Aromatic Amino Acid Auxotrophs Constructed by RecombinantMarker Exchange in Methylophilus methylotrophus AS1 Cells
Expressing the aroP-Encoded Transporter ofEscherichia coli�†‡
Yurgis A. V. Yomantas, Irina L. Tokmakova, Natalya V. Gorshkova, Elena G. Abalakina,Svetlana M. Kazakova, Evgueni R. Gak, and Sergey V. Mashko*
Ajinomoto-Genetika Research Institute, Moscow 117545, Russian Federation
Received 14 September 2009/Accepted 26 October 2009
The isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolicengineering of obligate methylotrophs, remains a rather complicated task. We describe a novel method ofconstructing mutants of the bacterium Methylophilus methylotrophus AS1 that are auxotrophic for aromaticamino acids. The procedure begins with the Mu-driven integration of the Escherichia coli gene aroP, whichencodes the common aromatic amino acid transporter, into the genome of M. methylotrophus. The resultingrecombinant strain, with improved permeability to certain amino acids and their analogues, was used formutagenesis. Mutagenesis was carried out by recombinant substitution of the target genes in the chromosomeby linear DNA using the FLP-excisable marker flanked with cloned homologous arms longer than 1,000 bp. M.methylotrophus AS1 genes trpE, tyrA, pheA, and aroG were cloned in E. coli, sequenced, disrupted in vitro usinga Kmr marker, and electroporated into an aroP carrier recipient strain. This approach led to the constructionof a set of marker-less M. methylotrophus AS1 mutants auxotrophic for aromatic amino acids. Thus, introduc-tion of foreign amino acid transporter genes appeared promising for the following isolation of desiredauxotrophs on the basis of different methylotrophic bacteria.
The nonpathogenic Gram-negative bacterium Methylophilusmethylotrophus is able to grow efficiently using C1 substrates(methanol, methylamine, or trimethylamine) as the sole sourceof carbon and energy, and it uses the ribulose monophosphatepathway for fixation of formaldehyde produced by the oxida-tion of methanol (36). Methanol has received considerableattention by the fermentation industry as an alternative sub-strate to the more generally used sugars from agriculturalcrops. It can be synthesized either from petrochemicals orrenewable resources, such as biogas (48), and therefore theproduction of methanol does not compete directly with humanfood supplies. Methylotrophs can therefore be considered po-tentially useful strains for industrial biotechnology. M. methylo-trophus AS1 is an obligate methylotroph originally isolatedfrom activated sludge, and it has been deposited in the Na-tional Collections of Industrial, Marine and Food Bacteria(NCIMB; no. 10515). This organism was extensively studied inthe 1970s and has been industrialized on a large scale for themanufacturing of single-cell proteins (SCP) from methanol(56, 63). During that period, a significant amount of researchwas conducted on the direct production of amino acids byfermentation from methanol (3, 58). Although initially prom-ising, these efforts ultimately proved relatively unsatisfactory
and impractical, due primarily to the rather poor set of genetictools that had been developed for methylotrophs.
Over the last 5 years, several genomes of methylotrophshave been sequenced (8, 20, 29, 37, 65, 67), and significantprogress in elucidating their metabolism has been achieved(14). The number of tools available for the genetic and meta-bolic engineering of methylotrophic bacteria has been ex-panded greatly (1, 15, 21, 43), and strategies to produce fineand bulk chemicals by methylotrophs have been described (5,42, 57, 61). All of these factors led to renewed interest in theconstruction of methylotrophic strain producers, and the largerknowledge base has enabled more targeted engineering ofthese bacteria (55).
Although M. methylotrophus AS1 has been extensively stud-ied with regard to the industrial scale production of SCP (56,63) and the oxidation of methanol at the initial stages of car-bon and energy metabolism (13, 28), there has been littlemetabolic analysis of amino acid biosynthesis in this organism.Moreover, selection of auxotrophic mutants of obligate methylo-trophs for broadening convenient genetic tools remains a par-ticularly complicated task (19). Although the isolation of sev-eral auxotrophs for M. methylotrophus AS1 has been described(6, 23, 40), their numbers are limited. Development of differentmethods for the isolation of the mutants did not lead to con-struction of a collection of auxotrophic mutants that couldassist in the investigation of amino acid biosynthetic pathwaysin M. methylotrophus AS1.
As for the L-lysine (Lys) synthesis, systematic research wascarried out by specialists at Ajinomoto Co., Inc., Japan, begin-ning with the investigation of the Lys biosynthetic pathway inM. methylotrophus AS1 (23, 61) and continuing with the con-
struction and improvement of a Lys producer (22, 24, 33, 34).This was followed by optimization of fed-batch fermentationfor overproduction of Lys from methanol (35).
The aim of our investigation was to generate strains basedon M. methylotrophus AS1 with the potential for industrialproduction of aromatic amino acids (AroAAs). It is known thatmutants with relaxed feedback inhibition of key biosyntheticenzymes should be isolated at the initial steps of the construc-tion of the amino acid producers and that the relevant degra-dation pathways should be blocked due to selection of thecorresponding auxotrophic strains (7, 31, 49).
In this study, a novel method for the construction of AroAAauxotrophic mutants of M. methylotrophus AS1 is described.This method is based on the introduction of a foreign geneencoding a specific amino acid transporter into the genome ofM. methylotrophus AS1. The resulting recombinant methylo-trophic strain, which possesses increased permeability to theAroAAs and their analogues, was mutated by recombination-mediated substitution of the target chromosomal genes ofaromatic pathways by a flippase recombinase (FLP)-excisablemarker from artificial linear DNA. This approach led to theconstruction of a set of M. methylotrophus AS1 marker-lessmutants with destroyed genes of AroAA biosynthesis. Thus,introduction of foreign amino acid transporter genes appearedpromising for the following isolation of desired auxotrophs onthe basis of different methylotrophic bacteria.
MATERIALS AND METHODS
Bacterial strains, plasmids, and cultivation conditions. Strains and plasmidsused in the study are shown in Table 1. M. methylotrophus cells were grown at37°C on minimal medium SEIIa (23) of the following composition: 1.9 g/literK2HPO4, 1.56 g/liter NaH2PO4 � 2H2O, 5 g/liter (NH4)2SO4, 200 mg/literMgSO4 � 7H2O, 72 mg/liter CaCl2 � 2H2O, 5 �g/liter CuSO4 � 5H2O, 25�g/liter MnSO4 � 5H2O, 23 �g/liter ZnSO4 � 7H2O, and 9.7 mg/liter FeCl3 �6H2O. Methanol was added to the liquid medium at a final concentration of 2%,or 1% methanol and 1.2% Bacto agar (Difco) were added to create the solid me-dium. Escherichia coli strains were cultured at 37°C in Luria-Bertani (LB) me-dium (52), on LB agar containing 1.2% Bacto agar, or on M9 minimal mediumsupplemented with 0.2% glucose. The following antibiotic concentrations wereused for M. methylotrophus: 100 �g/ml ampicillin (Ap), 2 �g/ml tetracycline (Tc),50 �g/ml streptomycin (Sm), 10 �g/ml kanamycin (Km), and 20 �g/ml chloram-phenicol (Cm). For E. coli strains, the following antibiotic concentrations wereused: 200 �g/ml Ap, 10 �g/ml Tc, and 50 �g/ml Km.
Bacterial mating. Plasmids were transferred into strains of M. methylotrophusAS1 by biparental mating using E. coli S17-1 bearing the respective plasmid asdescribed in reference 1.
DNA manipulation. Plasmid DNA isolation, E. coli transformation, restrictionenzyme digestion, ligation, end-blunting, and Southern hybridization were car-ried out as described by Sambrook and Russell (52). Restriction enzymes, T4DNA ligase, E. coli DNA polymerase Klenow fragment, calf intestine alkalinephosphatase, and Pfu polymerase were obtained from Fermentas (Lithuania),and P1vir phage-mediated generalized transduction experiments were carriedout as described by Miller (44).
PCR. All PCR analyses were performed using Pfu polymerase (Fermentas)with 1 �M of each primer, 200 �M of deoxynucleoside triphosphates, andtypically 200 ng of genomic DNA or 25 ng of plasmid DNA as a template.
Electroporation of M. methylotrophus AS1. The culture of M. methylotrophusAS1 was grown to an optical density at 600 nm of 1.0 in the SEII medium. Cellswere harvested from a 5-ml culture, placed on ice, and washed three times inice-cold distilled water. Finally, cells were resuspended in 100 �l ice-cold water,DNA was added (200 ng for plasmids; 500 ng for linear fragments), and elec-troporation was carried out in a 0.2-cm cuvette at 2.5 kV, 25 �F, and 200 �. Atotal of 1 ml of SEII medium was added, and the cells were grown for 3 h at 37°Cwith vigorous shaking. The electroporation efficiency was approximately 5.2 �104 transformants per 1 �g of plasmid DNA.
Construction of plasmids with aroP and brnQ. The plasmids and primers usedin this work are listed in Table 1 and in Table S1 in the supplemental material,respectively. The E. coli gene aroP (aroPEco) was amplified by PCR using chro-mosomal DNA from E. coli MG1655 as a template and the primers P24 and P25.The 1.9-kb PCR fragment containing aroP was digested with BamHI/EcoRI andligated into the broad-host-range cloning vector pAYCTER3 (1) digested withBamHI/EcoRI to obtain pAYCTER-aroPEco. The plasmid for integration ofaroP into the chromosome of M. methylotrophus AS1, pMIV5-[FRT-Kmr-FRT]-Mob-aroPEco, was constructed by blunt-end PCR cloning of the Pfu polymerase-amplified fragment containing aroPEco into the integrative plasmid pMIV5-[FRT-Kmr-FRT]-Mob (1), which was partially digested with PvuII (since two sitesfor PvuII are present in pMIV5-[FRT-Kmr-FRT]-Mob) and dephosphorylatedwith calf intestine alkaline phosphatase.
The E. coli gene brnQ was amplified using chromosomal DNA from E. coliMG1655 as a template and the primers P26 and P27. The 1.7-kb PCR fragmentcontaining brnQEco was digested with SmaI and ligated into the same restrictionsite as pAYCTER3 to create pAYCTER3-brnQEco.
Cloning of the genes from AroAA biosynthesis pathways of M. methylotrophusAS1. Chromosomal DNA of M. methylotrophus AS1 was partially digested withBsp143I (Sau3AI), and fragments greater than 2 kb were purified from agarosegels and ligated into the BglII site of pPW121 (51). The BglII restriction site islocated in the vector plasmid inside a specially modified structural part of the �cIrepressor gene. Cloning into this site leads to Tcr gene expression driven by thederepressed �PR-promoter, providing direct selection for the inserts. The result-ing ligation mixture was used to transform the appropriate E. coli mutant strainand select Tcr clones with complemented mutation. The plasmid DNA wasisolated from such clones, and after confirmation of the complementation eventby retransformation to the appropriate E. coli strain, the mutant strain wasfurther sequenced. The plasmids pPW-(trpEGDCMme), pPW-(tyrAMme), andpPW-(pheA-hisH-aroGMme) containing 5,227-bp, 2,445-bp, and 4,689-bp Bsp143I(Sau3AI) fragments, respectively, with genes for biosynthesis of AroAAs (Fig. 1)were selected using this approach.
Construction of plasmids and linear DNA fragments for mutagenesis. Toinactivate trpEMme, an FLP-excisable Kmr marker was amplified using Pfu poly-merase, primers P1 and P2 (see Table S1 in the supplemental material), andpKD4 as a template. The resultant 1.5-kb fragment carrying FRT-Kmr-FRT wasligated into the NcoI site of pPW-(trpEGDCMme) that had been blunted bythe Klenow fragment of E. coli DNA polymerase, and the plasmid pPW-(trpEGDCMme)-Km was constructed. A linear DNA fragment obtained afterdigestion of pPW-(trpEGDCMme)-Km by BamHI, and Eam1104I was used fortrpEMme mutant construction.
To inactivate tyrAMme, the 1.5-kb FRT-Kmr-FRT carrier fragment was ampli-fied from pKD4 by PCR analysis using primers P5 and P2 and ligated into theSwaI site of pPW-(tyrAMme). The resultant plasmid, pPW-(tyrAMme)-Km, wasdigested with SmaI and BamHI to obtain a linear fragment for the tyrAMme
mutant construction. The primer pairs P28/P29 and P30/P31 were used to am-plify the linear fragments containing FRT-Kmr-FRT flanked with “arms” ofdifferent lengths from pPW-(tyrAMme)-Km as a template in order to optimize themutant construction procedure.
To inactivate pheAMme, the linear fragment containing FRT-Kmr-FRT flankedwith arms of 1,000 bp in length was constructed by overlap extension PCR (18,66). The primers P8 and P9 were used for left arm amplification, P10/P11 wereused to amplify the right arm, and P12/P13 were employed for FRT-Kmr-FRTamplification. The primers P8 and P10 contain 21-nt regions at their 5� extrem-ities that are homologous to the 3� and 5� terminals of the FRT-Kmr-FRTmarker. The reaction was carried out for 30 cycles as follows: denaturation at95°C for 30 s, primer annealing at 58°C for 45 s, and primer extension at 72°C for2 min. Then, 50-ng samples of the left and right arm were used as primers, and50 ng of the fragment containing FRT-Kmr-FRT was used as a template toamplify the entire fragment. The reaction was carried out as follows: denatur-ation at 95°C for 1 min, primer annealing at 55°C for 1 min, and primer extensionat 72°C for 3 min.
For aroGMme, the left arm was amplified using the primers P14/P15, the rightarm was amplified using the primers P16/P17, and FRT-Kmr-FRT was amplifiedusing the primers P18/P19. Primers P14 and P16 contain 18- and 19-nt regions,respectively, at their 5� extremities that are homologous to the 5� and 3� terminalsof the FRT-Kmr-FRT marker. The reaction was carried out as follows: denatur-ation at 95°C for 30 s, primer annealing at 48°C for 45 s, and primer extension at72°C for 2 min. Finally, 50 ng of each resulting fragment was mixed together, andP15 and P17 were used for PCR amplification of the entire fragment. Thereaction was carried out as follows: denaturation at 95°C for 1 min, primerannealing at 48°C for 1 min, and primer extension at 72°C for 6 min.
Sequencing. The nucleotide sequencing of both strands was performed usingan ABI Prism 3100 genetic analyzer with a BigDye Terminator v3.1 cycle se-quencing kit (both from Applied Biosystems) according to the manufacturer’sinstructions. DNA sequencing data were analyzed using the Methylobacillusflagellatus KT genome database provided by GenBank (http://www.ncbi.nlm.nih.gov/nuccore/CP00284), and nucleotide and protein data were analyzed usingBLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Elimination of FRT-Kmr-FRT marker from the chromosome. pFLP31 wastransferred into strain M. methylotrophus with the FRT-Kmr-FRT marker in thechromosome by mobilization (using plasmid-encoded Apr as the selectivemarker), and the Kmr Apr colonies were isolated. The isolated colonies weresuspended at a final concentration of up to 107 cells/ml in 5 ml of SEIIa with Apliquid medium, and the suspension was heated at 42°C for 20 min, followed byincubation in a shaker at 37°C for 16 to 18 h to induce FLP recombinasesynthesis. The culture was then plated on nonselective SEIIa medium to obtain
individual colonies. These were analyzed for the occurrence of the Km and Apmarkers on the solid SEIIa medium in the presence of the appropriate antibiotic.In a typical experiment, approximately 80% of the resulting colonies were Kms
due to the FLP-mediated excision of the FRT-Kmr-FRT marker.Nucleotide sequence accession numbers. All determined nucleotide sequences
were deposited in the GenBank/EMBL/DDBJ databases. The accession numbersare as follows: GQ184460 for (trpEGDC)Mme, GQ184461 for tyrAMme, andGQ184459 for (pheA-hisH-aroG)Mme.
RESULTS
Unsuccessful attempts to isolate auxotrophic mutants of M.methylotrophus AS1 after MNNG treatment. Auxotrophic mu-tants have been difficult to isolate from obligate methylotrophs
TABLE 1. Strains and plasmids used in this study
Strain or plasmid Relevant characteristic(s) Source orreference
StrainsM. methylotrophus
AS1 Wild type NCIMB 10515AS1::aroPEco Modified strain AS1 containing the E. coli gene aroP integrated in the
chromosomeThis study
AS1::aroPEco-trpEMme::FRT TrpE mutant This studyAS1::aroPEco-tyrAMme::FRT TyrA mutant This studyAS1::aroPEco-pheAMme::FRT PheA mutant This studyAS1::aroPEco-aroGMme::FRT AroG mutant This studyAS1::aroPEco-trpEMme::FRT-
tyrAMme::FRTTrpE TyrA double mutant This study
E. coliTG1 F� �(lac-pro) supE thi hsd�5 �F� traD36 proAB lacIq lacZ�M15 VKM IMG-341S17-1 Tpr Smr; F� recA pro thi hsdR� hsdM RP4-2-Tc::Mu-Km::Tn7 ATCC 47055MG1655 F� �� ilvG rfb-50 rph-1 CGSC 6300JW1256 BW25113�trpE::kan 4JW2581 BW25113�tyrA::kan 4JW2580 BW25113�pheA::kan 4JW0737 BW25113�aroG::kan 4JW2582 BW25113�aroF::kan 4JW1694 BW25113�aroH::kan 4BW25113-�aroGFH BW25113�aroG �aroF �aroH with FLP-mediated Kmr excision This studyJW1256-�trpE JW1256 with FLP-mediated Kmr excision This studyJW2581-�tyrA JW2581 with FLP-mediated Kmr excision This studyJW2580-�pheA JW2580 with FLP-mediated Kmr excision This study
PlasmidspAYCTER3 Apr; IncQ broad-host-range cloning vector derived from pAYC32 1pAYCTER-aroPEco pAYCTER3 with the 1.9-kb BamHI-EcoRI PCR fragment containing the E. coli
gene aroPThis study
pAYCTER-brnQEco pAYCTER3 with the 1.7-kb SmaI PCR fragment containing the E. coli genebrnQ
This study
pMIV5-�FRT-Kmr-FRT-Mob Apr Kmr; pMIV5-Mob with the 1.5-kb HindIII-NdeI fragment from pKD4containing the Kmr gene-flanked FRT sites
1
pMIV5-�FRT-Kmr-FRT-Mob-aroPEco pMIV5-�FRT-Kmr-FRT- Mob with 1.9-kb PCR fragment containing the E. coligene aroP
This study
pFLP31 Apr Smr; pAYCTER3 with the 3.3-kb SmaI-BamHI fragment from pCP20containing the genes �cI857ts �cro-FLP
1
pPW121 Cloning vector 51pTP310 Tcr; pRK310 with the 5.7-kb BamHI fragment from pUC-MuAB containing the
genes MuAB ner cts1
pKD4 Kmr Apr; pANTS��FRT-Kmr-FRT 16pPW-(trpEGDC)Mme pPW121 with 5.2-kb Sau3A fragment containing M. methylotrophus genes
trpEGDCThis study
pPW-(tyrAMme) pPW121 with 2.4-kb Sau3A fragment containing the M. methylotrophus gene tyrA This studypPW-(pheA-hisH-aroG)Mme pPW121 with 4.6-kb Sau3A fragment containing M. methylotrophus genes pheA
hisH aroGThis study
VOL. 76, 2010 AROMATIC AUXOTROPH OF M. METHYLOTROPHUS WITH aroPEco 77
and particularly M. methylotrophus AS1 using standard meth-ods of mutagenesis (6, 19, 46). We attempted various formsof mutagenesis using N-methyl-N�-nitro-N-nitrosoguanidine(MNNG), not only the standard methods that are typicallyused for E. coli cells (44) but also methods with some specificmodifications described previously for M. methylotrophus AS1(23, 40) and for Methylobacillus flagellatus KT (62). No auxo-trophs deficient in AroAA biosynthesis were found among20,000 clones of the M. methylotrophus AS1 strain that weretreated with MNNG in the same manner. One of the explana-tions of that failure was that corresponding AroAAs added tothe medium might not permeate the bacterial cytoplasmicmembrane to allow sufficient mutant growth (19, 25, 59).
To test this hypothesis, we carried out a set of experimentsto determine the natural resistance of M. methylotrophus AS1to such toxic compounds as analogs of AroAAs. It is wellknown that the addition of a high concentration of some aminoacids (e.g., L-valine [Val]) can be detrimental to the growth ofthe strain due to the feedback inhibition of key enzymes par-ticipating in the biosynthesis of related amino acids (17). Thus,we tested the ability of M. methylotrophus AS1 to grow withexcess Val in the medium.
During our experiments, we found that M. methylotrophusAS1 had a basal level of resistance to the 5-methyltryptophanthat was significantly higher (more than a hundredfold) thantypical for E. coli strains. Moreover, M. methylotrophus AS1could grow in the presence of high concentrations of Val (upto 5 g/liter) that significantly exceeded values inhibitory forother Gram-negative bacteria. These results indirectly con-firmed suggestions regarding low permeability of the cyto-plasmic membrane of M. methylotrophus AS1 to amino acidsand their analogs.
Introduction of E. coli aroP increased the sensitivity of M.methylotrophus AS1 to analogs of AroAAs. We next introduceda recombinant plasmid with the E. coli gene aroP (aroPEco) inan attempt to increase the permeability of the AS1 membraneto AroAAs. As a member of the APC superfamily of trans-porters (69), AroPEco is the permease that transports Phe, Tyr,and Trp (11) across the inner membranes of E. coli bacteriausing the proton motive force mechanism (10).
As a control, another E. coli gene, brnQ, was used. BrnQEco
has a highly similar ortholog in Salmonella enterica serovarTyphimurium that belongs to the LIVCS family and is thoughtto function as a sodium/branched-chain amino acid (BCAA)symporter (47). BrnQEco likely corresponds to the LIV-IIBCAA transport system, which has been shown to transportLeu, Val, and Ile in E. coli (2).
The pAYCTER-aroPEco and pAYCTER3-brnQEco plasmidswere constructed and mobilized into M. methylotrophus AS1(see Materials and Methods). M. methylotrophus AS1 andtwo of its plasmid carrier derivatives possessing different E.coli transporter genes were plated on methanol-containingsolid medium supplemented with increased concentrationsof 5-methyltryptophan and Val as bacterial growth inhibitors(Table 2). The presence of aroPEco dramatically increased thesensitivity of M. methylotrophus cells to 5-methyltryptophan buthad no influence on the high level of Val resistance. In con-trast, brnQEco carrier methylotrophic cells clearly manifestedVal sensitivity in comparison to the control strain while retain-ing high resistance to the aromatics. Introduction of E. coliamino acid transporters therefore likely increased the perme-ability of the M. methylotrophus inner membrane for the cor-responding amino acids or their analogs. This approach ap-peared promising for construction of auxotrophic mutants.
We constructed a special recipient strain of M. methylotro-phus AS1 with aroPEco inserted into the bacterial chromosome.
FIG. 1. Genetic organization of the Bsp143I (Sau3AI) fragments containing the genes from aromatic amino acid biosynthesis pathways ofMethylophilus methylotrophus AS1 cloned in this work. The 5,227-bp Bsp143I fragment contains trpE, trpG, trpD, cds1, and trpC. The 4,689-bpBsp143I fragment contains the 3� part of serC, pheA, hisH, aroG, and the 5� part of pqqB. The 2,445-bp Bsp143I fragment contains tyrA and the5� part of aroA. The genes are shown as arrows, and the partial ORFs located on the fragment are indicated with apostrophes. The sites forrestrictases used for gene disruption and subcloning are shown.
TABLE 2. Resistance of M. methylotrophus AS1 strains to 5-methyl-tryptophan and Vala
M. methylotrophusstrain
Growth in the presence ofb:
5-Methyltryptophan(mg/liter) Val (g/liter)
0 5 10 50 0 1 2.5 5
AS1 pAYCTER3 AS1 pAYCTER-
aroPEco
� � �
AS1::aroPEco � � � AS1 pAYCTER-
brnQEco
� � �
a M. methylotrophus AS1 strains were grown on solid SEIIa medium.b , growth; �, no growth.
The plasmid pMIV5-[FRT-Kmr-FRT]-Mob-aroPEco carryingthe aroPEco gene and FLP-excisable Kmr marker in themini-Mu unit was used to integrate aroP into the M. methylo-trophus AS1 chromosome using the method of Abalakina et al.(1) based on the phage Mu-driven integration system. Thepresence of one copy of the integrated mini-Mu unit in thechromosome was confirmed by PCR and Southern hybrid-ization (results not shown). The Kmr marker was excised byFLP recombinase as previously described (1), and one of themarker-less Mu integrants that retained sensitivity to5-methyltryptophan (Table 2) was thereafter referred to asthe AS1::aroPEco strain.
Cloning of trpEGDMme genes via functional complementa-tion of the E. coli trpE mutant and inactivation of trpE inAS1::aroPEco. For the cloning of different M. methylotrophusAS1 aromatic pathway genes, corresponding E. coli strainsfrom the Keio collection were used, including in-frame, single-gene knockout mutants for all nonessential genes of E. coliK-12 (4).
Strain JW1256 from this collection carried the FLP-excis-able Kmr marker inserted into and replacing the central part ofthe first gene from the trpEDCBAEco operon. The Kmr markerwas excised, and the resulting E. coli JW1256-�trpE strain wasused as the recipient for isolation of trpEMme by complemen-tation of auxotrophicity. The selected Tcr, Trp transformantscontained the plasmid pPW-(trpEGDC)Mme with the inserted5.2-kb DNA fragment (Fig. 1). The nucleotide sequence anal-
ysis of this fragment revealed three directly aligned openreading frames (ORFs) encoding proteins that had a highlevel of homology (77.3% identity for TrpE, 83.3% forTrpG, and 85.5% for TrpD) with the known TrpEGD sub-units of anthranilate synthase, glutamine amidotransferase,and/or anthranilate phosphoribosyl transferase (EC 4.1.3.27/2.4.2.18) of Methylobacillus flagellatus KT.
trpEMme was disrupted by inserting a 1.5-kb DNA fragmentcontaining FRT-Kmr-FRT amplified from pKD4 (16). This in-sertion was designed such that after integration into the M.methylotrophus AS1::aroPEco chromosome and FLP-mediatedexcision of Kmr, the “scar” sequence with the residual FRT sitewould inactivate the trpEMme gene by in-frame insertion,thereby likely preventing polar effects (Fig. 2A).
M. methylotrophus AS1::aroPEco cells were electroporated withthe linear DNA fragment containing trpEMme::[FRT-Kmr-FRT],with more than 2 kb of the methylotrophic DNA flanking themarker at both ends. Kmr clones were then selected on methanol-containing medium supplemented with 100 mg/liter Trp. As acontrol, the standard AS1 strain was used as the recipient. Ap-proximately 100 Kmr clones auxotrophic for Trp were selected ina typical experiment with the AS1::aroPEco strain. We were un-able to obtain any recombinant Kmr clones under the same con-ditions when the control strain was used. PCR analysis confirmedthe inactivation of trpEMme due to recombination-mediatedmarker exchange in the chromosomes of 10 tested Kmr clones(Fig. 2B). The Kmr marker was removed from two clones of M.
FIG. 2. (A) Scheme of the trpE in-frame insertion mutant construction, structure of scar sequence, and in-frame fusion protein afterFLP-mediated excision of the Kmr gene. Positions of the primers (P1 and P2) used for PCR amplification of Kmr from pKD4 and of the primers(P3 and P4) used for verification of the mutation are indicated with arrows. (B) PCR verification of the trpE mutation is as follows: lane 1, DNAladder; lane 2, PCR of genomic DNA of M. methylotrophus AS1::aroPEco with primers P3/P4; lane 3, PCR of genomic DNA of M. methylotrophusAS1::aroPEco-(trpEMme::[FRT-Kmr-FRT]) with primers P3/P4; lane 4, PCR of genomic DNA of M. methylotrophus AS1::aroPEco-trpEMme::FRT withprimers P1/P4.
VOL. 76, 2010 AROMATIC AUXOTROPH OF M. METHYLOTROPHUS WITH aroPEco 79
methylotrophus AS1::aroPEco-(trpEMme::[FRT-Kmr-FRT]) usingan FLP recombinase expression plasmid. Eighty percent of theresulting clones were Kms. PCR analysis confirmed the elimina-tion of the Kmr marker and the presence of the 75-bp scar se-quence in trpEMme in the chromosomes of 10 tested Kms clones(Fig. 2). All obtained Kms clones remained auxotrophic for Trp.
In summary, we were able to obtain the stable marker-lessauxotrophic strain of M. methylotrophus AS1::aroPEco-trpEMme::FRT using (i) a specially constructed strain with thearoPEco transporter gene in the chromosome, (ii) linear DNAcarrying trpEMme that was inactivated by excisable Kmr, (iii) re-combination-mediated marker exchange between electroporatedDNA synthesized by PCR and the bacterial chromosome, and (iv)FLP-mediated marker curing.
Cloning and inactivation of a set of genes involved in aro-matic pathways and optimization of experimental procedures.The same approach was used for the cloning and inactivationof other genes from aromatic pathways, and construction ofauxotrophic mutants was based on the AS1::aroPEco strain.DNA fragments carrying tyrAMme, pheAMme, and aroGMme en-coding chorismate mutase/prephenate dehydrogenase (EC1.3.1.12/5.4.99.5), chorismate mutase/prephenate dehydratase(EC 4.2.1.51/5.4.99.5), and 3-deoxy-D-arabino-heptulosonate7-phosphate synthase (DAHPS) (EC 2.5.1.54) were initiallyselected from the library of M. methylotrophus AS1 genes bycomplementation of the corresponding mutations from E. coliK-12 strains.
The Kms variant of JW2581-�tyrAEco from the Keio collec-tion (4) was used for tyrAMme identification. The plasmid pPW-(tyrAMme) complemented the �tyrAEco mutation and containeda 2.4-kb fragment (Fig. 1). An ORF that is highly homologousto TyrA from M. flagellatus KT (60.4% identity) was found inthis fragment and disrupted by the insertion of the excisableKmr marker. This was followed by electroporation of the linearDNA fragment into M. methylotrophus AS1::aroPEco. Kmr
clones that could grow on methanol-containing medium sup-plemented with 100 mg/liter Tyr were selected, and this led tothe isolation of the tyrAMme::[FRT-Kmr-FRT] mutant. The dis-rupted structure of the gene was confirmed by PCR with P5/P2and P6/P7 primer pairs (data not shown).
The mutant construction procedure was optimized using thetyrAMme::[FRT-Kmr-FRT] linear DNA fragments, with themarker flanked by homologous DNA arms of different lengths.More than 10 of the desired clones were obtained per trialwhen the areas homologous to both target arms around theKmr marker were �1,000 bp.
It was possible to design the linear DNA fragments formutant construction using overlap extension PCR (26) withtarget arms of �1,000 bp. We later used this strategy for theconstruction of the pheAMme and aroGMme mutants (see be-low).
A double-auxotrophic M. methylotrophus AS1::aroPEco-trpEMme::FRT-tyrAMme::[FRT-Kmr-FRT] strain was also con-structed. The disrupted tyrAMme-carrying DNA fragment waselectroporated into the marker-less trpEMme mutant strain de-scribed above. Kmr clones grown on the methanol-containingmedium supplemented with 100 mg/liter each of Trp and Tyrwere then isolated.
pheAMme was isolated using E. coli JW2580-�pheAEco as therecipient strain for complementation. One of the recombinant
plasmids from the Tcr, Phe-selected clones carried a ratherlarge (4.7-kb) DNA fragment that contained three directlyaligned ORFs according to sequence analysis (Fig. 1). Thesewere homologous to PheA, HisH, and AroG from M. flagella-tus KT (63.4%, 71.9%, and 73.5% identity, respectively), wherethe corresponding genes were located in different parts of thebacterial genome.
In addition to pheAMme, the putative aroGMme gene thatlikely encodes DAHPS attracted our attention because its or-tholog, AroGEco, regulates the carbon flux into the generalaromatic pathway in E. coli (50). Moreover, only one gene forDAHPS, aroG, was annotated in the M. flagellatus KT genome.To test whether the putative gene indeed had aroGMme
function, a special E. coli strain was constructed. It is knownthat E. coli K-12 has three genes corresponding to theDAHPS isoenzymes aroGEco, aroFEco, and aroHEco; thesegenes code for proteins that are sensitive to feedback inhibition byPhe, Tyr, and Trp, respectively (50). Three strains from theKeio collection, JW0737 (�aroGEco::[FRT-Kmr-FRT]), JW2582(�aroFEco::[FRT-Kmr-FRT]), and JW1694 (�aroHEco::[FRT-Kmr-FRT]), were used to construct a new marker-less, DAHPS-negative strain. We removed the Kmr marker from the chromo-some of the initial recipient, JW0737, with consequent P1 ductionof two marked deletions from other strains. At each step,FLP-mediated marker excision finalized the stepwise processof chromosomal rearrangements. The resulting strain,BW25113-�aroGFHEco, could grow on minimal medium sup-plemented with glucose, all AroAAs, and p-aminobenzoic acid(PABA). The plasmid pPW-(pheA-hisH-aroG)Mme carryingthe DNA fragment from the putative operon restored thegrowth of E. coli BW25113-�aroGFHEco on the minimal me-dium containing glucose; this was likely due to compensationof absent E. coli DAHPS function by the expression ofaroGMme.
PCR-mediated overlap extension was used to construct twolinear DNA fragments that were electroporated into M. methylo-trophus AS1::aroPEco for inactivation of the pheAMme oraroGMme gene in the chromosome. For the electroporatedDNA fragment containing �pheAMme::[FRT-Kmr-FRT], Kmr
clones were obtained only on selective medium containingmethanol supplemented with all AroAAs and PABA. Wecould not obtain any clones when Phe was the sole aromaticsupplement. We propose that the polar effect of pheAMme
disruption could have led to untimely transcription terminationof the corresponding operon with aroGMme as the distal cis-tron. When the Kmr marker was excised, the residual scarrestoredtheORFin�pheAMme::FRT, andtheresultingmethylo-trophic strain AS1::aroPEco-�pheAMme::FRT could grow onmethanol-containing medium supplemented with 200 mg/literPhe alone.
The DNA fragment with �aroGMme::[FRT-Kmr-FRT] wasused for the construction of an auxotrophic mutant that couldgrow only when all essential aromatics were in the medium.Kmr removal did not change the nutrient demands of themarker-less strain. These results demonstrate unambiguouslythat only one gene encoding DAHPS, aroGMme, was present inthe M. methylotrophus AS1 genome, as had also been notedearlier for M. flagellatus KT.
Thus, by using M. methylotrophus AS1::aroPEco as the recip-ient, a set of auxotrophic mutants that possess disrupted
AroAA pathways was obtained using the optimized experi-mental procedure.
DISCUSSION
The isolation of auxotrophic mutants remains a rather com-plicated task for molecular genetic analysis of obligate methylo-trophs (19). Various contradictory explanations have beenoffered for the failure of the mutagenesis of certain methylo-trophic bacteria. Different groups have proposed either ex-tremely high toxicity of the mutagens used or the failure ofmutagens to permeate the cells (27). At the same time, a largegroup of experiments has directly or indirectly indicated thatthe inability of nutrient substances to permeate the methylo-trophic membrane was the main factor that hampered theisolation of the desired auxotrophs using selective medium (19,68). Indeed, the absence of intracellular transport of radioac-tively labeledGluwasconvincinglydemonstrated forM.methylo-trophus AS1 (68).
It is therefore not surprising that successful auxotrophicmutant isolation was obtained when changes in bacterial mem-branes occurred due to unknown mutations. It is likely thatthese changes took place when the strains became resistant toantibiotics (40) or when the selection of auxotrophic mutantsyielded mutants that were able to take up hydrophobic BCAAat high concentrations in the selective medium (62).
It is typically assumed that AroAAs can efficiently penetratethe bacterial cell membrane, as they are hydrophobic (41).Although their passive permeability is high, bacteria usuallyhave additional uptake systems. Gram-negative E. coli possessthe AroPEco system. This common transporter accepts all threeAroAAs, as well as several substrate-specific transport systems(Mtr, TnaB, TyrP, PheP) (31, 50). A common AroP system forall AroAA uptake has been described for Gram-positive bac-teria, in particular Corynebacterium glutamicum (30). At thesame time, there are no indications concerning the presence ofspecific amino acid transporter genes in the known genomes ofobligate methylotrophs.
Indeed, both significantly increased resistance of methylo-trophic bacteria to 5-methyltryptophan and resistance to highconcentrations of Val (which usually inhibits the growth ofGram-negative bacteria) were detected in our experiments.This resistance dramatically decreased after the introductionof the E. coli transporter genes aroP and brnQ in methylotro-phic cells. Here, we detected specificity of the heterologousproteins for transported substrates: AroPEco had decreasedresistance only to analogs of AroAAs, while BrnQEco haddecreased resistance only to Val.
Many E. coli genes are able to be expressed in M. methylo-trophus AS1 under the control of their native regulatory re-gions (1). We therefore anticipated the possible transcriptionof aroPEco and brnQEco in these methylotrophic cells and didnot carry out preliminary modifications of gene transporterpromoters, as this likely had to be done in another bacterialhost. Nevertheless, the fact that the protein products of thecloned genes acted as specific transporters integrated in theheterologous bacterial membrane could not have been pre-dicted in advance and was of experimental use.
We therefore recommend the introduction of foreign aminoacid transporter genes for the isolation of other desired methylo-
trophic auxotrophs. It seems possible to detect the functionalactivity of the foreign transporter by observing decreased re-sistance in the resulting recombinant strains to the structuralanalogs of the corresponding amino acids. The approach de-veloped in the present study may therefore be used for theisolation of mutants from different organisms.
The construction of mutants by recombination-mediatedmarker exchange was not original per se and has been used fordifferent methylotrophs in addition to M. methylotrophus AS1(6). This method included the introduction of suicide plasmidDNA that could not replicate in the corresponding bacterialhost and the substitution of the plasmid marker for the corre-sponding target in the bacterial chromosome. If the selectionpressure was absent, however, it was difficult to discriminatethe rare clones that carried the desired substitutions from themajority of variants that obtained the marker via single-cross-mediated integration of the whole suicide plasmid into thetarget gene. Use of linear DNA for marker exchange increasedthe selectivity of the isolation of the desired recombinantsbecause the marker could be inserted into the chromosomeonly through double-cross-mediated recombination.
Under standard conditions, linear DNA introduced into E.coli is degraded by the powerful RecBCD nuclease, and there-fore it is necessary to either use specially constructed recipientstrains, i.e., recBC and sbcB mutants (for an example, seereference 45), or inhibit the RecBCD and SbcCD nucleaseusing the � Gam protein, thereby preserving linear DNA andallowing it to be used as a substrate for recombination (re-viewed in references 12 and 53). The introduction of DNA intobacterial cells by electroporation, however, renders the prelim-inary inactivation of the RecBCD and SbcCD nuclease unnec-essary for successful participation of the introduced linearDNA in recombination-mediated rearrangements of the bac-terial chromosome, not only in E. coli but also in differentGram-negative bacteria (9, 38, 39).
In the present study, electroporation was used to allow re-combination-mediated insertion of the linear DNA fragmentinto the chromosome of M. methylotrophus AS1. It was shownthat a sufficient number of recombinant clones for geneticanalysis could be isolated using standard experimental condi-tions if the length of the flanked homology around the selectivemarker and its target gene in the bacterial chromosome was�1,000 bp. Thus, construction of a linear DNA fragment forfurther insertion could be achieved in vitro using a PCR-me-diated overlap extension procedure.
The synergy of the two experimentally achieved results, (i)obtaining the specialized recipient carrying the foreign trans-porter gene in its genome and (ii) optimization of the linearDNA fragment constructed for insertion mutagenesis, finallyled to isolation of M. methylotrophus AS1-based auxotrophicmutants with disrupted AroAA biosynthetic pathways. Thesemutants and the methods that we developed for their construc-tion were used later for the design of strains that overproducePhe from methanol (32, 60; Yomantas et al., unpublisheddata).
REFERENCES
1. Abalakina, E. G., I. L. Tokmakova, N. V. Gorshkova, E. R. Gak, V. Z.Akhverdyan, S. V. Mashko, and Y. A. V. Yomantas. 2008. Phage Mu-driventwo-plasmid system for integration of recombinant DNA in the Methylophi-lus methylotrophus genome. Appl. Microbiol. Biotechnol. 81:191–200.
VOL. 76, 2010 AROMATIC AUXOTROPH OF M. METHYLOTROPHUS WITH aroPEco 81
2. Anderson, J. J., and D. L. Oxender. 1978. Genetic separation of high- andlow-affinity transport systems for branched-chain amino acids in Escherichiacoli K-12. J. Bacteriol. 136:168–174.
3. Anthony, C. 1982. The commercial exploitation of methylotrophs, p. 328–348. In The biochemistry of methylotrophs. Academic Press, London, UnitedKingdom.
4. Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. Dat-senko, M. Tomita, B. L. Wanner, and H. Mori. 2006. Construction of Esch-erichia coli K-12 in-frame, single-gene knockout mutants: the Keio collec-tion. Mol. Sys. Biol. 2:2006.0008.
5. Belanger, L., M. M. Figueira, D. Bourque, L. Morel, M. Beland, L. Laramee,D. Groleau, and C. B. Míguez. 2004. Production of heterologous protein byMethylobacterium extorquens in high cell density fermentation. FEMS Micro-biol. Lett. 231:197–204.
6. Bohanon, M., C. Bastien, R. Yoshida, and R. Hanson. 1988. Isolation ofauxotrophic mutants of Methylophilus methylotrophus by modified-markerexchange. Appl. Environ. Microbiol. 54:271–273.
7. Bongaerts, J., M. Kramer, U. Muller, L. Raeven, and M. Wubbolts. 2001.Metabolic engineering for microbial production of aromatic amino acids andderived compounds. Metab. Eng. 3:289–300.
8. Chistoserdova, L., A. Lapidus, C. Han, L. Goodwin, L. Saunders, T. Brettin,R. Tapia, P. Gilna, S. Lucas, P. M. Richardson, and M. E. Lidstrom. 2007.Genome of Methylobacillus flagellatus, molecular basis for obligate methylo-trophy, and polyphyletic origin of methylotrophy. J. Bacteriol. 189:4020–4027.
9. Choi, K. H., A. Kumar, and H. P. Schweizer. 2006. A 10-min method forpreparation of highly electrocompetent Pseudomonas aeruginosa cells: ap-plication for DNA fragment transfer between chromosomes and plasmidtransformation. J. Microbiol. Methods 64:391–397.
10. Cosgriff, A. J., G. Brasier, J. Pi, C. Dogovski, J. P. Sarsero, and A. J. Pittard.2000. A study of AroP-PheP chimeric proteins and identification of a residueinvolved in tryptophan transport. J. Bacteriol. 182:2207–2217.
11. Cosgriff, A. J., and A. J. Pittard. 1997. A topological model for the generalaromatic amino acid permease, AroP, of Escherichia coli. J. Bacteriol. 179:3317–3323.
12. Court, D. L., J. A. Sawitzke, and L. C. Thomason. 2002. Genetic engineeringusing homologous recombination. Annu. Rev. Genet. 36:361–388.
13. Cox, J. M., D. J. Day, and C. Anthony. 1992. The interaction of methanoldehydrogenase and its electron acceptor, cytochrome cL in methylotrophicbacteria. Biochim. Biophys. Acta 1119:97–106.
14. Crowther, G. J., G. Kosaly, and M. E. Lidstrom. 2008. Formate as the mainbranch point for methylotrophic metabolism in Methylobacterium extorquensAM1. J. Bacteriol. 190:5057–5062.
15. Cue, D., H. Lam, R. L. Dillingham, R. S. Hanson, and M. C. Flickinger. 1997.Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotol-erant methylotroph. Appl. Environ. Microbiol. 63:1406–1420.
16. Datsenko, K., and B. L. Wanner. 2000. One-step inactivation of chromo-somal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad.Sci. U. S. A. 97:6640–6645.
17. De Felice, M., M. Levinthal, M. Iaccarino, and J. Guardiola. 1979. Growthinhibition as a consequence of antagonism between related amino acids:effect of valine in Escherichia coli K-12. Microbiol. Rev. 43:42–58.
18. Derbise, A., B. Lesic, D. Dacheux, J. M. Ghigo, and E. Carniel. 2003. A rapidand simple method for inactivating chromosomal genes in Yersinia. FEMSImmunol. Med. Microbiol. 38:113–116.
19. de Vries, G. E., U. Kues, and U. Stahl. 1990. Physiology and genetics ofmethylotrophic bacteria. FEMS Microbiol. Rev. 75:57–102.
20. Giovannoni, S. J., D. H. Hayakawa, H. J. Tripp, U. Stingl, S. A. Givan, et al.2008. The small genome of an abundant coastal ocean methylotroph. Envi-ron. Microbiol. 10:1771–1782.
21. Gliesche, C. G. 1997. Transformation of methylotrophic bacteria by electro-poration. Can. J. Microbiol. 43:197–201.
22. Gunji, Y., H. Ito, H. Masaki, and H. Yasueda. 2006. Characterization of aunique mutant lysE gene, originating from Corynebacterium glutamicum,encoding a product that induces L-lysine production in Methylophilus methylo-trophus. Biosci. Biotechnol. Biochem. 70:2927–2934.
23. Gunji, Y., N. Tsujimoto, M. Shimaoka, Y. Ogawa-Miyata, S. Sugimoto, andH. Yasueda. 2004. Characterization of the L-lysine biosynthetic pathway inthe obligate methylotroph Methylophilus methylotrophus. Biosci. Biotechnol.Biochem. 68:1449–1460.
24. Gunji, Y., and H. Yasueda. 2006. Enhancement of L-lysine production inmethylotroph Methylophilus methylotrophus by introducing a mutant LysEexporter. J. Biotechnol. 127:1–13.
25. Haber, C. L., L. N. Allen, S. Zhao, and R. S. Hanson. 1983. Methylotrophicbacteria: biochemical diversity and genetics. Science 221:1147–1153.
26. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989.Site-directed mutagenesis by overlap extension using the polymerase chainreaction. Gene 15:51–59.
27. Holloway, B. W., P. P. Kearny, and B. R. Lyon. 1987. The molecular geneticsof C1 utilizing microorganisms. Antonie van Leeuwenhoek 53:47–53.
28. Hothi, P., J. Basran, M. J. Sutcliffe, and N. S. Scrutton. 2003. Effects of
multiple ligand binding on kinetic isotope effects in PQQ-dependent meth-anol dehydrogenase. Biochemistry 42:3966–3978.
29. Hou, S., K. S. Makarova, J. H. Saw, P. Senin, B. V. Ly, et al. 2008. Completegenome sequence of the extremely acidophilic methanotroph isolate V4,Methylacidiphilum infernorum, a representative of the bacterial phylum Ver-rucomicrobia. Biol. Direct 3:26.
30. Ikeda, M., and R. Katsumata. 1994. Transport of aromatic amino acids andits influence on overproduction of the amino acids in Corynebacterium glu-tamicum. J. Ferment. Bioeng. 78:420–425.
31. Ikeda, M. 2006. Towards bacterial strains overproducing L-tryptophan andother aromatics by metabolic engineering. Appl. Microbiol. Biotechnol. 69:615–626.
32. Iomantas, Y. V., and E. G. Abalakina. 2002. Method for producing L-phenylalanine. U.S. patent 6350596.
33. Ishikawa, K., T. Asahara, Y. Gunji, H. Yasueda, and K. Asano. 2008. Dis-ruption of metF increased L-lysine production by Methylophilus methylotro-phus from methanol. Biosci. Biotechnol. Biochem. 72:1317–1324.
34. Ishikawa, K., Y. Gunji, H. Yasueda, and K. Asano. 2008. Improvement ofL-lysine production by Methylophilus methylotrophus from methanol via theEntner-Doudoroff pathway, originating in Escherichia coli. Biosci. Biotech-nol. Biochem. 72:2535–2542.
35. Ishikawa, K., Y. Toda-Murakoshi, F. Ohnishi, K. Kondo, T. Osumi, and K.Asano. 2008. Medium composition suitable for L-lysine production by Methylo-philus methylotrophus in fed-batch cultivation. J. Biosci. Bioeng. 106:574–579.
36. Jenkins, O., D. Byrom, and D. Jones. 1987. Methylophilus: a new genus ofmethanol-utilizing bacteria. Int. J. Syst. Bacteriol. 37:446–448.
37. Kane, S. R., A. Y. Chakicherla, P. S. Chain, R. Schmidt, M. W. Shin, T. C.Legler, K. M. Scow, F. W. Larimer, S. M. Lucas, P. M. Richardson, and K. R.Hristova. 2007. Whole-genome analysis of the methyl tert-butyl ether-de-grading beta proteobacterium Methylibium petroleiphilum PM1. J. Bacteriol.189:1931–1945.
38. Katashkina, J. I., Y. Hara, L. I. Golubeva, I. G. Andreeva, T. M. Kuvaeva,and S. V. Mashko. 2009. Use of the � Red-recombineering method forgenetic engineering of Pantoea ananatis. BMC Mol. Biol. 10:34.
39. Kilbane, J. J., II, and B. A. Bielaga. 1991. Instantaneous gene transfer fromdonor to recipient microorganism via electroporation. BioTechniques 10:354–365.
40. Kim, C. S., and T. K. Wood. 1997. Creating auxotrophic mutants in Methylo-philus methylotrophus AS1 by combining electroporation and chemical mu-tagenesis. Appl. Microbiol. Biotechnol. 48:105–108.
41. Kramer, R. 1994. Systems and mechanisms of amino acid uptake and excre-tion in prokaryotes. Arch. Microbiol. 162:1–13.
42. Lutke-Eversloh, T., C. N. Santos, and G. Stephanopoulos. 2007. Perspectivesof biotechnological production of L-tyrosine and its applications. Appl. Mi-crobiol. Biotechnol. 77:751–762.
43. Marx, C. J., and M. E. Lidstrom. 2001. Development of improved versatilebroad-host-range vectors for use in methylotrophs and other gram-negativebacteria. Microbiology 147:2065–2075.
44. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring HarborLaboratory, Cold Spring Harbor, NY.
45. Mochul’skaia, N. A., A. S. Mironov, and S. V. Mashko. 1994. Decrease in thelevel of DeoR-dependent repression of the deo operon as a result of inte-gration of foreign DNA fragments into the interoperator deoO1-deoO2 re-gion of the Escherichia coli chromosome. Genetika 30:1175–1183. (In Rus-sian.)
46. Moore, A. T., M. Nayudu, and B. W. Holloway. 1983. Genetic mapping inMethylophilus methylotrophus AS1. J. Gen. Microbiol. 129:785–799.
47. Ohnishi, K., A. Hasegawa, K. Matsubara, T. Date, T. Okada, and K. Kiri-tani. 1988. Cloning and nucleotide sequence of the brnQ gene, the structuralgene for a membrane-associated component of the LIV-II transport systemfor branched-chain amino acids in Salmonella typhimurium. Jpn. J. Genet.63:343–357.
48. Olah, G. A. 2005. Beyond oil and gas: the methanol economy. Angew. Chem.Int. Ed. Engl. 44:2636–2639.
49. Parekh, S., V. A. Vinci, and R. J. Strobel. 2000. Improvement of microbialstrains and fermentation processes. Appl. Microbiol. Biotechnol. 54:287–301.
50. Pittard, A. J. 1996. Biosynthesis of aromatic amino acids. p 458–484. In F. C.Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B.Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger(ed.), Escherichia coli and Salmonella: cellular and molecular biology. ASMPress, Washington, DC.
51. Puta, F., and R. Wambutt. 1992. Construction of a new Escherichia coli-Saccharomyces cerevisiae shuttle plasmid cloning vector allowing positiveselection for cloned fragments. Folia Microbiol. 37:193–198.
52. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratorymanual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Har-bor, NY.
53. Sawitzke, J. A., L. C. Thomason, N. Costantino, M. Bubunenko, S. Datta,and D. L. Court. 2007. Recombineering: in vivo genetic engineering in E.coli, S. enterica, and beyond. Methods Enzymol. 421:171–199.
54. Reference deleted.55. Schrader, J., M. Schilling, D. Holtmann, D. Sell, M. V. Filho, A. Marx, and J. A.
Vorholt. 2009. Methanol-based industrial biotechnology: current status and futureperspectives of methylotrophic bacteria. Trends Biotechnol. 27:107–115.
56. Senior, P. J., and J. Windass. 1980. The ICI single cell protein process.Biotechnol. Lett. 2:205–210.
57. Shen, P., H. Chao, C. Jiang, Z. Long, C. Wang, and B. Wu. 6 March 2009.Enhancing production of L-serine by increasing the glyA gene expression inMethylobacterium sp. MB200. J. Appl. Biochem. Biotechnol. [Epub ahead ofprint.]
58. Tani, Y. 1991. Production of useful chemicals by methylotrophs, p. 253–267.In I. Goldberg and J. S. Rokem (ed.), Biology of methylotrophs. Butter-worth-Heinemann, Cambridge, MA.
59. Tatra, P. K., and P. M. Goodwin. 1985. Mapping of some genes involved inC-1 metabolism in the facultative methylotroph Methylobacterium sp. strainAM1 (Pseudomonas AM1). Arch. Microbiol. 143:169–177.
60. Tokmakova, I. L., E. G. Abalakina, N. V. Gorshkova, and Y. A. V. Yomantas.2007. Method for inducing L-amino acid auxotrophy to a bacterium belong-ing to the genus Methylophilus. U.S. patent application RU2007111371 IPCC12N15/63.
61. Tsujimoto, N., Y. Gunjii, Y. Ogawa-Miyata, M. Shimaoka, and H. Yasueda.2006. L-Lysine biosynthetic pathway of Methylophilus methylotrophus andconstruction of an L-lysine producer. J. Biotechol. 124:327–337.
62. Tsygankov, Y. D., and S. M. Kazakova. 1987. Development of gene transfersystems in Methylobacillus flagellatus KT: isolation of auxotrophic mutants.Arch. Microbiol. 149:112–119.
63. Vasey, R. B., and K. A. Powell. 1984. Single-cell protein. Biotechnol. Genet.Eng. Rev. 2:285–310.
64. Reference deleted.65. Vuilleumier, S., L. Chistoserdova, M. C. Lee, F. Bringel, et al. 2009. Methylo-
bacterium genome sequences: a reference blueprint to investigate microbialmetabolism of C1 compounds from natural and industrial sources. PLoSOne 4:e5584. doi:10.1371/journal.pone.0005584.
66. Wach, A. 1996. PCR-synthesis of marker cassettes with long flanking homol-ogy regions for gene disruptions in S. cerevisiae. Yeast 12:259–265.
67. Ward, N., Q. Larsen, J. Sakwa, L. Bruseth, H. Khouri, et al. 2004. Genomicinsights into methanotrophy: the complete genome sequence of Methylococ-cus capsulatus (Bath). PLoS Biol. 2:1616–1628.
68. Windass, J. D., M. J. Worsey, E. M. Pioli, D. Pioli, P. T. Barth, K. T.Atherton, E. C. Dart, D. Byrom, K. Powell, and P. J. Senior. 1980. Improvedconversion of methanol to single-cell protein by Methylophilus methylotro-phus. Nature 287:396–401.
69. Young, G. B., D. L. Jack, D. W. Smith, and M. H. Saier. 1999. The aminoacid/auxin:proton symport permease family. Biochim. Biophys. Acta 1415:306–322.
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