer.
Materials and Methods
21
2.9 Transformations of bacteria
2.9.1 Chemical transformation of E. coli
Preparation of transformation competent E. coli cells and transformation of plasmid
DNA was performed according to the RbCl2 method of Hanahan (61).
2.9.2 Electroporation of E.coli
E. coli strain JM109 was used for electroporation in a MicroPulser (BioRad,
München, Germany). The preparation of electrocompetent cells and the
electrotransformation was performed following the manufacturer`s protocol (BioRad,
München, Germany).
2.9.3 Transformation of B. subtilis
Transformation of B. subtilis cells was conducted by the following procedure (4, 224):
the B. subtilis culture was grown in Paris-Medium until it reached a cell density of
OD580 = 1. The culture was divided into 0.5 mL aliquots, plasmid DNA (0.1-1 µg) was
added and the samples were incubated for 1-3 hours at 37°C. Selection for plasmid
bearing Bacillus cells was provided on LB-agar plates containing the suitable
antibiotic. For efficient DNA uptake of pBSMuL vectors by B. subtilis, the plasmid
DNA was SacI digested and self-ligated to generate plasmid multimers before adding
to the competent cells.
Paris-medium: 60 mM K2HPO4, 40 mM KH2PO4, 3 mM Na3-citrate, 1% (w/v) glucose,
20 mM K-glutamate, 2,2 mg/L Fe(III)NH4-citrate, 0,1% (w/v) casamino acids, 20 mg/L
tryptophan, 3 mM MgSO4
Materials and Methods
22
2.10 Polymerase chain reaction (PCR)
2.10.1 Standard PCR
Amplification of DNA-fragments was performed under standard PCR-conditions in a
reaction mix containing genomic or plasmid DNA (~1 ng) as the template, primer
oligonucleotides (each 50 pmol), dNTP’s (0.2 mM each), Taq- (2.5 U), Pfu-(2.5 U) or
TurboPfu-Polymerase (2.5-5 U) Buffers containing MgCl2 or MgSO4 were used as
recommended by the manufacturers. Conditions for standard PCR were: 1 x (2 min
95°C); 35 x (30 sec 95°C; 30 sec 58°C; 30 sec up to 3 min 72°C, depending on the
length of target gene), 1 x (7 min 72°C). If (Turbo-)Pfu-Polymerase was used, 1%
DMSO was added to the reaction mix. The PCR was performed using a Mastercycler
Gradient (Eppendorf, Hamburg, Germany). The resulting PCR-products were purified
by gel extraction using QIAEX II kit (Qiagen, Hilden, Germany).
2.10.2 Site directed mutagenesis
If a defined base exchange should be inserted close to the 5`- or 3`-ends of a target
gene, a standard PCR reaction was performed using mutagenesis oligonucelotides.
Other defined base exchanges were insetted into a target gene by using a modified
protocol of the Quik Change Site-Directed Mutagenesis Kit (Stratagene, Heidelberg,
Germany): a plasmid containing the target gene was extracted from an E. coli strain
with a functioning DNA-methylisation system (e.g. E. coli JM109) and used as a
template in a following PCR reaction to amplify the hole plasmid DNA. The standard
conditions were changed by the following modifications: Since the mutagenesis DNA
primers are complementary to each other, more template DNA (~10-100 ng) was
used to ensure proper product synthesis. TurboPfu-Polymerase (5 U), and a reduced
amount of PCR cycles (≤ 20) were chosen to minimize random point mutations. The
elongation steps were perfomed at 68°C, the elongation time depends on length of
the plasmid. After PCR reaction the sample was incubated with 10 U of restriction
enzyme DpnI for one hour at 37°C resulting in a complete elimination of methylised
Materials and Methods
23
template DNA. The remaining PCR product was directly transformed into E. coli
JM109 cells.
2.10.3 Saturation mutagenesis
To saturate a defined position in a protein with all possible amino acids and to
minimize the chance of stop codons and amino acid redundancy at the same time, a
standard PCR was performed using mutagenesis primer with wobbled bases for the
defined base position. This primer consists of a nucleotide mix of all 4 bases (dATP,
dCTP, dGTP and dTTP) in equimolar concentration for the first two codon positions
and a reduced base composition (only dCTP and dGTP) at the degenerated codon
position 3 (2).
2.10.4 Random mutagenesis
Random point mutations were introduced into a target gene by error-prone
polymerase chain reaction (ep-PCR) as described before (27, 226). The reaction mix
contains ~1 ng as the template, 50 pmol of each primer, 0.2 mM of each dNTP and
2.5 U Taq-Goldstar-Polymerase (Stratagene, Heidelberg, Germany). As reaction-
buffer the (NH4)2SO4-containing Taq-reaction-buffer from Fermentas (St. Leon-Rot,
Germany) was used. Each approach contains 6 mM MgCl2 and in addition 0.15- 0.3
mM MnCl2 yielding an average error rate of 1 up to 10 base substitutions per 1 kb.
The cycler conditions for ep-PCR were: 1 x (2 min 95°C); 30 x (30 sec 95°C; 30 sec
58°C; 30 sec up to 3 min 72°C, depending on the length of target gene) and 1 x (7
min 72°C).
2.10.5 Cassette mutagenesis of the PPX-domain of secA
To insert random point mutations only in the preprotein-crosslink domain of secA, a
method was used based on the QuickChange II Site-Directed Mutagenesis Kit from
Stratagene (Heidelberg, Germany). In the first step an ep-PCR of the PPX-domain
Materials and Methods
24
using primer pair ppx-up/ ppx-down and methylated plasmid DNA pBSMuL3secA
from E. coli JM109 as the template lead to a 350 bp fragment containing the library of
the PPX-domain of SecA. The DNA of gel extracted PPX-fragments was taken as the
primer pair in a second PCR amplifying the whole plasmid pBSMuL3secA resulting in
a plasmid mix of methylised wildtype plasmid and and generated plasmid variants.
To eliminate methylised wildtype secA (template) the PCR sample was incubated
with endonuclease DpnI. An aliquote of this sample was directly used for
electrotransformation into E. coli JM109 for plasmid library construction. A
schematical overview is given in Fig. 34 (Appendix).
2.11 Construction of integration plasmid pMCut
The 642 bp cutinase gene was amplified from E. coli expression plasmid pMac5-8
(176) by standard PCR using the primer pairs Cut_up and Cut_down containing
unique restriction sites for HindIII/EcoRI and BamHI/PaeI for cloning. The amplified
cutinase lacks a Bacillus ribosome binding site and an ATG-start codon. In a second
standard PCR a 430 bp fragment containing the strong constitutive HpaII promoter
sequence was amplified using oligonucleotides hpaII_up and hpaII_down containing
PaeI and HindIII restriction sites, respectively (Table 5) and the B. subtilis plasmid
pBSMuL1 (24) as the template. The 682 bp cutinase PCR-fragment and the 430 bp
HpaII promoter PCR-product were digested with endonuclease HindIII, ligated and
subsequently used as a DNA-template in another standard PCR using the primer pair
hpaII_up and Cut_down to amplify the promoter-cutinase gene-fusion. The resulting
1102 bp PCR-product was cloned into the unique PaeI site of Bacillus integration
vector pDG268-∆lacZ (Table 4). The resulting plasmid was analyzed by DNA-
sequencing and named pMCut.
2.12 DNA sequencing DNA sequencing was performed by SequiServe (Vaterstetten, Germany).
Materials and Methods
25
2.13 Enzyme assays
2.13.1 Lipolytic plate assay
Esterase indicator plates were prepared by addition of 15 ml of an emulsion of 50%
(v/v) tributyrin and 5% (w/v) gum arabic to 500mL of molten LB-agar with appropriate
antibiotica. Tributyrin was emulsified by sonication for 3 min at 75 W (duty cycle
100%) in a Branson 250 sonifier. Esterase and lipase activity is indicated by the
formation of clear halos around the colonies.
2.13.2 α-amylase activity plate assay
Starch agar (pH 6.8) was prepared by addition of 15 g of agar, 10 g of starch, 5 g of
gelatine, 3 g of beef extract to 1 L A. dest. After steam sterilisation the suitable
antibiotic was admixed. By adding 5 % (w/v) iodine solution (Lugol`s solution) to the
plate amylase activity is indicated by yellow halos around the colonies.
2.13.3 Lipolytic activity assay
Detection of lipolytic activity of cutinase was performed as described by Winkler and
Stuckmann using a spectrophotometric assay (209). As the substrate 30 mg
p-nitrophenyl-palmitate (pNPP) was dissolved in 10 mL isopropanol and mixed with
90 mL of Sørensen phosphate buffer (pH 8), supplemented with sodium desoxycholic
acid (207 mg) and gum arabic (100 mg). The final concentration of the substrate was
0.8 mM. The culture supernatant was separated from the cells by 5 min
centrifugation at 10.000 g and micro-filtration (membrane filter, pore size 0,22 µm,
Millipore Corp, Billerica, MA, USA). Culture supernatant (5-20 µL) was mixed with 2.5
mL of the substrate emulsion. After incubation at 37°C (15-30 min) the absorbance at
410 nm was recorded. The enzymatic activity was calculated using a molar
absorbtion coefficient of 15000 M-1·cm-1.
Materials and Methods
26
2.13.4 Esterase activity assay
For the detection of esterase activity of EstCL1 23.7 mg of pNPC (p-nitrophenyl-
caproat) was dissolved in ethanol (5 ml) and added to 95 ml of 100 mM potassium
phosphate buffer (pH 7.2) containing 10 mM MgSO4 to yield a final concentration of 1
mM pNPC. Culture supernatant (5-20 µL) was added to the substrate solution to give
a final volume of 1 ml, and the ∆OD410 was recorded for 15-30 min at 37°C. The
enzymatic activity was calculated using a molar absorbtion coefficient of
12500 M- 1·cm-1.
2.13.5 High throughput screening (HTS) assay for cutinase/EstCL1 secretion using a pipetting robot
B. subtilis colonies expressing SPs fused to the cutinase or EstCL1 encoded by
pBSMuL3-Cut-ss/ pBSMuL3-EstCL1-ss were transferred from LB-agar plates into
96er deepwell microtiter plates (DW-MTP, 2mL, Greiner Bio-One, Frickenhausen,
Germany). The strains were cultured in 1 mL LB-medium per well at 37°C using a
microplate shaker (600 rpm, TiMix 5, Edmund Bühler GmbH, Hechingen, Germany).
After 16 hours of growth, culture supernatant was prepared by centrifugation (30 min,
4°C, 5.000 g). Culture supernatants (200 µL) of each clone were carefully transferred
into a new MTP (0.5 mL, Greiner Bio-one, Frickenhausen, Germany).
The lipolytic activity of the supernatants against the substrate p-NPP for cutinase or
p-NPC for EstCL1 was determined by an automated lipolytic activity assay which was
integrated in a HTS process using a TECAN workstation Genesis 200 Freedom
(Tecan, Crailsheim, Germany):
In the first part of the Gemini V4.2 pipetting script “Ulf pNPP1_10 tiptouch_bis_10
Pl.gem (Fig. 35.A, Appendix)” 20 µL of each culture supernatant of one “source” plate
is diluted 10-fold in selective LB-medium provided in a 200 mL trough. The second
part of the Gemini script (Fig. 35.B, Appendix) defines the pipetting of the assay
plate: supernatant (20 µL of 10-fold diluted) are mixed with 180 µL of substrate
emulsion provided in a second 200 mL trough. After completing the Gemini script the
roboting arm (ROMA) is continuing the Process Manager program
Ulf_PnPP1_10V_Trog200 (Fig. 12) transporting the assay plate directly to the
preheated (30°C or 37°C) Genios spectrophotometer (Tecan, Crailsheim, Germany).
Materials and Methods
27
The changing of absorbance at 410 nm was detected by recording a 15 min kinetic at
30°C (cutinase) or 37°C (EstCL1).
2.14 Determination of protein concentration
The protein concentration was measured spectrophotometrically at 595 nm according
to the method of Bradford (19) using bovine serum albumin (BSA) as the standard.
2.15 TCA precipitation of proteins
1/10 volume of 1% (w/v) sodium dodecyl sulfate was added to the protein sample
and incubated for 5 min at RT. Proteins were precipitated with 1/10 volume of 70%
(v/v) trichloracetic acid (TCA) and incubated for 5 min at RT. After a centrifugation for
30 min (RT, 14000 rpm), samples were washed twice with ice-cold 80% (v/v) acetone
and dried at RT. Finally, proteins were resuspended in a suitable volume of SDS
loading buffer (10% Glycerol, 0,2% (v/v) SDS, 0,125 M Tris-HCL pH 6.8, 0,1% (w/v)
Bromphenol blue, 2% (v/v) β-Mercaptoethanol).
2.16 SDS-polyacryamide gel electrophoresis (SDS-PAGE)
The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a Mini-
PROTEAN 3 Cell (BioRad, München, Germany) under reducing conditions using a
5 % stacking gel and a 12-15% separating gel as described before (105). Protein
samples in SDS loading buffer were incubated 2-5 min at 95°C before separated by
SDS-PAGE. Afterwards, proteins were stained with Coomassie Brilliant Blue R250.
As the molecular weight standard the “Prestained Protein Marker” from Fermentas
(St. Leon-Roth, Germany) was used.
Materials and Methods
28
2.17 Cell culture and production of cutinase-specific antibodies by hybridoma
cells
Polyclonal anti-cutinase antibodies were produced by hybridoma cells (1) resulting
from the fusion of B-lymphocytes from immunized Balb/c mice with SP2/0 myeloma
cells (163) and secreted into the culture media. Cells were routinely cultured in
serum-free hybridoma media (Ex-Cell 610 – HSF, JRH biosciences, Lenexa, Kansas,
USA). Stock holding was performed in T-flask cultures (Greiner, Frickenhausen,
Germany) and under selection pressure with 400 µg mL-1 geniticin (G418, Gibco
BRL, Eggenstein, Germany) in the cultivation media. Batch cultivations for production
of the antibodies were all carried out in spinner flasks (100 – 300 mL working volume,
Techne, Cambridge, UK) with headspace aeration at agitation speed of 40 rpm.
Cultures were incubated at 37°C in an atmosphere of 95% air saturation, 5% CO2
and 99% humidity (Heraeus Instruments, Hanau, Germany). Viable cells were
counted whether manually by hemacytometer using the Trypan Blue exclusion
method (Roche Diagnostics, Mannheim, Germany) or by using an automated cell
viability analyzer (Vi-Cell XR, BeckmanCoulter, Inc., Miami, FL, USA). Initial cell
densities were 0.2 × 106 mL-1 for all cultivations and cell stock holding was
appropriately splitted to keep cell densities below 1.5 × 106 mL-1. Supernatants
containing anti-cutinase antibodies were recovered by centrifugation (200 × g, 10
minutes, 4°C) and kept refrigerated (4°C) for short-term usage or frozen (-80°C) for
long-term storage, respectively.
2.18 Immunodetection of proteins
For immunodetection proteins were concentrated by trichloroacetic acid precipitation,
separated by SDS-PAGE and transfered under semi dry conditions onto a
polyvinylidene difluoride (PVDF-) membrane in a Mini-PROTEAN 3 Cell (Biorad,
Munich, Germany). Blotting was carried out for 15 min at 15 mA followed by 20 min
at 300 mA using Dunn Carbonat buffer (10mM NaHCO3, 3 mM Na2CO3, 20% (v/v)
Methanol) as described before (46). After the transfer, blocking of the membrane was
achieved by incubation in TBST (50 mM Tris/HCL pH 6.8, 150 mM NaCl, 1 mM
Materials and Methods
29
MgCl2, 0.2% (v/v) Tween 20) containing 5% (w/v) milk powder. The following steps
were then carried out in TBST. For detection of cutinase protein a polyclonal
antiserum was used (1:1000 diluted) produced in hybridoma cells (2.17). For
detection of B. subtilis SecA a polyclonal antiserum was used (1:1000) raised against
SecA in a rabbit. A sample was kindly provided by M. Caspers (Prof. Dr. R. Freudl,
Bacterial Protein Secretion Group, IBT 1, Research Centre Jülich, Germany). For
protein detection horseradish peroxidase-labelled goat-anti-mouse antibody (BioRad,
München, Germany) was used as the second antibody. Detection was performed
with the ECL system (Amersham Pharmacia Biotech, England) using a luminograph
(LB 980, EG &G Berthold, Bad Wildbad, Germany) as recommended by the
manufacturer.
2.19 Purification of overexpressed cutinase in E.coli
The overexpression of cutinase from the fungus Fusarium solani pisi and the
following purification procedure was performed as follows: A preculture (50 mL) of
E. coli cells WK6 harbouring cutinase expression plasmid pMac5-8 (176) were grown
overnight in TB-medium (17 mM KH2PO4, 72 mM K2HPO4, 1,2 % (w/v) tryptone,
2,4 % (w/v) yeast extract, 0,4% (v/v) glycerine, pH 7,0) at 37°C. The main culture
was inoculated 1:1000 from the preculture and grown in TB-medium at 25°C until a
cell density of OD580= 1. After induction with a final IPTG concentration of 0.1 mM the
culture was incubated for further 16 hours at 25°C. After a centrifugation step (30
min, 4°C, ~14.000g) the accumulated cutinase in the periplasm was released by cold
osmotic shock: The cell pellet was resuspended in icecold digestion buffer (100 mM
Tris-HCl, 500 mM Sucrose, 1 mM EDTA). After centrifugation (30 min, 4°C,
~14.000g) the pH of the periplasmic fraction (supernatant) was lowered to 4.7 by
adding 50% (w/w) acetic acid. After overnight incubation at 4°C the precipitated
material was removed by centrifugation (30 min, 4°C, ~14.000g). The supernatant
was filtered through a cellulose-acetat filter (0.2 µm, Firma) and loaded on a
preequilibrated G25 Sephadex column (20 mM Tris-HCl, pH 7,6). The eluat was
loaded on a preequilibrated (20 mM Tris-HCl, pH 7,6) DEAE Sepharose Fast Flow-
column (Pfizer, Wien, Austria). After a desorbing step with a gradient (0-10%) of
elution buffer (20 mM Tris-HCl, 1 M NaCl, pH 7,6) the eluat was loaded on a
Materials and Methods
30
preequilibrated Q-Sepharose Fast Flow column (20 mM Tris-HCl, pH 7,6). After a
desorbing step with a step gradient (25%) of elution buffer (20 mM Tris-HCl, 1 M
NaCl, pH 7,6) the eluat was concentrated using a PL10 Centricon membrane (Pall,
Dreieich, Germany). In a final gel filtration step the sample was loaded on a
preequilibrated G200 Sephadex column (20 mM Tris-HCl, 150 mM NaCl, pH 7.6).
The eluat was concentrated using a PL10 Centricon membrane (Pall) followed by
buffer exchange to PBS buffer (4 mM KH2PO4, 16 mM Na2HPO4, 115 mM NaCl, pH
7,2) using a PD 10 column (Pfizer, Wien, Austria). Finally the sample was sterile
filtrated through a Pall Acrodisc Syring Filter (0,2 µm, Pall, Dreieich, Germany). The
purification of overexpressed cutinase was performed by E. Knieps-Grünhagen (Dr.
J. Hubbuch, Bioseparation group, IBT 2, Research Centre Jülich, Germany).
2.20 Pulse-chase protein labelling
Exponential phase cells of B. subtilis TEB1030 grown in S7 minimal medium (197) at
37°C were washed twice with methionine free S7 medium, normalized to an OD600 of
0.8 and incubated in methionine free S7 for 60 minutes at 33°C. Subsequently, the
cells (2.5ml) were labelled with 150µCi [35S]- methionine for 1 minute, followed by
chasing with a vast excess of non- radioactive methionine (2mg/ml). Samples (600µl)
for each strain were withdrawn at certain time points after the chase (10 sec, 30 sec
and 1 min) and precipitated overnight with ice- cold TCA (final concentration 8%).
Precipitates were washed three times in 80% acetone, resuspended in 100 µl lysis
buffer (10 mM Tris/HCl pH 8,0, 25 mM MgCl2, 200 mM NaCl, 5 mg/ml lysozyme) and
incubated at 37°C for 45 min. Lysis was completed by addition of 15µl of 10% (w/v)
SDS and heating for 10 min at 95°C. Immunoprecipitation and SDS-PAGE were
carried out as described by van Dijl et al. (194). For the immunoprecipitation, a
polyclonal antibody raised against cutinase in a rabbit was used. Radioactively
labelled Protein bands were visualized using a “Fuji BAS 1800 Bio Image Analyser.”
The pulse-chase experiments were performed by M. Caspers (Prof. Dr. R. Freudl,
Bacterial Protein Secretion Group, IBT 1, Research Centre Jülich, Germany).
Materials and Methods
31
2.21 Cell fractioning of B. subtilis
After a centrifugation step (30 min, 7.500 UpM, 4°C ) the cells were separated from
the culture supernatant. The cell pellet was resuspended in phosphat buffer (100 mM
K2HPO4, 100 mM KH2PO4) containing protease inhibitor “Complete” (1x, Roche,
Grenzach, Germany). Ultrasonic treatment (20 min, 75 W, “Branson Sonifer 250”,
Branson, Danbury, USA) and the incubation of the cells with lysozyme (10 mg/mL,
37°C, 30 min) was performed to ensure complete cell lysis. The fractionation in cell
wall, cell membrane and cytoplasm was performed to the protocol of Harwood et al.
(66).
2.22 Computational methods
Analyses of DNA, amino acid sequences and construction of plasmid maps were
carried out using the programs Edit Sequencer, Clone Manager for Windows 7.0,
Plasmid MapEnhancer 3.0 (SCIENTIFIC AND EDUCATIONAL SOFTWARE,
Durham, USA) and CS ChemDraw Pro 7.0 (CAMBRIDGESOFT, Cambridge, USA).
Sequence alignments, database searches and protein structure analysis were
performed using the standard programs of the NATIONAL CENTER FOR
BIOTECHNOLOGY INFORMATION (http://www.ncbi.nlm.nih.gov). DNA sequence
informations about B. subtilis 168 were obtained from the SubtiList-database
(http://genolist.pasteur.fr/SubtiList/). Signal peptide prediction of given amino acid
sequences were carried out using SignalP 3.0
(http://www.cbs.dtu.dk/services/SignalP/).
Results
32
3. Results
3.1 New expression and secretion vectors for Bacillus subtilis
3.1.1 Choice of expression host: B. subtilis versus E. coli
Today a large pool of cloning and expression plasmids is available for the gram-
negative bacterium Escherichia coli (e.g. pUC-Vectors, or the pET-vectors series
commercialized by Novagen, Madison, USA). Therefore, most protein expression
strategies in microbiological research focus on this organism. However, beside the
obvious advantages of the E. coli-systems, serious problems can occur during the
process of heterologous gene expression and purification: (i) low expression rates,
(ii) formation of inclusion bodies, (iii) improperly protein folding and/or (iv) toxicity
problems might oblige to change the expression host (8).
An alternative expression host also for large scale production of foreign proteins is
the Gram-positive bacterium Bacillus subtilis. One major advantage of the strain is its
classification as a GRAS – generally recognized as safe – organism free of any
endotoxin. Furthermore, in contrast to E. coli the bacterium B. subtilis offers an
efficient secretion apparatus guiding the expressed protein directly into the culture
supernatant (195); thereby bypassing the time-consuming cell disruption which
makes a subsequent protein purification much easier. In addition, in case of efficient
secretion the formation of inclusion bodies in the cytoplasm is reduced, leading to
higher amounts of properly folded and active enzymes. For all these reasons
B. subtilis has developed into an important expression strain frequently used in
industrial fermentations during the past decades (172). Various kinds of secretion
vectors have been published already for B. subtilis (106, 128); however, until now the
choice of available expression plasmids combining similar properties like E. coli-
systems is still limited.
The intended plasmids shall combine several advantages in comparison to available
Bacillus expression systems: an appropiate multiple cloning site consisting of 13
unique restriction sites, one or two strong constitutive promoters, a highly efficient
Results
33
signal sequence for protein secretion and the possibility to express proteins as His-
tagged fusions for easy detection and purification.
3.1.2 Construction of vectors pBSMuL1 and pBSMuL2
The multi-copy vectors pBSMuL1 and pBSMuL2 (Fig. 4) were constructed by
excision of the original cloning site (EcoRV, KpnI and HindIII) of pMA5 (36) by EcoRI
and HindIII double-digestion, blunting using the Klenow fragment and subsequent
religation resulting into an intermediate plasmid named pMA5∆MCS. The DNA-
fragment containing the new MCS of both vectors was constructed in vitro using
seven oligonucleotides (U1-U3, L1-L4; Table 5). A pool of all these oligonucleotides
(5 pmol each, dissolved in A. dest) with a final volume of 14 µL was incubated at 95-
100°C for 10 min and cooled down to room temperature afterwards. This sample was
directly used as a template in a standard PCR reaction using additional 50 pmol of
oligonucleotide U1 and L4 (Table 5) as flanking primers. The expected 201 bp DNA
fragment was amplified, flanked by VspI restriction sites (ATTAAT). After
gelextraction this product was digested using VspI and ligated into the NdeI-
linearized pMA5∆MCS plasmid; thereby eliminating the original NdeI site of the pMA5
vector. The resulting plasmid was sequenced and named pBSMuL1. In contrast to
the original pMA5 vector the cloning site is directly downstream of the strong HpaII
promoter; therefore, no additional cloning steps are necessary to get the target gene
under control of the promoter (36). To achieve an even higher expression and
secretion level a second constitutive promoter P59 (191) was inserted upstream of the
HpaII promoter. The P59 DNA-fragment was synthesized in vitro as described before
by using the oligonucleotides P59-up and P59-low (Table 5). The final 65 bp long
promoter DNA was cloned directly into the unique BstXI site of pBSMuL1. The
resulting plasmid was sequenced and named pBSMuL2 (Fig. 4).
Results
34
3.1.3 Overexpression, secretion and purification of His-tagged cutinase in
pBSMuL vectors
To confirm the functionality of these vectors, the cutinase from the fungus F. solani
pisi (51) was chosen as a heterologous enzyme for expression in B. subtilis. The
cutinase gene was amplified by a standard PCR from E. coli expression plasmid
pMac5-8 (176) using the primer pair cut1/cut2 (Table 5) containing restriction sites for
EcoRI and XhoI without including the cutinase stop codon at the 3`-end. The 0,7 kb
PCR product was cloned into the MCS of pBSMuL1 and pBSMuL2 to obtain an in
frame fusion of the cutinase gene and the 6x-His-sequence. The resulting plasmid
pBSMuL1-Cut was used to transform the lipase deficient B. subtilis strain TEB1030
(48) which lacks both genes for the extracellular lipolytic enzymes LipA and LipB as
Fig. 4: Plasmid map of the pBSMuL series: pBSMuL1 (7494 bp) and pBSMuL2 (7559 bp). (A) Overview of relevant elements: PHpaII, constitutive Gram-positive promoter; P59, second constitutive gram-positive promoter only present in pBSMuL2; MCS, multiple cloning site; ori E. coli, gene required for replication in E. coli; amp
r, β-lactamase
gene conferring ampicillin resistance in E. coli; repB, gene required for replication in B. subtilis; kan
r, kanamycin resistance gene; (B) Properties of the artificial MCS: rbs, ribosome binding site recognized in B. subtilis; sslipA, lipA signal sequence of B. subtilis for efficient protein secretion into the culture medium; the signal peptidase cleavage site (Ala-Lys-Ala) is marked with an arrow; His-Tag, C-terminal in-frame fusion to six histidine residues for protein purification.
PHpaII
MCS
EcoRI
Acc65I
KpnI
AccI
SalI
SmaI
HindIII
NotI
EagI
EcorV
XhoI
NdeI
pBSMuL
ori E. coli
repB
kanr
ampr
BamHI
rbs NdeI
.....TAATAAGGAGGACATATGAAATTTGTAAAAAGAAGGATCATTGCACTTGTAACAATTTTGATG
MetLysPheValLysArgArgIleIleAlaLeuValThrIleLeuMet
Acc65I
EcoRI KpnI
CTGTCTGTTACATCGCTGTTTGCGTTGCAGCCGTCAGCAAAAGCCGCCGAATTCGGTACC
LeuSerValThrSerLeuPheAlaLeuGlnProSerAlaLysAlaAlaGluPheGlyThr
XmaI EagI
SalI SmaI HindIII NotI EcoRV XhoI His-Tag
GTCGACCCCGGGAAGCTTGCGGCCGCGATATCTCTCGAGCACCACCACCACCACCACTGAAT.....
ValAspProGlyLysLeuAlaAlaAlalleSerLeuGluHisHisHisHisHisHisEnd
pBSMuL multiple cloning site (MCS)
signal peptidase
sslipA
yy
A
B
P59
Results
35
well as the extracellular proteases NprE and AprE. Consequently the strain TEB1030
shows no disturbing lipolytic background activity and less proteolytic degradation in
the supernatant. The resulting Bacillus strain expressing the cutinase was cultured in
selective LB-medium 20 hours at 37°C. About 25 µg of total protein of culture
supernatant was separated using SDS-polyacrylamide gelelectrophoresis (SDS-
PAGE) showing the successful overexpression and efficient secretion of 20 mg
cutinase per litre (Fig. 5, lane 1). Using the Ni-NTA Spin Column (Qiagen, Hilden,
Germany) as recommended by the manufacturer’s standard protocol, the
recombinant cutinase was purified to electrophoretic homogeneity as detected by
SDS-PAGE (Fig. 5, lane 4). The enzyme has been expressed and secreted in
catalytically active conformation as determined spectrophotometically using p-
nitrophenyl-palmitate (pNPP) as the substrate (209). The specific activity of the His-
tagged cutinase towards pNPP was 200 U/mg and thus similar to the native cutinase
(225 U/mg) expressed and purified from E. coli.
Fig. 5: Purification of His-Tag cutinase from the B. subtilis culture supernatant. The expression culture was grown 20 hours in LB-medium supplemented with kanamycin (50 µg/mL) at 37°C. 600 µL of culture supernatant (pH 8.2) was loaded on a Ni-NTA Spin Column (Qiagen, Hilden, Germany) and the His-tagged cutinase was purified as recommended by the manu-facturers standard protocol. 25 µg protein of the supernatant (lane 1) and comparable volumes of flow-through (lane 2), wash fraction (lane 3) and elution (lane 4) were concentrated using 70% trichloride acid precipitation and separated on a protein gel (12% SDS-PAGE) stained with coomassie brilliant blue. The protein band of the His-Tag cutinase is marked by an arrow. As a control 2 µg of purified native cutinase expressed in E. coli (22.2 kDa) was loaded as a positive control (lane +).
elu
tio
n
flo
w-t
hro
ug
h
mark
er
supern
ata
nt
wash f
racti
on
po
sit
ive
co
ntr
ol
55 kDa
45 kDa
35 kDa
25 kDa
15 kDa
M 1 2 3 4 +
Ni-NTA purification
Results
36
For higher production yields the second plasmid pBSMuL2 was tested for cutinase
overexpression and secretion. Because of the additional constitutive promoter P59 it
was possible to increase the secreted amount of mature cutinase three-fold.
Recombinant cutinase (60 mg/L) was detected in the culture supernatant by activity
determination using pNPP as the substrate (Fig. 6).
3.1.4 Stability of pBSMuL vectors in B. subtilis under non selective conditions
Both pBSMuL-plasmids multiply by the so-called rolling-circle type of replication
(repB from Staphylococcus aureus) which might lead to the disadvantage of
segregational instability (25) in non selective media. But especially industrial
applications require economical processes avoiding additional costs for antibiotics.
Therefore, we tested the stability of both plasmids under selective and non-selective
conditions as shown in Fig. 7:
Fig. 6: Expression and secretion efficiency of cutinase in Bacillus subtilis using vectors pBSMuL1 and pBSMuL2. The lipase-deficient strain Bacillus subtilis TEB1030 was transformed with cutinase overexpression plasmids pBSMuL1-Cut and pBSMuL2-Cut. As negative controls B. subtilis TEB1030 was transformed with vectors pBSMuL1 and pBSMuL2, respectively. All strains were incubated in selective LB-medium for 20 hours at 37°C and showed no significant growth differences. The culture supernatants were harvested by centrifugation. All culture supernatants were tested for lipolytic activity using the spectrophotometric pNPP-assay. The lipolytic activity was adapted to an OD580= 7. Standard deviations are indicated by error bars. The results represent data from five independent experiments.
0
2
4
6
8
10
12
14
pBSMuL1 pBSMuL2 pBSMuL1-Cut pBSMuL2-Cut
expression strains
U/mL
Results
37
Overnight cultures of both strains grown under selective conditions were used to
inoculate 25 mL of fresh LB-medium and cultured up to 24 hours at 37°C with and
without antibiotic selection: No reduction in cutinase secretion was detected under
non-selective growth conditions compared to the control Bacillus strain cultured
under antibiotic selective pressure. Even after inoculation of fresh LB-medium and
another 24 hours of growth at 37°C no differences were observed. However, after
another round of inoculation and cultivation in fresh medium the expression strain
started to loose cutinase activity due to plasmid instability as described for repB-
containing vectors (25). Therefore, even batch cultures grown under non-selective
conditions inoculated with selective starter cultures will lead to sufficient protein
production and secretion as demonstrated.
time (h)
25 µL 25 µL 25 µL
0- 24 25- 48 49- 72
A
time (h)
B
0
20
40
60
80
100
120
24 48 72
pBSMuL1+pBSMuL1-
pBSMuL2+pBSMuL2-
relative lipolyticactivity
(%)
time (h)
Fig. 7: Stability of pBSMuL vectors in B. subtilis. (A) After inoculation with 25µL from a preculture grown under selective pressure the Bacillus subtilis TEB1030 strains bearing cutinase overexpression plasmids pBSMuL1-Cut and pBSMuL2-Cut were grown three times in 25 mL LB-medium for 24 hours each with and without antibiotic selection. (B) The culture supernatants were harvested by centrifugation after 24 h, 48 h and 72 h of growth with (+) or without (-) kanamycin (50µg/ mL). At these time points the cultures showed no significant growth differences. All culture supernatants were tested for lipolytic activity using the spectrophotometric pNPP-assay. The lipolytic activity was adapted to an OD580= 7. The lipolytic activity of strains under selection pressure were defined as 100 %.The results represent means of two independent experiments.
Results
38
3.2 In search of the most functional signal peptide for heterologous protein
secretion in B. subtilis
In addition to the components of the secretion apparatus the signal peptides (SPs) of
the secreted proteins play a crucial role in the efficient translocation across the
cytoplasmic membrane (201). Tjalsma et al. used the computer tool SignalP to
predict essentially all secreted proteins in the Gram-positive bacterium B. subtilis
(188). Nevertheless, the prediction of secretion efficiency becomes even more
complicated when heterologous proteins are fused to the SP instead of the natural
secretion partner: former secretion studies in B. subtilis compared SP variants to the
wildtype when fused to a mature protein and revealed defects or differences for
export efficiency of the hybrid protein (13, 29, 130). But they could not detect this
effect for the same mature protein when fused to another SP. Therefore a strong
influence of the mature sequence was postulated for the secretion effect of a signal
peptide like described by Lehnhardt et al. (110).
Consequently one might suggest that a signal peptide and a secreted protein
constitute a unique unit, where the N-terminus of the mature protein, the so-called
signal-mature junction, plays an important role in the secretion efficiency making the
prediction of “good” SPs for heterologous protein secretion impossible. Therefore, the
high yield of industrially interesting proteins in heterologous fermentation hosts may
still be limited by the secretion efficiency due to the use of a “wrong” SP.
For the first time a strategy was chosen to bypass this limitation by generating a
library of all 173 native Sec-type B. subtilis SPs allowing to test each for export
efficiency in combination with a heterologous target protein.
3.2.1 Establishment of a high-throughput screening (HTS-) system to evaluate
secretion efficiency for heterologous proteins in B. subtilis
Next to the immense potential of automated screening processes for finding and
analysis of enzymes with improved or new properties for biotechnological
applications, HTS systems bear numerous factors leading to fluctuations that can be
critical to a successful screening especially for comparative studies. The most
relevant factors are (i) pipetting errors, (ii) different growth/expression rates if
Results
39
microorganisms (often E. coli) are used as cell factories mostly in combination with
an inducible expression system and (iii) instability of substrate and/or enzyme in the
performed assay.
Yet a screening in the Gram-positive bacterium B. subtilis provides even more
challenges in relation to E. coli: Most of the available vector systems are integrated
plasmids due to a higher transformation efficiency compared to replicating plasmids.
But if those are used for the screening of gene libraries, they cannot easily be
recovered for the purpose of necessary sequence analysis. Anyway, the general
transformation process is more extravagant, the common transformation rate is
significantly lower than in E. coli making the library construction a more time-
consuming procedure. In summary, the discovery and analysis of all factors that
reduce the screening capacity are important preconditions for an efficient HTS
system and are presented in this chapter.
3.2.1.1 Construction of a Bacillus vector system using cutinase as a secretion
reporter
In this study the heterologous lipoloytic enzyme cutinase from the fungus Fusarium
solani pisi was chosen as a secretion reporter for heterologous protein secretion in
B. subtilis, because of its eukaryotic origin and the availability of easy
spectrophotometrical activity assays (Fig. 8). Furthermore, cutinase itself and more
important the class of enzymes it belongs to – the so-called α/β-hydrolases – are of
high biotechnological importance (84, 121). For example the protease (subtilisin)
NO2
O (CH2)14
CH3
O
H2O
NO2
OH
+
HO (CH2 )14
CH3
O
p-nitrophenyl-palmitate (pNPP) p-nitrophenol palmitic acid
cutinase
Fig. 8: Schematic overview about the hydrolysis-reaction which the assay is based on. The absorbance at 410 nm was detected by recording a 15 min kinetic at 30°C. The formation of p-nitrophenol leads to a yellow colour of the assay solution whose intensity is a measure for activity increase.
Results
40
present in washing powder also belongs to the family of α/β-hydrolases showing high
structural similarities to cutinase used in this study (84, 117, 120, 121).
The first step in the optimization was to choose a suitable expression system for the
cutinase under the control of a constitutive promoter. Therefore the E. coli-B. subtilis
shuttle plasmid pBSMuL3 was constructed (Fig. 9): The artificial MCS was introduced
in the opposite direction to pBSMuL1
(compare Fig. 4) downstream from the
constitutive promoter PHpaII resulting in
ten unique restriction sites without a
Bacillus ribosome binding site or a signal
sequence. The two SacI restriction sites
flanking the Bacillus part of the vector
enable the formation of vector
multimeres with internal repeats (65)
which can be transformed efficiently
(~ 5X103 transformants/µg DNA) into
B. subtilis thereby suitable for gene
library construction. The cutinase gene
encoding the marker enzyme was
amplified by a standard PCR from E. coli
expression plasmid pMAC5-8 (176)
using the primer pair cut1/cut_low and
EcoRI/BamHI cloned into pBSMuL3 resulting in plasmid pBSMuL3-Cut. Once
transformed into B. subtilis no expression of cutinase occurred (data not shown)
since neither a rbs nor a start codon exists upstream from it. The cloning of different
SPs into the MCS of vector pBSMuL3-Cut in front of the cutinase gene results in
expression of a possibly transportable precursor form, thus allowing to study the
effects of any defined SP for cutinase export.
Fig. 9: Plasmid map of shuttle vector pBSMuL3 (7494 bp). PHpaII, constitutive Gram-positive promoter; MCS, multiple cloning site introduced in opposite direction as in pBSMuL1 (Fig. 4); ori E. coli, gene required for replication in E. coli; amp
r, β-lactamase gene conferring
ampicillin resistance in E. coli; repB, gene required for replication in B. subtilis; kan
r, kanamycin resistance gene. two SacI restriction sites for multimeres forming, required for high transformation rate in B. subtilis.
BamHI
ori E.coli
pBSMuL7494 bps
repB
kan r
amp r
pBSMuL37494 bps
repB
PHpaIIPHpaIIPHpaII
kan r
amp r
ori E.coli
MCSMCS
XhoI
EcoRV
NotI
HindIII
SmaI
SalI
KpnI
EcoRI
NdeI
SacI
SacI
Results
41
3.2.1.2 Analysis of growth differences in microtiter plates
A reliable screening system
claims an equal or at least a
similar growth of all cultures
compared. Since the
cultivation of B. subtilis was
intended to be performed in
DeepWell microtiter plates
(DW-MTP, 96 wells, 2 mL
total volume each), it was
imperative to analyse the
growth of B. subtilis with
respect to the minimization of
growth differences between
each well. Therefore
B. subtilis TEB1030 was
transformed with the cutinase
overexpression plasmid
pBSMuL3-Cut-ssLipA. The
resulting Bacillus strain was
able to secrete cutinase into the culture supernatant (see Fig. 17). About 660
colonies of this strain, grown not longer than 24 hours on selective LB-agar plates,
were transferred to DW-MTPs and incubated in 1 mL selective LB-medium each
(37°C, 600 rpm). At defined time points the cell density of one DW-MTP (= 95
cultures plus one blank without cells) was measured spectrophotometrically at a
wavelength of λ= 580 nm. The lowest growth variation was found in the time period
between 9 and 20 hours of growth (Fig. 10.A). With respect to practical lab work the
time point after 16 hours of growth was supposed to be suitable and chosen for a
more extended growth study: The cell density of 780 clones grown for 16 hours
under the same growth conditions distributed over eight DeepWell plates was
determined and revealed a standard deviation lower than 10 % (Fig. 10.B). In
addition no significant growth differences occurred between wells of the inner part
compared to wells of the outer regions of the plate (data not shown). This similar
0
1
2
3
4
5
6
0 100 200 300 400 500 600 700 800
0
1
2
3
4
5
6
0 10 20 30 40 500
20
40
60
80
100
120
A
Btime [h]
number of Bacillus clones
standarddeviation
[%]
cell growth [OD580]
cell growth [OD580]
Fig.10: Growth of B. subtilis in DeepWell microtiter plates. B. subtilis TEB1030 bearing plasmid pBSMuL3-Cut-ssLipA was incubated in 1 mL selective LB-medium per well at 37°C continiously shaking at 600 rpm. (A) The recorded cell density [OD580] is indicated by a blue line, the respective standard deviation (%) is indicated by a red line. Each value represents the average of 95 individual cultures of a DW-MTP. (B) The cell density [OD580] of 780 clones was recorded after 16 hours of growth. The mean value of OD580= 3,4 is indicated as a red line. The standard deviation was 9,6 %.
Results
42
growth behaviour of B. subtilis cultures under the chosen conditions in DeepWell
plates was considered to be sufficient for a reproducible high throughput screening.
3.2.1.3 Development of a HTS-process for detection of export efficiency using a
pipetting robot
Since the cutinase was chosen as the secretion reporter for HTS, the lipolytic activity
towards the standard substrate p-nitrophenyl-palmitate (p-NPP) had to be adapted to
an automated pipetting process in MTPs using a TECAN workstation Genesis 200
Freedom (Tecan, Crailsheim, Germany) as shown in Fig. 11. For this purpose a
pipetting program had to be designed and several parameter of the instruments had
to be coordinated in order to automate all required steps of the assay. (Fig. 12, 35). A
more detailed description is given under 2.13.5. The generated process was adapted
for the efficient MTP transport from the hotel (Fig. 11.B) to the pipetting station (Fig.
11.D), to the spectrophotometer (Fig. 11.E) and back to the hotel using the ROMA
(Fig. 11.C). Furthermore, a minimized pipetting programm containing permanent
B
D E A C
Fig. 11: The pipetting robot “TECAN workstation Genesis 200 Freedom”. Relevant features are (A) A commercially available computer system using Windows 2000 containing softwares of Facts, Gemini and Magellan. (B) A rotatable “hotel” to store and to release MTPs. (C) A roboting arm (ROMA) to grip, move and release MTPs. (D) A pipetting station containing eight needles (green coloured) for simultaneous uptake and release of liquids. (E) A heatable spectrophotometer “GENios” able to measure absorption, fluorescence or luminescence.
Results
43
washing steps with desalted water between the necessary pipetting and dilution
steps ensures a fast screening progress and avoids contaminations among the
different wells (Fig.12.B). Additionally the pipetted volumes were chosen to be no
smaller than 20 µL to minimize pipetting standard deviation to less then 3 % (data not
shown). However, the theoretically screenable number of 60 MTPs per day in a
maintenance-free process is clearly limited by the instability of the substrate pNPP in
the assay solution which leads to autohydrolysis of the substrate after five hours at
A
Fig. 12: Two different software scripts providing the complete pipetting process for one source plate. (A) the higher Process Manager programm “Ulf_PnPP1_10V_Trog200” and (B) the secondary Gemini Script “Ulf pNPP1_10 tiptouch bis 10 Pl.gem” controlling the pipetting station. For more details see text and chapter 2.13.5.
Results
44
RT (data not shown). This results in a screening process of 13 MTPs maximum
according to the time schedule calculated by the Facts software.
3.2.1.4 Validation of the automated HTS process in B. subtilis
The generated process was simulated for ten MTPs. The Facts software scheduled a
total run time of four hours (Fig. 13.A). Therefore the stability of cutinase stored at RT
up to four hours had to be defined. The supernatant of one batch culture B. subtilis
TEB1030 pBSMuL3-cut-sslipA was transferred into a MTP and used as the same
source plate for five measurements (Fig. 13.B) using the created process as shown
in Fig. 12.
B C
020406080
100120
0 1 2 3 4
incubation time at RT (h)
lipol
ytic
act
ivity
(%
)
A
Fig.13: Reproducibility and stability study of cutinase activity of B. subtilis culture supernatant over four hours performed with the pipetting robot. B. subtilis TEB1030 expressing cutinase on plasmid pBSMuL3-cut-sslipA was grown in 25 mL selective LB-medium for 20 hours and the culture supernatant was transferred into a MTP and used in the created HTS process. (A) Screenshot of the time schedule of a HTS process calculated from FACTS software for ten “source” plates. The time periodes of the ROMA in action is indicated by a red bar, those of the pipetting station and the GENios spectrophotometer in action are indicated by green and blue bars, respectively. The hole process was automatically interlocked optimizing the procedure time to 4 hours. (B) The HTS process for one source plate was started after incubating the supernatant for a defined time at RT. The absorbance at 410 nm was detected by recording a 15 min kinetic at 30°C in the GENios spectrophotometer. The cutinase activity of supernatant used for the HTS without incubation time was defined as 100 %. The standard deviation calculated of 94 wells each was always lower than 8 % and is indicated in error bars. (C) Magellan software transformes the raw data into a graph recorded by a GENios spectrophotometer. Exemplarily the recorded results for one MTP after 2 hours incubated at RT are shown as a screenshot. In each well the blue line indicates the increase of absorbance during 15 min of measurements. Wells A1 and A2 are negative controlls showing no lipolytic activity. The recored data are automatically transferred to Excel and are available for further calculations.
Results
45
The standard deviation of the assay was continiously less than 8 % (Fig. 13. B,C)
and even after four hours at RT the cutinase showed only minimal reduction of
lipolytic activity of about 5 % (Fig. 13. B).
A further approach was followed to define possible variations after retransformation
experiments. Thus the same process was performed with five “source” plates
containing supernatant that was collected from five different DW-MTPs inoculated
from different B. subtilis TEB1030 clones transformed with the same plasmid
pBSMuL3-cut-sslipA. This study confirmed the stability of the cutinase over the time
but revealed a significant increase in fluctuations of cutinase activity up to 25 % (Fig.
14).
3.2.2 Library construction and high-throughput screening of all Sec-type signal
sequences fused to cutinase.
In a so far unique approach the DNA sequences encoding for all presently identified
173 Sec-signal peptides (SPs) (188) ranging from 57 bp up to 138 bp were amplified
0
20
40
60
80
100
120
140
0 1 2 3 4
Fig. 14: Fluctuations in cutinase activity of different B. subtilis cells expressing and secreting wildtype cutinase after transformation with the same plasmid. B. subtilis TEB1030 was transformed with plasmid pBSMuL3-Cut-ssLipA. Single colonies grown on selective LB-agar plates were transferred each into one well of five DW-MTPs altogether and incubated in 1 mL selective LB-medium at 37°C continiously shaking at 600 rpm. After 16 hours of growth the supernatant was transferred into a new MTP. The HTS process for one “source” plate was started after incubation of the supernatant for a defined time at RT. The absorbance at 410 nm was detected by recording a 15 min kinetic at 30°C in the GENios spectrophotometer. The cutinase activity of supernatant without incubation time was defined as 100 %. The standard deviation calculated from 94 wells each (Two wells are blanks) was not higher than 25 % and is indicated by error bars. It represents growth differences, assay fluctuations and variations by plasmid copy numbers after retransformation.
incubation time at RT [h]
lipolytic
activity
[%]
Results
46
by standard PCR from genomic DNA of B. subtilis 168 using the specific primer pairs
(Table 12, Appendix) and extracted from agarose gel after electrophoresis (Fig.
15.A). All upstream primers contain a Bacillus consensus ribosom binding site (rbs)
and the ATG-start codon essential for efficient expression of cutinase in B. subtilis.
The plasmid pBSMuL3-Cut was used for in-frame cloning of all SPs in front of the
marker enzyme cutinase resulting in plasmid library pBSMuL3Cut-ss (Fig. 15.B).
Since the hpaII promotor and the rbs were also recognized in E. coli, the
prescreening was performed in E. coli JM109. Here, only the fusion of an amplified
SP, containing the rbs and the ATG-start codon and cutinase leads to expression of
cutinase protein and to the formation of halos around the E. coli colonies on tributyrin
A B C
D
cutinase BamH I EcoR I Hind III
pBSMuL3 ori E.coli
repB
kanr
ampr
MCS
7987 bp
P
ss
E F
genomic DNA B. subtilis ss
174 PCR reactions
Fig. 15: Schematic overview of amplification, cloning and screening of all Sec-type signal peptides (SPs) of B. subtilis. (A) PCR amplification of all SPs using specific primer pairs and genomic DNA from B. subtilis 168 as the template. (B) Cloning step into the E. coli–B. subtilis shuttle vector pBSMuL3Cut upstream of the heterologous secretion target cutinase. (C) Prescreening in E. coli JM109 on lipolytic indicator plates and plasmid isolation of halo-forming colonies. (D) Transformation of all plasmid encoded SP-cutinase fusions into B. subtilis TEB1030 and growth in MTPs. (E) Isolation of culture supernatant by centrifugation. (F) Automated spectrophotometric HTS-assay for cutinase quantification in isolated culture supernatants.
Results
47
indicator agar (Fig. 15.C). The plasmid DNA from halo-producing cells was isolated,
analyzed by restriction analysis or sequencing. The produced plasmid library
containing all B. subtilis Sec-signal sequences was transformed into B. subtilis
TEB1030, a strain lacking any disturbing extracellular lipolytic activity (48). After
growth on selective agar the colonies were transferred into Deepwell microtiter plates
and cultured for 16 hours in 1 mL LB-medium per well at 37°C using a microplate
shaker (Fig. 15.D). After a centrifugation step the culture supernatant of each clone
was carefully transferred into a new microplate for quantification of lipase activity
(Fig. 15.E). The lipolytic activity towards the standard substrate p-nitrophenyl-
palmitate (p-NPP) was quantified by the automated lipase activity assay using the
TECAN workstation Genesis 200 Freedom (Fig. 15.F) as described under 2.13.5.
3.2.3 The highest D-score does not necessarily predict the optimal signal
peptide for secretion of the heterologous cutinase.
The D-score calculated by the SignalP3.0 (9, 135) indicates the probability of an
amino acid sequence to function as a signal peptide. Therefore, high D-scores (0.8-
1.0) usually indicate high chances of secretion. Commonly, these sequences are
believed to work efficiently in protein translocation across the membrane. In contrast,
D-scores below 0.7 (0.5 - 0.7) indicate lower secretion efficiencies and values below
0.5 are commonly regarded as not recognized by the secretion machinery. However,
by evaluating the complete set of native signal peptides from B. subtilis we were able
to prove this generally accepted strategy to classify SPs.
Therefore SignalP 3.0 was used to determine the D-score for the first 70 amino acids
of all hybrid proteins (SP-cutinase) as shown in Table 13 (Appendix). In most cases
the SignalP-data confirms a signal peptide and the predicted cleavage site position
(C-site) in front of the +1 alanine to be recognized by a SPase I (Fig. 16).
Results
48
Among the plasmid library pBSMuL3Cut-ss 148 different SP-cutinase fusions were
successfully transferred into B. subtilis TEB1030. The screening of this signal peptide
library resulted in a clear ranking of all SPs as summarized in Figure 17 and Table 7.
The detailed results are given in Table 13 (Appendix).
AAGGAGGATATT ATG........................ GCTGAATTC GCG...
Met... .................... AlaGluPhe Ala...
Rbs Spacer
+1-1-3 -2
junction mature cutinaseSignal Sequence
N ++ H C
AAGCTT
A
B
1
0,8
0,6
0,4
0,2
0
10 20 30 40 50 60
score
Fig. 16: (A) Overall composition of signal peptides (SPs) fused to the mature junction of cutinase and (B) its computer analysis exercising SignalP 3.0. (A) The Bacillus ribosome binding site (rbs) and the spacer in front of the ATG-start codon ensure efficient mRNA-translation of the signal sequence gene-fusion. The signal peptides are devided into three domains: the positively charged N-region, the hydrophobic H-region and the C-region coding the recognition site for SPaseI. All SP-cutinase fusions contain the junction built of the amino acids alanine, glutamate and phenylalanine. The HindIII (AAGCTT) and EcoRI (GAACTT) restriction sites for cloning are underlined. (B) The graphical output of a SignalP 3.0 computer analysis is shown exemplarily for the first 70 amino acids of the hybrid protein SP-LipA cutinase calculated with the hidden Markov model (SignalP3-HMM ) for gram positive bacteria which was performed for all 173 hybrid proteins of SP fused to cutinase. The score represents the probability (1.0 = 100 %) for n-region (green line), h-region (dark blue line), c-region (turquoise blue line) and cleavage site (red line). The critical score of 0.5 for of an amino acid indicating significance to be part of a SP region is marked as a punctured line.
amino acid position of the hybrid protein
Results
49
This method to determine cutinase export based on an activity assay was verified by
immunoblotting experiments using cutinase-specific antibodies. Exemplarily, 7
different SP-cutinase fusions were analyzed by Western-blotting with the SP-fusions
of protease Epr (4.67 U/mL), unknown protein YncM (4.12 U/mL), lipase LipA (2.79
U/mL) and phosphodiesterase GlpQ (1.96 U/mL) chosen because of their relatively
high cutinase activity in the culture supernatant. Furthermore SP-fusions of α-
amylase AmyE (0.67 U/mL) leading to moderate cutinase activity and nuclease NucB
(0.34 U/mL) as well as protease WprA (0.12 U/mL) showing low cutinase activity in
the culture supernatant. This direct cutinase quantification on protein level (Fig. 18)
was in accordance of our activity based quantification of cutinase in the culture
medium (Table 7).
Fig. 17: Comparison of all 148 screened signal peptides (SPs) used for export of heterologous cutinase in B. subtilis. The SPs of Epr, YncM, LipA, GlpQ, AmyE, NucB and WprA which have been analyzed further by Western-blotting and pulse-chase experiments are highlighted in red. The lipolytic activity assay of the culture supernatants were performed at 30°C. The enzymatic activity was calculated using a molar absorbtion coefficient of 15000 M-1·cm-1. The results represent data of 12 independent experiments. The standard deviation was not higher than 25 %.
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 127 133 139 145
Sec-type SPs fused to cutinase
lip
oly
tic a
cti
vit
y [
U/m
L]
AmyE, 0.67
Epr, 4.67
YncM, 4.12
LipA, 2.79
GlpQ, 1.96
WprA, 0.12NucB, 0,34
Results
50
The size of cutinase identified in the supernatants corresponds to the purified mature
cutinase (22 kDa) isolated from recombinant E. coli. The exclusive occurence of
processed protein in B. subtilis culture supernatants indicates complete cleavage of
the SP by a SPaseI at the trans-site of the membrane. This processing results in
release of only mature cutinase into the medium and strongly indicates successful
secretion via the Sec-apparatus and cancels out cell lysis as an alternative
explanation. Interestingly, intracellularly expressed cutinase lacking a SP could not
be detected in the cytoplasm of B. subtilis or in the supernatant showing rapid
intracellular degradation of mature cutinase (Fig. 18, lane 2 and 3). Additionally,
when the cutinase was expressed with a SP, in all cases no precursor protein was
detectable in the cytoplasmic fraction (immunoblot not shown). However, a general
observation for the 25 best SPs for cutinase transport was the divergence of the
amino acid at position -3 in the C-region of the B. subtilis. consensus motif A-X-A in 8
cases to V-X-A. In contrast to that, only alanine was found at position -1. The C-
region is responsible for recognition and cleavage of the SP by one of the SPases I.
Thus at least for the fusion with the cutinase it is possible to define the most efficient
cleavage site as A/V-X-A.
Surprisingly, these results suggest that high secretion efficiencies can not simply be
correlated with known D-scores. Most of the best performing SPs identified in the
4 5 6 7 8 9 101 32
cutin
ase
C S
intracellularexpression
SP-Ep
r
SP-Y
ncM
SP-L
ipA
SP-G
lpQ
SP-Am
yE
SP-N
ucB
SP-W
prA
Figure 18: Immunodetection of cutinase in culture supernatants of B. subtilis expression strains. Western blot analysis of B. subtilis strains expressing different SP-cutinase fusions. Of each sample 20 µg protein were subjected to immunodetection by cutinase-specific antibodies produced in hybridoma cells. Lane 1: As positive control 1,5 µg purified cutinase was loaded. Lanes 2-3: intracellular expression of cutinase using vector pBSMuL3Cutintra; cellular fractions (C) and supernatants (S) were isolated by centrifugation. Lanes 4-10: Culture supernatants isolated from B. subtilis strains expressing various SP-cutinase fusions (SP-Epr, SP-YncM, SP-LipA, SP-GlpQ, SP-AmyE, SP-NucB and SP-WprA).
Results
51
screening show D-scores around 0.93 (e.g. Bpr, 0.936; YjfA, 0.924; Epr, 0.919) as
expected; nevertheless, SPs showing D-scores around 0.5 – 0.6 were also identified
(e.g. YncM, 0.507; Csn, 0.684) that work highly efficient in cutinase secretion (Table
7).
Furthermore, the screening revealed many SPs with high D-scores showing
moderate (e.g. GlpQ, AmyE), low (e.g. DacF) or no secretion at all (e.g. YodV) as
summarized in Table 7. In addition, further SP-characteristics like the number of
charged amino acids in the N-region as well as the overall hydrophobicity do not
correlate with secretion efficiencies, indicating no clear patterns or rules to predict the
No.
Name
Signal sequence
Secreted cutinase [U/mL]
a
Charge
N-region b
Hydro-
phobicity [% ]
c
D-
Score d
1 Epr MKNMSCKLVVSVTLFFSFLTIGPLAHA 4.67 2 62.96 0.919 2 YncM MAKPLSKGGILVKKVLIAGAVGTAVLFGTLSSGIPGLPAADA 4.12 4 76.19 0.507 3 YjfA MKRLFMKASLVLFAVVFVFAVKGAPAKA 3.84 3 78.57 0.924 4 YfhK MKKKQVMLALTAAAGLGLTALHSAPAAKA 3.67 3 68.97 0.906 5 Csn MKISMQKADFWKKAAISLLVFTMFFTLMMSETVFA 3.35 3 62.86 0.689 6 LytD MKKRLIAPMLLSAASLAFFAMSGSAQA 3.33 3 70.37 0.87 7 Bpr MRKKTKNRLISSVLSTVVISSLLFPGAAGA 2.97 5 56.67 0.936 8 WapA MKKRKRRNFK RFIAAFLVLA LMISLVPADVLA 2.88 8 65.63 0.918 9 BglC MKRSISIFITCLLITLLTMGGMIASPASA 2.87 2 65.52 0.839
10 LytB MKSCKQLIVCSLAAILLLIPSVSFA 2.83 2 64.00 0.916 11 LipA MKFVKRRIIALVTILMLSVTSLFALQPSAKA 2.79 4 64.52 0.874
15 PhrK MKKLVLCVSILAVILSGVA 2,53 2 73,68 0,669 … 26 GlpQ MRKNRILALFVLSLGLLSFMVTPVSA 1.96 3 69.23 0.921 … 65 AmyE MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASA 0.67 3 78.79 0.904 … 75 NucB MKKWMAGLFLAAAVLLCLMVPQQIQGASS 0.34 2 72.41 0.746 … 88 DacF MKRLLSTLLIGIMLLTFAPSAFA 0.14 2 73.91 0.909 89 TyrA MNQMKDTILLAGLGLIGGSIALA 0.13 0 73.91 0.466 90 LytF MKKKLAAGLTASAIVGTTLVVTPAEA 0.13 3 65.38 0.744 91 WprA MKRRKFSSVVAAVLIFALIFSLFSPGTKAAA 0.12 4 67.74 0.941 …
114 YnzA MELSFTKILVILFVGFLVFGPDKLPALG 0 0 78.57 0.468 115 YobV MKLERLLAMVVLLISKKQVQA 0 1 61.9 0.623 116 YocH MKKTIMSFVAVAALSTTAFGAHA 0 2 65.22 0.88 117 YodV MKVPKTMLLSTAAGLLLSLTATSVSA 0 2 61.54 0.927
a The lipolytic activity assay was performed at 30°C; the enzymatic activity was calculated using a molar extinction coefficient of 15000 M-1·cm-1. b The net charge of the N-region was calculated with amino acids aspartate and glutamate defined as -1; arginine and lysine defined as +1 and any other amino acid defined as 0. c The percentage of hydrophobic amino acids of each signal sequence was calculated with amino acids G, A, V, L, I, M, F, W and P defined as hydrophobic, any other amino acid being characterized as hydrophilic. d D-score calculated by SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) for the first 70 amino acids of the hybrid proteins
Table 7: Comparison of chosen screened signal sequences used for export of heterologous cutinase in B. subtilis. The results represent data of 12 independent experiments. The standard deviation was below 25 %. The complete data is listed in Table 13 (Appendix). The SPs which have been analyzed further by Western-blotting and pulse-chase experiments are highlighted in red.
Results
52
a The net charge of the N-region was calculated with amino acids aspartate and glutamate defined as -1; arginine and lysine defined as +1 and any other amino acid defined as 0. b The percentage of hydrophobic amino acids of the each signal sequence was calculated with amino acids G, A, V, L, I, M, F, W and P defined as hydrophobic, any other amino acid being characterized as hydrophilic. c D-score calculated by SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) for the first 70 amino acids of the hybrid proteins
optimal SP fusion-partner: One of the best SPs for cutinase secretion was SP-WapA
with 2,88 U/mL containing a huge number of 8 positive charges in the N-region. On
the other hand the SP-BglC has almost the same sequence length, shows an equal
cutinase export (2,87 U/mL) but possesses only 2 positively charged aa in the N-
region. It is remarkable that there is no direct dependency not even between the
length of the SPs and export efficiency. For instance the shortest screened SP-PhrK
(19 aa) shows a good export capacity of 2,5 U/mL, but the SP-YncM which
represents one of the longest sequences (42 aa) provides also an efficient cutinase
export of more than 4 U/mL. It is noteworthy that among the 15 best SPs many
sequence lengths occur around the average length for B. subtilis of about 28 aa.
Table 8: Signal peptides (SPs) not tolerated in B. subtilis when constitutively expressed in front of heterologous cutinase. C-regions which diverge drastically from the B. subtilis consensus cleavage site are highlighted in red.
No.
Name
SP: N- and H-region
SP: C-region
Charge N-region
a
Hydro-phobicity
[% ]b
D-Score
c
149 CwlD MRKKLKWLSFLLGFIILLFLFKYQ FSN 5 62,86 0.708 150 DltD MKKRFFGPIILAFILFAG AIA 3 85,71 0.653 151 FliL MKKKLMIILLIILIVIGALG AAA 3 86,96 0.777 152 LytR MRNERRKKKKTLLLTILTIIGLLVLGTGGYAYYLWH KAA 7 53,85 0.554 153 MreC MPNKRLMLLLLCIIILVAMI GFS 2 78,26 0.718 154 PbpB MIQMPKKNKFMNRGAAILSICFALFFFVILG RMA 5 70,59 0.704 155 PbpX MTSPTRRRTAKRRRRKLNKRGKLLFGLLAVMVCITI WNA 12 48,72 0.667 156 PhoA KKMSLFQNMKSKLLPIAAVSVLTAGIF AGA 4 66,67 0.745 157 PhrG MKRFLIGAGVAAVILSGW FIA 2 85,71 0.513 158 SpoIIP MRNKRRNRQIVVAVNGGKAVKAIFLFIVSLIVIFVL SGV 7 66,67 0.436 159 SpoIIR MKKTVIICIYIFLLLSG ALV 2 70 0.649 160 YdjN MKKRIILLLAVIIAAAAA GVA 3 85,71 0.557 161 YjcN MKKKTKIILSLLAALIVILIVLPVLSPVVFT ASS 4 70,59 0.801 162 YocA MKKKRKGCFAAAGFMMIFVF VIA 5 73,91 0.711 163 YolI MKKWIVLFLVLIAAAISIFVYVST GSE 1 70,37 0.638 164 YopL MKKLIMALVILGALGTSY ISA 2 71,43 0.605 165 YpmB MRKKALIFTVIFGIIFLAVLLVSASIYKS AMA 4 71,88 0.774 166 YqfZ MKRLTLVCSIVFILFILFYDLKIGTIPIQDLPVYE ASA 0 63,16 0.397 167 YrrL MYINQQKKSFFNKKRIILSSIVVLFLIIGG AFL 5 60,61 0.610 168 YrrR MKISKRMKLAVIAFLIVFFLLLLRL AEI 4 75 0.706 169 YrrS MSNNQSRYENRDKRRKANLVLNILIAIVSILIVV VAA 4 51,35 0.586 170 YunA MITDIFKPGCRKLCVFNMKGDYFVKVLLSALLLL LFA 3 64,86 0.338 171 YunB MPRYRGPFRKRGPLPFRYVMLLSVVFFILSTT VSL 6 62,86 0.690 172 YveB MNYIKAGKWLTVFLTFLGILLF IDL 1 72 0.568 173 YyaB MVYQTKRDVPVTLMIVFLILLIQ ADA 0 65,38 0.565
Results
53
25 SP-cutinase fusions were not expressed in B. subtilis (Table 8). Here, the
plasmids constructed in E. coli were not successfully transferred into the expression
and secretion strain B. subtilis TEB1030; by this, indicating a lethal effect. About 23
of these SPs do have calculated D-scores below 0.7 suggesting inefficient cleavage
of the signal peptide (Table 8). It can be speculated that these SP-cutinase fusion
proteins accumulate in the cytoplasmatic membrane, which finally prevents cell
growth.
3.2.4 Translocation efficiency is not solely responsible for the varying export
amounts of cutinase
The varying amounts of overexpressed cutinase in the supernatants when fused to
various signal peptides could be explained by different translocation efficiencies
across the cytoplasmic membrane via the Sec- translocase. Therefore, the
processing kinetics of selected hybrid proteins were analysed by pulse-chase
experiments. Since all newly synthesized protein precursors were labelled for a short
time with radioactive 35S-methionine, a defined pool of labelled proteins was
generated. In the next step, the processing of only the target precursor could be
detected by protein precipitation with specific antibodies raised against the cutinase.
If the C-region of a SP is recognized efficiently by a SPase I, the precursor form
should vanish rapidly and simultaneously the amount of mature cutinase protein is
expected to increase.
For these experiments, five SP-cutinase-fusion proteins (SP-AmyE, SP-GlpQ, SP-
LipA, SP-YncM and SP-Epr) with significant differences in cutinase export rate into
the supernatant were selected: two of which evoked the highest cutinase protein
amounts and lipolytic activities in the supernatants (SP- Epr and SP-YncM). Two
hybrid proteins resulted in significantly lower protein amounts and lipolytic activities
but were still counted among the members of the group of “efficient” signal peptides
with respect to the lipolytic activity in the supernatant (SP-LipA, SP-GlpQ). The fifth
hybrid protein yielded in a comparably low amount of cutinase protein and enzymatic
activity in the supernatant (SP-AmyE). In Fig. 19, the processing of cutinase
precursor proteins into the mature form by removal of the signal peptide is shown. A
strain carrying only the vector pBSMuL3 as a control shows no protein bands at all,
Results
54
demonstrating that the proteins observed in the other samples are indeed specific for
the cutinase precursor and the mature form.
Processing kinetics vary dramatically between the different signal peptides, with the
Epr and AmyE signal peptides mediating the fastest processing. For SP-Epr-
cutinase, no precursor is visible even after the shortest time point (10 seconds) of the
chase, while very low amounts of the precursor of the SP-AmyE-cutinase can be
detected after 10 seconds and 30 seconds of the chase. After one minute, no
precursor protein is detectable for the SP-AmyE- cutinase. In case of the SP-GlpQ
and SP-LipA fusion proteins, the amounts of the precursor after 10 seconds of the
chase are significantly higher than those observed for the SP-AmyE- cutinase and
decline rapidly over time. For the SP-LipA- fusion, processing into the mature form is
complete after one minute of the chase, whereas for the SP-GlpQ- fusion only very
low amounts of precursor are present at that time point. The YncM- fusion shows the
slowest export kinetic. After one minute of chase there is still more precursor than
mature form of the cutinase present, and even after 3 minutes of chase, large
amounts of the precursor form of this fusion were visible (data not shown).
10´´ 30´´ 1´ 10´´ 30´ ́ 1´
SP
-A
myE
SP
- YncM
SP
Epr
SP
-G
lpQ
10´´ 30´´ 1´ 10´ ́ 30´ ́ 1´
SP
- Lip
A
10´ ́ 30´ ́ 1´ 10´´ 30´ ́ 1´
contr
ol
10´´ 30´´ 1´ 10´´ 30´ ́ 1´
- - --10´´ 30´´ 1´ 10´ ́ 30´ ́ 1´
-
10´ ́ 30´ ́ 1´ 10´´ 30´ ́ 1´
1 2 3 4 5 6pm
Fig. 19: Analysis of the processing kinetics of cutinase precursor proteins with different SPs in B. subtilis TEB1030 via pulse-chase experiment. Kinetics were measured for strains with either the vector pBSMuL3 as control (lane 1) or cutinase fused to SP-AmyE, SP-GlpQ, SP-LipA, SP-YncM and SP-Epr on plasmid pBSMuL3Cut-ss (lane 2-6, respectively). The Bacilllus strains were pregrown in S7 minimal medium containing methionine at 37°C to an OD600nm of 0.8, washed twice and starved in S7 minimal medium without methionine for 60 min at 33 °C. Subsequently cells (2,5 mL) were labelled with 150µCi [35S]- methionine for 1 min, followed by chasing with vast excess of non-radioactive methionine. Samples (600µL) for each strain were withdrawn at certain time points (10 sec, 30 sec and 1min), precipitated with ice-cold TCA and further processed as described under 2.20. Sample volumes were equalized to 7.500 cpm. Positions of precursor form (p) and mature protein (m) are indicated.
Results
55
If the varying amounts of cutinase in the supernatant for the different signal peptide
cutinase fusion proteins would only be due to differences in the efficiency of protein
translocation across the cytoplasmic membrane, those fusions with the highest
amounts of protein in the supernatant should show the fastest processing kinetics.
However, this is clearly not the case. While the processing of the SP-Epr- fusion
protein, which shows the highest lipolytic acitivity and large amounts of cutinase
protein in the supernatant, indeed is very fast, the second “best” signal peptide with
respect to the amounts of cutinase protein and activity in the supernatant, YncM,
shows the slowest processing of all the proteins investigated in the pulse chase
experiment. Furthermore, the SP-AmyE fusion, which shows the lowest protein
amounts and lipolytic activity in the supernatant of the five proteins analysed in the
pulse- chase, is processed very rapidly. Thus translocation efficiency is not solely
responsible for varying amounts of secreted cutinase, present in the culture
supernatant.
3.2.5 High level production of cutinase in B. subtilis using the best identified
signal peptide
To analyse cutinase yield under batch culture conditions, B. subtilis TEB1030 strains
with cutinase fused to SP-Epr, SP-LipA and SP-AmyE on plasmid pBSMuL3-Cut-ss
were grown in Erlenmeyer flasks. Culture supernatants were analysed by SDS-
PAGE and a spectrophotometric lipase activity test. As shown on the SDS-gel (Fig.
20.A), there is a distinct cutinase band in all supernatants for cutinase fusions to a
SP compared to the negative control that corresponds to the calculated molecular
weight of about 22 kDa for the cutinase. The different amounts of cutinase were
confirmed by the lipolytic activities in the culture supernatant (Fig. 20.B). Additionally,
they show the same SP ranking as listed Fig. 17. According to the specific activity of
200 U/mg for cutinase against the substrate pNPP, about 35 mg cutinase can be
secreted into the medium by B. subtilis using the SP of Epr.
Results
56
B
A
1 2 3 4
cont
rol
SP-Epr
SP-LipA
SP-Am
yE
kDa
50
30
20
0
2
4
6
8
10
lipolyticactivity[U/mL]
1 2 3 4
Fig. 20: Cutinase production by B. subtilis via secretion into the medium. B. subtilis TEB1030 strains with either the vector pBSMuL3-Cut as control (lane 1) or cutinase fused to SP-Epr, SP-LipA, SP-AmyE on plasmid pBSMuL3Cut-ss (lane 2-4, respectively) were incubated in 250 mL flasks with 25 mL selective LB-medium for 20 hours at 37°C under continuous shaking (150 rpm) and showed no significant growth differences. The culture supernatants were isolated by centrifugation. (A) 20 µg of total proteins of each supernatant were concentrated using 70% trichloride acid precipitation and separated on a protein gel (15 % SDS-PAGE) stained with coomassie brilliant blue. The position of the cutinase band is marked with an arrow. The positions of molecular mass reference markers are indicated (kDa). (B) All culture supernatants were tested for lipolytic activity using the spectrophotometric pNPP-assay at 37°C. The lipolytic activity was adapted to an OD580= 7. Standard deviations are indicated by error bars. The results represent data from 3 independent experiments.
O cutinase palmitic acid nitrophenol - p ) NPP p ( palmitate - nitrophenyl - p O 3 H C 4
Results
57
3.3 One-step optimization of the heterologous esterase EstCL1 for secretion
using the SP-library
In the first approach using cutinase as the heterologous secretion reporter, every SP-
cutinase fusion was ligated and cloned separately to ensure the completeness of the
signal peptide fusion library (Fig. 15). Nevertheless, this procedure is tedious and
cost extensive. Therefore, in another approach we tested the possibility to use the
SP-library to find the optimal fusion partner easily in a standardized procedure of (i)
cloning, (ii) transformation and (iii) screening.
As positive control an equimolar mixture of all PCR-amplified SPs was cloned in front
of the cutinase gene. The ligation – theoretically containing all SP-cutinase fusions –
were transferred into B. subtilis TEB1030 and subsequently 930 randomly chosen
clones were screened for cutinase activity in the culture supernatant (data not
shown). The 12 best performing clones leading to secretion efficiencies of 4 – 5 U/mL
cutinase were isolated and sequenced. As the result, the 8 best performing SPs
according to Table 7 were identified again, with SP-YncM identified 3-times as well
as SP-LytD and SP-WapA both identified twice. Thereby it was demonstrated that
the fast random approach identifies the same SPs being highly efficient in cutinase
secretion.
In order to demonstrate the general applicability of this system, a second
heterologous protein was chosen for secretion optimization in B. subtilis. The
esterase EstCL1 of metagenomic origin was amplified from plasmid p11EstCL1 (109)
by a standard PCR using the primer pair EstCL1_up / EstCL1_low and cloned into
vector pBSMuL3 resulting in vector pBSMuL3-EstCL1. An equimolar mix of all
amplified SPs was cloned into plasmid pBSMuL3-EstCL1 resulting in the plasmid
library pBSMuL3-EstCL1-ssMix which was finally transformed into B. subtilis
TEB1030. The culture supernatants of more than 950 clones were tested in a
modified HTS assay using the TECAN workstation towards the standard esterase
substrate p-nitrophenyl-caproate (p-NPC). Plasmid DNA of ten clones showing
highest secretion efficiencies of EstCL1 was isolated and sequenced. Signal peptides
NprE and YfhK were identified twice; therefore, 8 SPs were characterized in more
detail (Fig. 21), with SP-YwmC being the most efficient one with respect to EstCL1-
secretion transporting up to 1400 U/L across the membrane.
Results
58
The measured activity corresponds to 17,5 mg protein/L calculated with a specific
activity of 80 U/mg against substrate pNPC determined at same conditions. Again,
like in cutinase secretion, the D-scores do not correlate in every case with the
secretion efficiencies of the target proteins. Furthermore, the best performing SPs in
EstCL1-secretion do not correlate with the SP-ranking of cutinase secretion (Fig. 21)
indicating the uniqueness of each hybrid protein.
Figure 21: Identification of the most efficient signal peptide in secretion of the heterologous esterase EstCL1 in B. subtilis. (A) The plasmid library pBSMuL3EstCL1-ssMix containing a mix of all Sec-type signal peptides fused to esterase EstCL1 was transferred into B. subtilis TEB1030. Culture supernatants of about 950 transformants were screened towards esterase activity using the HTS system described before with p-NPC as an esterase substrate. Plasmid DNA of ten clones showing highest activity was sequenced and tested again towards esterase activity. The increasing absorbance at 410 nm was detected by recording a 15 min kinetic at 37°C. The results represent data from three independent experiments. The standard deviation is indicated with error bars and was below 20 %. The D-score was calculated for the first 70 amino acids of each hybrid protein SP-EstCL1 using Computer program Signal P 3.0. (B) The ranking/secretion efficiency of the identified SPs are listed for SP-cutinase hybrid proteins according to Table 13 in the Appendix. The secretion efficiency of the best SP-Epr was defined as 100 % and is according to cutinase activity of 4,67 U/mL.
013,3078,6003,225,1
ranking 1126614541421188653
D-score
secretionefficiency
[%]
0,5880,7990,6950,8550,7250,7420,8170,875
SP-name
este
rase
Est
CL1
cutin
ase
YwmC YpjP YojL YwsB YfhK YxaK NprE YlxW
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
0,5
0,55
0,6
0,65
0,7
0,75
0,8
0,85
0,9
es
tera
se
ac
tiv
ity
[U/m
L]
D-s
co
re
A
B
Results
59
3.4. Directed Evolution of SP-AmyE to achieve high yield of cutinase export
As described before the secretion efficiency of the heterologous model enzyme
cutinase from the fungus Fusarium solani pisi in B. subtilis was improved by
identifying the best natural B. subtilis SP. Another possibility to increase the
functionality of a SP might be the usage of mutagenesis techniques. Directed
Evolution is a powerfull method for enzyme optimization based on random
mutagenesis of the target gene and does not stringently require detailed knowledge
of 3D-structure or reaction mechanism (54, 87, 125, 146, 159). So far Directed
Evolution was rarely used in combination with a screening in B. subtilis mainly due to
the lack of a suitable cloning and screening system resulting mostly in the choice of
E. coli as expression host.
But the establishment of an automated screening process for secretion (chaper 3.2)
offers the opportunity to screen large gene libraries in B. subtilis which is essentiell
for successful Directed Evolution approaches.
Here, two different non-recombinative methods were used to increase the export
capacity of a SP for the cutinase: i) The random insertion of mutations into a SP
using error-prone PCR and ii) saturation mutagenesis at defined regions of a SP
using wobbled oligonucleotides (2, 18, 27, 226).
After the construction of the corresponding signal sequence mutant library the
following cloning procedure using plasmid pBSMuL3-Cut and the automated
screening for lipolytic activity was performed as described in Chapter 3.2.
Based on the screening results summarized in Table 7 the SP of AmyE was chosen
for optimization by Directed Evolution showing a moderate secretion of cutinase
(0.67 U/mL). An overview of the detailed composition of the hybrid protein SP-AmyE-
cutinase and its computer analysis using SignalP 3.0 is given in Fig. 22.
m
Results
60
3.4.1 Random mutagenesis of SP-AmyE did not lead to increased amount of
exported cutinase
Random point mutations were generated performing an ep-PCR by variation of the
MnCl2-concentration in the reaction buffer. The ep-PCR was performed using primer
pair ss-AmyE-up/ -low and plasmid pBSMuL3-Cut-ssAmyE as the template. The
mutation rate was analysed for different concentrations of MnCl2. A compromise
between sufficient PCR product and a reasonably high error rate 0.35 mM MnCl2 was
chosen resulting in an error frequency of 10 base exchanges per kb DNA. This
results in about one mutation per SP. According to the size of 33 amino acids of SP-
AmyE the theoretical library size of about 6000 variants was calculated by using a
formula described elsewhere (7, 87) considering a 10 fold oversampling.
Nevertheless, the screening of more than 3200 clones revealed no significant
improvement of cutinase export compared to wild type SP-AmyE (data not shown).
Fig. 22: Composition of hybrid protein SP-AmyE-cutinase and its computer analysis exercising SignalP 3.0. (A) The signal peptid of AmyE consists of 33 amino acids and is devided into three domains: the N-region (light blue) with three positively charged amino acids, the hydrophobic H-region (black) and the C-region coding (green) the recognition site for SPaseI. The junction (J) between SP and mature protein is indicated in gray. (B) Computer analysis of first 70 amino acids of SP-AmyE fused to cutinase using Signal P 3.0. The graphical output from SignalP was calculating with the hidden Markov model (SignalP3-HMM ) for gram positive bacteria. The score represents the probability (1.0 = 100 %) for a cleavage site (red line), n- and c-region (light blue line) and the h-region (dark blue line).
+1
CN (7) H (23)
TSLLPLFAGFLLLFHLVLAGPAA A E F CutinaseA S AMFAKRFK
J
SPase I A
B
-1 +1 -2 -3
Results
61
3.4.2 Saturation mutagenesis of SP-AmyE revealed improved variants
Since the N- as well as the C-domain of a SP are known to have high impact on the
secretion level of the fusion partner (13, 28, 164, 170), both regions of SP-AmyE
(Fig. 22.A) were subjected to saturation mutagenesis. Additionally the Ala at the +1
position in the junction region was mutated because the most frequent amino acid
Ala at this position for all Bacillus Sec-type exoproteins does not necessarily
represent the most effective one for foreign protein export. Except for the C-region,
the saturation libraries were generated in a standard PCR reaction according to the
oligonucleotides listed in Table 9.
Table 9: Positions of SP-AmyE chosen for saturation mutagenesis. Randomization of bases was performed using an equimolar mix of all dNTPs (N) for the first two bases and an equimolar mix of only dCTP and dGTP (S) for the third base of a codon. Plasmid pBSMuL3-Cut-ssAmyE was used as the template.
Upstream Primer1 Downstream Primer1 Saturated position2 Saturated region of SP-AmyE
AmyE-2NNS-up AmyE-low Phe2
AmyE-3NNS-up AmyE-low Ala3
AmyE-4NNS-up AmyE-low Lys4
AmyE-5NNS-up AmyE-low Arg5
AmyE-6NNS-up AmyE-low Phe6
AmyE-7NNS-up AmyE-low Lys7
N-region
AmyE-up AmyE-low+1NNS Ala+13 +1 region
1 complete sequences listed in Table 5
2 positions as indicated in Fig. 25
3 Ala+1 is the first position of the mature protein as shown in Fig. 23
In order to create a saturation library covering the whole C-region, a mix of wobbled
oligonucleotides synthesized in cooperation with the biotechnological company
BioSpring AG (Frankfurt, Germany) was used. This single stranded (ss-) DNA mix
contains the whole sequence for SP-AmyE with partially wobbled bases encoding for
the three amino acids of the C-domain as listed in Table 10. The base compositions
were chosen to prefer the consensus motif of the C-region Ala-X-Ala (188) (ratio 9:1)
in the resulting SP-AmyE library.
Results
62
Table 10: Base composition of oligonucleotide mix to saturate the C-region of SP-AmyE. Randomization of bases was performed using an equimolar mix of all dNTPs (N) for the first two bases and an equimolar mix of only dCTP and dGTP (S) for the third base of a codon. “X” is defined as any aa.
1 positions as indicated in Fig. 23
2 ratio was chosen with preference for the consensus motif Ala-X-Ala in the C-region of a B. subtilis Sec-type SP
Every synthesized ss-DNA forms a hairpin at the 5`end that was used as a primer in
a Polymerase-reaction. This was perfomed by using the enzyme T7-Polymerase
(Fermentas, St. Leon-Rot, Germany) as described by the manufacturers protocoll
resulting in double stranded DNA ready for subsequent cloning.
The screening of saturation libraries of the C-domain (∼150 variants) as well as the
+1 position (∼260 variants) revealed no further enhancement of the secretion level
(Fig. 23.B,C). This result was confirmed by a pulse chase experiment performed with
the hybrid protein formed by wildtype SP-AmyE and cutinase: very low amounts of
the precursor of the SP-AmyE-cutinase protein could be detected after 10 seconds
and 30 seconds of the chase indicating no bottleneck in the signal peptidase
catalyzed liberation of the mature protein (Fig. 24).
However, of the C-domain three clones and of the +1 position six clones were
sequenced which were screened with the highest activity but still ranging in the
fluctuation range (25 %) of wild type activity. All sequenced clones showed the AXA-
motif of the C-domain of the SP-AmyE with the amino acids Cys, Val and Gly at
position –2 compared to Ser of wild type SP-AmyE. Thus this consensus motif for the
C-region in Bacillus subtilis might hardly be improvable at least in case of this hybrid
protein. Five of the six sequenced clones found in the +1 saturation library possessed
an Ala, one clone bore a Val at position +1 indicating a clear preference for the amino
acid Ala directly after the SP which can be regarded as the optimal amino acid.
position of SP-AmyE1 -3 (Ala) -2 (Ser) -1 (Ala)
base composition
GCC NNS NNS GCC NNS
ratio [%]2 90 10 100 90 10
resulting aa Ala X X Ala X
oligo-nucleotide mix
prefered aa motif
Ala X Ala
Results
63
Screening of the saturation libraries mutated in the N-region of SP-AmyE revealed
four interesting variants leading to highly efficient cutinase secretion (Fig. 25.B,C). In
order to cover the complete saturation library about 200 SP-variants were screened
per position (ten-fold oversampling), except for position R5. Here the library size was
60 SP-variants leading to a three-fold oversampling.
Fig. 23: Screening results after saturation mutagenesis generated in the C-region and +1 position of SP-AmyE fused to cutinase. The screening was performed using the automated high-throughput assay for lipolytic activity in B. subtilis culture supernatants using p-nitrophenyl-palmitate as the substrate. Amino acids are indicated by the one-letter code. (A) Amino acid sequence of SP-AmyE fused to cutinase. N- and H-regions are indicated by open boxes. The C-region and the +1 position are highlighted as red boxes. (B, C). The lipolytic activity of every single clone is shown as relative activity referring to the B. subtilis strain using wild-type SP-AmyE which is defined as 100 % and indicated as a black line. Amino acid substitutions of sequenced SP-variants are shown close to its data-spot, the respective amino acids of wildtype SP-AmyE are shown close to the black line indicating wildtype activity. The standard deviation of the screening system was 25 % and is indicated as a grey shaded field.
rela
tive
lipol
ytic
activ
ity[%
]
screened variants
0
20
40
60
80
100
120
140
0 50 100 150 200 250 300
VA
A
AA
A
+1
CN (7) H (23)
TSLLPLFAGFLLLFHLVLAGPAA A CutinaseA S AMFAKRFK
SPase I
E F
+1
C+1 position
BC-region
rela
tive
lipol
ytic
activ
ity[%
]
screened variants
0
20
40
60
80
100
120
140
0 50 100 150 200
AGAAVA
ACA
ASA A
A
Results
64
Changes at positions Phe2,
Lys4 and Phe6 were identified to
improve secretion of cutinase up
to three-fold compared to the
wild-type SP-AmyE. The best
four SP-variants were
sequenced, thereby identifying
two beneficial substitutions.
Amino acid exchanges from Phe
to Asp and Phe to Glu at
position Phe2 of the N-domain
both increased the secreted
amount of cutinase more than
three-fold. Therefore, the
exchange of the non polar
aromatic phenylalanine residue
to the negatively charged amino
acids aspartate or glutamate at position 2 leads to a drastically increased cutinase
export. Interestingly, by this exchange the net charge of the SP was reduced from +3
to +2. In contrast, a significant reduction in secretion occurred when the signal
peptide showed uncharged amino acids like alanine or threonine at this position (Fig.
25. B,C).
10´´ 30´´ 1´ 10´´ 30´´ 1´
SP
-A
myE
contr
ol
10´´ 30´´ 1´ 10´´ 30´´ 1´
-
1 2
p
m
Fig. 24: Processing kinetics of cutinase precursor protein with SP-AmyE in B. subtilis TEB1030 analysed by pulse-chase experiment. Kinetics were measured for strains with either the vector pBSMuL3 as control (lane 1) and cutinase fused to SP-AmyE on plasmid pBSMuL3Cut-SP-AmyE (lane 2). Both strains were treated as described in 2.20. Sample volumes were equalized to 7.500 cpm. Positions of precursor form (p) and mature protein (m) are indicated.
Results
65
100 2000 100 2000 100 2000
100 2000 100 200030 600
100
200
300
400
100
200
300
400
100
200
300
400
100
200
300
400
100
200
300
400
100
200
300
400
F2 A3 K4
R5 F6 K7
B
C
ASAMFAKRFK
N (7) H (21)
TSLLPLFAGFLLLFHLVLAGPAA A Cutinase
+1A
F2D F2E
F2A F2T
K4L
K4G
F6W
F6S
0
100
200
300
400
w t F2D F2E F2T F2A K4L K4G F6W F6S
Figure 25: Screening results of saturation mutagenesis libraries generated in the N-region of SP-AmyE fused to cutinase. (A) Amino acid sequence of SP-AmyE given in one-letter code. N-, H- and C-regions are indicated by open boxes. The saturated positions of the N-region are highlighted in red letters. (B) Detailed screening results of the saturation libraries at position 2-7 of SP-AmyE. The lipolytic activity of every single clone is given as relative activity refered to the B. subtilis strain using wild-type SP-AmyE which is defined as 100 %. Amino acid substitutions of sequenced SP-variants are given in the one-letter code close to its data-spot. (C) Reproduction of the secretion efficiency results for eight SP-variants in comparison to the wild-type SP-AmyE as reference. The results represent data from 7 independent experiments. The standard deviation is indicated by error bars. The amount of secreted cutinase fused to the wild-type SP-AmyE (wt) is defined as 100%. The results were confirmed by Western Blot analysis using cutinase specific antibodies (data not shown).
1. A +1
CN (7) H (23)
TSLLPLFAGFLLLFHLVLAGPAA CutinaseA S AMFAKRFK A E F
+1
C
lipolytic activity
[%]
Results
66
Another important position of the SP-AmyE N-region was identified to be Lys4. Most
of the SP-variants showed less than 50% of wild-type activity. However, the
substitution of Lys4 by a leucin residue leads to a three-fold increase in cutinase
secretion. The third important residue has been identified at position Phe6. The
substitution of Phe6 by tryptophan resulted in a two-fold improved export, whereas
the exchange to a serin residue had the opposite effect reducing the secreted
cutinase amount to less than 20%. At the positions Ala3, Arg5 and Lys7 no SP-
variants were identified showing significantly improved secretion amounts but seem
to be no critical position neither. All SP-variants showing improved properties were
confirmed by retransformation and repeated independent growth experiments (Fig.
25.C). Regarding the correlation between the change in export efficiency and the
change in the D-score caused by one defined amino acid exchange results in a
surprising finding for the sequenced variants (Fig. 26): for any amino acid exchanges
leading to higher cutinase export rates the D-score was at least minimally reduced up
to a strong decrease for variants whose amino acid Phe2 was changed to the polar
residues Asp or Glu. In summary, by using saturation mutagenesis in the N-region of
the SP-AmyE cutinase export was increased more than three-fold thus changing the
Fig. 26: Comparative analysis of export efficiency and calculated D-score for chosen variants of SP-AmyE with amino acid exchanges in the N-region. The SPs were found in the saturation library of the N-region of SP-AmyE (data taken from Fig. 25.C). The amount of secreted cutinase fused to the wild-type SP-AmyE is defined as 100%. Amino acid substitutions of sequenced SP-variants are given in the one-letter code. The D-score was calculated for the first 70 amino acids of each hybrid protein using Computer program Signal P 3.0. The D-score of hybrid protein cutinase and wild-type SP-AmyE was 0,904 and is indicated with a red bar.
0
100
200
300
400
0,92
0,86
0,88
0,9
0,94
0,96
D-s
core
Lipo
lytic
activ
ity(%
)
F6WF2A F2T F2D F2E K4G K4L F6S
different variants of SP-AmyE fused to cutinase
Results
67
SP of AmyE from a “lower class” SP (ranking No. 65, Table 7) to a “moderate“ SP”
(corresponding to ranking No. 23, Table 7) similar to SP-GlpQ (ranking No. 26, Table
7).
3.5 Coexpression and mutagenesis of B. subtilis secA to increase cutinase
export
The overexpression and secretion of heterologous proteins show major drawbacks
due to different bottlenecks in the secretory pathway (11, 20). The optimization of
SPs as described before comprises the efficient transport to the cell membrane as
welll the acceptable processing for release of the mature exoprotein outside the cell.
At least of the same importance is the essential protein SecA which is the central
component of the Sec pathway mediating the active translocation of proteins based
on interaction with the SP and the channel forming proteins. For some exoproteins
the overexpression of SecA had a positive effect on the secretion efficiency (111). To
study the influence of co-overexpressed SecA protein for cutinase export in a HTS
system, a suitable B. subtilis marker strain had to be constructed using the cutinase
of the fungus Fusarium solani pisi stably integrated into the chromosome as a fusion
with a Bacillus SP. In addition, variants of SecA were constructed by applying
random mutagenesis and coexpressed in the marker strain. Using the HTS assay
described under 3.2 the secA libraries were screened for enhanced cutinase export.
3.5.1 Construction and characterization of marker strain B. subtilis Marc1
The B. subtilis secretion marker-strain Marc1 was created by transforming B. subtilis
TEB1030 with the integration plasmid pMCut1 (2.11) which contains a DNA fragment
encoding for the hybrid protein SP-LipA-cutinase (Fig. 27.A). The correct double-
crossover integration of pMCut1 into the chromosomal locus amyE was confirmed by
an indicator plate assay using a starch-containing growth-medium (177) (data not
shown). A double crossover led to a phenotype expressing no Amylase E protein and
Results
68
thus no starch degredation on the indicator plate. In contrast to strain B. subtilis
TEB1030 the constructed marker strain B. subtilis Marc1 showed lipolytic activity in
the supernatant (Fig. 27.B). A further western blot analysis confirmed the presence of
cutinase in the supernatant of B. subtilis Marc1 (Fig. 27.C).
This marker strain shows a lipolytic activity of 18 U/L against p-NPP as the substrate
leading to a cutinase export amount of 90 µg protein/ L calculated with the specific
activity of 200 U/mg cutinase. The stability of the integrated cutinase was tested by
growth of B. subtilis Marc1 in 25 mL LB-medium under non selective conditions over
96 hours. After each 24 hours an aliquot was tested by a lipolytic activity assay as
well inoculated into fresh medium. The cutinase activity did not decrease over this
time period indicating a stable integration of the cutinase into the Bacillus
chromosome even under non selective growth conditions (data not shown).
Fig. 27: Construction and characterization of B. subtilis secretion marker strain Marc1. (A) E. coli-B. subtilis shuttle vector pMCut1 was integrated into the chromosomal amyE gene of B. subtilis TEB1030 via double crossover of two 500 bp flanking amyE fragments resulting in cutinase expressing strain B. subtilis Marc1. ss: signal sequence of LipA; The constitutive promoter PHpaII is symbolized as a rightangled black arrow; Cm: gene providing chloramphenicol resistance; the deletion of two genes of extracellular lipase A and B are indicated by crossed out yellow arrows. The expressed cutinase is recognized by its SP-LipA from the Sec machinery and translocated across the cell membrane. (B) B. subtilis TEB1030 and Marc1 were grown in 25 mL LB-medium for 20 hours and the culture supernatant was tested for lipolytic activity in a spectrophotometric assay using p-NPP as the substrate. Both cultures showed no significant growth differences. The lipolytic activity was adjusted to an OD580= 7. The data represent the average value of three independent experiments. The standard deviations were lower than 12 % and indicated by error bars. (C) Immunodetection of cutinase in culture supernatant of B. subtilis TEB1030 and Marc1 using cutinase-specific antibodies produced in hybridoma cells. From each supernatant a total protein of 20 µg was used.
pMCut1
chromosome
amyE gene
cutinase Cmss
lipA lipB
A
B. subtilis TEB1030 cell
B
0
5
10
15
20
TEB1030 Marc1
lipolytic activity[U/L]
C
cutinase
B. subtilis Marc1 cell
chromosome
lipA lipB
cutinase Cmss `amyEamyE`
Sec pore
Results
69
3.5.2 Co-overexpression of SecA improves cutinase secretion
Marker strain B. subtilis Marc1 was used to test the effect of overexpression of SecA
in addition to the chromosomal encoded secA controlled by its native promoter.
Therefore the secA gene containing its natural ribosome binding site was cloned into
plasmid pBSMuL3 resulting in pBSMuL3secA. To compare the export rate of
cutinase fused to SP-LipA with and without overexpressed SecA, B. subtilis Marc1
was transformed with pBSMuL3 and pBSMuL3secA (Fig. 28.A) and the culture
supernatants of both strains were tested for lipolytic activity. The plasmid encoded
expression of SecA increased the cutinase activity five-fold up to 100 U/L indicating a
protein export of 500 µg cutinase (Fig. 28.B). A Western blot analysis of the
Fig. 28: The effect of overexpressed SecA for cutinase export in marker strain Marc1. (A) Plasmid pBSMuL3secA overexpressing secA was transformed into B. subtilis Marc1. Vector features are described in Fig. 9. (B) B. subtilis Marc1 bearing empty plasmid pBSMuL3 and secA-overexpression plasmid pBSMuL3-secA were grown in 25 mL selective LB-medium for 20 hours and the culture supernatant was tested for lipolytic activity in the standard spectrophotometric assay. Both cultures showed no significant growth differences. The lipolytic activities were adjusted to an OD580= 7. The data represents means of four independent experiments. The standard deviations were lower than 10% and indicated by error bars. (C) Immunodetection of cutinase in culture supernatant (S) and cellular (C) SecA of B. subtilis TEB1030 (lane 2) and Marc1 (lane 3) after 20 hours. As a positive control 1,5 µg purified cutinase was used (lane 1). Cutinase-specific antibodies were produced in hybridoma cells. Polyclonal antibodies against B. subtilis SecA were produced in a rabbit. From each cell lysat/ supernatant a total protein amount of 20 µg was used.
amppBSMuL3
secA
repB
kan
MCS ori E.coli
secA2,5 kb
Transformation
A
control + secA
0
50
100
150
lipolyticactivity[U/L]
B C
1 2 3
cutinase
secA
S
C
cutinase
Marc1
B. subtilis Marc1 cell
lipA lipB
cutinase Cmss `amyEamyE`
secA
pBSMuL3secA
secA
cutin
ase
TEB1
030
Mar
c1
Results
70
supernatant confirmed the enhanced cutinase amount (Fig. 28C), a second smaller
band below the cutinase shows proteolytic degradation caused by one of the
extracellular proteases of B. subtilis. The immunodetection of cellular SecA revealed
an increased amount of SecA for the SecA-overexpression strain compared to the
control strain (Fig. 28C) confirming an overexpression of SecA in addition to the
chromosomal encoded SecA.
3.5.3 The precursor form of cutinase fused to SP-LipA is processed rapidly
under condition of SecA-overexpression.
The kinetic of precursor form processed into mature exoprotein is a quality control for
efficient translocation across the cell membrane. Therefore the cutinase export
kinetic of B. subtilis Marc1 with or without plasmid encoded secA was studied by a
pulse chase experiment (Fig. x): Control strain TEB1030 showed no signals
indicating high specificity of immunoprecipitation by using polyclonal cutinase
antibodies (lane 1). For TEB1030 containing plasmid encoded hybrid protein
cutinase-SP-LipA a precursor form is clearly visible up to 30 sec after moment of
chasing (lane 4). Nevertheless after 1 min all cutinase precursor is processed to
mature form reporting a rapid translocation process. Anyway Marc1 showed only the
1´10´´ 30´´
TE
B10
30
1
1´10´´ 30´´ 10´´1´10´´ 30´´
Mar
c1 +
secA
3
contr
ol4
Mar
c1 +
pB
SM
uL3
2
p
m
Fig. 29: Processing of precursor protein cutinase fused to SP-LipA in different B. subtilis strains. Kinetics were measured for the B. subtilis strains TEB1030 (lane 1), Marc1 bearing empty vector pBSMuL3 (lane 2) and Marc1 bearing secA-overexpression plasmid pBSMuL3-secA (lane 3). As a control B. subtilis TEB1030 bearing plasmid encoded hybrid protein cutinase-SP-LipA (pBSMuL3Cut-ss-LipA) was used (lane 4). The Bacilllus strains were pregrown in S7 minimal medium containing methionine at 37°C to an OD600nm of 0.8, washed twice and starved in S7 minimal medium without methionine for 60 min at 33 °C. Subsequently cells (2,5 mL) were labelled with 150µCi [35S]- methionine for 1 min, followed by chasing with vast excess of non-radioactive methionine. Samples (600µL) for each strain were withdrawn at certain time points (10 sec, 30 sec and 1min), precipitated with ice-cold TCA and further processed as described under 2.20. Sample volumes were equalized to 7.500 cpm. The different positions of unprocessed cutinase-SP-LipA precursor form (p) and the mature cutinase (m) are indicated.
Results
71
a pulse-chase experiment (Fig. 29): B. subtilis strain TEB1030 showed no signals
indicating high specificity of immunoprecipitation by using polyclonal cutinase
antibodies (lane 1). For control strain B. subtilis TEB1030 containing plasmid
encoded hybrid protein cutinase-SP-LipA the position of the precursor form is clearly
visible 10 sec after moment of chasing (lane 4). Anyway B. subtilis Marc1 with or
without overexpressed SecA showed at all measured time points only the mature
cutinase reporting an efficient processing (lane 2,3). This data suggested no export
bottleneck due to improper cleavage of SP-LipA at the trans-site of the membrane for
this B. subtilis secretion reporter strain.
3.5.4 A SecA variant leading to eight-fold enhanced protein export.
To achieve a further enhancement
of cutinase export in B. subtilis
Marc1, the strategy was to optimize
the recognition and binding affinity
of overexpressed SecA to the
hybrid protein SP-LipA-cutinase by
mutating the whole secA gene.
Therefore an ep-PCR was
performed to insert random point
mutations into the whole secA gene
by using primer pair secA_up/
secA_low and pBSMuL3secA as
the template. The generated secA
library pBSMuL3secA-ep was
finally transformed into B. subtilis
Marc1 and about 4300 B. subtilis
clones were screened for cutinase
activity. One SecA variant showed
Fig. 30:. The effect of two B. subtilis SecA variants for the export level of cutinase. B. subtilis Marc1 harbouring plasmid encoded secA and two secA mutants were grown in 1 mL selective LB-medium each in a DeepWell plate for 16 hours and the culture supernatant was tested for lipolytic activity in the automated spectrophotometric assay at 30°C described before. The data represents means of three independent experiments. The standard deviations were lower than 16 % and indicated by error bars. The amino acid exchanges are given in one letter code.
0
200
400
600
800
1000
SecA type variant + variant -wildtype
lipol
ytic
act
ivity
[U/L
]
amino acid exchange - A23T
K809D
A23TK809D
H289Y
Results
72
an eight-fold higher cutinase export than overexpressed wildtype SecA (Fig. 30). The
following sequencing revealed two amino acid exchanges at position 23 from alanine
to threonine and at position 809 from lysine to aspartate.
3.5.5 The improved export effect of SecA variant A23T, K809D was neutralised
by a further amino acid exchange H289Y in the PPX-domain
In a second approach only the preprotein-crosslink-(PPX-) domain was target of
mutagenesis. This domain is most likely responsible for the binding of SecA to the
signal peptide of a precursor protein as shown in former experiments (76, 144). To
mutate specifically the PPX-domain of secA by ep-PCR, a method was established
called “Quick Cassette Mutagenesis” based on the QuickChange II Site-Directed
Mutagenesis Kit from Stratagene (Heidelberg, Germany) as described under 2.10.5.
More than 2000 SecA variants were screened in Marc1 but showed no higher
cutinase export rate. Additionally the improved SecA variant A23T, K809D (Fig. 30)
was used for this “Quick Cassette Mutagenesis”: Under 350 screened PPX-variants
no further improvement could be observed. On the contrary, cutinase export of one
clone was decreased to wildtype level of overexpressed SecA. Interestingly, its
sequence showed a third amino acid substitution at position 289 from histidine to
tyrosine embedded in the SP binding domain (Fig. 31). In consequence this position
might be critical for the efficient interaction of SecA and the SP-LipA of the precursor.
Discussion
73
4. Discussion
Since many years Bacillus subtilis and related Bacilli like B. licheniformis are used as
expression and secretion host. The ability to express and secrete enzymes in high
amounts of up to 20 g/L into the culture medium as well as the classification as
GRAS, i.e. generally recognized as safe, organism free of any endotoxin, are
reasons for the wide use in industrial fermentation processes (172). In contrast to
high yield production of homologous proteins, heterologous enzyme secretion often
lead to disappointing low yields in Bacillus species due to numerous bottlenecks that
severely reduce the production capacity. In an already started process, identified
bottlenecks (e. g. inefficient targeting to the membrane, inefficient processing,
proteolytic degradation, misfolding, retention in the cell wall, inconvenient codon
usage, lack of suitable expression plasmids etc.) are targets of intensive studies to
eliminate or minimize them (11, 94, 114, 122, 180, 186, 208, 215).
This work focussed on the optimization of the secretion host B. subtilis for foreign
proteins with respect to the major role of the signal peptides for a successful
targeting to and across the cell membrane. Within the scope of this thesis a secretion
reporter system was established based on a biotechnological relevant lipolytic
enzyme of heterologous origin and (ii) adapted to an automated high throughput
screening (HTS) system in B. subtilis to (iii) analyse a generated library of all natural
B. subtilis signal peptides for optimal secretion efficiency of our target protein as well
as for other industrial applications. The established HTS system allowed (iv) the use
of Directed Evolution for the construction of mutant libraries of SPs or components of
the Sec pathway as a strategy for secretion enhancement. Another aspect of this
work deals with (v) the construction of useful Bacillus expression and secretion
vectors to broaden the range of biotechnological applications for B. subtilis.
Discussion
74
4.1 A new Bacillus vector series
pBSMuL vectors show a broad application spectrum
Although several new Bacillus plasmids were published over the last years (e.g.
(106, 108, 134, 147), there is a general need for stable and user-friendly expression
and secretion plasmids for B. subtilis (147, 208). Here, three multi-copy B. subtilis
expression vectors have been constructed, namely pBSMuL1,2,3.
For the first time, the pBSMuL series combines several convenient vector features for
B. subtilis comparable to available E. coli expression systems as shown in Table 11.
(i) The functionality for all vectors was demonstrated in this study using the
heterologous target genes cutinase and estCL1 as well as the homologous gene
secA. Additionally, the SP of pBSMuL1,2 can easily be cleaved off for intracellular
protein expression as shown in another study: plasmid pBSMuL2 was used for
intracellular overexpression of the homologous protein YtvA in B. subtilis resulting in
a production amount of more than 20 mg protein/L. The final yield after purification
was about 6 mg protein/L (Krauss, Gärtner, Jaeger, Eggert, unpublished results).
Since the pBSMuL vectors are based on an E. coli–B. subtilis shuttle plasmid (36,
228), the cloning procedure can be performed in E. coli. The E. coli part can be
cleaved off and the self-ligation of the remaining Bacillus vector fragment leads to
plasmid multimers, which are more efficiently uptaken by B. subtilis during
transformation (65). Consequently, this vector feature allows the generation and
screening of large gene libraries with one of the easiest transformation procedures
for B. subtilis (2.9.3) as done with vector pBSMuL3 (3.2, 3.4, 3.5) showing only a
slightly slower effectiveness than electroporation techniques (data not shown). Time-
consuming transformation methods like the “protoplasts” transformation can be
avoided (65).
Table 11: Properties and applications of the pBSMuL vector series
* C-terminal His-tag is optional
features constitutive promoter(s)
Bacillus
rbs MCS
signal peptide
His-tag multimeric
DNA formation
applications high level
expression high level
expression convenient
cloning secretion
intracellular expression
one-step purification
HTS-application
pBSMuL1 1 + + + + + * + pBSMuL2 2 + + + + + * + pBSMuL3 1 - + - + - +
Discussion
75
Especially pBSMuL3 is a promising tool for future projects using the SP-library for a
“one step optimization” of foreign secretory proteins in B. subtilis. This new strategy
was successfully applied for two heterologous lipolytic enzymes cutinase and EstCL1
(Fig. 20, 21) leading to secreted protein yields of 35 mg/L and 17,5 mg/L,
respectively. In comparison to results of other expression systems in B. subtilis used
for proteins of eukaryotic origin (21, 79, 133, 178, 206), the produced protein
amounts for the pBSMuL3 vector are quite satisfying. Moreover, since the second
promoter in the pBSMuL2 vector could increase three-fold the secretion yield for
cutinase up to 60 mg/L, the same insertion of this P59 promoter into the plasmid
pBSMuL3 could likely lead to an even higher production and secretion efficiency
which should be tested in the future.
Do pBSMuL vectors have the potential for industrial applications?
The construction of the pBSMuL series also addressed to industrial applications by
focussing on protein overexpression using constitutive promoters without the
necessity of induction, since common inducers like IPTG, xylose or heat represent
additional costs and can decrease dramatically the profit of a production system. This
money can be saved by the use of antibiotic-free growth medium, since a vector
stability was reported for pBSMuL1,2 overexpressing and secreting cutinase for at
least 48 hours in non selective medium without significant reduction (Fig. 7).
Nevertheless, a faster loss of the vector cannot be excluded for other target proteins
under non-selective growth conditions and/or under high cell density fermentation
due to enhanced selection for a plasmid-free cell culture. Moreover, in previous
studies it was reported that high secretion stress stimulates regulatory systems in
B. subtilis, which might have a negative influence in production yield (77, 78, 152).
In summary three multi-copy B. subtilis expression vectors for cloning of target genes
under control of one or two strong constitutive promoter(s) have been constructed.
The novel plasmid series enabled (i) convenient cloning into an artificial multiple
cloning site, (ii) high level overexpression without the necessity of induction, (iii)
generation of huge libraries for HTS processes, (iv) efficient secretion of
heterologous proteins, (v) application for the “one step optimization” by finding the
best natural SP for protein secretion and (vi) the easy one-step purification in the
Discussion
76
Gram-positive host B. subtilis. This system might be useful as an alternative in case
of serious problems (inclusion bodies, misfolding, low expression, toxicity)
concerning the well-established E. coli expression systems (8). However, an
industrial use for large-scale production needs to be chosen under fermentative
conditions in future projects.
4.2 A new secretion marker system for HTS processes in B. subtilis
As an alternative strategy to rational protein design (16) Directed Evolution enabled
enzyme design without much knowledge of structure or reaction mechanism. But the
analysis of large gene libraries requires a suitable High throughput screening (HTS)
assay, which allows a rapid and reliable determination of enzyme activity, a
significant reproducibility, a cheap and easily prepared reaction assay (176).
Lipolytic enzymes belong to the class of hydrolases. Some members of this group
show chemo- regio- and stereoselectivity, are very stable and active even in organic
solvent. Consequently they are of high commercial interest and are already used in
industrial production of detergents, food, pharmaceutics and agrochemicals (14, 15,
86, 88, 115). Since several reported HTS system were created successfully to detect
lipolytic enzymes with improved properties (49, 54, 85, 158), one of these
biocatalysts, the cutinase from Fusarium solani pisi (56), was chosen as a reporter
enzyme to analyse heterologous protein secretion in B. subtilis.
Since the secreted cutinase yield correlated with the lipolytic activity in the
supernatant in all measured cases, this lipolytic activity assay is a reliable method to
detect and compare easily the export capacity for cutinase in B. subtilis.
Chances and limits of the HTS system
The most important aspect of this automated HTS system, the reproducibility, was
good enough for a successful application using the lipase substrate pNPP due to an
overall standard deviation of about 10% including growth differences of B. subtilis,
stability of the assay and stability of secreted cutinase in Bacillus supernatant over
four hours (Fig. 13). The screening of mutant libraries in B. subtilis revealed an
Discussion
77
increased fluctuation in the cutinase activity up to a standard deviation of about 25%
(Fig. 14) most likely by a variation in the individual plasmid copy number (~50) per
cell after the transformation event. As a consequence of the performed mutant library
screening in B. subtilis, only clones with significantly improved or decreased cutinase
export (50 %) compared to wildtype activity were chosen for further analysis.
It is noteworthy that the incubation time of the Bacillus colonies on transformation
agar plates revealed to be a critical factor for reproducibility of cell growth in MTPs: if
Bacillus colonies were grown longer than 30 hours on agar plates before transferred
into the DW-MTP, the growth differences (compare Fig. 10) dramatically increased to
a standard deviation of more than 25 % (data not shown). Therefore it is crucial to
perform every step of the whole screening process stringently the same way to
guarantee the reproducibility and comparability of screened libraries.
In another study (38) the secreted cutinase showed almost the same activity even
over a time period of six hours incubated at -20°C, 4°C or RT confirming the enzyme
stability in the culture supernatant of B. subtilis TEB1030 that still harbours several
extracellular proteases (208). Nevertheless, processes for a duration of more than
five hours are not recommended due to substrate instability (chapter 3.2) resulting in
a maintenance free HTS process for maximal 1300 samples.
4.3 Improvement of heterologous protein secretion in B. subtilis by signal
sequence screening
Expression and survival of cutinase depends on the presence of a functional signal peptide
The generated Bacillus plasmid pBSMUL3-Cut, bearing only the cutinase gene
without its start codon, showed no cutinase expression since the vector lacks a
ribosome binding site and a start codon downstream from the promoter. This
property made plasmid pBSMuL3-Cut a versatile cloning and expression tool for the
comparison of cutinase export with different Bacillus Sec-type SPs. All 173 signal
sequences amplified by PCR (Table 12, Appendix) from the genome of B. subtilis
168 share a consensus sequence at the 5`-end encoding for a Bacillus rbs, a suitable
spacer region and the start codon ATG what is obligatory for a ribosomal translation
process (Fig. 16). Due to the phenomenon of SP-independent protein export (5, 187),
Discussion
78
the cutinase was overexpressed in B. subtilis on plasmid pBSMuL3-Cutintra lacking a
SP to test the occurrence of cutinase in the extracellular medium. But no cutinase
could be detected in the supernatant neither by immunodetection (Fig 18) nor by a
lipolytic activity assay (data not shown). Astonishingly, the cutinase was not even
detectable in the cytoplasmic fraction. This observation strongly suggests a massive
intracellular degradation as a consequence of a high sensitivity of the heterologous
cutinase to intracellular proteases in B. subtilis (67, 107). Contrary findings were
reported in previous studies for the production of foreign Bordetella pertussis toxin
subunits in B. subtilis: the secretion yield for most toxin subunits was low, whereas
the intracellular production without a SP expressed in the same vector revealed high
level production (73, 168, 169). One can speculate that these toxin subunits form a
more stable and protease resistant structure after intracellular expression compared
to cutinase, which might need a slower folding process to reach the final
conformation. This characteristic of the cutinase could explain the requirement for
secretion to prevent degradation (188). Furthermore, no precursor form of cutinase
was detected in the cytoplasmic when expressed as hybrid protein with different SPs.
This strongly supports the idea of a fast proteolytic degradation of precursor cutinase.
In conclusion, the intracellular transport for secretory cutinase seems to be a “race”
to the membran- the more efficient the binding of its fused SP to the targeting factors
(SRP, SecA) and the membrane, the higher is the amount of “surviving” precursor
cutinase that can be translocated across the cell membrane. This early secretion
step seems to be a major bottleneck for cutinase export.
The processing of cutinase precursor is not the limiting step
The observation that most of the functional SPs for cutinase export bear a suitable C-
terminal cleavage site suggested efficient processing (Table 13, Appendix). But the
simple analysis of a SP for a suitable C-region using prediction tools like SignalP
cannot guarantee an efficient processing rate of a precursor. For instance, the
precursor interacts with all involved Sec-components during the translocation
process across the membrane in an individual manner depending on its SP (131,
136, 178, 181, 189, 221, 227). Next, both SP and its mature protein might interact in
a unique way by its secondary structure (52, 145), thereby influencing the
Discussion
79
accessibility of the cleavage site (A-X-A) of the SP to a signal peptidase at the trans-
site of the membrane. But the C-region must be in an extended (β-) conformation for
an efficient cleavage event (141). Furthermore, the H-region must be of a suitable
length to guarantee the positioning of the C-region in the range of a SPase close to
the lipid headgroups at the trans-site of the membrane (72, 199). All these
possibilities, too complex yet to be considered in available prediction tools, could
disturb or prevent the suitable access of the C-region to the SPase leading to
insufficient processing and proteolytic degradation of the cutinase by extracellular
proteases (11).
Consequently, the processing kinetics of five selected SPs, showing different
cutinase export rates, were analysed by a pulse-chase experiment (Fig. 19). Even if
the processing rate of the precursors differed from each other significantly, in all
cases mature cutinase was visible indicating a successful processing event.
Remarkable is the observation that the secretion level of cutinase does not
corresponds to the processing rate. The SP-AmyE shows a similar fast export kinetic
than SP-Epr, although SP-AmyE results in a low, SP-Epr in a high secretion level of
cutinase. Moreover, SP-YncM leads to high yield of cutinase export but shows clearly
the slowest processing. In conclusion, the processing of all tested hybrid proteins is
efficient enough to provide cutinase secretion even if strong differences in the export
kinetics occurred. Hence, the processing of precursor to mature cutinase indicates no
export bottleneck for the secretion yield of cutinase in B. subtilis under the given
expression and culturing conditions.
Variations of the precursor synthesis rate might influence the secretion level
However, even if the chosen secretion marker system provides the same distance of
the gene to the promoter for all SP-cutinase fusion constructs, we cannot totally
exclude variations in cutinase export due to different precursor synthesis rate. Every
signal sequence fused to the cutinase gene leads to an individual mRNA, its
structure might influence the transcript stability or disturb the translation process
(112, 153). This effect could result in different amounts of precursor protein in the
cytoplasm and later result to different secretion yields of mature protein. A possible
feedback mechanism of a preprotein on translational level was postulated in earlier
Discussion
80
studies (174, 178, 190). Anyway, an additional analysis of transcriptional level was
not necessary during this study since the used secretion reporter system includes
this factor for export efficiency in the applied screening method.
In silico prediction of the most functional signal peptide for foreign proteins is
not yet possible
Our systematic approach using two different lipolytic enzymes as secretion targets
clearly demonstrate the inability to predict in silico an optimal SP for protein secretion
using the computer tools available so far. As one commonly used computer tool to
characterize a signal peptide, the SignalP-server generating the so-called D-score
was chosen (9, 135). There was no clear correlation between D-score and secretion
efficiency of the target proteins (Table 7, Fig. 21).
Since it was observed that defined alteration of net charge, hydrophobicity or length
of the SP could change secretion efficiency (13, 164), these criteria were analyzed in
the generated SP-library without clear correlation to the secretion amounts. Only the
overall composition of SPs – positively charged N-region, hydrophobic core region
and suitable cleavage site (A/VXA) – was obvious, in addition to this the SP-library
showed a strong heterogeneity in length, charge and hydrophobicity without
correlation to export efficiency (Table 13, Appendix).
Previous studies in B. subtilis compared SP-variants to the wildtype when fused to a
mature protein and revealed differences in export efficiency of the hybrid protein (13,
29, 130, 164). However, the effects could not be reproduced when other SPs were
fused to the same mature protein (29). Furthermore, secretion experiments in E. coli
reported an influence of the N-terminal part of the mature protein in secretion
efficiency, indicating the importance of an optimal interaction between signal peptide
and secretion target (110, 113, 118, 156, 210). Moreover, the junction between SP
and mature protein is a further critical variable that influences the secretion level of a
target protein (69, 93).
It looks like every fusion of a SP-mature protein forms a unique precursor interacting
with the components of the secretion apparatus suggesting that protein transport is
restricted severely by the efficiency of SP-recognition by all participating components
the translocation machinery.
Discussion
81
The new “one-step optimization” method could enable the secretion of any
target protein as demonstrated for EstCL1 in B. subtilis
The unpredictability of the secretion efficiency of a defined SP for an unknown
secretion target was discussed before. But the established HTS for B. subtilis offered
the screening of the complete SP library size in one step covering all possible
variations built of the generated pool of 173 Bacillus natural Sec-type SPs and the
target protein: and indeed, the best functional SPs for cutinase export, which were
found by a separately performed cloning procedure for each of the possible hybrid
protein, were found again in the combination of a one-cloning step and HTS. By this
new method for export optimization one can easily bypass the risk of low secretion
yield as a consequence of the “wrong” SP. This was successfully applied to the
secretion of a non-secretory protein EstCL1, an esterase of metagenomic origin.
Even if EstCL1 shows a pI of 4.5 suggesting no disturbing interaction in the cell wall
environment (171, 180, 186), its secretion into the growth medium up to 18 mg
protein/L is remarkable for two reasons. First, the encoding gene has a GC-content
of 60% which is significantly higher than the general GC-content of B. subtilis
(~ 50%) which might lead to expression problems by the wrong codon usage.
Second, the protein was defined as a non secretory protein due to the lack of any N-
terminal SP (109). Considering the minimal time expenditure of 1-3 weeks for the
complete cloning and screening process of EstCL1 to generate and test all natural
SPs, a general application of this “one-step optimization” method can be predicted for
B. subtilis for theoretically any heterologous protein production.
4.4 Saturation mutagenesis as a useful tool for the characterization and
optimization of a signal peptide
The HTS system enables Directed Evolution for secretion studies in B. subtilis
Besides the necessity for the overall criteria of a SP, no general statement could be
formulated so far how to make SPs more functional for a particular export target (29,
130, 182, 218, 219). Even if some studies performed a more systematic approach in
various Gram-positive bacteria (B. subtilis, Streptomyces lividans, Lactococcus lactis)
Discussion
82
by analysing a series of SP variants for export functionality (13, 29, 53, 130, 157,
225), the variants were small in number (< 30) and generated by site directed
mutagenesis.
In consequence, this lack of a generalized prediction for SP functionality claims for a
more random mutagenesis approach. So far, a systematic screening of mutant
libraries in B. subtilis with respect to secretion capacity was not found in literature.
Here, for the first time, Directed Evolution could be used in combination with a
systematic screening for secretion efficiency or differences in B. subtilis mainly due to
the establishment of a versatile Bacillus expression system with a sufficient
transformation rate for library construction (chapter 3.2).
The size of the signal peptide reduces the mutagenesis techniques
Among the already studied SPs of B. subtilis the SP of AmyE was a frequently used
object also for site directed mutagenesis. The screening of all SPs for cutinase
secretion revealed a medium level export for SP-AmyE (Fig. 17, Table 7) enabling
the detection of improvement and decrease in protein export. Therefore the SP-
AmyE was used as the target for random mutagenesis. Unfortunately, the classical
ep-PCR method (226), which was applied for enzyme improvement in many cases
(e.g. (49, 54, 85, 158) was not successful for the improvement of SP-AmyE after the
screening of more than 3000 B. subtilis clones. Regarding the shortness of the
encoding DNA of about 100 bp encoding for a SP, it is more likely that the highest
possible error rate of 1 base exchange/100 bp is not sufficient to generate enough
SP variants for a successful screening process. Especially since it was reported that
some amino acids are rarely possible to introduce by ep-PCR (50). However, for
future projects a more useful approach might be the so-called “hypermutagenic” PCR
that represents a modified ep-PCR. This method combines the use of varied
concentrations of the four different dNTPs in addition to MnCl2 what results in higher
mutation rates (196).
Discussion
83
The consensus motif A-X-A-A in the C-terminal part of a Bacillus SP is the most
functional sequence in the SP-AmyE for cutinase export
Tjalsma et al. (188) used the SignalP algorithm (135) to predict N-terminal signal
sequences. Furthermore, they analysed all found Sec-type SPs for amino acid
frequency around the SPase I cleavage site. A consensus motif “A-X-A-A” was
found, whereas the preference to alanine at positions –1 and -3 corresponds well to
the postulated interaction with the active site of a SPase I: according to the crystal
structure of an E. coli SPase I (141) the side chains of residues at the –1 and –3
positions are presumably bound in two hydrophobic substrate pockets (S1 and S3).
For this reason efficient binding occurs most likely only with uncharged and small
residues like alanine. The position -2 was predicted to allow any aa since its residue
points outwards from the SPase I. Accordingly, Tjalsma et al. found every aa except
cysteine and proline at position –2 for all Sec-type SPs. alanine was also the
preferred amino acid at the +1 position of the mature protein.
The question rised, if the most abundant amino acid sequence A-X-A-A is also the
most functional one for protein secretion. Using saturation mutagenesis and
subsequent HTS neither the C-region nor the +1 position of the SP-AmyE (A-S-A-A)
could be significantly improved for cutinase export (Fig. 23). On the contrary, the
screening results confirmed clearly the consensus motif since the most efficient
clones of the C-region as well as the +1 position showed wildtype activity and
contained the consensus motif A-X-A-A except for one case, which showed only a
slight deviation to sequence A-X-A-V. The presence of only alanine at the –1 position
corresponds to the findings in previous studies (164, 200) and to the postulated
interaction with a SPase I (141). More surprisingly was the finding of the amino acid
exchange to a cysteine at the position –2 in one case due to the total absence of
cysteine at this position in all Bacillus Sec-type SPs (188). But one has to be careful
with final conclusions for two reasons: first, the presence of alanine at the +1 position
might lead to a second cleavage site (Fig. 22) since a shift to another C-region was
observed for SP-AmyE by change of its amino acid sequence (164). Furthermore,
even if unlikely, amino acid exchanges at the N-terminus of the mature cutinase
might have an influence on enzyme activity. This can be critical since the cutinase
export was determined only indirectly by a lipolytic activity assay.
Discussion
84
Saturation mutagenesis revealed unpredictable amino acid exchanges in the N-
region for a higher functionality of SP-AmyE
A powerful method to generate all possible variants of a protein at a defined amino
acid position is called “Site-specific saturation mutagenesis”. This method introduces
all possible base triplets at a given codon position, thereby resulting in the formation
of all 20 amino acids at this position of the protein (2). Since the N-region of a SP is
important for functional interaction with SecA (3, 29) and the phospholipids of the cell
membrane (23, 203), each position of the N-region of SP-AmyE was completely
saturated and screened for cutinase export functionality (Fig. 25). The screening
revealed that the positions Ala3, Arg5 and Lys7 showed neither significantly
improved secretion but seem to be no critical position nor. On the contrary, the most
critical position for efficient cutinase export is position 4 since most of the
corresponding SP variants showed strong reduced export functionality. Interestingly,
among three of four sequenced improved SP-variants (F2D, F2E, K4L, F6W) a
reduction of the net charge of the SP-AmyE from +3 to +2 was observed. On the one
hand, this reduced positively charged N-terminus might suggest a less effective
interaction of the SP to its binding partners (3, 23) resulting in less secretion as
reported in a previous study (29). On the other hand this phenomenon of less
positive residues and improved secretion was observed before: in a secretion study
of the Gram-positive bacterium Streptomyces lividans a charge reduction from +3 to
+2 in the SP resulted in doubling of secreted α-amylase inhibitor tendamistat.
Furthermore, introduction of more positive charges significantly decreased secretion.
The authors (53) could exclude transcriptional effects and suggested a modulation of
precursor synthesis similar to the postulated feedback mechanism described before
(178). Furthermore, an additional lysine in the N-region of a SP drastically reduced
the export rate of a nuclease in Lactococcus lactis (157). An influence of the SP
charge variation on the synthesis of precursor protein was also observed in E. coli
(10, 80, 184) and might be the reason for the improved cutinase export by the SP-
AmyE variants with reduced net charge in B. subtilis.
However, possible effects on transcriptional level cannot be excluded for the cutinase
precursor. In another study the analysis of SP-AmyE variants for secretion of β-
Discussion
85
lactamase in B. subtilis revealed an increased secretion level that was caused by a
higher mRNA synthesis (130, 132).
Another aspect was the prediction of a distinct effect by one of the amino acid
exchanges for cutinase export. Thus the export efficiency of the variants was tested
for correlation to the D-score calculated by SignalP (Fig. 26). The D-score represents
the probability of a particular amino acid to function as a SP or not. Astonishingly,
there was no correlation between protein export and the D-score for any of the four
improved SPs. On the contrary, the D-scores were decreased in all cases indicating
a lower probability of a functional SP. Next, The prediction for decreased secretion
appeared to be only slightly better since there was a correlation in two of the four
examined variants.
Anyway, these result indicated again a clear lack for a suitable prediction of SP
optimization by in silico analysis using computer tools like SignalP and show a useful
application of Directed Evolution for significant improvement of protein export by
saturation mutagenesis in relevant parts of a SP.
4.5 Improvement of heterologous protein secretion in B. subtilis by co-
overexpression of SecA
Chaperone-like function of SecA might increase cutinase secretion in B. subtilis The translocation motor protein SecA is an essential component of the general
secretion pathway in bacteria and therefore of special interest. Here, the influence of
overexpressed SecA to secretion could be observed since a functional B. subtilis
reporter strain for export efficiency was constructed. This marker strain B. subtilis
Marc1 contains a chromosomal integrated cutinase fused to SP-LipA and showed a
stable cutinase secretion of about 90 µg protein/L (Fig. 27). This comparatively low
export level due to the integrated one-copy plasmid was useful since it suggested a
non-saturated Sec machinery without induction of secretion stress. And indeed, in a
pulse-chase experiment no cutinase precursor was detectable indicating efficient and
complete translocation of cutinase (Fig. 29). Although the Sec machinery was
obviously not overloaded, the constitutive overexpression of SecA in B. subtilis
Discussion
86
Marc1 led to a five-fold higher cutinase export (Fig. 28). In a previous study the
overexpression of SecA had a similar positive effect on the secretion of the
homologous levansucrase (111). The authors explained this by a low binding affinity
of the SP to SecA but argued against a requirement of an export-specific intracellular
chaperon since the absence of a SecB homologue in B. subtilis and the existence of
a self-stabilizing intermediate folding state of levansucrase under cytosolic
conditions. However, in contrast to the homologous levansucrase showing low affinity
of its SP to SecA (111), the heterologous cutinase was fused to the efficient SP-LipA
(Table 7, Fig. 17) what claims for another explanation. Interestingly, however, the
expression of SecA is strictly regulated in B. subtilis and reaches the maximum at the
end of the exponential growth phase and not, as one might expect, later in the actual
secretion phase during stationary growth (71) indicating a possible function of SecA
in the early stage of secretion. This idea is supported by the observation that SecA
exists next to a membrane-associated form also in a soluble form in the cell (35,
116). Additionally, no homologous protein to the export-specific intracellular
chaperone SecB of E. coli (43) could be found in B. subtilis (188). In consequence,
SecA might not only serve as the translocation motor but function itself as an
intracellular chaperone for secretory proteins. According to this study, SecA could
prevent the strong intracellular proteolytic degradation of the cutinase that was
shown before (Fig. 18).
Histidine 289 in the PPX-domain of SecA plays a key role for secretion of
hybrid protein SP-LipA-cutinase
The random mutagenesis of SecA and the subsequent screening in B. subtilis Marc1
yielded the SecA variant A23T, K809D showing an eight-fold export improvement of
cutinase compared to overexpressed wildtype SecA (Fig. 30). Alanine at position 23
is located in the nucleotide binding domain 1 (NBF1), which forms together with the
NBF2 the essential nucleotide binding site for ATP hydrolysis (Fig. 31). Lysine at
position 809 is located in the helical scaffold domain (HSD). The exact mechanism of
how the SecA variant A23T, K809D led to the improved export of precursor SP-LipA-
cutinase is hard to define and highly speculative. So far one might suggest that the
SecA protein becomes more active by the amino acid exchange at the nucleotide
Discussion
87
binding site resulting in a generally increased protein export. But first observations
did not detect an increase in the total amount of secretory proteins in this B. subtilis
Marc1 strain (data not shown). Another possible explanation is a higher binding
affinity of this SecA variant A23T, K809D for the N-terminal part of the precursor
protein SP-LipA-cutinase since an increased affinity of SecA to a preprotein was
reported before to have strong influence on the export efficiency (111) which has to
be investigated further.
However a further amino acid exchange
in the SecA variant A23T, K809D,
H289Y reduced the positive effect of
SecA A23T, K809D for the cutinase
export back to wildtype level indicating
an inhibitory effect for the binding event
of SecA to the hybrid protein (Fig. 30).
Interestingly, this non-conservative
amino acid exchange from the basic
histidine to an uncharged, but polar
tyrosine at position 289 is located in the
PPX-domain, that provides binding of the
precursor to SecA (144). It is noteworthy
that a hyperactive SecA variant H309Y
was found in E. coli, that could suppress
signal peptide defects. This amino acid
exchange corresponds to position 288 in
the PPX-domain of B. subtilis SecA
(139) which is in direct contact to amino acid H289. However, this result confirmed
the central role of the PPX-domain of SecA for precursor binding and suggested a
key role for histidine 289 for efficient binding and translocation of hybrid protein SP-
LipA-cutinase.
Homologous recombination disturbs the screening for SecA variants During the HTS for plasmid encoded SecA variants to improve cutinase secretion in
B. subtilis Marc1 the occurrence of false positive clones was frequently observed:
several B. subtilis clones showed an increased cutinase activity in the automated
N B F I-
d o m a in
P P X -d o m a in
L y s 8 0 9
H is 2 8 9
Fig. 31: Localisation of amino acid substitutions in the protein structure of B.
subtilis SecA that effected cutinase secretion. Structure of monomeric B. subtilis SecA modified after Hunt et al. (76). Highlighted in defined colours are the PPX-domain (blue), the NBFI-(pink), the NBFII-domain (brown) and the HSD-domain (green). Relevant positions of amino acid substitutions are indicated in red. The C- and N-terminus of SecA are indicated in white.
Discussion
88
lipolytic activity assay up to 40-fold, immunoblotting confirmed the strong increase of
secreted cutinase (data not shown). A subsequent sequence analysis of the
corresponding plasmids revealed an open reading frame encoding for cutinase fused
to SP-LipA instead of SecA downstream from the promoter. Since the B. subtilis
Marc1 strain was tested for a stable genome-integrated cutinase, the only possible
explanation was a homologous recombination between the plasmid pBSMuL3-secA
and the integrated cutinase cassette pMCut1. In a previous study the minimal
requirement for detectable homologous recombination was determined as 70 bp of
homology (98). The DNA analysis in B. subtilis Marc1 harbouring pBSMuL3-secA
revealed a homology of 400 bp due to the use of the same DNA fragment containing
the HpaII promoter. This could at least explain the high expression and secretion of
cutinase in the corresponding B. subtilis clones. The permanent chance of
recombination using the pBSMuL vectors in B. subtilis Marc1 strongly suggests the
use of either another integration vector for construction of a secretion reporter strain
or the use of other overexpression vectors in B. subtilis Marc1 with the total lack of
homologous DNA regions between plasmid and chromosome. Furthermore, another
possible source of homologous recombination is the secA gene itself, since plasmid
encoded SecA coexists to the chromosomally encoded SecA in B. subtilis Marc1
(Fig. 28) and shows 2500 bp of homology for possible crossing over events. For
intended future projects this additional critical factor must be considered since it can
decrease the efficiency of the HTS system.
4.6 High level secretion for any foreign protein in B. subtilis- prospects for a
general strategy
In this work, it turned out, that the best SP for the secretion of one target protein was
not automatically also the best for the secretion of another target protein. The
complex interaction of SP, mature protein and all participating Sec components might
explain the lack of correlation between the D-score calculated by SignalP to the
measured secretion efficiency. Based on the results for cutinase and EstCL1
secretion, novel computer tools need to be generated to identify the complex
interaction patterns between SP and mature part of the desired secretion target
protein.
Discussion
89
0
1000
2000
3000
4000
5000
6000
7000
8000
LB-medium TB-medium
SP-AmyE SP-YncMSP-Epr
Alip
olyt
icac
tivity
[U/L
]
B
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
0 5 10 15 20
growth time [h]
TB-medium
LB-medium
Sca
ttere
d lig
ht in
tens
ity I-
I 0[r
el. U
nits
]
However, our strategy to screen a complete natural SP-library fused to the secretion
target using the “one-step optimization” represents a powerful tool for a fast and easy
export optimization of virtually any target protein. It bypasses the single cloning and
screening procedure method saving up to 6-12 months of lab work. Furthermore, the
used B. subtilis strains were grown in simple LB-medium during all experiments
Fig. 32: Comparison of cutinase expressing B. subtilis strains in LB- and TB-medium using the on-line light scattering measurement technique for shaken MTPs. B. subtilis TEB1030 strains bearing plasmid pBSMuL3 encoding for hybrid proteins of cutinase and different SPs (SP-AmyE, SP-Epr, SP-YncM) were grown in a MTP (37°C, 995 rpm, 200 µL total volume each, inoculated with 10 µL of overnight culture) in LB- or TB-medium. The time point for the lipolytic activity assay after 20 hours is indicated by a red arrow. The results represent data from two independent experiments. (A) The growth behaviour was recorded over 20 hours using an excitation filter of 620 nm for continuous detection of the scattered light intensity difference from initial value (I-I0). For more details concerning the continuous on-line measuring technique see Samorski et al. (166). (B) The lipolytic activity assay of the culture supernatant was performed at 37°C in a MTP as described before.
Discussion
90
concerning secretion capacity. It could be possible to enhance the secretion yield by
improving the growth conditions for B. subtilis. This was tested for cutinase secretion
in B. subtilis using a new on-line measuring technique for shaken MTPs. The
experiments were performed by Dipl. Ing. Frank Kensy of the Department of
Biochemical Engineering of the RWTH Aachen University (Prof. J. Büchs, Aachen,
Germany). B. subtilis strains secreting cutinase with SPs of Epr, YncM and AmyE
were grown in LB-medium and TB-medium (Fig. 32). The growth as well as the
cutinase export was three-fold increased using TB-medium. However, these results
also suggest a further possible enhancement of protein yield for other targets.
Various industrially used Bacillus media could be tested for even higher cell growth
(148, 204).
Considering all aspects of SP-screening, Directed Evolution and growth
improvement, a general strategy could be formulated to optimize SPs for
heterologous proteins (Fig. 33). This strategy is speculative since it is based on the
assumption, that the improved export effects observed in single experiments might
be accumulative leading to an 8-fold total export improvement:
in a first step of a general optimization strategy it might be possible to enhance the
secretion yield up to three-fold compared to commonly used B. subtilis SPs (SP-
AmyE, SP-AprE or SP-SacB) as shown for the cutinase using SP-Epr resulting in 35
mg secreted protein/L.
The second step could be a Directed Evolution approach to function as a “fine-
tuning” of the best natural SP. Based on the saturation mutagenesis results of SP-
AmyE, the production yield of cutinase could be additionally increased two-fold up to
70 mg protein/L.
Finally, before an intended commercial application, the growth conditions could be
adapted to achieve an even higher secretion yield as shown for cutinase production
leading to a further 1.5-fold enhanced secretion by using a different growth medium
up to 100 mg protein/L.
Discussion
91
For the future, this general strategy will be tested for cutinase and other heterologous
targets in B. subtilis. But the established system might not only be limited to
B. subtilis- an application for other Gram-positive bacteria is intended and comprises
an interesting and very promising future perspective.
1 2 3
secr
etio
nyi
eld
[%]
different optimisation steps
frequently used SP
one-step optimization
Directed Evolution
growth conditions
0
100
300
500
700
900
1100
3-fold
2-fold
1,5-fold
1-2
1-2
estim
ated
time
sche
dule
[wee
ks]
4-6
Fig. 33: Idealized model of a general strategy to optimize secretion yields of heterologous proteins. The model is speculative since independent results of the export optimization of the cutinase are combined to an addititve model. The secretion yield is based on secretion level defined as 100% calculated from Table 13 as a mean of three commonly used natural SPs of B. subtilis for heterologous protein production (178), namely SP-AmyE, SP-AprE and SP-SacB. (1) In the first step the best natural SP of B. subtilis for the target protein is found in the SP-library (3.3). (2) A further enhancement of this SP can be obtained by using Directed Evolution strategies (3.4). (3) In another optimization phase the growth can be optimized to enhance the production yield (e.g. growth media). The presented time schedule for the complete optimization procedure with a total time of eight weeks is based on average rates observed for these experiments.
Summary
92
5. Summary Bacillus species are commonly used for industrial production of many biocatalysts.
Despite its powerful export machinery guiding up to 25 g/L of homologous
exoproteins directly into the growth medium, the Gram-positive host B. subtilis often
shows disappointingly low secretion yields of foreign proteins. This thesis deals with
new strategies to overcome some of the frequently described bottlenecks for
heterologous protein production in B. subtilis with respect to industrial applications.
(1) Due to a lack of stable and user-friendly expression plasmids for B. subtilis
comparable to available E. coli systems, a new B. subtilis vector series
pBSMuL1-3, for cloning of target genes under control of one or two strong
constitutive promoter(s), has been constructed. The novel plasmid series
provides a (i) convenient multiple cloning site, (ii) high level overexpression
without the necessity of induction, (iii) generation of libraries for HTS
processes, (iv) efficient secretion of heterologous proteins, and (v) an easy
one-step purification in the Gram-positive host B. subtilis. The functionality of
the plasmids was successfully tested for cutinase and EstCL1 export during
this project.
(2) One major limitation for secretion of foreign proteins is the use of unfavorable
signal peptides (SPs) resulting in inefficient secretion and unsatisfying low
production yields. The main goal of this work was to bypass this secretion
handicap by a so far new strategy called SP-screening. In a first step a
functional secretion reporter system for B. subtilis was generated using the
biotechnological relevant heterologous enzyme cutinase. For the first time, a
pool of all natural B. subtilis SPs was constructed, fused to the cutinase and
screened for export efficiency of cutinase. The screening results revealed a
ranking among the different SPs showing the SP of Epr being the best one
leading to a secretion yield of 35 mg protein/L. Pulse-chase analysis of the
processing kinetics of chosen SPs revealed the targeting of the precursor to
the translocase as the critical step for secretion of the cutinase. Comparative
analysis of all screened SPs with respect to computer prediction tool SignalP
and different relevant criteria of a SP indicated that the in silico prediction of
the best functional SP for a distinct protein is not possible so far due to the
Summary
93
complex interaction of a hybrid protein with all participating components of the
general secretion pathway.
(3) The convenient adaptation of the secretion reporter system to a high
throughput screening (HTS) process in B. subtilis using a pipetting robot
enabled the application of a new “one step optimisation” method to find the
best natural SP for export of any heterologous target in only one cloning and
screening event. This was demonstrated successfully for the export of the
cutinase and a second heterologous enzyme, the non secretory protein
EstCL1 leading to a production yield of 17,5 mg secreted protein/L.
(4) Once established, this HTS system using the cutinase as a secretion marker
was successfully applied to screen for improved signal peptides variants of
SP-AmyE generated by Directed Evolution. Several SP variants were detected
showing a three-fold improved export functionality for the cutinase.
(5) Most of the Sec components play a crucial role for a functioning Sec
machinery in B. subtilis. The most important one is the central motorprotein
SecA which mediates the translocation of precursor proteins through the Sec
pore across the cell membrane. To detect its effect on cutinase export, a
secretion reporter strain B. subtilis Marc1 was constructed containing an
integrated cassette expressing the cutinase fused to the signal peptide of
LipA. Intracellular overexpressed SecA in B. subtilis Marc1 improved the
cutinase export level five-fold most likely by functioning as an intracellular
chaperone protecting the proteolytically sensitive cutinase precursor. A secA
mutant library was generated by Directed Evolution and its screening for
secretion efficiency revealed the SecA variant A23, D809 showing an eight-
fold improved cutinase export. Furthermore, the amino acid histidine at
position 289 in the preprotein binding domain of SecA was shown to be critical
for the effective binding and export of the hybrid protein SP-LipA-cutinase
since the exchange to tyrosine (H809Y) had a severe export reduction as the
consequence.
Zusammenfassung
94
6. Zusammenfassung
Unter den Gram-positiven Bakterien befinden sich verschiedene Vertreter der
Gattung Bacillus, die schon seit Jahrzehnten für die industrielle Produktion von
zahlreichen Biokatalysatoren genutzt werden. Der bekannteste unter ihnen ist
sicherlich Bacillus subtilis, der aufgrund seines leistungsfähigen Sekretionsapparates
zwar in der Lage ist, große Mengen an homologen Exportproteinen ins Nähmedium
zu sekretieren, doch sind die Exportausbeuten an heterologen Proteinen oft
enttäuschend gering. Im Rahmen der vorliegenden Arbeit lag der Schwerpunkt auf
der Entwicklung und Anwendung neuer Strategien, um die häufig auftretenden
Engpässe bei der heterologen Proteinproduktion in B. subtilis zu umgehen. Der
Aspekt der industriellen Anwendung sollte dabei im Vordergrund stehen.
(1) Im Vergleich zu erhältlichen Expressionssystemen für E. coli besteht immer
noch ein Mangel an stabil replizierenden und anwenderfreundlichen
Expressionsplasmiden für B. subtilis. Aus diesem Grund wurde eine neue
Vektorserie pBSMuL1-3 für B. subtilis entwickelt. Diese erlaubt das Klonieren
von Zielgenen unter der Kontrolle von einem oder zwei starken konstitutiven
Promotoren. Die neue Plasmidserie enthält (i) eine großzügige „Multiple
Cloning Site“, sorgt für (ii) eine starke Überexpression ohne die Notwendigkeit
der Induktion, ermöglicht (iii) das Erstellen von Genbänken für HTS Prozesse,
sorgt für (iv) effiziente Sekretion von heterologen Proteinen und ermöglicht (v)
eine einfache Ein-Schritt Proteinaufreinigung aus dem Kulturüberstand von
B. subtilis. Die Funktionalität der Plasmide konnte im Laufe dieser Arbeit für
die beiden heterologen Enzyme Cutinase und EstCL1 gezeigt werden.
(2) Ein wichtiger limitierender Faktor für die Sekretion heterologer Proteine in
B. subtilis stellt die Wahl eines „falschen“ Signalpeptides (SP) dar, was zu
ineffizienter Sekretion und damit zu ungenügender Proteinausbeute führt. Um
dieses Sekretionshemmnis zu umgehen, wurde eine neue Strategie
entwickelt, das sogenannte SP-Screening. Zunächst wurde ein funktionelles
Sekretionsreporter-System für B. subtilis entwickelt, was auf der lipolytischen
Aktivität des heterologen, biotechnologisch relevanten Enzyms Cutinase
basiert. In dieser Arbeit wurde zum ersten Mal überhaupt ein „Pool“ aller
bisher bekannten SP aus B. subtilis erstellt, vor die Cutinase kloniert und auf
Zusammenfassung
95
Exporteffizienz der Cutinase getestet. Es ergab sich eine Rangliste unter den
verschiedenen Signalpeptiden, wobei sich das Signalpeptid von Epr als das
beste erwies. Es führte zu einer Sekretionsausbeute von 35 mg Cutinase/L.
Anhand von Pulse-Chase Experimenten wurde die Prozessierungskinetik des
Vorläuferproteins der Cutinase, fusioniert mit verschiedenen SP, analysiert. Es
stellte sich heraus, dass der Transport des Vorläuferproteins zur Membran der
kritische Schritt bei der Sekretion der Cutinase ist. Durch vergleichende
Analyse aller getesteter SP unter Einbeziehung des Computerprogrammes
SignalP und verschiedener Kriterien von SP konnte gezeigt werden, dass
aufgrund der sehr komplexen Interaktion des Fusionsproteines mit allen
beteiligten Komponenten des Sekretionsweges die in siliko-Vorhersage des
effizientesten SP für ein beliebiges Zielprotein bislang nicht möglich ist.
(3) Die gelungene Integration des Sekretionsreporter-Systems in einen
automatisierten Hochdurchsatz-Screening- (HTS-) Prozess für B. subtilis
erlaubte die Anwendung einer Ein-Schritt-Optimierungsmethode; um das
beste aller SP für die Sekretion eines beliebigen Zielproteins in einem
einzigen Klonierungs- und Screeningverfahrens zu ermitteln. Diese neue
Methode konnte erfolgreich für den Export der Cutinase als auch für den
Export eines weiteren heterologen Enzyms, das nicht sekretorische Protein
EstCL1, angewandt werden, von dem über 17 mg Protein/L sekretiert wurde.
(4) Desweiteren konnte das entwickelte HTS-System genutzt werden, um
verbesserte Varianten des SP von AmyE zu finden, die mittels ungerichteter
Mutagenese generiert wurden. Mehrere SP-Varianten von AmyE wurden
gefunden, die eine dreifach gesteigerte Exportfunktionalität für die Cutinase
zeigten.
(5) Fast alle Komponenten des Sec-Apparates sind essentiell für einen
funktionellen Proteinexport. Die Schlüsselrolle spielt das zentrale Motorprotein
SecA, welches für den aktiven Transport des Vorläuferproteins über die
Zellmembran sorgt. Um den Effekt von SecA auf den Export der Cutinase zu
untersuchen, wurde der Sekretionsreporter-Stamm B. subtilis Marc1
konstruiert, der eine ins Genom integrierte Kassette trägt, die für das
Fusionsprotein aus Cutinase und dem SP von LipA codiert. Die
Überexpression von SecA im Cytoplasma von B. subtilis Marc1 erhöhte die
Exporteffizienz der Cutinase um ein fünffaches. Hierbei wirkt SecA
Zusammenfassung
96
wahrscheinlich als intrazelluläres Chaperon, welches das Vorläuferprotein der
Cutinase vor proteolytischem Abbau schützt. Eine SecA-Genbank wurde
mittels ungerichteter Mutagenese hergestellt und auf verbesserte Sekretion
der Cutinase getestet. Es wurde die SecA-Variante A23, D809 gefunden, die
zu einer achtfachen Exportsteigerung führte. Desweiteren zeigte sich die
Aminosäure Histidin an Position 289 in der Präprotein-Bindedomäne von
SecA als kritisch für das effektive Binden und den Transport des
Vorläuferproteins der Cutinase zur Translokase, da der Aminosäureaustausch
zum Tyrosin (H809Y) einen erheblichen Exportrückgang zu Folge hatte.
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Appendix
111
8. Appendix
Table 12: Oligonucleotides used for PCR-amplification of all Sec-dependent signal sequences from genomic DNA of B.subtilis 168. All upstream primers start with the consensus sequence 5´-ATATAAGCTTAAGGAGGATATTATG-3´ containing the EcoRI-restriction site, the Bacillus ribosome binding site and the ATG-start codon for optimal translation. All downstream primers possess the consensus sequence 3´-ATATGAATTCAGC-5´ containing the HindIII restriction site and the GCT-triplett coding for alanine at the +1 position of all signal sequences. In some primers silent mutations are introduced avoiding additional EcoRI or HindIII restriction sites. These base substitutions are highlighted in bold letters. The signal sequences are listed in alphabetical order.
Signal
sequence
Upstream primer (5'→→→→3')
Downstream primer (5'→→→→3')
AbnA ATATAAGCTTAAGGAGGATATTATGAAAAAGAAAAAAACATGG ATATGAATTCAGCTGCCTCTGCGGGAGCAGCAGA AmyE ATATAAGCTTAAGGAGGATATTATGTTTGCAAAACGATTCAAA ATATGAATTCAGCAGCACTCGCAGCCGCCGGTCC AprE ATATAAGCTTAAGGAGGATATTATGAGAAGCAAAAAATTGTGG ATATGAATTCAGCAGCCTGCACAGACATGTTGCT AspB ATATAAGCTTAAGGAGGATATTATGAAACTGGCAAAAAGAGTA ATATGAATTCAGCCGCTTTCGCTGTGATTGCCAG BglC ATATAAGCTTAAGGAGGATATTATGAAACGGTCAATCTCTATT ATATGAATTCAGCTGCTGATGCCGGCGAAGCTAT BglS ATATAAGCTTAAGGAGGATATTATGCCTTATCTGAAACGAGTG ATATGAATTCAGCAGCTGAGGCAGTAGCAGTGAC Bpr ATATAAGCTTAAGGAGGATATTATGAGGAAAAAAACGAAAAAC ATATGAATTCAGCTGCCCCGGCTGCTCCCGGAAA CccA ATATAAGCTTAAGGAGGATATTATGAAATGGAACCCGCTTATT ATATGAATTCAGCTCCTTTTACTGATAAAAAGAA CitH ATATAAGCTTAAGGAGGATATTATGGGAAATACTCGTAAAAAA ATATGAATTCAGCAACGTCTGCCAGCTCTTTTTG CotC ATATAAGCTTAAGGAGGATATTATGAAAAATCGGCTCTTTATT ATATGAATTCAGCAGATTTTGCGGCTTGGACAGT Csn ATATAAGCTTAAGGAGGATATTATGAAAATCAGTATGCAAAAA ATATGAATTCAGCCGCAAAAACCGTTTCGCTCAT CwlD ATATAAGCTTAAGGAGGATATTATGAGGAAAAAACTTAAATGG ATATGAATTCAGCATTGCTGAACTGATACTTGAA DacB ATATAAGCTTAAGGAGGATATTATGCGCATTTTCAAAAAAGCA ATATGAATTCAGCAGCATGTGCTGTATTCACATT DacF ATATAAGCTTAAGGAGGATATTATGAAACGTCTTTTATCCACT ATATGAATTCAGCTGCAAATGCAGACGGTGCAAA DltD ATATAAGCTTAAGGAGGATATTATGAAAAAGCGTTTTTTCGGT ATATGAATTCAGCTGCGATGGCGCCTGCGAATAG Epr ATATAAGCTTAAGGAGGATATTATGAAAAACATGTCTTGCAAA ATATGAATTCAGCCGCATGAGCGAGAGGGCCTAT FliL ATATAAGCTTAAGGAGGATATTATGAAGAAAAAGTTAATGATC ATATGAATTCAGCAGCCGCCGCCCCGAGAGCACC FliZ ATATAAGCTTAAGGAGGATATTATGAAAAAGAGTCAATATTTT ATATGAATTCAGCTGCCGCGGCAGCAGCAATCGG GlpQ ATATAAGCTTAAGGAGGATATTATGAGAAAAAATAGAATACTG ATATGAATTCAGCTGCCGACACTGGCGTTACCAT LipA ATATAAGCTTAAGGAGGATATTATGAAATTTGTAAAAAGAAGG ATATGAATTCAGCAGCGGCTTTTGCTGACGGCTG LipB ATATAAGCTTAAGGAGGATATTATGAAAAAAGTACTTATGGCA ATATGAATTCAGCAGCTTTTGCGCCAGACGGCGG LytB ATATAAGCTTAAGGAGGATATTATGAAATCTTGCAAACAATTG ATATGAATTCAGCTGCAAAAGAAACTGATGGAAT LytC ATATAAGCTTAAGGAGGATATTATGCGTTCTTATATAAAAGTC ATATGAATTCAGCGGCCAAAGCTGTTGGCACAAA LytD ATATAAGCTTAAGGAGGATATTATGAAAAAGAGACTAATCGCA ATATGAATTCAGCTGCCTGGGCAGAACCAGACAT LytE ATATAAGCTTAAGGAGGATATTATGAAAAAGCAAATCATTACA ATATGAATTCAGCTGCAAATAACGCTCCTAAAAC LytF ATATAAGCTTAAGGAGGATATTATGAAAAAGAAATTAGCAGCA ATATGAATTCAGCTGCTTCAGCTGGTGTCACTAC LytR ATATAAGCTTAAGGAGGATATTATGAGAAACGAACGCAGAAAA ATATGAATTCAGCAGCTGCTTTATGCCATAAGTA Mdr ATATAAGCTTAAGGAGGATATTATGGACACAACAACAGCAAAA ATATGAATTCAGCCGCGGTGGCAACAATCGTATT MotB ATATAAGCTTAAGGAGGATATTATGGCGAGAAAAAAGAAGAAG ATATGAATTCAGCGCTGCTCGCGTACAGCACAAT Mpr ATATAAGCTTAAGGAGGATATTATGAAATTAGTTCCAAGATTC ATATGAATTCAGCCGCTTTTGCCGGTACGCCAAA MreC ATATAAGCTTAAGGAGGATATTATGCCGAATAAGCGGTTAATG ATATGAATTCAGCCGAAAATCCAATCATAGCCAC NprB ATATAAGCTTAAGGAGGATATTATGCGCAACTTGACCAAGACA ATATGAATTCAGCAGCTGAGGCATGTGTTACAAA NprE ATATAAGCTTAAGGAGGATATTATGGGTTTAGGTAAGAAATTG ATATGAATTCAGCAGCCTGAACACCTGGCAGGCT NucB ATATAAGCTTAAGGAGGATATTATGAAAAAATGGATGGCAGGC ATATGAATTCAGCCGAAGATGCGCCTTGGATCTG Pbp ATATAAGCTTAAGGAGGATATTATGAAAAAAAGCATAAAACTT ATATGAATTCAGCGGCAAGCGCAGCCTGATGCAT PbpB ATATAAGCTTAAGGAGGATATTATGATTCAAATGCCAAAAAAG ATATGAATTCAGCTGCCATTCTCCCCAGGATGAC PbpD ATATAAGCTTAAGGAGGATATTATGACCATGTTACGAAAAATA ATATGAATTCAGCAGCGATAACTGTAAATGCAAA PbpX ATATAAGCTTAAGGAGGATATTATGACAAGCCCAACCCGCAGA ATATGAATTCAGCAGCATTCCAAATCGTAATGCA Pel ATATAAGCTTAAGGAGGATATTATGAAAAAAGTGATGTTAGCT ATATGAATTCAGCTGCGTTCGCGCCAGCTGGAGT PelB ATATAAGCTTAAGGAGGATATTATGAAACGACTTTGTTTATGG ATATGAATTCAGCACCTAATGCTTTTCCAGGCAA PenP ATATAAGCTTAAGGAGGATATTATGAAGTTGAAAACTAAAGCG ATATGAATTCAGCTGCTTCGGCATGTGTTGAGTT PhoA ATATAAGCTTAAGGAGGATATTATGAAAAAAATGAGTTTGTTT ATATGAATTCAGCAGCTCCGGCAAAGATTCCAGC PhoB ATATAAGCTTAAGGAGGATATTATGAAAAAATTCCCGAAGAAA ATATGAATTCAGCGGCGCTGGCTTCAGGCACACT PhrA ATATAAGCTTAAGGAGGATATTATGAAATCTAAATGGATGTCA ATATGAATTCAGCTGCATGAACCATCACCTGAGT PhrC ATATAAGCTTAAGGAGGATATTATGAAATTGAAATCTAAGTTG ATATGAATTCAGCCGCATTAGCAGAAACGCCAGC PhrF ATATAAGCTTAAGGAGGATATTATGAAATTGAAGTCTAAACTA ATATGAATTCAGCTGCAATAGTTGTTGCCACGAA PhrG ATATAAGCTTAAGGAGGATATTATGAAAAGATTTCTGATTGGC ATATGAATTCAGCCGCAATAAACCAACCTGATAA PhrK ATATAAGCTTAAGGAGGATATTATGAAAAAACTTGTGCTTTGC ATATGAATTCAGCAGCTACTCCACTTAAAATCAC RpmG ATATAAGCTTAAGGAGGATATTATGAGAAAAAAGATTACGTTA ATATGAATTCAGCCGCTGATGCAGAGCTCTTCAT SacB ATATAAGCTTAAGGAGGATATTATGAACATCAAAAAGTTTGCA ATATGAATTCAGCCGCAAACGCTTGAGTTGCGCC SacC ATATAAGCTTAAGGAGGATATTATGAAAAAGAGACTGATTCAA ATATGAATTCAGCTGCATCTGCCGAAAATGCCAT SleB ATATAAGCTTAAGGAGGATATTATGAAGTCCAAAGGATCGATT ATATGAATTCAGCCGAAAAGGCAGAGATCGTTTC SpoIID ATATAAGCTTAAGGAGGATATTATGAAACAATTCGCAATCACA ATATGAATTCAGCGGCCCCCGCTTCCTTATTATG SpoIIP ATATAAGCTTAAGGAGGATATTATGAGAAATAAACGCAGAAAC ATATGAATTCAGCCACACCGGATAGAACAAAAAT SpoIIQ ATATAAGCTTAAGGAGGATATTATGAGAGAGGAAGAAAAGAAA ATATGAATTCAGCAAGGACAGCTGTTAAAATGAC
Appendix
112
Signal
sequence
Upstream primer (5'→→→→3')
Downstream primer (5'→→→→3')
SpoIIR ATATAAGCTTAAGGAGGATATTATGAAAAAAACAGTAATCATT ATATGAATTCAGCTACGAGCGCTCCGGATAATAA TasA ATATAAGCTTAAGGAGGATATTATGGGTATGAAAAAGAAATTG ATATGAATTCAGCTGCCCATGTTCCTCCTCCAAC TyrA ATATAAGCTTAAGGAGGATATTATGAATCAAATGAAAGATACA ATATGAATTCAGCGGCTAGGGCAATCGAACCGCC Vpr ATATAAGCTTAAGGAGGATATTATGAAAAAGGGGATCATTCGC ATATGAATTCAGCAGCCGGAGCTGCCTGAACGCC WapA ATATAAGCTTAAGGAGGATATTATGAAAAAAAGAAAGAGGCGA ATATGAATTCAGCTGCTAGTACATCGGCTGGCAC WprA ATATAAGCTTAAGGAGGATATTATGAAACGCAGAAAATTCAGC ATATGAATTCAGCCGCTGCAGCTTTGGTTCCCGG XynA ATATAAGCTTAAGGAGGATATTATGTTTAAGTTTAAAAAGAAT ATATGAATTCAGCTGCAGAGGCGGTTGCCGAAAA YbbC ATATAAGCTTAAGGAGGATATTATGAGAAAAACAATATTCGCT ATATGAATTCAGCAGCAGAGGCAGCGGTTATCGT YbbE ATATAAGCTTAAGGAGGATATTATGAAAACAAAGACACTGTTC ATATGAATTCAGCTGCGAAGGTTTCATTTGGCGC YbbR ATATAAGCTTAAGGAGGATATTATGGATAAATTCTTAAACAAC ATATGAATTCAGCGCTGTTAACCGCCACATAAAG YbdG ATATAAGCTTAAGGAGGATATTATGAAAACATTATGGAAAGTC ATATGAATTCAGCCGAGACGGATACAAGCAAAAC YbdN ATATAAGCTTAAGGAGGATATTATGGTGAAAAAATGGCTTATT ATATGAATTCAGCAGCTGATGCCGAATACGTAAA YbfO ATATAAGCTTAAGGAGGATATTATGAAACGAATGATAGTGAGA ATATGAATTCAGCAGCACGGGCTGAGGCTGAAAA YbxI ATATAAGCTTAAGGAGGATATTATGAAAAAATGGATATATGTT ATATGAATTCAGCTGCGTGGACGGAGAAGCCGCC YckD ATATAAGCTTAAGGAGGATATTATGAAACGAATAACCATCAAC ATATGAATTCAGCGGCCTCAGCTGTACCGGTCAG YdbK ATATAAGCTTAAGGAGGATATTATGAAACTTTTTAATCGGAAG ATATGAATTCAGCACTGATGACAATCCGTTTAAT YddT ATATAAGCTTAAGGAGGATATTATGAGAAAGAAAAGAGTTATT ATATGAATTCAGCTGCAGAAGCGTAACCTGCAGG YdhT ATATAAGCTTAAGGAGGATATTATGTTTAAGAAACATACGATC ATATGAATTCAGCTGCTAAAACAGCAGACGCAAG YdjM ATATAAGCTTAAGGAGGATATTATGTTGAAGAAAGTCATTTTA ATATGAATTCAGCCGCACTGGCATCTGATGAAAA YdjN ATATAAGCTTAAGGAGGATATTATGAAAAAAAGAATCATATTA ATATGAATTCAGCCGCAACCCCCGCGGCAGCTGC YfhK ATATAAGCTTAAGGAGGATATTATGAAAAAGAAACAAGTAATG ATATGAATTCAGCAGCTTTTGCTGCGGGAGCGGA YfjS ATATAAGCTTAAGGAGGATATTATGAAGTGGATGTGTTCAATA ATATGAATTCAGCCGCCTGTGCTGCACCTCCGGC YfkD ATATAAGCTTAAGGAGGATATTATGATGAAAAAGCTATTTCAT ATATGAATTCAGCCGCGTGGATGGGCTGAACGCC YfkN ATATAAGCTTAAGGAGGATATTATGAGAATACAGAAAAGACGA ATATGAATTCAGCTGCATGAATGGGTGGTGTTGG YhaK ATATAAGCTTAAGGAGGATATTATGCGCACATGGAAACGTATA ATATGAATTCAGCTGCCAGTGCTGCGTAAAACAA YhcR ATATAAGCTTAAGGAGGATATTATGCTGTCTGTCGAAATGATA ATATGAATTCAGCAGCTTCGAACGTGTACATTAC YhdC ATATAAGCTTAAGGAGGATATTATGAAATCCCTGCCGTATACC ATATGAATTCAGCAGCCATTGACACGATAATCAA YhfM ATATAAGCTTAAGGAGGATATTATGAAAAAAATAGTGGCAGCC ATATGAATTCAGCCGCGTCTACCGATTGATACAC YhjA ATATAAGCTTAAGGAGGATATTATGAAAAAAGCGGCGGCGGTT ATATGAATTCAGCGGCTTCCGCCACATGGCCAGC YjcM ATATAAGCTTAAGGAGGATATTATGAAAAAAGAATTACTTGCT ATATGAATTCAGCTGCAAAAACTTCATTTGTTGA YjcN ATATAAGCTTAAGGAGGATATTATGAAAAAGAAAACTAAAATT ATATGAATTCAGCCGAAGAAGCTGTAAAGACAAC YjdB ATATAAGCTTAAGGAGGATATTATGAATTTCAAAAAAACGGTT ATATGAATTCAGCAGCTGAAGCTACCCCGCTGAC YjfA ATATAAGCTTAAGGAGGATATTATGAAAAGACTGTTTATGAAG ATATGAATTCAGCCGCCTTGGCGGGTGCACCTTT YjiA ATATAAGCTTAAGGAGGATATTATGGCAGCACAGACTGATTAC ATATGAATTCAGCAGAGAACACAAAGAGTACGAA YknX ATATAAGCTTAAGGAGGATATTATGAAAAAAGTCTGGATCGGA ATATGAATTCAGCGGCGCTGCCGCTTGTCGGAGC YkoJ ATATAAGCTTAAGGAGGATATTATGCTCAAGAAAAAATGGATG ATATGAATTCAGCGGCAAACGCGTTGTAGCTGAA YkvT ATATAAGCTTAAGGAGGATATTATGACAACGAAATTCACTGCT ATATGAATTCAGCAATTTTGGCTGCAGGCATGAA YkvV ATATAAGCTTAAGGAGGATATTATGTTGACGAAGCGCTTGCTT ATATGAATTCAGCAGCTTGTGCCGCACCTGGAAA YkwD ATATAAGCTTAAGGAGGATATTATGAAGAAAGCATTTATTTTA ATATGAATTCAGCCGCTGATGCTTGCTGTACGCC YlaE ATATAAGCTTAAGGAGGATATTATGAAGAAAACATTTGTAAAA ATATGAATTCAGCTGCGCTGGCTGCATCAGGACC YlbL ATATAAGCTTAAGGAGGATATTATGCTACGTAAAAAACATTTT ATATGAATTCAGCCGCTTCTCCCGGTTTTGTAAT YlqB ATATAAGCTTAAGGAGGATATTATGAAAAAAATCGGTTTATTG ATATGAATTCAGCAGCGTCTGCTTGCTGTGCCGG YlxF ATATAAGCTTAAGGAGGATATTATGTCCGGCAAAAAGAAAGAA ATATGAATTCAGCACCAGCAGCCCAAAGAACAAT YlxW ATATAAGCTTAAGGAGGATATTATGAGGGGCAAATCAGCAGTC ATATGAATTCAGCAGCCGCGCTTTTGTTGTTTTC YlxY ATATAAGCTTAAGGAGGATATTATGTACAAAAAATTTGTACCG ATATGAATTCAGCTGCCCCTATATAATCAAGTGC YncM ATATAAGCTTAAGGAGGATATTATGGCGAAACCACTATCAAAA ATATGAATTCAGCAGCGTCTGCCGCGGGTAAACC YndA ATATAAGCTTAAGGAGGATATTATGAGATTCACTAAGGTAGTT ATATGAATTCAGCTGCTTGTGCCGTTAATGGAAA YnfF ATATAAGCTTAAGGAGGATATTATGATTCCACGCATAAAAAAA ATATGAATTCAGCTGCCAAAACTTCAGTAGCGCC YngK ATATAAGCTTAAGGAGGATATTATGAAGGTTTGCCAAAAGTCG ATATGAATTCAGCCGCATTGGCCATAAACGGAAC YnzA ATATAAGCTTAAGGAGGATATTATGGAATTGAGCTTCACAAAA ATATGAATTCAGCGCCAAGCGCCGGCAGTTTATC YoaW ATATAAGCTTAAGGAGGATATTATGAAAAAGATGTTGATGTTA ATATGAATTCAGCGGCCGACGCTTCCCCTACATG YobB ATATAAGCTTAAGGAGGATATTATGAAGATTAGGAAAATCTTA ATATGAATTCAGCAGCCAATGCGGGAACCGCGGA YobV ATATAAGCTTAAGGAGGATATTATGAAACTGGAACGTTTGTTA ATATGAATTCAGCAGCCTGCACTTGTTTTTTGCT YocA ATATAAGCTTAAGGAGGATATTATGAAGAAAAAGAGAAAAGGC ATATGAATTCAGCGGCGATGACAAAGACAAAAAT YocH ATATAAGCTTAAGGAGGATATTATGAAGAAGACGATTATGTCC ATATGAATTCAGCAGCGTGAGCTCCGAATGCAGT YodV ATATAAGCTTAAGGAGGATATTATGAAGGTTCCAAAAACAATG ATATGAATTCAGCAGCCGACACCGAGGTTGCTGT YojL ATATAAGCTTAAGGAGGATATTATGAAAAAGAAGATTGTAGCC ATATGAATTCAGCTGCTTCCGCGGGTGCTGCGGC YolA ATATAAGCTTAAGGAGGATATTATGAAGAAGAGAATTACATAT ATATGAATTCAGCCGCTTTTGCTTTTGATGAATC YolC ATATAAGCTTAAGGAGGATATTATGAAGAAGAGGCTAATCGGA ATATGAATTCAGCTGCTTCGATTAAAGTAATACC YolI ATATAAGCTTAAGGAGGATATTATGAAAAAGTGGATTGTTTTA ATATGAATTCAGCTTCGCTACCTGTAGAAACATA YomL ATATAAGCTTAAGGAGGATATTATGAGAAAGAAAAGAGTTATT ATATGAATTCAGCTGCAGTAGCGTAACCTGCAGG YopL ATATAAGCTTAAGGAGGATATTATGAAAAAACTTATTATGGCT ATATGAATTCAGCTGCACTTATGTAAGAAGTGCC YoqH ATATAAGCTTAAGGAGGATATTATGAAACGATTTATTTTAGTT ATATGAATTCAGCGGCGTTTGTTTGAATGGGATA YoqM ATATAAGCTTAAGGAGGATATTATGAAATTAAGAAAAGTATTG ATATGAATTCAGCAGCGAATGCAGGAGAAGCAGA YpbG ATATAAGCTTAAGGAGGATATTATGAAGCTATCAGTGAAAATT ATATGAATTCAGCTGCTGTTGCATACATTTTTGC YpcP ATATAAGCTTAAGGAGGATATTATGAATAATAATAAACTATTG ATATGAATTCAGCGGCCGTGGCAAAAAAGGCCCG YpjP ATATAAGCTTAAGGAGGATATTATGAAATTGTGGATGAGAAAG ATATGAATTCAGCCGCCATAAGGGCAGCCGGAGG YpmB ATATAAGCTTAAGGAGGATATTATGAGAAAAAAAGCATTAATA ATATGAATTCAGCTGCCATGGCTGATTTATAAAT YpmS ATATAAGCTTAAGGAGGATATTATGAATAAGTGGAAGCGACTG ATATGAATTCAGCCACCTGAGCCTGTTCCCCCGG YpuA ATATAAGCTTAAGGAGGATATTATGAAGAAAATTTGGATTGGA ATATGAATTCAGCGGCATCCGCGAGACTGACCTT YpuD ATATAAGCTTAAGGAGGATATTATGGGAAGAATAAAAACCAAG ATATGAATTCAGCTCCAATTGCTGTCGGAACATC YqfZ ATATAAGCTTAAGGAGGATATTATGAAGCGTCTCACCTTAGTA ATATGAATTCAGCTGCTGATGCTTCATAGACAGG YqgA ATATAAGCTTAAGGAGGATATTATGAAGCAAGGAAAATTTTCT ATATGAATTCAGCAGCCTCTGCTTTTCCTTTAGG
Appendix
113
Signal
sequence
Upstream primer (5'→→→→3')
Downstream primer (5'→→→→3')
YqxI ATATAAGCTTAAGGAGGATATTATGTTTAAGAAATTACTTTTA ATATGAATTCAGCAGCTTTGGCATGTCCATCCAA YqxM ATATAAGCTTAAGGAGGATATTATGTTTCGATTGTTTCACAAT ATATGAATTCAGCAGCGCTTGTATCATCGGAAAA YqzC ATATAAGCTTAAGGAGGATATTATGACAAAACGGGGCATTCAG ATATGAATTCAGCAGCAGCGGCCTGGTCTTCGTC YqzG ATATAAGCTTAAGGAGGATATTATGATGATCAAACAATGTGTG ATATGAATTCAGCTGCGTGAGCGGCTGTGGTGCC YraJ ATATAAGCTTAAGGAGGATATTATGACATTGACTAAACTGAAA ATATGAATTCAGCGGCAAGTGCCTGACTGGAAAA YrrL ATATAAGCTTAAGGAGGATATTATGTATATCAATCAGCAAAAA ATATGAATTCAGCTAAAAATGCCCCGCCAATGAT YrrR ATATAAGCTTAAGGAGGATATTATGAAGATATCGAAACGAATG ATATGAATTCAGCGATTTCTGCAAGTCTCAGCAG YrrS ATATAAGCTTAAGGAGGATATTATGAGCAATAATCAATCTCGT ATATGAATTCAGCCGCTGCTACTACGACAATTAG YrvJ ATATAAGCTTAAGGAGGATATTATGAACAAGAAATACTTTGTC ATATGAATTCAGCAGCTGTAACGGATGAAAAGGT YuaB ATATAAGCTTAAGGAGGATATTATGAAACGCAAATTATTATCT ATATGAATTCAGCTTCAGCCGCGAAAGAAGCTGT YunA ATATAAGCTTAAGGAGGATATTATGATTACTGACATTTTTAAG ATATGAATTCAGCTGCAAACAAAAGGAGAAGAAG YunB ATATAAGCTTAAGGAGGATATTATGCCAAGATATCGCGGCCCT ATATGAATTCAGCAAGGCTGACCGTTGTTGACAG YurI ATATAAGCTTAAGGAGGATATTATGACAAAAAAAGCATGGTTT ATATGAATTCAGCCGCGCTTGCTGAAGCTGCTGG YusW ATATAAGCTTAAGGAGGATATTATGCATTTGATCAGAGCAGCC ATATGAATTCAGCTCCTTCTGCCTGATGCTGGTC YvbX ATATAAGCTTAAGGAGGATATTATGAAAAAATGGCTGATCATA ATATGAATTCAGCTGCCTTCGCTTCTCCTTTTGT YvcE ATATAAGCTTAAGGAGGATATTATGAGAAAGAGTTTAATTACA ATATGAATTCAGCCGCCGATGCAGTTTTACTTGT YveB ATATAAGCTTAAGGAGGATATTATGAACTATATAAAAGCAGGC ATATGAATTCAGCTAAGTCGATAAACAGCAATAT YvgO ATATAAGCTTAAGGAGGATATTATGAAACGTATTCGTATCCCA ATATGAATTCAGCAGCGGAAGCAAAGGACAAAGG YvgV ATATAAGCTTAAGGAGGATATTATGAAAAAGAAACAGCAGTCT ATATGAATTCAGCGACAATGGCTGCTAGCAATAC YvnB ATATAAGCTTAAGGAGGATATTATGAGGAAATACACGGTTATT ATATGAATTCAGCGCCGCCAGACAAAACGGAAAG YvpA ATATAAGCTTAAGGAGGATATTATGAAAAAAATCGTGTCTATC ATATGAATTCAGCTGCAAAAACGGTTGATGGCTG YvpB ATATAAGCTTAAGGAGGATATTATGAAAACACTGCGAACTCTA ATATGAATTCAGCTGCATCTATTTTAAGTCCGAA YwaD ATATAAGCTTAAGGAGGATATTATGAAAAAGCTGTTGACTGTC ATATGAATTCAGCAGCGTGCGCGGCAGGGGTGAC YwcI ATATAAGCTTAAGGAGGATATTATGAAACGTTTGCTTGTATCT ATATGAATTCAGCGGCACGCGCAGTTTTTCTGTC YwdK ATATAAGCTTAAGGAGGATATTATGAAAGTTTTTATCATATTA ATATGAATTCAGCGCCGAATGCTCCGAGTCCCAC YweA ATATAAGCTTAAGGAGGATATTATGCTAAAAAGAACTTCATTC ATATGAATTCAGCTGCATGAGCTTGGCCCGAAGG YwfM ATATAAGCTTAAGGAGGATATTATGAAGGGGAATATTTACAGC ATATGAATTCAGCTGCCTGAACGGTTCCCGTGGT YwgB ATATAAGCTTAAGGAGGATATTATGAAAATGAAATCAGGAATG ATATGAATTCAGCAGCCTGTACCGGCAGCCGTGA YwjE ATATAAGCTTAAGGAGGATATTATGAAGGTATTTATCGTGATT ATATGAATTCAGCGGCACGGCCCATGAAGATATC YwmB
ATATAAGCTTAAGGAGGATATTATGAAGAAAAAACAAGTAAGC
ATATGAATTCAGCCGCATGAATGGTATGGAAAACAGCAATGACGAAGCTCAACA
YwmC ATATAAGCTTAAGGAGGATATTATGAAGAAAAGATTTTCACTG ATATGAATTCAGCTGCAAAAGCAGGTGAAGTTAA YwmD ATATAAGCTTAAGGAGGATATTATGAAAAAATTGCTGGCTGCC ATATGAATTCAGCGGCAAAAGACGGGGAGGCAAT YwoF ATATAAGCTTAAGGAGGATATTATGAGAAAATGGTATTTTATT ATATGAATTCAGCCGCTTTTGTCTTATCATAAAC YwqC ATATAAGCTTAAGGAGGATATTATGGGAGAATCTACGAGCTTA ATATGAATTCAGCAGCAGTGGCAGCCGCGGTTAC YwqO ATATAAGCTTAAGGAGGATATTATGAAGTTTTTGCTGTCGGTC ATATGAATTCAGCGTCCAGCCTCCTGTTTGGATA YwsB ATATAAGCTTAAGGAGGATATTATGAACAAACCAACAAAACTA ATATGAATTCAGCAGCATGGATCGTGCCAGCGCC YwtC ATATAAGCTTAAGGAGGATATTATGAAATTTGTCAAAGCCATC ATATGAATTCAGCAGCTGACATAAACATGAACAC YwtD ATATAAGCTTAAGGAGGATATTATGAACACACTGGCAAACTGG ATATGAATTCAGCAGCTTCCGCAATCTCCGCTTT YwtF ATATAAGCTTAAGGAGGATATTATGGAAGAACGATCACAGCGC ATATGAATTCAGCAGCATAGGCGCCGACAGAGCC
YxaK ATATAAGCTTAAGGAGGATATTATGGTCAAGTCATTTCGTATGAAA ATATGAATTCAGCCGCATAAGCAGCTGTCGGCTG
YxiA ATATAAGCTTAAGGAGGATATTATGTTCAACCGATTGTTCCGT ATATGAATTCAGCTGCATAGACGGAGTTTGGGAG YxiT ATATAAGCTTAAGGAGGATATTATGAAATGGAATAACATGCTG ATATGAATTCAGCCGCTTGAACTGCTTTTAAAGA YyaB ATATAAGCTTAAGGAGGATATTATGGTTTATCAAACGAAGAGA ATATGAATTCAGCGGCATCTGCTTGTATCAGTAA YybN ATATAAGCTTAAGGAGGATATTATGAATAAATTCCTGAAGAGC ATATGAATTCAGCGGCTTTTATAAAATTACTACT YycP ATATAAGCTTAAGGAGGATATTATGAAAAAGTGGATGATTACC ATATGAATTCAGCGCTTTTGAGAGGAGAAATGAA
Table 13: Comparison of all screened signal sequences used for export of heterologous cutinase in B. subtilis. The results represent data of 12 independent experiments.
No.
Name
Signal sequence
Secreted cutinase [U/mL]
a
Charge
N-region b
Hydro-
phobicity [% ]
c
D-
Score d
1 Epr MKNMSCKLVVSVTLFFSFLTIGPLAHA 4.67 2 62.96 0.919 2 YncM MAKPLSKGGILVKKVLIAGAVGTAVLFGTLSSGIPGLPAADA 4.12 4 76.19 0.507 3 YjfA MKRLFMKASLVLFAVVFVFAVKGAPAKA 3.84 3 78.57 0.924 4 YfhK MKKKQVMLALTAAAGLGLTALHSAPAAKA 3.67 3 68.97 0.906 5 Csn MKISMQKADFWKKAAISLLVFTMFFTLMMSETVFA 3.35 3 62.86 0.689 6 LytD MKKRLIAPMLLSAASLAFFAMSGSAQA 3.33 3 70.37 0.87 7 Bpr MRKKTKNRLISSVLSTVVISSLLFPGAAGA 2.97 5 56.67 0.936 8 WapA MKKRKRRNFK RFIAAFLVLA LMISLVPADVLA 2.88 8 65.63 0.918 9 BglC MKRSISIFITCLLITLLTMGGMIASPASA 2.87 2 65.52 0.839
10 LytB MKSCKQLIVCSLAAILLLIPSVSFA 2.83 2 64.00 0.916 11 LipA MKFVKRRIIALVTILMLSVTSLFALQPSAKA 2.79 4 64.52 0.874 12 YckD MKRITINIITMFIAAAVISLTGTAEA 2.79 2 65.38 0.839 13 Pel MKKVMLATALFLGLTPAGANA 2.67 2 76.19 0.843 14 YnfF MIPRIKKTICVLLVCFTMLSVMLGPGATEVLA 2.61 3 68.75 0.699
Appendix
114
No.
Name
Signal sequence
Secreted cutinase [U/mL]
a
Charge N-region
b
Hydro-phobicity
[% ]c
D-Score
d
15 PhrK MKKLVLCVSILAVILSGVA 2.53 2 73.68 0.669 16 YbdN MVKKWLIQFAVMLSVLSTFTYSASA 2.51 2 60.00 0.853 17 YobB MKIRKILLSSALSFGMLISAVPALA 2.49 3 72.00 0.904 18 YddT MRKKRVITCVMAASLTLGSLLPAGYASA 2.41 4 60.71 0.901 19 YhfM MKKIVAAIVVIGLVFIAFFYLYSRSGDVYQSVDA 2.30 2 64.71 0.286 20 BglS MPYLKRVLLLLVTGLFMSLFAVTATASA 2.25 2 71.43 0.911 21 Vpr MKKGIIRFLLVSFVLFFALSTGITGVQAAPA 2.20 3 74.19 0.877 22 AprE MRSKKLWISLLFALTLIFTMAFSNMSVQA 2.19 3 62.07 0.886 23 YjdB MNFKKTVVSALSISALALSVSGVASA 2.10 2 61.54 0.928 24 YbbE MKTKTLFIFSAILTLSIFAPNETFA 2.09 2 60.00 0.825 25 PhrC MLKSKLFVICLAAAAIFTAAGVSANA 2.00 3 69.23 0.862 26 GlpQ MRKNRILALFVLSLGLLSFMVTPVSA 1.96 3 69.23 0.921 27 SacC MKKRLIQVMIMFTLLLTMAFSADA 1.95 3 66.67 0.86 28 YurI MTKKAWFLPLVCVLLISGWLAPAASASA 1.94 2 75.00 0.871 29 PhoB MKKFPKKLLPIAVLSSIAFSSLASGSVPEASA 1.92 4 62.50 0.926 30 PenP MKLKTKASIKFGICVGLLCLSITGFTPFFNSTHAEA 1.92 4 55.56 0.708 31 YfkD MMKKLFHSTLIVLLFFSFFGVQPIHA 1.87 2 69.23 0.735 32 YvpA MKKIVSILFMFGLVMGFSQFQPSTVFA 1.87 2 70.37 0.717 33 YdjM MLKKVILAAFILVGSTLGAFSFSSDASA 1.83 2 67.86 0.832 34 AbnA MKKKKTWKRFLHFSSAALAAGLIFTSAAPAEA 1.82 6 59.38 0.898 35 YwjE MKVFIVIMIIVVIFFALILLDIFMGRA 1.80 1 88.89 0.638 36 YqgA MKQGKFSVFLILLLMLTLVVAPKGKAEA 1.64 2 71.43 0.807 37 LipB MKKVLMAFIICLSLILSVLAAPPSGAKA 1.62 2 75.00 0.914 38 FliZ MKKSQYFIVFICFFVLFSVHPIAAAAA 1.61 2 70.37 0.684 39 DacB MRIFKKAVFVIMISFLIATVNVNTAHA 1.57 3 66.67 0.876 40 SacB MNIKKFAKQATVLTFTTALLAGGATQAFA 1.51 3 62.07 0.866 41 YrvJ MNKKYFVLIVCIIFTSALFPTFSSVTA 1.49 2 59.26 0.762 42 YlaE MKKTFVKKAMLTTAAMTSAALLTFGPDAASA 1.47 4 61.29 0.93 43 Pbp MKKSIKLYVAVLLLFVVASVPYMHQAALA 1.46 3 68.97 0.733 44 YbxI MKKWIYVVLVLSIAGIGGFSVHA 1.44 2 73.91 0.612 45 YolA MKKRITYSLLALLAVVAFAFTDSSKAKA 1.44 3 57.14 0.834 46 YqxI MFKKLLLATSALTFSLSLVLPLDGHAKA 1.41 2 71.43 0.837 47 YoaW MKKMLMLAFTFLLALTIHVGEASA 1.36 2 70.83 0.821 48 NprB MRNLTKTSLLLAGLCTAAQMVFVTHASA 1.33 2 57.14 0.85 49 YlxF MSGKKKESGKFRSVLLIIILPLMFLLIAGGIVLWAAG 1.33 4 75.68 0.563 50 YbfO MKRMIVRMTLPLLIVCLAFSSFSASARA 1.31 3 64.29 0.86 51 YlqB MKKIGLLFMLCLAALFTIGFPAQQADA 1.30 2 74.07 0.838 52 SpoIID MKQFAITLSVLCALILLVPTLLVIPFQHNKEAGA 1.28 1 67.65 0.718 53 YwmC MKKRFSLIMMTGLLFGLTSPAFA 1.17 3 69.57 0.912 54 YvbX MKKWLIIAVSLAIAIVLFMYTKGEAKA 1.09 2 70.37 0.627 55 YkvV MLTKRLLTIYIMLLGLIAWFPGAAQA 1.02 2 76.92 0.808 56 YlxY MYKKFVPFAVFLFLFFVSFEMMENPHALDYIGA 0.98 2 69.70 0.488 57 XynA MFKFKKNFLVGLSAALMSISLFSATASA 0.95 3 64.29 0.913 58 SleB MKSKGSIMACLILFSFTITTFINTETISAFS 0.94 2 51.61 0.651 59 YbbC MRKTIFAFLTGLMMFGTITAASA 0.92 2 69.57 0.861 60 YxiT MKWNNMLKAAGIAVLLFSVFAYAAPSLKAVQA 0.90 2 71.88 0.707 61 LytC MRSYIKVLTMCFLGLILFVPTALA 0.90 2 70.83 0.786 62 PhrA MKSKWMSGLLLVAVGFSFTQVMVHA 0.86 2 68.00 0.74 63 YkvT MTTKFTALAVFLLCFMPAAKI 0.77 1 71.43 0.715 64 CotC MKNRLFILICFCVICLFLSFGQPFFPSMILTVQAAKS 0.68 2 64.86 0.787 65 AmyE MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASA 0.67 3 78.79 0.904 66 NprE MGLGKKLSVAVAASFMSLSISLPGVQA 0.62 2 70.37 0.832 67 YolC MKKRLIGFLVLVPALIMSGITLIEA 0.58 3 76.00 0.727 68 YqzG MMIKQCVICLSLLVFGTTAAHA 0.57 1 63.64 0.829 69 YndA MRFTKVVGFLSVLGLAAVFPLTAQA 0.52 2 76.00 0.895 70 YfjS MKWMCSICCAAVLLAGGAAQA 0.49 1 71.43 0.845 71 YvcE MRKSLITLGLASVIGTSSFLIPFTSKTASA 0.43 2 56.67 0.891 72 YkwD MKKAFILSAAAAVGLFTFGGVQQASA 0.42 2 73.08 0.688 73 Mdr MDTTTAKQASTKFVVLGLLLGILMSAMDNTIVATA 0.41 1 60.00 0.595 74 YwfM MKGNIYSLFVLIAAFFWGTTGTVQA 0.36 1 68.00 0.669 75 NucB MKKWMAGLFLAAAVLLCLMVPQQIQGASS 0.34 2 72.41 0.746 76 YqxM MFRLFHNQQKAKTKLKVLLIFQLSVIFSLTAAICLQFSDDTSA 0.33 5 51.16 0.457 77 YkoJ MLKKKWMVGLLAGCLAAGGFSYNAFA 0.30 3 73.08 0.712 78 Mpr MKLVPRFRKQWFAYLTVLCLALAAAVSFGVPAKA 0.30 4 70.59 0.886 79 YpuA MKKIWIGMLAAAVLLLMVPKVSLADA 0.29 2 80.77 0.782 80 TasA MGMKKKLSLGVASAALGLALVGGGTWA 0.29 3 77.78 0.782 81 YwmD MKKLLAAGIIGLLTVSIASPSFA 0.26 2 73.91 0.789 82 YwtD MNTLANWKKFLLVAVIICFLVPIMTKAEIAEA 0.25 2 68.75 0.763 83 YdbK MKLFNRKVTLVSLILMAVFQFFMALIIKRIVIS 0.22 3 69.70 0.699 84 YfkN MRIQKRRTHVENILRILLPPIMILSLILPTPPIHA 0.21 3 62.86 0.654 85 YwaD MKKLLTVMTMAVLTAGTLLLPAQSVTPAAHA 0.16 2 67.74 0.883 86 YpjP MKLWMRKTLVVLFTIVTFGLVSPPAALMA 0.15 3 75.86 0.842
Appendix
115
No.
Name
Signal sequence
Secreted cutinase [U/mL]
a
Charge
N-region b
Hydro-
phobicity [% ]
c
D-
Score d
87 RpmG MRKKITLACKTCGNRNYTTMKSSASA 0.14 3 30.77 0.536 88 DacF MKRLLSTLLIGIMLLTFAPSAFA 0.14 2 73.91 0.909 89 TyrA MNQMKDTILLAGLGLIGGSIALA 0.13 0 73.91 0.466 90 LytF MKKKLAAGLTASAIVGTTLVVTPAEA 0.13 3 65.38 0.744 91 WprA MKRRKFSSVVAAVLIFALIFSLFSPGTKAAA 0.12 4 67.74 0.941 92 YbbR MDKFLNNRWAVKIIALLFALLLYVAVNS 0.11 1 67.86 0.612 93 YhjA MKKAAAVLLSLGLVFGFSYGAGHVAEA 0.09 2 74.07 0.577 94 YjiA MAAQTDYKKQVVGILLSLAFVLFVFS 0.08 1 65.38 0.567 95 PbpD MTMLRKIIGWILLLCIIPLFAFTVIA 0.08 1 80.77 0.777 96 YjcM MKKELLASLVLCLSLSPLVSTNEVFA 0.08 1 57.69 0.776 97 YhaK MRTWKRIPKTTMLISLVSPFLLITPVLFYAALA 0.08 4 66.67 0.725 98 PelB MKRLCLWFTVFSLFLVLLPGKALG 0.07 2 75.00 0.857 99 SpoIIQ MREEEKKTSQVKKLQQFFRKRWVFPAIYLVSAAVILTAVL 0.07 5 52.50 0.408
100 MotB MARKKKKKHEDEHVDESWLVPYADILTLLLALFIVLYASS 0.07 6 50.00 0.307 101 YdhT MFKKHTISLLIIFLLASAVLA 0.06 2 71.43 0.701 102 YbdG MKTLWKVLKIVFVSLAALVLLVSVS 0.06 2 72.00 0.783 103 LytE MKKQIITATTAVVLGALFA 0.05 2 68.42 0.502 104 PhrF MKLKSKLLLSCLALSTVFVATTIA 0.05 3 58.33 0.807 105 YhcR MLSVEMISRQNRCHYVYKGGNMMRRILHIVLITALMFLNVMYTFEA 0.04 4 54.35 0.359 106 CccA MKWNPLIPFLLIAVLGIGLTFFLSVKG 0.03 1 81.48 0.745 107 CitH MGNTRKKVSVIGAGFTGATTAFLIAQKELADV 0.02 3 59.38 0.43 108 AspB MKLAKRVSALTPSTTLAITAKA 0.02 3 54.55 0.897 109 YknX MKKVWIGIGIAVIVALFVGINIYRSAAPTSGSA 0.01 2 72.73 0.585 110 YhdC MKSLPYTIALLFCGLIIVSMA 0 1 71.43 0.673 111 YlbL MLRKKHFSWMLVILILIAVLSFIKLPYYITKPGEA 0 3 65.71 0.501 112 YlxW MRGKSAVLLSLIMLIAGFLISFSFQMTKENNKSAA 0 2 60 0.614 113 YngK MKVCQKSIVRFLVSLIIGTFVISVPFMANA 0 3 66.67 0.878 114 YnzA MELSFTKILVILFVGFLVFGPDKLPALG 0 0 78.57 0.468 115 YobV MKLERLLAMVVLLISKKQVQA 0 1 61.9 0.623 116 YocH MKKTIMSFVAVAALSTTAFGAHA 0 2 65.22 0.88 117 YodV MKVPKTMLLSTAAGLLLSLTATSVSA 0 2 61.54 0.927 118 YojL MKKKIVAGLAVSAVVGSSMAAAPAEA 0 3 73.08 0.8 119 YomL MRKKRVITCVMAASLTLGSLLPAGYATA 0 4 60.71 0.884 120 YoqH MKRFILVLSFLSIIVAYPIQTNA 0 2 65.22 0.744 121 YoqM MKLRKVLTGSVLSLGLLVSASPAFA 0 3 68 0.911 122 YpbG KLSVKIAGVLTVAAAAMTAKMYATA 0 2 68 0.707 123 YpcP MNNNKLLLVDGMALLFRAFFATA 0 1 69.57 0.57 124 YpmS NKWKRLFFILLAINFILAAGFVALVLLPGEQAQV 0 3 76.47 0.701 125 YpuD MGRIKTKITILLVLLLLLAGGYMYINDIELKDVPTAIG 0 3 65.79 0.409 126 YqzC MTKRGIQAFAGGIILATAVLAAVFYLTDEDQAAA 0 2 67.65 0.518 127 YraJ MTLTKLKMLSMLTVMIASLFIFSSQALA 0 2 64.29 0.898 128 YuaB MKRKLLSSLAISALSLGLLVSAPTASFAAE 0 3 63.33 0.909 129 YusW MHLIRAAGAVCLAVVLIAGCRFNEDQHQAEG 0 1 61.29 0.421 130 YvgO MKRIRIPMTLALGAALTIAPLSFASA 0 3 73.08 0.89 131 YvgV MKKKQQSSAKFAVILTVVVVVLLAAIV 0 3 66.67 0.682 132 YvnB MRKYTVIASILLSFLSVLSGG 0 2 61.9 0.669 133 YvpB MKTLRTLCVLMILSGVIFFGLKIDA 0 2 68 0.775 134 YwcI MKRLLVSLRVWMVFLMNWVTPDRKTARA 0 2 60.71 0.577 135 YwdK MKVFIILGAINALLAVGLGAFG 0 1 90.91 0.553 136 YweA MLKRTSFVSSLFISSAVLLSILLPSGQAHA 0 2 60 0.923 137 YwgB MKMKSGMEQAVSVLLLLSRLPVQA 0 2 62.5 0.656 138 YwmB MKKKQVSHAIIISVMLSFVIAVFHTIHA 0 3 60.71 0.598 139 YwoF MRKWYFILLAGVLTSVILAFVYDKTKA 0 2 62.96 0.667 140 YwqC MGESTSLKEILSTLTKRILLIMIVTAAATA 0 -1 56.67 0.576 141 YwqO MKFLLSVIAGLLILALYLFWKVQPPVWI 0 1 82.14 0.569 142 YwsB MNKPTKLFSTLALAAGMTAAAAGGAGTIHA 0 2 70 0.662 143 YwtC MKFVKAIWPFVAVAIVFMFMSA 0 2 86.36 0.718 144 YwtF MEERSQRRKKKRKLKKWVKVVAGLMAFLVIAAGSVGAYA 0 9 56.41 0.675 145 YxaK MVKSFRMKALIAGAAVAAAVSAGAVSDVPAAKVLQPTAAYA 0 3 73.17 0.679 146 YxiA MFNRLFRVCFLAALIMAFTLPNSVYA 0 2 69.23 0.873 147 YybN MNKFLKSNFRFLLAAALGISLLASSNFIKA 0 3 63.33 0.81 148 YycP MKKWMITIAMLILAGIALFVFISPLKS 0 2 77.78 0.725 a The lipolytic activity assay was performed at 30°C; the enzymatic activity was calculated using a molar
absorbtion coefficient of 15000 M-1
·cm-1
. b The net charge of the N-region was calculated with amino acids aspartate and glutamate defined as -1; arginine
and lysine defined as +1 and any other amino acid defined as 0. c The percentage of hydrophobic amino acids of the each signal sequence was calculated with amino acids G, A,
V, L, I, M, F, W and P defined as hydrophobic, any other amino acid being characterized as hydrophilic. d D-score calculated by SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/)
Appendix
116
pW TsecA
10000 bps
A) ep-PCR of PPX-domain
pW TsecA/
pMutsecA
C) DpnI digestion of wildtype plasmid
pMutsecA
10000 bps
pW TsecApW TsecA//pMutsecA
PPX-cassetteas primer pair
B) PCR of whole plasmid
pW TsecA
10000 bps
A B
Fig.35: Overview of the complete Gemini pipetting Script ”Ulf_PnPP1_10_tiptouch_bis_10PL”. The script was generated using software Gemini Version 4.2 (Tecan, Crailsheim, Germany) and integrated in the Process Manager Script “Ulf_PnPP_1_10V_Trog2000” providing an automated HTS process using the pipetting robot “TECAN workstation Genesis 200 Freedom (Tecan, Crailsheim, Germany. (A) First part of the pipetting script providing a 1:10 dilution of B. subtilis culture supernatants in one MTP (96 wells) using fresh LB-medium. (B) Second part of the pipetting script providing the filling of the assay plate with assay solution and diluted supernatants.
Fig. 34: Cassette mutagenesis to generate a library of the PPX-domain of SecA. A) ep-PCR of the PPX-domain with primers ppx_up and ppx_down using methylated plasmid DNA (pBSMuL3secA) from E.coli strain JM109 as the template lead to 350 bp DNA fragments containing the library of the PPX-domain of SecA. The used PCR conditons resulted in an error rate of 5-8 bases per kb DNA. B) The DNA of gel extracted PPX-fragments were taken as the primer pair in a second PCR amplifiying the whole plasmid pBSMuL3secA resulting in a plasmid mix of methylised wildtype plasmid and generated plasmid variants. C) To eliminate methylised wildtype secA (template) the PCR sample was incubated with endonuclease DpnI. An aliquote of this sample was directly used for electrotransformation into E.coli JM109 for plasmid library construction described before.
Lebenslauf
Lebenslauf
Name: Brockmeier Vorname: Ulf Geburtsdatum: 18.11.1973 Geburtsort: Münster Familienstand: ledig Schulausbildung: 1980-1982 Marienschule, kathol. Grundschule, Ochtrup 1982-1984 St.-Josef-Schule, kathol. Grundschule, Ahaus 1984-1988 Alexander-Hegius-Gymnasium in Ahaus 1988-1993 Gymnasium am Ostring, Bochum
Abschluss: Allgemeine Hochschulreife Zivildienst: 1993-1994 St. Josef Hospital Bochum Hochschulausbildung: 10/1994-04/2001 Diplomstudiengang der Fachrichtung Biologie an der Ruhr-Universität Bochum 04/2001-04/2002 Diplomarbeit am Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum
Thema: Die Sekretionswege der lipolytischen Enzyme LipA und LipB von Bacillus subtilis
seit 08/2002 Promotion am Institut für Molekulare Enzymtechnologie der Heinrich-Heine Universität Düsseldorf im Forschungszentrum Jülich
Thema: New strategies to optimize the secretion capacity for heterologous proteins in Bacillus subtilis
Anstellungen: 06/2001-04/2002 Studentische Hilfskraft an der Ruhr-Universität Bochum (Lehrstuhl Biologie der Mikroorganismen) 08/2005-04/2006 Wissenschaftlicher Angestellter an der Heinrich-Heine Universität
Düsseldorf (Institut für Molekulare Enzymtechnologie) Stipendien: 08/2002-07/2005 Stipendium des Europäischen Graduiertenkollegs der Ruhr-
Universität Bochum (EGC 795): Regulatory Circuits in Cellular Systems: Fundamentals and Biotechnological Applications