WO2014185430A1 - L-アミノ酸の製造法 - Google Patents
L-アミノ酸の製造法 Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/14—Glutamic acid; Glutamine
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
Definitions
- the present invention relates to a method for producing L-amino acids using coryneform bacteria.
- L-amino acids are industrially useful as additives for animal feed, ingredients for seasonings and foods and drinks, amino acid infusions, and the like.
- L-amino acids are industrially produced, for example, by fermentation using various microorganisms capable of producing L-amino acids.
- methods for producing L-amino acids by fermentation include a method using a wild-type microorganism (wild strain), a method using an auxotrophic strain derived from a wild strain, and various drug-resistant mutant strains derived from a wild strain. And a method using a strain having characteristics of both an auxotrophic strain and a metabolic control mutant.
- Escherichia coli has at least two inorganic phosphate uptake systems (Non-patent Document 1).
- One is a low-affinity inorganic phosphate transporter (Pit) system, and the other is a high-affinity phosphate-specific transporter (Pst).
- PitA and pitB genes are known as genes encoding the Pit system.
- the pstSCAB gene is known as a gene encoding the Pst system, and the product of the pstSCAB gene forms a complex and functions as the Pst system.
- the relationship between the activity of these phosphate transporters and L-amino acid production has not been known.
- An object of the present invention is to develop a novel technique for improving L-amino acid producing ability of coryneform bacteria and to provide an efficient method for producing L-amino acid.
- the present inventor has improved the ability of L-amino acid production of coryneform bacteria by modifying coryneform bacteria so that the activity of the phosphate transporter is increased.
- the present invention has been completed.
- the present invention can be exemplified as follows.
- a method for producing an L-amino acid comprising culturing a coryneform bacterium having L-amino acid-producing ability in a medium, and collecting the L-amino acid from the medium, A method wherein the bacterium has been modified to increase the activity of a phosphate transporter.
- the method, wherein the activity of a phosphate transporter is increased by increasing the expression of a gene encoding a phosphate transporter.
- the method, wherein the gene is a pitA gene.
- the pitA gene is the DNA described in (a) or (b) below: (A) DNA having the base sequence shown in SEQ ID NO: 5 or 25, (B) A DNA that hybridizes with a probe that can be prepared from a complementary sequence of the nucleotide sequence shown in SEQ ID NO: 5 or 25 or a complementary sequence thereof under stringent conditions and that encodes a protein having phosphate transporter activity.
- the pitA gene is DNA encoding the protein described in (A) or (B) below: (A) a protein having the amino acid sequence shown in SEQ ID NO: 6 or 26, (B) a protein having an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acid residues and having phosphate transporter activity in the amino acid sequence shown in SEQ ID NO: 6 or 26 . [6] The method, wherein the expression of the gene is increased by increasing the copy number of the gene and / or modifying an expression regulatory sequence of the gene.
- a method for producing an L-amino acid comprising culturing a coryneform bacterium having L-amino acid-producing ability in a medium, and collecting the L-amino acid from the medium,
- the bacterium has a mutant pitA gene encoding a phosphate transporter having a mutation in which an amino acid residue corresponding to the phenylalanine residue at position 246 of SEQ ID NO: 6 is substituted with an amino acid residue other than phenylalanine
- the method of the present invention is a method for producing an L-amino acid, comprising culturing a coryneform bacterium having L-amino acid-producing ability in a medium, and collecting the L-amino acid from the medium.
- the coryneform bacterium used in this method is also referred to as “the bacterium of the present invention”.
- the bacterium of the present invention is a coryneform bacterium having an ability to produce L-amino acids, modified so that the activity of a phosphate transporter is increased.
- bacteria having L-amino acid-producing ability refers to the production and recovery of a target L-amino acid when cultured in a medium. Bacteria that have the ability to accumulate in the medium or in the cells to the extent possible.
- the bacterium having L-amino acid-producing ability may be a bacterium capable of accumulating a larger amount of the target L-amino acid in the medium than the unmodified strain.
- Non-modified strains include wild strains and parent strains.
- the bacterium having L-amino acid-producing ability is a bacterium that can accumulate the target L-amino acid in an amount of 0.5 g / L or more, more preferably 1.0 g / L or more in the medium. May be.
- L-amino acids include basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L-leucine, glycine, etc.
- Aliphatic amino acids amino acids which are hydroxymonoaminocarboxylic acids such as L-threonine and L-serine, cyclic amino acids such as L-proline, aromatic amino acids such as L-phenylalanine, L-tyrosine and L-tryptophan, L- Examples thereof include sulfur-containing amino acids such as cysteine, L-cystine and L-methionine, acidic amino acids such as L-glutamic acid and L-aspartic acid, and amino acids having an amide group in the side chain such as L-glutamine and L-asparagine.
- the bacterium of the present invention may have an ability to produce two or more amino acids.
- L-amino acids are L-amino acids unless otherwise specified.
- the L-amino acid may be a free form, a salt thereof, or a mixture thereof. That is, the term “L-amino acid” in the present invention may mean a free L-amino acid, a salt thereof, or a mixture thereof.
- the salt include sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and potassium salt.
- L-lysine may be free L-lysine, L-lysine sulfate, L-lysine hydrochloride, L-lysine carbonate, or a mixture thereof.
- L-glutamic acid may be free L-glutamic acid, sodium L-glutamate (MSG), ammonium L-glutamate, or a mixture thereof.
- coryneform bacteria examples include bacteria belonging to genera such as Corynebacterium genus, Brevibacterium genus, and Microbacterium genus.
- coryneform bacteria include the following species. Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum
- coryneform bacteria include the following strains. Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium melassecola ATCC 17965 Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Corynebacterium glutamicum (Brevibacterium divaricatum) ATCC 14020 Corynebacterium glutamicum (Brevibacterium flavum) ATCC 13826, ATCC 14067, AJ124
- corynebacteria belonging to the genus Brevibacterium has been classified as a genus of corynebacteria, but bacteria integrated into the genus corynebacteria (Int. J. Syst. Bacteriol., 41, 255 (1991)) are also available. included.
- Corynebacterium stationis which was previously classified as Corynebacterium ammoniagenes, includes bacteria that have been reclassified as Corynebacterium stationis by 16S rRNA sequencing (Int. J Syst. Evol. Microbiol., 60, 874-879 (2010)).
- strains can be sold, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States States of America). That is, a registration number corresponding to each strain is given, and it is possible to receive a sale using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of American Type Culture Collection.
- the bacterium of the present invention may inherently have L-amino acid-producing ability or may have been modified to have L-amino acid-producing ability.
- a bacterium having L-amino acid-producing ability can be obtained, for example, by imparting L-amino acid-producing ability to the bacterium as described above, or by enhancing the L-amino acid-producing ability of the bacterium as described above. .
- L-amino acid-producing ability can be imparted or enhanced by a method conventionally used for breeding amino acid-producing bacteria such as coryneform bacteria or Escherichia bacteria (Amino Acid Fermentation, Academic Publishing Center, Inc., 1986). (May 30, 1st edition issued, see pages 77-100). Examples of such methods include acquisition of auxotrophic mutants, acquisition of L-amino acid analog-resistant strains, acquisition of metabolic control mutants, and recombination with enhanced activity of L-amino acid biosynthetic enzymes. The creation of stocks. In the breeding of L-amino acid-producing bacteria, properties such as auxotrophy, analog resistance, and metabolic control mutation that are imparted may be single, or two or more.
- L-amino acid biosynthetic enzymes whose activities are enhanced in breeding L-amino acid-producing bacteria may be used alone or in combination of two or more.
- imparting properties such as auxotrophy, analog resistance, and metabolic control mutation may be combined with enhancing the activity of biosynthetic enzymes.
- An auxotrophic mutant, an analog resistant strain, or a metabolically controlled mutant having L-amino acid production ability is subjected to normal mutation treatment of the parent strain or wild strain, and the auxotrophic, analog It can be obtained by selecting those exhibiting resistance or metabolic control mutations and having the ability to produce L-amino acids.
- Normal mutation treatments include X-ray and ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), etc. Treatment with a mutagen is included.
- the L-amino acid-producing ability can be imparted or enhanced by enhancing the activity of an enzyme involved in the target L-amino acid biosynthesis. Enhancing enzyme activity can be performed, for example, by modifying bacteria so that expression of a gene encoding the enzyme is enhanced. Methods for enhancing gene expression are described in WO00 / 18935 pamphlet, European Patent Application Publication No. 1010755, and the like. A detailed method for enhancing the enzyme activity will be described later.
- the L-amino acid-producing ability can be imparted or enhanced by reducing the activity of an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of the target L-amino acid to produce a compound other than the target L-amino acid. It can be carried out.
- an enzyme that catalyzes a reaction that produces a compound other than the target L-amino acid by branching from the biosynthetic pathway of the target L-amino acid includes enzymes involved in the degradation of the target amino acid. It is. A method for reducing the enzyme activity will be described later.
- L-amino acid-producing bacteria and methods for imparting or enhancing L-amino acid-producing ability are given below.
- any of the modifications exemplified below for imparting or enhancing the properties of L-amino acid-producing bacteria and L-amino acid-producing ability may be used alone or in appropriate combination.
- L-glutamic acid producing bacteria examples include strains in which the activity of one or more enzymes selected from L-glutamic acid biosynthetic enzymes are enhanced.
- enzymes include, but are not limited to, glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citric acid Synthase (gltA), methyl citrate synthase (prpC), phosphoenol pyruvate carbocilase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenol pyruvate synthe
- the parentheses are abbreviations for genes encoding the enzymes (the same applies to the following description).
- these enzymes it is preferable to enhance the activity of one or more enzymes selected from, for example, glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl citrate synthase.
- Examples of coryneform bacteria modified to increase the expression of glutamate synthetase gene include those disclosed in WO99 / 07853.
- the L-glutamic acid-producing bacterium or the parent strain for deriving it is selected from enzymes selected from enzymes that catalyze reactions that branch off from the biosynthetic pathway of L-glutamic acid to produce compounds other than L-glutamic acid. Examples include strains in which the activity of the above enzymes is reduced or deficient.
- Such enzymes include, but are not limited to, isocitrate lyase (aceA), ⁇ -ketoglutarate dehydrogenase (sucA, odhA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (IlvG), acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), succinate dehydrogenase (sdhABCD), 1-pyrroline-5-carboxylate dehydrogenase (putA ).
- aceA isocitrate lyase
- sucA ⁇ -ketoglutarate dehydrogenase
- pta phosphotransacetylase
- ack acetate kinase
- ack acetohydroxy acid synthase
- Coryneform bacteria with reduced or deficient ⁇ -ketoglutarate dehydrogenase activity and methods for obtaining them are described in WO2008 / 075483.
- Specific examples of coryneform bacteria with reduced or deficient ⁇ -ketoglutarate dehydrogenase activity include the following strains.
- L-glutamic acid-producing bacteria or parent strains for inducing the same also include strains in which both ⁇ -ketoglutarate dehydrogenase (sucA) activity and succinate dehydrogenase (sdh) activity are reduced or deficient (Japanese Patent Application Laid-Open (JP-A) 2010-041920).
- specific examples of such strains include, for example, an odhAsdhA double-deficient strain of Corynebacterium glutamicum ATCC14067 (Corynebacterium glutamicum 8L3G ⁇ SDH strain) (Japanese Patent Laid-Open No. 2010-041920).
- examples of L-glutamic acid-producing bacteria or parent strains for deriving the same also include strains modified to enhance D-xylulose-5-phosphate-phosphoketolase and / or fructose-6-phosphate phosphoketolase activity. (Special Table 2008-509661). Either one or both of D-xylulose-5-phosphate-phosphoketolase activity and fructose-6-phosphate phosphoketolase activity may be enhanced. In the present specification, D-xylulose-5-phosphate phosphoketolase and fructose-6-phosphate phosphoketolase may be collectively referred to as phosphoketolase.
- D-xylulose-5-phosphate-phosphoketolase activity is the consumption of phosphoric acid to convert xylulose-5-phosphate into glyceraldehyde-3-phosphate and acetyl phosphate, and one molecule of H 2 O Means the activity of releasing. This activity is measured by the method described in Goldberg, M. et al. (Methods Enzymol., 9,515-520 (1966)) or L. Meile (J. Bacteriol. (2001) 183; 2929-2936). be able to.
- fructose-6-phosphate phosphoketolase activity means that phosphoric acid is consumed, fructose 6-phosphate is converted into erythrose-4-phosphate and acetyl phosphate, and one molecule of H 2 O is released. Means activity. This activity is measured by the method described in Racker, E (Methods Enzymol., 5, 276-280 (1962)) or L. Meile (J. Bacteriol. (2001) 183; 2929-2936). be able to.
- Examples of methods for imparting or enhancing L-glutamic acid-producing ability for coryneform bacteria include methods for imparting resistance to organic acid analogs and respiratory inhibitors, and methods for imparting sensitivity to cell wall synthesis inhibitors. .
- Examples of such methods include a method for imparting monofluoroacetic acid resistance (Japanese Patent Laid-Open No. 50-113209), a method for imparting adenine resistance or thymine resistance (Japanese Patent Laid-Open No. 57-065198), and a method of weakening urease activity.
- JP 52-038088 method for imparting malonic acid resistance (JP 52-038088), method for imparting resistance to benzopyrone or naphthoquinones (JP 56-1889), imparting HOQNO resistance
- a method for imparting resistance to ⁇ -ketomalonic acid Japanese Patent Laid-Open No. 57-2689
- a method for imparting guanidine resistance Japanese Patent Laid-Open No. 56-35981
- imparting sensitivity to penicillin And a method Japanese Patent Laid-Open No. 4-88994
- Such resistant or sensitive bacteria include the following strains. Corynebacterium glutamicum (Brevibacterium flavum) AJ3949 (FERM BP-2632; see JP 50-113209) Corynebacterium glutamicum AJ11628 (FERM P-5736; see JP-A-57-065198) Corynebacterium glutamicum (Brevibacterium flavum) AJ11355 (FERM P-5007; see JP 56-1889) Corynebacterium glutamicum AJ11368 (FERM P-5020; see JP-A-56-1889) Corynebacterium glutamicum (Brevibacterium flavum) AJ11217 (FERM P-4318; see JP-A-57-2689) Corynebacterium glutamicum AJ11218 (FERM P-4319; see JP 57-2689) Corynebacterium glutamicum (Brevibacterium flavum) AJ11564 (FERM P-
- Examples of a method for imparting or enhancing L-glutamic acid producing ability for coryneform bacteria include a method for enhancing expression of the yggB gene and a method for introducing a mutant yggB gene having a mutation introduced into the coding region ( WO2006 / 070944).
- the yggB gene encodes a mechanosensitive channel.
- the yggB gene of Corynebacterium glutamicum ATCC13032 corresponds to the complementary sequence of the sequences 1,336,091 to 1,337,692 in the genome sequence registered in the NCBI database as Genbank Accession No. NC_003450, and is also called NCgl1221.
- the YggB protein encoded by the yggB gene of Corynebacterium glutamicum ATCC13032 is registered as GenBank accession No. NP_600492.
- the nucleotide sequence of the yggB gene of Corynebacterium glutamicum 2256 (ATCC 13869) and the amino acid sequence of the YggB protein encoded by the same gene are shown in SEQ ID NOs: 21 and 22, respectively.
- mutant yggB gene used herein examples include the yggB gene having the following mutations.
- the YggB protein encoded by the mutant yggB gene is also referred to as a mutant YggB protein.
- the yggB gene not having the mutation and the YggB protein encoded by the same gene are also referred to as a wild-type yggB gene and a wild-type YggB protein, respectively.
- Examples of the wild type YggB protein include a protein having the amino acid sequence shown in SEQ ID NO: 22.
- the C-terminal side mutation is a mutation introduced into a part of the base sequence of the region encoding the sequence of amino acid numbers 419 to 533 of SEQ ID NO: 22.
- the C-terminal mutation is not particularly limited as long as the mutation is introduced into at least a part of the base sequence of the above region, but preferably has an insertion sequence (hereinafter also referred to as “IS”) or a transposon inserted therein.
- the C-terminal mutation may be any of those accompanied by amino acid substitution (missense mutation), those having a frameshift mutation introduced by insertion of the IS or the like, and those having a nonsense mutation introduced.
- a mutant yggB gene having a C-terminal mutation for example, IS is inserted at a position encoding valine residue at position 419 of SEQ ID NO: 22, which is more than that of the wild-type YggB protein (SEQ ID NO: 22).
- a yggB gene encoding a mutant YggB protein having a short total length of 423 amino acid residues can be mentioned (Japanese Patent Laid-Open No. 2007-222163).
- the nucleotide sequence of this mutant yggB gene (V419 :: IS) and the amino acid sequence of the mutant YggB protein encoded by the same gene are shown in SEQ ID NOs: 23 and 24, respectively.
- examples of the C-terminal mutation include a mutation that substitutes proline existing in the region of amino acid numbers 419 to 533 of SEQ ID NO: 22 with another amino acid.
- the YggB protein encoded by the yggB gene has five transmembrane regions.
- the transmembrane regions are amino acid numbers 1 to 23 (first transmembrane region), 25 to 47 (second transmembrane region), and 62 to 84 (third membrane), respectively. This corresponds to the region of through region), 86 to 108 (fourth membrane penetration region), and 110 to 132 (fifth membrane penetration region).
- the yggB gene may have a mutation in the region encoding these transmembrane regions.
- the mutation in the transmembrane region is preferably a mutation including substitution, deletion, addition, insertion or inversion of one or several amino acids, and is not accompanied by a frameshift mutation and a nonsense mutation.
- Mutations in the transmembrane region include mutations in which one or several amino acids are inserted between the leucine residue at position 14 and the tryptophan residue at position 15 in the amino acid sequence shown in SEQ ID NO: 22, and the alanine residue at position 100 And the like, and the mutation that substitutes the alanine residue at position 111 to another amino acid residue.
- the above “one or several” specifically means preferably 1 to 20, more preferably 1 to 10, further preferably 1 to 5, particularly preferably 1 to 3. .
- the mutant yggB gene is mutated into a region encoding an amino acid residue corresponding to the amino acid residue at the above position in SEQ ID NO: 22. As long as it has.
- which amino acid residue is “the amino acid residue corresponding to the amino acid residue at the above position in SEQ ID NO: 22” is determined based on the amino acid sequence of the wild type YggB protein and SEQ ID NO: 22 It can be determined by alignment with the amino acid sequence.
- mutant phosphate transporters described later and variants of genes encoding the same can be applied mutatis mutandis.
- Amino acid number X of SEQ ID NO: 22 may be read as “X position of SEQ ID NO: 22”.
- Examples of the method for imparting or enhancing L-glutamine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-glutamine biosynthesis enzymes is increased.
- Examples of such an enzyme include, but are not limited to, glutamate dehydrogenase (gdhA) and glutamine synthetase (glnA).
- the method for imparting or enhancing L-glutamine production ability is, for example, selected from an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-glutamine to produce a compound other than L-glutamine.
- an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-glutamine to produce a compound other than L-glutamine.
- a method of modifying the bacterium so that the activity of the further enzyme is reduced can also be mentioned.
- Such an enzyme is not particularly limited, and includes glutaminase.
- L-glutamine-producing bacteria or parent strains for inducing them examples include coryneform bacteria (EP1229121, EP1424398) with enhanced activity of glutamate dehydrogenase (gdhA) and / or glutamine synthetase (glnA), and coryneforms with reduced glutaminase activity Type bacteria (Japanese Patent Laid-Open No. 2004-187684). Enhancement of glutamine synthetase activity can also be achieved by disruption of the glutamine adenylyltransferase gene (glnE) or the PII regulatory protein gene (glnB) (EP1229121).
- gdhA glutamate dehydrogenase
- glnA glutamine synthetase
- glnA glutaminase activity Type bacteria
- Enhancement of glutamine synthetase activity can also be achieved by disruption of the glutamine adenylyltransferase gene (g
- Corynebacterium glutamicum (Brevibacterium flavum) AJ11573 (FERM P-5492, JP 56-161495) Corynebacterium glutamicum (Brevibacterium flavum) AJ11576 (FERM BP-10381, JP 56-161495) Corynebacterium glutamicum (Brevibacterium flavum) AJ12212 (FERM P-8123, JP 61-202694)
- L-proline producing bacteria examples include a strain in which the activity of one or more enzymes selected from L-proline biosynthesis enzymes are enhanced.
- enzymes involved in L-proline biosynthesis include glutamate 5-kinase, ⁇ -glutamyl-phosphate reductase, and pyrroline-5-carboxylate reductase.
- the proB gene German Patent No. 3127361 encoding glutamate kinase which is desensitized to feedback inhibition by L-proline can be preferably used.
- examples of L-proline-producing bacteria or parent strains for inducing them also include strains in which the activity of an enzyme involved in L-proline degradation is reduced.
- examples of such an enzyme include proline dehydrogenase and ornithine aminotransferase.
- L-threonine producing bacteria examples include a strain in which the activity of one or more enzymes selected from L-threonine biosynthetic enzymes are enhanced.
- enzymes include, but are not limited to, aspartokinase III (lysC), aspartate semialdehyde dehydrogenase (asd), aspartokinase I (thrA), homoserine kinase (thrB), threonine synthase ( threonine synthase) (thrC), aspartate aminotransferase (aspartate transaminase) (aspC).
- the L-threonine biosynthesis gene may be introduced into a strain in which threonine degradation is suppressed.
- the activity of the L-threonine biosynthetic enzyme is inhibited by the final product L-threonine. Therefore, in order to construct an L-threonine-producing bacterium, it is preferable to modify the L-threonine biosynthetic gene so as not to receive feedback inhibition by L-threonine.
- the thrA, thrB, and thrC genes constitute a threonine operon, and the threonine operon forms an attenuator structure. Expression of the threonine operon is inhibited by isoleucine and threonine in the culture medium, and is suppressed by attenuation.
- Enhanced expression of the threonine operon can be achieved by removing the leader sequence or attenuator in the attenuation region (Lynn, S. P., Burton, W. S., Donohue, T. J., Gould, R. M., um Gumport, R. I., and Gardner, J. F. J. Mol. Biol. 194: 59-69 1987 (1987); WO02 / 26993; WO2005 / 049808; WO2005 / 049808; WO2003 / 097839 ).
- the threonine operon may be constructed so that a gene involved in threonine biosynthesis is expressed under the control of a lambda phage repressor and promoter (see European Patent No. 0593792).
- Bacteria modified so as not to be subjected to feedback inhibition by L-threonine can also be obtained by selecting a strain resistant to ⁇ -amino- ⁇ -hydroxyvaleric acid (AHV), which is an L-threonine analog.
- HAV ⁇ -amino- ⁇ -hydroxyvaleric acid
- the threonine operon modified so as not to be subjected to feedback inhibition by L-threonine is improved in the expression level in the host by increasing the copy number or being linked to a strong promoter.
- An increase in copy number can be achieved by introducing a plasmid containing a threonine operon into the host.
- An increase in copy number can also be achieved by transferring the threonine operon onto the host genome using a transposon, Mu phage, or the like.
- the thrA gene encoding aspartokinase homoserine dehydrogenase I of E. coli has been revealed (nucleotide numbers 337-2799, GenBank accession NC_000913.2, gi: 49175990).
- the thrA gene is located between the thrL gene and the thrB gene in the chromosome of E. coli K-12.
- the thrB gene encoding homoserine kinase of Escherichia coli has been elucidated (nucleotide numbers 2801 to 3733, GenBank accession NC_000913.2, gi: 49175990).
- the thrB gene is located between the thrA gene and the thrC gene in the chromosome of E. coli K-12.
- the thrC gene encoding threonine synthase from E.coli has been elucidated (nucleotide numbers 3734 to 5020, GenBank accession NC_000913.2, gi: 49175990).
- the thrC gene is located between the thrB gene and the yaaX open reading frame in the chromosome of E. coli K-12.
- thrA * BC operon containing a mutant thrA gene encoding an aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine and a wild type thrBC gene is known in the threonine-producing strain E. coli VKPM B-3996. It can be obtained from plasmid pVIC40 (US Pat. No. 5,705,371).
- the rhtA gene of E. coli is present at 18 minutes of the E. coli chromosome close to the glnHPQ operon, which encodes an element of the glutamine transport system.
- the rhtA gene is the same as ORF1 (ybiF gene, nucleotide numbers 764 to 1651, GenBank accession number AAA218541, gi: 440181), and is located between the pexB gene and the ompX gene.
- the unit that expresses the protein encoded by ORF1 is called rhtA gene (rht: resistant toosehomoserine andeonthreonine (resistant to homoserine and threonine)).
- the asd gene of E. coli has already been clarified (nucleotide numbers 3572511 to 3571408, GenBank accession NC_000913.1, gi: 16131307), and can be obtained by PCR using primers prepared based on the nucleotide sequence of the gene ( White, TJ et al., Trends Genet., 5, 185 (1989)).
- the asd gene of other microorganisms can be obtained similarly.
- the aspC gene of E. ⁇ ⁇ coli has already been clarified (nucleotide numbers 983742 to 984932, GenBank accession NC_000913.1, gi: 16128895), and obtained by PCR using a primer prepared based on the nucleotide sequence of the gene be able to.
- the aspC gene of other microorganisms can be obtained similarly.
- coryneform bacteria having L-threonine-producing ability examples include Corynebacterium acetoacidophilum AJ12318123 (FERM BP-1172) (see US Patent No. 5,188,949).
- L-lysine producing bacteria examples include a strain in which the activity of one or more enzymes selected from L-lysine biosynthetic enzymes are enhanced.
- enzymes include, but are not limited to, dihydrodipicolinate synthase (dapA), aspartokinase III (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate Diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (US Pat. No.
- phosphoenolpyrvate carboxylase ppc
- aspartate semialdehyde dehydrogenase phosphoenolpyrvate carboxylase
- Asd aspartate semialdehyde dehydrogenase
- aspartate aminotransferase aspartate transaminase
- aspC diaminopimelate epi Diaminopimelate epimerase
- dapF diaminopimelate epi Diaminopimelate epimerase
- dapD tetrahydrodipicolinate succinylase
- dapE succinyl-diaminopimelate deacylase
- aspartase aspA (195) ).
- dihydrodipicolinate reductase diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and
- the activity of one or more enzymes selected from succinyl diaminopimelate deacylase is enhanced.
- a gene (cyo) (EP 1170376 A) involved in energy efficiency, a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) ( US Pat. No. 5,830,716), ybjE gene (WO2005 / 073390), or combinations thereof may have increased expression levels.
- Aspartokinase III (lysC) is subject to feedback inhibition by L-lysine.
- a mutant lysC gene encoding aspartokinase III that has been desensitized to feedback inhibition by L-lysine is used. It may be used (US Pat. No.
- the L-lysine-producing bacterium or the parent strain for deriving it is selected from enzymes selected from enzymes that catalyze reactions that branch off from the biosynthetic pathway of L-lysine to produce compounds other than L-lysine. Examples include strains in which the activity of the above enzymes is reduced or deficient. Such enzymes include, but are not limited to, homoserine dehydrogenase, lysine decarboxylase (US Pat. No. 5,827,698), and malic enzyme (WO2005 / 010175). .
- L-lysine-producing bacteria or parent strains for inducing them include mutants having resistance to L-lysine analogs.
- L-lysine analogs inhibit the growth of bacteria such as Enterobacteriaceae and coryneform bacteria, but this inhibition is completely or partially released when L-lysine is present in the medium.
- the L-lysine analog is not particularly limited, and examples thereof include oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), ⁇ -methyllysine, and ⁇ -chlorocaprolactam.
- Mutant strains having resistance to these lysine analogs can be obtained by subjecting bacteria to normal artificial mutation treatment.
- coryneform bacteria having L-lysine-producing ability include, for example, AEC resistant mutant strains (Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ11082 (NRRL B-11470) strain, etc .; Japanese Patent Publication No.
- L-leucine L-homoserine, L-proline
- Mutants requiring amino acids such as L-serine, L-arginine, L-alanine and L-valine (US Pat. No. 3,708) 395 and 3854272); DL- ⁇ -amino- ⁇ -caprolactam, ⁇ -amino-lauryllactam, aspartic acid analogs, sulfa drugs, quinoids, mutant strains resistant to N-lauroylleucine; oxaloacetic acid Mutants exhibiting resistance to decarboxylase inhibitors or respiratory enzyme inhibitors (Japanese Patent Laid-Open Nos.
- JP 50-53588, 50-31093, 52-102498, 53-9394) JP, 53-86089, 55-9783, 55-9759, 56-32995, 56-39778, 56-39778, JP 53-43591, JP-B 53-1833); mutants requiring inositol or acetic acid (JP 55-9784, JP 56-8692); fluoropyruvic acid or 34 ° C or higher Mutants exhibiting sensitivity to the temperature of JP-A-55-9783 and JP-A-53-86090; Mutants resistant to Ji glycol (U.S. Pat. No. 4,411,997) and the like.
- L-arginine producing bacteria examples include a strain in which the activity of one or more enzymes selected from L-arginine biosynthesis enzymes are enhanced.
- enzymes include, but are not limited to, N-acetylglutamate synthase (argA), N-acetylglutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetylornithine
- Examples thereof include transaminase (argD), ornithine carbamoyltransferase (argF), arginosuccinate synthetase (argG), arginosuccinate lyase (argH), and carbamoyl phosphate synthetase (carAB).
- N-acetylglutamate synthase (argA) gene examples include mutant N-acetylglutamate synthase in which amino acid residues corresponding to the 15th to 19th positions of the wild type are substituted and feedback inhibition by L-arginine is released. It is preferable to use a gene to be encoded (European Application Publication No. 1170361).
- examples of L-arginine-producing bacteria or parent strains for inducing them also include strains having resistance to amino acid analogs and the like.
- Such strains include, for example, coryneform bacterial strains that have L-histidine, L-proline, L-threonine, L-isoleucine, L-methionine, or L-tryptophan requirements in addition to 2-thiazolealanine resistance.
- coryneform bacterial strains that have L-histidine, L-proline, L-threonine, L-isoleucine, L-methionine, or L-tryptophan requirements in addition to 2-thiazolealanine resistance.
- Coryneform bacterial strain resistant to ketomalonic acid, fluoromalonic acid or monofluoroacetic acid Japanese Patent Laid-Open No.
- coryneform bacterial strain resistant to argininol Japanese Examined Patent Publication No. 62-24075
- Coryneform bacterial strain resistant to X-guanidine X is a fatty acid or fatty chain derivative
- Arginine Hydroxamate and 6-Azauracil A coryneform bacterium strain having resistance Japanese Patent Laid-Open No. 57-150381.
- Specific examples of coryneform bacteria having the ability to produce L-arginine include the following strains.
- L-citrulline and L-ornithine-producing bacteria share a biosynthetic pathway with L-arginine.
- N-acetylglutamate synthase argA
- N-acetylglutamylphosphate reductase argC
- ornithine acetyltransferase argJ
- N-acetylglutamate kinase argB
- acetylornithine transaminase argD
- WO 2006-35831 By increasing the enzyme activity of deacetylase (argE), the ability to produce L-citrulline and / or L-ornithine can be imparted or enhanced (WO 2006-35831).
- L-histidine producing bacteria examples include strains in which the activity of one or more enzymes selected from L-histidine biosynthetic enzymes are enhanced.
- examples of such an enzyme include, but are not limited to, ATP phosphoribosyltransferase (hisG), phosphoribosyl-AMP cyclohydrolase (hisI), phosphoribosyl-ATP pyrophosphohydrolase (hisI), phosphoribosylformimino-5-aminoimidazole carboxamide ribonucleoside.
- tide isomerase (hisA), amide transferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), and histidinol dehydrogenase (hisD).
- hisA tide isomerase
- hisH amide transferase
- hisC histidinol phosphate aminotransferase
- hisB histidinol phosphatase
- hisD histidinol dehydrogenase
- L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are known to be inhibited by L-histidine. Therefore, the ability to produce L-histidine can be imparted or enhanced, for example, by introducing a mutation that confers resistance to feedback inhibition in the ATP phosphoribosyltransferase gene (hisG) ( Russian Patent No. 2003677 and No. 2). 2119536).
- L-cysteine producing bacteria examples include strains in which the activity of one or more enzymes selected from L-cysteine biosynthetic enzymes are enhanced.
- enzymes are not particularly limited, and include serine acetyltransferase and 3-phosphoglycerate dehydrogenase.
- Serine acetyltransferase activity can be enhanced, for example, by introducing a mutant cysE gene encoding a mutant serine acetyltransferase resistant to feedback inhibition by cysteine into bacteria.
- Mutant serine acetyltransferases are disclosed, for example, in JP-A-11-155571 and US Patent Publication No. 20050112731. Further, the 3-phosphoglycerate dehydrogenase activity can be enhanced, for example, by introducing a mutant serA gene encoding a mutant 3-phosphoglycerate dehydrogenase resistant to feedback inhibition by serine into a bacterium. Mutant 3-phosphoglycerate dehydrogenase is disclosed, for example, in US Pat. No. 6,180,373.
- the L-cysteine-producing bacterium or the parent strain for deriving it is selected from enzymes selected from enzymes that catalyze reactions that branch off from the biosynthetic pathway of L-cysteine to produce compounds other than L-cysteine. Examples include strains in which the activity of the above enzymes is reduced or deficient. Examples of such enzymes include enzymes involved in the degradation of L-cysteine.
- the enzyme involved in the degradation of L-cysteine is not particularly limited, and examples thereof include cysteine desulfhydrase (aecD) (Japanese Patent Laid-Open No. 2002-233384).
- examples of L-cysteine-producing bacteria or parent strains for inducing them include strains with enhanced L-cysteine excretion system and strains with enhanced sulfate / thiosulfate transport system.
- Proteins of the L-cysteine excretion system include proteins encoded by the ydeD gene (JP 2002-233384), proteins encoded by the yfiK gene (JP 2004-49237), emrAB, emrKY, yojIH, acrEF, bcr, And each protein encoded by each gene of cusA (Japanese Patent Laid-Open No.
- sulfate / thiosulfate transport system protein examples include proteins encoded by the cysPTWAM gene cluster.
- coryneform bacterium having L-cysteine-producing ability a coryneform bacterium in which intracellular serine acetyltransferase activity is increased by retaining a serine acetyltransferase in which feedback inhibition by L-cysteine is reduced (Japanese Patent Application Laid-Open No. 2005-133867). 2002-233384).
- L-methionine producing bacteria examples include L-threonine-requiring strains and mutants having resistance to norleucine (Japanese Patent Laid-Open No. 2000-139471).
- examples of L-methionine-producing bacteria or parent strains for deriving them also include strains that retain mutant homoserine transsuccinylase that is resistant to feedback inhibition by L-methionine (Japanese Patent Laid-Open No. 2000-139471). , US20090029424).
- L-methionine is biosynthesized with L-cysteine as an intermediate, L-methionine production ability can be improved by improving L-cysteine production ability (Japanese Patent Laid-Open No. 2000-139471, US20080311632).
- L-leucine producing bacteria examples include strains in which the activity of one or more enzymes selected from L-leucine biosynthesis enzymes are enhanced.
- examples of such an enzyme include, but are not limited to, an enzyme encoded by a gene of leuABCD operon.
- a mutant leuA gene US Pat. No. 6,403,342
- isopropyl malate synthase from which feedback inhibition by L-leucine has been released can be suitably used.
- Coryneform bacteria having L-leucine-producing ability include, for example, Corynebacterium amicglutamicum (Brevibacterium lactofermentum) AJ3718 (FERM P-2516), which is resistant to 2-thiazolealanine and ⁇ -hydroxyleucine, and is auxotrophic for isoleucine and methionine. Is mentioned.
- Examples of the method for imparting or enhancing L-isoleucine producing ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-isoleucine biosynthesis enzymes is increased.
- Examples of such an enzyme include, but are not limited to, threonine deaminase and acetohydroxy acid synthase (JP-A-2-458, FR 0356739, and US Pat. No. 5,998,178).
- Coryneform bacteria having the ability to produce L-isoleucine include coryneform bacteria in which a brnE gene encoding a branched-chain amino acid excretion protein is amplified (JP 2001-169788), and L-isoleucine by protoplast fusion with L-lysine producing bacteria.
- Coryneform bacterium imparted with productivity JP-A 62-74293
- coryneform bacterium enriched with homoserine dehydrogenase JP-A 62-91193
- threonine hydroxamate resistant strain JP-A 62-195293
- ⁇ -Ketomalone resistant strain JP 61-15695
- methyl lysine resistant strain JP 61-15696
- Corynebacterium glutamicum (Brevibacterium flavum) AJ12149 (FERM BP-759) (US Pat. No. 4,656,135).
- L-valine producing bacteria examples include a strain in which the activity of one or more enzymes selected from L-valine biosynthesis enzymes is enhanced.
- enzymes include, but are not limited to, enzymes encoded by genes of ilvGMEDA operon and ilvBNC operon.
- ilvBN encodes acetohydroxy acid synthase
- ilvC encodes isomeroreductase (WO 00/50624).
- the ilvGMEDA operon and the ilvBNC operon are subject to expression suppression (attenuation) by L-valine, L-isoleucine, and / or L-leucine.
- the threonine deaminase encoded by the ilvA gene is an enzyme that catalyzes the deamination reaction from L-threonine to 2-ketobutyric acid, which is the rate-limiting step of the L-isoleucine biosynthesis system. Therefore, for L-valine production, it is preferable that the ilvA gene is disrupted and the threonine deaminase activity is reduced.
- the L-valine-producing bacterium or the parent strain for deriving it is selected from an enzyme that catalyzes a reaction that produces a compound other than L-valine by branching from the biosynthetic pathway of L-valine.
- a strain in which the activity of the above enzyme is reduced is also mentioned.
- enzymes include, but are not limited to, threonine dehydratase involved in L-leucine synthesis and enzymes involved in D-pantothenic acid synthesis (International Publication No. 00/50624).
- L-valine-producing bacteria or parent strains for inducing them include strains having resistance to amino acid analogs and the like.
- Such strains include, for example, L-isoleucine and L-methionine requirement, coryneform bacterial strains resistant to D-ribose, purine ribonucleoside, or pyrimidine ribonucleoside and capable of producing L-valine.
- L-alanine producing bacteria examples include coryneform bacteria lacking H + -ATPase (Appl Microbiol Biotechnol. 2001 Nov; 57 (4): 534-40) and aspartic acid ⁇ -Coryneform bacteria with enhanced decarboxylase activity (JP 07-163383 A).
- L-tryptophan producing bacteria L-phenylalanine producing bacteria, L-tyrosine producing bacteria>
- methods for imparting or enhancing L-tryptophan production ability, L-phenylalanine production ability, and / or L-tyrosine production ability include biosynthesis of L-tryptophan, L-phenylalanine, and / or L-tyrosine.
- Biosynthetic enzymes common to these aromatic amino acids are not particularly limited, but 3-deoxy-D-arabinohepturonic acid-7-phosphate synthase (aroG), 3-dehydroquinate synthase (aroB) Shikimate dehydrogenase (aroE), shikimate kinase (aroL), 5-enolic acid pyruvylshikimate 3-phosphate synthase (aroA), chorismate synthase (aroC) (European Patent No. 763127). Expression of genes encoding these enzymes is controlled by a tyrosine repressor (tyrR), and the activity of these enzymes may be enhanced by deleting the tyrR gene (European Patent No. 763127).
- tyrR tyrosine repressor
- L-tryptophan biosynthesis enzyme examples include, but are not limited to, anthranilate synthase (trpE), tryptophan synthase (trpAB), and phosphoglycerate dehydrogenase (serA).
- trpE anthranilate synthase
- trpAB tryptophan synthase
- serA phosphoglycerate dehydrogenase
- L-tryptophan production ability can be imparted or enhanced by introducing DNA containing a tryptophan operon.
- Tryptophan synthase consists of ⁇ and ⁇ subunits encoded by trpA and trpB genes, respectively.
- anthranilate synthase is subject to feedback inhibition by L-tryptophan
- a gene encoding the enzyme into which a mutation that releases feedback inhibition is introduced may be used.
- phosphoglycerate dehydrogenase is feedback-inhibited by L-serine
- a gene encoding the enzyme into which a mutation that releases feedback inhibition is introduced may be used to enhance the activity of the enzyme.
- L-tryptophan-producing ability is imparted or enhanced by increasing the expression of an operon consisting of malate synthase (aceB), isocitrate lyase (aceA), and isocitrate dehydrogenase kinase / phosphatase (aceK). (WO2005 / 103275).
- the L-phenylalanine biosynthetic enzyme is not particularly limited, and examples thereof include chorismate mutase and prefenate dehydratase. Chorismate mutase and prefenate dehydratase are encoded by the pheA gene as a bifunctional enzyme. Since chorismate mutase-prefenate dehydratase is feedback-inhibited by L-phenylalanine, in order to enhance the activity of the enzyme, a gene encoding the enzyme into which a mutation that releases feedback inhibition is introduced may be used.
- the L-tyrosine biosynthetic enzyme is not particularly limited, and examples thereof include chorismate mutase and prephenate dehydrogenase. Chorismate mutase and prefenate dehydrogenase are encoded by the tyrA gene as a bifunctional enzyme. Since chorismate mutase-prefenate dehydrogenase is feedback-inhibited by L-tyrosine, to enhance the activity of the enzyme, a gene encoding the enzyme into which a mutation that releases feedback inhibition is introduced may be used.
- the L-tryptophan, L-phenylalanine, and / or L-tyrosine producing bacterium may be modified so that biosynthesis of aromatic amino acids other than the target aromatic amino acid is lowered.
- L-tryptophan, L-phenylalanine, and / or L-tyrosine-producing bacteria may be modified so that the by-product uptake system is enhanced.
- By-products include aromatic amino acids other than the desired aromatic amino acid. Examples of genes encoding uptake systems of by-products include, for example, uptake systems of tnaB and mtr, which are L-tryptophan uptake systems, and pheP, L-tyrosine, which are genes encoding uptake systems of L-phenylalanine. TyrP, which is a gene coding for (EP1484410).
- Coryneform bacteria having the ability to produce L-tryptophan include Corynebacterium glutamicum AJ12118 (FERM BP-478 Patent 01688102) resistant to sulfaguanidine, strains into which tryptophan operon has been introduced (JP 63240794), coryneform bacteria And a strain into which a gene encoding shikimate kinase derived therefrom has been introduced (Japanese Patent Laid-Open No. 01994749).
- coryneform bacteria having the ability to produce L-phenylalanine include, for example, Corynebacterium amicglutamicum BPS-13 strain FER (FERM BP-1777), Corynebacterium glutamicum K77 (FERM BP-2062) having reduced phosphoenolpyruvate carboxylase or pyruvate kinase activity Corynebacterium glutamicum K78 (FERM BP-2063) (European Patent Publication No. 331145, Japanese Patent Laid-Open No. 02-303495) and tyrosine-requiring strain (Japanese Patent Laid-Open No. 05-049489).
- coryneform bacteria having the ability to produce L-tyrosine include Corynebacterium glutamicum AJ11655 (FERM P-5836) (Japanese Patent Publication No. 2-6517), Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ12081 (FERM P-7249) -70093).
- examples of a method for imparting or enhancing L-amino acid-producing ability include a method of modifying a bacterium so that the activity of discharging L-amino acid from the bacterium cell is increased.
- the activity to excrete L-amino acids can be increased, for example, by increasing the expression of a gene encoding a protein that excretes L-amino acids.
- genes encoding proteins that excrete various amino acids include b2682 gene (ygaZ), b2683 gene (ygaH), b1242 gene (ychE), and b3434 gene (yhgN) (Japanese Patent Laid-Open No. 2002-300874) .
- examples of a method for imparting or enhancing L-amino acid producing ability include a method for modifying bacteria so that the activity of a protein involved in sugar metabolism or a protein involved in energy metabolism is increased.
- Proteins involved in sugar metabolism include proteins involved in sugar uptake and glycolytic enzymes.
- genes encoding proteins involved in sugar metabolism include glucose 6-phosphate isomerase gene (pgi; WO 01/02542 pamphlet), phosphoenolpyruvate synthase gene (pps; EP 877090 specification) , Phosphoenolpyruvate carboxylase gene (ppc; WO 95/06114 pamphlet), pyruvate carboxylase gene (pyc; WO 99/18228 pamphlet, European application 1092776), phosphoglucomutase gene (Pgm; WO 03/04598 pamphlet), fructose diphosphate aldolase gene (pfkB, fbp; WO 03/04664 pamphlet), pyruvate kinase gene (pykF; WO 03/008609 pamphlet), transaldolase Gene (talB; WO03 / 008611 pamphlet), fumarase residue Child (
- non-PTS sucrose uptake gene gene csc; European Application Publication No. 149911 pamphlet
- sucrose utilization gene scrAB operon; International Publication No. 90/04636 pamphlet
- genes encoding proteins involved in energy metabolism include a transhydrogenase gene (pntAB; US Pat. No. 5,830,716), a cytochrome bo type oxidase (cyoB; European Patent Application Publication No. 1070376) Is mentioned.
- the gene used for breeding the above-mentioned L-amino acid-producing bacteria is not limited to the above-exemplified genes or genes having a known base sequence, as long as the function of the encoded protein is not impaired. May be.
- a gene used for breeding an L-amino acid-producing bacterium is an amino acid in which one or several amino acids at one or several positions are substituted, deleted, inserted or added in the amino acid sequence of a known protein. It may be a gene encoding a protein having a sequence.
- the description regarding the phosphate transporter gene and the variant of the phosphate transporter described later can be applied mutatis mutandis.
- the bacterium of the present invention has been modified to increase phosphate transporter activity.
- the bacterium of the present invention can be obtained by modifying a coryneform bacterium having L-amino acid-producing ability so that the phosphate transporter activity is increased.
- the bacterium of the present invention can also be obtained by imparting or enhancing L-amino acid-producing ability after modifying the coryneform bacterium so that the phosphate transporter activity is increased.
- the bacterium of the present invention may have acquired L-amino acid-producing ability by being modified so that the phosphate transporter activity is increased.
- the modification for constructing the bacterium of the present invention can be performed in any order.
- phosphate transporter refers to a protein having phosphate transporter activity.
- phosphate transporter activity refers to an activity of taking inorganic phosphate (Pi) into the cell from outside the cell.
- a phosphate transporter As a phosphate transporter, a low-affinity phosphate-specific transporter (Pst) system or a high-affinity phosphate-specific transporter (Pst) system is used. Is mentioned.
- Examples of a gene encoding a phosphate transporter include a pitA gene encoding a Pit system, a pitB gene, and a pstSCAB gene encoding a Pst system (Non-patent Document 1). The Pst system functions as a complex of four proteins (product of the pstSCAB gene).
- the activity of either the Pit system or the Pst system may be increased.
- it is preferable to increase the activity of the Pit system and it is more preferable to increase the activity of the PitA protein that is the pitA gene product.
- the pitA gene of Escherichia coli K12 MG1655 strain corresponds to the sequence of positions 3635665 to 3637164 in the genome sequence registered as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990) in the NCBI database.
- the pitA gene of Escherichia coli K12 MG1655 is synonymous with ECK3478 and JW3460.
- the nucleotide sequence of the pitA gene of the MG1655 strain and the amino acid sequence of the PitA protein encoded by the same gene are shown in SEQ ID NOs: 1 and 2, respectively.
- the pitA gene of Pantoea ananatis LMG20103 strain corresponds to the complementary sequence of positions 1397898 to 1395514 in the genome sequence registered as GenBank accession NC_013956 (VERSION NC_013956.2 GI: 332139403) in the NCBI database.
- the nucleotide sequence of the pitA gene of Pantoea ananatis LMG20103 and the amino acid sequence of the PitA protein encoded by this gene are shown in SEQ ID NOs: 3 and 4, respectively.
- the pitA gene of Corynebacterium glutamicum ATCC13032 corresponds to a complementary sequence of the 481391 to 482776 positions in the genome sequence registered as GenBank accession NC_003450 (VERSION NC_003450.3 GI: 58036263) in the NCBI database.
- the pitA gene of Corynebacterium glutamicum ATCC13032 is synonymous with Cgl0460.
- the nucleotide sequence of the pitA gene of Corynebacterium glutamicum ATCC13032 and the amino acid sequence of the PitA protein encoded by the gene are shown in SEQ ID NOs: 25 and 26, respectively.
- the nucleotide sequence of the pitA gene of Corynebacterium glutamicum 2256 (ATCC 13869) and the amino acid sequence of the PitA protein encoded by the gene are shown in SEQ ID NOs: 5 and 6, respectively.
- the phosphate transporter may be a variant of the above-mentioned phosphate transporter, for example, various PitA proteins, as long as it has phosphate transporter activity. Such variants may be referred to as “conservative variants”. Examples of conservative variants include homologues and artificially modified forms of the above-mentioned phosphate transporters such as various PitA proteins.
- the gene encoding the homologue of the PitA protein can be easily obtained from a public database by BLAST search or FASTA search using the base sequence (SEQ ID NO: 1, 3, 5, or 25) of the pitA gene as a query sequence, for example. be able to.
- the gene encoding the PitA protein homolog can be obtained, for example, by PCR using a bacterial or yeast chromosome as a template and oligonucleotides prepared based on these known gene sequences as primers.
- the gene encoding a conservative variant of phosphate transporter may be, for example, the following gene. That is, as long as the phosphate transporter gene encodes a protein having phosphate transporter activity, one or several amino acids at one or several positions in the amino acid sequence are substituted, deleted, inserted, Alternatively, it may be a gene encoding a protein having an added amino acid sequence. In this case, the phosphate transporter activity is usually 70% or more, preferably 80% or more, more preferably 90% or more with respect to the protein before one or several amino acids are substituted, deleted, inserted or added. % Or more can be maintained.
- one or several differs depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, but specifically, preferably 1-20, more preferably 1-10. It means 1 to 5, more preferably 1 to 5, particularly preferably 1 to 3.
- substitution, deletion, insertion, or addition of one or several amino acids described above is a conservative mutation that maintains the protein function normally.
- a typical conservative mutation is a conservative substitution.
- Conservative substitution 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.
- substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from 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, 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 substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Trp to Phe or Tyr, substitution
- the gene having a conservative mutation as described above is 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99%, based on the entire amino acid sequence. It may be a gene encoding a protein having a homology of at least% and having phosphate transporter activity. In the present specification, “homology” means “identity”.
- the phosphate transporter gene is a protein having a phosphate transporter activity that hybridizes under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a complementary sequence to the whole or a part of the base sequence. It may be DNA encoding. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more between DNAs having homology.
- the probe used for the hybridization may be a part of a complementary sequence of a gene.
- a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template.
- a DNA fragment having a length of about 300 bp can be used as the probe. More specifically, when a DNA fragment having a length of about 300 bp is used as the probe, the hybridization washing conditions include 50 ° C., 2 ⁇ SSC, and 0.1% SDS.
- the phosphate transporter gene may be one in which any codon is replaced with an equivalent codon as long as it encodes a protein having phosphate transporter activity.
- the phosphate transporter gene may be modified so as to have an optimal codon according to the codon usage frequency of the host to be used.
- the percentage sequence identity between two sequences can be determined using, for example, a mathematical algorithm.
- a mathematical algorithm include Myers and Miller (1988) CABIOS 4: 11 17 algorithm, Smith et aldv (1981) Adv. Appl. Math. 2: 482 local homology algorithm, Needleman and Wunsch (1970) J. Mol. Biol. 48: 443 453 homology alignment algorithm, Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444 2448 similarity search method, Karlin and Altschul ⁇ (1993) Proc. Natl. Acad. Sci. USA 90: 5873 5877, an improved algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264.
- sequence comparison for determining sequence identity can be performed.
- the program can be appropriately executed by a computer.
- Such programs include, but are not limited to, the PC / Gene program CLUSTAL (available from Intelligents, Mountain View, Calif.), The ALIGN program (Version 2.0), and Wisconsin Genetics Software Package, Version 8 (Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA) GAP, BESTFIT, BLAST, FASTA, and TFASTA. Alignment using these programs can be performed using initial parameters, for example.
- CLUSTAL program Higgins et al. (1988) Gene 73: 237 244 (1988), Higgins et al.
- Gapped BLAST (BLAST 2.0) can be used to obtain an alignment with a gap added for the purpose of comparison.
- PSI-BLASTA (BLAST 2.0) can be used to perform iterative searches that detect distant relationships between sequences.
- BLAST 2.0 For Gapped BLAST and PSI-BLAST, see Altschul et al. (1997) Nucleic Acids Res. 25: 3389.
- the initial parameters of each program eg, BLASTN for nucleotide sequences, BLASTX for amino acid sequences
- the alignment may be performed manually.
- sequence identity between two sequences is calculated as the ratio of residues that match between the two sequences when the two sequences are aligned for maximum matching.
- genes and protein variants can be applied mutatis mutandis to any proteins such as L-amino acid biosynthesis enzymes and the genes encoding them.
- Protein activity increases “means that the activity per cell of the protein is increased relative to unmodified strains such as wild strains and parental strains. Note that “increasing protein activity” is also referred to as “enhancing protein activity”. “Protein activity increases” specifically means that the number of molecules per cell of the protein is increased and / or the function per molecule of the protein compared to an unmodified strain. Is increasing. That is, “activity” in the case of “increasing protein activity” means not only the catalytic activity of the protein, but also the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. May be. The activity of the protein is not particularly limited as long as it is increased compared to the non-modified strain.
- the protein activity is increased 1.5 times or more, 2 times or more, or 3 times or more compared to the non-modified strain.
- “the protein activity increases” means not only to increase the activity of the protein in a strain that originally has the activity of the target protein, but also to the activity of the protein in a strain that does not originally have the activity of the target protein. Including granting.
- a suitable protein may be introduced after the activity of the target protein originally possessed by the host is weakened and / or deleted.
- Modification that increases the activity of the protein is achieved, for example, by increasing the expression of the gene encoding the protein.
- increasing gene expression is also referred to as “enhanced gene expression”.
- the expression of the gene may be increased 1.5 times or more, 2 times or more, or 3 times or more, for example, as compared to the unmodified strain.
- increasing gene expression means not only increasing the expression level of a target gene in a strain that originally expresses the target gene, but also in a strain that originally does not express the target gene. Including expressing a gene. That is, “increasing gene expression” includes, for example, introducing the gene into a strain that does not hold the target gene and expressing the gene.
- An increase in gene expression can be achieved, for example, by increasing the copy number of the gene.
- Increase in gene copy number can be achieved by introducing the gene into the host chromosome.
- Introduction of a gene into a chromosome can be performed, for example, using homologous recombination (Miller I, J. H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Only one copy of the gene may be introduced, or two copies or more may be introduced.
- multiple copies of a gene can be introduced into a chromosome by performing homologous recombination with a sequence having multiple copies on the chromosome as a target. Examples of sequences having many copies on a chromosome include repetitive DNA sequences (inverted DNA) and inverted repeats present at both ends of a transposon.
- homologous recombination may be performed by targeting an appropriate sequence on a chromosome such as a gene unnecessary for L-amino acid production.
- Homologous recombination is, for example, the Red-driven integration method (Datsenko, K. A, and Wanner, B. L. Proc. Natl. Acad. Sci. U S A. 97: 6640-6645 (2000) ), A method using a linear DNA, a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, a method using a suicide vector that does not have a replication origin and functions in a host, or a phage It can be performed by the transduction method used.
- the gene can also be randomly introduced onto the chromosome using transposon or Mini-Mu (Japanese Patent Laid-Open No. 2-109985, US Pat. No. 5,882,888, EP805867B1).
- the increase in gene copy number can be achieved by introducing a vector containing the target gene into the host.
- a DNA fragment containing a target gene can be linked to a vector that functions in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to increase the copy number of the gene. it can.
- a DNA fragment containing a target gene can be obtained, for example, by PCR using a genomic DNA of a microorganism having the target gene as a template.
- a vector capable of autonomous replication in a host cell can be used as the vector.
- the vector is preferably a multicopy vector.
- the vector preferably has a marker such as an antibiotic resistance gene.
- the vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid.
- vectors capable of autonomous replication in coryneform bacteria include, for example, pHM1519 (Agric, Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol.
- plasmids having improved drug resistance genes plasmid pCRY30 described in JP-A-3-210184; plasmid pCRY21 described in JP-A-2-72876 and US Pat. No. 5,185,262.
- the gene may be retained in the bacterium of the present invention so that it can be expressed.
- the gene may be introduced so as to be expressed under the control of a promoter sequence that functions in the bacterium of the present invention.
- the promoter may be a host-derived promoter or a heterologous promoter.
- the promoter may be a native promoter of a gene to be introduced or a promoter of another gene. As the promoter, for example, a stronger promoter as described later may be used.
- each gene when two or more genes are introduced, each gene may be retained in the bacterium of the present invention so that it can be expressed.
- all the genes may be held on a single expression vector, or all may be held on a chromosome.
- each gene may be separately hold
- an operon may be constructed by introducing two or more genes.
- the gene to be introduced is not particularly limited as long as it encodes a protein that functions in the host.
- the introduced gene may be a host-derived gene or a heterologous gene.
- the increase in gene expression can be achieved by improving the transcription efficiency of the gene.
- Improvement of gene transcription efficiency can be achieved, for example, by replacing a promoter of a gene on a chromosome with a stronger promoter.
- strong promoter is meant a promoter that improves transcription of the gene over the native wild-type promoter.
- artificially redesigned P54-6 promoter (Appl. Microbiol.
- the promoter activity can be increased by bringing the -35 and -10 regions in the promoter region closer to the consensus sequence (WO 00/18935).
- Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotickpromoters in biotechnology. Biotechnol. Annu. Rev.,. 1, 105-128 (1995)).
- the increase in gene expression can be achieved by improving the translation efficiency of the gene.
- Improvement of gene translation efficiency can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)) of the gene on the chromosome with a stronger SD sequence.
- SD Shine-Dalgarno
- RBS ribosome binding site
- a stronger SD sequence is meant an SD sequence in which the translation of mRNA is improved over the originally existing wild-type SD sequence.
- RBS of gene 10 derived from phage T7 can be mentioned (Olins P. O. et al, Gene, 1988, 73, 227-235).
- substitution of several nucleotides in the spacer region between the RBS and the start codon, particularly the sequence immediately upstream of the start codon (5'-UTR), or insertion or deletion contributes to mRNA stability and translation efficiency. It is known to have a great influence, and the translation efficiency of a gene can be improved by modifying them.
- a site that affects gene expression such as a promoter, an SD sequence, and a spacer region between the RBS and the start codon is also collectively referred to as an “expression control region”.
- the expression regulatory region can be determined using a promoter search vector or gene analysis software such as GENETYX.
- GENETYX gene analysis software
- These expression control regions can be modified by, for example, a method using a temperature sensitive vector or a Red driven integration method (WO2005 / 010175).
- Improvement of gene translation efficiency can also be achieved, for example, by codon modification.
- codon modification when performing heterologous expression of a gene, the translation efficiency of the gene can be improved by replacing rare codons present in the gene with synonymous codons that are used more frequently. Codon substitution can be performed, for example, by a site-specific mutagenesis method in which a target mutation is introduced into a target site of DNA. Alternatively, gene fragments in which codons have been replaced may be fully synthesized. The frequency of codon usage in various organisms can be found in the “Codon Usage Database” (http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)) Is disclosed.
- the increase in gene expression can be achieved by amplifying a regulator that increases gene expression or by deleting or weakening a regulator that decreases gene expression.
- the modification that increases the enzyme activity can be achieved, for example, by enhancing the specific activity of the enzyme.
- Enzymes with enhanced specific activity can be obtained by searching for various organisms, for example.
- a highly active type may be obtained by introducing a mutation into a conventional enzyme.
- the enhancement of specific activity may be used alone or in any combination with the above-described method for enhancing gene expression.
- the activity of a phosphate transporter can be increased, for example, by allowing a host to retain a phosphate transporter gene encoding a phosphate transporter having a “specific mutation”.
- the phosphate transporter having the “specific mutation” is also referred to as a mutant phosphate transporter, and the gene encoding it is also referred to as a mutant phosphate transporter gene.
- the phosphate transporter having no “specific mutation” is also referred to as a wild-type phosphate transporter, and the gene encoding it is also referred to as a wild-type phosphate transporter gene.
- the mutant phosphate transporter having the “specific mutation” may have a higher specific activity than the wild-type phosphate transporter.
- Examples of the wild-type phosphate transporter include PitA protein having no “specific mutation” (wild-type PitA protein).
- Examples of the mutant phosphate transporter include a PitA protein having a “specific mutation” (mutant PitA protein).
- a gene encoding a wild type PitA protein is also referred to as a wild type pitA gene, and a gene encoding a mutant PitA protein is also referred to as a mutant pitA gene.
- Examples of the wild-type PitA protein include various PitA proteins exemplified above and conservative variants thereof that do not have “specific mutation”. That is, the mutant phosphate transporter may be the same as any protein selected from, for example, the various PitA proteins exemplified above and conservative variants thereof, except for having a “specific mutation”.
- the mutant phosphate transporter may be a protein having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 26 except that it has a “specific mutation”.
- the mutant phosphate transporter has one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 26 except that it has a “specific mutation”. It may be a protein having an amino acid sequence containing substitutions, deletions, insertions, or additions.
- the mutant phosphate transporter is preferably 80% or more, preferably 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 26, except that it has a “specific mutation”. May be a protein having an amino acid sequence having homology of 90% or more, more preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more.
- “homology” means “identity”.
- the mutant phosphate transporter is a variant that has a “specific mutation” in the various PitA proteins exemplified above and further contains a conservative mutation at a place other than the “specific mutation”. It may be.
- the mutant phosphate transporter has a “specific mutation” in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 26, and other than the “specific mutation”. It may be a protein having an amino acid sequence further including substitution, deletion, insertion, or addition of one or several amino acids at a position.
- one or several specifically means preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3.
- One or several amino acid substitutions, deletions, insertions or additions described above are conservative mutations in which the function of the protein is maintained normally.
- a typical conservative mutation is a conservative substitution.
- Conservative substitution 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.
- substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from 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, 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 substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Trp to Phe or Tyr, substitution
- Examples of the “specific mutation” include a mutation in which an amino acid residue corresponding to the phenylalanine residue at position 246 in SEQ ID NO: 6 is substituted with an amino acid residue other than phenylalanine.
- the amino acid residue after substitution may be any amino acid other than phenylalanine as long as it is a natural amino acid. Lysine, glutamic acid, tyrosine, valine, isoleucine, serine, aspartic acid, asparagine, glutamine, arginine, cysteine, methionine, tryptophan , Glycine, alanine and histidine, serine being particularly preferred.
- the “X position” in the amino acid sequence means the X position from the N terminal of the amino acid sequence, and the amino acid residue at the N terminal is the amino acid residue at the first position.
- the position of an amino acid residue shows a relative position, The absolute position may be moved back and forth by deletion, insertion, addition, etc. of an amino acid. That is, “the amino acid residue corresponding to the phenylalanine residue at position 246 of SEQ ID NO: 6” means that when one amino acid residue on the N-terminal side from position 246 in SEQ ID NO: 6 is deleted, the N-terminal Means the 245th amino acid residue.
- amino acid residue corresponding to the phenylalanine residue at position 246 of SEQ ID NO: 6 is counted from the N terminus when one amino acid residue is inserted at the N-terminal side from position 246 in SEQ ID NO: 6. The 247th amino acid residue.
- amino acid residue is “an amino acid residue corresponding to the phenylalanine residue at position 246 of SEQ ID NO: 6” is aligned with the amino acid sequence of SEQ ID NO: 6 Can be determined.
- the alignment can be performed using, for example, known gene analysis software. Specific software includes DNA Solutions from Hitachi Solutions and GENETYX from Genetics (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24 (1), 72-96, 1991; Barton GJ et) al., Journal of molecular biology, 198 (2), 327-37. 1987).
- the “amino acid residue corresponding to the phenylalanine residue at position 246 in SEQ ID NO: 6” is phenylalanine. It may not be a residue. That is, for example, “mutation in which the amino acid residue corresponding to the phenylalanine residue at position 246 of SEQ ID NO: 6 is substituted with a serine residue” includes the wild-type acidic phosphate transporter in the amino acid sequence shown in SEQ ID NO: 6.
- amino acid residue corresponding to the phenylalanine residue at position 246 is a phenylalanine residue
- it is not limited to a mutation that substitutes the phenylalanine residue with a serine residue, and is shown in SEQ ID NO: 6 in the wild-type acidic phosphate transporter
- the mutant phosphate transporter gene can be obtained by modifying the wild-type phosphate transporter gene so that the encoded phosphate transporter has a “specific mutation”.
- the wild-type phosphate transporter gene may be a host-derived gene into which the mutant phosphate transporter gene is introduced, or a heterologous gene.
- Modification of DNA can be performed by a known method. Specifically, for example, as a site-specific mutation method for introducing a target mutation into a target site of DNA, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, H. A. Eds. , Stockton press (1989); Carter, P., Meth.
- the mutant phosphate transporter gene can also be obtained by chemical synthesis.
- the coryneform bacterium can retain the mutant phosphate transporter gene.
- the method for introducing the mutant phosphate transporter gene into the coryneform bacterium is not particularly limited, and a conventionally known method can be used.
- the mutant phosphate transporter gene can be introduced into a coryneform bacterium in the same manner as the above-described method for increasing the copy number of a gene.
- the wild-type phosphate transporter gene of the coryneform bacterium may be modified by natural mutation or mutagen treatment so that the encoded phosphate transporter has a “specific mutation”.
- the bacterium of the present invention may or may not have a wild type phosphate transporter gene.
- the bacterium of the present invention may have one or more copies of the mutant phosphate transporter gene.
- the method of transformation is not particularly limited, and a conventionally known method can be used.
- recipient cells are treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162) and methods for introducing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E .., 1997. Gene 1: 153-167) can be used.
- DNA-receptive cells such as those known for Bacillus subtilis, actinomycetes, and yeast, can be made into protoplasts or spheroplasts that readily incorporate recombinant DNA into recombinant DNA.
- Introduction method (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, M. J., Ward, J. M. and Hopwood, O. A. 1978. Nature 274: 398-400; Hinnen, A., Hicks, J. B. and Fink, G. R. 1978. Proc. Natl.Acad. Sci. USA 75: 1929-1933) can also be applied.
- the electric pulse method Japanese Patent Laid-Open No. 2-207791 reported for coryneform bacteria can be used.
- the increase in protein activity can be confirmed by measuring the activity of the protein.
- the phosphate transporter activity can be measured, for example, by measuring the incorporation of inorganic phosphate by a known method (R. M. Harris et al., Journal of Bacteriology, Sept. 2001, p5008-5014).
- the increase in protein activity can also be confirmed by confirming that the expression of the gene encoding the protein has increased.
- An increase in gene expression can be confirmed by confirming that the transcription amount of the gene has increased, or by confirming that the amount of protein expressed from the gene has increased.
- the transcription amount of the gene has increased by comparing the amount of mRNA transcribed from the gene with an unmodified strain such as a wild strain or a parent strain.
- Methods for assessing the amount of mRNA include Northern hybridization, RT-PCR, etc. (Sambrook, J., et al., Molecular Cloning A Laboratory Manual / Third Edition, Cold spring Harbor Laboratory Press, Cold spring Harbor (USA ), 2001).
- the amount of mRNA may be increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more, compared to the unmodified strain.
- the amount of protein can be increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more as compared to the unmodified strain.
- the above-described techniques for increasing the activity of a protein can enhance the activity of an arbitrary protein, such as an L-amino acid biosynthetic enzyme, and can detect an arbitrary gene, such as an arbitrary protein. It can be used to enhance the expression of the encoding gene.
- an arbitrary protein such as an L-amino acid biosynthetic enzyme
- Protein activity decreases means that the activity per cell of the protein is decreased compared to wild-type strains and parental unmodified strains, and the activity is completely lost. including. Specifically, “the activity of the protein is decreased” means that the number of molecules per cell of the protein is decreased and / or the function per molecule of the protein compared to the unmodified strain. Means that it is decreasing. In other words, “activity” in the case of “decrease in protein activity” means not only the catalytic activity of the protein but also the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. May be. Note that “the number of molecules per cell of the protein is decreased” includes a case where the protein does not exist at all.
- the function per molecule of the protein is reduced includes the case where the function per molecule of the protein is completely lost.
- the activity of the protein is not particularly limited as long as it is lower than that of the non-modified strain. For example, it is 50% or less, 20% or less, 10% or less, 5% or less, or 0, compared to the non-modified strain. %.
- the modification that reduces the activity of the protein is achieved, for example, by reducing the expression of a gene encoding the protein.
- Gene expression decreases includes the case where the gene is not expressed at all.
- the expression of the gene is reduced is also referred to as “the expression of the gene is weakened”. Gene expression may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to an unmodified strain.
- the decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof.
- Reduction of gene expression can be achieved, for example, by modifying an expression regulatory sequence such as a gene promoter or Shine-Dalgarno (SD) sequence.
- the expression control sequence is preferably modified by 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. Further, part or all of the expression regulatory sequence may be deleted.
- reduction of gene expression can be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (such as inducers and inhibitors) involved in transcription and translation control, proteins (such as transcription factors), nucleic acids (such as siRNA), and the like.
- the modification that decreases the activity of the protein can be achieved, for example, by destroying a gene encoding the protein.
- Gene disruption can be achieved, for example, by deleting part or all of the coding region of the gene on the chromosome.
- the entire gene including the sequences before and after the gene on the chromosome may be deleted.
- the region to be deleted may be any region such as an N-terminal region, an internal region, or a C-terminal region as long as a decrease in protein activity can be achieved.
- the longer region to be deleted can surely inactivate the gene.
- it is preferable that the reading frames of the sequences before and after the region to be deleted do not match.
- gene disruption is, for example, introducing an amino acid substitution (missense mutation) into a coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting 1 to 2 bases. It can also be achieved by introducing a frameshift mutation (Journal of Biological Chemistry 272: 8611-8617 (1997) Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833 -20839 (1991)).
- gene disruption can be achieved, for example, by inserting another sequence into the coding region of the gene on the chromosome.
- the insertion site may be any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated.
- Other sequences are not particularly limited as long as they reduce or eliminate the activity of the encoded protein, and examples include marker genes such as antibiotic resistance genes and genes useful for L-amino acid production.
- Modifying a gene on a chromosome as described above includes, for example, deleting a partial sequence of the gene and preparing a deleted gene modified so as not to produce a normally functioning protein.
- the host is transformed with the recombinant DNA containing, and the homologous recombination is caused between the deletion type gene and the wild type gene on the chromosome, thereby replacing the wild type gene on the chromosome with the deletion type gene. Can be achieved.
- the recombinant DNA can be easily manipulated by including a marker gene in accordance with a trait such as auxotrophy of the host.
- the modification that reduces the activity of the protein may be performed by, for example, a mutation treatment.
- Mutation treatment includes X-ray irradiation or ultraviolet irradiation, or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), methylmethanesulfonate (MMS), etc.
- MNNG N-methyl-N′-nitro-N-nitrosoguanidine
- EMS ethyl methanesulfonate
- MMS methylmethanesulfonate
- the decrease in the activity of the protein can be confirmed by measuring the activity of the protein.
- the decrease in gene expression can be confirmed by confirming that the transcription amount of the gene has decreased, or confirming that the amount of protein expressed from the gene has decreased.
- the amount of transcription of the gene has been reduced by comparing the amount of mRNA transcribed from the same gene with that of the unmodified strain.
- methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, and the like (Molecular cloning (Cold spring spring Laboratory Laboratory, Cold spring Harbor (USA), 2001)).
- the amount of mRNA may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the unmodified strain.
- the amount of protein may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the unmodified strain.
- the gene has been destroyed by determining part or all of the nucleotide sequence, restriction enzyme map, full length, etc. of the gene according to the means used for the destruction.
- the above-described method for reducing the activity of a protein involves reducing the activity of any protein, for example, an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of the target L-amino acid to produce a compound other than the target L-amino acid. , And can be used to reduce the expression of any gene, for example, a gene encoding any of these proteins.
- the method of the present invention comprises a method for producing an L-amino acid comprising culturing the bacterium of the present invention in a medium and collecting L-amino acid from the medium. is there.
- the medium to be used is not particularly limited as long as the bacterium of the present invention can grow and the target L-amino acid is produced.
- a normal medium used for culturing bacteria such as coryneform bacteria can be used.
- a medium containing a carbon source, a nitrogen source, a phosphate source, a sulfur source, and other components selected from various organic components and inorganic components as necessary can be used.
- the type and concentration of the medium component may be appropriately set according to various conditions such as the type of bacteria used and the type of amino acid to be produced.
- the carbon source examples include glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, waste molasses, starch hydrolyzate, biomass hydrolyzate and other sugars, acetic acid, fumaric acid, citric acid, Examples include organic acids such as succinic acid, alcohols such as glycerol, crude glycerol, and ethanol, and fatty acids.
- the carbon source one type of carbon source may be used, or two or more types of carbon sources may be used in combination.
- the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, soybean protein degradation product, ammonia, and urea. Ammonia gas or ammonia water used for pH adjustment may be used as a nitrogen source. As the nitrogen source, one kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be used in combination.
- the phosphoric acid source examples include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphoric acid.
- phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate
- phosphate polymers such as pyrophosphoric acid.
- the phosphoric acid source one type of phosphoric acid source may be used, or two or more types of phosphoric acid sources may be used in combination.
- the sulfur source include inorganic sulfur compounds such as sulfate, thiosulfate, and sulfite, and sulfur-containing amino acids such as cysteine, cystine, and glutathione.
- the sulfur source one kind of sulfur source may be used, or two or more kinds of sulfur sources may be used in combination.
- organic and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamin B1, vitamin B2, vitamin B6 and nicotine Examples include vitamins such as acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components such as peptone, casamino acid, yeast extract, and soybean protein degradation products containing these.
- inorganic salts such as sodium chloride and potassium chloride
- trace metals such as iron, manganese, magnesium and calcium
- vitamin B1, vitamin B2, vitamin B6 and nicotine include vitamins such as acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components such as peptone, casamino acid, yeast extract, and soybean protein degradation products containing these.
- vitamins such as acid, nicotinamide, and vitamin B12
- amino acids amino acids
- nucleic acids amino acids
- organic components such as peptone, casamino acid, yeast extract, and soybean
- L-lysine producing bacteria often have an enhanced L-lysine biosynthetic pathway and weakened L-lysine resolution. Therefore, when culturing such L-lysine-producing bacteria, for example, one or more amino acids selected from L-threonine, L-homoserine, L-isoleucine, and L-methionine are supplemented to the medium. Is preferred.
- L-glutamic acid when L-glutamic acid is produced by coryneform bacteria, it is preferable to limit the amount of biotin in the medium, or to add a surfactant or penicillin to the medium.
- Culture conditions are not particularly limited as long as the bacterium of the present invention can grow and the target L-amino acid is produced.
- the culture can be performed, for example, under normal conditions used for culture of bacteria such as coryneform bacteria.
- the culture conditions may be appropriately set according to various conditions such as the type of bacteria used and the type of amino acid to be produced.
- Cultivation can be performed aerobically using a liquid medium.
- the culture can be performed by aeration culture or shaking culture.
- the culture temperature may be, for example, 20 to 40 ° C, preferably 25 to 37 ° C.
- the pH of the medium may be adjusted to 5 to 8, for example.
- an inorganic or organic acidic or alkaline substance, ammonia gas, or the like can be used.
- the culture period may be, for example, 15 hours to 90 hours.
- the culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof.
- cultivation may be performed by dividing into seed culture and main culture. In that case, the culture conditions of the seed culture and the main culture may or may not be the same.
- both seed culture and main culture may be performed by batch culture.
- seed culture may be performed by batch culture
- main culture may be performed by fed-batch culture or continuous culture.
- L-glutamic acid when producing L-glutamic acid, it is also possible to carry out the culture while precipitating L-glutamic acid in the medium using a liquid medium adjusted to conditions under which L-glutamic acid is precipitated.
- the conditions for precipitation of L-glutamic acid include, for example, pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, and particularly preferably pH 4.0. (European Patent Application Publication No. 1078989).
- a method of fermenting basic amino acid using bicarbonate ion and / or carbonate ion as a main counter ion of basic amino acid may be used.
- basic amino acids can be produced while reducing the amount of sulfate ions and / or chloride ions that have been conventionally used as counter ions for basic amino acids.
- L-amino acids from the fermentation broth is usually performed by ion exchange resin method (Nagai, H. et al., Separation Science and Technology, 39 (16), 3691-3710), precipitation method, membrane separation method 9-164323, Japanese Patent Laid-Open No. 9-173792), a crystallization method (WO2008 / 078448, WO2008 / 078646), and other known methods can be combined.
- ion exchange resin method Naagai, H. et al., Separation Science and Technology, 39 (16), 3691-3710
- precipitation method membrane separation method 9-164323
- Japanese Patent Laid-Open No. 9-173792 Japanese Patent Laid-Open No. 9-173792
- a crystallization method WO2008 / 078448, WO2008 / 078646
- the recovered L-amino acid may be a free form, a salt thereof, or a mixture thereof.
- the salt include sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and potassium salt.
- ammonium L-glutamate in the fermentation broth is crystallized by adding an acid, and equimolar sodium hydroxide is added to the crystals to obtain sodium L-glutamate (MSG).
- MSG sodium L-glutamate
- you may decolorize by adding activated carbon before and after the crystallization see Industrial crystallization of sodium glutamate, Journal of the Seawater Society of Japan, Vol. 56, No. 5, Tetsuya Kawakita).
- L-amino acid is precipitated in the medium, it can be recovered by centrifugation or filtration.
- the L-amino acid precipitated in the medium may be isolated together after crystallization of the L-amino acid dissolved in the medium.
- the recovered L-amino acid may contain bacterial cells, medium components, moisture, and bacterial metabolic byproducts in addition to the L-amino acid.
- the purity of the recovered L-amino acid may be, for example, 50% or more, preferably 85% or more, particularly preferably 95% or more (JP1214636B, USP5,431,933, USP4,956,471, USP4,777,051, USP4,946,654, USP5,840,358, USP6,238,714, US2005 / 0025878)).
- L-amino acid is L-glutamic acid
- sodium L-glutamate crystals can be used as an umami seasoning.
- the sodium L-glutamate crystals may be used as a seasoning by mixing with nucleic acids such as sodium guanylate and sodium inosinate having an umami taste.
- One aspect of the method of the present invention is a method for producing an L-amino acid comprising culturing a coryneform bacterium having L-amino acid-producing ability in a medium and collecting the L-amino acid from the medium. And the bacterium has a mutant pitA gene encoding a phosphate transporter having a mutation in which an amino acid residue corresponding to the phenylalanine residue at position 246 of SEQ ID NO: 6 is substituted with an amino acid residue other than phenylalanine. It is the method characterized by having.
- the above description of the bacterium of the present invention and the method of the present invention can be applied mutatis mutandis.
- the amino acid residue corresponding to the phenylalanine residue at position 246 of SEQ ID NO: 6 is preferably substituted with a serine residue.
- the coryneform bacterium is preferably Corynebacterium glutamicum.
- the produced L-amino acid may be any amino acid, but is preferably L-glutamic acid.
- Example 1 Glu production culture using a pitA-enhanced strain
- Glu production was performed using a Glu-producing strain of C. glutamicum with enhanced expression of the pitA gene, and the enhanced expression of the pitA gene contributed to Glu production. The effect was evaluated.
- the strains used are as follows. C. glutamicum 2256 ⁇ ldhA ⁇ sucA yggB * / pVK9 C. glutamicum 2256 ⁇ ldhA ⁇ sucA yggB * / pVK9-Plac-pitA
- a DNA fragment for ldhA gene deletion was amplified using 2256 strain chromosomal DNA as a template and using a pair of primers 1 and 2 and a pair of primers 3 and 4 respectively.
- PCR was performed using primers 5 and 6 using a mixture of equal amounts of the two amplified fragments as a template to obtain a DNA fragment to which the two fragments were bound.
- the obtained DNA fragment was treated with SalI and introduced into the SalI site of pBS4S (WO2005 / 113745) to construct an ldhA deletion plasmid.
- the ldhA gene was deleted by inserting this ldhA deletion plasmid into the chromosome of 2256 strain and then dropping it.
- a DNA fragment for sucA gene deletion was amplified using 2256 strain chromosomal DNA as a template and using a pair of primers 7 and 8 and a pair of primers 9 and 10, respectively.
- PCR was performed using primers 11 and 12 using a mixture of equal amounts of the two amplified fragments as a template to obtain a DNA fragment to which the two fragments were bound.
- the obtained DNA fragment was treated with BamHI and introduced into the BamHI site of pBS3 (WO2006 / 070944) to construct a plasmid for sucA deletion.
- the sucA gene was deleted by inserting this sucA deletion plasmid into the chromosome of the 2256 ⁇ ldhA strain and then dropping it.
- a Glu-producing strain containing the yggB gene with an IS mutation (V419 :: IS) was obtained.
- the nucleotide sequence of the yggB gene containing the IS mutation (V419 :: IS) and the amino acid sequence of the YggB protein encoded by the same gene are shown in SEQ ID NOs: 23 and 24, respectively.
- the obtained Glu production strain was designated as 2256 ⁇ ldhA ⁇ sucA yggB * strain.
- a pitA expression plasmid (pVK9-Plac-pitA) was constructed by the following method. First, the pitA gene fragment was amplified using primers 2 3 and 14 using 2256 strain chromosomal DNA as a template. Next, the amplified fragment was ligated to pVK9 plasmid (US2006-0141588) treated with bamHI and pstI using in-fusion (TaKaRa INC.) To construct a pitA expression plasmid. The constructed pitA expression plasmid was designated as pVK9-Plac-pitA.
- the constructed pVK9-Plac-pitA and pVK9 as a vector control were respectively introduced into the Glu-producing bacterium 2256 ⁇ ldhA ⁇ sucA yggB * strain to construct 2256 ⁇ ldhA ⁇ sucA yggB * / pVK9-Plac-pitA strain and 2256 ⁇ ldhA ⁇ sucA yggB * / pVK9 strain.
- a medium having the above composition adjusted to pH 8.0 with KOH was prepared, sterilized by an autoclave (115 ° C., 15 min) and subjected to culture.
- Example 2 Glu production culture using pitA mutant
- Glu production was performed using a Glu producing strain of C. glutamicum in which mutation was introduced into the pitA gene, and the effect of mutation of pitA gene on Glu production. was evaluated.
- strains used are as follows. C. glutamicum 2256 ⁇ ldhA ⁇ sucA yggB * C. glutamicum 2256 ⁇ ldhA ⁇ sucA yggB * pitAmut
- the plasmid pBS4S-pitAmut for pitA mutation introduction was constructed by the following method. Primers 15 and 16 using the chromosomal DNA of Glu-producing strain B3 containing a mutation (Phe246Ser ttc ⁇ tcc) in which the phenylalanine residue at position 246 of the PitA protein is replaced by a serine residue in the coding region of the pitA gene PCR was performed to amplify the pitA gene fragment having the above mutation. Next, the amplified fragment was ligated to BamHI and PstI-treated pBS4S plasmid using in-fusion (TaKaRa INC.) To construct a pitA mutation-introducing plasmid. The constructed plasmid for introducing pitA mutation was designated as pBS4S-pitAmut.
- the constructed pBS4S-pitAmut was inserted into the chromosome of the Glu-producing bacterium 2256 ⁇ ldhA ⁇ sucA yggB * strain and then dropped to construct a 2256 ⁇ ldhA ⁇ sucA yggB * pitAmut strain in which a mutation was introduced into the pitA gene.
- the pitA mutant strain 2256 ⁇ ldhA ⁇ sucA yggB * pitAmut strain was constructed using a mutation-introducing plasmid constructed using the chromosomal DNA of the Glu acid-producing bacterium B3 strain as a template.
- PrimeSTAR registered trademark
- Mutagenesis Basal Kit manufactured by Takara Bio Inc. It can also be constructed using the prepared plasmid for mutagenesis.
- PCR is performed using primers 15 and 16 using a chromosomal DNA of a wild strain such as C. glutamicum 2256 strain (ATCC 13869) as a template to amplify a pitA gene fragment having no mutation.
- the amplified fragment is ligated to the pBS4S plasmid treated with BamHI and PstI using in-fusion (TaKaRa INC.)
- a plasmid containing the wild type pitA gene sequence is constructed using this plasmid as a template.
- PCR is performed using an appropriate primer that changes T at position 737 of the pitA gene to C according to the instructions for the PrimeSTAR (registered trademark) Mutagenesis Basal Kit. Plasmid pBS4S-pitAmut can be constructed. Using this, the same pitA mutant can be constructed.
- a medium having the above composition adjusted to pH 8.0 with KOH was prepared, sterilized by an autoclave (115 ° C., 15 min) and subjected to culture.
- L-amino acid producing ability of coryneform bacteria can be improved, and L-amino acids can be produced efficiently.
- SEQ ID NO: 1 Base sequence of pitA gene of E. coli MG1655
- SEQ ID NO: 2 Amino acid sequence of PitA protein of E. coli MG1655
- SEQ ID NO: 3 Base sequence of pitA gene of Pantoea ananatis LMG20103
- SEQ ID NO: 4 PitA of Pantoea ananatis LMG20103
- Amino acid sequence of the protein SEQ ID NO: 5 Nucleotide sequence of the pitA gene of Corynebacterium glutamicum 2256 (ATCC 13869)
- SEQ ID NO: 6 Amino acid sequence of the PitA protein of Corynebacterium glutamicum 2256 (ATCC 13869)
- SEQ ID NO: 7 to 20 Primer
- SEQ ID NO: 21 Corynebacterium
- SEQ ID NO: 22 The amino acid sequence of the YggB protein of
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Abstract
Description
[1]
L-アミノ酸生産能を有するコリネ型細菌を培地で培養すること、および該培地よりL-アミノ酸を採取すること、を含むL-アミノ酸の製造法であって、
前記細菌が、リン酸トランスポーターの活性が増大するように改変されていることを特徴とする、方法。
[2]
リン酸トランスポーターをコードする遺伝子の発現を上昇させることにより、リン酸トランスポーターの活性が増大した、前記方法。
[3]
前記遺伝子がpitA遺伝子である、前記方法。
[4]
前記pitA遺伝子が、下記(a)又は(b)に記載のDNAである、前記方法:
(a)配列番号5または25に示す塩基配列を有するDNA、
(b)配列番号5または25に示す塩基配列の相補配列又は同相補配列から調製され得るプローブとストリンジェントな条件下でハイブリダイズし、かつ、リン酸トランスポーター活性を有するタンパク質をコードするDNA。
[5]
前記pitA遺伝子が、下記(A)又は(B)に記載のタンパク質をコードするDNAである、前記方法:
(A)配列番号6または26に示すアミノ酸配列を有するタンパク質、
(B)配列番号6または26に示すアミノ酸配列において、1若しくは数個のアミノ酸残基の置換、欠失、挿入、または付加を含むアミノ酸配列を有し、かつ、リン酸トランスポーター活性を有するタンパク質。
[6]
前記遺伝子の発現が、該遺伝子のコピー数を高めること、及び/又は該遺伝子の発現調節配列を改変することによって上昇した、前記方法。
[7]
配列番号6の246位のフェニルアラニン残基に相当するアミノ酸残基がフェニルアラニン以外のアミノ酸残基に置換される変異を有するリン酸トランスポーターをコードする変異型pitA遺伝子を前記細菌に保持させることにより、リン酸トランスポーターの活性が増大した、前記方法。
[8]
配列番号6の246位のフェニルアラニン残基に相当するアミノ酸残基が、セリン残基に置換されたことを特徴とする、前記方法。
[9]
L-アミノ酸生産能を有するコリネ型細菌を培地で培養すること、および該培地よりL-アミノ酸を採取すること、を含むL-アミノ酸の製造法であって、
前記細菌が、配列番号6の246位のフェニルアラニン残基に相当するアミノ酸残基がフェニルアラニン以外のアミノ酸残基に置換される変異を有するリン酸トランスポーターをコードする変異型pitA遺伝子を保持していることを特徴とする、方法。
[10]
配列番号6の246位のフェニルアラニン残基に相当するアミノ酸残基が、セリン残基に置換されたことを特徴とする、前記方法。
[11]
前記細菌が、コリネバクテリウム属細菌である、前記方法。
[12]
前記コリネ型細菌が、コリネバクテリウム・グルタミカムである、前記方法。
[13]
前記L-アミノ酸が、L-グルタミン酸である、前記方法。
[14]
前記L-グルタミン酸が、L-グルタミン酸アンモニウムまたはL-グルタミン酸ナトリウムである、前記方法。
[15]
配列番号6の246位のフェニルアラニン残基に相当するアミノ酸残基がセリン残基に置換される変異を有するリン酸トランスポーターをコードするDNA。
[16]
配列番号6の246位のフェニルアラニン残基に相当するアミノ酸残基がセリン残基に置換される変異を有するリン酸トランスポーターをコードする変異型pitA遺伝子を保持しているコリネ型細菌。
本発明の細菌は、リン酸トランスポーターの活性が増大するように改変された、L-アミノ酸生産能を有するコリネ型細菌である。
本発明において、「L-アミノ酸生産能を有する細菌」とは、培地で培養したときに、目的とするL-アミノ酸を生成し、回収できる程度に培地中または菌体内に蓄積する能力を有する細菌をいう。L-アミノ酸生産能を有する細菌は、非改変株よりも多い量の目的とするL-アミノ酸を培地に蓄積することができる細菌であってよい。非改変株としては、野生株や親株が挙げられる。また、L-アミノ酸生産能を有する細菌は、好ましくは0.5g/L以上、より好ましくは1.0g/L以上の量の目的とするL-アミノ酸を培地に蓄積することができる細菌であってもよい。
コリネバクテリウム・アセトアシドフィラム(Corynebacterium acetoacidophilum)
コリネバクテリウム・アセトグルタミカム(Corynebacterium acetoglutamicum)
コリネバクテリウム・アルカノリティカム(Corynebacterium alkanolyticum)
コリネバクテリウム・カルナエ(Corynebacterium callunae)
コリネバクテリウム・グルタミカム(Corynebacterium glutamicum)
コリネバクテリウム・リリウム(Corynebacterium lilium)
コリネバクテリウム・メラセコーラ(Corynebacterium melassecola)
コリネバクテリウム・サーモアミノゲネス(コリネバクテリウム・エフィシエンス)(Corynebacterium thermoaminogenes (Corynebacterium efficiens))
コリネバクテリウム・ハーキュリス(Corynebacterium herculis)
ブレビバクテリウム・ディバリカタム(コリネバクテリウム・グルタミカム)(Brevibacterium divaricatum (Corynebacterium glutamicum))
ブレビバクテリウム・フラバム(コリネバクテリウム・グルタミカム)(Brevibacterium flavum (Corynebacterium glutamicum))
ブレビバクテリウム・イマリオフィラム(Brevibacterium immariophilum)
ブレビバクテリウム・ラクトファーメンタム(コリネバクテリウム・グルタミカム)(Brevibacterium lactofermentum (Corynebacterium glutamicum))
ブレビバクテリウム・ロゼウム(Brevibacterium roseum)
ブレビバクテリウム・サッカロリティカム(Brevibacterium saccharolyticum)
ブレビバクテリウム・チオゲニタリス(Brevibacterium thiogenitalis)
コリネバクテリウム・アンモニアゲネス(コリネバクテリウム・スタティオニス)(Corynebacterium ammoniagenes (Corynebacterium stationis))
ブレビバクテリウム・アルバム(Brevibacterium album)
ブレビバクテリウム・セリナム(Brevibacterium cerinum)
ミクロバクテリウム・アンモニアフィラム(Microbacterium ammoniaphilum)
Corynebacterium acetoacidophilum ATCC 13870
Corynebacterium acetoglutamicum ATCC 15806
Corynebacterium alkanolyticum ATCC 21511
Corynebacterium callunae ATCC 15991
Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060,ATCC 13869,FERM BP-734
Corynebacterium lilium ATCC 15990
Corynebacterium melassecola ATCC 17965
Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC 13868
Corynebacterium glutamicum (Brevibacterium divaricatum) ATCC 14020
Corynebacterium glutamicum (Brevibacterium flavum) ATCC 13826, ATCC 14067, AJ12418(FERM BP-2205)
Brevibacterium immariophilum ATCC 14068
Corynebacterium glutamicum (Brevibacterium lactofermentum) ATCC 13869
Brevibacterium roseum ATCC 13825
Brevibacterium saccharolyticum ATCC 14066
Brevibacterium thiogenitalis ATCC 19240
Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872
Brevibacterium album ATCC 15111
Brevibacterium cerinum ATCC 15112
Microbacterium ammoniaphilum ATCC 15354
L-グルタミン酸生産菌又はそれを誘導するための親株としては、L-グルタミン酸生合成系酵素から選択される1またはそれ以上の酵素の活性が増強された株が挙げられる。そのような酵素としては、特に制限されないが、グルタメートデヒドロゲナーゼ(gdhA)、グルタミンシンテターゼ(glnA)、グルタメートシンテターゼ(gltBD)、イソシトレートデヒドロゲナーゼ(icdA)、アコニテートヒドラターゼ(acnA, acnB)、クエン酸シンターゼ(gltA)、メチルクエン酸シンターゼ(prpC)、ホスホエノールピルベートカルボシラーゼ(ppc)、ピルベートデヒドロゲナーゼ(aceEF, lpdA)、ピルベートキナーゼ(pykA, pykF)、ホスホエノールピルベートシンターゼ(ppsA)、エノラーゼ(eno)、ホスホグリセロムターゼ(pgmA, pgmI)、ホスホグリセレートキナーゼ(pgk)、グリセルアルデヒド-3-フォスフェートデヒドロゲナーゼ(gapA)、トリオースフォスフェートイソメラーゼ(tpiA)、フルクトースビスフォスフェートアルドラーゼ(fbp)、ホスホフルクトキナーゼ(pfkA, pfkB)、グルコースフォスフェートイソメラーゼ(pgi)、6-ホスホグルコン酸デヒドラターゼ(edd)、2-ケト-3-デオキシ-6-ホスホグルコン酸アルドラーゼ(eda)、トランスヒドロゲナーゼが挙げられる。なお、カッコ内は、その酵素をコードする遺伝子の略記号である(以下の記載においても同様)。これらの酵素の中では、例えば、グルタメートデヒドロゲナーゼ、クエン酸シンターゼ、ホスホエノールピルベートカルボキシラーゼ、及びメチルクエン酸シンターゼから選択される1またはそれ以上の酵素の活性を増強するのが好ましい。
Corynebacterium glutamicum (Brevibacterium lactofermentum) L30-2株 (特開2006-340603号明細書)
Corynebacterium glutamicum (Brevibacterium lactofermentum) ΔS株 (国際公開95/34672号パンフレット)
Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ12821 (FERM BP-4172;フランス特許公報9401748号明細書参照)
Corynebacterium glutamicum (Brevibacterium flavum) AJ12822 (FERM BP-4173;フランス特許公報9401748号明細書)
Corynebacterium glutamicum AJ12823 (FERM BP-4174;フランス特許公報9401748号明細書)
Corynebacterium glutamicum L30-2株 (特開2006-340603号)
Corynebacterium glutamicum (Brevibacterium flavum) AJ3949 (FERM BP-2632;特開昭50-113209参照)
Corynebacterium glutamicum AJ11628 (FERM P-5736;特開昭57-065198参照)
Corynebacterium glutamicum (Brevibacterium flavum) AJ11355 (FERM P-5007;特開昭56-1889号公報参照)
Corynebacterium glutamicum AJ11368 (FERM P-5020;特開昭56-1889号公報参照)
Corynebacterium glutamicum (Brevibacterium flavum) AJ11217 (FERM P-4318;特開昭57-2689号公報参照)
Corynebacterium glutamicum AJ11218 (FERM P-4319;特開昭57-2689号公報参照)
Corynebacterium glutamicum (Brevibacterium flavum) AJ11564 (FERM P-5472;特開昭56-140895公報参照)
Corynebacterium glutamicum (Brevibacterium flavum) AJ11439 (FERM P-5136;特開昭56-35981号公報参照)
Corynebacterium glutamicum H7684 (FERM BP-3004;特開平04-88994号公報参照)
Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ11426 (FERM P-5123;特開平56-048890号公報参照)
Corynebacterium glutamicum AJ11440 (FERM P-5137;特開平56-048890号公報参照)
Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ11796 (FERM P-6402;特開平58-158192号公報参照)
C末端側変異は、配列番号22のアミノ酸番号419~533の配列をコードする領域の塩基配列の一部に導入された変異である。C末端側変異は、上記領域の塩基配列中の少なくとも一部に変異が導入される限り特に制限されないが、インサーションシーケンス(以下、「IS」ともいう)やトランスポゾンが挿入されたものが好ましい。C末端側変異は、アミノ酸置換を伴うもの(ミスセンス変異)や、上記IS等の挿入によってフレームシフト変異が導入されたもの、ナンセンス変異が導入されたものの何れでもよい。C末端側変異を有する変異型yggB遺伝子として、具体的には、例えば、配列番号22の419位のバリン残基をコードする箇所にISが挿入され、野生型YggBタンパク質(配列番号22)よりも短い全長423アミノ酸残基の変異型YggBタンパク質をコードするyggB遺伝子が挙げられる(特開2007-222163)。この変異型yggB遺伝子(V419::IS)の塩基配列、及び同遺伝子がコードする変異型YggBタンパク質のアミノ酸配列を、それぞれ配列番号23および24に示す。また、C末端側変異として、配列番号22のアミノ酸番号419~533の領域内に存在するプロリンを他のアミノ酸に置換する変異も挙げられる。
yggB遺伝子がコードするYggBタンパク質は、5個の膜貫通領域を有していると推測されている。配列番号22の野生型YggBタンパク質のアミノ酸配列において、膜貫通領域はそれぞれ、アミノ酸番号1~23(第1膜貫通領域)、25~47(第2膜貫通領域)、62~84(第3膜貫通領域)、86~108(第4膜貫通領域)、110~132(第5膜貫通領域)の領域に相当する。yggB遺伝子は、これら膜貫通領域をコードする領域内に変異を有していてよい。膜貫通領域の変異は、1若しくは数個のアミノ酸の置換、欠失、付加、挿入又は逆位を含む変異であって、フレームシフト変異およびナンセンス変異を伴わないものが望ましい。膜貫通領域の変異としては、配列番号22に示されるアミノ酸配列において、14位のロイシン残基と15位のトリプトファン残基間に1又は数個のアミノ酸を挿入する変異、100位のアラニン残基を他のアミノ酸残基へ置換する変異、111位のアラニン残基を他のアミノ酸残基へ置換する変異などが挙げられる。尚、上記「1又は数個」とは、具体的には、好ましくは1~20個、より好ましくは1~10個、さらに好ましくは1~5個、特に好ましくは1~3個を意味する。
L-グルタミン生産能を付与又は増強するための方法としては、例えば、L-グルタミン生合成系酵素から選択される1またはそれ以上の酵素の活性が増大するように細菌を改変する方法が挙げられる。そのような酵素としては、特に制限されないが、グルタミン酸デヒドロゲナーゼ(gdhA)やグルタミンシンセターゼ(glnA)が挙げられる。
Corynebacterium glutamicum (Brevibacterium flavum) AJ11573 (FERM P-5492、特開昭56-161495)
Corynebacterium glutamicum (Brevibacterium flavum) AJ11576 (FERM BP-10381、特開昭56-161495)
Corynebacterium glutamicum (Brevibacterium flavum) AJ12212 (FERM P-8123、特開昭61-202694)
L-プロリン生産菌又はそれを誘導するための親株としては、L-プロリン生合成系酵素から選択される1またはそれ以上の酵素の活性が増強された株が挙げられる。L-プロリン生合成に関与する酵素としては、グルタミン酸5-キナーゼ、γ‐グルタミル-リン酸レダクターゼ、ピロリン-5-カルボキシレートレダクターゼが挙げられる。酵素活性の増強には、例えば、L-プロリンによるフィードバック阻害が解除されたグルタメートキナーゼをコードするproB遺伝子(ドイツ特許第3127361号)が好適に利用できる。
L-スレオニン生産菌又はそれを誘導するための親株としては、L-スレオニン生合成系酵素から選択される1またはそれ以上の酵素の活性が増強された株が挙げられる。そのような酵素としては、特に制限されないが、アスパルトキナーゼIII(lysC)、アスパラギン酸セミアルデヒドデヒドロゲナーゼ(asd)、アスパルトキナーゼI(thrA)、ホモセリンキナーゼ(homoserine kinase)(thrB)、スレオニンシンターゼ(threonine synthase)(thrC)、アスパラギン酸アミノトランスフェラーゼ(アスパラギン酸トランスアミナーゼ)(aspC)が挙げられる。これらの酵素の中では、アスパルトキナーゼIII、アスパラギン酸セミアルデヒドデヒドロゲナーゼ、アスパルトキナーゼI、ホモセリンキナーゼ、アスパラギン酸アミノトランスフェラーゼ、及びスレオニンシンターゼから選択される1またはそれ以上の酵素の活性を増強するのが好ましい。L-スレオニン生合成系遺伝子は、スレオニン分解が抑制された株に導入してもよい。
L-リジン生産菌又はそれを誘導するための親株としては、L-リジン生合成系酵素から選択される1またはそれ以上の酵素の活性が増強された株が挙げられる。そのような酵素としては、特に制限されないが、ジヒドロジピコリン酸シンターゼ(dihydrodipicolinate synthase)(dapA)、アスパルトキナーゼIII(aspartokinase III)(lysC)、ジヒドロジピコリン酸レダクターゼ(dihydrodipicolinate reductase)(dapB)、ジアミノピメリン酸デカルボキシラーゼ(diaminopimelate decarboxylase)(lysA)、ジアミノピメリン酸デヒドロゲナーゼ(diaminopimelate dehydrogenase)(ddh)(米国特許第6,040,160号)、ホスホエノールピルビン酸カルボキシラーゼ(phosphoenolpyrvate carboxylase)(ppc)、アスパラギン酸セミアルデヒドデヒドロゲナーゼ(aspartate semialdehyde dehydrogenease)(asd)、アスパラギン酸アミノトランスフェラーゼ(aspartate aminotransferase)(アスパラギン酸トランスアミナーゼ(aspartate transaminase))(aspC)、ジアミノピメリン酸エピメラーゼ(diaminopimelate epimerase)(dapF)、テトラヒドロジピコリン酸スクシニラーゼ(tetrahydrodipicolinate succinylase)(dapD)、スクシニルジアミノピメリン酸デアシラーゼ(succinyl-diaminopimelate deacylase)(dapE)、及びアスパルターゼ(aspartase)(aspA)(EP 1253195 A)が挙げられる。これらの酵素の中では、例えば、ジヒドロジピコリン酸レダクターゼ、ジアミノピメリン酸デカルボキシラーゼ、ジアミノピメリン酸デヒドロゲナーゼ、ホスホエノールピルビン酸カルボキシラーゼ、アスパラギン酸アミノトランスフェラーゼ、ジアミノピメリン酸エピメラーゼ、アスパラギン酸セミアルデヒドデヒドロゲナーゼ、テトラヒドロジピコリン酸スクシニラーゼ、及びスクシニルジアミノピメリン酸デアシラーゼから選択される1またはそれ以上の酵素の活性を増強するのが好ましい。また、L-リジン生産菌又はそれを誘導するための親株では、エネルギー効率に関与する遺伝子(cyo)(EP 1170376 A)、ニコチンアミドヌクレオチドトランスヒドロゲナーゼ(nicotinamide nucleotide transhydrogenase)をコードする遺伝子(pntAB)(米国特許第5,830,716号)、ybjE遺伝子(WO2005/073390)、またはこれらの組み合わせの発現レベルが増大していてもよい。アスパルトキナーゼIII(lysC)はL-リジンによるフィードバック阻害を受けるので、同酵素の活性を増強するには、L-リジンによるフィードバック阻害が解除されたアスパルトキナーゼIIIをコードする変異型lysC遺伝子を利用してもよい(米国特許5,932,453号明細書)。また、ジヒドロジピコリン酸合成酵素(dapA)L-リジンによるフィードバック阻害を受けるので、同酵素の活性を増強するには、L-リジンによるフィードバック阻害が解除されたジヒドロジピコリン酸合成酵素をコードする変異型dapA遺伝子を利用してもよい。
L-アルギニン生産菌又はそれを誘導するための親株としては、L-アルギニン生合成系酵素から選択される1またはそれ以上の酵素の活性が増強された株が挙げられる。そのような酵素としては、特に制限されないが、N-アセチルグルタミン酸シンターゼ(argA)、N-アセチルグルタミルフォスフェートレダクターゼ(argC)、オルニチンアセチルトランスフェラーゼ(argJ)、N-アセチルグルタメートキナーゼ(argB)、アセチルオルニチントランスアミナーゼ(argD)、オルニチンカルバモイルトランスフェラーゼ(argF)、アルギノコハク酸シンテターゼ(argG)、アルギノコハク酸リアーゼ(argH)、カルバモイルフォスフェートシンテターゼ(carAB)が挙げられる。N-アセチルグルタミン酸シンターゼ(argA)遺伝子としては、例えば、野生型の15位~19位に相当するアミノ酸残基が置換され、L-アルギニンによるフィードバック阻害が解除された変異型N-アセチルグルタミン酸シンターゼをコードする遺伝子を用いると好適である(欧州出願公開1170361号明細書)。
Corynebacterium glutamicum (Brevibacterium flavum) AJ11169(FERM BP-6892)
Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ12092(FERM BP-6906)
Corynebacterium glutamicum (Brevibacterium flavum) AJ11336(FERM BP-6893)
Corynebacterium glutamicum (Brevibacterium flavum) AJ11345(FERM BP-6894)
Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ12430(FERM BP-2228)
L-シトルリンおよびL-オルニチンは、L-アルギニンと生合成経路が共通している。よって、N-アセチルグルタミン酸シンターゼ(argA)、N-アセチルグルタミルリン酸レダクターゼ(argC)、オルニチンアセチルトランスフェラーゼ(argJ)、N-アセチルグルタミン酸キナーゼ(argB)、アセチルオルニチントランスアミナーゼ(argD)、および/またはアセチルオルニチンデアセチラーゼ(argE)の酵素活性を上昇させることによって、L-シトルリンおよび/またはL-オルニチンの生産能を付与または増強することができる(国際公開2006-35831号パンフレット)。
L-ヒスチジン生産菌又はそれを誘導するための親株としては、L-ヒスチジン生合成系酵素から選択される1またはそれ以上の酵素の活性が増強された株が挙げられる。そのような酵素としては、特に制限されないが、ATPホスホリボシルトランスフェラーゼ(hisG)、ホスホリボシル-AMPサイクロヒドロラーゼ(hisI)、ホスホリボシル-ATPピロホスホヒドロラーゼ(hisI)、ホスホリボシルフォルミミノ-5-アミノイミダゾールカルボキサミドリボタイドイソメラーゼ(hisA)、アミドトランスフェラーゼ(hisH)、ヒスチジノールフォスフェイトアミノトランスフェラーゼ(hisC)、ヒスチジノールフォスファターゼ(hisB)、ヒスチジノールデヒドロゲナーゼ(hisD)が挙げられる。
L-システイン生産菌又はそれを誘導するための親株としては、L-システイン生合成系酵素から選択される1またはそれ以上の酵素の活性が増強された株が挙げられる。そのような酵素としては、特に制限されないが、セリンアセチルトランスフェラーゼや3-ホスホグリセレートデヒドロゲナーゼが挙げられる。セリンアセチルトランスフェラーゼ活性は、例えば、システインによるフィードバック阻害に耐性の変異型セリンアセチルトランスフェラーゼをコードする変異型cysE遺伝子を細菌に導入することにより増強できる。変異型セリンアセチルトランスフェラーゼは、例えば、特開平11-155571や米国特許公開第20050112731に開示されている。また、3-ホスホグリセレートデヒドロゲナーゼ活性は、例えば、セリンによるフィードバック阻害に耐性の変異型3-ホスホグリセレートデヒドロゲナーゼをコードする変異型serA遺伝子を細菌に導入することにより増強できる。変異型3-ホスホグリセレートデヒドロゲナーゼは、例えば、米国特許第6,180,373号に開示されている。
L-メチオニン生産菌又はそれを誘導するための親株としては、L-スレオニン要求株や、ノルロイシンに耐性を有する変異株が挙げられる(特開2000-139471)。また、L-メチオニン生産菌又はそれを誘導するための親株としては、L-メチオニンによるフィードバック阻害に対して耐性をもつ変異型ホモセリントランスサクシニラーゼを保持する株も挙げられる(特開2000-139471、US20090029424)。なお、L-メチオニンはL-システインを中間体として生合成されるため、L-システインの生産能の向上によりL-メチオニンの生産能も向上させることができる(特開2000-139471、US20080311632)。
L-ロイシン生産菌又はそれを誘導するための親株としては、L-ロイシン生合成系酵素から選択される1またはそれ以上の酵素の活性が増強された株が挙げられる。そのような酵素としては、特に制限されないが、leuABCDオペロンの遺伝子にコードされる酵素が挙げられる。また、酵素活性の増強には、例えば、L-ロイシンによるフィードバック阻害が解除されたイソプロピルマレートシンターゼをコードする変異leuA遺伝子(米国特許第6,403,342号)が好適に利用できる。
L-イソロイシン生産能を付与又は増強するための方法としては、例えば、L-イソロイシン生合成系酵素から選択される1またはそれ以上の酵素の活性が増大するように細菌を改変する方法が挙げられる。そのような酵素としては、特に制限されないが、スレオニンデアミナーゼやアセトヒドロキシ酸シンターゼが挙げられる(特開平2-458号, FR 0356739, 及び米国特許第5,998,178号)。
L-バリン生産菌又はそれを誘導するための親株としては、L-バリン生合成系酵素から選択される1またはそれ以上の酵素の活性が増強された株が挙げられる。そのような酵素としては、特に制限されないが、ilvGMEDAオペロンやilvBNCオペロンの遺伝子にコードされる酵素が挙げられる。ilvBNはアセトヒドロキシ酸シンターゼを、ilvCはイソメロリダクターゼ(国際公開00/50624号)を、それぞれコードする。なお、ilvGMEDAオペロンおよびilvBNCオペロンは、L-バリン、L-イソロイシン、および/またはL-ロイシンによる発現抑制(アテニュエーション)を受ける。よって、酵素活性の増強のためには、アテニュエーションに必要な領域を除去または改変し、生成するL-バリンによる発現抑制を解除するのが好ましい。また、ilvA遺伝子がコードするスレオニンデアミナーゼは、L-イソロイシン生合成系の律速段階であるL-スレオニンから2-ケト酪酸への脱アミノ化反応を触媒する酵素である。よって、L-バリン生産のためには、ilvA遺伝子が破壊等され、スレオニンデアミナーゼ活性が減少しているのが好ましい。
L-アラニン生産菌又はそれを誘導するための親株としては、H+-ATPaseを欠失しているコリネ型細菌(Appl Microbiol Biotechnol. 2001 Nov;57(4):534-40)やアスパラギン酸β-デカルボキシラーゼ活性が増強されたコリネ型細菌(特開平07-163383)が挙げられる。
L-トリプトファン生産能、L-フェニルアラニン生産能、および/またはL-チロシン生産能を付与又は増強するための方法としては、例えば、L-トリプトファン、L-フェニルアラニン、および/またはL-チロシンの生合成系酵素から選択される1またはそれ以上の酵素の活性が増大するように細菌を改変する方法が挙げられる。
本発明の細菌は、リン酸トランスポーター活性が増大するように改変されている。本発明の細菌は、L-アミノ酸生産能を有するコリネ型細菌を、リン酸トランスポーター活性が増大するように改変することにより取得できる。また、本発明の細菌は、リン酸トランスポーター活性が増大するようにコリネ型細菌を改変した後に、L-アミノ酸生産能を付与または増強することによっても得ることができる。なお、本発明の細菌は、リン酸トランスポーター活性が増大するように改変されたことにより、L-アミノ酸生産能を獲得したものであってもよい。本発明の細菌を構築するための改変は、任意の順番で行うことができる。
以下に、タンパク質の活性を増大させる手法について説明する。
以下に、タンパク質の活性を低下させる手法について説明する。
本発明の方法は、本発明の細菌を培地で培養すること、および該培地よりL-アミノ酸を採取すること、を含むL-アミノ酸の製造法である。
本実施例では、pitA遺伝子の発現が増強されたC. glutamicumのGlu生産株を用いてGlu生産を行い、pitA遺伝子の発現増強がGlu生産に与える影響について評価した。
C. glutamicum 2256ΔldhAΔsucA yggB*/pVK9
C. glutamicum 2256ΔldhAΔsucA yggB*/pVK9-Plac-pitA
C. glutamicum 2256株(ATCC 13869)を親株に用いて、以下の方法で、モデルGlu生産株として2256ΔldhAΔsucA yggB*株を構築した。使用したプライマーを表1に示す。
2256ΔldhAΔsucA yggB*/pVK9-Plac-pitA株および2256ΔldhAΔsucA yggB*/pVK9株を用いて、Glu生産培養を行った。用いた培地の組成を表2に示す。
培養(前培養および本培養)は、坂口フラスコに培地を20mL張り込み、CaCO3を50g/Lになるように添加して、31.5℃のボックスシェーカーで振とうして行なった。最初に、前培養として、培地1を用いて上記の株のそれぞれを24時間培養した。次いで、得られた前培養液2mLを培地2に植菌し、植菌から2時間後にTween40(終濃度4g/L)を添加してメイン培養を行った。サンプリングは植菌から17時間後に行った。残存糖およびグルタミン酸は、AS-310(旭化成)を用いて定量した。
結果を表3に示す。表3中、「RS」は残存糖量を、「Glu」はグルタミン酸量を示す。本実施例により、C. glutamicumにおいてpitA遺伝子の発現を上昇させることで、C. glutamicumの生育とGlu生産性が向上することが明らかとなった。よって、pitA遺伝子がコードするリン酸トランスポーターの活性を増大させることは、グルタミン酸等のアミノ酸生産に有効であると考察された。
本実施例では、pitA遺伝子に変異を導入したC. glutamicumのGlu生産株を用いてGlu生産を行い、pitA遺伝子の変異がGlu生産に与える影響について評価した。
C. glutamicum 2256ΔldhAΔsucA yggB*
C. glutamicum 2256ΔldhAΔsucA yggB* pitAmut
実施例1で構築したモデルGlu生産株であるC. glutamicum 2256ΔldhAΔsucA yggB*を親株として、pitA遺伝子に変異が導入された2256ΔldhAΔsucA yggB* pitAmut株を構築した。使用したプライマーを表4に示す。
2256ΔldhAΔsucA yggB*株および2256ΔldhAΔsucA yggB* pitAmut株を用いて、Glu生産培養を行った。用いた培地の組成を表5に示す。
培養(前培養および本培養)は、坂口フラスコに培地を20mL張り込み、CaCO3を50g/Lになるように添加して、31.5℃のボックスシェーカーで振とうして行なった。最初に、前培養として、培地3を用いて上記の株のそれぞれを24時間培養した。次いで、得られた前培養液2mLを培地3に植菌し、植菌から2時間後にTween40(終濃度4g/L)を添加してメイン培養を行った。サンプリングは植菌から21.5時間後に行った。残存糖およびグルタミン酸は、AS-310(旭化成)を用いて定量した。
結果を表6に示す。表6中、「RS」は残存糖量を、「Glu」はグルタミン酸量を示す。本実施例により、C. glutamicumにおいてpitA遺伝子に変異(Phe246Ser)を導入することで、C. glutamicumの生育とGlu生産性が向上することが明らかとなった。よって、本pitA変異(Phe246Ser)はグルタミン酸等のアミノ酸生産に有効であると考察された。
配列番号1:E. coli MG1655のpitA遺伝子の塩基配列
配列番号2:E. coli MG1655のPitAタンパク質のアミノ酸配列
配列番号3:Pantoea ananatis LMG20103のpitA遺伝子の塩基配列
配列番号4:Pantoea ananatis LMG20103のPitAタンパク質のアミノ酸配列
配列番号5:Corynebacterium glutamicum 2256 (ATCC 13869)のpitA遺伝子の塩基配列
配列番号6:Corynebacterium glutamicum 2256 (ATCC 13869)のPitAタンパク質のアミノ酸配列
配列番号7~20:プライマー
配列番号21:Corynebacterium glutamicum 2256 (ATCC 13869)のyggB遺伝子の塩基配列
配列番号22:Corynebacterium glutamicum 2256 (ATCC 13869)のYggBタンパク質のアミノ酸配列
配列番号23:Corynebacterium glutamicum 2256 (ATCC 13869)のyggB遺伝子(V419::IS)の塩基配列
配列番号24:Corynebacterium glutamicum 2256 (ATCC 13869)のYggBタンパク質(V419::IS)のアミノ酸配列
配列番号25:Corynebacterium glutamicum ATCC13032のpitA遺伝子の塩基配列
配列番号26:Corynebacterium glutamicum ATCC13032のPitAタンパク質のアミノ酸配列
配列番号27、28:プライマー
Claims (16)
- L-アミノ酸生産能を有するコリネ型細菌を培地で培養すること、および該培地よりL-アミノ酸を採取すること、を含むL-アミノ酸の製造法であって、
前記細菌が、リン酸トランスポーターの活性が増大するように改変されていることを特徴とする、方法。 - リン酸トランスポーターをコードする遺伝子の発現を上昇させることにより、リン酸トランスポーターの活性が増大した、請求項1に記載の方法。
- 前記遺伝子がpitA遺伝子である、請求項2に記載の方法。
- 前記pitA遺伝子が、下記(a)又は(b)に記載のDNAである、請求項3に記載の方法:
(a)配列番号5または25に示す塩基配列を有するDNA、
(b)配列番号5または25に示す塩基配列の相補配列又は同相補配列から調製され得るプローブとストリンジェントな条件下でハイブリダイズし、かつ、リン酸トランスポーター活性を有するタンパク質をコードするDNA。 - 前記pitA遺伝子が、下記(A)又は(B)に記載のタンパク質をコードするDNAである、請求項3または4に記載の方法:
(A)配列番号6または26に示すアミノ酸配列を有するタンパク質、
(B)配列番号6または26に示すアミノ酸配列において、1若しくは数個のアミノ酸残基の置換、欠失、挿入、または付加を含むアミノ酸配列を有し、かつ、リン酸トランスポーター活性を有するタンパク質。 - 前記遺伝子の発現が、該遺伝子のコピー数を高めること、及び/又は該遺伝子の発現調節配列を改変することによって上昇した、請求項2~5のいずれか一項に記載の方法。
- 配列番号6の246位のフェニルアラニン残基に相当するアミノ酸残基がフェニルアラニン以外のアミノ酸残基に置換される変異を有するリン酸トランスポーターをコードする変異型pitA遺伝子を前記細菌に保持させることにより、リン酸トランスポーターの活性が増大した、請求項1~6のいずれか1項に記載の方法。
- 配列番号6の246位のフェニルアラニン残基に相当するアミノ酸残基が、セリン残基に置換されたことを特徴とする、請求項7に記載の方法。
- L-アミノ酸生産能を有するコリネ型細菌を培地で培養すること、および該培地よりL-アミノ酸を採取すること、を含むL-アミノ酸の製造法であって、
前記細菌が、配列番号6の246位のフェニルアラニン残基に相当するアミノ酸残基がフェニルアラニン以外のアミノ酸残基に置換される変異を有するリン酸トランスポーターをコードする変異型pitA遺伝子を保持していることを特徴とする、方法。 - 配列番号6の246位のフェニルアラニン残基に相当するアミノ酸残基が、セリン残基に置換されたことを特徴とする、請求項9に記載の方法。
- 前記コリネ型細菌が、コリネバクテリウム属細菌である、請求項1~10のいずれか一項に記載の方法。
- 前記コリネ型細菌が、コリネバクテリウム・グルタミカムである、請求項11に記載の方法。
- 前記L-アミノ酸が、L-グルタミン酸である、請求項1~12のいずれか一項に記載の方法。
- 前記L-グルタミン酸が、L-グルタミン酸アンモニウムまたはL-グルタミン酸ナトリウムである、請求項13に記載の方法。
- 配列番号6の246位のフェニルアラニン残基に相当するアミノ酸残基がセリン残基に置換される変異を有するリン酸トランスポーターをコードするDNA。
- 配列番号6の246位のフェニルアラニン残基に相当するアミノ酸残基がセリン残基に置換される変異を有するリン酸トランスポーターをコードする変異型pitA遺伝子を保持しているコリネ型細菌。
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Also Published As
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KR20150099809A (ko) | 2015-09-01 |
BR112015007916A2 (pt) | 2015-09-29 |
US9506094B2 (en) | 2016-11-29 |
EP2868745A1 (en) | 2015-05-06 |
PE20150681A1 (es) | 2015-05-15 |
US20150307907A1 (en) | 2015-10-29 |
EP2868745B1 (en) | 2017-06-21 |
EP2868745A4 (en) | 2015-09-02 |
KR101773755B1 (ko) | 2017-09-01 |
CN105008532A (zh) | 2015-10-28 |
MY185322A (en) | 2021-05-04 |
CN105008532B (zh) | 2017-07-21 |
JPWO2014185430A1 (ja) | 2017-02-23 |
PH12015500792B1 (en) | 2015-06-15 |
BR112015007916B1 (pt) | 2023-04-04 |
JP5831669B2 (ja) | 2015-12-09 |
PH12015500792A1 (en) | 2015-06-15 |
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