Genetically modified microorganism and method for producing organic acid

Abstract

A genetically modified microorganism comprising a gene capable of expressing a variant YeeX protein or a homolog thereof in which one to several amino acids are substituted, inserted and / or deleted in the amino acid sequence of a wild-type YeeX protein or a homolog thereof has improved organic acid production capacity compared to the microorganism before the genetic modification.

Description

Technical Field

[0001] The present invention relates to a genetically modified microorganism capable of producing a high amount of organic acid and a method for producing an organic acid using the genetically modified microorganism. Background Art

[0002] Organic acids obtained using microbial fermentation production processes are widely used in industry. For example, succinic acid is used in a variety of products such as pharmaceutical and food additives and bath preparations, while acetic acid is widely used as a food and reagent. As a method for producing organic acids including succinic acid and acetic acid using microorganisms, Patent Document 1 discloses a method in which, in order to reduce the effects of fermentation inhibitors when using sugar solutions obtained from non-edible resources as raw materials, microorganisms are used in which the expression of at least one gene selected from the group consisting of the following genes is enhanced compared to a non-modified strain: genes encoding phosphoglycerate kinase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and fumarate hydratase, respectively; genes encoding proteins constituting the phosphotransferase system; and genes encoding oxidative stress response factors.

[0003] Furthermore, 3-hydroxyadipic acid (IUPAC name: 3-hydroxyhexanedioic acid), α-hydrogenated hexanedioic acid (IUPAC name: (E)-hex-2-enedioic acid), and adipic acid (IUPAC name: hexanedioic acid), all dicarboxylic acids with 6 carbon atoms, are also attracting industrial attention. These can be used as raw materials for polyamides by polymerizing with polyamines. Furthermore, by adding amino groups to their ends to form lactams, they can also be used independently as raw materials for polyamides. Patent Document 2 discloses a method for producing 3-hydroxyadipic acid and other substances using microorganisms: a method for introducing a nucleic acid encoding a polypeptide involved in the production of 3-hydroxyadipic acid and α-hydrogenated hexanedioic acid, or a genetically modified microorganism in which the expression of the polypeptide is enhanced, and a method for producing a substance using the microorganism.

[0004] Meanwhile, YeeX proteins are classified as DUF496 family proteins. While little information is known about YeeX proteins, Non-Patent Document 1 confirms the expression of YeeX proteins in Escherichia coli by two-dimensional SDS-PAGE analysis. Patent Document 3 describes YeeX proteins as an example of proteins that assist in protein production. However, no literature describes the effects of YeeX proteins on organic acid production.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Laid-Open No. 2017-192325

[0008] Patent Document 2: WO2019 / 107516

[0009] Patent Document 3: US2007 / 0298418

[0010] Non-patent literature

[0011] Non-patent document 1: FEMS Microbiology Letters, vol. 169, pp. 375-382 (1998) Summary of the Invention

[0012] Problems to be solved by the invention

[0013] In the production of chemical products such as organic acids using microorganisms, improving the yield of products is a challenge. Specifically, introducing mutations into microorganisms to improve the production capacity of chemical products such as organic acids is a challenge.

[0014] Means for solving problems

[0015] The present inventors focused on genes encoding YeeX proteins, whose functions are unknown, and conducted intensive research to address the aforementioned issues. As a result, they discovered that microorganisms containing genes capable of expressing mutant YeeX proteins or homologs thereof have enhanced production capacity for chemicals, including organic acids, leading to the completion of the present invention.

[0016] Specifically, the present invention is composed of the following (1) to (10).

[0017] (1) A genetically modified microorganism comprising a gene capable of expressing a mutant YeeX protein or a homolog thereof in which one or more amino acids are substituted, inserted, and / or deleted in the amino acid sequence of a wild-type YeeX protein or a homolog thereof.

[0018] (2) The genetically modified microorganism according to (1), wherein the variant YeeX protein or its homolog is a variant YeeX protein or its homolog after substitution, insertion and / or deletion of one or more amino acids in the region corresponding to amino acid residues 74 to 100 in the amino acid sequence of SEQ ID NO: 1.

[0019] (3) The genetically modified microorganism according to (1) or (2), wherein the variant YeeX protein or its homolog has a mutation in which the alanine at position 84 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0020] (4) The genetically modified microorganism according to any one of (1) to (3), which has a gene capable of expressing the mutant YeeX protein or a homolog thereof in its genome.

[0021] (5) The genetically modified microorganism according to (4), wherein the gene capable of expressing the wild-type YeeX protein or its homolog on the genome is mutated or replaced with a gene capable of expressing the mutant YeeX protein or its homolog.

[0022] (6) The genetically modified microorganism according to any one of (1) to (5), wherein the mutant YeeX protein or a homolog thereof has a mutation in which alanine at position 84 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine.

[0023] (7) The genetically modified microorganism according to any one of (1) to (6), wherein the microorganism has an ability to produce an organic acid.

[0024] (8) A genetically modified microorganism according to any one of (1) to (7), wherein the microorganism is a microorganism belonging to the genus Serratia, Escherichia, Actinobacillus, Basfia, Pseudomonas, Hafnia, Acinetobacter, Shimwellia or Aerobacter.

[0025] (9) A method for producing an organic acid, comprising culturing the genetically modified microorganism described in (7) or (8) in a culture medium containing a carbon source as a fermentation raw material.

[0026] (10) The method for producing an organic acid according to (9), wherein the organic acid is succinic acid, acetic acid, 3-hydroxyadipic acid, α-hydrogenated adiponic acid and / or adipic acid.

[0027] Effects of the Invention

[0028] The genetically modified microorganisms of the present invention contain a gene capable of expressing a mutant YeeX protein or a homolog thereof, and thus can produce chemicals such as organic acids at a higher yield than the microorganisms before genetic modification. DETAILED DESCRIPTION

[0029] Hereinafter, the present invention will be described in more detail. However, the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the gist of the present invention.

[0030] The genetically modified microorganism of the present invention is characterized by comprising a gene capable of expressing a mutant YeeX protein or a homolog thereof. Furthermore, the method for producing an organic acid of the present invention is characterized by culturing the genetically modified microorganism.

[0031] YeeX protein belongs to the DUF496 family of proteins. "YeeX" refers to the gene encoding the YeeX protein. HHPred (Homology Detection & Structure Prediction by HMM-HMM Comparison) search results, based on secondary structure prediction of the amino acid sequence of SEQ ID NO: 1, indicate that the YeeX protein has a structure similar to that of a transcriptional regulator.

[0032] A homolog of a YeeX protein refers to a wild-type protein that has a high sequence identity to the amino acid sequence identified as the YeeX protein and is presumed to have a function or structure similar to that of the YeeX protein. The YeeX protein utilized in the present invention preferably exhibits a sequence identity of 50% or greater, more preferably 55% or greater, even more preferably 70% or greater, even more preferably 80% or greater, even more preferably 90% or greater, and particularly preferably 95% or greater, relative to the amino acid sequence of SEQ ID NO: 1.

[0033] Examples of YeeX proteins or homologs thereof include the YeeX protein from Escherichia coli (NCBI Protein ID: NP_416511, SEQ ID NO: 1), a homolog of the YeeX protein from Serratia grimesii (NCBI Protein ID: HCJ99940, SEQ ID NO: 2), a homolog of the YeeX protein from Acinetobacter baumanii (NCBI Protein ID: AAL09094, SEQ ID NO: 3), a homolog of the YeeX protein from Actinobacillus succinogenes (NCBI Protein ID: WP_012072666, SEQ ID NO: 4), and a homolog of the YeeX protein from Aerobacter cloacae (NCBI Protein ID: WP_012072667, SEQ ID NO: 5). ID: WP_115875767, sequence number 5), a homolog of the YeeX protein from Basfia succiniciproducens (Protein ID: WP_011200744, sequence number 6), a homolog of the YeeX protein from Hafnia paralvei (Protein ID: WP_004089583, sequence number 7), a homolog of the YeeX protein from Pseudomonas aeruginosa (Protein ID: MXH34489, sequence number 8), a homolog of the YeeX protein from Shimwellia blattae (Protein ID: WP_002439990, sequence number 9), etc.

[0034] In the present invention, "sequence homology" refers to the ratio (percentage) of identical amino acids or bases relative to all amino acid sequences (including amino acids as translation start points) or base sequences (including start codons) of overlapping (overlap) in the best alignment (alignment) when a gap is introduced or not introduced in two amino acid sequences or base sequences, calculated according to formula (1). Sequence homology can be easily studied using the algorithm BLAST (Basic Local Alignment Search Tool) commonly used in the art. For example, regarding BLAST, anyone can use it on websites such as NCBI (National Center for Biotechnology Information) or KEGG (Kyoto Encyclopedia of Genes and Genomes) and use default parameters to easily study sequence homology.

[0035] Sequence homology (%) = number of identical sequences (ignoring the gaps) / length of the shorter sequence (excluding the length of the gap) × 100 ···Formula (1).

[0036] According to formula (1), when the sequence identity between the amino acid sequences described in SEQ ID NOs. 1 to 9 was calculated using the Genetyx function (% Identity Matrix), the lowest sequence identity value was 54.46% between SEQ ID NOs. 4 and 9, indicating that the amino acid sequences described in SEQ ID NOs. 1 to 9 share at least 50% sequence identity with each other. The results of the sequence identity calculation using Genetyx are shown in Table 1. In Table 1 below, the leftmost number represents the sequence number.

[0037] [Table 1]

[0038]

[0039] The gene encoding the YeeX protein or its homolog is not particularly limited, as long as it is the amino acid sequence described in SEQ ID NOs. 1 to 9 or a base sequence that can be translated into the amino acid sequence of these homologs. It can be determined by referring to the codons corresponding to each amino acid (standard genetic code). In this case, the base sequence can also be redesigned using codons commonly used in the host microorganism used in the present invention.

[0040] Specific examples of the base sequences of genes encoding polypeptides having the amino acid sequences described in SEQ ID NOs. 1 and 2 include the base sequences described in SEQ ID NOs. 10 and 11, respectively.

[0041] One characteristic of the YeeX protein and its homologs is that the region corresponding to amino acid residues 74 to 100 in the amino acid sequence of SEQ ID NO: 1 is predicted to form an α-helical structure, and the arrangement shares high homology between species. Protein secondary structure can be easily studied by analyzing the amino acid sequence using the well-known web application Quick2D, etc. Another characteristic of the YeeX protein and its homologs is that the amino acid residue corresponding to position 84 in the amino acid sequence of SEQ ID NO: 1 is alanine. Multiple alignments of the YeeX proteins and their homologs having the amino acid sequences of SEQ ID NOs: 1 to 9 shown in Table 2 demonstrate that the region corresponding to amino acid residues 74 to 100, which are predicted to form an α-helical structure, and the alanine corresponding to position 84 in the amino acid sequence of SEQ ID NO: 1 are conserved.

[0042] [Table 2]

[0043]

[0044] The mutant YeeX protein or homolog thereof of the present invention is characterized by having a mutation resulting from substitution, insertion, and / or deletion of one to several amino acids, specifically one to ten, preferably one to five, more preferably one to three, even more preferably one to three, even more preferably one or two, and particularly preferably one amino acid, in the amino acid sequence of the wild-type YeeX protein or homolog thereof. The site of mutation is not particularly limited, but is preferably a region corresponding to amino acid residues 74 to 100 in the amino acid sequence of SEQ ID NO: 1, which has high interspecies sequence homology and is predicted to form an α-helical structure. More preferably, it is a region corresponding to amino acid residues 74 to 94, even more preferably, a region corresponding to amino acid residues 75 to 88, even more preferably, a region corresponding to amino acid residues 82 to 88, and particularly preferably, alanine at amino acid residue 84.

[0045] When the mutation site in the mutant YeeX protein or its homolog is alanine, which corresponds to amino acid residue 84 in the amino acid sequence of SEQ ID NO: 1, the mutation is preferably substituted with an amino acid other than alanine. The amino acid other than alanine is any amino acid residue, preferably valine, leucine, isoleucine, proline, glycine, methionine, or phenylalanine, which are hydrophobic amino acid residues, more preferably valine, leucine, or isoleucine, and even more preferably valine.

[0046] The method for expressing the mutant YeeX protein or its homolog in the genetically modified microorganism of the present invention is not particularly limited. Specific examples include the following methods: introducing a mutation into a gene encoding an endogenous YeeX protein or its homolog by a known method; introducing a gene encoding a YeeX protein or its homolog using an expression vector capable of autonomously replicating in a microorganism; introducing a gene encoding a mutant YeeX protein or its homolog using a method such as homologous recombination in the genome of a microorganism; or placing a gene encoding an endogenous YeeX protein or its homolog in a modified microorganism. In the case of a gene encoding a variant YeeX protein or a homolog thereof, the following methods are preferred in the genetically modified microorganisms of the present invention: a method in which the gene is assembled into an expression vector autonomously replicable in the microorganism and introduced into the microorganism; or a method in which a gene encoding a variant YeeX protein or a homolog thereof is introduced into the genome of the microorganism by introducing a mutation into a gene encoding an endogenous YeeX protein or a homolog thereof, homologous recombination into the genome of the microorganism, or the like, thereby replacing the gene encoding the variant YeeX protein or a homolog thereof in the genome of the host microorganism.

[0047] The microorganism used in the present invention is not particularly limited as long as it is a genetically modified microorganism having a gene encoding a mutant YeeX protein or a homolog of a mutant YeeX protein. Preferably, it is a microorganism having the ability to produce a chemical product, more preferably a microorganism having the ability to produce an organic acid or an amino acid, and even more preferably a microorganism having the ability to produce an organic acid. Specifically, it is preferably selected from the genus Serratia, Escherichia, Actinobacillus, Basfia, Pseudomonas The present invention also provides a microorganism belonging to the group consisting of the genus Pseudomonas, the genus Hafnia, the genus Acinetobacter, the genus Shimwellia or the genus Aerobacter, more preferably a microorganism belonging to the genus Serratia, the genus Escherichia, the genus Actinobacillus or the genus Basfia, and particularly preferably a microorganism belonging to the genus Serratia or the genus Escherichia.

[0048] When the genetically modified microorganism of the present invention has the ability to produce organic acids, it is characterized by having superior organic acid productivity compared to the microorganism before genetic modification by including a gene capable of expressing a mutant YeeX protein or a homolog thereof. Here, "excellent organic acid productivity" means that, under the same host microorganism and the same fermentation conditions, the organic acid is produced at a higher yield than a microorganism strain containing a gene capable of expressing only the wild-type YeeX protein of SEQ ID NO: 1, or a microorganism strain in which the gene capable of expressing the YeeX protein is defective. In the method for producing organic acids using the genetically modified microorganism of the present invention, the acetic acid yield is calculated according to formula (2). The succinic acid yield, 3-hydroxyadipic acid yield, α-hydrogenated adipate yield, or adipic acid yield are calculated by replacing the acetic acid in formula (2) with succinic acid, 3-hydroxyadipic acid, α-hydrogenated adipate, or adipic acid, respectively.

[0049] Yield (%) = acetic acid (mol) / carbon source consumption (mol) × 100···Formula (2).

[0050] In the present invention, when a gene encoding a mutant YeeX protein to be expressed is incorporated into an expression vector, the expression vector preferably comprises a promoter, a ribosome binding sequence, a gene encoding the protein to be expressed, and a transcription termination sequence.

[0051] When assembling a gene encoding a variant YeeX protein into the genome of a microorganism, the genome assembly nucleic acid preferably comprises a promoter, a ribosome binding sequence, a gene encoding the protein to be expressed, and a transcription termination sequence, so that the gene encoding the variant YeeX protein or its homolog is assembled to replace the wild-type YeeX protein or its homolog that the microorganism has traditionally harbored. A gene that controls promoter activity may also be included.

[0052] The promoter used in the present invention is not particularly limited as long as it can express the enzyme in the microorganism, and examples thereof include gap promoter, trp promoter, lac promoter, tac promoter, and T7 promoter.

[0053] In the present invention, when using an expression vector to introduce genes or express proteins, there is no particular limitation as long as it can replicate autonomously in the microorganism. Examples include pBBR1MCS vector, pBR322 vector, pMW vector, pET vector, pRSF vector, pCDF vector, pACYC vector, and derivatives of the above vectors.

[0054] In the present invention, when a nucleic acid for genome assembly is used to introduce a gene or express a protein, site-specific homologous recombination is used for introduction. The method for site-specific homologous recombination is not particularly limited, and examples thereof include a method using λ Red recombinase and the sacB gene (Biosci. Biotechnol. Biochem. 2007; 71(12): 2905-2911) and a method using λ Red recombinase and FLP recombinase (Proc. Natl. Acad. Sci. USA 2000; 97(12): 6640-6645).

[0055] The method for introducing an expression vector or a nucleic acid for genome assembly is not particularly limited as long as it is a method for introducing a nucleic acid into a microorganism, and examples thereof include: electroporation (J. Bacteriol. 1988; 170: 2796-2801.); calcium ion method (J. Mol. Biol. 1970; 53(1): 159-162.); and the like.

[0056] In the case where the genetically modified microorganism of the present invention has the ability to produce organic acids, the organic acid produced is not particularly limited as long as it is an organic acid that the microorganism can produce and accumulate in the culture medium. Specifically, carboxylic acids such as acetic acid, succinic acid, formic acid, pyruvic acid, fumaric acid, malic acid, oxaloacetic acid, citric acid, levulinic acid, 3-oxoadipic acid, 3-hydroxyadipic acid, α-hydrogenated adipate, adipic acid, and 2,5-furandicarboxylic acid can be listed. Among these carboxylic acids, acetic acid, succinic acid, 3-hydroxyadipic acid, α-hydrogenated adipate, and / or adipic acid are preferred from the perspective of significantly observing the effects of the present invention. In this specification, 3-hydroxyadipic acid is sometimes referred to as 3HA, α-hydrogenated adipate is sometimes referred to as HMA, and adipic acid is sometimes referred to as ADA.

[0057] Organic acids are produced via the reaction pathways inherent in the genetically modified microorganisms of the present invention. Specifically, 3HA, HMA, and ADA are produced via the reaction pathway shown in Scheme 1 below. When fermenting these organic acids, microbial strains expressing enzymes catalyzing Reaction A, Reaction B, Reaction C, Reaction D, Reaction E, Reaction F, and Reaction G are used.

[0058] [Chemical Formula 1]

[0059]

[0060] Route 1 above is an example of the reaction pathway required for producing 3HA, HMA, and / or ADA. Here, Reaction A represents the reaction of producing 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA. Reaction B represents the reaction of producing 3-hydroxyadipyl-CoA from 3-oxoadipyl-CoA. Reaction C represents the reaction of producing 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA. Reaction D represents the reaction of producing adipyl-CoA from 2,3-dehydroadipyl-CoA. Reaction E represents the reaction of producing 3HA from 3-hydroxyadipyl-CoA. Reaction F represents the reaction of producing HMA from 2,3-dehydroadipyl-CoA. Reaction G represents the reaction of producing ADA from adipyl-CoA.

[0061] Specific examples of the enzymes that catalyze these reactions include acyltransferases that catalyze reaction A, 3-oxoadipyl-CoA reductases that catalyze reaction B, enoyl-CoA hydratases that catalyze reaction C, enoyl-CoA reductases that catalyze reaction D, and CoA transferases that catalyze reactions E, F, and G.

[0062] Specific examples of genes encoding an enzyme that catalyzes Reaction A include acyltransferase pcaF (NCBI Gene ID: 1041755, SEQ ID NO: 13) derived from Pseudomonas putida KT2440 strain.

[0063] Specific examples of genes encoding an enzyme that catalyzes Reaction B include 3-oxoadipyl-CoA reductase from Serratia marcescens ATCC13880 (NCBI Gene ID: JMPQ01000047.1, SEQ ID NO: 14).

[0064] Specific examples of genes encoding an enzyme that catalyzes Reaction C include enoyl-CoA hydratase paaF (NCBI Gene ID: 1046932, SEQ ID NO: 15) derived from Pseudomonas putida KT2440 strain.

[0065] Specific examples of genes encoding an enzyme that catalyzes Reaction D include enoyl-CoA reductase dcaA (NCBI-Protein ID: AAL09094.1, SEQ ID NO: 16) derived from Acinetobacter baylyi strain ADP1.

[0066] Specific examples of genes encoding enzymes that catalyze reactions E, F, and G include full-length contiguous sequences comprising pcaI and pcaJ (NCBI Gene IDs: 1046613, 1046612, SEQ ID NOs: 17, 18) from Pseudomonas putida strain KT2440. The polypeptides encoded by pcaI and pcaJ catalyze reactions E, F, and G by forming a complex.

[0067] The gene encoding the enzyme catalyzing reactions A to G may be a gene that the microorganism has always possessed, or may be artificially introduced. The method for introducing the gene is not particularly limited, and methods such as assembling the gene into an expression vector that can autonomously replicate in the microorganism and introducing it into the microorganism, or assembling the gene into the genome of the microorganism can be used.

[0068] In the present invention, the genetically modified microorganism is cultured in a culture medium containing a carbon source usable by a conventional microorganism as a fermentation feedstock, preferably a liquid culture medium, to produce an organic acid. In addition to the carbon source usable by the genetically modified microorganism, a culture medium further containing a nitrogen source, inorganic salts, and, as needed, organic micronutrients such as amino acids or vitamins may also be used. As long as these nutrient sources are present, both natural and synthetic culture media may be used.

[0069] Fermentation raw materials refer to raw materials that can be metabolized by the genetically modified microorganism. "Metabolism" refers to the conversion of a chemical substance absorbed from outside the cell by the microorganism or generated from other chemical substances inside the cell into other chemical substances through an enzymatic reaction. As a carbon source, sugars are preferably used. In addition, in addition to sugars, as long as it can be used as a single carbon source for the growth of the above-mentioned genetically modified microorganisms, it can be preferably used. Specific examples of preferred carbon sources include: monosaccharides such as glucose, fructose, galactose, mannose, xylose, arabinose, etc.; disaccharides such as sucrose formed by the combination of these monosaccharides; polysaccharides; and starch saccharification liquid, molasses, cellulose-containing biomass saccharification liquid, etc. containing these sugars.

[0070] Furthermore, when producing 3HA, HMA, and / or ADA, 3-hydroxyadipic acid, α-hydrogenated adipate, and / or adipic acid can be efficiently produced by adding succinic acid as a substrate for CoA transferase in addition to the sugars listed above.

[0071] The carbon sources listed above may be used alone or in combination. In the addition of the carbon source, the concentration of the carbon source in the culture medium is not particularly limited and can be appropriately set according to the type of carbon source, but preferably, the concentration is 5 to 300 g / L for sugars and 0.1 to 100 g / L for succinic acid.

[0072] Examples of nitrogen sources include ammonia gas, ammonia water, ammonium salts, urea, nitrates, and other supplementary organic nitrogen sources such as oil residues, soybean hydrolyzates, casein decomposition products, other amino acids, vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as tryptone, various fermentation cells and their hydrolyzates. The concentration of the nitrogen source in the culture medium is not particularly limited, but is preferably 0.1 to 50 g / L.

[0073] As inorganic salts used for culturing the genetically modified microorganism, for example, phosphates, magnesium salts, calcium salts, iron salts, and manganese salts can be appropriately added and used.

[0074] Regarding the culture conditions for genetically modified microorganisms producing organic acids, the culture medium composition, culture temperature, stirring rate, pH, aeration rate, inoculum size, etc., can be appropriately adjusted or selected based on the type of genetically modified microorganism and external conditions. If foaming occurs during liquid culture, a defoaming agent such as mineral oil, silicone oil, or a surfactant can be added to the culture medium as appropriate.

[0075] After the genetically modified microorganism culture produces an organic acid to a recoverable amount, the product produced can be recovered. For example, the culture can be stopped at a point when the cumulative amount increases appropriately, and the product can be isolated according to conventional methods for extracting fermentation products from the culture. Specifically, after separating the microorganisms by centrifugation or filtration, the product can be isolated from the culture by column chromatography, ion exchange chromatography, activated carbon treatment, crystallization, membrane separation, distillation, or the like. More specifically, examples include, but are not limited to, methods in which an acidic component is added to a salt of the product to recover the precipitate; methods in which the culture is concentrated using a reverse osmosis membrane or rotary evaporator to remove water and increase the concentration of the product, followed by recovery by distillation; methods in which the product and / or its salt is crystallized by cooling crystallization or adiabatic crystallization, followed by centrifugation or filtration to obtain crystals of the product and / or its salt; methods in which an alcohol is added to the culture to convert sugars into esters, followed by distillation to recover the esters, and then hydrolyzing the product to obtain the product. In addition, these recovery methods can be appropriately selected and optimized according to the physical properties of the product.

[0076] Example

[0077] (Reference Example 1) Preparation of Nucleic Acid for Assembling the Variant YeeX Protein Homolog Recorded in SEQ ID NO: 12 into the Genome of Serratia grimesii (S. grimesii) Strain NBRC13537

[0078] To introduce the gene encoding the variant YeeX protein homolog into the genome of S. grimesii, a method utilizing λRed recombinase and the sacB gene was used. The nucleic acid sequence required for assembling the nucleic acid into the genome was obtained by nucleic acid synthesis (manufactured by Genewiz). This nucleic acid sequence includes the upstream region 840b of the gene encoding the wild-type YeeX protein homolog on the genome of S. grimesii, the sacB gene, and the downstream region 840b of the gene encoding the kanamycin resistance gene and the wild-type YeeX protein homolog (SEQ ID NO: 19). Primers (SEQ ID NOs: 20 and 21) were designed for PCR amplification of the nucleic acid fragment obtained by nucleic acid synthesis, and PCR reactions were performed according to conventional methods. The resulting fragment was purified by agarose gel electrophoresis and a nucleic acid column purification kit (manufactured by Cytiva) for use in the preparation of the variant strain.

[0079] (Reference Example 2) Preparation of Plasmid 1

[0080] The pBBR1MCS-2 vector (MEKovach, (1995), Gene 166:175-176), which is autonomously replicable in Escherichia coli and S. grimesii, was digested with XhoI to generate pBBR1MCS-2 / XhoI. To incorporate a constitutively expressing promoter into this vector, primers (SEQ ID NOs: 23 and 24) were designed for PCR amplification of the upstream region 200b (SEQ ID NO: 22) of gapA (NCBI Gene ID: NC_000913.3) using genomic DNA from Escherichia coli (E. coli) str. K-12 substr. MG1655 as a template. PCR was performed according to conventional methods. The resulting fragment was ligated with pBBR1MCS-2 / XhoI using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Co., Ltd.) and introduced into E. coli DH5α. This plasmid was extracted from the obtained recombinant strain, and the base sequence of the plasmid was confirmed by a conventional method, and designated as pBBR1MCS-2::Pgap. Next, pBBR1MCS-2::Pgap was cleaved with ScaI to obtain pBBR1MCS-2::Pgap / ScaI.

[0081] To amplify the gene encoding the enzyme that catalyzes reaction A, primers (SEQ ID NOs: 25 and 26) were designed for PCR amplification of the full length of the acyltransferase gene pcaF (NCBI Gene ID: 1041755, SEQ ID NO: 13) using genomic DNA from pseudomonas putida strain KT2440 as a template. PCR reactions were performed according to conventional methods. The resulting fragment was ligated to pBBR1MCS-2::Pgap / ScaI using the In-Fusion HD Cloning Kit and introduced into the Escherichia coli DH5α strain. The plasmid was extracted from the resulting recombinant strain, and the plasmid, whose base sequence was confirmed by conventional methods, was designated pBBR1MCS-2::AT.

[0082] Next, pBBR1MCS-2::AT was cut using HpaI to obtain pBBR1MCS-2::AT / HpaI. In order to amplify the gene encoding the enzymes for catalytic reactions E, F, and G, primers (sequence numbers 27 and 28) for PCR amplification of the full-length continuous sequence comprising the CoA transferase genes pcaI and pcaJ (NCBI GeneID: 1046613, 1046612, sequence numbers 17 and 18) were designed using the genomic DNA of the Pseudomonas putida KT2440 strain as a template. PCR reactions were performed according to conventional methods. The resulting fragment and pBBR1MCS-2::AT / HpaI were connected using the In-Fusion HD Cloning Kit and introduced into E. coli DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose base sequence was confirmed by conventional methods was set to pBBR1MCS-2::ATCT.

[0083] pBBR1MCS-2::ATCT was cut using ScaI to obtain pBBR1MCS-2::ATCT / ScaI. In order to amplify the nucleic acid encoding the 3-oxoadipyl-CoA reductase that catalyzes reaction B, primers (sequence numbers 29 and 30) for amplifying the nucleic acid described in sequence number 14 were designed using the genomic DNA of the Serratia marcescens ATCC13880 strain as a template, and a PCR reaction was performed according to a conventional method. The resulting fragment and pBBR1MCS-2::ATCT / ScaI were connected using In-Fusion HD Cloning Kit (manufactured by Takara BioCo., Ltd.) and introduced into E. coli DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was set as plasmid 1.

[0084] (Reference Example 3) Preparation of Plasmid 2

[0085] pMW119 (manufactured by Nippon Gene Co., Ltd.) was digested with SacI to obtain pMW119 / SacI. To incorporate a constitutive expression promoter into this vector, primers (SEQ ID NOs: 31 and 32) were designed for PCR amplification of the upstream region 200b (SEQ ID NO: 22) of gapA (NCBI Gene ID: NC_000913.3) using genomic DNA from E. coli str. K-12 substr. MG1655 as a template, and a PCR reaction was performed according to conventional methods. The resulting fragment was ligated to pMW119 / SacI using the In-Fusion HD Cloning Kit (manufactured by TakaraBio Co., Ltd.) and introduced into E. coli DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid, whose base sequence was confirmed by conventional methods, was designated pMW119::Pgap.

[0086] Next, pMW119::Pgap was cut using SphI to obtain pMW119::Pgap / SphI. In order to amplify the gene encoding the enzyme that catalyzes reaction C, primers (sequence numbers 33 and 34) for PCR amplification of the full length of the enoyl-CoA hydratase gene paaF (NCBI Gene ID: 1046932, sequence number 15) were designed using the genomic DNA of the pseudomonas putida KT2440 strain as a template, and a PCR reaction was performed according to a conventional method. The resulting fragment and pMW119::Pgap / SphI were connected using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Co., Ltd.) and introduced into E. coli DH5α. The plasmid was extracted from the resulting recombinant strain and the base sequence was confirmed by a conventional method. The resulting plasmid was set to pMW119::EH.

[0087] pMW119::EH was cut using HindIII to obtain pMW119::EH / HindIII. In order to amplify the gene encoding the enzyme that catalyzes reaction D, primers (sequence numbers 35 and 36) for PCR amplification of the full length of dcaA (NCBI-Protein ID: AAL09094.1, sequence number 16) from Acinetobacter baylyi ADP1 strain were designed, and PCR reaction was performed according to a conventional method. The resulting fragment and pMW119::EH / HindIII were connected using In-Fusion HD Cloning Kit (Takara Bio Co., Ltd. system) and imported into E. coli DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was set as plasmid 2.

[0088] (Reference Example 4) Preparation of a plasmid (plasmid 3) for expressing the mutant YeeX protein homolog described in SEQ ID NO: 12

[0089] pMW119::Pgap described in Reference Example 3 was cleaved with KpnI to obtain pMW119::Pgap / KpnI. Primers (SEQ ID NOs: 38 and 39) were designed for PCR amplification of the full-length gene encoding the variant YeeX protein homolog described in SEQ ID NO: 12 (SEQ ID NO: 37), and PCR reactions were performed according to conventional methods. The resulting fragment was ligated with pMW119::Pgap / KpnI using the In-Fusion HD Cloning Kit and introduced into E. coli DH5α. This plasmid was extracted from the resulting recombinant strain, and the base sequence was confirmed by conventional methods, designated as plasmid 3.

[0090] (Reference Example 5) Preparation of Nucleic Acid for Integrating the Variant YeeX of SEQ ID NO: 44 into the Genome of Escherichia coli (E. coli) MG1655 Strain

[0091] In order to introduce the gene encoding the variant YeeX into the genome of E. coli MG1655 strain, a method utilizing λ Red recombinase and sacB gene was used. The nucleic acid sequence required for assembling the nucleic acid into the genome was obtained by nucleic acid synthesis (manufactured by Genewiz). The nucleic acid sequence included the upstream region 500b of the gene encoding wild-type YeeX on the genome of the E. coli strain, the sacB gene, and the downstream region 500b of the gene encoding the kanamycin resistance gene and wild-type YeeX (sequence number 45). Primers (sequence numbers 46 and 47) were designed for PCR amplification of the nucleic acid fragment obtained by nucleic acid synthesis, and PCR reactions were performed according to conventional methods. The obtained fragments were purified by agarose gel electrophoresis and a nucleic acid column purification kit (manufactured by Cytiva) for use in the preparation of variant strains.

[0092] (Reference Example 6) Preparation of a plasmid (plasmid 4) for expressing the mutant YeeX described in SEQ ID NO: 44

[0093] The pCDF-1b plasmid was digested with KpnI to obtain pCDF-1b / KpnI. Primers (SEQ ID NOs. 49 and 50) were designed for PCR amplification of the gene encoding the variant YeeX protein homolog described in SEQ ID NO. 44, as well as the entire upstream and downstream regions (SEQ ID NO. 48) of the gene. PCR reactions were performed according to conventional methods. The resulting fragments were ligated with pCDF-1b / KpnI using the In-Fusion HD Cloning Kit and introduced into E. coli DH5α. This plasmid was extracted from the resulting recombinant strain, and the sequence was confirmed using conventional methods, designated plasmid 4.

[0094] (Comparative Example 1) Cultivation of a S. grimesii strain having a gene encoding the protein described in SEQ ID NO: 2

[0095] As the parent strain having the wild-type YeeX protein homolog described in SEQ ID NO: 2, Serratia grimesii NBRC13537 was used, from which the gene encoding the glucose transporter PtsG (SEQ ID NO: 40) and the genes encoding the pyruvate kinases PykF and PykA (SEQ ID NOs: 41 and 42) were deleted.

[0096] A platinum ring of the S. grimesii parent strain was inoculated into 5 mL of LB medium adjusted to pH 7 (10 g / L Bacto tryptone (manufactured by Difco Laboratories), 5 g / L Bacto yeast extract (manufactured by Difco Laboratories), and 5 g / L sodium chloride), and cultured at 30°C and 120 min-1 with shaking for 18 hours. 0.15 mL of this culture solution was added to 15 mL of medium I (10 g / L glucose, 1 g / L ammonium sulfate, 50 mM potassium phosphate, 0.025 g / L magnesium sulfate, 0.0625 mg / L iron sulfate, 2.7 mg / L manganese sulfate, 0.33 mg / L calcium chloride, 1.25 g / L sodium chloride, 2.5 g / L Bacto tryptone, 1.25 g / L Bacto yeast extract) adjusted to pH 6.5 in a test tube with a screw cap, and cultured at 30°C, 120 min-1, and shaking for 48 hours.

[0097] After centrifugation and separation of bacterial cells from the culture solution, the supernatant was treated with a Millex-GV membrane (0.22 μm, PVDF, manufactured by Merck), and the permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 3.

[0098] [Quantitative Analysis Conditions of Glucose, Acetic Acid, and Succinic Acid by HPLC]

[0099] HPLC: Shimazu Prominence (manufactured by Shimadzu Corporation)

[0100] Column: Shodex Sugar SH1011 (Showa Denko K.K.), 300 mm long, 8 mm inner diameter, 6 μm particle size

[0101] Mobile phase: 0.05M sulfuric acid aqueous solution

[0102] Flow rate: 0.6 mL / min

[0103] Column temperature: 65°C

[0104] Detector: RI.

[0105] [Quantitative Analysis Conditions for 3HA, HMA, and ADA by LC-MS / MS]

[0106] HPLC: 1290 Infinity (manufactured by Agilent Technologies)

[0107] Column: Synergi hydro-RP (manufactured by Phenomenex), length 100 mm, inner diameter 3 mm, particle size 2.5 μm

[0108] Mobile phase: 0.1% formic acid aqueous solution / methanol = 70 / 30

[0109] Flow rate: 0.3 mL / min

[0110] Column temperature: 40°C

[0111] LC detector: DAD (210 nm)

[0112] MS / MS: Triple-Quad LC / MS (manufactured by Agilent Technologies)

[0113] Ionization method: ESI negative mode.

[0114] (Comparative Example 2) Preparation and Cultivation of S. grimesii / yeeX-deficient Strain with a Deficient Gene Encoding YeeX Protein

[0115] The pKD46 plasmid required for expressing the λRed recombinase was introduced into the S. grimesii strain described in Comparative Example 1 by electroporation. Following introduction, the strain was cultured at 30°C in LB agar medium containing 500 μg / mL ampicillin. The nucleic acid fragment prepared in Reference Example 1 was introduced into the resulting strain by electroporation. Following introduction, the strain was cultured at 30°C in LB agar medium containing 25 μg / mL kanamycin. The resulting recombinant strain was an S. grimesii / pKD46 / yeeX-deficient strain in which the full-length gene sequence encoding the YeeX protein homolog had been replaced with the recombination cassette sequence. To remove the pKD46 plasmid from the S. grimesii / pKD46 / yeeX-deficient strain, a platinum loopful of the strain was inoculated into 5 mL of LB medium (10 g / L Bacto Tryptone (Difco Laboratories), 5 g / L Bacto Yeast Extract (Difco Laboratories), and 5 g / L sodium chloride) and cultured at 37°C with shaking at 120 min for 48 hours. 10 μL of this culture was then cultured on LB agar at 30°C, and colonies lacking ampicillin resistance were selected to obtain the S. grimesii / yeeX-deficient strain.

[0116] A platinum loop of the strain was inoculated into 5 mL of LB medium (10 g / L Bacto tryptone (manufactured by Difco Laboratories), 5 g / L Bacto yeast extract (manufactured by Difco Laboratories), 5 g / L sodium chloride) containing 25 μg / mL kanamycin adjusted to pH 7, and cultured at 30°C and 120 min-1 with shaking for 18 hours. 0.15 mL of this culture solution was added to 15 mL of medium I (10 g / L glucose, 1 g / L ammonium sulfate, 50 mM potassium phosphate, 0.025 g / L magnesium sulfate, 0.0625 mg / L iron sulfate, 2.7 mg / L manganese sulfate, 0.33 mg / L calcium chloride, 1.25 g / L sodium chloride, 2.5 g / L Bacto tryptone, 1.25 g / L Bacto yeast extract) containing 25 μg / mL kanamycin adjusted to pH 6.5 in a test tube with a screw cap, and cultured at 30°C with shaking at 120 min-1 for 48 hours.

[0117] After centrifugation and separation of bacterial cells from the culture solution, the supernatant was treated with a Millex-GV membrane (0.22 μm, PVDF, manufactured by Merck), and the permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 3.

[0118] (Example 1) Preparation and Cultivation of a S. grimesii / YeeX Variant Possessing a Gene Encoding a Variant YeeX Protein Homolog (SEQ ID NO: 12)

[0119] To amplify the full length of a nucleic acid sequence (SEQ ID NO: 43), encompassing the upstream region 840b of the gene encoding the wild-type YeeX protein homolog in the genome of S. grimesii, the gene encoding the variant YeeX protein homolog described in SEQ ID NO: 12, and the downstream region 840b of the gene encoding the wild-type YeeX protein homolog, a PCR reaction was performed according to conventional methods using primers of SEQ ID NO: 20 and 21. The resulting fragment was purified by agarose gel electrophoresis and a nucleic acid column purification kit (manufactured by Cytiva) and used to generate variant strains.

[0120] This nucleic acid fragment (SEQ ID NO: 43) was introduced into the S. grimesii / yeeX-deficient strain containing the pKD46 plasmid prepared in Comparative Example 2 by electroporation. Following introduction, the strain was cultured at 30°C in Medium I agar containing 50 g / L sucrose. The resulting colonies were cultured at 30°C in LB agar and LB agar containing 25 μg / mL kanamycin, and strains lacking kanamycin resistance were selected. The resulting strain is a S. grimesii / YeeX variant strain obtained by replacing the gene encoding the YeeX protein homolog (SEQ ID NO: 2) without alanine substitution at position 84 of the amino acid sequence of SEQ ID NO: 1 with a gene encoding a variant YeeX protein homolog (SEQ ID NO: 12) in which alanine substitution at position 84 of the amino acid sequence of SEQ ID NO: 1 is replaced with valine.

[0121] This strain was cultured using the same method as in Comparative Example 1. The supernatant, after centrifugation and bacterial separation from the culture, was treated with a Millex-GV (0.22 μm, PVDF, manufactured by Merck) membrane. The permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 3.

[0122] (Comparative Example 3) Preparation and Culture of S. grimesii Strain Carrying Plasmid 1

[0123] Plasmid 1 prepared in Reference Example 2 was introduced into the S. grimesii strain described in Comparative Example 1 by electroporation. After introduction, the strain was cultured in LB agar medium containing 25 μg / mL kanamycin at 30° C. The resulting recombinant strain was designated S. grimesii / plasmid 1.

[0124] This strain was cultured using the same method as in Comparative Example 2. The supernatant, after centrifugation and bacterial separation from the culture, was treated with a Millex-GV (0.22 μm, PVDF, manufactured by Merck), and the permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 3.

[0125] (Example 2) Preparation and cultivation of S. grimesii / YeeX mutants harboring plasmid 1

[0126] Plasmid 1 prepared in Reference Example 2 was introduced into the S. grimesii / YeeX variant prepared in Example 1 by electroporation. After introduction, the strain was cultured at 30°C in LB agar medium containing 25 μg / mL kanamycin. The resulting recombinant strain was designated the S. grimesii / plasmid 1 / YeeX variant.

[0127] This strain was cultured using the same method as in Comparative Example 2. The supernatant, after centrifugation and bacterial separation from the culture, was treated with a Millex-GV (0.22 μm, PVDF, manufactured by Merck), and the permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 3.

[0128] (Example 3) Preparation and cultivation of S. grimesii strains harboring plasmid 1 and plasmid 3

[0129] Plasmid 3 prepared in Reference Example 4 was introduced into the S. grimesii / plasmid 1 strain prepared in Comparative Example 3 by electroporation. After introduction, the strain was cultured at 30°C in LB agar medium containing 25 μg / mL kanamycin and 500 μg / mL ampicillin. The resulting recombinant strain was designated S. grimesii / plasmid 1 / plasmid 3.

[0130] A platinum loop of the strain was inoculated into 5 mL of LB medium (10 g / L Bacto tryptone (manufactured by Difco Laboratories), 5 g / L Bacto yeast extract (manufactured by Difco Laboratories), 5 g / L sodium chloride) adjusted to pH 7 and containing 25 μg / mL kanamycin and 500 μg / mL ampicillin, and cultured at 30°C and 120 min-1 with shaking for 18 hours. 0.15 mL of this culture solution was added to 15 mL of medium I (10 g / L glucose, 1 g / L ammonium sulfate, 50 mM potassium phosphate, 0.025 g / L magnesium sulfate, 0.0625 mg / L iron sulfate, 2.7 mg / L manganese sulfate, 0.33 mg / L calcium chloride, 1.25 g / L sodium chloride, 2.5 g / L Bacto tryptone, 1.25 g / L Bacto yeast extract) containing 25 μg / mL kanamycin and 500 μg / mL ampicillin adjusted to pH 6.5 in a test tube with a screw cap, and cultured at 30°C with shaking at 120 min-1 for 48 hours.

[0131] The supernatant, obtained after centrifugation and separation of bacterial cells from the culture solution, was treated with a Millex-GV membrane (0.22 μm, PVDF, manufactured by Merck). The permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 3.

[0132] (Comparative Example 4) Preparation and Culture of S. grimesii Strains Possessing Plasmid 1 and Plasmid 2

[0133] Plasmid 2 prepared in Reference Example 3 was introduced into the S. grimesii / plasmid 1 strain prepared in Comparative Example 3 by electroporation. After introduction, the strain was cultured at 30°C in LB agar medium containing 25 μg / mL kanamycin and 500 μg / mL ampicillin. The resulting recombinant strain was designated the S. grimesii / plasmid 1 / plasmid 2 strain.

[0134] This strain was cultured using the same method as in Example 3. The supernatant, after centrifugation and separation of bacterial cells from the culture, was treated with a Millex-GV (0.22 μm, PVDF, manufactured by Merck) membrane, and the permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 3.

[0135] (Example 4) Preparation and cultivation of S. grimesii / YeeX mutants harboring plasmids 1 and 2

[0136] Plasmid 2 prepared in Reference Example 3 was introduced into the S. grimesii / YeeX variant prepared in Example 1 by electroporation. After introduction, the strain was cultured at 30°C in LB agar medium containing 25 μg / mL kanamycin and 500 μg / mL ampicillin. The resulting recombinant strain was designated the S. grimesii / plasmid 1 / plasmid 2 / YeeX variant.

[0137] This strain was cultured using the same method as in Example 3. The supernatant, after centrifugation and separation of bacterial cells from the culture, was treated with a Millex-GV (0.22 μm, PVDF, manufactured by Merck) membrane, and the permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 3.

[0138] [Table 3]

[0139]

[0140] It is apparent from the results of Comparative Examples 1 and 2 and Example 1 that the S. grimesii / YeeX mutant strain having a gene encoding a mutant YeeX protein homolog (SEQ ID NO: 12) in its genome produces organic acids such as succinic acid and acetic acid at a higher yield than the S. grimesii parent strain and the S. grimesii / YeeX-deficient strain having a gene encoding a wild-type YeeX protein homolog (SEQ ID NO: 2) in its genome.

[0141] The results of Comparative Example 3 and Example 2 show that even the S. grimesii / plasmid 1 / YeeX variant strain having plasmid 1 expressing the enzymes required for 3HA production has improved fermentation production yields of organic acids such as succinate, acetic acid, 3HA, and HMA compared to the parent strain having plasmid 1.

[0142] In Example 3, even in the S. grimesii / plasmid 1 / plasmid 3 strain, which harbors a gene encoding a wild-type YeeX protein homolog (SEQ ID NO: 2) in its genome and plasmid 3 and plasmid 1 expressing a mutant YeeX protein homolog (SEQ ID NO: 12), an improvement in organic acid yield was observed. This demonstrates that even in S. grimesii strains expressing both a wild-type YeeX protein homolog and a mutant YeeX protein homolog by introducing a mutant YeeX protein homolog using an expression plasmid, the effects of the present invention can be achieved.

[0143] According to the results of Comparative Example 4 and Example 4, it was found that even the S. grimesii / plasmid 1 / plasmid 2 / YeeX variant strain having plasmid 1 and plasmid 2 expressing the enzymes required for ADA production had significantly improved yields of organic acids such as succinic acid, acetic acid, 3HA, HMA and ADA compared to the parent strain having plasmid 1 and plasmid 2.

[0144] (Comparative Example 5) Cultivation of an E. coli strain harboring a gene encoding a wild-type YeeX protein (SEQ ID NO: 1)

[0145] Escherichia coli MG1655 was used as the parent strain harboring the wild-type YeeX protein described in SEQ ID NO: 1. This strain was cultured using the same method as in Comparative Example 1. The supernatant, after centrifugation of the culture broth to separate bacterial cells, was treated with a Millex-GV (0.22 μm, PVDF, manufactured by Merck) membrane, and the permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 4.

[0146] (Comparative Example 6) Preparation and Cultivation of E. coli / yeeX-deficient Strain in Which the Gene Encoding YeeX Protein is Deficient

[0147] The pKD46 plasmid required for expressing the λRed recombinase was introduced into the E. coli strain described in Comparative Example 5 by electroporation. Following introduction, the strain was cultured at 30°C in LB agar medium containing 50 μg / mL ampicillin. The nucleic acid fragment prepared in Reference Example 5 was introduced into the resulting strain by electroporation. Following introduction, the strain was cultured at 30°C in LB agar medium containing 25 μg / mL kanamycin. The resulting recombinant strain was an E. coli / pKD46 / yeeX-deficient strain in which the full-length gene sequence encoding YeeX was replaced with the recombination cassette sequence. To remove the pKD46 plasmid from the E. coli / pKD46 / yeeX-deficient strain, a platinum loopful of the strain was inoculated into 5 mL of LB medium (10 g / L Bacto Tryptone (Difco Laboratories), 5 g / L Bacto Yeast Extract (Difco Laboratories), and 5 g / L sodium chloride) and cultured at 37°C with shaking at 120 min⁻¹ for 48 hours. 10 μL of this culture was then cultured on LB agar at 30°C, and colonies lacking ampicillin resistance were selected to obtain the E. coli / yeeX-deficient strain.

[0148] This strain was cultured using the same method as in Comparative Example 2. The supernatant, after centrifugation and bacterial separation from the culture, was treated with a Millex-GV (0.22 μm, PVDF, manufactured by Merck) membrane. The permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 4.

[0149] (Example 5) Preparation and Cultivation of E. coli / YeeX Mutant Strain Possessing a Gene Encoding a Mutant YeeX Protein (SEQ ID NO: 44)

[0150] To amplify the full length of a nucleic acid sequence (SEQ ID NO: 48), encompassing the upstream region 500b of the gene encoding the wild-type YeeX protein (SEQ ID NO: 1) on the E. coli genome, the gene encoding the mutant YeeX protein described in SEQ ID NO: 44, and the downstream region 500b of the gene encoding the wild-type YeeX protein, a PCR reaction was performed according to conventional methods using primers of SEQ ID NO: 46 and 47. The resulting fragment was purified by agarose gel electrophoresis and a nucleic acid column purification kit (manufactured by Cytiva) and used to prepare mutant strains.

[0151] This nucleic acid fragment (SEQ ID NO: 48) was introduced by electroporation into the E. coli / yeeX-deficient strain harboring the pKD46 plasmid prepared in Comparative Example 6. Following introduction, the strain was cultured at 30°C on Medium I agar containing 50 g / L sucrose. The resulting colonies were cultured at 30°C on LB agar and LB agar containing 25 μg / mL kanamycin, and strains lacking kanamycin resistance were selected. The resulting strain is an E. coli / YeeX mutant strain in which the gene encoding the wild-type YeeX protein of SEQ ID NO: 1 on the genome is replaced with a gene encoding a mutant YeeX protein (SEQ ID NO: 44) in which the alanine at position 84 of the amino acid sequence of SEQ ID NO: 1 is substituted with valine.

[0152] This strain was cultured using the same method as in Comparative Example 1. The supernatant, after centrifugation and bacterial separation from the culture, was treated with a Millex-GV (0.22 μm, PVDF, manufactured by Merck) membrane. The permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 4.

[0153] (Comparative Example 7) Preparation and Culture of E. coli Strain Carrying Plasmid 1

[0154] Plasmid 1 prepared in Reference Example 2 was introduced into the E. coli strain described in Comparative Example 5 by electroporation. After introduction, the strain was cultured in LB agar medium containing 25 μg / mL kanamycin at 30° C. The resulting recombinant strain was designated E. coli / plasmid 1 strain.

[0155] This strain was cultured using the same method as in Comparative Example 2. The supernatant, after centrifugation and bacterial separation from the culture, was treated with a Millex-GV (0.22 μm, PVDF, manufactured by Merck) membrane. The permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 4.

[0156] (Example 6) Preparation and cultivation of E. coli / YeeX mutants harboring plasmid 1

[0157] Plasmid 1 prepared in Reference Example 2 was introduced into the E. coli / YeeX variant prepared in Example 5 by electroporation. After introduction, the strain was cultured at 30°C in LB agar medium containing 25 μg / mL kanamycin. The resulting recombinant strain was designated the E. coli / Plasmid 1 / YeeX variant.

[0158] This strain was cultured using the same method as in Comparative Example 2. The supernatant, after centrifugation and bacterial separation from the culture, was treated with a Millex-GV (0.22 μm, PVDF, manufactured by Merck) membrane. The permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 4.

[0159] (Example 7) Preparation and Culture of E. coli Strains Carrying Plasmid 1 and Plasmid 4

[0160] Plasmid 4 prepared in Reference Example 6 was introduced into the E. coli / plasmid 1 strain prepared in Comparative Example 7 by electroporation. After introduction, the strain was cultured at 30°C in LB agar medium containing 25 μg / mL kanamycin and 50 μg / mL streptomycin. The resulting recombinant strain was designated E. coli / plasmid 1 / plasmid 4.

[0161] A platinum loop of the strain was inoculated into 5 mL of LB medium (10 g / L Bacto tryptone (manufactured by Difco Laboratories), 5 g / L Bacto yeast extract (manufactured by Difco Laboratories), 5 g / L sodium chloride) adjusted to pH 7 and containing 25 μg / mL kanamycin and 50 μg / mL streptomycin, and cultured at 30°C and 120 min-1 with shaking for 18 hours. 0.15 mL of this culture solution was added to 15 mL of medium I (10 g / L glucose, 1 g / L ammonium sulfate, 50 mM potassium phosphate, 0.025 g / L magnesium sulfate, 0.0625 mg / L iron sulfate, 2.7 mg / L manganese sulfate, 0.33 mg / L calcium chloride, 1.25 g / L sodium chloride, 2.5 g / L Bacto tryptone, 1.25 g / L Bacto yeast extract) containing 25 μg / mL kanamycin and 50 μg / mL streptomycin adjusted to pH 6.5 in a test tube with a screw cap, and cultured at 30°C, 120 min-1, with shaking for 48 hours.

[0162] After centrifugation and separation of bacterial cells from the culture solution, the supernatant was treated with a Millex-GV membrane (0.22 μm, PVDF, manufactured by Merck), and the permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 4.

[0163] (Comparative Example 8) Preparation and Culture of E. coli Strains Carrying Plasmid 1 and Plasmid 2

[0164] Plasmid 2 prepared in Reference Example 3 was introduced into the E. coli / plasmid 1 strain prepared in Comparative Example 7 by electroporation. After introduction, the strain was cultured at 30°C in LB agar medium containing 25 μg / mL kanamycin and 50 μg / mL ampicillin. The resulting recombinant strain was designated the E. coli / plasmid 1 / plasmid 2 strain.

[0165] A platinum loop of the strain was inoculated into 5 mL of LB medium (10 g / L Bacto tryptone (manufactured by Difco Laboratories), 5 g / L Bacto yeast extract (manufactured by Difco Laboratories), 5 g / L sodium chloride) adjusted to pH 7 and containing 25 μg / mL kanamycin and 50 μg / mL ampicillin, and cultured at 30°C and 120 min-1 with shaking for 18 hours. 0.15 mL of this culture solution was added to 15 mL of medium I (10 g / L glucose, 1 g / L ammonium sulfate, 50 mM potassium phosphate, 0.025 g / L magnesium sulfate, 0.0625 mg / L iron sulfate, 2.7 mg / L manganese sulfate, 0.33 mg / L calcium chloride, 1.25 g / L sodium chloride, 2.5 g / L Bacto tryptone, 1.25 g / L Bacto yeast extract) containing 25 μg / mL kanamycin and 50 μg / mL ampicillin adjusted to pH 6.5 in a test tube with a screw cap, and cultured at 30°C with shaking at 120 min-1 for 48 hours.

[0166] After centrifugation and separation of bacterial cells from the culture solution, the supernatant was treated with a Millex-GV membrane (0.22 μm, PVDF, manufactured by Merck), and the permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 4.

[0167] (Example 8) Preparation and Culture of E. coli / YeeX Mutant Strain Possessing Plasmid 1 and Plasmid 2

[0168] Plasmid 2 prepared in Reference Example 3 was introduced into the E. coli / YeeX variant prepared in Example 5 by electroporation. Following introduction, the strain was cultured at 30°C in LB agar medium containing 25 μg / mL kanamycin and 50 μg / mL ampicillin. The resulting recombinant strain was designated the E. coli / plasmid 1 / plasmid 2 / YeeX variant.

[0169] This strain was cultured using the same method as in Comparative Example 8. The supernatant, after centrifugation and separation of bacterial cells from the culture solution, was treated with a Millex-GV (0.22 μm, PVDF, manufactured by Merck). The permeate was analyzed by HPLC and LC-MS / MS. The results of quantitative analysis of organic acids accumulated in the culture supernatant and the yield of organic acids calculated using formula (2) are shown in Table 4.

[0170] [Table 4]

[0171]

[0172] It is apparent from the results of Comparative Examples 5 and 6 and Example 5 that the E. coli / YeeX mutant strain having a gene encoding the mutant YeeX protein (SEQ ID NO: 44) in its genome produced organic acids such as succinic acid and acetic acid at higher yields than the E. coli parent strain and the E. coli / YeeX-deficient strain having a gene encoding the wild-type YeeX protein (SEQ ID NO: 1) in its genome.

[0173] The results of Comparative Example 7 and Example 6 show that even the E. coli / plasmid 1 / YeeX mutant strain having plasmid 1 expressing the enzymes required for 3HA production has improved fermentation production yields of organic acids such as succinic acid, acetic acid, and 3HA compared to the parent strain having plasmid 1.

[0174] In Example 6, even in the E. coli / plasmid 1 / plasmid 4 strain, which harbors a gene encoding the wild-type YeeX protein (SEQ ID NO: 1) in its genome and plasmids 4 and 1 expressing the mutant YeeX protein (SEQ ID NO: 44), an improvement in organic acid yield was observed. This demonstrates that the effects of the present invention can be achieved even in E. coli strains expressing both the wild-type and mutant YeeX proteins by introducing a mutant YeeX protein using an expression plasmid.

[0175] According to the results of Comparative Example 8 and Example 8, it was found that even the E. coli / plasmid 1 / plasmid 2 / YeeX mutant strain having plasmid 1 and plasmid 2 expressing the enzymes required for ADA production had significantly improved yields of organic acids such as succinic acid, acetic acid, 3HA, HMA, and ADA compared to the parent strain having plasmid 1 and plasmid 2. Sequence Listing <110> Toray Industries, Inc. <120> Genetically modified microorganism and method for producing organic acid <130> 21094WO01 <160> 50 <170> PatentIn version 3.5 <210> 1 <211> 109 <212> PRT <213> Escherichia coli MG1655 <400> 1 Met Glu Thr Thr Lys Pro Ser Phe Gln Asp Val Leu Glu Phe Val Arg 1 5 10 15 Leu Phe Arg Arg Lys Asn Lys Leu Gln Arg Glu Ile Gln Asp Val Glu 20 25 30 Lys Lys Ile Arg Asp Asn Gln Lys Arg Val Leu Leu Leu Asp Asn Leu 35 40 45 Ser Asp Tyr Ile Lys Pro Gly Met Ser Val Glu Ala Ile Gln Gly Ile 50 55 60 Ile Ala Ser Met Lys Gly Asp Tyr Glu Asp Arg Val Asp Asp Tyr Ile 65 70 75 80 Ile Lys Asn Ala Glu Leu Ser Lys Glu Arg Arg Asp Ile Ser Lys Lys 85 90 95 Leu Lys Ala Met Gly Glu Met Lys Asn Gly Glu Ala Lys 100 105 <210> 2 <211> 107 <212> PRT <213> Serratia grimesii NBRC13537 <400> 2 Met Lys Met Asp Asn Ala Asn Lys Pro Ser Phe Gln Asp Val Leu Glu 1 5 10 15 Phe Val Arg Met Phe Arg Arg Lys Asn Lys Leu Gln Arg Glu Ile Ile 20 25 30 Asp Asn Glu Lys Lys Val Arg Asp Asn Gln Lys Arg Val Leu Leu Leu 35 40 45 Asp Asn Leu Ser Glu Tyr Ile Lys Pro Gly Met Ser Ile Glu Asp Val 50 55 60 Gln Gly Ile Ile Gly Asn Met Arg Ser Asp Tyr Glu Asp Arg Val Asp 65 70 75 80 Asp Tyr Ile Ile Lys Asn Ala Asp Leu Ser Lys Glu Arg Arg Glu Leu 85 90 95 Ser Lys Lys Leu Lys Ala Met Gly Glu Val Lys 100 105 <210> 3 <211> 94 <212> PRT <213> Acinetobacter baumannii <400> 3 Phe Arg Arg Lys Asn Lys Leu Gln Arg Glu Ile Gln Asp Ile Glu Lys 1 5 10 15 Lys Ile Arg Asp Asn Gln Lys Arg Val Leu Leu Leu Asp Asn Leu Ser 20 25 30 Asp Tyr Ile Lys Pro Gly Met Ser Val Glu Ala Ile Gln Gly Ile Ile 35 40 45 Ala Ser Met Lys Ser Asp Tyr Glu Asp Arg Val Asp Asp Tyr Ile Ile 50 55 60 Lys Asn Ala Glu Ile Ser Lys Glu Arg Arg Asp Ile Ser Lys Lys Leu 65 70 75 80 Lys Ala Met Gly Glu Met Lys His Ala Asp Val Lys Ala Glu 85 90 <210> 4 <211> 114 <212> PRT <213> Actinobacillus succinogenes 130Z <400> 4 Met Glu Asn Val Asn Lys Gln Ser Phe Gln Glu Val Leu Glu Tyr Val 1 5 10 15 Arg Ile Asn Arg Gln Arg Asn Lys Leu Leu Arg Glu Ile Gly Asp Cys 20 25 30 Glu Arg Lys Ile Arg Asp Asn Lys Lys Arg Val Leu Leu Leu Asp Asn 35 40 45 Leu Thr Asp Tyr Ile Gln Asp Asn Met Thr Ile Glu Asp Ile Arg Ala 50 55 60 Ile Ile Asn Asn Met His Asp Asp Tyr Glu Asn Arg Val Asp Asp Tyr 65 70 75 80 Val Ile Lys Ala Ala Glu Leu Ser Lys Gln Arg Arg Asp Leu Lys Thr 85 90 95 Arg Met Lys Glu Leu Lys Ala Ser His Ala Ala Leu Ala Lys Lys Gly 100 105 110 Light Glu <210> 5 <211> 111 <212> PRT <213> Aerobacter cloacae <400> 5 Met Glu Thr Thr Lys Pro Ser Phe Gln Asp Val Leu Glu Phe Val Arg 1 5 10 15 Leu Phe Arg Arg Lys Asn Lys Leu Gln Arg Glu Ile Gln Asp Val Glu 20 25 30 Light Light Ile Arg Asp Asn Gln Light Arg Val Leu Leu Leu Asp Asn Leu 35 40 45 Ser Asp Tyr Ile Lys Pro Gly Met Ser Val Glu Ala Ile Gln Gly Ile 50 55 60 Ile Ala Ser Met Lys Ser Asp Tyr Glu Asp Arg Val Asp Asp Tyr Ile 65 70 75 80 Ile Lys Asn Ala Glu Leu Ser Lys Glu Arg Arg Asp Ile Ser Lys Lys 85 90 95 Leu Lys Val Met Gly Glu Ile Lys Asn Gly Glu Ala Lys Gly Glu 100 105 110 <210> 6 <211> 114 <212> PRT <213> Basfia succiniciproducens <400> 6 Met Glu Asn Val Asn Lys Gln Ser Phe Gln Asp Val Leu Glu Tyr Val 1 5 10 15 Arg Leu Tyr Arg Leu Arg Asn Lys Leu Leu Arg Asp Ile Gly Asp Asn 20 25 30 Asp Arg Lys Ile Arg Asp Asn Gln Lys Arg Val Leu Leu Leu Asp Asn 35 40 45 Leu Ser Gln Tyr Ile Thr Asn Asp Met Ser Val Glu Asp Ile Arg Ala 50 55 60 Ile Ile Glu Asn Met Arg Asp Asp Tyr Glu Gly Arg Val Asp Asp Tyr 65 70 75 80 Met Ile Arg Asn Ala Asp Leu Ser Lys Glu Arg Arg Glu Ile Lys Glu 85 90 95 Light Met Light Ala Gln Light Light Ala His Ala Glu Leu Leu Light Light Ala 100 105 110 Aspen Aspen <210> 7 <211> 107 <212> PRT <213> Hafnia paralvei <400> 7 Met Glu Asn Val Asn Lys Pro Thr Phe Gln Asn Val Leu Glu Phe Val 1 5 10 15 Arg Met Phe Arg Arg Lys Asn Lys Leu Gln Arg Glu Ile Val Asp Asn 20 25 30 Glu Lys Lys Ile Arg Asp Asn Gln Lys Arg Val Leu Leu Leu Asp Asn 35 40 45 Leu Ser Glu Tyr Ile Lys Pro Gly Met Ser Val Glu Ala Ile Gln Ala 50 55 60 Ile Ile Ala Asp Met Arg Gly Asn Tyr Glu Asp Arg Val Asp Asp Tyr 65 70 75 80 Ile Ile Lys Asn Ala Asp Leu Ser Lys Glu Arg Arg Glu Leu Ser Lys 85 90 95 Light Leu Light Ala Leu Gly Glu Gly Glu Ser Light 100 105 <210> 8 <211> 107 <212> PRT <213> Pseudomonas aeruginosa <400> 8 Met Glu Thr Thr Lys Pro Ser Phe Gln Asp Val Leu Glu Phe Val Arg 1 5 10 15 Leu Tyr Arg Arg Lys Asn Lys Leu Gln Arg Glu Ile Gln Asp Val Glu 20 25 30 Lys Lys Ile Arg Asp Asn Gln Lys Arg Val Leu Leu Leu Asp Asn Leu 35 40 45 Ser Asp Tyr Ile Lys Pro Gly Met Ser Val Glu Ala Ile Gln Gly Ile 50 55 60 Ile Ala Ser Met Lys Ser Asp Tyr Glu Asp Arg Val Asp Asp Tyr Ile 65 70 75 80 Ile Lys Asn Ala Glu Leu Ser Lys Glu Arg Arg Asp Ile Ser Lys Lys 85 90 95 Leu Lys Val Met Gly Glu Ala Lys Val Glu Gly 100 105 <210> 9 <211> 112 <212> PRT <213> Shimwellia blattae <400> 9 Met Glu Asn Thr Lys Pro Ser Phe Gln Asp Val Leu Glu Phe Val Arg 1 5 10 15 Leu Phe Arg Arg Lys Asn Lys Leu Gln Arg Glu Ile Gln Asp Val Glu 20 25 30 Lys Lys Ile Arg Asp Asn Gln Lys Arg Val Leu Leu Leu Asp Asn Leu 35 40 45 Ser Asp Tyr Ile Lys Pro Gly Met Thr Val Glu Ala Ile Gln Gly Ile 50 55 60 Ile Ala Ser Met Lys Ser Asp Tyr Glu Asp Arg Val Asp Asp Tyr Ile 65 70 75 80 Ile Lys Asn Ala Glu Leu Ser Lys Glu Arg Arg Asp Ile Ser Lys Lys 85 90 95 Leu Lys Val Met Gly Glu Met Lys Asn Gln Asp Pro Glu Ala Lys Ala 100 105 110 <210> 10 <211> 330 <212> DNA <213> Escherichia coli MG1655 <400> 10 atggaaacta ccaagccttc attccaggac gtactggaat ttgttcgtct gttccgtcgt 60 aagaacaaac tgcaacgtga aattcaggac gttgagaaaa agatccgtga caaccagaag 120 cgcgtcctgc tgctggacaa cctgagcgat tacatcaagc cggggatgag cgttgaagca 180 atccagggca tcatcgccag catgaaaggt gactatgaag atcgcgttga cgattacatc 240 atcaaaaatg ccgagctctc caaagaacgc cgcgatatct caaaaagct gaaagctatg 300 ggcgaaatga aaaacggcga agcgaagtaa 330 <210> 11 <211> 324 <212> DNA <213> Serratia grimesii NBRC13537 <400> 11 atgaagatgg ataatgcaaa taagccgagt ttccaggacg ttctggagtt tgtgcgtatg 60 ttccgccgta aaaataagct gcaacgcgaa attatcgaca acgaaaagaa agttcgtgat 120 aaccaaaagc gtgtgctgct actcgacaac ctgagtgagt acatcaagcc aggcatgagc 180 attgaagacg ttcagggcat cattggcaac atgcgcagcg actatgaaga tcgcgttgat 240 gactacatca tcaaaaatgc cgatctgtct aaagaacgtc gcgaactgtc caaaaagctg 300 aaagctatgg gcgaagtgaa gtaa 324 <210> 12 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 12 Met Lys Met Asp Asn Ala Asn Lys Pro Ser Phe Gln Asp Val Leu Glu 1 5 10 15 Phe Val Arg Met Phe Arg Arg Lys Asn Lys Leu Gln Arg Glu Ile Ile 20 25 30 Asp Asn Glu Lys Lys Val Arg Asp Asn Gln Lys Arg Val Leu Leu Leu 35 40 45 Asp Asn Leu Ser Glu Tyr Ile Lys Pro Gly Met Ser Ile Glu Asp Val 50 55 60 Gln Gly Ile Ile Gly Asn Met Arg Ser Asp Tyr Glu Asp Arg Val Asp 65 70 75 80 Asp Tyr Ile Ile Lys Asn Val Asp Leu Ser Lys Glu Arg Arg Glu Leu 85 90 95 Sir Lys Lys Leu Lys Ala Met Gly Glu Val Lys 100 105 <210> 13 <211> 774 <212> DNA <213> Pseudomonas putida KT2440 <400> 13 atgccgcgat atatcgatgt gcaggcgccc gaacatggcg ttcagctcat taccctgcaa 60 cggcccgagg ccttgaatgc cctgtgcacc gagctactgg cagaactggc cgctgcgctg 120 caggctgccg ggaacgacga gcatgtccgt gccacagtga ttaccggcag cgccaaggca 180 ttcgccgcag gcgccgacat ccgcgagatg gccgatcgcg acctggtcgg catcctcaat 240 gacccgcg tagcgcattg gcaaagcatc gccgcattg ccaaaccgct gattgctgca 300 gtcaacggct atgccctggg tggcggttgc gaactggcaa tgtgcgccga catcgtcatc 360 gccagtaccg acgcccgttt cggccagccg gaaatcaacc ttggcatcat ccccggtgct 420 ggcggcaccc agcgcctgtt acgtgccgtc ggtaagccgt tggccatgca gatggtgctg 480 acgggggaag ccatcactgc cctccgcgcc gcctggtcag cgaaatcacc 540 cagcccgaac tcaccgtaga acgcgccatg caggttgccc gcagcatcgc cgccaaagcg 600 ccgctggctg tgcgcctggc caaggaggcg ttactgaagg ccggtgatac cgacctggcc 660 agcggcctgc gcttcgagcg ccatgccttc acctgctgg cgggcaccgc cgaccgcgat 720 gaaggcatcc gcgccttcca ggaaaagcgc caggcccgct tccaagggcg ctga 774 <210> 14 <211> 1530 <212> DNA <213> Serratia marcescens ATCC13880 <400> 14 atggcagaaa gtaatgcggc aattcaatcg gctgcgatta tcggcgcggg aacgatgggc 60 agaggcatcg cttatctttt cgcgcaaaaa ggcattcgca cggtgcttta taatcgcaac 120 ggcaataccc tcaatcaggc tcgcgaatat atcgcgcaag acctgaacaa gaaagtcgaa 180 cagggcaaga tcgcgctgca ggataaaggc gcggtgctgg ccaatctaat gttcacttca 240 gtgtttgagg ccatcgccga cagcgagctg gtgatagaaa ccatcgccga gcaagaacaa accaaacttg aggtgctggc ggccatcgcc gcggtggtca agcccgacac gctgatcgcc 360 accatacct cctcactgtc gcttaacaag ctggctactg cggtgacgca cagcgaacgc tttatcggtt tgcatttttt caaccccgcg ccgctgatga agctgattga aatcattccg 480 gcctacttta ccgcgcacgc caccaccga cgctgccgcc aactggtggc ggcgttgggg 540 aaacacgatg tcgtctgcca ggccacgccg gggttcatcg tcaatcgcat ggcccgcccc 600. tactacctgg aagggttccg cctgttgga gaacacgtgg cgcgcgcggc tcagatcgac cgcgccctca aggccggcgg gcgcttccgc atggggccgc tcgagctgac cgattttatc 720 ggccaagaca tcaactatca ggtcagtcgg caaatctggc aggatatgca atacgacccg cgctataccc ccggtcatct gcagcgttca ctggtcgatg ccggtctgtt ggggaaaaag 840 aacggccgct cctattttgc cgccgaaga accgccccgc cggtgacggc cgccagcaat gcagacgtcg agacgctgcg cgtttacggc gcaccctt tttttaccct gttacagcag 960 cgagccgcgc ttcagtggcc acagctgcgc gtggaacaac ggccggcatt accggggctg 1020 gggtcggccg tccagatcaa tgacgctttc accgtcagca tcaccgatgg ccgcacggcg 1080 agccaactgg ccgagcagac ggcagcggat gcctttgtgg tcgatgtcgc cctgaactac 1140 gccgacacga cgtatctggc ggcggcgcac agccgccacg cctctgcggc caataaggcg 1200 ctgtttttac gcctgctgca cacggcaatc ccgcaggttg aatttatcaa ggactctccg 1260 gcgcttatcg tcgcccgcgt cctcagcagc ctgatcaatg agtcggtgat catggtggaa 1320 agcggcgtct gcagccggga agacatcgat gtcgccgccg tcgcgggcgt taactacgcc 1380 ggcggcattt tcgactggct cggcaaactg ggggagaaaa acgtcaggac aacgctgagc 1440 aatctggctc agctgctgca cgcggcgcgc tatgcgccgc attacaccct tctgcacgcc 1500 gcgcaaccgg cgctgacgac cacgccttaa 1530 <210> 15 <211> 774 <212> DNA <213> Pseudomonas putida KT2440 <400> 15 atgccgcgat atatcgatgt gcaggcgccc gaacatggcg ttcagctcat taccctgcaa 60 cggcccgagg ccttgaatgc cctgtgcacc gagctactgg cagaactggc cgctgcgctg 120 caggctgccg ggaacgacga gcatgtccgt gccacagtga ttaccggcag cgccaaggca 180 ttcgccgcag gcgccgacat ccgcgagatg gccgatcgcg acctggtcgg catcctcaat 240 gacccgcgcg tagcgcattg gcaaagcatc gccgcattcg ccaaaccgct gattgctgca 300 gtcaacggct atgccctggg tggcggttgc gaactggcaa tgtgcgccga catcgtcatc 360 gccagtaccg acgcccgttt cggccagccg gaaatcaacc ttggcatcat ccccggtgct 420 ggcggcaccc agcgcctgtt acgtgccgtc ggtaagccgt tggccatgca gatggtgctg 480 acgggggaag ccatcactgc cctccgcgcc cagcaggccg gcctggtcag cgaaatcacc 540 cagcccgaac tcaccgtaga acgcgccatg caggttgccc gcagcatcgc cgccaaagcg 600 ccgctggctg tgcgcctggc caaggaggcg ttactgaagg ccggtgatac cgacctggcc 660 agcggcctgc gcttcgagcg ccatgccttc accctgctgg cgggcaccgc cgaccgcgat 720 gaaggcatcc gcgccttcca ggaaaagcgc caggcccgct tccaagggcg ctga 774 <210> 16 <211> 1155 <212> DNA <213> Acinetobacter baylyi ADP1 <400> 16 atgattcgcg atgaagggat gttgcaacaa ttactttcga caatacgaga ttttgtaaaa 60 aatgaattga ttcctcgaga gcatgaagtt gcagaaaagg attgtattcc tgaagatatt 120 attcagcaaa tgcgagaact aggcctattt ggtttaacca ttcccgaaga atacggtgga 180 cttggaatca caatggaaga agaggtgaat gtcgcgtttg aacttggtca gacatcccca 240 gcatttcgtt cattgattgg cacaaataat ggcattggtt caagtggttt aatcattgat 300 ggaactgagg agcaaaaaca gaaatatttg ccacgttatg ccagtggaga aattattggt 360 tcattttgct taactgaacc agaagcgggt tcagatgctg cctctttaaa aacgacagcg 420 gtaaaagatg gtgatttcta catattaaat ggaaccaagc gttttattac caatgcaccg 480 catgcagcaa catttaccgt aatggcacgc accaatccag caattaaagg ggcaggtgga 540 atttctgctt ttttggtaga agccaataca ccaggtatca cactaggcaa aatagatcag 600 aaaatgggac aaaaaggctc tcatacctgt gatgtgattt ttgaaaattg tcgagtacct 660 gcatctgcat taattggtgg cgttgaaggc gttggtttta aaacagcaat gaaagtgctg 720 gataaaggcc gtctacatat tggtgcatat agtgtaggtg ttgcagagcg tatgttgaat 780 gatgcactac attatgctgt cgagcgtaag cagtttggtc aacccattgc aaattttcaa 840 ttgattcaag ccatgctggc agactctaaa gccgaaattt atgcggctaa atgtatggtt 900 ttggatgcag cacgtcgccg tgatgaaggc caaaatatta gtacagaagc ctcatgtgcc 960 aagatgtttg caacagaaat gtgtggtcga gtcgcagatc gctgtgtgca aattcatggt 1020 ggggcaggtt acatcagtga atattcgatt gagcgttttt atcgtgacgt gcgtttattc 1080 cgtctttatg agggtacgac ccaagttcag caaattatta ttgccaaaaa tatgattaag 1140 gaagtgacgt cctaa 1155 <210> 17 <211> 696 <212> DNA <213> Pseudomonas putida KT2440 <400> 17 ttgatcaata aaacgtacga gtccatcgcc agcgcggtgg aagggattac cgacggttcg 60 accatcatgg tcggtggctt cggcacggct ggcatgccgt ccgagctgat cgatggcctc 120 attgccaccg gtgcccgcga cctgaccatc atcagcaaca acgccggcaa cggcgagatc 180 ggcctggccg ccctgctcat ggcaggcagc gtgcgcaagg tggtctgctc gttcccgcgc 240 cagtccgact cctacgtgtt cgacgaactg taccgcgccg gcaagatcga gctggaagtg 300 gtcccgcagg gcaacctggc cgagcgtatc cgcgccgcag gctccggcat tggtgcgttc 360 ttctcgccaa ccggctacgg caccctgctg gccgagggca aggaaacccg tgagatcgat 420 ggccgcatgt acgtgctgga aatgccgctg cacgccgact tcgcactgat caaggcgcac 480 aagggtgacc gttggggcaa cctgacctac cgcaaggccg cccgcaactt cggcccgatc 540 atggccatgg ctgccaagac cgccatcgcc caggtcgacc aggtcgtcga actcggtgaa 600 ctggacccgg aacacatcat caccccgggt atcttcgtcc agcgcgtggt cgccgtcacc 660 ggtgctgccg cttcttcgat tgccaaagct gtctga 696 <210> 18 <211> 642 <212> DNA <213> Pseudomonas putida KT2440 <400> 18 atgaccatca ccaaaaagct ctcccgcacc gagatggccc aacgcgtggc cgcagacatc 60 Met-Thr-Ile-His-Pro-Lys-Lys-Ala-Leu-Ser-Ala-Thr-Glu-Met-Ala-Asn-Ala-Val-Ala-Gln-Asp-Ile 60 caggaaggcg cgtacgtaaa cctgggcatc ggcgcaccga ccctggtggc caactacctg 120 Gln-Glu-Gly-Ala-Val-Thr-Lys-Pro-Trp-Gly-Ile-Gly-Ala-Thr-Asp-Pro-Trp-Val-Ala-Asn-Tyr-Leu 120 ggcgacaagg aagtgttcct gcacagcgag aacggcctgc tgggcatggg cccaagccct 180 Gly-Asp-Lys-Lys-Val-Phe-Leu-His-Ser-Glu-Asn-Gly-Leu-Trp-Gly-Met-Gly-Pro-Lys-Pro-Leu 180 gcgccgggcg aggaagacga tgacctgatc aacgccggca agcagcacgt caccctgctg 240 Ala-Ala-Gly-Glu-Glu-Asp-Asp-Leu-Ile-Asn-Ala-Gly-Lys-Gln-His-Val-Thr-Leu-Leu 240 accggtggtg ccttcttcca ccatgccgat tcgttctcga tgatgcgtgg cggccacctg 300 Thr-Gly-Gly-Ala-Phe-Phe-Ser-His-Met-Ala-Asp-Phe-Val-Ser-Asp-Asp-Ala-Val-Ala-His-Leu 300 gacatcgctg tactgggcgc cttccaggtg tcggtcaagg gcgacctggc caactggcac 360 Asp-Ile-Ala-Tyr-Trp-Gly-Ala-Phe-Gln-Val-Ser-Val-Lys-Gly-Asp-Leu-Ala-Asn-Trp-His 360 acgggtgccg aaggctcgat cccggccgta ggcggtgcaa tggacctggc caccggcgcc 420 Thr-Gly-Ala-Glu-Ala-Arg-Ile-Pro-Gly-Arg-Gly-Val-Asn-Gly-Asp-Leu-Ala-Thr-Gly-Ala 420 cgccaggtgt tcgtgatgat ggaccacctg accaagaccg gcgaaagcaa gctggtgccc 480 Ala-Gln-Val-Phe-Val-Asp-Asp-Gly-Thr-Leu-Thr-Lys-Thr-Gly-Glu-Lys-Gln-Leu-Val-Pro 480 gagtgcacct acccgctgac cggtatcgct tgcgtcagcc gcatctacac cgacctggcc 540 Glu-Cys-Thr-Tyr-Pro-Leu-Thr-Gly-Ile-Ala-Cys-Val-Ser-Ala-Ile-Tyr-Thr-Asp-Leu-Ala 540 gtactggaag tgacacctga agggctgaaa gtggtcgaaa tctgcgcgga catcgacttt 600 Val-Tyr-Trp-Lys-Val-Asp-Thr-Glu-Gly-Leu-Lys-Val-Val-Glu-Asn-Leu-Arg-Gly-Ile-Asp-Phe 600 gacgagctgc agaaactcag tggcgtgccg ctgatcaagt ga 642 Asp-Glu-Leu-Gln-Lys-Ser-Ser-Trp-Ala-Cys-Ala-Asp-Ile-Lys 642 <210> 19<210> 19 <211> 4998<211> 4998 <212> DNA<212> DNA <213> 人工序列 <213> Artificial Sequence <220> <220> <223> Source of DNA <400> 19 ttggttgaac gctttttggc ttaatggcct tttgaacacc acaacactga ggaaaatgaa 120. atgaaaacaa aaagtagcct ggttttattg ctgccactgg cgttaagttt cgcggccttt ggtggagagt tcagcggcaa agtcattaag ctggggcgtcg accccaccta tccgccgttg 180 gaatacaaga ctccacaggg tgcgctgacc ggattcggtg tcgatattgc gcaggcaatg 300. tgcgatcaaa tgcaggccaa atgcatttgg gtcgaaagca gttgggatgg gatgatcccg gggttgcagg caaaaaagtt tgacgccatt gcctcgtcca tgaccattac gccgcagcgt 360 caggcgcaaa tagccttctc ggataaagtg tccaatgccc cggcacggtt ggtagccccgt 420 aaaggcagcg atctgcaacc taccgcggct tcgctgaag gcaaatccgt tggcgtacaa 540. gcggatcca gccaggaagc ttacgccaac gcgctatggc gaccagctgg ggtcaatgtg gtgtcctacc aaagccagca ggaagccaat gaagatttgg tcaatggacg gttggatgcg tcactgttgg ccagtgtcag cgccagtgag tttttccata cgcctgccgg gaaggatttt 660 gcctttaccg gtgctgagct caatgacagc aaatatttcg gtacggcga cggtattggg 720 ttgcgtaaag aggatacggc attgctcaat gcatttaatg ccgcgctgaa agcgatcatc 780 gccaacggca cttataagaa agtgaacgat aaatactttg attttgacgt gtatggttca 840 gggcaataag atcctttta accatcaca tatacctgcc gttcactatt atttagtgaa 900 atgagatatt atgatatttt ctgaattgtg attaaaagg caacttatg cccatgcaac 960 agaaactata aaaatacag agaatgaaaa gaacagata gattttttag ttctttaggc 1020 ccgtagtctg caatccttt tatcaaca agaggaaaa tagaccagtt 1080 gcaatccaaa cgagagtcta atagaatgag gtcgaaaagt aaatcgcgcg ggtttgttac 1140 tgataaagca ggcaagacct aaatgtgta aagggcaag tgtatacttt ggcgtcaccc 1200 cttacatatt ttaggttttttgtg cgtaactaac tgccatctt caacaggag 1260 ggctggaag agcagaccgc taacacagta cataaaaaag gagacatga cgatgaacat 1320 caaaagttt gcaaacaag cacagtatt aaccttact accgcactgc tggcaggagg 1380 cgcaactcaa gcgtttgcga aagaaacgaa ccaaaagcca tataaggaaa catacggcat 1440 ttcccatt acacgccatg atatgctgca aatccctgaa cagcaaaaaa atgaaaaata 1500 tcaagttcct gaattcgatt cgtccacaat taaaaatatc tcttctgcaa aaggcctgga 1560 cgtttgggac agctggccat tacaaaacgc tgacggcact gtcgcaaact atcacggcta 1620 ccacatcgtc tttgcattag ccggagatcc taaaaatgcg gatgacacat cgatttacat 1680 gttctatcaa aaagtcggcg aaacttctat tgacagctgg aaaaacgctg gccgcgtctt 1740 taaagacagc gacaaattcg atgcaaatga ttctatccta aaagaccaaa cacaagaatg 1800 gtcaggttca gccacattta catctgacgg aaaaatccgt ttattctaca ctgatttctc 1860 cggtaaacat tacggcaaac aaacactgac aactgcacaa gttaacgtat cagcatcaga 1920 cagctctttg aacatcaacg gtgtagagga ttataaatca atctttgacg gtgacggaaa 1980 aacgtatcaa aatgtacagc agttcatcga tgaaggcaac tacagctcag gcgacaacca 2040 tacgctgaga gatcctcact acgtagaaga taaaggccac aaatacttag tatttgaagc 2100 2160 tggcaaaagc acatcattct tccgtcaaga aagtcaaaaa cttctgcaaa gcgataaaaa 2220 acgcacggct gagttagcaa acggcgctct cggtatgatt gagctaaacg atgattacac 2280 actgaaaaaa gtgatgaaac cgctgattgc atctaacaca gtaacagatg aaattgaacg 2340 cgcgaacgtc tttaaaatga acggcaaatg gtacctgttc actgactccc gcggatcaaa 2400 aatgacgatt gacggcatta cgtctaacga tatttacatg cttggttatg tttctaattc 2460 tttaactggc ccatacaagc cgctgaacaa aactggcctt gtgttaaaaa tggatcttga 2520 tcctaacgat gtaaccttta cttactcaca cttcgctgta cctcaagcga aagcaacaa 2580 2640 tgcgccaagc ttcctgctga acatcaaagg caagaaaaca tctgttgtca aagacagcat 2700 ccttgaacaa ggacaattaa cagttaacaa ataaaaacgc aaaagaaaat gccgatatcc 2760 tattggcatt ttcttttatt tcttatcaac ataaaggtga atcccatatg aactatataa 2820 aagcaggcaa atggctaacc gtattcctaa cctttttagg atattgctg tttatcgact 2880 tgtcgactct agaggatcct gtgtaggctg gagctgctc gaagttccta tactttctag 2940 agaataggaa cttcggaata ggaacttca gatcccctca cgctgccgca agcactcagg 3000 gcgcaagggc tgctaagga agcggaacac gtagaaagcc agtccgcaga aacggtgctg 3060 accccggatg aatgtcagct actgggctat ctggacaagg gaaacgcaa gcgcaagg 3120 aaagcaggta gcttgcagtg gcttacatg gcgatagcta gactggggcgg ttttatggac 3180 agcaagcgaa ccggaattgc cagctggggc gcctctggt aaggttggga agccctgcaa 3240 agtaaactgg atggctttct tgccgccaag gatctgatgg cgcagggat caatctga 3300 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc 3360 tccggccgct tgggtggaga gctattcgg ctatgactgg gcacacaga caatcggctg 3420 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcagac 3480 cgacctgtcc gtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc 3540 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg 3600 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga 3660 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg 3720 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg 3780 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt 3840 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc 3900 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg 3960 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga 4020 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc 4080 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc 4140 gaaatgaccg agatacaggt ttgtggcggt caggcacaac actcatcgcc ctatcttact 4200 cactttgctg tccggacaaa aacccgaagc gcctgcttca tgatctgacc ccctattcgc 4260 gcccggtagc agtacagacc ttgagcaggt cgcccagttc ttccagttgt tgtgtcagct 4320 ccatgctcag ccagacataa ccgtaaattg gcgcttcgcc gtgctgactg acactggctt 4380 cctgcatcag cgttttcagt tcagcggcga tttcgcttag ctcgcctgcc acgacagatt 4440 gttgcgcttg tgggccatta cgcatcgtgt ccgccaatga ttcaagcgac cgcagcgtca 4500 gcaattgcgc actgcgcagg gttttcgcat tcagcataat gaaatgggtt tcacgcgagg 4560 cccaataagc gtccgccaac agctccagag tacagaccag attgcggctc aacgtttgta 4620 ccgcttcaaa tacagccgga ggaatatggg tttctttact gctgggaaca atcaggccgc 4680 gcaatttgac cacctgattc aagagatctt tcaactgcgg ttccaaccgc ggccgctcta 4740 tcatgttcgg tgacaataa gcaccgtaga ttttactcgc gctttgcaag caatccgcca 4800 tctgcatacg ccacagaatg taggcgcgct gagggtaaat gctggtgaac aacaacgcca 4860 gcaacgaacc aaaaattacg tcaccactgc gccacaacgc ggtatgcatg tcacctgccc 4920 ccgcgccaca aaccaccgcc agtgtaatcc ccaccaacag tgccatatag ggccgcttac 4980 cgagtgtcag atagccgc 4998 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 20 ttggttgaac gctttttggc 20 <210> twenty one <211> 18 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty one gcggctatct gacactcg 18 <210> twenty two <211> 200 <212> DNA <213> Escherichia coli str. K-12 substr. MG1655 <400> twenty two cgtaattgcc ctttaaaatt cggggcgccg accccatgtg gtctcaagcc caaaggaaga 60 gtgaggcgag tcagtcgcgt aatgcttagg cacaggattg atttgtcgca atgattgaca 120 cgattccgct tgacgctgcg taaggttttt gtaattttac aggcaacctt ttatcacta 180 acaaatagct ggtggaatat 200 <210> twenty three <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty three taccgtcgac ctcgacgtaa ttgcccttta 30 <210> twenty four <211> 41 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty four ggccccccct cgagtcatta agtactatat tccaccagct a 41 <210> 25 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 25 ctggtggaat atatgcacga cgtattcatc tg 32 <210> 26 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 26 ctcgagtcat taagtgttaa ctcaaacccg ctcgatggcc a 41 <210> 27 <211> 55 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 27 gagcgggttt gagttcaatt gtagctggtg gaatatttga tcaataaaac gtacg 55 <210> 28 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 28 gagtcattaa gtgtttcact tgatcagcgg cacg 34 <210> 29 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 29 ctggtggaat atagtactat ggcagaaagt aatgc 35 <210> 30 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 30 ttccaccagc taagtgctag cttaaggcgt ggtcgtcag 39 <210> 31 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 31 cggccagtga attcgagctc cgtaattgcc ctttaaaatt 40 <210> 32 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 32 gatccccggg taccgcatgc tatattccac cagctatttg t 41 <210> 33 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 33 gctggtggaa tatagatgcc gcgatatatc gatg 34 <210> 34 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 34 attacgccaa gcttgtcagc gcccttggaa gcgg 34 <210> 35 <211> 50 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 35 gggcgctgac aagcttagct ggtggaatat atgattcgcg atgaagggat 50 <210> 36 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 36 tgattacgcc aagcttttag gacgtcactt ccttaa 36 <210> 37 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA fragments <400> 37 atgaagatgg ataatgcaaa taagccgagt ttccaggacg ttctggagtt tgtgcgtatg 60 ttccgccgta aaaataagct gcaacgcgaa attatcgaca acgaaaagaa agttcgtgat 120 aaccaaaagc gtgtgctgct actcgacaac ctgagtgagt acatcaagcc aggcatgagc 180 attgaagacg ttcagggcat cattggcaac atgcgcagcg actatgaaga tcgcgttgat 240 gactacatca tcaaaaatgt cgatctgtct aaagaacgtc gcgaactgtc caaaaagctg 300 aaagctatgg gcgaagtgaa gtaa 324 <210> 38 <211> 50 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 38 aaatagctgg tggaatatag catgcatgaa gatggataat gcaaataagc 50 <210> 39 <211> 46 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 39 cgactctaga ggatccccgg gtaccttact tcacttcgcc catagc 46 <210> 40 <211> 477 <212> PRT <213> Serratia grimesii NBRC13537 <400> 40 Met Phe Lys Asn Ala Phe Ala Asn Leu Gln Lys Val Gly Lys Ser Leu 1 5 10 15 Met Leu Pro Val Ser Val Leu Pro Ile Ala Gly Ile Leu Leu Gly Val 20 25 30 Gly Ser Ala Asn Phe Ser Trp Leu Pro Ala Val Val Ser His Val Met 35 40 45 Ala Glu Ala Gly Gly Ser Val Phe Ala Asn Met Pro Leu Ile Phe Ala 50 55 60 Ile Gly Val Ala Leu Gly Phe Thr Asn Asn Asp Gly Val Ser Ala Leu 65 70 75 80 Ala Ala Val Val Ala Tyr Gly Ile Met Val Lys Thr Met Ala Val Val 85 90 95 Ala Pro Leu Val Leu His Leu Pro Ala Glu Glu Ile Ala Ala Lys His 100 105 110 Leu Ala Asp Thr Gly Val Leu Gly Gly Ile Ile Ser Gly Ser Ile Ala 115 120 125 Ala Tyr Met Phe Asn Arg Phe Phe Arg Ile Gln Leu Pro Glu Tyr Leu 130 135 140 Gly Phe Phe Ala Gly Lys Arg Phe Val Pro Ile Ile Ser Gly Leu Ala 145 150 155 160 Ala Ile Val Leu Gly Val Val Leu Ser Phe Ile Trp Pro Pro Ile Gly 165 170 175 Thr Ala Ile Gln Thr Phe Ser Gln Trp Ala Ala Tyr Gln Asn Pro Val 180 185 190 Val Ala Phe Gly Ile Tyr Gly Val Val Glu Arg Ala Leu Val Pro Phe 195 200 205 Gly Leu His His Ile Trp Asn Val Pro Phe Gln Met Gln Ile Gly Glu 210 215 220 Tyr Thr Asn Ala Ala Gly Gln Val Phe His Gly Asp Ile Pro Arg Tyr 225 230 235 240 Met Ala Gly Asp Pro Thr Ala Gly Lys Leu Ser Gly Gly Phe Leu Phe 245 250 255 Lys Met Tyr Gly Leu Pro Ala Ala Ala Ile Ala Ile Trp His Ser Ala 260 265 270 Lys Pro Glu Asn Arg Ala Lys Val Gly Gly Ile Met Ile Ser Ala Ala 275 280 285 Leu Thr Ser Phe Leu Thr Gly Ile Thr Glu Pro Ile Glu Phe Ser Phe 290 295 300 Met Phe Val Ala Pro Ile Leu Tyr Ala Ile His Ala Ile Leu Ala Gly 305 310 315 320 Leu Ala Phe Pro Ile Cys Ile Leu Leu Gly Met Arg Asp Gly Thr Ser 325 330 335 Phe Ser His Gly Leu Ile Asp Phe Ile Val Leu Ser Gly Asn Ser Ser 340 345 350 Lys Ile Trp Leu Phe Pro Ile Val Gly Ile Ile Tyr Gly Leu Val Tyr 355 360 365 Tyr Thr Ile Phe Arg Val Leu Ile Ala Lys Leu Asp Leu Lys Thr Pro 370 375 380 Gly Arg Glu Asp Thr Val Ser Glu Gln Val Ala Gln Gly Gly Ser Glu 385 390 395 400 Met Ser Ala Ala Leu Val Gln Ala Phe Gly Gly Lys Glu Asn Ile Thr 405 410 415 Asn Leu Asp Ala Cys Ile Thr Arg Leu Arg Val Ser Val Ala Asp Val 420 425 430 Ser Lys Val Asp Gln Ala Gly Leu Lys Lys Leu Gly Ala Ala Gly Val 435 440 445 Val Val Ala Gly Ser Gly Val Gln Ala Ile Phe Gly Thr Lys Ser Asp 450 455 460 Asn Leu Lys Thr Asp Met Asp Glu Tyr Ile Arg Asn His 465 470 475 <210> 41 <211> 470 <212> PRT <213> Serratia grimesii NBRC13537 <400> 41 Met Lys Lys Thr Lys Ile Val Cys Thr Ile Gly Pro Lys Thr Glu Ser 1 5 10 15 Glu Glu Met Leu Thr Asn Leu Leu Asn Ala Gly Met Asn Val Met Arg 20 25 30 Leu Asn Phe Ser His Gly Asp Tyr Glu Glu His Gly Asn Arg Ile Lys 35 40 45 Asn Met Arg Ala Val Met Ala Lys Thr Gly Gln Asn Ala Gly Ile Leu 50 55 60 Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr Met Lys Leu Glu Gly Gly 65 70 75 80 Lys Asp Ala Ala Leu Val Ala Gly Gln Thr Phe Thr Phe Thr Thr Asp 85 90 95 Gln Ser Val Ile Gly Asn Asn Glu Arg Val Ala Val Thr Tyr Ala Gly 100 105 110 Phe Ser Ala Asp Leu Lys Ile Gly Asn Thr Val Leu Val Asp Asp Gly 115 120 125 Leu Ile Gly Met Glu Val Thr Asn Val Thr Glu Asn Glu Val Val Cys 130 135 140 Lys Val Leu Asn Ser Gly Asp Leu Gly Glu Asn Lys Gly Val Asn Leu 145 150 155 160 Pro Gly Val Ser Ile Gln Leu Pro Ala Leu Ala Glu Lys Asp Lys Arg 165 170 175 Asp Leu Ile Phe Gly Cys Glu Gln Gly Val Asp Phe Val Ala Ala Ser 180 185 190 Phe Ile Arg Lys Arg Ser Asp Val Leu Glu Ile Arg Glu His Leu Lys 195 200 205 Ala His Gly Gly Glu Gln Ile Gln Ile Ile Ser Lys Ile Glu Asn Gln 210 215 220 Glu Gly Leu Asn Asn Phe Asp Glu Ile Leu Glu Ala Ser Asp Gly Ile 225 230 235 240 Met Val Ala Arg Gly Asp Leu Gly Val Glu Ile Pro Val Glu Glu Val 245 250 255 Ile Phe Ala Gln Lys Met Met Ile Glu Lys Cys Asn Arg Ala Arg Lys 260 265 270 Val Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Ile Lys Asn Pro 275 280 285 Arg Pro Thr Arg Ala Glu Ala Gly Asp Val Ala Asn Ala Ile Leu Asp 290 295 300 Gly Thr Asp Ala Val Met Leu Ser Gly Glu Ser Ala Lys Gly Lys Tyr 305 310 315 320 Pro Leu Glu Ala Val Thr Ile Met Ala Thr Ile Cys Glu Arg Thr Asp 325 330 335 Arg Val Met Pro Ser Arg Ile Asp Ser Leu Asn Asp Asn Arg Lys Leu 340 345 350 Arg Ile Thr Glu Ala Val Cys Arg Gly Ala Val Glu Thr Ala Glu Lys 355 360 365 Leu Asp Ala Pro Leu Ile Val Val Ala Thr Ser Gly Gly Lys Ser Ala 370 375 380 Lys Ser Val Arg Lys Tyr Phe Pro Asn Ala Val Ile Leu Ala Leu Thr 385 390 395 400 Thr Asn Glu Val Thr Ala His Gln Leu Ile Leu Ser Lys Gly Val Ile 405 410 415 Pro Gln Met Val Lys Glu Ile Ala Ser Thr Asp Asp Phe Tyr Arg Ile 420 425 430 Gly Lys Glu Ala Ala Leu Ala Ser Gly Leu Ala Gln Lys Gly Asp Val 435 440 445 Val Val Met Val Ser Gly Ala Leu Val Pro Ser Gly Thr Thr Asn Thr 450 455 460 Ala Ser Val His Val Leu 465 470 <210> 42 <211> 480 <212> PRT <213> Serratia grimesii NBRC13537 <400> 42 Met Ser Arg Arg Leu Arg Arg Thr Lys Ile Val Thr Thr Leu Gly Pro 1 5 10 15 Ala Thr Asp Arg Asp Asn Asn Leu Glu Lys Ile Ile Ala Ala Gly Ala 20 25 30 Asn Val Val Arg Leu Asn Phe Ser His Gly Ser Ala Glu Asp His Gln 35 40 45 Ala Arg Ala Asp Lys Val Arg Glu Ile Ala Ala Lys Leu Gly Arg His 50 55 60 Val Ala Ile Leu Gly Asp Leu Gln Gly Pro Lys Ile Arg Val Ser Thr 65 70 75 80 Phe Lys Glu Gly Lys Ile Phe Leu Asn Ile Gly Asp Lys Phe Leu Leu 85 90 95 Asp Ala Asn Met Ser Lys Gly Glu Gly Asp Lys Glu Lys Val Gly Ile 100 105 110 Asp Tyr Lys Gly Leu Pro Ala Asp Val Val Pro Gly Asp Val Leu Leu 115 120 125 Leu Asp Asp Gly Arg Val Gln Leu Lys Val Leu Glu Val Gln Gly Met 130 135 140 Lys Val Phe Thr Glu Val Thr Val Gly Gly Pro Leu Ser Asn Asn Lys 145 150 155 160 Gly Ile Asn Lys Leu Gly Gly Gly Leu Ser Ala Glu Ala Leu Thr Glu 165 170 175 Lys Asp Lys Ala Asp Ile Val Thr Ala Ala Lys Ile Gly Val Asp Tyr 180 185 190 Leu Ala Val Ser Phe Pro Arg Thr Gly Glu Asp Leu Asn Tyr Ala Arg 195 200 205 Arg Leu Ala Arg Asp Ala Gly Cys Asn Ala Lys Ile Val Ser Lys Val 210 215 220 Glu Arg Ala Glu Ala Val Cys Ser Asp Glu Ala Met Asp Asp Ile Ile 225 230 235 240 Leu Ala Ser Asp Val Val Met Val Ala Arg Gly Asp Leu Gly Val Glu 245 250 255 Ile Gly Asp Pro Glu Leu Val Gly Ile Gln Lys Lys Leu Ile Arg Arg 260 265 270 Ala Arg Thr Leu Asn Arg Ala Val Ile Thr Ala Thr Gln Met Met Glu 275 280 285 Ser Met Ile Thr Asn Pro Met Pro Thr Arg Ala Glu Val Met Asp Val 290 295 300 Ala Asn Ala Val Leu Asp Gly Thr Asp Ala Val Met Leu Ser Ala Glu 305 310 315 320 Thr Ala Ala Gly Gln Tyr Pro Ala Glu Thr Val Ala Ala Met Ala Arg 325 330 335 Val Cys Leu Gly Ala Glu Lys Ile Pro Ser Ile Asn Val Ser Lys His 340 345 350 Arg Leu Asp Val Gln Phe Asp Asn Ile Glu Glu Ala Ile Ala Met Ser 355 360 365 Ser Met Tyr Ala Ala Asn His Leu Lys Gly Val Thr Ala Leu Ile Ala 370 375 380 Met Thr Glu Ser Gly Arg Thr Ala Leu Met Met Ser Arg Ile Ser Ser 385 390 395 400 Gly Leu Pro Ile Phe Ala Met Ser Arg His Glu His Thr Leu Asn Leu 405 410 415 Thr Ala Leu Tyr Arg Gly Val Thr Pro Val Tyr Phe Asp Ser His Glu 420 425 430 Asp Gly Val Ile Ala Ala Asn Asp Ala Val Asn Arg Leu Arg Asp Lys 435 440 445 Gly Phe Leu Val Ser Gly Asp Leu Val Ile Val Thr Gln Gly Asp Val 450 455 460 Met Glu Thr Val Gly Thr Thr Asn Thr Ser Arg Ile Leu Arg Val Glu 465 470 475 480 <210> 43 <211> 2137 <212> DNA <213> The snowstorm <220> <223> Source of DNA <400> 43 ttggttgaac gctttttggc ttaatggcct tttgaacacc acaacactga ggaaaatgaa 120. atgaaaacaa aaagtagcct ggttttattg ctgccactgg cgttaagttt cgcggccttt ggtggagagt tcagcggcaa agtcattaag ctggggcgtcg accccaccta tccgccgttg 180 gaatacaaga ctccacaggg tgcgctgacc ggattcggtg tcgatattgc gcaggcaatg 300. tgcgatcaaa tgcaggccaa atgcatttgg gtcgaaagca gttgggatgg gatgatcccg gggttgcagg caaaaaagtt tgacgccatt gcctcgtcca tgaccattac gccgcagcgt 360 caggcgcaaa tagccttctc ggataaagtg tccaatgccc cggcacggtt ggtagccccgt 420 aaaggcagcg atctgcaacc taccgcggct tcgctgaaag gcaaatccgt tggcgtacaa 480 cagggatcca gccaggaagc ttacgccaac gcgctatggc gaccagctgg ggtcaatgtg 540 gtgtcctacc aaagccagca ggaagccaat gaagatttgg tcaatggacg gttggatgcg 600 tcactgttgg ccagtgtcag cgccagtgag tttttccata cgcctgccgg gaaggatttt 660 gcctttaccg gtgctgagct caatgacagc aaatatttcg gtatcggcga cggtattggg 720 ttgcgtaaag aggatacggc attgctcaat gcatttaatg ccgcgctgaa agcgatcatc 780 gccaacggca cttataagaa agtgaacgat aaatactttg attttgacgt gtatggttca 840 gggcaataac ggcggattgg cgggaaacaa aaaataaaag ggccaggcaa atgcccggcc 900 cgataaaaac ggttacttca cttcgcccat agctttcagc tttttggaca gttcgcgacg 960 ttctttagac agatcgacat ttttgatgat gtagtcatca acgcgatctt catagtcgct 1020 gcgcatgttg ccaatgatgc cctgaacgtc ttcaatgctc atgcctggct tgatgtactc 1080 actcaggttg tcgagtagca gcacacgctt ttggttatca cgaactttct tttcgttgtc 1140 gataatttcg cgttgcagct tatttttacg gcggaacata cgcacaaact ccagaacgtc 1200 ctggaaactc ggcttatttg cattatccat cttcataccc ttgctttagt aatacacaga 1260 ttcatttgct tacggccaca atgataccaa gatacaggtt tgtggcggtc aggcacaaca 1320 ctcatcgccc tatcttactc actttgctgt ccggacaaaa acccgaagcg cctgcttcat 1380 gatctgaccc cctattcgcg cccggtagca gtacagacct tgagcaggtc gcccagttct 1440 tccagttgtt gtgtcagctc catgctcagc cagacataac cgtaaattgg cgcttcgccg 1500 tgctgactga cactggcttc ctgcatcagc gttttcagtt cagcggcgat ttcgcttagc 1560 tcgcctgcca cgacagattg ttgcgcttgt gggccattac gcatcgtgtc cgccaatgat 1620 tcaagcgacc gcagcgtcag caattgcgca ctgcgcaggg ttttcgcatt cagcataatg 1680 aaatgggttt cacgcgaggc ccaataagcg tccgccaaca gctccagagt acagaccaga 1740 ttgcggctca acgtttgtac cgcttcaaat acagccggag gaatatgggt ttctttactg 1800 ctgggaacaa tcaggccgcg caatttgacc acctgattca agagatcttt caactgcggt 1860 tccaaccgcg gccgctctat catgttcggt gacaaataag caccgtagat tttactcgcg 1920 ctttgcaagc aatccgccat ctgcatacgc cacagaatgt aggcgcgctg agggtaaatg 1980 ctggtgaaca acaacgccag caacgaacca aaaattacgt caccactgcg ccacaacgcg 2040 gtatgcatgt cacctgcccc cgcgccacaa accaccgcca gtgtaatccc caccaacagt 2100 gccatatagg gccgcttacc gagtgtcaga tagccgc 2137 <210> 44 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 44 Met Glu Thr Thr Lys Pro Ser Phe Gln Asp Val Leu Glu Phe Val Arg 1 5 10 15 Leu Phe Arg Arg Lys Asn Lys Leu Gln Arg Glu Ile Gln Asp Val Glu 20 25 30 Lys Lys Ile Arg Asp Asn Gln Lys Arg Val Leu Leu Leu Asp Asn Leu 35 40 45 Ser Asp Tyr Ile Lys Pro Gly Met Ser Val Glu Ala Ile Gln Gly Ile 50 55 60 Ile Ala Ser Met Lys Gly Asp Tyr Glu Asp Arg Val Asp Asp Tyr Ile 65 70 75 80 Ile Lys Asn Val Glu Leu Ser Lys Glu Arg Arg Asp Ile Ser Lys Lys 85 90 95 Leu Lys Ala Met Gly Glu Met Lys Asn Gly Glu Ala Lys 100 105 <210> 45 <211> 4301 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA Fragment <400> 45 tgctggagtt gcaaatcaat gcatactggg ccacgcgccc cagccatttc gtgttattga 60 acgcgcaaaa acttcgtgat acccagcaca tgatgcagca aatactgctg agccttgttc 120 atgcgctgta cgaaggtaat ccgcagccgg tttttgccaa tacggaaaaa ttgaacgatg 180 ctgtggaaga gctgcgtcag ttgctcaata accaccatga cctgaaggtt gtggaaacac 240 caatctatgg ttatgtgtgg ctgaacatgg aaacggcgca tcagcttgag ttgctatcga 300 atctgatttg ccgggccttg cgcaaataat tcctgaactt cagaatcatc ttgctgctgc 360 ttcgattcag caaggataaa gggtatgata gtgaaaaggg ataaaagcat tgtcatctgc 420 <h2 style=";text-align:left;direction:ltr">ggcagctatg agtaatgttg gccctaacga atagcggttg cttaaacgaa tccgactctc 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acattatcag gggtataaaa gatccttttt aacccatcac atatacctgc cgttcactat 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tatttagtga aatgagatat tatgatattt tctgaattgt gattaaaaag gcaactttat 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcccatgcaa cagaaactat aaaaaataca gagaatgaaa agaaacagat agatttttta 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gttctttagg cccgtagtct gcaaatcctt ttatgatttt ctatcaaaca aaagaggaaa 720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atagaccagt tgcaatccaa acgagagtct aatagaatga ggtcgaaaag taaatcgcgc 780<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gggtttgtta ctgataaagc aggcaagacc taaaatgtgt aaagggcaaa gtgtatactt 840<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tggcgtcacc ccttacatat tttaggtctt tttttattgt gcgtaactaa cttgccatct 900<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tcaaacagga gggctggaag aagcagaccg ctaacacagt acataaaaaa ggagacatga 960<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acgatgaaca tcaaaaagtt tgcaaaacaa gcaacagtat taacctttac taccgcactg 1020<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctggcaggag gcgcaactca agcgtttgcg aaagaaacga accaaaagcc atataaggaa 1080<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acatacggca tttcccatat tacacgccat gatatgctgc aaatccctga acagcaaaaa 1140<h2 style=";text-align:left;direction:ltr"> aatgaaaat atcaagttcc tgaattcgat tcgtccacaa ttaaaaat ctcttctgca aaaggcctgg acgtttgggga cagctggcca ttacaaaacg ctgacggcac tgtcgcaaac tatcacggct accacatcgt ctttgcatta gccggagatc ctaaaaatgc ggatgacaca 1380. tcgatttaca tgttctatca aaaagtcggc gaaacttcta ttgacagctg gaaaaacgct ggccgcgtct ttaagacag cgacaaattc gatgcaaatg attctatcct aaaagaccaa 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 actgatttct ccggtaaaca ttacggcaaa caaacactga caactgcaca agttaacgta 1620. 1620. 1620. 1620. 1620. 1620. 1620. 1620. 1620. 1620. 1620. 1620 ggtgacggaa aaacgtatca aaatgtacag cagttcatcg atgaaggcaa ctacagctca ggcgacaacc atacgctgag agatcctcac tacgtagaag ataaaggcca caaatactta gtatttgaag caaacactgg aactgaag ggctaccaag gcgaagaatc tttatttaac aaagcatact atggcaaaag cacatcattc ttccgtcaag aaagtcaaaa acttctgcaa agcgataaaa aacgcacggc tgagttagca aacggcgctc tcggtatgat tgagctaaac 1980 - 1980 - 1980 - 1980 - 1980 - 1980 - 1980 - gaaattgaac gcgcgaacgt ctttaaaatg aacggcaaat ggtacctgtt cactgactcc cgcggatcaa aaatgacgat tgacggcatt acgtctaacg atatttacat gcttggttat gtttctaatt ctttaactgg cccatacaag ccgctgaaca aaactggcct tgtgttaaaa atggatcttg atcctacga tgtaaccttt acttactcac acttcgctgt acctcaagcg 2280. aaaggaaca atgtcgtgat tacaagctat atgacaaaca gaggattcta cgcagacaaa caatcaacgt ttgcgccaag cttcctgctg aacatcaaag gcaagaaaac atctgttgtc aaagacagca tccttgaaca aggacaatta acagttaca aataaaacg caaaagaaa tgccgatatc ctattggcat tttcttttat ttcttatcaa cataaaggtg aatcccatat 2520. aaagcaggca aatggctac cgtattccta acctttttag accttttgc gtttatcgac ttgtcgactc tagaggatcc tgtgtaggct ggagctgctt cgaagttcct 2580 atactttcta gagaatagga acttcggaat aggaacttca agatcccctc acgctgccgc 2640 aagcactcag ggcgcaaggg ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag 2700 aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca 2760 agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg 2820 gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg 2880 aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcaggggga 2940 tcaagatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg 3000 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 3060 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 3120 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta 3180 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 3240 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 3300 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 3360 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 3420 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 3480 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc 3540 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 3600 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 3660 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 3720 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga 3780 ctctggggtt cgaaatgacc gttcccgttt tattcaatga gggttgcccg gcaaccctca 3840 ttgctcattg attcttatct gtgtatcacc gtcatcattc tcatccgaga accaatcgaa 3900 attaacaaca gccttcttct gtatgcagca aggcaaaaag ttctgtaact ccattgttat 3960 taactgcact ggttactaac acgttgtgcg ctccagcttc ccgtaaccaa cttttcacca 4020 aagatatttg ttccatgctg gctaaatctg ctttggttac tactccaatg accgggtgat 4080 tcatggcccg gtagggcgtt tttacctcta attcctgctt cagcgctgag attcctgctt 4140 gttgccagaa aagcaggcac ctgccgtctg aacggtatcg atccggaagc gtatctgcgc 4200 catattctga gcatactgcc ggaatggccc tccaaccgtg ttgacgaact cctgccatgg 4260 aacgtagttc tcaccaataa ataagcgtca atacggtgct c 4301 <210> 46 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 46 tgctggagtt gcaaatcaat gc 22 <210> 47 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 47 gagcaccgta ttgacgctta tt 22 <210> 48 <211> 1330 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA fragments <400> 48 tgctggagtt gcaaatcaat gcatactggg ccacgcgccc cagccatttc gtgttattga 60 acgcgcaaaa acttcgtgat acccagcaca tgatgcagca aatactgctg agccttgttc 120 atgcgctgta cgaaggtaat ccgcagccgg ttttgccaa tacggaaaaa ttgaacgatg 180 ctgtggaaga gctgcgtcag ttgctcaata accaccatga cctgaaggtt gtggaaacac 240 caatctatgg tttgtgtgg ctgaacatgg aaacggcgca tcagcttgag ttgctatcga 300 atctgatttg ccgggccttg cgcaaataat tcctgaactt cagaatcatc ttgctgctgc 360 ttcgattcag caaggataaa gggtatgata gtgaaaaggg ataaaagcat tgtcatctgc 420 ggcagctatg agtaatgttg gcctaacga atagcggttg cttaaacgaa tccgactctc 480 acattatcag gggtataaaa atggaaacta ccaagccttc attccaggac gtactggaat 540 ttgttcgtct gttccgtcgt aagaacaaac tgcaacgtga aattcaggac gttgagaaaa 600 agatccgtga caaccagaag cgcgtcctgc tgctggacaa cctgagcgat tacatcaagc 660 cggggatgag cgttgaagca atccagggca tcatcgccag catgaaaggt gactatgaag 720 atcgcgttga cgattacatc atcaaaaatg ccgagctctc caaagaacgc cgcgatatct 780 ccaaaaagct gaaagctatg ggcgaaatga aaaacggcga agcgaagtaa ttcccgtttt 840 attcaatgag ggttgcccgg caaccctcat tgctcattga ttcttatctg tgtatcaccg 900 tcatcattct catccgagaa ccaatcgaaa ttaacaacag ccttcttctg tatgcagcaa 960 ggcaaaaagt tctgtaactc cattgttatt aactgcactg gttactaaca cgttgtgcgc 1020 tccagcttcc cgtaaccaac ttttcaccaa agatatttgt tccatgctgg ctaaatctgc 1080 tttggttact actccaatga ccgggtgatt catggcccgg tagggcgttt ttacctctaa 1140 ttcctgcttc agcgctgaga ttcctgcttg ttgccagaaa agcaggcacc tgccgtctga 1200 acggtatcga tccggaagcg tatctgcgcc atattctgag catactgccg gaatggccct 1260 ccaaccgtgt tgacgaactc ctgccatgga acgtagttct caccaataaa taagcgtcaa 1320 tacggtgctc 1330 <210> 49 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 49 accaccatca cgtgggtacc tgctggagtt gcaaatcaat gc 42 <210> 50 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 50 cgtcgtcatc attcgaaccg gagcaccgta ttgacgctta tt 42

Claims

1. A genetically modified microorganism comprising a gene capable of expressing a mutant YeeX protein having a mutation in which only alanine at position 84 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine, leucine, phenylalanine, isoleucine, or methionine, The microorganism is a microorganism belonging to the genus Escherichia.

2. A genetically modified microorganism comprising a gene capable of expressing a mutant YeeX protein homolog having a mutation in which only alanine at position 87 in the amino acid sequence of SEQ ID NO: 2 is substituted with valine, The microorganism is a microorganism belonging to the genus Serratia.

3. The genetically modified microorganism according to claim 1, wherein the mutant YeeX protein has a mutation in which only alanine at position 84 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine. 4 . The genetically modified microorganism according to claim 1 , wherein the microorganism has an ability to produce an organic acid.

5. A method for producing an organic acid, comprising culturing the genetically modified microorganism according to claim 4 in a culture medium containing a carbon source as a fermentation raw material to produce the organic acid, The organic acid is succinic acid, acetic acid, 3-hydroxyadipic acid, α-hydrogenated adiponic acid and / or adipic acid.

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