JP2013000064A - Recombinant microorganism, and method for producing alanine using the recombinant microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for identifying a gene associated with an alanine excretion transporter and improving alanine secretion productivity in an microorganism capable of producing the alanine by utilizing the gene.SOLUTION: The recombinant microorganism is obtained by modifying a microorganism capable of producing the L-alanine so as to have enhanced expression of ygaW gene or a gene that is functionally equivalent to the ygaW gene. The gene encodes a protein having the L-alanine excretion transporter function.

Description

  The present invention relates to a recombinant microorganism modified so that expression of a predetermined gene is enhanced, and a method for producing alanine using the recombinant microorganism.

  Mass transport through the cell membrane is very important for maintaining the physiological functions and homeostasis of cells, and is an essential function regardless of prokaryotic or eukaryotic cells. In other words, in order to maintain life activity, cells take in various nutrients from the outside world, while metabolites and waste products generated in the metabolic process, as well as harmful substances that have entered the cell, are released outside the cell. Discharge. These substance transports are carried out in an energy-dependent manner via transporter proteins localized in the cell membrane.

  As for transporters, transporters that use harmful substances such as antibiotics and heavy metals as exhaust substrates have been studied since the 1980s, and transporters that discharge amino acids, sugars, and other nutrients in the last 10 years. The existence of has been revealed at the genetic level. In particular, with regard to amino acids, Corynebacterium glutamicum, which is used for the production of many amino acids by fermentation, reported that a lysine excretion transporter exists in 1996 (Non-patent Document 1). Over 10 efflux transporters have been identified at the genetic level in E. coli. As substrates of these efflux transporters, many substances such as threonine, sulfur-containing amino acids, basic amino acids, acidic amino acids, branched chain amino acids, aromatic amino acids and analogs thereof are known.

  On the other hand, regarding alanine, it has been observed that alanine (Ala) is secreted and produced in bacterial wild strains of a wide range of genus species and bacterial mutants modified by metabolic engineering (Patent Document 1, Non-Patent Document 2). ~ 4). In addition, Ala-producing strains are bred using recombinant technology from Escherichia coli that originally does not secrete Ala (Non-patent Document 5).

  Based on the above, it is considered that many bacteria in the natural world have universally an alanine excretion transporter, but at present there are no reports on the substance of any bacterial species.

  On the other hand, in amino acid fermentation at the industrial level, the improvement in productivity based on the enhancement of the function of the amino acid synthesis metabolic pathway is approaching the limit, and in recent years, attention has been paid to amino acid excretion transporters. That is, as an application study of amino acid excretion transporters, it is expected that productivity of amino acid-producing bacteria will be improved by enhancing the excretion function by high expression of the exhaust transporters. If an alanine excretion transporter can be identified, this can be applied to improve alanine productivity.

US 5,559,016

Vrljic M, Sahm H & Eggeling L (1996) A new type of transporter with a new type of cellular function: L-lysine export from Corynebacterium glutamicum. Mol Microbiol 22: 815-826. Kinoshita S, Udaka S & Shimono M (1957) Studies on the amino acid fermentation part I. Production of L-glutamic acid by various microorganisms.J Gen Appl Microbiol 3: 193-205. Hashimoto S & Katsumata R (1998) L-Alanine fermentation by an alanine racemase-deficient mutant of the DL-alanine hyperproducing bacterium Arthrobacter oxydans HAP-l.J Ferment Bioeng 86: 385-390. Hols P, Kleerebezem M, Schanck AN, Ferain T, Hugenholtz J, Delcour J & de Vos WM (1999) Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering. Nat Biotechnol 17: 588-592. Zhang X, Jantama K, Moore JC, Shanmugam KT & Ingram LO (2007) Production of L-alanine by metabolically engineered Escherichia coli.Appl Microbiol Biot 77: 355-366.

  Therefore, the present invention identifies a gene involved in an alanine excretion transporter and uses this to improve a recombinant production of alanine, and a method for producing alanine using the recombinant microorganism. It is intended to provide.

  As a result of intensive studies by the present inventors in order to achieve the above-mentioned object, a gene encoding a protein involved in the L-alanine excretion transporter can be identified, and by using this, L-alanine production can be performed. It has been found that the ability to produce alanine secretion in microorganisms having the ability can be greatly improved, and the present invention has been completed.

The present invention includes the following.
(1) A recombinant microorganism modified so that expression of the ygaW gene or a gene functionally equivalent to the gene is enhanced with respect to a microorganism having L-alanine producing ability.

(2) The recombinant microorganism according to (1), wherein the ygaW gene and a gene functionally equivalent to the gene are any of the following (a) to (c): A gene encoding a protein comprising the amino acid sequence shown in (2) (b) coding for a protein having an amino acid sequence having 70% or more sequence similarity to the amino acid sequence shown in SEQ ID NO: 2 and having an L-alanine excretion transport function (C) encodes a protein that can hybridize under stringent conditions to a complementary strand of DNA consisting of all or part of the base sequence shown in SEQ ID NO: 1 and has an L-alanine excretion transport function Genes

  (3) The gene functionally equivalent to the ygaW gene has four transmembrane domains consisting of amino acid sequences having a total sequence similarity of 80% or more with respect to the amino acid sequences shown in SEQ ID NOs: 33, 34, 35 and 36. The recombinant microorganism according to (1), which is a gene encoding a protein having a region.

  (4) The recombinant microorganism according to (1), wherein the functionally equivalent gene is a gene derived from an organism other than E. coli.

  (5) The recombinant microorganism according to (1), wherein the microorganism having L-alanine producing ability is originally a microorganism having L-alanine producing ability.

  (6) The microorganism having the ability to produce L-alanine is a microorganism into which a gene involved in the L-alanine biosynthetic pathway is introduced into a microorganism that does not inherently have the ability to produce L-alanine. (1) The recombinant microorganism according to the above.

  (7) The recombinant microorganism according to (1), wherein the microorganism having the ability to produce L-alanine is Escherichia coli.

  (8) A method for producing L-alanine, wherein the recombinant microorganism according to any one of (1) to (7) above is cultured in a medium, and L-alanine is recovered from the medium during or after completion of the culture.

  According to the present invention, L-alanine secretion production ability in microorganisms having L-alanine production ability can be greatly improved. Therefore, productivity in the fermentation production of L-alanine can be greatly improved by utilizing the recombinant microorganism according to the present invention.

It is a characteristic view which shows the result of having carried out multiple alignment analysis about the ygaW gene and its homolog at the amino acid sequence level. It is a characteristic view which shows the result of having quantified L-alanine and L-alanyl-L-alanine contained in the culture supernatant in MG1655 strain and MLA301 strain by HPLC. It is the photograph which imaged the state when each strain | stump | stock containing mutant strains LAX12 and LAX16 was grown on a 50 μg / ml L-Ala-containing plate medium and a 3 mM Ala-Ala-containing plate medium. It is a characteristic view which shows the cell growth curve in the culture medium containing L-alanine and the culture medium containing Ala-Ala of LAX12 strain | stump | stock, LAX16 strain | stump | stock, and their parent strain, MLA301 strain | stump | stock. It is a characteristic view which shows the result of having measured the L-alanine density | concentration inside and outside a cell about LAX12 strain | stump | stock, LAX16 strain | stump | stock, and their parent strain, MLA301 strain | stump | stock. It is a characteristic view which shows the result of having analyzed the product of the ytfF gene and the ygaW gene using the TMHMM program. It is a characteristic view which shows the result of having measured the L-alanine density | concentration inside and outside a cell about each transformant. It is a characteristic view which shows the result of having measured the L-alanine density | concentration inside and outside a cell about the ygaW gene deficient strain and the strain | stump | stock which introduce | transduced the ygaW gene into the said deficient strain. FIG. 5 is a characteristic diagram showing a growth curve as a result of measuring extracellular L-alanine concentration over time for MG1655 introduced with recombinant plasmid pYgaW and MG1655 introduced with vector pSTV29. It is an electrophoresis photograph which shows the result of having analyzed the transcription level in the presence or absence of Ala-Ala using RT-PCR for the ygaW, ytfF, yddG and yeaS genes. About MG1655 introduced with vector pSTV29, MG1655 introduced with pAlaD and MG1655 introduced with pAlaD-YgaW, as a result of measuring extracellular L-alanine concentration with time, results of measuring glucose consumption with time and results of each strain It is a characteristic view which shows a growth curve.

Hereinafter, the present invention will be described in detail.
The recombinant microorganism according to the present invention is modified so that expression of a specific gene is enhanced with respect to a microorganism having L-alanine producing ability. This recombinant microorganism exhibits a very excellent ability to produce L-alanine compared to the ability to produce L-alanine in the microorganism (host microorganism) before recombination.

Microorganism having L-alanine producing ability In the present invention, the microorganism having L-alanine producing ability means the ability to produce L-alanine to the extent that it can be recovered from the cell or from the medium when cultured in the medium. . That is, even if L-alanine cannot be recovered from the culture medium when a certain microorganism is cultured in the culture medium, the microorganism has the ability to produce L-alanine as long as L-alanine is accumulated in the cells of the microorganism. include.

  The microorganism having L-alanine producing ability may be a wild-type microorganism that has been isolated and identified from the natural environment and has not undergone genetic modification (gene transfer, gene disruption, homologous recombination), etc. However, it may be a mutant microorganism in which a mutation is introduced into a wild-type microorganism. The mutation may be a naturally introduced mutation or an artificially introduced mutation. Furthermore, microorganisms having L-alanine producing ability include microorganisms that have been modified to give L-alanine producing ability to microorganisms that do not inherently have L-alanine producing ability (for example, recombinant Body).

  As a microorganism having L-alanine producing ability, Arthrobacter oxydans disclosed in Non-Patent Document 3 can be cited as an example of a microorganism having originally L-alanine producing ability. In addition, microorganisms that have L-alanine-producing ability include Corynebacterium gelatinosum, Micrococcus sodonensis, Clostridium sp. Cryptococcus albicans, Rhizopus nigricans, Rhizopus japonicus, Penicillium trzebinskianum, Mucor and the like.

  Examples of microorganisms that have been modified to impart L-alanine producing ability include the Lactococcus lactis mutants disclosed in Non-Patent Document 4 above. This Lactococcus lactis mutant strain is deficient in the L-lactate dehydrogenase gene in wild-type Lactococcus lactis, introduced an alanine dehydrogenase gene derived from Bacillus sphaericus so that it can be expressed, and further D-alanine which is an optical isomer of L-alanine In order to suppress production, the alanine racemase gene is deleted. The Lactococcus lactis mutant strain modified in this way can synthesize L-alanine with high optical purity (> 99%) as the final product of sugar metabolism.

  Furthermore, examples of microorganisms that have been modified to impart L-alanine-producing ability include Escherichia coli mutants disclosed in Non-Patent Document 5 above. This Escherichia coli mutant can synthesize alanine as a product in fermentation using glycol by replacing the inherent D-lactate dehydrogenase gene with an alanine dehydrogenase gene derived from Geobacillus stearothermophilus.

  Modifications that give L-alanine-producing ability are not limited to the above-mentioned Lactococcus lactis and Escherichia coli, but can be similarly applied to other microorganisms belonging to the genus Lactococcus and other microorganisms belonging to the genus Escherichia. Can do. Further, the modification that imparts the ability to produce L-alanine can be similarly performed on bacteria such as Bacillus genus, Pseudomonas dacunhae, Zymomonas mobilis, Corynebacterium glutamicum, Arthrobacter and the like. Furthermore, modification that imparts the ability to produce L-alanine can be similarly performed on yeasts (fungi) such as the genus Saccharomyces.

  On the other hand, in order to confer L-alanine producing ability to a microorganism that does not inherently have L-alanine producing ability, a method of introducing a gene involved in the L-alanine biosynthetic pathway in an expressible manner can be mentioned. Examples of genes involved in the L-alanine biosynthetic pathway include alanine dehydrogenase gene, alanine racemase gene, alanine aminotransferase, and various aminotransferases. Among these genes involved in the L-alanine biosynthetic pathway, by introducing one or more genes into a microorganism that does not inherently have the ability to produce L-alanine, the microorganism obtains the ability to produce L-alanine. Will be.

  The method for introducing a gene involved in the L-alanine biosynthetic pathway in an expressible manner is not particularly limited, and the gene is cloned into a vector such as an appropriate plasmid, and a host microorganism is obtained using the obtained recombinant vector. A method for transformation is mentioned. The vector that can be used can be appropriately selected depending on the host microorganism, without individually demonstrating available vectors. Moreover, a conventionally well-known method can be used suitably also as the transformation method using a vector. Examples of transformation methods include calcium chloride method, competent cell method, protoplast or spheroplast method, and electric pulse method.

  In addition, when a gene involved in the L-alanine biosynthetic pathway is introduced so that it can be expressed, the gene may be introduced into the chromosomal DNA of the host microorganism or present in the host microorganism in the state of a vector having the gene. You may let them. The number of copies of the gene to be introduced is not limited at all, and the gene may be introduced by a single copy or the gene may be introduced by multiple copies.

  On the other hand, in order to confer L-alanine-producing ability to microorganisms that do not inherently have L-alanine-producing ability, other compounds are produced using L-alanine as a substrate in the metabolic pathway involving L-alanine. The method of inhibiting reaction is mentioned. As an example, there is a method of reducing or eliminating the activity of an enzyme that catalyzes a reaction that produces another compound using L-alanine as a substrate in a metabolic pathway involving L-alanine. Enzymes that catalyze reactions that produce other compounds using L-alanine as a substrate in metabolic pathways involving L-alanine include alanine racemase and three major aminotransferases (YfbQ involved in L-alanine synthesis in E. coli). , YfdZ, AvtA) and their homologues. By deleting genes encoding these enzymes, the activity of the enzymes can be reduced or deleted, and as a result, L-alanine can be highly accumulated.

  The method for reducing or eliminating the activity of the enzyme is not particularly limited. For example, a mutation may be introduced into a gene encoding the enzyme by a usual mutation treatment method or a genetic engineering technique. Examples of the mutation treatment method include a method of irradiating with X-rays and ultraviolet rays, a method of treating with a mutation agent such as N-methyl-N′-nitro-N-nitrosoguanidine, and the like. The site for introducing a mutation may be a so-called coding region in a gene or an expression control region such as a promoter for controlling the expression of the gene.

Genes involved in L-alanine efflux transporter In the present invention, the above-described microorganisms capable of producing L-alanine are modified so that expression of a novel gene involved in L-alanine efflux transporter is enhanced. Here, the novel gene is a gene identified as a ygaW gene in E. coli. That is, the ability to produce L-alanine can be improved by modifying the microorganism having L-alanine producing ability to enhance the expression of the ygaW gene or a gene functionally equivalent to the gene. it can.

  In addition, L-alanine-producing ability may be imparted to microorganisms that do not inherently have L-alanine-producing ability after modification so that expression of a novel gene involved in L-alanine excretion transporter is enhanced. Then, modification that enhances the expression of a novel gene involved in the L-alanine excretion transporter and impartation of L-alanine production ability may be performed simultaneously.

  Here, examples of the modification that enhances the expression of the gene include a method of modifying the expression control region of the gene concerned and a method of introducing the gene so that the gene can be expressed. In addition, these methods can be combined to enhance gene expression. Confirmation that the gene expression is improved as compared with the parent strain, for example, a wild strain or an unmodified strain, is not particularly limited, but a method for comparing the amount of mRNA of the gene with a wild type or an unmodified strain. Can be mentioned. More specifically, the expression level of a specific gene can be quantitatively compared by techniques such as Northern hybridization and RT-PCR.

  In addition, when enhancing the expression of the ygaW gene or a gene functionally equivalent to the gene, it is sufficient that at least the expression level of the gene is statistically significantly increased as compared to the wild strain or the unmodified strain. . In particular, the expression level of the gene is preferably set as appropriate so that the amount of L-alanine secretory production increases.

  The ygaW gene is a gene registered in a database as a gene whose function is unknown in E. coli. The nucleotide sequence of this ygaW gene and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 1 and 2, respectively. However, the ygaW gene in Escherichia coli is not limited to those specified by SEQ ID NOs: 1 and 2, but may be a gene having a different base sequence or amino acid sequence but having a paralogous relationship or a narrowly homologous relationship. .

  Further, the ygaW gene in E. coli is not limited to those specified in SEQ ID NOs: 1 and 2, and is, for example, 70% or more, preferably 80% or more, more preferably 90% with respect to the amino acid sequence of SEQ ID NO: 2. %, More preferably 95% or more of the amino acid sequence having a sequence similarity, and may encode a protein that functions as an L-alanine excretion transporter. The sequence similarity value can be calculated by a BLASTN or BLASTX program that implements the BLAST algorithm (default setting). In addition, the value of sequence similarity was calculated by comparing the total of amino acid residues that completely matched when paired amino acid sequences were subjected to pair-wise alignment analysis and amino acid residues having similar physicochemical functions. Calculated as a percentage of the total number of all amino acid residues.

  Furthermore, the ygaW gene in Escherichia coli is not limited to those specified in SEQ ID NOs: 1 and 2, for example, one or several amino acids are substituted, deleted, or inserted into the amino acid sequence of SEQ ID NO: 2. Alternatively, it may be a protein that has an added amino acid sequence and encodes a protein that functions as an L-alanine excretion transporter. Here, the term “several” refers to, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 10, and most preferably 2 to 5.

  Furthermore, the ygaW gene in Escherichia coli is not limited to those specified by these SEQ ID NOs: 1 and 2. For example, a string may be added to all or part of the complementary strand of DNA consisting of the base sequence of SEQ ID NO: 1. It may encode a protein that hybridizes under a gentle condition and functions as an L-alanine efflux transporter. The term “stringent conditions” as used herein means the conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and is determined appropriately with reference to, for example, Molecular Cloning: A Laboratory Manual (Third Edition). can do. Specifically, the stringency can be set according to the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution.

  On the other hand, a gene functionally equivalent to the ygaW gene is a gene that encodes a protein that is derived from an organism other than E. coli (for example, γ-proteobacteria) and functions as an L-alanine efflux transporter. Here, a gene that is derived from an organism other than E. coli and encodes a protein that functions as an L-alanine excretion transporter means a gene in a broad sense of a homolog or a gene in an ortholog.

  Such a gene functionally equivalent to the ygaW gene can be identified by searching a database storing genomic DNA sequence information related to organisms other than E. coli. Here, examples of the database include databases such as MBGD, GenBank, DDBJ, and EMBL. However, when searching for a gene functionally equivalent to the ygaW gene, there is no limitation on the database to be used. In particular, as the database, it is preferable to use an MBGD equipped with a system that searches one genome information for one species. A search for a gene functionally equivalent to the ygaW gene using a database can be performed based on the sequence similarity value described above using the nucleotide sequence of SEQ ID NO: 1 and / or the amino acid sequence of SEQ ID NO: 2 as a query sequence. In such a search, the sequence similarity described above is synonymous with homology. In such a search, for example, a homology of 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, most preferably 95% or more with respect to the amino acid sequence of SEQ ID NO: 2. A gene encoding a protein having an amino acid sequence having (sequence similarity) can be specified as a gene functionally equivalent to the ygaW gene.

  A gene functionally equivalent to the ygaW gene has, for example, an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added to the amino acid sequence of SEQ ID NO: 2, It may encode a protein that functions as an alanine excretion transporter. Here, the term “several” refers to, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 10, and most preferably 2 to 5.

  Furthermore, it hybridizes under stringent conditions to a gene functionally equivalent to the ygaW gene, for example, all or part of the complementary strand of DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and L-alanine It may encode a protein that functions as an efflux transporter. The term “stringent conditions” as used herein means the conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and is determined appropriately with reference to, for example, Molecular Cloning: A Laboratory Manual (Third Edition). can do. Specifically, the stringency can be set according to the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution.

  More specifically, a gene functionally equivalent to the ygaW gene is not particularly limited, but the following genes stored in the MBGD database can be raised. That is, examples of genes functionally equivalent to the ygaW gene include, for example, the SF2698 gene (base sequence: SEQ ID NO: 3, amino acid sequence: SEQ ID NO: 4) derived from Shigella flexneri 301, the STM2800 gene derived from Salmonella typhimurium LT2; ATCC 700720 ( Base sequence: SEQ ID NO: 5, amino acid sequence: SEQ ID NO: 6), CKO_04017 gene derived from Citrobacter koseri ATCC BAA-895 (base sequence: SEQ ID NO: 7, amino acid sequence: SEQ ID NO: 8), ENT638_3150 gene derived from Enterobacter sp. Nucleotide sequence: SEQ ID NO: 9, amino acid sequence: SEQ ID NO: 10), KPN_02999 gene derived from Klebsiella pneumoniae ATCC 700721 (base sequence: SEQ ID NO: 11, amino acid sequence: SEQ ID NO: 12), ESA_00597 gene derived from Enterobacter sakazakii ATCC BAA-894 ( Base sequence: SEQ ID NO: 13, amino acid sequence: SEQ ID NO: 14), YPO0544 gene derived from Yersinia pestis CO92 (base sequence: SEQ ID NO: 15, amino acid) Acid sequence: SEQ ID NO: 16), SPRO_0751 gene derived from Serratia proteamaculans 568 (base sequence: SEQ ID NO: 17, amino acid sequence: SEQ ID NO: 18), VC1828 gene derived from Vibrio cholerae N16961 (base sequence: SEQ ID NO: 19, amino acid sequence: sequence) No. 20), DD1591_0599 gene derived from Dickeya zeae Ech1591 (base sequence: SEQ ID NO: 21, amino acid sequence: SEQ ID NO: 22), ECA3826 gene derived from Erwinia carotovora SCRI1043 (base sequence: SEQ ID NO: 23, amino acid sequence: SEQ ID NO: 24), VF_0967 gene derived from Vibrio fischeri ES114 (base sequence: SEQ ID NO: 25, amino acid sequence: SEQ ID NO: 26), PBPRA2535 gene derived from Photobacterium profundum SS9 (base sequence: SEQ ID NO: 27, amino acid sequence: SEQ ID NO: 28), derived from Sodalis glossinidius morsitans SG0439 gene (base sequence: SEQ ID NO: 29, amino acid sequence: SEQ ID NO: 30) and Rhodobacter sph aeroides 2.4.1; RSP_3651 gene derived from ATCC BAA-808 (base sequence: SEQ ID NO: 31, amino acid sequence: SEQ ID NO: 32).

  These listed genes are summarized in gene name, species, e-value, coincidence at the amino acid sequence level, and sequence similarity at the amino acid sequence level.

  Each gene shown in the above table encodes a protein having a sequence similarity of 70% or more with respect to the amino acid sequence of the protein encoded by the ygaW gene, and the E-value is a very small value (at most 2.00e). It can be said that there is a high probability that it is significant as a homologue of the ygaW gene from an evolutionary and statistical point of view. In particular, among the genes shown in the above table, those encoding a protein having 70% or more identity to the amino acid sequence of the protein encoded by the ygaW gene are homologs having functions very similar to those of the ygaW gene. It can be said that there is a high probability.

  By the way, when the protein encoded by the ygaW gene was analyzed using the TMHMM (Transmembrane Hidden Markov Model (Krogh A et al. (2001) J Mol Biol 305, 567-580)) program, four transmembrane domains were identified. can do. Among the genes shown in the above table, the top 13 genes (SF2698, CKO_04017, STM2800, ENT638_3150, KPN_02999, ESA_00597, YPO0544, SPRO_0751, VC1828, ECA3826, DD1591_0599, PBPRA2535 and VF_0967) are subjected to multiple alignment analysis. And the result of having analyzed about four transmembrane regions is shown in FIG. In FIG. 1, four regions with thick lines are transmembrane regions. In addition, in FIG. 1, the amino acid residues indicated by circles in the amino acid sequence of the protein encoded by the ygaW gene maintain the function as an L-alanine excretion transporter, as shown in Examples described later in detail. It has been suggested that this is a very important amino acid. The multiple alignment analysis uses the Clustal W program (Thompson JD et al. (1994) Nucleic acids Res 22, 4673-4680).

  As can be seen from FIG. 1, between these genes, the sequence similarity in the four transmembrane regions (TMS1 to TMS4) is very high. That is, it can be said that the four transmembrane regions found in the ygaW gene are highly conserved even in genes functionally equivalent to the ygaW gene. Table 2 shows the results of calculating the identity and similarity at the amino acid sequence level for these four transmembrane regions TMS1 to TMS4. In Table 2, the “total” column indicates the identity and similarity when all four transmembrane regions TMS1 to TMS4 are calculated together.

  As can be seen from Table 2, the similarity value in these transmembrane regions is very high even for genes (eg RSP_3651) that have low identity and similarity when comparing the whole proteins. Show. Specifically, each gene shown in Table 2 encodes a protein having a transmembrane region having a sequence similarity of 80% or more in total with respect to the transmembrane region in the protein encoded by the ygaW gene.

  From the analysis results shown in Table 2, a gene functionally equivalent to the ygaW gene has a transmembrane region having a total of 80% or more sequence similarity to the transmembrane region in the protein encoded by the ygaW gene. In addition, it can be called a gene encoding a protein that functions as an L-alanine excretion transporter. Here, in the gene functionally equivalent to the ygaW gene, the transmembrane region is preferably 85% or more in total, more preferably 90% or more, and most preferably 95% or more in total compared to the ygaW gene. Has sequence similarity.

  The amino acid sequences of the four transmembrane regions in the protein encoded by the ygaW gene are shown in SEQ ID NOs: 33, 34, 35 and 36, respectively. As described above, the sequence similarity with these amino acid sequences can be calculated by a BLASTN or BLASTX program that implements the BLAST algorithm (default setting).

  In particular, in the amino acid sequence of the protein encoded by the ygaW gene shown in SEQ ID NO: 2, the 34th serine and the 125th glycine are used as L-alanine excretion transporters, as shown in Examples described later in detail. It is suggested that it is a very important amino acid in maintaining the function. Therefore, even in a gene functionally equivalent to the ygaW gene, it is preferable that the serine corresponding to the 34th serine and the glycine corresponding to the 125th glycine in the amino acid sequence shown in SEQ ID NO: 2 are conserved.

  By the way, the ygaW gene and the gene functionally equivalent to the gene are not limited to those specified by the specific base sequence or amino acid sequence described above, and naturally or artificially introduced with mutations. It may be specified by a base sequence or amino acid sequence different from the specific base sequence or amino acid sequence. When artificially introducing a mutation, a conventionally known site-specific mutation method can be applied. According to this method, a desired mutation can be introduced into the coding region of the ygaW gene or a gene functionally equivalent to the gene, and an amino acid residue at a specific site is substituted, deleted, inserted or added. A mutant gene encoding a protein having an amino acid sequence can be obtained. A mutant gene can also be obtained by employing a method of randomly introducing mutations.

  Whether or not a given gene encodes a protein involved in the L-alanine excretion transporter is determined by transforming a microorganism having L-alanine production ability using the gene and the obtained transformed microorganism. This can be confirmed by culturing in a medium and quantifying L-alanine excreted in the medium. That is, when the L-alanine excretion in the transformed microorganism is significantly increased compared to the L-alanine excretion in the microorganism before transformation, the given gene is involved in the L-alanine excretion transporter It can be determined that it encodes the protein to be processed.

Recombinant microorganism The recombinant microorganism according to the present invention is a microorganism modified so that expression of the ygaW gene and / or a gene functionally equivalent to the gene is enhanced relative to a microorganism capable of producing L-alanine. means.

  For example, the modification for enhancing the expression of the ygaW gene can be performed by increasing the copy number of the ygaW gene in the cell by using, for example, a gene recombination technique. For example, a DNA fragment containing the ygaW gene may be ligated with an expression vector that functions in a host microorganism, preferably a multi-copy vector, to produce a recombinant DNA, which is introduced into the microorganism and transformed.

  The expression vector that can be used is not particularly limited, and examples thereof include a plasmid type vector and a chromosomal transfer type vector that can be integrated into the genome of the host organism. The expression vector is not particularly limited, and any available expression vector may be appropriately selected according to the host microorganism. Examples of expression vectors include plasmid DNA, bacteriophage DNA, retrotransposon DNA, and artificial chromosome DNA (YAC).

  Examples of plasmid DNA include YCp type E. coli-yeast shuttle vectors such as pRS413, pRS414, pRS415, pRS416, YCp50, pAUR112 or pAUR123, YEp type E. coli-yeast shuttle vectors such as pYES2 or YEp13, pRS403, pRS404, pRS405, pRS406, YIp type E. coli-yeast shuttle vectors such as pAUR101 or pAUR135, plasmids derived from E. coli (pBR322, pBR325, pUC18, pUC19, pUC118, pUC119, pTV118N, pTV119N, pBluescript, pHSG298, pHSG396 or pTrc99A, ColE-based plasmids, pACYC177 or pACYC184 P15A-based plasmids such as, pMW118, pMW119, pMW101-based plasmids such as pMW218 or pMW219), Agrobacterium-derived plasmids (eg pBI101 etc.), Bacillus subtilis-derived plasmids (eg pUB110, pTP5 etc.), etc. Examples of the phage DNA include λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP), φX174, M13mp18 or M13mp19. . Examples of retrotransposons include Ty factor. Examples of YAC vectors include pYACC2. Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.

  In the expression vector, the ygaW gene and / or a gene functionally equivalent to the gene needs to be incorporated into the vector in a state where it can be expressed. The expression possible state means that the ygaW gene and / or the gene is expressed so that the ygaW gene and / or the gene functionally equivalent to the gene is expressed under the control of a predetermined promoter in the host organism. It means that a functionally equivalent gene and a promoter are linked and incorporated into a vector. Expression vectors include ygaW gene and / or a gene functionally equivalent to the gene, promoter and terminator, cis elements such as enhancers, splicing signal, poly A addition signal, selection marker, ribosome binding sequence (SD Sequence) and the like. Examples of selection markers include antibiotic resistance genes such as ampicillin resistance gene, kanamycin resistance gene, and hygromycin resistance gene.

  Moreover, a conventionally well-known method can be used suitably also as the transformation method using an expression vector. Examples of transformation methods include calcium chloride method, competent cell method, protoplast or spheroplast method, and electric pulse method.

  On the other hand, increasing the copy number of the ygaW gene and / or gene functionally equivalent to the gene means that multiple copies of the ygaW gene and / or gene functionally equivalent to the gene exist on the chromosomal DNA of the microorganism. Can also be achieved. To introduce multiple copies of the ygaW gene and / or a gene functionally equivalent to the gene into the chromosomal DNA of a microorganism, homologous recombination is performed using a sequence present in the chromosomal DNA as a target. Can do.

  Further, the expression enhancement of the ygaW gene and / or a gene functionally equivalent to the gene may be performed by adding an expression regulatory sequence such as an endogenous or introduced ygaW gene and / or a promoter of a gene functionally equivalent to the gene, It can also be achieved by a method of replacing with a gene capable of higher expression, a method of introducing a regulator that increases the expression of a predetermined gene, and the like. The promoter capable of high expression is not particularly limited, and examples thereof include lac promoter, trp promoter, trc promoter, pL promoter and the like. Moreover, it is also possible to introduce a mutation into the expression control region of the endogenous or introduced ygaW gene and / or a gene functionally equivalent to the gene, and modify it so that it can be expressed at a higher level.

  Note that enhancement of expression of the ygaW gene and / or a gene functionally equivalent to the gene can also be achieved by extending the survival time of the mRNA or preventing the intracellular degradation of the transport protein.

  The recombinant microorganism according to the present invention has improved L-alanine excretion activity by being modified so that expression of the ygaW gene and / or a gene functionally equivalent to the gene is enhanced. That is, the amount of L-alanine excreted into the medium when the recombinant microorganism according to the present invention is cultured is modified so that expression of the ygaW gene and / or a gene functionally equivalent to the gene is enhanced. Compared with the amount of L-alanine in the parent strain of, it is significantly improved.

L-alanine production method L-alanine is produced by culturing the above-described recombinant microorganism according to the present invention in a medium, accumulating L-alanine in the medium, and collecting L-alanine contained in the medium. can do.

  As a medium used for the culture, a normal medium containing a carbon source, a nitrogen source, inorganic salts, and other organic micronutrients such as amino acids and vitamins as necessary can be used. Either synthetic or natural media can be used. Any type of carbon source and nitrogen source may be used as long as the strain to be cultured is available.

  As the carbon source, saccharides such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysate and molasses can be used. In addition, organic acids such as acetic acid and citric acid, and alcohols such as ethanol alone Or it can use together with another carbon source. As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, and ammonium acetate, nitrates, and the like can be used. Organic micronutrients include amino acids, vitamins, fatty acids, nucleic acids, and peptone, casamino acids, yeast extracts, soybean protein breakdown products, etc. containing these, and auxotrophic mutations that require amino acids for growth. When using a strain, it is preferable to supplement the required nutrients. As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like can be used.

  In the case of a recombinant microorganism produced using Escherichia coli imparted with L-alanine producing ability, the culture is preferably controlled at a culture temperature of 35 to 37 ° C. and a pH of 7.0 to 7.5. Under such conditions, L-alanine can be accumulated in the culture medium by preferably culturing for about 8 to 10 hours.

  The method for recovering L-alanine from the culture solution after completion of the culture is not particularly limited, and may be performed according to a known recovery method. For example, L-alanine can be recovered by a method of concentrating crystallization after removing the cells from the culture solution, ion exchange chromatography or the like.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limited to a following example.
[Example]
In this example, first, an L-Ala excretion-deficient mutant strain lacking L-Ala excretion ability was obtained by chemical mutation treatment. Subsequently, two novel genes (ygaW, ytfF) and two known genes (yddG, yeaS) having an L-Ala excretion function were identified using as an index whether or not the phenotype of the excretion ability was complementary. Their function and transcriptional analysis were performed, and it was clarified that ygaW gene encodes a physiologically important L-Ala efflux transporter among four genes. Moreover, it was clarified that the enhancement of the excretion function by amplification of the ygaW gene improves L-Ala secretion productivity in E. coli Ala production strains.

<Strategy for acquiring mutants lacking L-Ala discharge ability>
1) Peptide feeding method
In C. glutamicum, it was shown that a lysE-deficient strain first identified as a gene encoding an amino acid excretion transporter is highly sensitive to lysine-containing peptides. In response to this result, Thr and Ile efflux transporter-deficient mutant strains were obtained by isolating strains sensitive to peptides containing threonine (Thr) and isoleucine (Ile). In this peptide feeding method, the mutant strain lacking the target amino acid excretion transporter cannot excrete the amino acid derived from the incorporated peptide in the presence of the peptide containing the amino acid, causing excessive accumulation in the cell. As a result, it is based on the principle of causing growth inhibition (peptide sensitivity). In this example, in order to identify the L-Ala efflux transporter of Escherichia coli, first, an attempt was made to obtain an L-Ala efflux-deficient mutant lacking the L-Ala efflux transporter using this peptide feeding method. It was.

2) Preparation of mutants lacking L-Ala metabolic ability
There are two metabolic pathways for L-Ala: a pathway for producing L-Ala from pyruvate and a pathway for converting L-Ala to D-Ala. The synthesis reaction of L-Ala is catalyzed by three types of aminotransferases (AvtA, YfbQ and YfdZ). In addition, it is known that the synthesis of D-Ala is catalyzed by two types of alanine racemase (Alr and DadX). These aminotransferase and racemase reactions are all known to be reversible. Therefore, even if cells lack L-Ala excretion ability, L-Ala is metabolized by these enzymatic reactions, so there is no possibility of excessive accumulation in the cells and sensitivity to L-Ala-containing peptides. There was sex. In fact, when E. coli wild-type strain MG1655 was cultured in a minimal medium supplemented with L-alanyl-L-alanine (Ala-Ala), an L-Ala-containing dipeptide, L-Ala produced by hydrolysis of the incorporated dipeptide was metabolized. And disappeared from the culture system (FIG. 2A). Therefore, in the present Example, in obtaining an L-Ala excretion-deficient mutant, a mutant MLA301 in which all two alanine racemases and three aminotransferases were deleted was prepared. Since this strain lacks L-Ala and D-Ala synthases, it showed L-Ala and D-Ala requirements. When this mutant strain was cultured in a minimal medium supplemented with Ala-Ala, approximately twice the concentration of L-Ala accumulated in the medium as the added Ala-Ala disappeared. Since this L-Ala remained at a constant level and did not decrease, it was clarified that this mutant strain lacks the ability to metabolize L-Ala (FIG. 2B).

  2A and 2B show the results of quantifying L-alanine contained in the culture supernatant of MG1655 strain and MLA301 strain by HPLC, respectively. In FIG. 2A and FIG. 2B, the open triangle indicates the Ala-Ala concentration remaining in the medium, and the open circle indicates the L-alanine concentration in the medium.

More specifically, first, E. coli MB2795 strain (alr :: FRT, dadX :: FRT) lacking two types of alanine racemase (Alr and DadX) was prepared (Strych U et al., (2001) FEMS Microbiol. Lett. 196: 93-98). This MB2795 strain was transformed with the vector pYfdZ18cs-KM (Yoneyama H et al., (2011) Biosci. Biotechnol. Biochem. 75, 930-938) for disrupting the yfdZ gene encoding L-alanine synthase. . The obtained transformant was cultured at 42 ° C. overnight in Luria liquid medium (Luria broth) containing 50 μg / mL D-alanine and 6.25 μg / mL kanamycin. Then, the recombinant in which the vector was incorporated was selected by culturing at 37 ° C. in a Luria solid medium containing 50 μg / mL D-alanine and 6.25 μg / mL kanamycin. Subsequently, the yfdZ gene disruption strain was selected using kanamycin resistance and chloramphenicol sensitivity as indices. The obtained yfdZ gene-disrupted strain was transformed with a vector pCP20 (Cherepanov PP & Wackernagel W (1995) Gene 158: 9-14) having a site-specific recombinase gene (FLP gene), and a kanamycin resistance cassette was The FRT sequence was left in the yfdZ gene by deletion. Next, the avtA gene and the yfbQ gene were sequentially destroyed by a transduction method using P1vir phage. In this case, E. coli HYE008 (avtA :: GM, yfbQ :: KM, Ala -) at was used as a donor, Luria solid medium containing 12.5 [mu] g / mL of gentamicin and 12.5 [mu] g / mL kanamycin, 50 [mu] g / mL AvtA gene-disrupted strain and yfbQ gene-disrupted strain were selected in the presence of D-alanine.

  L-alanine requirement of the obtained mutant strain MLA301 is determined using a minimal solid medium containing 50 μg / mL L-alanine (containing 50 μg / mL D-alanine) or a minimal solid medium containing no L-alanine. (Including 50 μg / mL D-alanine). The result is FIG. 3 described above.

  The left side of FIG. 3 is a photograph of a plate medium containing 50 μg / ml L-Ala and 50 μg / ml D-Ala, and the right side of FIG. 3 is 3 mM Ala-Ala and 50 μg / ml D It is the photograph which imaged the plate culture medium containing -Ala. All are 44 hours after the start of culture. In FIG. 3, the section 1 is the MG1655 strain, and the section 2 is the MLA301 strain.

  The disruption of the avtA gene, yfbQ gene and yfdZ gene was verified by PCR using the following pair of primers. Primers for avtA gene confirmation: 5′-GGAATTCCGAGCATGGCGACGATAA-3 ′ (SEQ ID NO: 37) and 5′-GGAATTCCAGTGCATGGATGTCGAG-3 ′ (SEQ ID NO: 38). Primers for yfbQ gene confirmation: 5′-CGGGATCCCGATCAGAACAATTCACT-3 ′ (SEQ ID NO: 39) and 5′-CGGGATCCCGACGTATGATGACATC-3 ′ (SEQ ID NO: 40). Primers 5′-CAGGATCCTGAAGGCTGATGACCAG-3 ′ (SEQ ID NO: 41) and 5′-CCGGATCCGGTACTTTTGCCCTGATG-3 ′ (SEQ ID NO: 42) for confirming the yfdZ gene.

<Induction of Ala-Ala sensitive mutants and evaluation of L-Ala excretion ability>
1) Induction of Ala-Ala sensitive mutants from L-alanine metabolizing mutants
L-Ala excretion-deficient mutants were isolated by treating Escherichia coli MLA301 lacking L-Ala and D-Ala metabolic ability with nitrosoguanidine, followed by concentration of penicillin in the presence of Ala-Ala. This was done by selecting strains that were highly sensitive to Ala-Ala on a minimal plate medium. As a result of replica evaluation of about 3,000 colonies, 22 strains (Ala-Ala sensitive mutant strains) that grew on a plate medium containing 50 μg / ml L-Ala and showed a phenotype that could not grow on a plate medium containing 3 mM Ala-Ala I got it. These mutant strains were named LAX1 to LAX22. FIG. 3 shows the growth of two typical mutants LAX12 and LAX16 on a plate medium containing 50 μg / ml L-Ala and 3 mM Ala-Ala.

  The left side of FIG. 3 is a photograph of a plate medium containing 50 μg / ml L-Ala and 50 μg / ml D-Ala, and the right side of FIG. 3 is 3 mM Ala-Ala and 50 μg / ml D It is the photograph which imaged the plate culture medium containing -Ala. All are 44 hours after the start of culture. In FIG. 3, section 1 is the MG1655 strain, section 2 is the MLA301 strain, section 3 is the LAX12 strain, and section 4 is the LAX16 strain.

2) Evaluation of sensitivity of Ala-Ala sensitive mutants to Ala-Ala
The Ala-Ala sensitive mutants LAX12 and LAX16 and their parent strain MLA301 were examined for sensitivity (MIC) to Ala-Ala using a minimal plate medium and found to be 0.039, 0.156 and> 10 mg / ml, respectively. These results revealed that these mutants were highly sensitive to Ala-Ala. In addition, as a result of examining the sensitivity to Ala-Ala in a minimal liquid medium, both mutant strains showed growth similar to the parent strain MLA301 in a medium supplemented with 50 μg / ml L-Ala, whereas 3 mM Ala-Ala Since growth was suppressed in the supplemented medium compared to the parent strain, it was revealed that both mutant strains showed Ala-Ala sensitivity even in the liquid medium (FIG. 4).

  In FIG. 4, A shows a cell growth curve in a medium containing 50 μg / ml L-alanine and 50 μg / ml D-Ala, and B contains 3 mM Ala-Ala and 50 μg / ml D-Ala. The cell growth curve in the culture medium which shows is shown. In FIG. 4, the open circle is the cell growth curve of the LAX12 strain, the open triangle is the cell growth curve of the LAX16 strain, and the open square is the cell growth curve of the MLA301 strain.

3) Intracellular and extracellular L-Ala levels of Ala-Ala sensitive mutants in the presence of Ala-Ala The resulting mutants are highly sensitive to Ala-Ala because the L-Ala efflux transporter is mutated As a result, it was predicted that the function was lost and excessive L-Ala accumulated in the cells. To test this hypothesis, Ala-Ala sensitive mutants LAX12, LAX16 and parental MLA301 cells were incubated in a minimal medium containing 6 mM Ala-Ala, and the intracellular and extracellular L-Ala concentrations were measured over time. (FIG. 5). The intracellular L-Ala level of MLA301 increased rapidly and then decreased to the basal level (about 40 mM). On the other hand, the intracellular L-Ala levels of both mutants increased rapidly, similar to that of the parent strain, but then reached a plateau of about 150-190 mM without decreasing. The steady-state intracellular L-Ala level of the mutant strain was much higher than the basal level of the parent strain, and conversely, the extracellular L-Ala level was lower in the mutant strain than that of the parent strain. The L-Ala excretion rates of LAX12, LAX16, and MLA301 calculated from time-dependent changes in extracellular L-Ala levels are 133, 137, and 180 nmol / mg of dry cell weight / min, respectively. Noh was about 25% lower than the parent strain. From the above results, it was suggested that the Ala-Ala sensitive mutant had a functional disorder in the L-Ala efflux transporter.

  5A shows the result of measuring intracellular L-alanine concentration by HPLC, and FIG. 5B shows the result of measuring extracellular L-alanine concentration by HPLC. In FIG. 5, open circles indicate the LAX12 strain, open triangles indicate the LAX16 strain, and open squares indicate the MLA301 strain.

<Identification and functional analysis of L-Ala efflux transporter gene>
1) Cloning of a gene that complements the mutation of the Ala-Ala sensitive mutant As described above, it is considered that the Ala-Ala sensitive mutant became highly sensitive to Ala-Ala due to the lack of the L-Ala efflux transporter. Thus, we attempted to obtain a gene encoding the L-Ala efflux transporter by performing shotgun cloning using the return of Ala-Ala sensitivity to insensitivity as an index. Cloning was performed by transforming a genomic library prepared from the chromosomal DNA of the wild-type E. coli strain into LAX12 strain.

  As a result, a large number of colonies capable of growing on the Ala-Ala-containing minimal plate medium were obtained. Next, plasmids were extracted from 11 independent clones and subjected to restriction enzyme analysis, subcloning and sequencing of DNA fragments inserted into the vector. As a result of collating the nucleotide sequence information of the resulting insert fragment with the entire genome sequence of Escherichia coli MG1655 registered in Japan DNA Data Bank (http://www.ddbj.nig.ac.jp), Ala-Ala Four genes (yeaS, yddG, ytfF, ygaW) that complement the sensitivity of the sensitive mutants were identified. Of the four species, yeaS and yddG are known genes and have been reported to encode leucine and aromatic amino acid excretion transporters, respectively. Since both genes encode amino acid excretion transporters, it was speculated that L-Ala is their minor substrate. On the other hand, ytfF and ygaW are genes with unknown functions, and it was predicted on the database that they encode inner membrane proteins. In fact, when these gene products were analyzed using the TMHMM program, it was predicted that YtfF had 10 transmembrane domains and YgaW had 4 transmembrane domains (FIG. 6). Therefore, it was suggested that these proteins with unknown functions are also involved in L-Ala excretion as transporters.

2) Evaluation of L-Ala excretion function of complementary genes The four identified genes (yeaS, yddG, ytfF, ygaW) were cloned into the medium copy vector pSTV29, respectively, and plasmids pYeaS, pYddG, pYtfF and pYgaW were prepared. . DNA fragments containing each gene were amplified by PCR using the chromosome of MG1655 strain as a template. In PCR for amplifying the yeaS gene, a pair of primer sets: yeaS-F and yeaS-R were used. In PCR for amplifying the yddG gene, a pair of primer sets: yddG-F and yddG-R were used. In PCR for amplifying the ytfF gene, a pair of primer sets: ytfF-F and ytfF-R were used. In PCR for amplifying the ygaW gene, a pair of primer sets: ygaW-F and ygaW-R were used. The base sequence of the primer used is listed below (underlined part is BamHI recognition sequence).
yeaS-F: 5'-CG GGATCC CGACGTCAGCGAAAGTGC-3 '(SEQ ID NO: 43)
yeaS-R: 5'-CG GGATCC CAGAAAGGCGTTGAGCG-3 '(SEQ ID NO: 44)
yddG-F: 5'-CG GGATCC AGTGCTTGCTTCGGGG-3 '(SEQ ID NO: 45)
yddG-R: 5'-CG GGATCC ATAGACTTCGGCAGCGTG-3 '(SEQ ID NO: 46)
ytfF-F: 5'-CG GGATCC CGGCACTGAAAAGCGTCG-3 '(SEQ ID NO: 47)
ytfF-R: 5'-CG GGATCC AGAGAGCGCCAGTTCACC-3 '(SEQ ID NO: 48)
ygaW-F: 5'-CG GGATCC CGTTACCTCACCCCCAAAC-3 '(SEQ ID NO: 49)
ygaW-R: 5'-CG GGATCC CGCGAATGGGACGTACCG-3 '(SEQ ID NO: 50)

  The DNA fragment amplified by PCR was treated with BamHI and cloned into the BamHI site of pSTV29. It was confirmed that when the obtained plasmids pYeaS, pYddG, pYtfF and pYgaW were separately transformed into the Ala-Ala sensitive mutant LAX12, the Ala-Ala sensitivity was restored to insensitivity. To assess whether the recovery is due to the L-Ala excretion function of the cloned gene, cells of the LAX12 strain introduced with the recombinant plasmid were incubated in a minimal medium containing 6 mM Ala-Ala. The L-Ala level inside and outside the cell was measured (FIG. 7).

  As a result, the intracellular L-Ala level was lower in the recombinant plasmid-introduced strain than in the LAX12 strain (LAX12 / pSTV29) having the vector pSTV29 (Fig. 6A). On the other hand, the extracellular L-Ala level was higher in the recombinant plasmid-introduced strain than in the LAX12 / pSTV29 strain (FIG. 7B). The L-Ala excretion rates of the parent strain (MLA301) and the Ala-Ala sensitive mutant strain (LAX12) were calculated from the time course of extracellular L-Ala levels. The results were 181 and 135 nmol / mg of dry cell weight / min, respectively. It was. In contrast, the excretion rates of the transformants in which the cloned genes (yeaS, yddG, ytfF, ygaW) were introduced into LAX12 were 141, 153, 163, 226 nmol / mg of dry cell weight / min. , Both were higher than LAX12. In particular, the excretion rate of the transformant having the ygaW gene was higher than that of the parent strain MLA301. From these results, it was shown that the cloned gene products (YgaW, YtfF, YddG, YeaS) have L-Ala excretion ability, thereby complementing the Ala-Ala sensitive mutation of LAX12.

  FIG. 7A shows the change over time in the L-alanine concentration in the cell, and FIG. 7B shows the change over time in the L-alanine concentration outside the cell. In Fig. 7, the open circle is LAX12 with pSTV29 installed, the black circle is LAX12 with pYeaS installed, the black triangle is LAX12 with pYddG installed, and the black square has pYtfF installed The black diamond is LAX12 with pYgaW. In FIG. 7, the white triangle is MLA 301 into which pSTV29 is introduced.

3) Effect of ygaW gene deficiency on L-Ala excretion of L-Ala metabolic deficient mutants It was clarified that all four complementary genes obtained changed L-Ala levels inside and outside of the LAX12 strain. However, it was found that amplification of the ygaW gene caused the most remarkable change among them (FIG. 7). From this, it was speculated that the ygaW gene encodes a major L-Ala efflux transporter. If so, the deficiency of the ygaW gene should result in significant changes in the intracellular and extracellular L-Ala levels. In order to test this hypothesis, a mutant MLA301ΔygaW in which the ygaW gene was disrupted in the MLA301 strain lacking the ability to metabolize L-Ala was prepared.

  First, pYgaW was treated with NruI and then self-ligated to prepare a plasmid pΔYgaW from which the NruI segment including the 401 bp segment and the 12 bp downstream segment in the ygaW gene was removed. The plasmid pΔYgaW retains a putative stem-loop structure existing downstream of the ygaW gene, and is considered to have no effect on the expression of the gene located downstream of the ygaW gene. Next, the BamHI fragment of plasmid pΔYgaW was excised and cloned into the BamHI site of pTH18cs1 (Hashimoto-Gotoh, T. et al., 2000, Gene 241: 185-191) to prepare pTH18cΔYgaW.

  Next, Escherichia coli MLA301 strain was transformed with the obtained pTH18cΔYgaW. Transformants were cultured overnight at 42 ° C. in L liquid medium containing 50 μg / ml D-alanine, 6.25 μg / ml gentamicin, 6.25 μg / ml kanamycin and 12.5 μg / ml chloramphenicol. Thereafter, selection was carried out at 37 ° C. on an L solid medium containing 50 μg / ml D-alanine, 6.25 μg / ml gentamicin, 6.25 μg / ml kanamycin and 12.5 μg / ml chloramphenicol. Subsequently, a mutant MLA301ΔygaW in which the ygaW gene was disrupted was obtained by selection using chloramphenicol sensitivity as an index.

  First, when the sensitivity of MLA301ΔygaW to Ala-Ala was examined, it was found that it showed the same sensitivity (MIC, 0.039 mg / ml) as LAX12. Next, the intracellular and extracellular L-Ala levels were measured in the presence of 6 mM Ala-Ala (FIG. 8).

  As a result, it was found that the intracellular L-Ala level of MLA301ΔygaW, which is a ygaW gene-deficient strain, was much higher than that of the parent strain, and the extracellular L-Ala level was lower than that of the parent strain. In addition, changes in intracellular and extracellular L-Ala levels caused by this ygaW gene deficiency were counteracted by introducing a recombinant plasmid pYgaW having the ygaW gene (FIG. 8). The time course of these extracellular L-Ala levels was almost the same as that of the LAX12 strain.

  From the above results, it was found that the ygaW gene product YgaW greatly contributed to E. coli L-Ala excretion. On the other hand, a disrupted strain was also produced for the ytfF gene, which is another function-unknown gene, and a similar experiment was performed. However, no significant difference was found between the parent strain MLA301 and the parent strain MLA301.

  FIG. 8A shows the time course of intracellular L-alanine concentration, and FIG. 8B shows the time course of extracellular L-alanine concentration. In FIG. 8, white circles indicate MLA301 strain, white triangles indicate MLA301ΔygaW, and white squares indicate MLA301ΔygaW into which pYgaW has been introduced.

  Further, when the nucleotide sequences of the ygaW gene in the LAX12 and LAX16 strains were determined, it was revealed that they were substitution mutant ygaW genes encoding mutant ygaW proteins having G125E mutation and S34F mutation, respectively. That is, in the amino acid sequence of the protein encoded by the ygaW gene shown in SEQ ID NO: 2, the 34th serine and the 125th glycine are amino acids that are very important for maintaining the function as an L-alanine excretion transporter. It was suggested that This 34th serine is contained in the transmembrane region located closest to the N-terminal among the four transmembrane regions contained in the protein encoded by the ygaW gene (see FIG. 1). The 125th glycine is contained in the transmembrane region located on the most C-terminal side among the four transmembrane regions contained in the protein encoded by the ygaW gene (see FIG. 1).

  As indicated by the loss of function due to amino acid substitution in these ygaW proteins, amino acid substitution that impairs the similarity of amino acids in the transmembrane region was shown to be a factor that significantly reduces the alanine transport ability. On the other hand, it is widely recognized that regions directly connected to such functions are highly conserved as a result of selective pressure in a direction that does not allow substitution in terms of molecular evolution. From this point of view, it can be said that those having a high sequence similarity of 80% or more and a low e-value in the amino acid sequence of this transmembrane region are highly likely to have an alanine transport function like YgaW.

4) Ala secretion of wild-type Escherichia coli by amplification of ygaW gene As a result of evaluating the L-Ala levels inside and outside the cells of the transformants having 4 cloned genes in the presence of Ala-Ala, each gene product was found to be L- It became clear that it was involved in the discharge of Ala (Fig. 7). However, this condition is an environment where a level of L-Ala that is much higher than the physiological intracellular L-Ala level exists, and does not reflect the ability to excrete L-Ala under physiological conditions. Therefore, in order to examine the physiological function of each gene product, the Ala excretion ability of transformants having these genes was examined using a wild strain having a normal intracellular amino acid level. Wild strain MG1655 into which recombinant plasmids pYeaS, pYddG, pYtfF and pYgaW were separately introduced was cultured in a minimal liquid medium.

  As a result, Ala was secreted into the medium only when the wild strain that originally did not secrete Ala introduced the ygaW gene (FIG. 9). This also showed that ygaW gene among the four genes contributes the most to Ala excretion even under physiological conditions.

  FIG. 9 shows only the results for MG1655 introduced with the recombinant plasmid pYgaW and MG1655 introduced with the vector pSTV29, and does not show MG1655 introduced with the recombinant plasmids pYeaS, pYddG and pYtfF. FIG. 9A shows the results of measuring the extracellular L-alanine concentration over time, and FIG. 9B shows the growth curve of each strain.

<Expression control analysis by RT-PCR>
For the four genes ygaW, ytfF, yddG and yeaS genes, the transcription levels in the presence or absence of Ala-Ala were analyzed using RT-PCR (FIG. 10). RT-PCR was performed by extracting total RNA from MLA301 cells incubated with Ala-Ala for 5 minutes and using it as a template. The reverse transcription reaction was performed using 300 ng of total RNA as a template and 2.5 U of AMV reverse transcriptase XL (Takara Bio Inc.). The reaction conditions were 30 ° C. for 10 minutes, 42 ° C. for 30 minutes, 95 ° C. for 5 minutes, and 4 ° C. for 5 minutes. PCR was performed using 0.5 μl of the obtained cDNA (amount corresponding to 15 ng of total RNA) and using Quick Taq HS DyeMix (manufactured by Toyobo). ygaWRT-F / ygaWRT-R, ytfFRT-F / ytfFRT-R, yddGRT-F / yddGRT-R and yeaSRT-F / yeaSRT-R are used as primer sets for PCR for ygaW, ytfF, yddG and yeaS genes, respectively. used. For comparison, gapART-F / gapART-R was used as a primer set for PCR of the gapA gene. The temperature conditions for PCR were 94 ° C for 2 minutes, 30 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 68 ° C for 30 seconds. For 5 minutes at 68 degrees. PCR products were analyzed by electrophoresis using 2% agarose gel.

The base sequences of the primers used are listed below.
yeaSRT-F 5'-CGGTTATCTTGCGGCCTG-3 '(SEQ ID NO: 51)
yeaSRT-R 5'-TTGCAGCGTCGCCAGTCG-3 '(SEQ ID NO: 52)
yddGRT-F 5'-AGGCGGTGACAATGGGTTAC-3 '(SEQ ID NO: 53)
yddGRT-R 5'-TTTAATCATGACGGGCGTGC-3 '(SEQ ID NO: 54)
ytfFRT-F 5'-GCAGGGTTGATGTGGGGG-3 '(SEQ ID NO: 55)
ytfFRT-R 5'-GAATGACCACCGGCAGGG-3 '(SEQ ID NO: 56)
ygaWRT-F 5'-TTCTCACCGCAGTCACGC-3 '(SEQ ID NO: 57)
ygaWRT-R 5'-CTGCTGGTAACGGCTGAC-3 '(SEQ ID NO: 58)
gapART-F 5'-TGAATGGCAAACTGACTGGTATGGC-3 '(SEQ ID NO: 59)
gapART-R 5'-AACCGGTTTCGTTGTCGTACCAGGA-3 '(SEQ ID NO: 60)
As a result, as shown in FIG. 10, it was found that the transcription of ygaW, ytfF and yeaS genes was promoted in the presence of Ala-Ala. Among them, the transcription of the ygaW gene was most strongly promoted. On the other hand, no change was observed in the transcription level of yddG regardless of the presence or absence of incubation with Ala-Ala. From these results, it was suggested that expression of ygaW, ytfF and yeaS genes was induced by an increase in intracellular L-Ala level in the presence of Ala-Ala.

  In FIG. 10, lanes 3, 5, 7, 9 and 11 are the results when the MLA301 strain was cultured in a minimal medium containing 6 mM Ala-Ala, and lanes 2, 4, 6, 8 and 10 These are the results when the MLA301 strain was cultured in a minimal medium not containing 6 mM Ala-Ala. Lane 1 is a molecular weight marker. Lanes 2 and 3 are the results of RT-PCR analysis of the ygaW gene, lanes 4 and 5 are the results of RT-PCR analysis of the ytfF gene, and lanes 6 and 7 are the results of RT-PCR analysis of the yddG gene. Lanes 8 and 9 are the results of RT-PCR analysis of the yeaS gene, and lanes 10 and 11 are the results of RT-PCR analysis of the gapA gene.

<Application of ygaW gene to Ala fermentation production>
Since Ala secretion was observed in the transformant in which the ygaW gene cloned into the multicopy vector was introduced into the wild-type E. coli strain, it was decided whether or not ygaW gene amplification could be applied to Ala fermentation production. For this purpose, the alanine dehydrogenase gene (alaD) (Kuroda, S. et al., 1990, Biochemistry 29: 1009-1015) derived from Bacillus sphaericus, which performs reductive amination of pyruvate, and the multicopy of the ygaW gene It was cloned on the vector pSTV29 and introduced into E. coli wild strain MG1655.

First, a gene fragment containing the open reading frame region of the alaD gene derived from Bacillus sphaericus ATCC10208 and the downstream region of the alaD gene was amplified by PCR. In this PCR, BSalaD-F (5'-GATGTTTAGATAAATGAAGATTGGTATTCC-3 '(SEQ ID NO: 61)) and BSalaD-R (5'-AAA CTGCAG CTAATCCACCATAAATG-3' (SEQ ID NO: 62) are used as a pair of primers, and the underlined portion recognizes PstI. Array) was used. The promoter region of the ldh gene derived from Streptococcus bovis JCM5802 was amplified by PCR. It is known that the promoter region of this ldh gene functions in the bacterium. In this PCR, as a pair of primers, SBPldh-F (5′-GCT GGATCC ATAAATTGATGAATCAC-3 ′ (SEQ ID NO: 63), the underlined portion is a BamHI recognition sequence) and SBPldh-R (5′-GGAATACCAATCTTCATTTATCTAAACATC-3 ′ (SEQ ID NO: 64 )) And used.

  Thereafter, the obtained mixture of the two PCR products was fused by PCR using as a template. In this PCR, SBPldh-F and BSalaD-R were used as primer sets. The obtained PCR product was treated with BamHI and PstI, and then cloned into pSTV29 after treatment with BamHI and PstI. The obtained plasmid was designated as pAlaD.

  Next, the ygaW gene fragment amplified by PCR using the above primer sets ygaW-F and ygaW-R was treated with BamHI and cloned into the BamHI site in pAlaD so as to be in the opposite direction to the alaD gene. The obtained plasmid was designated as pAlaD-YgaW.

  An Ala fermentation production test was performed using E. coli wild strain MG1655 transformed with pAlaD-YgaW. Ala fermentation production was performed anaerobically using an L liquid medium (pH 7.2) supplemented with 0.5% glucose, 1% ammonium chloride and 100 mM MOPS. As a result, it was observed that the Ala production amount of the strain into which both alaD and ygaW genes were introduced was higher than that of the strain into which only the alaD gene was introduced (FIG. 11A). Accordingly, the yield to sugar (g / g glucose) was also improved from 22.5% (MG1655 / pAlaD) to 32.7% (MG1655 / pAlaD-YgaW).

  On the other hand, there was no significant difference between the two strains in terms of growth and glucose consumption (FIGS. 11B and C). From this result, it was proved that the enhancement of Ala discharge ability by amplification of the ygaW gene leads to the improvement of the production efficiency of the Ala production strain.

  In FIG. 11, the white circle is MG1655 into which the vector pSTV29 is introduced, the white triangle is MG1655 into which pAlaD is introduced, and the white square is MG1655 into which pAlaD-YgaW is introduced. FIG. 11A shows the results of measuring the L-alanine concentration of each strain over time, FIG. 11B shows the results of measuring the glucose consumption of each strain over time, and FIG. 11C shows the growth curve of each strain.

Claims (8)

  A recombinant microorganism modified so that expression of the ygaW gene or a gene functionally equivalent to the gene is enhanced relative to a microorganism having L-alanine producing ability. The recombinant microorganism (a) shown in SEQ ID NO: 2 according to claim 1, wherein the ygaW gene and a gene functionally equivalent to the gene are any of the following (a) to (c): A gene encoding a protein containing an amino acid sequence (b) a gene encoding a protein comprising an amino acid sequence having 70% or more sequence similarity to the amino acid sequence shown in SEQ ID NO: 2 and having an L-alanine excretion transport function ( c) A gene encoding a protein capable of hybridizing under stringent conditions to a complementary strand of DNA consisting of all or part of the base sequence shown in SEQ ID NO: 1 and having an L-alanine excretion transport function   The gene functionally equivalent to the ygaW gene has four transmembrane regions consisting of amino acid sequences having a sequence similarity of 80% or more in total with respect to the amino acid sequences shown in SEQ ID NOs: 33, 34, 35 and 36. The recombinant microorganism according to claim 1, which is a gene encoding a protein.   The recombinant microorganism according to claim 1, wherein the functionally equivalent gene is a gene derived from an organism other than E. coli.   The recombinant microorganism according to claim 1, wherein the microorganism having L-alanine-producing ability is originally a microorganism having L-alanine-producing ability.   The microorganism having L-alanine-producing ability is a microorganism in which a gene involved in the L-alanine biosynthetic pathway is introduced into a microorganism that does not inherently have L-alanine-producing ability. The recombinant microorganism described.   The recombinant microorganism according to claim 1, wherein the microorganism capable of producing L-alanine is Escherichia coli.   A method for producing L-alanine, comprising culturing the recombinant microorganism according to any one of claims 1 to 7 in a medium, and recovering L-alanine from the medium during or after completion of the culture.

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