Enzymes can dramatically increase the rate of chemical reactions while acting stereoselective and regioselective and are therefore very attractive for industry. However, only few enzymes taken from nature work under the often harsh conditions required by industrial applications. Proteins with desired attributes can be obtained either by searching through the largely unknown naturally occurring species or by improving or altering already characterized proteins. Today, much research effort is devoted to adjusting various enzyme attributes to technical demands and to exploring nonnatural functions of enzymes.
Two basic approaches are used to optimize enzymes to fullfill desired prop-erties: rational design and directed evolution Fig. 36.1.
1.1. Rational Design
To optimize a protein by rational design, the structure of the protein or a close homolog should be known. Better results can be obtained, when information about mechanism and function of the protein are available. Scientists planning to redesign an enzyme for the first time should be aware that integration of knowledge into the design process is very time intensive and requires train-ing. Variants are planned based on intuition, sequence alignments and in silico modeling. Amino acid exchanges required for optimization are introduced into the gene of the original protein via site-directed mutagenesis. Finally, over-expressed variants require further characterization.
1.2. Directed Evolution
Despite advances in our understanding of protein structure and function this knowledge is by far not sufficient to tailor amino acid exchanges. From this perspective it is very appealing to use random design approaches such as com-binatorial strategies to search sequence space for beneficial mutations without prior knowledge of structure or function.
From natural evolution we have learned that organisms and accordingly natu-rally occurring proteins are constantly changing to adapt to new environmental
36Directed Protein Evolution
Sabine C. Stebel, Annette Gaida, Katja M. Arndt, and Kristian M. Müller
requirements and microorganisms can even cometabolize xenobiotica they were never adapted to. Directed evolution mimics the Darwinian principles of muta-tion and selection underlying evolution in nature. In general the term “directed evolution” is used to sum up different techniques for the creation of protein variants by low frequency introduction of point mutations, their selection or screening for the desired properties, and the recombination of beneficial muta-tions found in different clones. Directed evolution normally consists of repeated cycles of mutagenesis, screening or selection, and recombination Fig. 36.1.
1.3. Semi-Rational Design
The experimental combination of rational design and directed evolution is called semi-rational design.
This chapter describes and explains methods used in directed evolution experiments and presents successful applications.
2. Methods for the Creation of Diversity
Crucial to any evolutionary optimization strategy is a suitable library to start with and efficient means of screening or preferably selecting optimized variants from these libraries. A library of mutants is constructed at the genetic level from which the protein library is translated, thus any protein selected can be identified by its DNA sequence. Multiple methods are available to construct such DNA libraries which conceptually can be divided into three groups Table 36.1.
2.1. Random Diversity by Disturbed DNA Replication
2.1.1. Chemical and Physical MutationThe first group comprises methods generating random diversity by disturbed DNA replication.
Physical and chemical mutagens like UV irradiation or alkylating agents were the first employed to purposely damage DNA. UV irradiation generates radicals or is absorbed by the double bond of pyrimidine bases (typically thymi-dines), which thus excited, can form dimers with neighboring pyrimidine bases. These dimers when not successfully repaired lead to random incorporation of
Fig. 36.1. Schema showing how directed evolution and rational design are connected
Chapter 36 Directed Protein Evolution 633
nucleotides. Alkylation agents like ethylenemethanesulfonat (EMS) alkylate e.g., the oxygens of guanidine at position 6 leading to a transition from GC to AT (1). Hydroxylamine hydroxylates specifically the NH2 group of cytosine. The resulting hydroxylaminocytosine pairs with adenine leading to a transition from CG to TA (1).
2.1.2. Mutator StrainsConceptually simple but hard to control regarding the mutation rate are Escherichia coli K12 mutator strains like XL1-red from Stratagene, which are deficient in three DNA repair pathways (mutS, mutD, and mutt) increasing the mutation rate up to 5,000 times compared to the wild-type strain.
2.1.3. Error Prone PCR (epPCR)The most popular method, because it is easy to control and implement, is the generation of random point mutations by error prone PCR (epPCR) (2). Taq-DNA-polymerase is prone to errors during polymerase chain reaction. The normal error rate of Taq-DNA-Polymerase (1 × 10−4 to 2 × 10−5 errors
Table 36.1. Three ways to construct DNA libraries.
Random diversity by disturbed DNA replication
Controlled levels of random diversity at specific positions Diversity by recombination
• Chemical and physical mutation
• Mutator strains
• epPCR
• Random insertion / deletion mutagenesis (PSM, RAISE, RID)
per nucleotide) is by far not enough to be used for the construction of a combina-torial library by means of PCR. However it can be easily enhanced by add-ing small amounts of Mn2+ ions, which replace the natural cofactor Mg2+. If necessary, the mutation rate can be further enhanced by using a biased dNTP mixture with unequal representation of nucleotides.
2.1.4. Sequence Saturation Mutagenesis (SESAM)In the above described methods to mutagenize DNA typically only one nucle-otide per codon is changed, thus in average every amino acid can be changed to only about 5 out of 19 possible amino acids. Sequence saturation mutagen-esis (SESAM) (3) overcomes this by substituting nucleotides with a universal base (deoxyinosine, dITP) Fig. 36.2. In the first step a PCR is performed with a 5′ biotinylated primer and dATPαS in addition to the four natural dNTPs. dATPαS is similar to the natural dATP but carries a sulfur atom instead of an oxygen at the α-phosphate. The resulting phosphate bond is cleaved by iodine and the biotinylated fragments are isolated from the fragment mixture by binding of biotin to immobilized streptavidin. The cleaved and isolated frag-ments are tailed with dITP by terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the repetitive random addition of deoxyribonucleotides to the 3′ end of single and double stranded
Sequence Saturation Mutagenesis (SESAM)
PCR in presence of dATPαS with biotinylated 5 �primer
DNA. The resulting elongated strand is extended to full length on long and biotinylated ssDNA templates without amplification of this template. The reverse primer, which can not bind to the long ssDNA template, anneals to the newly synthesized strand and dsDNA is generated that contains a nucleotide analog in one strand and randomly inserted nucleotides at the corresponding position in the complementary strand.
To replace the dITP with standard nucleotides an additional PCR is carried out where the full length genes containing dITP serve as template.
2.1.5. Methods of Random Insertion/DeletionRandom diversity can also be generated by transposons like Tn4430 in pen-tapeptide scanning mutagenesis (PSM) (4) Fig. 36.3. Tn4430 contains two restriction sites for KpnI 5 bp from both termini. Tn4430 duplicates 5 bp of its target site during transposition. Therefore, if Tn4430 is removed from the target plasmid by digest with KpnI it leaves a 15 bp fingerprint consisting of two times 5 bp from the transposon ends and 5 bp from the duplicated target-site. The gene of interest, which is flanked by two unique restriction sites,
Pentapeptide Scanning Mutagenesis (PSM)
A
B
Transposase Site-specific recombinase
GGGGTACCGCCAGCA … TGCTGGCGGTACCCC
… N1N2N3N4N5N6N7N8N9N10N11
KpnI KpnI
Insertion of tn4430
+
BA KpnI
BA
A B
A B
Separate target gene with tn4430from target gene without tn4430and backbone with and without tn4430by agarose gel
KpnI
KpnI KpnI
A
B
KpnIKpnI
… N1−6 GGGGTACCCC N2−11…
Reclone in a vector without KpnI site anddigest with KpnI
is removed from the plasmid and genes with transposons are separated from those without by agarose gel and cloned into a new vector backbone.
2.1.5.1. Random Insertional-Deletion Strand Exchange Mutagenesis (RAISE): RAISE (5) Fig. 36.4 is a modified version of DNase shuffling (see the follow-ing). After DNA digest the generated fragments are incubated with terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the repetitive random addition of deoxyribonucleotides to the 3' end of single and double stranded DNA. During reassembly of the randomly elongated fragments deletions, insertions, or exchanges are generated depend-ent of the length of the random insert.
2.1.5.2. Random Insertion and Deletion Mutagenesis (RID): RID (6) Fig. 36.5 can insert randomly up to 16 random bases at random positions along a whole gene. To perform RID the gene of interest is first flanked with two different unique restriction sites and cut by these two enzymes, e.g., EcoRI at the 5' end and HindIII at the 3' end. In the next step a synthetic oligonu-cleotide linker that has a HindIII restriction site at the 5' end and a comple-mentary sticky end at the 3' end is ligated to the 5' end of the gene leaving
Random Insertional-deletion StrandExchange mutagenesis (RAISE)
Fragmentation by DNaseI digest
Addition of random nucleotides by TdT
Reassembly by a primerless ‘PCR’
Reassembly by a primerless ‘PCR’
Fig. 36.4. Random insertional-deletion strand exchange mutagenesis (RAISE)
Chapter 36 Directed Protein Evolution 637
a nick as the 3' end does not have a mono-phosphate and thus will not link to the 3' end of dsDNA. The gene is again digested by HindIII and cyclized. The DNA strand containing the nick is degraded by T4 DNA polymerase resulting in circular ssDNA that is randomly cut once with ceric ammonium nitrate–EDTA complex (Ce(IV)-EDTA). Random anchors are ligated to both ends. Both anchors contain a unique restriction site e.g., a BciVI site,additional nucleotides to be inserted and a random tail for the hybridization with the unknown 3' end ssDNA and 5' end respectively. The ssDNA is filled in and the ends are blunted if necessary. After renewed cyclization this dsDNA
PCR fill-in reactionCleavage with BciVIblunting of endscyclizationEcoRI
EcoRI
EcoRIHind III
Hind IIIHind III
Digest with Hind III and EcoRIEcoRI Hind IIII
Fig. 36.5. Random insertion and deletion (RID)
638 S. C. Stebel et al.
ring is cut with EcoRI and HindIII thus generating the original gene including a randomly inserted sequence.
2.2. Controlled Levels of Random Diversity at Specific Positions
The techniques described in Section 2.1 randomly generate diversity along the whole gene. The second group of methods encompasses techniques that insert controlled levels of random diversity at specific positions. In principle all the methods of the second group are based on the incorporation of synthetic DNA sequences into the coding gene. The synthesis of oligonucleotides or even whole genes allows the complete control over identity, position, and level of mutation. Most commonly used are degenerate oligonucleotides, which at defined positions contain degenerate codons and can be bought from most suppliers. With such oli-gonucleotides specific positions in a gene can be completely randomized.
2.3. Diversity by Recombination
2.3.1. Homologous Recombination Methods
2.3.1.1. DNA Shuffling and its Derivates: A big disadvantage of the methods described above is the indiscriminate incorporation of beneficial as well as deleterious mutations. If proteins are to be optimized only by point mutations at some stage the disadvantageous mutations will reduce or completely overlay the beneficial effects of advantageous mutations. In nature this problem is solved by sexual recombination allowing for out crossing of deleterious mutations. The third group of methods comprises approaches mimicking this natural sexual recombination. Unlike in nature where only the genes of two parents are crossed, shuffling can recombine the genes of multiple parents. The first method becoming popular for homologous DNA recombination in the test tube was DNA shuffling (7) Fig. 36.6. In DNA shuffling, the DNA is first digested by DNase I. The digested fragments of the desired size are extracted from an agarose gel and reassembled by a primerless ‘PCR’ reac-tion in which the fragments serve each other as primer and template at the same time (recursive PCR). The reconstructed and recombined genes are afterwards amplified using a standard PCR reaction. Nucleotide exchange and excision technology (NExT) (8,9) Fig. 36.7 is similar to the DNase digest but much easier to control. NExT uses a statistical fragmentation by incorporation of uridine (dUTP) during PCR, followed by digestion with uracildeglycosylase (UDG) and NaOH instead of DNase I. Additionally NExT uses vent polymerase for the reassembly reaction. Vent is a proofread-ing polymerase with strand displacement activity. In case of multiple prim-ing events on one strand, vent can remove these oligonucleotides and read on significantly improving yield of reassembled genes.
2.3.1.2. Staggered Extension Process (StEP): STEP (10) Fig. 36.8 is based on fast template switches owing to very short elongation times during a PCR reaction. As a consequence partially elongated strands are generated that can switch the paren-tal template and thus lead to recombination of different parental genes.
A further development of StEP is the recombination-dependent exponential amplification PCR (RDA-PCR) (11) Fig. 36.9. In RDA-PCR the library is divided into two groups, which are amplified with two different primer pairs adding different overhangs, e.g., group 1 is amplified using primers A and B and group 2 with primer C and D. These two groups are then mixed, divided into two new groups and a StEP reaction is carried out but this time with primer pairs A/D
Chapter 36 Directed Protein Evolution 639
Fig. 36.6. DNA shuffling
DNA shuffling
Library of randomly mutated genes or pool of homologous genes
Random fragmentation by digest with DNaseI
Reassembly by primerless PCR:Annealing
Library of hybrid genes
Extension
Denaturation and annealing
Extension
Fig. 36.7. Nucleotide exchange and excision technology (NExT)
Nucleotide Exchange and Excision Technology (NExT)
Library of randomly mutated genesor pool of homologous genes
PCR incorporating dUTP
U
U U U
U UU
Removal of uridine by UDGBreaking of DNA backbone by NaOH
Reassembly by primerless PCR withstrand displacement activity by vent polymerase
Library of hybrid genes
Extension
Denaturation and annealing
Extension
640 S. C. Stebel et al.
Staggered Extension Process (StEP)
Library of randomly mutated genes or pool of homologous genes
and B/C thus ensuring that only chimeric genes with at least onecross over are amplified. Unfortunately, only chimeric genes with odd numbers of crossovers can be amplified using this method.
2.3.1.3. Random Chimeragenesis on Transient Templates (RACHITT): RACHITT (12) Fig. 36.10 is conceptually similar to StEP. In RACHITT a
Chapter 36 Directed Protein Evolution 641
Random Chimeragenesis on Transient Templates (RACHITT)
Library of randomly mutated genesor pool of homologous genes
Random fragmentation by digest with DNaseI
+Uracil containing template strand
Hybridization
Removal of flaps and fill in raction by Pfu polymerase
Removal of template strand by uracil-DNA-glycosylase
Generation of dsDNA by PCR
Fig. 36.10. Random chimeragenesis on transient templates (RACHITT)
parental DNA strand containing dUTP is generated and fragments of the opposite strand anneal to this parental template. Nonannealed overhangs are digested by the exonuclease activity of Pfu DNA polymerase, which also fills in the remaining gaps. The generated fragments are ligated and the uridine containing parental strand is rendered unproductive by digest with endonucle-ase V. The resulting chimeric strand is amplified by PCR.
2.3.1.4. Recombined Extension on Truncated Templates (RETT): Eukaryotic RNA in contrast to DNA doesn’t contain any introns and thus is shorter and more suited for DNA shuffling than eukaryotic DNA. A recombination method more likely useful for eukaryotic genes than the previously described ones is recombined extension on truncated templates (RETT) (13) Fig. 36.11 as it uses RNA. Fragments can be generated by using random primers for reverse transcription or by unidirectional serial truncation of cDNA with exonuclease III. A specific primer is afterwards annealed to complementary ssDNA fragmentation and extended by PCR. Short fragments extended from this specific primer (like in StEP) are annealed to another ssDNA fragment and thus switch templates. This extension is repeated until full length genes are generated, which then are used to generate dsDNA by PCR.
2.3.1.5. Mutagenic and Unidirectional Reassembly Method (MURA): Truncated genes can lead to altered attributes of the enzyme coded by this gene, e.g., specificity and thermal stability. Very similar to RETT is mutagenic and
642 S. C. Stebel et al.
unidirectional reassembly method (MURA) (14) Fig. 36.12. In contrast to RETT MURA starts with dsDNA, which is digested by DNaseI like in DNA-shuffling. The resulting fragments however are not reassembled by a primerless PCR but like in RETT the reassembly is achieved using unidirectional primers contain-ing appropriate restriction sites (MURA primer). This unidirectional reassem-bly additionally to template switches results in N- or C- terminally truncated mutants depending on the location of the primer.
2.3.1.6. Multiplex-PCR-Based Recombination (MUPREC): Only applicable to homologous templates is multiplex-PCR-based recombination (MUPREC) (15) Fig. 36.13. Multiplex PCR (16) is a variant of PCR, which simultaneously amplifies many targets of interest in one reaction by using more than one pair of primers. In this case two different genes are amplified with multiple 5′ and multiple 3′ primers containing point mutations to generate mutated gene fragments of different lengths. These fragments are afterwards mixed and recombined in a reassembly PCR. For this method primers have to be designed
Fig. 36.11. Recombined extension on truncated templates (RETT)
Recombined Extensions of Truncated Templates(RETT)
Reverse transcription withrandom primers Serial deletion by Exonuclease III
Specific primer is annealedand extended
Repeated until full length ssDNAis generated
Reverse transcription
Chapter 36 Directed Protein Evolution 643
Mutagenic and Unidirectional ReassemblyMethod (MURA)
Fragmentation of parental gene
MURA primer
Unidirectional reassembly
Blunting of ends
Digest with enzyme contained in MURA primer
Fig. 36.12. Mutagenic and unidirectional reassembly method (MURA)
with comparable melting temperatures. In multiplex-PCR the amount of each primer used influences the frequency of the mutations incorporated.
2.3.1.7. Random-Priming in vitro Recombination (RPR): A much earlier published variation of MUPREC is random-priming in vitro recombination (RPR) (17) that instead of specially designed primers uses random primers of identical length, which results in fragments of many different sizes that can be reassembled by primerless PCR reaction.
2.3.1.8. In vitro Heteroduplex Formation and in vivo Repair: In vitro heteroduplex formation and in vivo repair (18) Fig. 36.14 is a method that is mainly useful for the recombination of large genes or whole operons as this method has not the size limitations of PCR based methods. If cells are transformed with partially overlapping inserts with included mismatches, each mismatch between the two DNA strands can be repaired independently by combination with homologous regions und this results in chimeric sequences. The E. coli mismatch MutHLS repair system consists of three components: MutS for the recognition of mismatches, MuL is a molecular matchmaker that activates the nicking endonuclease MutH. MutH can bind to GATC sequences and nicks the DNA, which is then unwound by helicase II. The mismatch gap is filled in by polymerase III by copying the complementary DNA strand.
In vitro heteroduplex formation and in vivo repair
PCR
Denaturation and annealing
Ligation and cloning
Fig. 36.14. In vitro heteroduplex formation and in vivo repair
Chapter 36 Directed Protein Evolution 645
2.3.2. Nonhomologous Recombination Methods
2.3.2.1. Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY)/SCRATCHY: It was shown that introns mostly occur at positions with low intermolecular interaction, pointing to proteins being com-posed of building blocks of domains developed earlier in evolution (19). Nonhomologous recombination or family shuffling explores these largely unknown possibilities.
In contrast to the above mentioned recombination methods, where gene reassembly is based on high homology among the genes to be recombined incremental truncation for the creation of hybrid enzymes (ITCHY) (20,21) Fig. 36.15 allows recombination of nonhomologous templates. Two genes, one incrementally truncated from the 5′ end, the other from the 3′ end are
Incremental Truncation for the Creation of HybridEnzymes (ITCHY)
Nsi I Nsi IA D
Linearization of vector by digest with A/B and C/D
Nsi I Nsi I
B C
Incremental truncation by exonuclease
Nsi I Nsi I
Nsi I Nsi I
Nsi I Nsi I
Blunting of endsDigest with Nsi I and ligation
Nsi I
Nsi I
Nsi I
Fig. 36.15. Incremental truncation for the creation of hybrid enzymes (ITCHY)
646 S. C. Stebel et al.
ligated thus creating chimeras with one cross over. The gene length of the generated chimeras is not conserved and recombination mostly occurs at nonstructurally related sites. The chimeric libraries generated by ITCHY can be afterwards shuffled by the above mentioned methods thus enhancing the cross over rate. This combination of ITCHY technology with DNA shuffling is called SCRATCHY (22).
2.3.2.2. Sequence Homology-Independent Protein Recombination (SHIPREC): A further development of the basic ITCHY idea is sequence homology-independent protein recombination (SHIPREC) (23) Fig. 36.16. In contrast to ITCHY SHIPCREC generates chimeras that have the cross over at similar struc-tural positions. In SHIPREC two genes are fused together via a linker containing a unique restriction site and digested with DNaseI. Fragments of the size of the single genes are selected by agarose gel electrophoresis and blunted. The blunted chimeras are afterwards circularized and cut at the linker position thus placing the gene fragment corresponding to the C-terminal part of the protein coded by the first gene behind the gene fragment of the second gene that codes for to the N-terminal part of the second protein. Limiting the size of fragments to the size of the original genes ensures that the chimeras cross at similar parental structures.
2.3.2.3. Synthetic Shuffling or Assembly of Designed Oligonucleotides (ADO): Synthetic shuffling (24) Fig. 36.17 is a combination of the use of degenerate oligonucleotides and DNA shuffling. Instead of fragment-
Blunting of endsSelection of fragments in the size ofthe single original genes by agarose ge
Fragmentation e.g. by DNaseI digestA
A
A
Digest with A
N-Term. C-Term. N-Term. C-Term.
N-Term. C-Term.
Fig. 36.16. Sequence homology-independent protein recombination (SHIPREC)
Chapter 36 Directed Protein Evolution 647
Synthetic shuffling
x xx x xx x x xx x x x xx x x
x xx x x
x xx x
x xx x
xx x
xx x
x xx x
x x x
x xx
x
Diverse syntheticoligonucleotides
Reassembly by primerless PCR
x xx x x x x xxx x x x x x x x x x x x xx
x xx x xx x xxxx x x x x x x xxx x xx
x xxxxx x x x xx x x x x x x x xxx x xx
Fig. 36.17. Synthetic shuffling
ing genes the fragments are designed as degenerate oligonucleotides containing all variations wanted and then reassembled by a primerless PCR reaction. This method unites the benefits of rational design with the statistical approach of shuffling. In principle Assembly of Designed Oligonucleotides (ADO) (25) is identical to synthetic shuffling but takes into account conserved regions that can be used as linkers for homolo-gous recombination.
2.3.2.4. Degenerate Oligonucleotide Gene Shuffling (DOGS): DOGS (26) Fig. 36.18 was designed to decrease the amount of parental DNA reas-sembled from shuffling procedures. For DOGS complementary degenerate primers are designed for conserved motives found in the candidate genes. Each of these segments is flanked by primers and individually amplified. For the reassembly procedure the library of fragments can be put together at different ratios generating many biased libraries containing no parental genes. Conceptually Structure-based Combinatorial Protein Engeneering (SCOPE) (27) is identical to DOGS but the fragments generated are not based only on sequence identity but additionally on variable connections among structural elements.
2.3.2.5. Sequence-Independent Site-Directed Chimeragenesis (SISDC): SISDC (28) Fig. 36.19 is additionally to DOGS and SCOPE taking into account the above mentioned assumption that proteins are assembled from earlier developed building blocks. These building blocks are calculated by an algorithm called SCHEMA (19). At the interconnections between these building blocks proteins can be fragmented and exchanged between fami-lies. For this specific fragmentation consensus sequences at these points of contact between the building blocks are determined and marker tags are
648 S. C. Stebel et al.
Degenerate Oligonucleotide Gene Shuffling(DOGS)
Library of randomly mutated genesor pool of homologous genes
Design of degenerate primer pairs upon conserved motives
inserted. These marker tags are inserted by PCR primers into the gene like the DNA fragments are generated in DOGS. Each building block thus is amplified by itself and reassembled again sequentially to the full parental genes. Each tag consists mainly of a recognition site for BaeI ([10/15] ACNNNNGTAYC[12/7]), which digests dsDNA at the 5′ end before the tenth random position and at the 3′ end after the twelfth random position thus leaving a five nucleotide overhang at both ends. The custom-made random positions of the BaeI recognition are different for each target site. The five nucleotides in front of and after BaeI are designed correspond-ing to the consensus sequence of the respective building block ensuring correct order of reassembly of the chimeric genes. To eliminate chimeras with uncut tags, a SmaI site is introduced in the downstream part of the BaeI site.
2.3.2.6. Exon Shuffling: In eukaryotes crossover reactions often occur in introns creating new combinations of exons. These rearrangements lead to new genes with altered functions during evolution. The natural process of recombining exons from unrelated genes is called exon shuffling. In vitro exon shuffling (29) is carried out analog to DOGS or SCOPE but is the best suited method for rearrangement of eukaryotic genes. The exons of multiple related genes are amplified with oligonucleotides that determine the order of reassembly like in SISDC. Each exon is amplified by itself and new genes can be reassembled like using a building set in a reassembly PCR.
Chapter 36 Directed Protein Evolution 649
3. Applications
3.1. Mutator Strain + Selection
E. coli mutator strains belong to the earliest methods used to randomly mutate DNA. In 1985 Liao et al. used a mutD5 E. coli mutator strain to generate thermostable enzyme variants of the kanamycin nucleotidyltransferase (30).
Kanamycin nucleotidyltransferase is the enzyme responsible for resistance against the antibiotic kanamycin. Liao cloned the kanamycin resistance first into a shuttle vector and propagated it in an E. coli mutD5 mutator strain. Afterwards the gene was transferred into Bacillus stearothermophilus. The wild type kanamycin nucleotidyltransferase only confers resistance to kan-amycin up to 55°C but Liao was able to identify mutants conferring resistance to B. stearothermohilus at 65°C. All these mutants carried the amino acid exchange D80Y and those carrying the additional mutation T130K even con-ferred resistance up to 70°C.
Esterases can effectively discriminate among stereoisomers and are therefore in use for the production of optical pure compounds. In 1997 Bornscheuer successfully enhanced the enantioselectivity of an esterase from Pseudomonas fluorescens by passages of the gene through the muta-tor strain XL1-Red (31). He was able to increase the enantiomeric excess from 0% up to 25%.
3.1.1. Chemical MutagenesisSubtilisin is a mesophilic alkaline serine endo protease of great commercial value. It is used in food and leather processing, and in laundry detergents to remove protein stains from clothing (24). It is highly desirable to enhance wash performance at low temperatures to save energy and reduce the wear on textiles. Thus, cold adapted subtilisins are of high interest. Taguchi et al. used hydroxylamine to chemically mutagenize the gene of subtilisin BPN' from Bacillus amyloliquefaciens und engineered a cold adapted protease, which had a 2-fold higher activity at 10°C and only three amino acid substitutions (32). Kano et al. generated with the same methodology applied by Taguchi a subtili-sin BPN' mutant active even at 1°C, which only had one amino acid exchange (V84I) (33).
3.1.2. epPCR + ScreeningOrganic solvents can cause serine proteases to catalyse unusual reactions that are normally not possible in aqueous solutions. Thus serine proteases are promising candidates for the catalysis of unusual chemical reactions. Chen et al. adapted subtilisin E to be active in a dimethylformamide (DMF) solution by sequential rounds of error prone PCR (34). Sequential rounds of error prone PCR generated a mutant enzyme containing 10 amino acid exchanges which was in DMF nearly as active as the wild-type subtilisin E in aqueous solvent. One year later You et al. further enhanced the cata-lytic activity of subtilisin E, already active in DMF, 16 times by the same strategy (35).
3.1.3. Synthetic ShufflingNess et al. succeeded to generate subtilisin variants with 2- to 3-fold higher thermostability and activity at pH 10 (36), but these enzymes could not be pro-duced in useful amounts owing to autoproteolysis. To circumvent this problem they fine tuned these enzymes to be less active at production conditions (pH 7). Based on the library of subtilisin genes from the previous study (36) they created synthetic oligonucleotides that were used to assemble a new library of subtili-sin genes (24). By this approach the authors found active and highly chimeric enzyme variants that showed the desired attributes they had not been able to obtain by other directed-evolution methods.
Chapter 36 Directed Protein Evolution 651
3.1.4. DNA Shuffling + SelectionE. coli β-galactosidase encoded by the lacZ gene is highly specific for β-d-galactosyl substrates and acts only weakly on ONPG (o-nitrophenyl β-d-fucopyranosides), PNPG (p-nitrophenyl β-d-fucopyranosides) or β-d-fucosyl moieties. Although enzymes are very specific in their choice of substrate Zhang et al. changed the specificity of β-galactosidase from β-d-galactosyl substrates to ONPG by only six amino acid exchanges. The resulting enzyme variant preferred ONPG 1,000-fold and PNPG 66-fold over the former β-d-galactosyl substrate (37).
3.1.5. Rational DesignRecombinant proteins are useful as pharmaceuticals and a multi-billion dollar market. These proteins comprise cytokines, growth factors, enzymes, antibod-ies and the number of approved pharmaceutical proteins increases every year. Many of these proteins cause serious side-effects and complications, which limit their clinical usefulness. Frequently companies started to modify proteins to either enhance their therapeutic properties or to facilitate their production.
One antibody used in the treatment of metastatic breast cancer is Herceptin (Genentech/Roche) an anti-HER2 monoclonal antibody. Herceptin was devel-oped from the murine monoclonal antibody 4D5 (38), which specifically inhibits the proliferation of human tumor cells that overexpress the human epidermal growth factor receptor 2 oncoprotein p185Her2. The application of this antibody was severely limited by a human anti-mouse antibody immune response. Thus, antibody 4D5 was humanized using preassembled oligonu-cleotides containing the antigen binding loops from Ab 4D5 and the human variable and IgG constant regions.
3.1.6. ShufflingDengue fever (DF) and the severe dengue haemorrhagic fever (DHF) are the most important arboviral diseases and they are transmitted by the mosquito Aedes aegypti. Up to now no specific treatment for dengue diseases besides supportive intensive care exists. Dengue exists in four serotypes (DEN-1-4) and infection with one serotype only induces resistance against this specific serotype. It is hypothesized that secondary infection with another dengue type can lead to DHF by cross-reaction of antibodies of the dengue type the person is already resistant against. Therefore it is important to have a vaccine that is effective against all four dengue variants (39).
A DNA vaccine consists of a plasmid containing DNA of an infectious organism under a eukaryotic promoter. If such a plasmid is injected into a muscle this leads to the synthesis of the coded protein of the infectious organism. The presence of this protein inside the cell leads to an immune response leading to resistance against this organism. Apt et al. (40) developed such a DNA vaccine that confers immunity against all four dengue types by shuffling of codon optimized dengue envelope genes. The DNA vaccine combines epitopes from all four dengue types. This DNA vaccine was suc-cessfully tested in mice and rhesus macaques (41).
Further application of directed evolution in vaccine development could be recombination of the antigen-coding genes from different serovars to improve immunogenicity or crossprotective range of vaccines.
Bioremediation is the process of using microorganisms to degrade danger-ous chemicals from the environment. Bioremediation is the fastest developing
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area of environmental restoration. Microorganisms can be used to degrade various chemicals such as hydrocarbons, polychlorated biphenyls, pesticides, metals and much more. By engineering enzymes and metabolic pathways it is possible to enlarge the substrate range that can be metabolized and to acceler-ate metabolization (42).
Pentachlorophenol (PCP) is a compound that was used and is still used in developing countries as fungicide, wood preservative and as herbicide. PCP is restricted in the US since 1984 as it can affect the endocrine system of verte-brate life forms and may lead to immune system dysfunction.
PCP can be metabolized by Sphingobium chlorophenolicum, a gram- negative bacterium isolated from PCP contaminated soil. By employing three rounds of genome shuffling Dai et al. (43) developed strains able to grow in presence of 6–8 M PCP whereas the wild-type bacterium only grows up to concentrations of 0.6 mM. These newly developed strains are able to degrade PCP whereas the wild-type does not.
Parathion or diethyl parathion are very potent insecticides and acaricides. Parathion is a cholinesterase inhibitor and can be absorbed through the skin. If incorporated it disrupts neural function by inhibiting the essential enzyme acetylcholinesterase. Cho et al. (44) improved by two rounds of directed evolution with DNA shuffling the hydrolysis of methyl parathion by organo-phosphorus hydrolase (OPH) 25-fold. OPH is a bacterial enzyme that degrades a wide range of neurotoxic organophosphate nerve agents and could be also developed to degrade the chemical weapons sarin and soman.
3.1.7. Family ShufflingCytokines are small molecules that are secreted by cells upon immune stimuli. They play important roles in the regulation and mediation of immu-nity, inflammation, and hematopoiesis. Cytokines possess antiviral and anti-proliferating activities and thus could have therapeutic value in the treatment of diseases. Chang et al. (45) shuffled a family of over 20 human interferon-a-genes (Hu-IFN-α) and screened for variants with antiviral and antiproliferative properties in murine cells. After two rounds of shuffling and selection they obtained clones that where even more active than the native murine IFNαs. The shuffled clones were up to 250 fold more active than the single IFNαs originally used for shuffling.
3.1.8. ITCHYGlutathione acetyltransferases (GST) play an important role in many cellular processes e.g., detoxification by conjugating electrophilic compounds to the tripeptide glutathione (GSH). GSTs are ubiquitous in aerobes and form a superfamily of species-independent classes that share a common protein fold.
Griswold et al. (46) created an ITCHY-library of chimeric enzymes of human GSTθ-1-1 (hGSTTT1-1) and rat GSTθ-2-2 (rGSTT2-2), that only share 54.3% amino acid identity and exhibit different substrate specificities. The ITCHY library gave rise to variants with improved kcat with the substrate used for selection compared to either of the parental enzymes and additionally showed activity on ethacrynic acid, a compound recognized by neither paren-tal enzyme. This combination of a human with nonhuman enzymes to form active chimeras shows that this method could be used for the humanization of proteins with therapeutic values that show no conserved framework allowing for rational grafting.
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3.1.9. SHIPRECCytochromes are proteins that contain heme groups and are responsible for the transport of electrons. P450 is a family of membrane-bound cytochromes with an absorption maximum of 450 nm when complexed with CO. One of the major roles of the cytochrome P450 system is the detoxification of harmful substances.
Sieber et al. (23) produced hybrids of two cytochromes, which share only 16% amino acid sequence identity. They created a library of sequences of the membrane-associated human cytochrome P450 and a soluble bacterial P450 (the heme domain of cytochrome P450 from Bacillus megaterium) with single crossovers all along the aligned genes. Two functional P450 hybrids were selected that showed improved solubility in the bacterial cytoplasm.
3.1.10. Enzyme Truncation + Error prone PCR (+Shuffling)On the first view it seems counterintuitive to truncate an enzyme to enhance its overall stability and activity. Hecky et al. (47,48), however, improved β-lactamase by structural perturbation and compensation. Here, structural per-turbation and compensation means that the enzyme is first truncated N- or C-terminally until in vivo function is abolished and the truncation is compen-sated by directed evolution for activity. Afterwards identified mutations are studied in wild-type background. β-lactamase, the enzyme responsible for resistance against β-lactam antibiotics, was N-terminally truncated by five amino acids. After three rounds of error prone PCR, shuffling und selection they found truncated clones that where more active than the wild type protein. Mutations found in the structural perturbed background were inserted into wild type background resulting an overall stabilization of the protein. These mutations increased thermal stability, chemical stability, activity, and shifted the thermal optimum from 35°C to 50°C while maintaining full activity at low temperatures.
The same principle was true for chloramphenicol acetyltransferase (CAT), which is the protein responsible for inactivation of the antibiotic chloramphen-icol. An N-terminally 10 amino acids truncated CAT variant could be rescued with seven rounds of directed evolution comprising error prone PCR, NExT shuffling, and selection at 37°C (8,9). After insertion of the mutations found into the wild type background the already high melting temperature of 71°C was further increased by 6°C while activity at room temperature was doubled. Furthermore, solubility and chemical stability in guanidine was enhanced (Stebel et al., in preparation).
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