JP2016192902A - Method for manufacturing alginic acid-assimilating microorganism and target substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microorganism that can assimilate alginic acid, and a method for producing a target substance by using the microorganism by a fermentation method.SOLUTION: A method for manufacturing a target substance in which a genus Vibrio bacterium that has a target substance production ability and alginic acid assimilation ability is cultured in culture medium containing a carbon source obtained from seaweed, the target substance is created and accumulated in the culture medium, and the target substance is recovered from the culture medium.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a novel microorganism serving as a microbial host at the time of fermentation production of useful substances when using seaweed biomass using bacteria.

  Fermentation methods using microorganisms are used for mass production of substances used in fuels such as ethanol, foods such as L-amino acids, feeds, and pharmaceuticals, which are industrially useful.

  As main raw materials for fermentation using microorganisms, sugars produced from food grains such as sugar cane and corn, that is, glucose, fructose, sucrose, molasses, starch hydrolysate, and the like are used.

  In addition, cereals and oils derived from non-food terrestrial biomass such as sugarcane, corn stalks and leaves, and Jatropha oil are also used by microorganisms due to the increase in grain prices due to the increase in food demand due to the global population increase in recent years. It is industrially useful as the main raw material source for fermentation.

  However, while non-food terrestrial biomass production does not compete directly with food grain production, it competes indirectly with food grain production in terms of farmland, agricultural water, fertilizer use, etc. It is pointed out that there is a problem (Non-Patent Document 1).

  Therefore, attention has been focused on marine biomass centered on seaweed as the main raw material for fermentation using microorganisms. Marine biomass can be produced without the use of farmland, agricultural water, and fertilizers, and among the algae, brown algae, including non-food parts, is twice the sugar cane and five times the maize fermented main ingredient production capacity per unit area It is known that there is (Non-patent Document 2). Brown algae store significant amounts of sugars such as alginic acid, glucose and mannitol, which are polymerized polysaccharides of D-mannuronic acid and L-guluronic acid.

  However, when saccharides accumulated in brown algae are used as the main raw material for fermentation, it is difficult to assimilate by microorganisms the alginic acid contained as the main component in the saccharides in brown algae. (Non-patent document 3, Non-patent document 4, Patent document 1, and Patent document 2). That is, it is considered that obtaining microorganisms capable of assimilating alginic acid is important in producing useful substances by fermentation using microorganisms from marine biomass from brown algae.

  If a microorganism that can assimilate alginic acid as a single carbon source and can grow rapidly can be newly isolated and obtained, it is useful as a host microorganism for fermentation using microorganisms that produce useful substances from marine biomass. There is no known method for producing a target substance by fermentation using Vibrio spp.

International patent application WO 2009/046375 International patent application WO 2011/024858

Bringezu, et al. (2009) United Nations Environmental Program Somerville, et al. (2010) Science. 329, p790-792. Takeda, et al. (2011) Energy Environ. Sci. 4 p2575-2581 Wargacki, et al. (2012) Science. 335.p308-313

  An object of the present invention is to provide a microorganism capable of assimilating alginic acid and a method for producing a target substance by fermentation using the microorganism.

  The inventor of the present invention has isolated Vibrio spp. That can assimilate alginic acid from the digestive tract of Sazae (scientific name: Turbo cornutus), which is considered to be ingested on a daily basis by brown algae. Furthermore, the inventors have found that amino acids can be produced by imparting amino acid-producing ability to vibrio bacteria capable of assimilating alginic acid.

[1] A bacterium belonging to the genus Vibrio having an ability to assimilate alginate, wherein the 16S ribosomal RNA sequence has a sequence having a homology of 95% or more with SEQ ID NO: 1.
[2] The Vibrio bacterium as described above, wherein the 16S ribosome has SEQ ID NO: 1.
[3] The Vibrio bacterium as described above, which can grow using alginic acid as a single carbon source.
[4] The Vibrio spp. Described above, wherein the Vibrio spp. Is NITE P-01635 strain.
[5] A Vibrio bacterium having an ability to produce a target substance and having an ability to assimilate alginate is cultured in a medium containing a carbon source extracted from a carbon source obtained from a seaweed-derived biomass seaweed, A method for producing a target substance by a fermentation method, characterized in that the target substance is produced and accumulated in a medium, and the target substance is recovered from the medium.
[6] The method as described above, wherein the bacterium having the ability to assimilate alginate is a Vibrio bacterium having a 16S ribosome sequence having a sequence having a homology of 95% or more with SEQ ID NO: 1.
[7] The method described in the above, wherein the bacterium having the ability to assimilate alginic acid is NITE P-01635 strain.
[8] The target substance is L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L-leucine, glycine, L-threonine, Selected from L-serine, L-proline, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L-cystine, L-methionine, L-glutamic acid, L-aspartic acid, L-glutamine and L-asparagine The method as described above, wherein said method is one or more amino acids.
[9] The method described in the above, wherein the target substance is ethanol.
[10] The target substance is isopropyl alcohol, acetone, propylene, 1,3-butanediol, 1,4-butanediol, 1-propanol, 1,3-propanediol, 1,2-propanediol, ethylene glycol, And the method as described above, selected from the group consisting of isobutanol.
[11] The method described above, wherein the bacterium is a Vibrio bacterium having enhanced activity of an L-amino acid biosynthesis enzyme.
[12] The method described in the above, wherein the bacterium is a Vibrio bacterium with enhanced activity of a protein that excretes L-amino acids.
[13] The method described above, wherein the protein that excretes the L-amino acid is ybjL.
[14] The method as described above, wherein the seaweed is a large seaweed contained in brown algae, red algae, and green algae.
[15] The method as described above, wherein the carbon source obtained from the seaweed is one or more carbon sources selected from alginic acid, cellulose, mannitol, pectin, galacturonic acid, carrageenan, and agar.
[16] The method as described above, wherein the saccharide extract obtained from the seaweed contains alginic acid.
[17] The method as described above, wherein the saccharide extract obtained from the seaweed contains alginic acid, and the culture is carried out after the hydrolysis of alginic acid by pretreatment with an alginic acid hydrolase or acid. .

  Production of useful substances from marine biomass becomes possible.

16S rRNA gene sequence comparison between Vibrio sp.SA2 and Vibrio rumoiensis Type strain (upper sequence is Vibrio sp.SA2 strain, lower sequence is Vibrio rumoiensis type strain S-strain, dotted frame is due to gap, solid frame is Difference due to replacement). Molecular phylogenetic tree based on the 16S rRNA gene full-length sequence of Viibrio sp. SA2 (numbers next to the branches indicate bootstrap values). Test tube culture results of Vibrio sp. SA2 strain in a sodium alginate minimal medium (1: Turbidity of Vibrio sp. SA2 strain at a wavelength of 600 nm, 2: Turbidity of MG1655 strain at a wavelength of 600 nm). Maximum specific growth rate in minimal liquid medium with glucose or sodium alginate alone as carbon source.

<1> Bacteria of the present invention The Vibrio bacterium of the present invention is a bacterium belonging to the genus Vibrio having an alginate-assimilating ability, and the 16S ribosomal RNA sequence has a sequence showing 95% or more homology with SEQ ID NO: 1. Vibrio bacteria.

  In the present invention, alginic acid means a polymerized polysaccharide of D-mannuronic acid and L-guluronic acid. As used herein, the term “alginic acid” refers to β-D-mannuronic acid (M) and α-L-guluronic acid (G), also referred to as β-1,4′-mannurono-1,4′-L-guluroglycan. ) Is a polysaccharide in which the pyranose rings of two uronic acids are mainly β1 → 4 linked. In alginic acid, β-D-mannuronic acid homopolymer (hereinafter also referred to as “poly (M)” or “M block”), α-L-guluronic acid homopolymer (hereinafter referred to as “poly (G)”) or There is a region of a heteropolymer of β-D-mannuronic acid and α-L-guluronic acid (hereinafter also referred to as “poly (MG)” or “MG block”). The alginic acid of the present invention is preferably obtained from seaweed.

In the present invention, “seaweed” refers to green algae plants such as Aosa and Aonori, red algae plants such as Amanori and Ogonori, brown algae plants such as Kombu, Alame, Kajime, Wakame, Honda Walla, etc., and a plurality of species selected from these Refers to a combination of Particularly preferred are brown algae plants, which are macroalgae called Macroalgae. Brown algae plants are preferred in that they store a large amount of mannitol, a sugar alcohol, as an initial assimilation product of photosynthesis. The brown algae plants that can be used in the present invention include the order of the brown algae class Cytosoniphonales, Ectocarpales, Dictyosiphonales, Chordariales, Ralphsiales, Ralphsiales, (Desmaresiales), Compositae (Laminariales), Musselid (Cuteralials), Usbaogi (Syringoderales), Sphazelarias, sigmoidea (Dicyotales), Tiropteris (es) Fucales), including but those belonging to Durubiaera th (Durvilaeales), preferably Laminaria japonica (Laminaria japonica)
It is. Macombu has a very high production volume as marine biomass and is large in size, and can be stably supplied in large quantities as a raw material for producing the target substance.

  In the present invention, the seaweed can be either raw or dried, preferably raw. By using raw seaweed, it is possible to omit the drying process of seaweed with extremely high energy consumption, and there is almost no need to add water separately in the process of obtaining a seaweed saccharified solution, which is advantageous. In the present invention, the seaweed may be swollen as necessary.

  In the present invention, the seaweed may be fragmented as necessary. The seaweed can be fragmented by a known method, for example, using a scissors, a mixer, ultrasonic treatment, a French press, a stone mortar, a mortar, a homogenizer, glass beads, a milling machine, etc. until the desired size is obtained. Can be cut into pieces.

Carbon sources such as alginic acid, cellulose, mannitol, pectin, galacturonic acid, carrageenan and agar contained in seaweed are extracted from seaweed by the following method.
(1) Extraction process After thoroughly washing the raw seaweed, alkali is added and heated to solubilize alginic acid in the alga body. Alginic acid contained in seaweed produces minerals and salts in seawater and is filled between cell walls in an insoluble jelly state. By adding a sodium salt to seaweed, the insoluble salt of alginic acid is replaced with water-soluble sodium alginate and is eluted out of the alga.
(2) Filtration step When the alginic acid is sufficiently dissolved, the insoluble component is removed by filtration to obtain a sodium alginate solution. Due to the viscosity of the solubilized alginic acid, the extract is highly viscous. In order to filter this, a large amount of water is added to dilute to lower the viscosity.
(3) Precipitate An acid is added to an aqueous solution of sodium alginate to lower the pH and precipitate again as insoluble alginic acid. If a calcium salt is used instead of an acid, it can also be precipitated as insoluble calcium alginate.
(4) Dehydrated and precipitated alginic acid is washed thoroughly and dried to obtain alginic acid. If this alginic acid is neutralized with an alkali, it becomes an alginate. If sodium is used as the alkali used for neutralization, it becomes sodium alginate, and if potassium is used, it becomes potassium alginate.

  The alginic acid obtained in the above process is desirably used as a carbon source for the medium after performing an enzymatic degradation reaction with an alginate lyase or a hydrolysis reaction with an acid. For example, in order to obtain a carbon source as a main raw material for fermentation from seaweed, an acid such as inorganic acid such as hydrochloric acid or phosphoric acid, sulfonic acid such as sulfuric acid, carboxylic acid such as acetic acid or formic acid is added to seaweed. By the acid treatment, a seaweed saccharified solution in which decomposition of polysaccharides in seaweed, that is, saccharification is promoted can be obtained.

In addition, to obtain a carbon source as the main raw material for fermentation from seaweed, enzymatic treatment with an enzyme having the enzymatic activity of alginate lyase (EC.4.2.2.3), λ-carrageenase (Lambda-carrageenase, EC 3.2.1.162) or pectinase It is desirable to do. By performing the enzyme treatment, a seaweed saccharified solution in which decomposition of polysaccharides in seaweed, that is, saccharification is promoted can be obtained.
The above-mentioned seaweed fragmentation, swelling, acid treatment, and enzyme treatment steps for obtaining the seaweed saccharified solution may be performed independently and continuously, or any of the steps may be performed simultaneously. Also good. Further, the order of these steps may be changed.

As the alginate lyase, the above-described enzyme can be used, and it can be directly expressed in cells and used. Alternatively, alginate lyase isolated separately may be reacted with a processed seaweed to obtain alginate.

  As the pectinase, polygalacturonase, pectin lyase, pectin esterase, pectin methyl esterase and the like can be used.

  Cellulose contained in seaweed can be decomposed by adding cellulase after extracting and separating polysaccharides containing alginic acid. In the present invention, cellulase is also referred to as endo-1,4-β-glucanase, and means an enzyme that mainly hydrolyzes β1 → 4 glucoside bonds of cellulose to produce cellobiose. Cellulase is present in higher plants, bacteria, filamentous fungi, wood-rotting fungi, mollusks and the like. In the present invention, the origin of cellulase is not limited, and those extracted and purified from microorganisms or those produced by molecular biology can also be used.

  The microorganism of the present invention is a microorganism that can assimilate alginate purified by the extraction process exemplified above. In the present invention, alginates mean sodium alginate and potassium alginate, and the microorganism of the present invention is preferably a microorganism capable of growing in a minimal medium using alginic acid and alginate as a carbon source. For example, a microorganism capable of assimilating alginic acid in the medium at 0.5 g / L or more, desirably 1 g / L or more, more desirably 2.5 g / L or more is preferable. In the present invention, a medium containing a minimum amount of components necessary for vegetative growth of microorganisms is defined as a minimum medium.

  The microorganism of the present invention belongs to the genus Vibrio, and the species name is selected from Vibrio rumoiensis or Vibrio alginovora.

  The microorganism of the present invention is characterized in that the 16S ribosomal RNA sequence has a sequence having a homology of 95% or more with SEQ ID NO: 1, but preferably 96% or more, 97% or more, 98% or more, 99% or more. It is preferable to have properties. Whether or not it has a defined 16S ribosomal RNA can be determined, for example, by comparing the base sequence data of the 16S ribosomal RNA gene with the sequence data of a known species, comparing the sequence data of a known species and performing phylogenetic analysis. it can. The phylogenetic analysis and the phylogenetic tree creation method are performed, for example, according to the following procedure.

  First, genomic DNA as a template is extracted from bacteria. Methods for extracting DNA from bacteria are known and any method may be used. In general, a method of treating cells with a cell wall degrading enzyme such as lysozyme, a physical destruction method using glass beads, a treatment method of repeating freeze-thawing, and the like are used. Commercially available reagents for DNA extraction can also be used. Genomic DNA does not necessarily have to be extracted in an intact state. Therefore, it is possible to appropriately select a method that has a low possibility of sample contamination, is easy to operate, and can be performed quickly.

  Next, the target DNA encoding 16S ribosomal RNA is amplified by polymerase chain reaction (PCR). The primer sequences used in PCR can be designed as appropriate so that at least target DNAs encoding 16S ribosomal RNAs of all known bacteria belonging to the family Lacnospiridae can be amplified, but are usually conserved across species. A primer (universal primer) consisting of the above sequences is used. The PCR conditions are not particularly limited, and can be appropriately selected within the range usually used. The reaction can be performed using a commercially available PCR reagent according to the attached instructions.

  A DNA fragment amplified by PCR is purified using a spin column or the like as necessary, and then its base sequence is determined. The base sequence can be determined according to a standard method.

  From the determined base sequence, a known sequence showing the highest homology can be extracted by performing a homology search with a known bacterial 16S ribosomal DNA sequence using an appropriate gene sequence database and homology search program. it can. For example, BLAST and FASTA can be used through the homepage of the Japan DNA Data Bank (DDBJ). If blastn or fasta is selected as a program, the determined base sequence is used as a query, 16S rRNA (Prokaryotes) is selected as a search target database, and a search is performed, a known sequence showing high homology is extracted and output. . Any other gene sequence database can be used as long as it contains a dataset of bacterial 16S ribosomal RNA gene sequences. Other than the above-mentioned homology search programs known per se can also be used.

  A molecular evolutionary phylogenetic tree can be estimated based on the base sequence of the amplified DNA, and the taxonomic position of the isolated bacterium can be specified. Molecular evolutionary phylogenetic tree analysis software is also available on the Internet and can be used (CLUSTAL W etc.). As a result of phylogenetic tree analysis, when the isolated bacterium is in the same cluster as the bacterium belonging to the family Lacnospiridae, the bacterium can be identified as a bacterium belonging to the genus Vibrio of the present invention.

  The microorganism of the present invention can be obtained from the seaweed and the organism that grows using seaweed as a food source. For example, it can be obtained from Sazae, abalone, sea cucumber, horse mackerel, hailfish, red sea bream, spatula and the like.

  In one aspect, the bacterium of the present invention comprises a 16S ribosomal RNA gene comprising a nucleic acid sequence having 95% or more homology with the nucleic acid sequence represented by SEQ ID NO: 1, preferably the nucleic acid sequence represented by SEQ ID NO: 1. It has a 16S ribosomal RNA gene. Here, SEQ ID NO: 1 shows the sequence of the 16S ribosomal RNA gene of one of the bacteria of the present invention (NITE P-01635 strain) belonging to the genus Vibrio described later. Homology between nucleic acid sequences is calculated using the above homology search program BLAST

  It can be confirmed that the bacterium has the 16S ribosomal RNA gene using the above-described method for analyzing the base sequence of the 16S ribosomal RNA gene. Alternatively, in accordance with the detection reagent and detection method of the present invention described in detail below, using a primer or probe that can specifically detect the gene, a method known per se (PCR, Southern blotting, DNA array, etc.) is used. I can do it.

  An example of a strain of the bacterium of the present invention isolated by the present inventors (NITE P-01635 strain, also referred to herein as Vibrio sp. SA2) is the accession number NITE P-01635, which is an independent administrative corporation product. It is deposited in Japan with the Evaluation Technology Infrastructure Organization (Room No. 2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture) (Contract date: June 13, 2013).

  In addition, it is preferable that the microorganism of the present invention is further modified so as to have a substance-producing ability.

  In the present invention, the target substance is not limited as long as the target substance is produced using the microorganism of the present invention. For example, L-amino acid, nucleic acid, ethanol, isopropyl alcohol, acetone, propylene, 1,3-butanediol, 1,4-butanediol, 1-propanol, 1,3-propanediol, 1,2-propanediol, ethylene glycol, and isobutanol are preferred.

In the present invention, L-amino acid means L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L-
Leucine, glycine, L-threonine, L-serine, L-proline, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L-cystine, L-methionine, L-glutamic acid, L-aspartic acid, L -Means one or more amino acids selected from glutamine and L-asparagine.

  In the present invention, the nucleic acid includes purine nucleosides and purine nucleotides. Purine nucleosides include inosine, guanosine, xanthosine, and adenosine. The bacterium of the present invention may have the ability to produce one kind of purine nucleoside, or may have the ability to produce two or more kinds of purine nucleosides. In the present invention, one kind of purine nucleoside may be produced, or two or more kinds of purine nucleosides may be produced. Purine nucleotides include 5'-phosphate esters of purine nucleosides. Examples of 5'-phosphate esters of purine nucleosides include inosinic acid (inosine-5'-phosphate ester; IMP), guanylic acid (guanosine-5'-phosphate ester; GMP), xanthylic acid (xanthosine-5'-). Phosphate ester; XMP), and adenylic acid (adenosine-5′-phosphate ester; AMP).

  In the present invention, the following method is selected for modification to have L-amino acid production ability.

<Amino acid producing bacteria>
Vibrio bacteria having L-amino acid-producing ability can be obtained by imparting L-amino acid-producing ability to a wild strain of Vibrio bacteria as described above. In order to confer L-amino acid-producing ability, an auxotrophic mutant strain, an L-amino acid analog resistant or metabolically controlled mutant strain, which has been used for breeding conventional coryneform bacteria, Escherichia bacteria, etc., L- Breeding can be carried out using a method of creating a recombinant strain with enhanced activity of an amino acid biosynthesis enzyme (Amino Acid Fermentation Co., Ltd. Publishing Center). In breeding L-amino acid-producing bacteria, imparting properties such as auxotrophy, L-amino acid analog resistance, and metabolic control mutations may be combined with enhancement of the activity of L-amino acid biosynthetic enzymes.
Hereinafter, a method for imparting various amino acid-producing ability will be exemplified.

L-lysine producing bacteria For example, L-lysine producing bacteria are L-homoserine, or mutant strains requiring L-threonine and L-methionine (Japanese Patent Publication Nos. 48-28078 and 56-6499), inositol or acetic acid. Mutants that require oxidase (JP 55-9784, JP 56-8692), or oxalidine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine, γ-methyllysine, α- It can be bred as a mutant strain resistant to chlorocaprolactam, DL-α-amino-ε-caprolactam, α-amino-lauryllactam, aspartic acid-analog, sulfa drug, quinoid, or N-lauroylleucine. In particular, it is preferable to breed as a mutant having resistance to S- (2-aminoethyl) -L-cysteine (AEC) as an L-lysine analog.

  Mutation treatment methods for obtaining mutants from Vibrio spp. Include ultraviolet irradiation, or mutations commonly used for mutation treatments such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or nitrous acid. The method of processing with an agent is mentioned. Moreover, Vibrio bacteria having L-amino acid-producing ability can also be obtained by selecting natural mutants of Vibrio bacteria.

An L-amino acid analog-resistant mutant strain can be obtained by, for example, inoculating a mutated Vibrio bacterium into an agar medium containing various concentrations of an L-amino acid analog and selecting a strain that forms a colony. it can.

  In addition, the auxotrophic mutant strain is formed by forming a colony of Vibrio bacteria on an agar medium containing a target nutrient substance (for example, L-amino acid) and replicating the colony to an agar medium not containing the nutrient substance. It can be obtained by selecting a strain that cannot grow on an agar medium containing no substance.

  Next, a method for imparting or enhancing L-lysine production ability by enhancing the activity of the L-lysine biosynthesis enzyme will be exemplified below.

  The ability to produce L-lysine can be imparted, for example, by enhancing dihydrodipicolinate synthase activity and / or aspartokinase activity.

  In order to enhance the dihydrodipicolinate synthase activity and / or aspartokinase activity of Vibrio spp., The gene fragment encoding dihydrodipicolinate synthase and / or the gene fragment encoding aspartokinase functions in Vibrio A recombinant DNA is prepared by ligating with a vector to be used, preferably a multicopy vector, and this is introduced into a host of Vibrio bacteria and transformed. As a result of an increase in the copy number of the gene encoding dihydrodipicolinate synthase and / or the gene encoding aspartokinase in the cells of the transformed strain, the activities of these enzymes are enhanced. Hereinafter, dihydrodipicolinate synthase may be abbreviated as DDPS, aspartokinase as AK, and aspartokinase III as AKIII.

  Any microorganism that can express DDPS activity and AK activity among microorganisms belonging to the genus Vibrio can be used as a microorganism that provides a gene encoding DDPS and a gene encoding AK. The microorganism may be either a wild strain or a mutant derived therefrom. Specific examples include E. coli (Escherichia coli) K-12 strain and Vibrio natrigens IFO15636 strain. A gene encoding DDPS derived from bacteria belonging to the genus Escherichia (dapA, Richaud, F. et al. J. Bacteriol., 297 (1986)) and a gene encoding AKIII (lysC, Cassan, M., Parsot, C., Cohen , GN and Patte, JC, J. Biol. Chem., 261, 1052 (1986)), the base sequences have been elucidated, and primers were synthesized based on the base sequences of these genes. These genes can be obtained by a PCR method using a chromosomal DNA of a microorganism such as E. coli K-12 or Vibrio natrigens IFO15636.

Vibrio gene can be obtained by using the following GenBank database.
Vibrio cholerae O1 biovar eltor str.N16961 chromosome I, complete sequence; AE003852
Vibrio cholerae O1 biovar eltor str.N16961 chromosome II, complete sequence; AE003853
Vibrio parahaemolyticus RIMD 2210633 chromosome I, complete sequence; BA000031
Vibrio parahaemolyticus RIMD 2210633 chromosome II, complete sequence; BA000032
Vibrio fischeri ES114 chromosome I, complete sequence; CP000020
Vibrio fischeri ES114 chromosome II, complete sequence; CP000021
Vibrio vulnificus CMCP6 chromosome I, complete sequence; AE016795
Vibrio vulnificus CMCP6 chromosome II, complete sequence; AE016796
Vibrio vulnificus YJ016 chromosome I, complete sequence; BA000037
Vibrio vulnificus YJ016 chromosome II, complete sequence; BA000038

Examples of Vibrio bacteria that are known to have alginic acid lyase activity and can degrade alginic acid include the following microbial species:
Vibrio alginolytics
Vibrio splendidus
Vibrio kanaloaei
Vibrio pomeroyi
Vibrio chagasii
Vibrio lentus
Vibrio cyclitrophicus
Vibrio crassostreae
Vibrio halitiocoli

  Vibrio gene can be obtained by referring to the GenBank database ID shown in the following table. Specifically, enter the Assembly ID shown in the table below from the NCBI URL http://www.ncbi.nlm.nih.gov/ and download each Assembly ID from the WGS (Whole Genome Sequence) Project page. The gene of the genus Vibrio can be obtained.

  DDPS and AK used in the present invention are preferably those that are not subject to feedback inhibition by L-lysine. Wild-type DDPS derived from Vibrio is known to undergo feedback inhibition by L-lysine, and wild-type AKIII from Vibrio is known to undergo inhibition and feedback inhibition by L-lysine. Therefore, it is preferable that dapA and lysC introduced into Vibrio bacteria encode DDPS and AKIII having a mutation that cancels feedback inhibition by L-lysine, respectively.

  In the present invention, DDPS and AK do not necessarily need to be mutated. For example, it is known that DDPS derived from Corynebacterium is not subject to feedback inhibition by L-lysine.

  In addition, homologs may exist for genes encoding aspartokinase, and the gene source is not limited as long as it has aspartokinase activity.

Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to clearly quantify this condition, for example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97%. 60 ° C., 1 × S, which is a condition in which DNAs having the above homology hybridize and DNAs having lower homology do not hybridize, or washing conditions of normal Southern hybridization
SC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, salt concentration and temperature corresponding to 0.1% SDS once, more preferably 2-3 times The conditions to wash | clean are mentioned.

  Aspartokinase activity can be measured by the method described in Miyajima, R et al; The Journal of Biochemistry (1968), 63 (2), 139-148. In addition, the genes are not limited to wild-type genes, and as long as they have aspartokinase activity, one or several amino acids at one or more positions in the amino acid sequence encoded by the above open reading frame of each gene It may be a mutant or artificially modified protein that encodes a protein having an amino acid sequence including substitution, deletion, insertion, or addition. Here, “1 or several” varies depending on the position and type of the protein in the three-dimensional structure of the amino acid residue, but specifically 1 to 20, preferably 1 to 10, more preferably 1 to 10. Mean 5. The substitution, deletion, insertion, or addition of one or several amino acids is a conservative mutation that maintains the above function. A conservative mutation is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr.

  Typical conservative mutations are conservative substitutions. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys. , Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg Substitution, Glu to Gly, Asn, Gln, Lys or Asp substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe Substitution, Leu to Ile, Met, Val or Phe, Lys to Asn, Glu, Gln, His or Arg, Met to Ile, Leu, Val or Phe, Phe to Trp, Tyr, Met, Ile or Leu substitution, Ser to Thr or Ala substitution, Thr to Ser or Ala substitution, Trp to Phe or Tyr substitution, Tyr to His, Phe or Trp substitution, and Val To Met, Ile or Leu It is.

  80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more homology to the entire amino acid sequence encoded by the open reading frame of each gene, and A sequence encoding a protein having aspartokinase activity can also be used. For the homology of amino acid sequences, for example, the algorithm BLAST (Proc. Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA (Methods Enzymol., 183, 63 (1990)) by Karlin and Altschul) is used. Can be determined. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed (see http://www.ncbi.nlm.nih.gov).

  The plasmid used for gene cloning may be any plasmid that can be replicated in microorganisms such as Escherichia bacteria. Specific examples include pBR322, pTWV228, pMW119, and pUC19.

  Any vector that functions in the genus Vibrio can be used as long as it is a plasmid that can autonomously replicate in the genus Vibrio. Any vector plasmid having an ori derived from pUC, pACYC184, or IncQ can be used. As a marker gene used for selection, a Tn903-derived kanamycin resistance gene, a Tn9-derived chloramphenicol resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, or the like can be used.

  In order to prepare a recombinant DNA by ligating dapA and lysC and a vector that functions in Vibrio bacteria, the vector is cleaved with a restriction enzyme that matches the ends of the DNA fragment containing dapA and lysC. Ligation is usually performed using a ligase such as T4 DNA ligase. dapA and lysC may be mounted on separate vectors or on the same vector.

  Examples of DNA encoding a mutant dihydrodipicolinate synthase that is not subject to feedback inhibition by L-lysine include DNA encoding a protein having a sequence in which the histidine residue at position 118 is substituted with a tyrosine residue. As a DNA encoding a mutant aspartokinase that is not subject to feedback inhibition by L-lysine, the threonine residue at position 352 is replaced with an isoleucine residue, the glycine residue at position 323 is replaced with an asparagine residue, 318 Examples include DNA encoding AKIII having a sequence in which the methionine at the position is replaced with isoleucine (see US Pat. Nos. 5,610,010 and 6,040,160 for these variants). Mutant DNA can be obtained by site-specific mutagenesis such as PCR.

  Wide host range plasmids RSFD80, pCAB1, and pCABD2 are known as plasmids containing mutant dapA encoding mutant mutant dihydrodipicolinate synthase and mutant lysC encoding mutant aspartokinase (USA) Patent No. 6040160). Escherichia coli JM109 strain (US Pat. No. 6,040,160) transformed with RSFD80 was named AJ12396, and this strain was established on October 28, 1993 at the Institute of Biotechnology, Ministry of International Trade and Industry. Deposited with the Patent Biological Depositary Center, National Institute of Product Evaluation Technology) under the deposit number FERM P-13936, transferred to the international deposit under the Budapest Treaty on November 1, 1994, under the deposit number of FERM BP-4859 Deposited at RSFD80 can be obtained from AJ12396 strain by a known method.

  Any method may be used for introducing the recombinant DNA prepared as described above into Vibrio bacteria so long as sufficient transformation efficiency can be obtained. For example, the electroporation method (Canadian Journal of Microbiology, 43. 197 (1997)).

  Enhancement of DDPS activity and / or AK activity can also be achieved by the presence of multiple copies of dapA and / or lysC on the chromosomal DNA of Vibrio bacteria. Introducing dapA and / or lysC in multiple copies onto the chromosomal DNA of Vibrio bacteria can be carried out by homologous recombination using a sequence present in multiple copies on the chromosomal DNA as a target. As a sequence present in multiple copies on chromosomal DNA, repetitive DNA, inverted repeats present at the end of a transposable element, and the like can be used. Alternatively, as disclosed in JP-A-2-109985, dapA and / or lysC can be mounted on a transposon and transferred to introduce multiple copies onto chromosomal DNA. In any method, as a result of an increase in the copy number of dapA and / or lysC in the transformant, DDPS activity and / or AK activity is amplified.

  Amplification of DDPS activity and / or AK activity can be achieved by replacing expression control sequences such as dapA and / or lysC promoters with strong ones in addition to the gene amplification described above (JP-A-1-215280). No. publication). For example, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter of lambda phage, PL promoter, tet promoter, amyE promoter, spac promoter and the like are known as strong promoters. By replacing these promoters, DDPS activity and / or AK activity is amplified by enhancing the expression of dapA and / or lysC. Substitution of expression regulatory sequences may be combined with increasing copy number of dapA and / or lysC.

DNA cleavage, ligation, other preparation of chromosomal DNA, PCR, plasmid DNA
As methods for preparing, transforming, setting oligonucleotides used as primers, etc., ordinary methods well known to those skilled in the art can be employed. These methods are described in Sambrook, J., Fritsch, EF, and Maniatis, T., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, (1989) and the like.

  In addition to enhancing DDPS activity and / or AK activity, the activity of other enzymes involved in L-lysine biosynthesis may be enhanced. Examples of such enzymes include dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (above, WO 96/40934 pamphlet), phosphoenolpyruvate carboxylase (ppc ) (Japanese Patent Laid-Open No. 60-87788), Aspartate aminotransferase (aspC) (Japanese Patent Publication No. 6-2002028), Diaminopimelate epimerase (dapF) (Japanese Patent Laid-Open No. 2003-135066), Aspartate semialdehyde dehydration Examples thereof include enzymes of the diaminopimelate pathway such as elementary enzyme (asd) (WO 00/61723 pamphlet), and enzymes of the aminoadipate pathway such as homoaconite hydratase (Japanese Patent Laid-Open No. 2000-157276). The parentheses after the enzyme name are gene names (the same applies to the following description).

  The Vibrio bacterium of the present invention may be a bacterium having enhanced L-lysine production ability by enhancing L-lysine excretion activity. For example, L-lysine excretion activity can be increased by increasing the expression level of the ybjE gene and increasing the expression level of the lysE gene (Japanese Patent Laid-Open No. 2005-237379, WO97 / 23697 pamphlet).

  Furthermore, the bacterium of the present invention further functions negatively in the activity of an enzyme that catalyzes a reaction that produces a compound other than an L-amino acid by branching from the L-amino acid biosynthetic pathway, or in the synthesis or accumulation of an L-amino acid. The enzyme activity may be reduced or deficient. In the production of L-lysine, examples of such enzymes include homoserine dehydrogenase, lysine decarboxylase (cadA, ldcC), malic enzyme, and the like, and a strain in which the activity of the enzyme is reduced or deleted is disclosed in WO95 / 23864, It can be constructed with reference to WO96 / 17930 pamphlet and WO2005 / 010175 pamphlet.

  As a method for reducing or eliminating these enzyme activities, a mutation that reduces or eliminates the activity of the enzyme in the cell is applied to the gene of the enzyme on the genome by a usual mutation treatment method or gene recombination technique. What is necessary is just to introduce. Such mutations can be introduced, for example, by deleting a gene encoding an enzyme on the genome by genetic recombination or by modifying an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence. Achieved. Also, introducing amino acid substitutions (missense mutations) into the region encoding the enzyme on the genome, introducing stop codons (nonsense mutations), and introducing frameshift mutations that add or delete one or two bases. It can also be achieved by deleting a part or the entire region of the gene (J. Biol. Chem. 272: 8611-8617 (1997)). In addition, a gene encoding a mutant enzyme in which all or part of the coding region has been deleted is constructed, and a normal gene on the genome is replaced with the gene by homologous recombination, transposon, or IS factor. By introducing it into the gene, the enzyme activity can be reduced or eliminated.

For example, in order to introduce a mutation that reduces or eliminates the activity of the above enzyme by genetic recombination, the following method is used. A partial gene of the target gene is modified to produce a mutant gene that does not produce a normally functioning enzyme, transformed into a bacterium belonging to the genus Vibrio with the DNA containing the gene, and the mutant gene and genome By causing recombination with a gene, the target gene on the genome can be replaced with a mutant. Such gene replacement using homologous recombination is a method called “Red-driven integration” (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97 : 6640-6645 (2000)), a combination of Red driven integration method and λ phage-derived excision system (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)) And a method using a linear DNA such as WO2005 / 010175) and a method using a plasmid containing a temperature-sensitive replication origin (US Pat. No. 6,303,383; Japanese Patent Laid-Open No. 05-007491). Moreover, site-directed mutagenesis by gene replacement using homologous recombination as described above can also be performed using a plasmid that does not have replication ability on the host.

  The above-described method for enhancing the enzyme activity involved in L-lysine biosynthesis and the method for reducing the enzyme activity can be similarly applied to breeding other L-amino acid-producing bacteria. Hereinafter, methods for breeding other L-amino acid-producing bacteria will be described.

  An L-tryptophan-producing bacterium can be constructed by, for example, modifying an anthranilate synthase activity, a phosphoglycerate dehydrogenase activity, or a tryptophan synthase activity so that one or more activities are enhanced. Here, since anthranilate synthase and phosphoglycerate dehydrogenase are subjected to feedback inhibition by L-tryptophan and L-serine, respectively, the enzyme activity can be further enhanced by retaining a desensitized mutant enzyme. it can. Specifically, for example, the anthranilate synthase gene (trpE) and / or the phosphoglycerate dehydrogenase gene (serA) is mutated so as not to be subjected to feedback inhibition, and the obtained mutant gene belongs to the genus Vibrio. By introducing into a bacterium, a bacterium that retains the desensitizing enzyme can be obtained. (See pamphlet of International Publication No.94 / 08031)

  A bacterium into which a recombinant DNA containing a tryptophan operon has been introduced is also a suitable L-tryptophan-producing bacterium. Specifically, a method for introducing a tryptophan operon containing a gene encoding a desensitized anthranilate synthase can be mentioned. (JP 57-71397, JP 62-244382, US Pat. No. 4,371,614). Moreover, L-tryptophan production ability can also be improved or imparted by enhancing expression of a gene (trpBA) encoding tryptophan synthase among tryptophan operons. Tryptophan synthase consists of α and β subunits and is encoded by trpA and trpB, respectively.

  A suitable L-tryptophan-producing bacterium can also be obtained by deleting trpR, which is a repressor of tryptophan operon, or by introducing a mutation into trpR (US Pat. No. 4,371,614, International Publication No. WO2005 / 056776). ).

  Furthermore, L-tryptophan-producing bacteria can also be constructed by modifying them so as to have L-phenylalanine and L-tyrosine-requiring traits.

  In addition, as L-tryptophan-producing bacteria, strains with increased 3-phosphoserine phosphatase (serB) activity (US4,371,614) and strains with increased phosphoenolpyruvate carboxykinase (pckA) may also be used. Can be built.

L-tryptophan, L-phenylalanine, and L-tyrosine are all aromatic amino acids and have a common biosynthetic system. As a gene encoding an aromatic amino acid biosynthetic enzyme, deoxyarabino-heptulonic acid phosphate synthase (aroG), 3-dehydroquinate synthase (aroB), shikimate dehydratase, shikimate kinase (aroL), 5-enolic acid pyruvylshikimate 3-phosphate synthase (aroA), chorismate synthase (aroC). Therefore, the ability to produce aromatic amino acids can be improved by making multiple copies of genes encoding these enzymes on a plasmid or genome. In addition, these genes are known to be controlled by a tyrosine repressor (tyrR), and the biosynthetic enzyme activity of aromatic amino acids may be increased by deleting the tyrR gene. (See European Patent No. 763127) In addition, in the case of enhancing each amino acid-producing ability, biosynthetic systems other than the target aromatic amino acid may be weakened. For example, when the target amino acid is L-tryptophan, the L-phenylalanine biosynthesis system and the L-tyrosine biosynthesis system may be weakened. (US4,371,614)

  Moreover, since 3-deoxy-D-arabinohepturonic acid-7-phosphate synthase (aroF, aroG) receives feedback inhibition by an aromatic amino acid, it may be modified so as not to receive feedback inhibition. For example, when Escherichia coli aroF is used, L-aspartic acid at position 147 from the N-terminal or L-serine at position 181 from the other amino acid residue, and in the case of aroG, L-aspartic acid at position 146 from the N-terminus One amino acid residue of 147th L-methionine, 150th L-proline or 202th L-alanine, or two amino acid residues of 157th L-methionine and 219th L-alanine to other amino acids An aromatic amino acid-producing bacterium can be obtained by introducing a mutant aroF or aroG gene substituted in to a host. (EP0488424)

  As an L-phenylalanine-producing bacterium, in addition to the above-described modification, a strain in which tyrA and tyrR are deleted, a strain in which a phenylalanine excretion gene yddG or a yedA gene is amplified can be used.

  A preferable bacterium belonging to the genus Vibrio having L-threonine-producing ability can be obtained by modifying the L-threonine biosynthetic enzyme so as to enhance it. The genes encoding L-threonine biosynthetic enzymes include aspartokinase III gene (lysC), aspartate semialdehyde dehydrogenase gene (asd), aspartokinase I gene (thrA) encoded by the thr operon, homoserine kinase Gene (thrB) and threonine synthase gene (thrC). Two or more of these genes may be introduced. The L-threonine biosynthesis gene may be introduced into a bacterium belonging to the genus Vibrio in which threonine degradation is suppressed. For example, threonine degradation can be suppressed by reducing threonine dehydrogenase activity.

  The enzyme activity of the L-threonine biosynthetic enzyme is suppressed by the final product, L-threonine. Therefore, in order to construct an L-threonine-producing bacterium, it is desirable to modify the L-threonine biosynthetic gene so that it is not subject to feedback inhibition by L-threonine. The thrA, thrB, and thrC genes constitute the threonine operon, but the threonine operon forms an attenuator structure, and the expression of the threonine operon is inhibited by isoleucine and threonine in the culture medium. The expression is suppressed by attenuation. Therefore, this modification can be achieved by removing the leader sequence of the attenuation region or the attenuator (Lynn, SP, Burton, WS, Donohue, TJ, Gould, RM, Gumport, RI, and Gardner, JFJ Mol. Biol. 194: 59-69 (1987); WO 02/26993 pamphlet; WO 2005/049808 pamphlet).

  Moreover, in order to modify Vibrio bacteria so as not to receive feedback inhibition by L-threonine, resistance may be imparted to α-amino-β-hydroxyvaleric acid (AHV).

  Further, aspartokinase III gene (lysC) is desirably a gene modified so as not to be subjected to feedback inhibition by L-lysine. The lysC gene modified so as not to receive such feedback inhibition can be obtained from the genes described above.

In addition to the L-threonine biosynthetic enzymes, strengthening the genes related to glycolysis, TCA cycle, respiratory chain, genes controlling the expression of these genes, and sugar uptake genes are also useful for breeding L-threonine producing bacteria. Is preferred. These genes effective for L-threonine production include transhydronase gene (pntAB) (European Patent 733712), phosphoenolpyruvate carboxylase gene (pepC) (International Publication No. 95/06114 pamphlet), phospho Examples include the enol pyruvate synthase gene (pps) (European Patent No. 877090), the pyruvate carboxylase gene of Coryneform bacteria or Bacillus bacteria (International Publication No. 99/18228, European Application Publication No. 1092776).

  Moreover, strengthening the expression of genes that confer resistance to L-threonine and / or genes that confer resistance to L-homoserine, or confer L-threonine resistance and / or L-homoserine resistance to the host Is also suitable. Examples of genes that confer resistance include rhtA gene (Res. Microbiol. 154: 123-135 (2003)), rhtB gene (European Patent Application Publication No. 0994190), and rhtC gene (European Patent Application Publication No. 1013765). ), YfiK, yeaS gene (European Patent Application Publication No. 1016710). For methods for imparting L-threonine resistance to a host, the methods described in European Patent Application Publication No. 0994190 and International Publication No. 90/04636 can be referred to.

In order to construct a Vibrio bacterium having L-glutamic acid-producing ability, it is desirable to modify so that expression of a gene encoding an enzyme involved in L-glutamic acid biosynthesis is increased. Examples of enzymes involved in L-glutamate biosynthesis include glutamate dehydrogenase (hereinafter also referred to as “GDH”) (gdhA), glutamine synthetase (glnA), glutamate synthase (gltAB), citrate synthase (gltA), phosphoenols. Pyruvate carboxylase (ppc), pyruvate carboxylase, pyruvate dehydrogenase (aceEF,
lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglycerum mutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3- Examples thereof include phosphate dehydrogenase (gapA), triose phosphate isomerase (tpiA), furtose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose phosphate isomerase (pgi) and the like. Among these enzyme genes, one or more of CS, PEPC and GDH are preferable, and all three are more preferable. (US Pat. Nos. 6,197,559, 6,331,419, European Patent 0999282)

  Further, the 6-phosphogluconate dehydratase activity, 2-keto-3-deoxy-6-phosphogluconate aldolase activity, or both of these activities may be modified (European Patent Application Publication No. 1352966). The yhfK gene (WO2005 / 085419 pamphlet) and ybjL gene (WO2008 / 133161 pamphlet) encoding a protein that excretes L-glutamic acid and the ybjL gene derived from Escherichia coli are represented by SEQ ID NO. The number 3 is shown.

  Further, as a bacterium belonging to the genus Vibrio having L-glutamic acid-producing ability, a bacterium having a reduced or deficient activity of an enzyme that catalyzes a reaction that branches off from the biosynthetic pathway of L-glutamic acid to produce other compounds May be. Examples of the enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-glutamic acid to produce a compound other than L-glutamic acid include 2-oxoglutarate dehydrogenase, isocitrate lyase, phosphate acetyltransferase, acetate kinase, acetohydroxyacid synthase Acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase and the like. Among these, it is particularly preferable to reduce or eliminate 2-oxoglutarate dehydrogenase activity, and strains that have reduced or lacked 2-oxoglutarate dehydrogenase activity are disclosed in European Patent Nos. 0952221, 0955368, and US Pat. No. 5,378,616. It can be constructed by reference.

  As a bacterium having the ability to produce L-histidine, a bacterium having an increased expression level of a gene encoding an enzyme of the L-histidine biosynthesis pathway may be used. Examples of genes encoding L-histidine biosynthetic enzymes include ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (hisI), phosphoribosyl-ATP pyrophosphohydrolase (phosphoribosy L-ATP pyrophosphohydrolase) hisIE), phosphoribosylformimino-5-aminoinidazole carboxyamide ribotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase (hisH) hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD) and the like.

  L-histidine-producing bacteria can also be obtained by conferring resistance to sulfaguanidine, D, L-1,2,4-triazole-3-alanine, and streptomycin (Russia 2119536).

  In order to construct L-cysteine-producing bacteria, serine acetyltransferase is modified so that cystathionine-β-lyase activity is reduced (Japanese Patent Laid-Open No. 2003-169668), and feedback inhibition by L-cysteine is reduced. It is preferable to modify so that (Japanese Patent Laid-Open No. 11-155571)

  In order to obtain L-arginine-producing bacteria, α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamic acid, S- (2-aminoethyl) -cysteine, α-methylserine, β-2-thienylalanine Or modification to have resistance to sulfaguanidine. It is also suitable as a method for breeding L-arginine-producing bacteria to have a mutation resistant to feedback inhibition by L-arginine and to have a highly active N-acetylglutamate synthase.

  In addition, as a Vibrio bacterium having L-arginine-producing ability, a bacterium having an increased expression level of a gene encoding an enzyme involved in L-arginine biosynthesis can be used. For example, L-arginine biosynthetic enzymes include N-acetylglutamate synthase (argA), N-acetylglutamyl phosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetyl Examples include ornithine transaminase (argD), acetylornithine deacetylase (argE) ornithine carbamoyltransferase (argF), argininosuccinate synthase (argG), and argininosuccinate lyase (argH) carbamoyl phosphate synthase (carAB). Among them, the N-acetylglutamate synthase gene (argA) is a mutant gene that encodes a mutant enzyme in which the amino acid sequence corresponding to the 15th to 19th positions of the wild type is substituted and feedback inhibition by L-arginine is released. It is more preferable to use (European Application Publication No. 1170361).

  The L-leucine-producing bacterium inactivates the branched chain amino acid transaminase encoded by the ilvE gene and increases the activity of the aromatic amino acid transaminase encoded by the tyrB gene (Japanese Patent Laid-Open No. 2004-024259), or 4- It can be obtained by modifying to have azaleucine or 5,5,5-trifluoroleucine resistance. Furthermore, the feedback inhibition of isopropylmalate synthase by L-leucine is modified for desensitization (European Patent No. 1067191), so as to be resistant to β-2-thienylalanine and β-hydroxyleucine. A suitable L-leucine-producing bacterium can also be constructed by modifying to (US Pat. No. 5,763,231).

L-isoleucine-producing bacterium is resistant to 6-dimethylaminopurine (Japanese Patent Laid-Open No. 5-304969)
L-isoleucine hydroxamate resistance (Japanese Patent Laid-Open No. 5-130882), thiisoleucine resistance (Japanese Patent Laid-Open No. 5-130882), DL-ethionine resistance (Japanese Patent Laid-Open No. 5-130882), or arginine hydroxamate It can be obtained by imparting resistance (Japanese Patent Laid-Open No. 5-130882). In addition, recombinant Vibrio bacteria can be obtained by using a plasmid to enhance a gene encoding threonine deaminase or acetohydroxy acid synthase, which is an L-isoleucine biosynthetic enzyme (JP-A-2-458, JP-A-2-42988). Gazette, JP-A-8-47397) and the like.

  The L-valine-producing bacterium is modified so as to have a mutation requiring lipoic acid for growth or / and a mutation deficient in proton ATPase, as described in WO96 / 06926, or at least ilvG, It can be constructed by expressing each gene of ilvM, ilvE and ilvD and introducing a DNA fragment containing the ilvGMEDA operon into the cell. The ilvGMEDA operon is subject to expression regulation (attenuation) of the operon by L-valine and / or L-isoleucine and / or L-leucine, so that the expression suppression by the produced L-valine is released. It is preferable that a region necessary for nuation is removed or mutated (US Pat. No. 5,998,178). The ilvGMEDA operon preferably does not express threonine deaminase activity.

  In addition, the L-amino acid-producing bacterium used in the present invention may have a gene that is involved in sugar uptake, sugar metabolism (glycolysis), and energy metabolism in addition to a gene encoding a specific biosynthetic enzyme. Good.

  Examples of genes involved in sugar metabolism include genes encoding glycolytic enzymes and sugar uptake genes. Glucose 6-phosphate isomerase gene (pgi; WO 01/02542 pamphlet), phosphoenolpyruvate Synthase gene (pps; European application 877090), phosphoglucomutase gene (pgm; WO 03/04598 pamphlet), fructose diphosphate aldolase gene (fba; WO 03/04664 pamphlet), pyruvate Kinase gene (pykF; WO 03/008609 pamphlet), transaldolase gene (talB; WO 03/008611 pamphlet), fumarase gene (fum; WO 01/02545 pamphlet), phosphoenolpyruvate synthase gene ( pps; European Application Publication No. 877090 pamphlet), non-PTS sucrose uptake gene Gene (csc; European Application Publication No. 149911 pamphlet) and sucrose utilization gene (scrAB operon; International Publication No. 90/04636 pamphlet).

  Examples of genes involved in energy metabolism include a transhydrogenase gene (pntAB; US Pat. No. 5,830,716) and a cytochromoe bo type oxidase gene (cyoB European Patent Application Publication 1070376).

<Isopropyl alcohol-producing bacteria>
Examples of a method for imparting or enhancing isopropyl alcohol production ability include a method of modifying a microorganism so that the activity of one or more enzymes selected from isopropyl alcohol biosynthesis enzymes is increased. Examples of such enzymes include, but are not limited to, acetoacetate decarboxylase, isopropyl alcohol dehydrogenase, CoA transferase, and thiolase (WO2009 / 008377A1). In particular, it is preferable to enhance the activities of all these four enzymes. Examples of the method for imparting or enhancing the ability to produce isopropyl alcohol include a method of modifying a microorganism so that the activity of GntR (gntR) is reduced. GntR refers to a transcription factor that negatively regulates the expression of an operon encoding the metabolic system of gluconic acid. Specifically, the operon encodes a gluconic acid uptake system and a gluconic acid phosphorylase. For example, Escherichia coli has two gluconic acid metabolic systems, GntI and GntII, but GntR suppresses the expression of both.

  The microorganism having the ability to produce isopropyl alcohol may be modified so that the activity of lactate dehydrogenase is reduced. By such modification, the production of lactic acid can be suppressed and isopropyl alcohol can be produced efficiently even under culture conditions in which oxygen supply is limited. Culture conditions with limited oxygen supply are generally 0.02 vvm to 2.0 vvm (vvm; aeration volume [mL] / liquid volume [mL] / hour [minute] when only air is used as the gas. ), Which means a rotational speed of 200 to 600 rpm.

<Acetone-producing bacteria>
Acetone is a precursor of isopropyl alcohol in isopropyl alcohol production. Therefore, the acetone production ability can be imparted or enhanced by partially using a method for imparting or enhancing isopropyl alcohol production ability. For example, the ability to produce acetone increases the activity of one or more enzymes selected from the above-exemplified isopropyl alcohol biosynthetic enzymes other than isopropyl alcohol dehydrogenase, ie, acetoacetate decarboxylase, CoA transferase, and thiolase. Thus, it can be imparted or enhanced by modifying the microorganism.

<Ethanol-producing bacteria>
Moreover, as a microorganism capable of producing ethanol, a lactate dehydrogenase gene (ldhA) deficient and a mutation introduced with a pyruvate decarboxylase gene (pdc) and an alcohol dehydrogenase gene (adhB) derived from Zymomonas mobilis Strains, and strains in which the phosphoenolpyruvate carboxylase gene (ppc) of the strain is further deleted (J Mol Microbiol Biotechnol 2004, 8, 243-254). Moreover, as a microorganism capable of producing ethanol, a pyruvate / formate lyase gene (pfl) and a fumarate reductase gene (frd) are deleted, and Zymomonas mobilis (Zymomonas
mobilis) -derived pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase gene (adhB) have also been introduced (Ann NY Acad Sci. 2008, 1125, 363-372).

<2> Production method of the present invention The production method of the target substance of the present invention is a medium containing a carbon source obtained from seaweed, which is a microorganism belonging to the genus Vibrio bred to have the target substance-producing ability by the above-mentioned method. In this method, the target substance is produced and accumulated in the medium by culturing, and the target substance is recovered from the medium or cells.

  As a medium to be used, a medium conventionally used in fermentation production of a target substance using microorganisms can be used. That is, a normal medium containing a carbon source, a nitrogen source, inorganic ions, and other organic components as required can be used. Here, as the carbon source, a carbon source obtained from seaweed can be used.

Examples of the carbon source obtained from seaweed include alginic acid, cellulose, glucose, mannitol, pectin, galacturonic acid, carrageenan, and agar. Brown algae contain alginate, mannitol and glucose in a ratio of 5: 8: 1 (20 January 2012 VOL335 SCIENCE)
As long as the medium contains a carbon source obtained from seaweed, other sugar sources such as glucose, sucrose, fructose, glycerol, and ethanol may be contained.

As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used. As an organic trace nutrient source, it is desirable to contain an appropriate amount of a required substance such as vitamin B1, L-homoserine or a yeast extract. In addition to these, a small amount of potassium phosphate, magnesium sulfate, iron ion, manganese ion or the like is added as necessary. Moreover, when culturing the microorganism of the present invention, it is more preferable to contain a certain concentration of salt. The salt may be a salt of the target substance combined with counter ions, or salt (may be NaCl). The medium used in the present invention is a carbon source, a nitrogen source, inorganic ions, and other organic substances as required. As long as it is a medium containing a trace amount component, either a natural medium or a synthetic medium may be used.

  In addition, L-amino acids that improve growth and productivity may be added. For example, in the case of L-lysine fermentation, L-threonine, L-homoserine, and L-isoleucine are used. In the case of L-threonine fermentation, L-isoleucine, L-lysine, L-glutamic acid, and L-homoserine are used in L-tryptophan fermentation. , L-phenylalanine, L-tyrosine and the like are preferably added. The addition concentration is about 0.01-10 g / L.

  The culture is preferably carried out under aerobic conditions for 1 to 7 days, the culture temperature is 24 ° C. to 37 ° C., and the pH during the culture is preferably 5 to 9. In addition, an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used for pH adjustment. The L-amino acid can be recovered from the fermentation broth by combining an ion exchange resin method, a precipitation method and other known methods. In addition, when L-amino acid accumulates in the microbial cell, for example, the microbial cell is crushed by ultrasonic waves, and the microbial cell is removed by centrifugation. Can be recovered.

  In the present invention, NaCl is preferably present in the medium at 0.5% or more, preferably 0.5-4%, more preferably 0.7-3.5%.

<Example 1> Acquisition of Vibrio sp. SA2 strain Sazae collected in the Kasumura area of Nanao City, Ishikawa Prefecture was used for screening for new microorganisms capable of assimilating alginate (collected location: latitude north: 37.0981 east longitude: sea near 137.0499 ).

  Sterilization of Daigo artificial seawater SP solution (manufactured by Wako Pure Chemical Industries, Ltd., see below for composition) sterilized by autoclaving at 120 ° C and 0.15 MPa for 20 minutes. The object was suspended in blue ase by scooping 1 loop. The suspension of the gastrointestinal tract in the sodium alginate selective agar medium (see below for the composition) containing only sodium alginate with a polymerization degree of 2000 or more (first grade reagent 300-400cP, catalog number: 192-09995 manufactured by Wako Pure Chemical Industries, Ltd.) Was appropriately diluted with Daigo artificial seawater and plated. A stationary culture was performed at a culture temperature of 25 ° C. for a culture time of 75 hours to confirm the formation of a large number of colonies.

  The colony is scraped with ASE, suspended in physiological saline, washed 3 times with physiological saline, then plated on a freshly prepared sodium alginate selection agar medium, and incubated at 37 ° C for 50 hours incubation time. Then, the strain in which colony formation was confirmed was further subjected to SI using a sodium alginate selection agar medium, and suspended in 4 mL of a medium in which 15 g / L NaCl was additionally added to the LB liquid medium, and cultured in a test tube. Culture at 37 ° C with a reciprocating agitation speed of 120rpm. When the absorbance at 600nm reaches an OD of about 0.3, add an equal amount of sterile 20% glycerol and freeze at -80 ° C to obtain a glycerol stock. Prepared and stored in ultra-low temperature storage. The strain that formed a single colony earliest was named SA2 strain.

Daigo artificial seawater SP composition
MgCl ₂・ 6H ₂ O 9.474g / L
CaCl ₂・ 2H ₂ O 1.328g / L
Na ₂ SO ₄ 3.505g / L
KCl 0.597g / L
NaHCO ₃ 0.171g / L
KBr 0.085g / L
Na ₂ B ₄ O ₇・ 10H ₂ O 0.034g / L
SrCl ₂ 0.012g / L
NaF 3mg / L
LiCl 1mg / L
KI 0.07mg / L
CoCl ₂・ 6H ₂ O 0.0002mg / L
AlCl ₃・ 6H ₂ O 0.008mg / L
FeCl ₃・ 6H ₂ O 0.006mg / L
Na ₂ WO ₄・ 2H ₂ O 0.0002mg / L
(NH ₄ ) ₆ Mo ₇ O ₂₄・ 4H ₂ O 0.0008mg / L
NaCl 20.747g / L

Sodium alginate selection agar medium Composition Sodium alginate 5g / L
Na ₂ HPO ₄ 6 g / L
KH ₂ PO ₄ 3 g / L
NaCl 0.5 g / L
NH ₄ Cl 1 g / L
NaCl 21.247g / L
MgSO ₄・ 7H ₂ O 0.246g / L
Thiamine ・ HCl 10mg / L
MgCl ₂・ 6H ₂ O 9.474g / L
CaCl ₂・ 2H ₂ O 1.328g / L
Na ₂ SO ₄ 3.505g / L
KCl 0.597g / L
NaHCO ₃ 0.171g / L
KBr 0.085g / L
Na ₂ B ₄ O ₇・ 10H ₂ O 0.034g / L
SrCl ₂ 0.012g / L
NaF 3mg / L
LiCl 1mg / L
KI 0.07mg / L
CoCl ₂・ 6H ₂ O 0.0002mg / L
AlCl ₃・ 6H ₂ O 0.008mg / L
FeCl ₃・ 6H ₂ O 0.006mg / L
Na ₂ WO ₄・ 2H ₂ O 0.0002mg / L
(NH ₄ ) ₆ Mo ₇ O ₂₄・ 4H ₂ O 0.0008mg / L
Bacto Agar 15g / L

<Example 2> Molecular phylogenetic analysis of Vibrio sp. SA2 strain
Using a PurElute Bacterial Genomid Kit (manufactured by Edgebio) from cells obtained by static culture of 50 μL of glycerol stock of SA2 strain on an agar medium supplemented with 15 g / L NaCl in LB liquid medium at 37 ° C. for 16 hours. Total genomic DNA was extracted. The obtained total genomic DNA was analyzed with the next-generation sequencer Miseq (manufactured by Illumina), and BLAST analysis (non-patent literature Altshul, et al. '' Gapped BLAST and
PSI-BLAST: new generation of protein database search programs '' (1997) Nucleic
Acids Research. 25.p3389-3402), the full length 16S rRNA gene sequence was obtained (SEQ ID NO: 1). As a result, SA2 strain showed 99.5% homology in comparison with Vibrio rumoiensis type strain S-1 strain (DSM19141 strain) and 16S rRNA gene full-length sequence (difference due to gap of 2 bases and difference due to substitution of 6 bases). Including Fig. 1).

Molecular phylogenetic tree estimation software MEGA ver.5.0 (non-patent document Tamura, et al. '' MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods '''(2011) Mol.Biol.Evol.28.p2731-2739) (Non-Patent Document Saitou, et al.''The neighbor-joining method: a new method for reconstructing phylogenic trees''
(1987) Mol. Biol. Evol. 4. p406-425), a molecular phylogenetic tree was prepared (FIG. 2). Kimura 2-parameter model was used as the base substitution model used in the neighborhood binding method (Non-Patent Document imura.``A simple method for evaluating evolutionary rates of base substitutions through comparative studies of nucleotide sequences '' (1980) J. Mol. .Evol.16.p111-120
See). In addition, in order to evaluate the reliability of the tree shape of the prepared molecular phylogenetic tree, we verified it by the bootstrap method 1000 times. (See Felsenstein. Confidence limits on phylogenics: an approach using the bootstrap "(1985) Evolution.39.p783-791)

  Molecular phylogenetic analysis revealed that the SA2 strain belongs to a cluster formed by Vibrio species. It was supported that the SA2 strain belongs to a cluster formed by Vibrio rumoiensis, Vibrio litoralis, Vibrio casei with a bootstrap value of 98%. The SA2 strain also formed a cluster with Vibrio rumoiensis, which was supported with a bootstrap value of 89%. The above results revealed that the SA2 strain is a closely related species of the genus Vibrio. Therefore, the SA2 strain was named Vibrio sp. SA2 strain (NITE P-01635 strain).

<Example 3> Identification of Vibrio sp. SA2 strain species by DNA-DNA hybridization
According to BLAST analysis, Vibrio sp. SA2 strain (NITE P-01635 strain) had homology of the full-length 16S rRNA gene of 99.5% with Vibrio rumoiensis, 97.9% with Vibrio litoralis, and 96.3% with Vibrio casei. Vibrio sp. SA2 strain may be the same type of microorganism as Vibrio rumoiensis because it has been reported that microbial species with homology of the full-length 16S rRNA gene of less than 98.7% can be judged as different species. . (Stackebrandt, et al. “Taxonomic parameters revisited: tarnished gold standards” (2006) Microbiol. Today. 33.p152-155.)

  Therefore, the inventors identified the species of Vibrio sp. SA2 strain by DNA-DNA hybridization test by exchanging the fixed side DNA and the probe side DNA between Vibrio sp. SA2 strain and Vibrio rumoiensis type strain three times. Carried out. (Yoshiaki Kawamura, `` Systematic Classification and Identification of Bacteria '' (2000) The Japanese Journal of Bacteriology. 55.p545-584, edited by Kenichiro Suzuki, `` Classification and Identification of Microorganisms '' Molecular Genetics and Molecular Biology ”(2000) p34-47)

  As a result, the genomic DNA of Vibrio sp. SA2 strain and Vibrio rumoiensis type strain showed a value of 36-39% and an average of 37.5% homology (Table 2 below). At present, microbial species are defined as homologous strains showing homology values of 70% or more as a result of comparison of homology values by DNA-DNA hybridization test (Wayne, et al. Report of the ad). hoc committee on reconciliation of approaches to bacterial systematics. "(1987) Int. J. Syst. Bacteriol. 37.p463-464.). Therefore, Vibrio sp. SA2 strain was found to be a novel species of microorganism belonging to another species from Vibrio rumoiensis type strain.

<Example 4> Test tube culture of Vibrio sp. SA2 strain in a minimal medium As a characteristic of a conventional microorganism strain belonging to Vibrio rumoiensis, it cannot grow because it is too hot at 40 ° C and does not have alginate lyase activity. It has been reported that there is no ability to assimilate alginate (Garrity, et al. (2005) Bergey's manual of systematic bacteriology 2nd Edition Part B p526-527). Therefore, the inventors of the present invention used a glycerol stock of Vibrio sp. SA2 strain (NITE P-01635 strain) and a glycerol stock of Escherichia coli MG1655 strain (ATCC 47076 strain) as a comparative control strain in an agar medium supplemented with LB NaCl 15 g / L. The cells were plated one by one and statically cultured at 37 ° C. for 16 hours as a seed culture. After seed culture, the cells grown on the agar medium were scraped with ase and suspended in 1 mL of sterile physiological saline, and the turbidity at an absorbance of 600 nm was measured with a spectrophotometer U-2000 (manufactured by Hitachi). Thereafter, inoculation was performed in 5 mL of a minimal liquid medium supplemented with sodium alginate as the only carbon source shown below as the main culture so that the turbidity at an absorbance of 600 nm was 0.05. Using a constant temperature shaking culture apparatus TVS062CA (manufactured by Advantech Co., Ltd.), the test tube was cultured for 9 hours continuously at 37 ° C. and 70 rpm. As a result, significant growth of Vibrio sp. SA2 strain (NITE P-01635 strain) by assimilation of alginic acid was observed. Therefore, it was shown that the Vibrio sp. SA2 strain is a novel microbial strain having an alginate assimilation ability different from the microbial strain belonging to the conventional Vibrio rumoiensis.

Sodium alginate minimal liquid medium Composition Sodium alginate 2.5g / L
Na ₂ HPO ₄ 6 g / L
KH ₂ PO ₄ 3 g / L
NH ₄ Cl 1 g / L
NaCl 15.5g / L
MgSO ₄・ 7H ₂ O 0.246g / L
Thiamine ・ HCl 10mg / L

<Example 5> Specific maximum growth rate measurement in minimal medium of Vibrio sp. SA2 strain
Plate glycerol stock of Vibrio sp. SA2 strain (NITE P-01635 strain) and glycerol stock of Escherichia coli MG1655 strain (ATCC 47076 strain) as a comparison strain on agar medium supplemented with LB NaCl 15g / L, 50μL each, As a seed culture, static culture was performed at a culture temperature of 37 ° C. for 16 hours. After seed culture, the cells grown on the agar medium were scraped with ase and suspended in 1 mL of sterile physiological saline, and the turbidity at an absorbance of 600 nm was measured with a spectrophotometer U-2000 (manufactured by Hitachi). Thereafter, inoculation was carried out in 5 mL of a minimal liquid medium supplemented with sodium alginate as the only carbon source shown above so that the turbidity at an absorbance of 600 nm was 0.05. Using a constant temperature shaking culture apparatus TVS062CA (manufactured by Advantech Co., Ltd.), the test tube culture was performed continuously at 37 ° C. and 70 rpm for 24 hours.

The same medium in the test tube culture was used for the main culture, and the following two types of M9 minimal medium with glucose as a single carbon source were used. Was done. The results are shown in Table 3 and FIG. From the obtained growth data, the specific maximum growth rate under each condition was measured (FIG. 4). As a result, it was clarified that the specific maximum growth rate of Vibrio sp. SA2 strain in the liquid culture medium with minimum sodium alginate was about twice the specific growth rate of Escherichia coli MG1655 strain in the glucose M9 liquid medium.

Glucose M9 liquid medium Composition glucose 2.5g / L
Na ₂ HPO ₄ 6 g / L
KH ₂ PO ₄ 3 g / L
NH ₄ Cl 1 g / L
NaCl 0.5g / L
MgSO ₄・ 7H ₂ O 0.246g / L
Thiamine ・ HCl 10mg / L

Glucose M9 NaCl liquid medium Composition glucose 2.5g / L
Na ₂ HPO ₄ 6 g / L
KH ₂ PO ₄ 3 g / L
NH ₄ Cl 1 g / L
NaCl 15.5g / L
MgSO ₄・ 7H ₂ O 0.246g / L
Thiamine ・ HCl 10mg / L

<Example 6> Measurement of viable temperature of Vibrio sp. SA2 strain in a sodium alginate minimal medium
Plate glycerol stock of Vibrio sp. SA2 strain (NITE P-01635 strain) and glycerol stock of Escherichia coli MG1655 strain (ATCC 47076 strain) as a comparison strain on agar medium supplemented with LB NaCl 15g / L, and seed. As culture, static culture was performed at a culture temperature of 37 ° C. for 16 hours. Also, 50 μL of glycerol stock of Vibrio rumoiensis S-1 strain (DSM 19141 strain) was plated on an agar medium supplemented with LB NaCl 15 g / L, and statically cultured as a seed culture at a culture temperature of 31.5 ° C. for 16 hours. After seed culture, the cells grown on the agar medium were scraped with ase and suspended in 1 mL of sterile physiological saline, and the turbidity at an absorbance of 600 nm was measured with a spectrophotometer U-2000 (manufactured by Hitachi). Thereafter, inoculation was carried out in 5 mL of a minimal liquid medium supplemented with sodium alginate as the only carbon source shown above so that the turbidity at an absorbance of 600 nm was 0.05. Using a constant-temperature shaking culture apparatus TVS062CA (manufactured by Advantech Co., Ltd.), the culture temperature condition was changed from 37 ° C. to 43 ° C. by 1 ° C., and the test tube culture was performed continuously at 70 rpm for 24 hours. As a result, as shown in Table 4, Vibrio sp. SA2 strain (NITE P-01635 strain) grew well in a sodium alginate minimal liquid medium at a culture temperature of 40 ° C., and Vibrio sp. SA2 strain (NITE P-01635 strain). ) Was shown to be a microorganism capable of assimilating sodium alginate at 40 ° C. From the above experimental results, it can be seen that Vibrio sp. SA2 strain is a new microbial strain belonging to a new microbial species having an ability to assimilate alginate that can grow at a high temperature of 40 ° C., which is different from the microbial strain belonging to Vibrio rumoiensis. Indicated.

<Example 7> L-glutamic acid-producing ability imparted to Vibrio sp. SA2 strain Genomic DNA of Escherichia coli MG1655 strain was extracted using PurElute Bacterial Genomic kit (manufactured by Edgebio). In order to heterologously express the YbjL protein shown in SEQ ID NO: 3 in the Vibrio sp. SA2 strain using the obtained genomic DNA as a template, PCR reaction was performed with the primers shown in SEQ ID NO: 4 and SEQ ID NO: 5, and the amplified DNA The plasmid pMW219-ybjL was ligated to the vector pMW219 (Nippon Gene) digested with restriction enzymes HindIII and SalI using the In-Fusion HD Cloning Kit (Takara Bio) Built.

  Vibrio sp. SA2 strain was cultured in 40 mL of LB NaCl 15 g / L liquid medium in two Sakaguchi flasks at a culture temperature of 37 ° C. and a stirring speed of 120 rpm until the OD600 nm was around 0.5. 80 mL of the obtained culture broth was collected by centrifugation under conditions of a rotation speed of 7000 rpm, an operation time of 7 min, and a temperature of 4 ° C. Sterilized after washing twice with 15 mL of sterilized 2 mM HEPES, 100 mM Sucrose, 5 mM CaCl2 solution chilled on ice (suspended, centrifuged at 7000 rpm, operating time 7 min, temperature 4 ° C) The suspension was suspended in 1 mL of a 10% glycerol solution to obtain an electrocompetent cell of Vibrio sp. SA2. PMW219-ybjL plasmid DNA (1 μg or more) precipitated with ethanol was added to 100 μL of Vibrio sp. SA2 strain, and electroporated at 1.8 kV 25 μF to introduce pMW219-ybjL into Vibrio sp. SA2. 0.5 mL of LB NaCl 15 g / L liquid medium was added to the electroporated electrocompetent cell, and the culture was allowed to stand at 37 ° C. for 2.5 hours for recovery culture.After that, 15 g of LB NaCl containing 40 mg / L kanamycin was added. Colony formation was confirmed by static culture for 24 hours on / L agar medium. The obtained colonies were further SI-treated with LB NaCl 15 g / L agar medium containing 40 mg / L kanamycin and suspended in 4 mL of LB NaCl 15 g / L liquid medium containing 40 mg / L kanamycin. went. Cultivate the tube at 37 ° C with a reciprocating stirring speed of 120rpm. When the OD at an absorbance of 600nm reaches about 0.3, add an equal amount of sterile 20% glycerol and freeze at -80 ° C to obtain a glycerol stock. The glycerol stock of the strain prepared and obtained was named the glycerol stock of Vibrio sp. SA2 / pMW219-ybjL strain.

  5 g of sodium alginate was added to 100 mL of 3N sulfuric acid, followed by heating at 65 ° C. for 3 hours. The obtained sodium alginate hydrolyzate solution was adjusted to pH 7.0 by adding KOH, and a sodium alginate hydrolyzate sugar solution was obtained. The following sodium alginate hydrolyzate minimal liquid medium was prepared using the obtained sodium alginate hydrolyzate sugar solution.

Sodium alginate hydrolyzate minimal liquid medium Composition Sodium alginate hydrolyzate 2.5g / L
Na ₂ HPO ₄ 6 g / L
KH ₂ PO ₄ 3 g / L
NH ₄ Cl 1 g / L
NaCl 15.5g / L
MgSO ₄・ 7H ₂ O 0.246g / L
Thiamine ・ HCl 10mg / L
Kanamycin 40mg / L

  Thaw glycerol stock of Vibrio sp. SA2 / pMW219-ybjL strain, apply 100 μL each uniformly to LB NaCl 15 g / L agar medium containing 40 mg / L kanamycin, and allow to stand at 30 ° C for 48 hours did. Approximately 1/4 amount of the cells on the obtained plate was suspended in 0.5 mL of physiological saline, and the turbidity at a wavelength of 600 nm was measured with a spectrophotometer U-2000 (manufactured by Hitachi). The obtained suspension containing the fungus is inoculated into a 5 mL sodium alginate hydrolyzate-minimum liquid medium filled in an L-shaped test tube with a volume of turbidity at a wavelength of 600 nm of 0.05, and shaken. The cells were cultured for 22 hours at 34 ° C. with a rotating stirring speed of 70 rpm in a culturing apparatus TN-1506 (manufactured by Advantech Toyo Co., Ltd.). After completion of the culture, the amount of L-glutamic acid accumulated in the medium was measured using Biotech Analyzer AS310 (manufactured by Sakura Seiki Co., Ltd.).

  The results are shown in Table 5. As shown in Table 5, a strain obtained by introducing pMW219-ybjL to L-glutamic acid-producing ability by introducing pMW219-ybjL into a Vibrio sp. SA2 strain isolated as a microorganism capable of assimilating alginic acid as a single carbon source A significant amount of L-glutamic acid was accumulated in a medium with a single carbon source.

Claims (17)

  A Vibrio bacterium having an ability to assimilate alginate, wherein the 16S ribosomal RNA sequence has a sequence having a homology of 95% or more with SEQ ID NO: 1.   The Vibrio bacterium according to claim 1, wherein the 16S ribosome has SEQ ID NO: 1.   The vibrio bacterium according to claim 1 or 2, characterized in that it can grow on alginic acid as a single carbon source.   The Vibrio bacterium according to any one of claims 1 to 3, wherein the Vibrio bacterium is NITE P-01635 strain.   Vibrio bacteria having the ability to produce the target substance and having the ability to assimilate alginic acid are cultured in a medium containing a carbon source extracted from seaweeds, and the target substance is produced and accumulated in the medium. A method for producing a target substance by fermentation, wherein the substance is recovered.   6. The method according to claim 5, wherein the Vibrio bacterium having the ability to assimilate alginate is a bacterium having a 16S ribosome sequence having a sequence having a homology of 95% or more with SEQ ID NO: 1.   6. The method according to claim 5, wherein the Vibrio bacterium having an ability to assimilate alginate is a bacterium having a 16S ribosome sequence of SEQ ID NO: 1.   The method according to claim 5, wherein the bacterium belonging to the genus Vibrio having an ability to assimilate alginic acid is NITE P-01635 strain.   The target substance is L-lysine, L-ornithine, LL-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L-leucine, glycine, L-threonine, L -Selected from serine, L-proline, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L-cystine, L-methionine, L-glutamic acid, L-aspartic acid, L-glutamine and L-asparagine The method according to any one of claims 5 to 8, which is one or more amino acids.   The method according to claim 5, wherein the target substance is ethanol.   The target substance is isopropyl alcohol, acetone, propylene, 1,3-butanediol, 1,4-butanediol, 1-propanol, 1,3-propanediol, 1,2-propanediol, ethylene glycol, and isobutanol 9. The method according to any one of claims 5 to 8, wherein the method is selected from the group consisting of:   The method according to any one of claims 5 to 9, wherein the bacterium is a Vibrio bacterium having enhanced activity of an L-amino acid biosynthesis enzyme.   The method according to any one of claims 5 to 9, wherein the bacterium is a Vibrio bacterium having an enhanced activity of a protein that excretes L-amino acids.   14. The method according to claim 13, wherein the protein excreting L-amino acid is ybjL.   The method according to claim 5, wherein the seaweed is a large seaweed contained in brown algae, red algae, and green algae.   The carbon source obtained from the seaweed is at least one carbon source selected from alginic acid, cellulose, mannitol, pectin, galacturonic acid, carrageenan, and agar. The method according to one item.   The saccharide extract obtained from the seaweed contains alginic acid, and the culture is performed after the hydrolysis of alginic acid is performed by an enzymatic degradation reaction with an alginate lyase or a hydrolysis reaction with an acid. The method according to any one of 16.