WO2010032698A1 - 植物由来原料から乳酸を生産する方法及び乳酸生産細菌 - Google Patents
植物由来原料から乳酸を生産する方法及び乳酸生産細菌 Download PDFInfo
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- WO2010032698A1 WO2010032698A1 PCT/JP2009/065957 JP2009065957W WO2010032698A1 WO 2010032698 A1 WO2010032698 A1 WO 2010032698A1 JP 2009065957 W JP2009065957 W JP 2009065957W WO 2010032698 A1 WO2010032698 A1 WO 2010032698A1
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- lactic acid
- escherichia coli
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- strain
- sucrose
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
Definitions
- the present invention relates to a method for producing lactic acid from plant-derived materials and lactic acid-producing bacteria.
- Lactic acid is a useful substance that has recently attracted attention as an intermediate for polymer raw materials, agricultural chemicals, and pharmaceuticals. Lactic acid is classified into L-lactic acid and D-lactic acid. Polylactic acid currently industrially produced is L-lactic acid polymer, and D-lactic acid has been attracting attention as an intermediate for polymer raw materials, agricultural chemicals and pharmaceuticals in recent years. is there. In nature, there are microorganisms that efficiently produce lactic acid, such as lactic acid bacteria and filamentous fungi. Lactic acid production methods using them include Lactobacillus delbrueckii, etc., as microorganisms that produce L-lactic acid efficiently, and D-lactic acid efficiently. As a well-produced microorganism, a method using a microorganism of the genus Sporolactobacillus is known. However, in any application, it is a fact that lactic acid as a raw material requires high optical purity.
- sucrose PTS Phosphoenolpyruvate: Carbohydrate Phosphotransferase System
- sucrose non-PTS sucrose non-PTS
- sucrose PTS Phosphoenolpyruvate: Carbohydrate Phosphotransferase System
- sucrose non-PTS sucrose non-PTS
- the microorganism takes sucrose as it is, and then breaks it down into glucose and fructose.
- sucrose PTS when the microorganism takes up sucrose, it phosphorylates sucrose and converts it to sucrose-6-phosphate. And it decomposes into glucose-6-phosphate and fructose inside the microorganism.
- fructose derived from sucrose first appears inside the microorganism in a form that is not phosphorylated.
- this non-phosphorylated fructose hereinafter referred to as non-phosphorylated fructose
- the fructose must be isomerized to glucose or phosphorylated.
- the microorganism is Escherichia coli (except for Escherichia coli that can assimilate some sucrose)
- the literature suggests that the activity of isomerizing non-phosphorylated fructose into glucose and phosphorylating fructose are both very low (FEMS Yeast Res, Vol. 5, pp.
- cscA it is known that the production of amino acids derived from phosphoenolpyruvate (PEP), such as tryptophan, is further improved by introducing genes of cscA, cscB, cscK, and cscR (see, for example, 2007-49993).
- PEP phosphoenolpyruvate
- An object of the present invention is to provide a lactic acid-producing bacterium and a lactic acid production method that are useful for efficiently assimilating sucrose and producing lactic acid from sucrose more efficiently.
- the present invention provides a lactic acid-producing bacterium and a lactic acid production method. That is, the present invention is as follows.
- One or more genes including repressor protein (cscR), sucrose hydrolase (cscA), fructokinase (cscK), and sucrose permeation) including at least a sucrose hydrolase gene in the sucrose non-PTS gene group A combination of an enzyme (cscB) and a combination of sucrose hydrolase (cscA), fructokinase (cscK) and sucrose permease (cscB)), and a system for enhancing lactic acid production by genetic recombination E. coli with lactic acid production.
- cscR repressor protein
- cscA sucrose hydrolase
- cscK fructokinase
- sucrose permeation including at least a sucrose hydrolase gene in the sucrose non-PTS gene group A combination of an enzyme (cscB) and a combination of sucrose hydrolase (cscA), fructokinase (cscK) and sucrose permease (cs
- [2] The lactic acid-producing Escherichia coli according to [1], wherein only the sucrose hydrolase gene is included in the sucrose non-PTS gene group, and a lactic acid production-enhancing system by genetic recombination is provided.
- [3] The lactic acid-producing Escherichia coli according to [1] or [2], wherein the lactic acid-producing Escherichia coli further has a fructose metabolic capacity improving system.
- [4] The lactic acid-producing Escherichia coli according to any one of [1] to [3], wherein the lactic acid production-enhancing system includes inactivation or reduction of pyruvate formate lyase activity.
- the lactic acid-producing Escherichia coli according to any one of [1] to [4], wherein the lactic acid production-enhancing system includes enhancement of NADH-dependent lactic acid dehydrogenase activity for producing D-lactic acid or L-lactic acid.
- the lactic acid production enhancement system includes enhancement of D-lactate dehydrogenase activity and inactivation or reduction of FAD-dependent D-lactate dehydrogenase activity inherent in the E. coli [1] to [ 4] The lactic acid-producing Escherichia coli according to any one of [4].
- the lactic acid production-enhancing system is capable of enhancing L-lactate dehydrogenase activity and inactivating at least one of D-lactate dehydrogenase activity and FMN-dependent L-lactate dehydrogenase activity inherent in the E. coli.
- the lactic acid-producing Escherichia coli according to any one of [1] to [4], comprising reduction.
- sucrose hydrolase gene is derived from an Escherichia coli O157 bacterium.
- a method for producing lactic acid comprising producing lactic acid from a plant-derived raw material containing sucrose using the lactic acid-producing E. coli according to any one of [1] to [15].
- the lactic acid-producing bacterium of the present invention comprises one or more genes (including a repressor protein (cscR), a sucrose hydrolase (cscA), a fructokinase (including a sucrose hydrolase gene) in the sucrose non-PTS gene group. cscK) and sucrose permease (cscB) and a combination of sucrose hydrolase (cscA), fructokinase (cscK) and sucrose permease (cscB)), and by genetic recombination Lactic acid-producing Escherichia coli equipped with a lactic acid production enhancing system.
- the lactic acid production method of the present invention is a lactic acid production method comprising producing lactic acid from a plant-derived raw material containing sucrose using the lactic acid-producing bacterium.
- the lactic acid-producing bacterium of the present invention comprises one or more genes (including a repressor protein (cscR), a sucrose hydrolase (cscA), a fructokinase (including a sucrose hydrolase gene) in the sucrose non-PTS gene group. cscK) and sucrose permease (cscB), and a combination of sucrose hydrolase (cscA), fructokinase (cscK) and sucrose permease (cscB)), and a system for enhancing lactic acid production Therefore, fructose derived from sucrose can be phosphorylated and incorporated into the cells, and the fructose is converted into lactic acid using a lactic acid production enhancing system.
- cscR repressor protein
- cscA sucrose hydrolase
- a fructokinase including a sucrose hydrolase gene
- sucrose non-PTS gene group sucrose non-PTS gene group.
- sucrose non-PTS gene groups that have one or more genes including at least sucrose hydrolase and have produced substances using sucrose as a carbon source have been reported so far. It has not been.
- sucrose-derived fructose is increased by introducing one or more kinds of sucrose non-PTS genes in an imperfect state, that is, at least one gene containing at least a sucrose hydrolase gene into lactic acid-producing Escherichia coli. It has been found that it is utilized in efficiency, and that productivity is remarkably increased as compared with the prior art. As a result, lactic acid can be obtained efficiently and in a short time from sucrose, which is derived from plants and is inexpensive and industrially valuable.
- the lactic acid-producing bacterium of the present invention can assimilate sucrose and its fructose fructose to produce lactic acid regardless of the presence or absence of glucose, which is another sugar resource. Lactic acid can be produced without waiting for a decrease or depletion of the sugar substrate, which is more efficient.
- glucose uptake is generally preferred over fructose, and it is known that fructose is not fully metabolized in the presence of glucose.
- sugar metabolism is a basic function for living organisms.
- the sucrose non-PTS gene group in the present invention refers to a gene group involved in a non-PTS system in the sucrose utilization pathway of microorganisms. Specifically, it is a gene group composed of a repressor protein (cscR), sucrose hydrolase (cscA), fructokinase (cscK), and sucrose permease (cscB). In the present invention, at least one of them may contain at least cscA.
- cscR repressor protein
- cscA sucrose hydrolase
- cscK fructokinase
- cscB sucrose permease
- cscA a combination of cscA and cscK, a combination of cscA and cscB, a combination of cscA and cscR, a combination of cscA, cscB and cscR, cscA And a combination of cscK and cscR.
- a combination of a repressor protein (cscR), a sucrose hydrolase (cscA), a fructokinase (cscK), and a sucrose permease (cscB) is selected from a combination of genes of a sucrose non-PTS gene group to be introduced.
- sucrose hydrolase cscA
- fructokinase cscK
- sucrose permease cscB
- the sucrose hydrolase (invertase, CscA) in the present invention is classified into enzyme number 3.2.1.26 based on the report of the International Biochemical Union (IUB) enzyme committee.
- IUB International Biochemical Union
- This enzyme is an enzyme that is not originally possessed by E. coli such as the K12 strain, and is one of enzymes in non-PTS metabolic pathways including a proton cotransporter, invertase, fructokinase, and a sucrose-specific repressor ( Canadian Journal of Microbiology, (1991) vol. 45, pp 418-422).
- sucrose outside the cell is decomposed into glucose and fructose on the cell membrane and released to the outside of the cell via glucose PTS and fructose PTS. Phosphorylated and taken up into the cytoplasm.
- fructose can be supplied to the fructose metabolism system in bacteria to enable assimilation using a glycolysis system.
- the sucrose hydrolase (invertase, CscA) gene introduced into the host bacterium of the present invention has a base sequence of a gene encoding sucrose hydrolase (invertase, CscA) obtained from an organism having this enzyme.
- DNA or a synthetic DNA sequence synthesized based on its known base sequence can be used. Preferred are: Erwinia, Porteus, Proteus, Vibrio, Agrobacterium, Rhizobium, Staphylococcus (Staphylococcus) ), And those derived from Bifidobacterium and Escherichia. Examples include DNA having the base sequence of a gene derived from Escherichia coli O157 strain.
- DNA having a base sequence of a gene derived from Escherichia coli O157 strain.
- a signal sequence for transferring cscA to the periplasm of the microbial cell is added to cscA.
- the gene for the repressor protein (CscR) introduced into the host bacterium of the present invention includes DNA having the base sequence of the gene encoding the repressor protein (CscR) obtained from an organism having this enzyme, or a known one thereof
- a synthetic DNA sequence synthesized based on the base sequence can be used. Preferred are: Erwinia, Porteus, Proteus, Vibrio, Agrobacterium, Rhizobium, Staphylococcus (Staphylococcus) ), And those derived from Bifidobacterium and Escherichia.
- Examples include DNA having the base sequence of a gene derived from Escherichia coli O157 strain. Particularly preferred is DNA having a base sequence of a gene derived from Escherichia coli O157 strain.
- fructokinase (CscK) gene to be introduced into the host bacterium of the present invention
- a synthetic DNA sequence synthesized based on the base sequence can be used.
- Preferred are: Erwinia, Porteus, Proteus, Vibrio, Agrobacterium, Rhizobium, Staphylococcus (Staphylococcus) ), And those derived from Bifidobacterium and Escherichia.
- Examples include DNA having the base sequence of a gene derived from Escherichia coli O157 strain. Particularly preferred is DNA having a base sequence of a gene derived from Escherichia coli O157 strain.
- sucrose permease (CscB) gene introduced into the host bacterium of the present invention DNA having a base sequence of a gene encoding sucrose permease (CscB) obtained from an organism having this enzyme, or a known one thereof A synthetic DNA sequence synthesized based on the base sequence of can be used. Preferred are: Erwinia, Porteus, Proteus, Vibrio, Agrobacterium, Rhizobium, Staphylococcus (Staphylococcus) ), And those derived from Bifidobacterium and Escherichia. Examples include DNA having the base sequence of a gene derived from Escherichia coli O157 strain. Particularly preferred is DNA having a base sequence of a gene derived from Escherichia coli O157 strain.
- sucrose assimilation means the ability of sucrose to be reduced in molecular weight or polymerized as it is, preferably reduced in molecular weight and taken into the living body, or metabolically converted to another substance.
- assimilation includes decomposition that lowers the molecular weight of sucrose. Specifically, it includes breaking down sucrose into D-glucose and D-fructose.
- the fact that the metabolic capacity of fructose is improved refers to a state in which the uptake of fructose into cells is increased.
- the system for improving the metabolic capacity of fructose means a structure for improving the metabolic capacity of fructose.
- the “host” means the E. coli that becomes the lactic acid-producing E. coli of the present invention as a result of introduction of one or more genes from outside the cell.
- the numerical range shown in this specification indicates a range including the described numerical value as a minimum value and a maximum value, respectively.
- the lactic acid production enhancing system in the present invention refers to a structure for improving the lactic acid production ability introduced or modified by genetic recombination.
- a lactic acid production-enhancing system may be any system that increases the original amount of lactic acid production in the target Escherichia coli.
- Preferable examples include inactivation, reduction or enhancement of enzyme activities involved in lactic acid production activity, or combinations thereof. Thereby, in combination with the above-mentioned CscA activity, lactic acid can be effectively produced from sucrose even in Escherichia coli that originally has no sucrose utilization ability.
- the term “by gene recombination” means that a change in the base sequence is caused by the insertion of another DNA into the base sequence of the native gene, or the substitution, deletion or combination of a part of the gene. It may be included as long as it is, for example, it may be obtained as a result of mutation.
- FruR activity refers to the amount of protein produced by expression of a gene controlled by FruR, or the function of the protein quantified.
- “reduction” means that the activity of the enzyme or FruR is significantly reduced as compared with the state before the treatment due to genetic recombination of the gene encoding the enzyme or transcription factor FruR. Refers to the state of being.
- FruR activity refers to the amount of protein produced by expression of a gene controlled by FruR, or the function of the protein quantified.
- inactivation or reduction of pyruvate formate lyase (Pfl) activity enhancement of NADH-dependent lactate dehydrogenase activity to produce D-lactic acid or L-lactic acid, or both From the viewpoint of reducing by-products and increasing the amount of lactic acid produced (see WO2005 / 033324 for inactivation or reduction of pyruvate formate lyase (Pfl) activity) NADH-dependent D-lactic acid dehydrogenase
- Yang et al. See Metab. Eng. Vol. 1 (2), pp141-152 (1999)).
- the pyruvate formate lyase (Pfl) in the present invention is classified into enzyme number 2.3.154 according to the report of the International Biochemical Union (I.U.B.) Enzyme Committee, and formate acetyltransferase. It is also called an enzyme.
- This enzyme is a general term for enzymes that reversibly catalyze the reaction of producing formic acid from pyruvic acid.
- the NADH-dependent lactate dehydrogenase in the present invention includes D-lactate dehydrogenase (LdhA) and L-lactate dehydrogenase (Ldh2).
- LdhA refers to an enzyme derived from Escherichia coli that produces D-lactic acid and NAD from pyruvic acid and NADH.
- Ldh2 refers to an enzyme that produces L-lactic acid and NAD from pyruvate and NADH, and examples thereof include an enzyme derived from Bifidobacterium longum.
- the enhancement of lactate dehydrogenase activity means that the enzyme produced by a gene encoding LdhA or Ldh2 is significantly recombined by genetic recombination of the gene encoding LdhA or Ldh2, as compared with the state before the treatment. It refers to the state where the activity has increased.
- lactic acid has optical isomers of D-lactic acid and L-lactic acid
- NADH-dependent D-lactic acid dehydrogenase or NADH-dependent L-lactic acid dehydrogenase is used to increase the production amount of either optical isomer.
- a system including the enhancement of the activity is sometimes referred to as “D-lactic acid production enhanced system” or “L-lactic acid production enhanced system”. Therefore, the type of lactic acid production enhancement system can be selected as appropriate depending on the type of lactic acid of interest.
- the D-lactic acid production enhanced system further includes inactivation or reduction of the FAD-dependent D-lactic acid dehydrogenase (Dld) activity inherent in the E. coli. It may be a thing. More preferably, the enhanced D-lactic acid production system inactivates or reduces the FAD-dependent D-lactate dehydrogenase (Dld) activity inherent in the E. coli and inactivates pyruvate formate lyase (Pfl) activity.
- Dld FAD-dependent D-lactic acid dehydrogenase
- NADH-dependent D-lactate dehydrogenase activity Activation or reduction and / or enhancement of NADH-dependent D-lactate dehydrogenase activity, and inactivation or reduction of Dld activity, inactivation or reduction of Pfl activity, It is most preferable that both have NADH-dependent D-lactate dehydrogenase (LdhA) activity derived from E. coli.
- LdhA NADH-dependent D-lactate dehydrogenase
- the L-lactic acid production-enhanced system uses the FMN-dependent L-lactate dehydrogenase (LldD) activity or D-lactate dehydrogenase (LdhA) activity inherent in the E. coli. Inactivation or reduction may be further included, and preferably, LldD activity and LdhA activity are inactivated or reduced at the same time. More preferably, at least one of pfl activity, lld activity and ldhA activity is inactivated or reduced, and NADH-dependent L-lactate dehydrogenase activity is enhanced. Most preferably, all of Pfl activity, LldD activity, and LdhA activity are inactivated or reduced, and the BAD bacteria-derived NADH-dependent L-lactate dehydrogenase activity is enhanced.
- LldD FMN-dependent L-lactate dehydrogenase
- LdhA D-lactate dehydrogenase
- the FMN-dependent L-lactate dehydrogenase (LldD) in the present invention is an enzyme classified as enzyme number 1.1.2.3 according to the report of the International Biochemical Union (IUB) enzyme committee. is there. This enzyme is a general term for enzymes that catalyze the reaction of producing pyruvic acid from L-lactic acid.
- MT-10934 / pGlydhA described in WO2005 / 033324 can be exemplified as an example of a bacterium with enhanced LdhA activity and inactivated or reduced Pfl activity.
- a gene encoding LdhA or Ldh2 is linked to a promoter of a gene responsible for expression of a protein involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis. It is effective to incorporate it into an expression plasmid in such a state and introduce it into a desired bacterium.
- the promoter of the gene responsible for the expression of the protein involved in glycolysis, nucleic acid biosynthesis or amino acid biosynthesis is a strong promoter that constantly functions in bacteria, preferably in Escherichia coli, and It refers to a promoter that is less susceptible to suppression of expression even in the presence of glucose.
- the bacterium thus obtained has an accumulated amount of D-lactic acid or L-lactic acid as compared with that in which the expression of ldhA or ldh2 is not enhanced when producing D-lactic acid or L-lactic acid under aeration conditions. As a result, the impurity pyruvic acid concentration decreases, and the optical purity of D-lactic acid or L-lactic acid can be improved.
- the FAD-dependent D-lactate dehydrogenase (Dld) in the present invention is a generic term for enzymes that catalyze a reaction for producing pyruvate from D-lactic acid in the presence of oxidized flavin adenine dinucleotide as a coenzyme. .
- Dld activity in the present invention is inactivated or reduced and / or Pfl activity is inactivated or reduced and / or LdhA activity is enhanced
- WO2005 / Examples include Escherichia coli MT-10994 (FERM BP-10058) strain described in No. 033324.
- the promoter of a gene responsible for the expression of a protein involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis is a strong promoter that constantly functions in a microorganism and can be expressed even in the presence of glucose.
- promoters that are difficult to be suppressed include glyceraldehyde 3-phosphate dehydrogenase (hereinafter sometimes referred to as GAPDH) promoter and serine hydroxymethyltransferase promoter.
- GAPDH glyceraldehyde 3-phosphate dehydrogenase
- the promoter in the present invention means a site where RNA polymerase having sigma factor binds and initiates transcription.
- the GAPDH promoter derived from Escherichia coli is represented by base numbers 397 to 440 in the base sequence information of GenBank accession number X02662.
- the gene encoding LdhA in the present invention is expressed on the genome by using a promoter of a gene that controls expression of a protein involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis, and has Pfl activity
- Escherichia coli MT-10994 (FERM BP- described in WO2005 / 033324) may be used. 10058)
- a strain can be exemplified.
- Escherichia coli MT-10994 is expressed by functionally linking the ldhA gene to the GAPDH promoter on the genome, and PflB and Dld are inactivated by gene disruption.
- This strain is the deposit number of FERM BP-10058, and the center of the National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology, 1-1-1, Higashi 1-1-1 Tsukuba, Ibaraki Prefecture, is the It has been deposited since March 19, 2004, based on the Budapest Treaty concerning international recognition.
- the lactic acid-producing bacterium of the present invention further includes a fructose metabolic capacity improving system.
- a fructose metabolic capacity improving system examples include those by enhancing phosphorylation ability or fructose uptake ability in the fructose metabolic pathway.
- the enhancement of the phosphorylation ability in the fructose metabolic pathway is the addition of fructose-1-phosphate kinase activity, and the enhancement of the fructose uptake ability is derived from the reduction of FruR activity. To more preferable.
- “Granting” or “enhancement” of the ability in the present invention refers to a promoter of an enzyme gene possessed by the host bacterium on the genome in addition to introducing a gene encoding the enzyme from outside the host bacterium. It includes those in which the enzyme gene is strongly expressed by enhancing the activity or replacing it with another promoter.
- “enhancement” of phosphorylation ability means that the amount of phosphorylated substrate or the amount of metabolites derived from the phosphorylated substrate is significantly increased by improving the activity of phosphorylase.
- the enhancement of the fructose uptake ability in the present invention means that the enzyme activity controlled by FruR is significantly lower than that before the treatment due to genetic recombination of the gene encoding FruR.
- the activity of the enzyme in the present invention may be the activity measured by any existing measurement system.
- Fructose-1-phosphate kinase (FruK) in the present invention is classified into enzyme number 2.7.1.56 according to the report of the International Biochemical Union (I.U.B.) Enzyme Committee. This enzyme is also called kinase 1. While fructose uptake in the presence of glucose is generally suppressed in bacteria such as E. coli, enhanced FruK expression promotes fructose uptake in the presence of glucose, and in D-lactic acid producing bacteria The knowledge that it contributes to the improvement of the productivity of D-lactic acid has not been seen so far.
- the fructose-1-phosphate kinase (FruK) gene introduced into the host bacterium of the present invention includes a nucleotide sequence of a gene encoding fructose-1-phosphate kinase (FruK) obtained from an organism having this enzyme. Or a synthetic DNA sequence synthesized based on a known base sequence thereof can be used. Preferred are those derived from Escherichia, Pseudomonas, Aerobacter, Clostridium, especially those derived from Escherichia Examples thereof include DNA having a base sequence of a gene derived from Escherichia coli MG1655 strain. Particularly preferred is a DNA having a base sequence of a gene derived from Escherichia coli MG1655 strain.
- FruR in the present invention controls the expression of a group of genes constituting a fructose PTS pathway, that is, a fructose operon, which is a system in which a microorganism phosphorylates fructose and takes it into cells.
- a fructose operon which is a system in which a microorganism phosphorylates fructose and takes it into cells.
- Disrupting the FruR gene is known to suppress the activity of synthesizing phosphoenolpyruvate (PEP), a phosphate donor to fructose.
- PEP phosphoenolpyruvate
- the FruR gene whose expression is reduced in the present invention may be any gene originally possessed by the host bacterium, and may be DNA having the base sequence of the FruR gene originally possessed by the host bacterium, or a known base sequence thereof. It may be a synthetic DNA sequence incorporated on the basis.
- sucrose hydrolase and fructose-1-phosphate kinase are obtained by introduction of a gene encoding each protein derived from Escherichia coli O157 or Escherichia coli MG1655. Can do. By using genes derived from these bacteria, functional expression can be ensured.
- a bacterium imparted with an enzyme activity refers to a bacterium imparted with the enzyme activity from outside the cell body to the cell body by some method.
- These bacteria can be produced, for example, using a method such as introducing a gene encoding the enzyme and protein from outside the cell body into the cell body using a gene recombination technique. Preparation of genomic DNA necessary for introducing a gene from outside the cell into the cell, DNA cleavage and ligation, transformation, PCR (Polymerase Chain Reaction), design of oligonucleotides used as primers, synthesis, etc. This can be done by conventional methods well known to those skilled in the art. These methods are described in Sambrook, J. et al. , Et. al. , “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor ab Laboratory Press, (1989).
- a bacterium with reduced enzyme activity refers to a bacterium whose native activity is impaired by some method from the outside of the cell body to the inside of the cell body, similarly to the bacterium imparted with the above activity.
- These bacteria can be produced, for example, by destroying genes encoding the enzymes and proteins (gene disruption).
- gene disruption refers to the introduction of a mutation in the base sequence of a gene, the insertion of another DNA, or the deletion of a certain part of a gene so that the function of the gene cannot be exhibited. Show.
- the gene cannot be transcribed into mRNA and the structural gene is not translated, or the transcribed mRNA is incomplete, resulting in a mutation or deletion in the amino acid sequence of the translated structural protein. The function cannot be performed.
- the gene-disrupted strain can be produced by any method as long as a disrupted strain that does not express the enzyme or protein is obtained.
- Various methods of gene disruption have been reported (natural breeding, addition of mutagen, UV irradiation, irradiation, random mutation, transposon, site-specific gene disruption), but only certain genes can be disrupted Thus, gene disruption by homologous recombination is preferred. Homologous recombination techniques are described in J. Bacteriol. 161, 1219-1221 (1985) and J. MoI. Bacteriol. , 177, 1511-1519 (1995) and Proc. Natl. Acad. Sci. U. S. A, 97, 6640-6645 (2000). These methods and their applications can be easily implemented by engineers in the same industry.
- E. coli means E. coli that can have the ability to produce lactic acid from plant-derived materials by using any means regardless of whether or not it originally has the ability to produce lactic acid from plant-derived materials.
- the Escherichia coli into which each of the above genes is introduced may not have a normal ability to produce lactic acid, and any Escherichia coli may be used as long as the above genes can be introduced and changed. More preferably, it can be Escherichia coli preliminarily imparted with the ability to produce lactic acid, whereby lactic acid can be produced more efficiently.
- sucrose utilization ability can be imparted to Escherichia coli which originally does not have sucrose utilization ability, and lactic acid can be efficiently produced from sucrose. Examples of such Escherichia coli that does not originally have sucrose utilization ability include K12 strain, B strain, C strain and strains derived therefrom.
- pyruvate formate lyase (Pfl) activity described in, for example, WO 2005/033324 is inactivated or reduced, and NADH-dependent D-derived from Escherichia coli.
- Escherichia coli with enhanced lactate dehydrogenase (LdhA) activity, or E. coli with further FAD-dependent D-lactate dehydrogenase (Dld) activity in addition to these properties, or malate dehydrogenase (Mdh) activity examples include E. coli that has been inactivated or reduced, in which Pfl activity is inactivated or reduced, and / or Dld activity has been inactivated or reduced.
- a promoter for expressing various genes may be any promoter as long as it can control the expression of any of the above genes, but it is a strong promoter that constantly functions in a microorganism and in the presence of glucose. Promoters that are less susceptible to suppression of expression are preferred, and specific examples include the promoter of glyceraldehyde 3-phosphate dehydrogenase (hereinafter sometimes referred to as GAPDH) and the promoter of serine hydroxymethyltransferase.
- GAPDH glyceraldehyde 3-phosphate dehydrogenase
- serine hydroxymethyltransferase serine hydroxymethyltransferase.
- any means usually used for this purpose can be used without particular limitation, and examples thereof include gene disruption by homologous recombination and the like.
- the lactic acid production method of the present invention includes producing lactic acid from a plant-derived raw material containing sucrose using the lactic acid-producing bacterium, that is, contacting the lactic acid-producing bacterium with a plant-derived raw material containing sucrose. And a recovery step of recovering lactic acid obtained by contact.
- the plant-derived material used in the lactic acid production method is a carbon source obtained from a plant, and is not particularly limited as long as it is a plant-derived material containing sucrose. In the present invention, it refers to organs such as roots, stems, trunks, branches, leaves, flowers or seeds, plants containing them, degradation products of these plant organs, and further from plant bodies, plant organs, or degradation products thereof.
- organs such as roots, stems, trunks, branches, leaves, flowers or seeds, plants containing them, degradation products of these plant organs, and further from plant bodies, plant organs, or degradation products thereof.
- those that can be used as a carbon source in culture by microorganisms are also included in plant-derived materials.
- carbon sources included in such plant-derived materials generally include sugars such as starch, glucose, fructose, xylose, and arabinose, or vegetative decomposition products and cellulose containing a large amount of these components.
- sugars such as starch, glucose, fructose, xylose, and arabinose
- vegetative decomposition products and cellulose containing a large amount of these components.
- a hydrolyzate etc. or these combinations can be mentioned
- the glycerin or fatty acid derived from vegetable oil may also be included in the carbon source in this invention.
- Examples of plant-derived materials in the present invention can preferably include crops such as cereals, corn, rice, wheat, soybeans, sugar cane, beet, cotton, etc., or a combination thereof, There are no particular restrictions on raw products, juice, pulverized products, and the like. Moreover, the form of only the above-mentioned carbon source may be sufficient.
- the contact between the lactic acid-producing bacteria and the plant-derived raw material in the contact step is generally performed by culturing the lactic acid-producing bacteria in a medium containing the plant-derived raw material.
- the contact density between the plant-derived raw material and the lactic acid-producing bacterium varies depending on the activity of the lactic acid-producing bacterium, but generally, the initial sugar concentration in terms of glucose is 20 with respect to the total mass of the mixture as the concentration of the plant-derived raw material in the medium. From the viewpoint of bacterial glucose tolerance, the initial sugar concentration can be 15% by mass or less.
- Each of these other components may be added in an amount usually added to the microorganism medium, and is not particularly limited.
- the content of lactic acid-producing bacteria in the medium varies depending on the type and activity of the bacteria, but in general, the initial bacterial concentration is preferably 0.1% by mass to 30% by mass with respect to the culture solution, which is preferable from the viewpoint of controlling culture conditions. May be 1 mass% to 10 mass%.
- a medium used for culturing lactic acid-producing bacteria a medium containing a carbon source, a nitrogen source, inorganic ions, and organic trace elements required by microorganisms to produce lactic acid, nucleic acids, vitamins, etc. There is no limit.
- the carbon source saccharides such as glucose, fructose and molasses, organic acids such as fumaric acid, citric acid and succinic acid, alcohols such as methanol, ethanol and glycerol, and others are appropriately used.
- organic nitrogen sources such as organic ammonium salts, inorganic ammonium salts, ammonia gas, ammonia water, and the like, organic nitrogen sources such as protein hydrolysates, and the like are appropriately used.
- the inorganic ions magnesium ions, phosphate ions, potassium ions, iron ions, manganese ions, and others are appropriately used as necessary.
- a culture medium used for this invention if the point used for industrial production is considered, a liquid culture medium is preferable.
- a medium supplemented with two or more amino acids can be mentioned.
- a medium supplemented with two or more amino acids means a medium containing at least two kinds of naturally occurring amino acids, yeast extract, casamino acid, peptone, whey, molasses, corn steep liquor
- the culture medium containing the hydrolyzate of natural products or natural product extracts such as
- a medium containing at least one kind selected from yeast extract, peptone, whey, molasses and corn steep liquor, or a mixture thereof is preferably 0.5% by mass to 20% by mass, and preferably 2% by mass. To 15% by mass is more preferable.
- the addition of corn steep liquor can provide a great effect. In this case, it may be better to add no salt such as ammonium sulfate.
- the medium is usually a liquid medium.
- the culture conditions vary depending on the prepared bacterial cells and the culture apparatus, but in general, the culture temperature is preferably 20 ° C. to 40 ° C., more preferably 25 ° C. to 35 ° C., and the pH is NaOH, NH 3 or the like. It is preferable to adjust and culture at 4 to 9, preferably 6.0 to 7.2, more preferably 6.5 to 6.9.
- the culture time is not particularly limited, but is a time necessary for the bacterial cells to sufficiently grow and to produce lactic acid.
- a culture tank capable of controlling temperature, pH, aeration conditions, and stirring speed.
- the culturing of the present invention is not limited to using a culture tank.
- seed culture may be performed in advance as a preculture, and this may be inoculated into a medium in a culture tank prepared in advance.
- the microorganisms obtained in the present invention are cultured to produce lactic acid, it is not necessary to perform aeration at all, but it is better to perform aeration in order to obtain a more preferable result.
- the aeration condition referred to here does not necessarily require air to pass through the culture medium, and depending on the shape of the culture tank, the top surface ventilation may be used to ventilate the air layer above the culture medium while stirring the culture medium appropriately. It means that a gas containing oxygen is allowed to flow into the culture tank.
- the dissolved oxygen concentration changes depending on the combination of internal pressure, stirring blade position, stirring blade shape, and stirring speed. Therefore, the following are optimal using lactic acid productivity and the amount of organic acids other than lactic acid as indicators.
- Conditions can be determined. For example, when culturing in a relatively small culture tank such as the culture device BMJ-01 manufactured by ABLE, when using 500 g of the culture solution, the air is stirred at 0.005 L / min to 0.5 L / min at normal pressure. Preferable results can be obtained under aeration conditions that can be achieved at a speed of 50 rpm to 500 rpm, more preferably 0.05 L / min to 0.25 L / min at normal pressure and a stirring speed of 100 rpm to 400 rpm.
- This condition is a condition that enables oxygen supply that can be achieved under conditions where the aeration stirring condition is water at a temperature of 30 ° C. and the oxygen transfer rate coefficient k La is 1 / h or more and 400 / h or less at normal pressure. It is.
- the aeration conditions described above do not need to be consistently performed from the beginning of the culture to the end, and preferable results can be obtained by performing it in part of the culture process.
- lactic acid obtained by this contact is recovered, and is generally performed by recovering lactic acid from the culture obtained by the above culture.
- the culture in the present invention refers to the cells produced by the above-described method, the culture solution, and the processed product thereof.
- a conventionally known method can be used from the culture solution.
- the bacterial cells are removed by centrifugation, then acidified and directly distilled, or lactide is formed. Distillation method, esterification after adding alcohol and catalyst, distillation method, extraction method in organic solvent, separation method using ion exchange column, concentration method by electrodialysis, and a combination of these methods. Can be adopted.
- the bacterial cells produced by the method of the present invention produce an enzyme group suitable for the production of lactic acid, it is possible to further produce and recover lactic acid using this enzyme group. Considered part of the method of recovery.
- Example 1 ⁇ Preparation of Escherichia coli MG1655 strain dld gene deletion strain> The entire base sequence of the genomic DNA of Escherichia coli is known (GenBank accession number U00096), and the base sequence of a gene encoding the FAD-dependent D-lactate dehydrogenase of Escherichia coli (hereinafter sometimes abbreviated as dld) is also reported. (Genbank accession number M10038).
- CAACACCCAAGCTTTCGCG SEQ ID NO: 1
- TTCCACTCCCTTGTGTGGGGC SEQ ID NO: 2
- AACTGCAGGAATTACGGATGGCAGGAG SEQ ID NO: 3
- TGTGTCTAGTG primer SEQ ID NO: 3
- Genomic DNA of Escherichia coli MG1655 strain was prepared by the method described in Current Protocols in Molecular Biology (JohnWiley & Sons), and the obtained genomic DNA was used as a template, and the primers of SEQ ID NO: 1 and SEQ ID NO: 2 were usually used.
- PCR is performed under the following conditions to amplify a DNA fragment of about 1.4 kbp (hereinafter sometimes referred to as a dld-L fragment), and PCR is performed under normal conditions using the primers of SEQ ID NO: 3 and SEQ ID NO: 4.
- a DNA fragment of about 1.2 kbp hereinafter sometimes referred to as a dld-R fragment
- the obtained dld-L fragment and dld-R fragment were digested with restriction enzymes HindIII and PstI and PstI and XbaI, respectively.
- This fragment was mixed with a fragment obtained by digesting a temperature sensitive plasmid pTH18cs1 (Hashimoto-Gotoh, T., et.al., Gene, Vol.241 (1), pp185-191 (2000)) with HindIII and XbaI.
- DH5 ⁇ competent cells DNA-903, Toyobo Co., Ltd.
- Escherichia coli MG1655 strain can be obtained from the American Type Culture Collection, which is a cell / microorganism / gene bank.
- Example 2 The plasmid pTH ⁇ dld obtained in Example 1 was transformed into MG1655 strain at 30 ° C. to obtain a transformant that grew on an LB agar plate containing 10 ⁇ g / ml of chloramphenicol. The obtained transformant was applied to an agar plate and cultured at 30 ° C. overnight. Next, it was applied to an LB agar plate containing 10 ⁇ g / ml of chloramphenicol so as to obtain these cultured cells, and colonies that grew at 42 ° C. were obtained. Further, the operation of obtaining a single colony growing at 42 ° C. was repeated once again, and a clone in which the entire plasmid was integrated into the chromosome by homologous recombination was selected. This clone was confirmed to have no plasmid in the cytoplasm.
- the clone was spread on an LB agar plate, cultured at 30 ° C. overnight, then inoculated into LB liquid medium (3 ml / tube), and cultured with shaking at 42 ° C. for 3 to 4 hours. This was appropriately diluted (about 10 ⁇ 2 to 10 ⁇ 6 ) so that a single colony was obtained, applied to an LB agar plate, and cultured overnight at 42 ° C. to obtain a colony. 100 colonies were picked up randomly from the colonies that appeared, and each was grown on an LB agar plate and an LB agar plate containing 10 ⁇ g / ml chloramphenicol. I picked a clone.
- dld deletion strain an about 2.0 kb fragment containing dld is amplified from the chromosomal DNA of these target clones by PCR, a strain lacking the dld gene region is selected, and a clone satisfying the above is designated as a dld deletion strain, The resulting strain was named MG1655 ⁇ dld strain.
- Example 3 ⁇ Escherichia coli MG1655pflB, dld gene deletion strain preparation> The entire base sequence of Escherichia coli genomic DNA is known (GenBank accession number U00096), and the base sequence of the gene (pflB) encoding Escherichia coli pyruvate formate lyase is also reported (Genbank accession number X08035). ).
- this DNA fragment (hereinafter referred to as pflB-L fragment) And a DNA fragment of about 1.3 kbp (hereinafter sometimes referred to as a pflB-R fragment) is amplified by PCR under the normal conditions using the primers of SEQ ID NO: 7 and SEQ ID NO: 8. did.
- This DNA fragment was separated and collected by agarose electrophoresis, and the pflB-L fragment was digested with HindIII and SphI, and the pflB-R fragment was digested with SphI and PstI, respectively.
- the two digested fragments were reacted with HindIII and PstI digests of the temperature sensitive plasmid pTH18cs1 (GenBank accession number AB019610) with T4 DNA ligase, and then transformed into Escherichia coli DH5 ⁇ competent cell (Toyobo Co., Ltd. Sakai DNA-903). After conversion, a plasmid containing two fragments, a 5 ′ upstream vicinity fragment and a 3 ′ downstream vicinity fragment of the pflB gene, was obtained and named pTH ⁇ pfl.
- the obtained plasmid pTH ⁇ pfl was transformed into the MG1655 ⁇ dld strain obtained in Example 2 to obtain a transformant that grew at 30 ° C. on an LB agar plate containing 10 ⁇ g / ml of chloramphenicol.
- the obtained transformant was applied to an agar plate and cultured at 30 ° C. overnight. Next, it was applied to an LB agar plate containing 10 ⁇ g / ml of chloramphenicol so as to obtain these cultured cells, and colonies that grew at 42 ° C. were obtained.
- the MG1655 ⁇ dld strain in which the pfl gene was disrupted was obtained according to the same method as in Example 2, and named MG1655 ⁇ pfl ⁇ dld strain.
- PCR is performed under normal conditions using a combination of SEQ ID NO: 9 and SEQ ID NO: 10, and SEQ ID NO: 11 and SEQ ID NO: 12 (about 800 bp (hereinafter referred to as mdh-L fragment). And a DNA fragment of about 1,000 bp (hereinafter sometimes referred to as mdh-R fragment) was amplified. This DNA fragment was separated and collected by agarose electrophoresis, and the mdh-L fragment was digested with KpnI and BamHI, and the mdh-R fragment was digested with BamHI and XbaI, respectively.
- the two digested fragments were reacted with KpnI and XbaI digests of the temperature sensitive plasmid pTH18cs1 (GenBank accession number AB019610) with T4 DNA ligase, and then transformed into Escherichia coli DH5 ⁇ competent cell (Toyobo Co., Ltd. Sakai DNA-903). After conversion, a plasmid containing two fragments, a 5 ′ upstream fragment and a 3 ′ downstream fragment, of the gene encoding mdh was obtained, and this plasmid was designated as pTH ⁇ mdh.
- Plasmid pTH ⁇ mdh was transformed into the Escherichia coli MG1655 ⁇ pfl ⁇ dld strain obtained in Example 3, and the MG1655 ⁇ pfl ⁇ dld strain in which the mdh gene was disrupted was obtained in the same manner as in Example 2. This strain was designated as MG1655 ⁇ pfl ⁇ dld ⁇ mdh strain.
- the 5 'end was phosphorylated. Further, the temperature sensitive plasmid pTH18cs1 was digested with SmaI and then dephosphorylated with alkaline phosphatase. The above two phosphorylated fragments and the dephosphorylated plasmid were reacted with T4 DNA ligase, and then transformed into Escherichia coli DH5 ⁇ competent cell (Toyobo Co., Ltd. DNA-903), and the aspA gene 5 A plasmid containing two fragments, a 'upstream neighboring fragment and a 3' downstream neighboring fragment, was obtained, and this plasmid was designated as pTH ⁇ asp.
- Plasmid pTH ⁇ asp was transformed into the Escherichia coli MG1655 ⁇ pfl ⁇ dld ⁇ mdh strain obtained in Example 4, and finally the MG1655 ⁇ pfl ⁇ dld ⁇ mdh strain in which the aspA gene was disrupted was obtained, which was designated as MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp.
- the detailed method for obtaining this strain was in accordance with the method described in Example 2 of the present invention.
- Example 6 ⁇ Replacement of ldhA promoter with GAPDH promoter on the genome of Escherichia coli MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp strain> The nucleotide sequence of the Escherichia coli ldhA gene has already been reported (GenBank accession number U36928).
- GAPDH glyceride 3-phosphate dehydrogenase
- D-lactate dehydrogenase gene (ldhA)
- GGAATTCCCGGAGAAAGTCTTATGAAACT (SEQ ID NO: 19)
- CCCAAGCTTTTAAACCAGTTCGGTCGGC (SEQ ID NO: 20) were obtained by PCR using the genomic DNA of Escherichia coli MG1655 strain as a template.
- the obtained DNA fragment was digested with restriction enzymes EcoRI and HindIII to obtain a D-lactate dehydrogenase (ldhA) gene fragment of about 1.0 kbp.
- the above two DNA fragments and the fragment obtained by digesting plasmid pUC18 with restriction enzymes EcoRI and HindIII were mixed and ligated using ligase, and then Escherichia coli DH5 ⁇ competent cell (Toyobo Co., Ltd. DNA-903). And a transformant that grows on an LB agar plate containing 50 ⁇ g / mL of ampicillin was obtained. The obtained colonies were cultured overnight at 30 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pGAP-ldhA was recovered from the obtained cells.
- AAGGTACCACCACCAGAGCGTTCTCAAGC SEQ ID NO: 21
- GCTCTAGATTCTCCAGTGATGTTTGAATCAC SEQ ID NO: 22
- GTCTAGAGCAATGATCTACACGATTCG (SEQ ID NO: 23) prepared based on the sequence information of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter of Escherichia coli MG1655 strain and the ldhA gene of Escherichia coli MG1655 strain were prepared.
- PCR was performed using the previously prepared plasmid pGAPldhA as a template using AACTGCAGGTTCGTTCTCATACACGTCC (SEQ ID NO: 24) to obtain a DNA fragment of about 850 bp consisting of the GAPDH promoter and the region near the start codon of the ldhA gene.
- the fragments obtained above were digested with restriction enzymes KpnI and XbaI, XbaI and PstI, respectively, and this fragment was mixed with a fragment obtained by digesting the temperature sensitive plasmid pTH18cs1 with KpnI and PstI and ligated using ligase. Thereafter, DH5 ⁇ competent cells (Toyobo Co., Ltd., DNA-903) were transformed at 30 ° C. to obtain transformants that grew on LB agar plates containing 10 ⁇ g / ml of chloramphenicol. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 ⁇ g / ml of chloramphenicol, and the plasmid was recovered from the obtained bacterial cells and named pTH-GAPldhA.
- the obtained plasmid pTH-GAPldhA was transformed into the Escherichia coli MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp strain obtained in Example 5, and cultured at 30 ° C. overnight on an LB agar plate containing 10 ⁇ g / ml of chloramphenicol. Got.
- the obtained transformant was inoculated into an LB liquid medium containing 10 ⁇ g / ml of chloramphenicol and cultured at 30 ° C. overnight. Next, it was applied to an LB agar plate containing 10 ⁇ g / ml of chloramphenicol so as to obtain these cultured cells, and colonies that grew at 42 ° C. were obtained.
- the obtained colonies were cultured overnight in an LB liquid medium not containing chloramphenicol at 30 ° C., and further applied to an LB agar plate not containing chloramphenicol to obtain colonies that grew at 42 ° C.
- GAPldhA genome insertion strain 100 colonies were picked up randomly from the colonies that appeared, and each was grown on an LB agar plate containing no chloramphenicol and an LB agar plate containing 10 ⁇ g / ml chloramphenicol. Selected clones. Furthermore, an approximately 800 bp fragment containing the GAPDH promoter and the ldhA gene is amplified from the chromosomal DNA of these target clones by PCR, a strain in which the ldhA promoter region is replaced by the GAPDH promoter is selected, and a clone satisfying the above is selected as MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / It was named GAPldhA genome insertion strain.
- Example 7 ⁇ Escherichia coli MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp ⁇ fruR / GAPldhA genome insertion strain>
- the entire base sequence of the genomic DNA of Escherichia coli is known (GenBank accession number U00096), and the base sequence of the fruR gene of Escherichia coli MG1655 has already been reported. That is, the fruR gene is described in 88028 to 89032 of the Escherichia coli MG1655 strain genomic sequence described in GenBank accession number U00096.
- TACTGCAGATCTCATAATACCGCCTCTGG SEQ ID NO: 25
- GCTCTAGATATAGCCATTGTACTGGTATGG SEQ ID NO: 26
- TATCTAGATGCTCGCGCTGTAGCTAGG Synthesized.
- fruR About 950 bp (hereinafter referred to as fruR) is obtained by performing PCR under normal conditions using the genomic DNA of Escherichia coli MG1655 strain as a template and the above primers in combination of SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.
- a DNA fragment of about 880 bp (hereinafter also referred to as a fruR-R fragment). This DNA fragment was separated and recovered by agarose electrophoresis, and the fruR-L fragment was digested with PstI and XbaI, and the fruR-R fragment was digested with XbaI and EcoRI, respectively.
- the plasmid pTH ⁇ fruR was transformed into the Escherichia coli MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion strain obtained in Example 6, and the MG1655 ⁇ pfl ⁇ dld ⁇ mdhAspG strain obtained by disrupting the fruR gene was obtained in the same manner as in Example 2.
- This strain was named as MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp ⁇ fruR / GAPldhA genome insertion strain.
- Example 8 ⁇ Construction of Escherichia coli O157-derived sucrose hydrolase (invertase) gene expression vector and the expression vector transformant>
- the amino acid sequence of Escherichia coli O157 invertase and the nucleotide sequence of the gene have already been reported. That is, the gene (cscA) encoding invertase is described in 3274383 to 3275816 of the Escherichia coli O157 strain genomic sequence described in GenBank accession number AE005174.
- GAPDH glyceraldehyde 3-phosphate dehydrogenase
- the genomic DNA of Escherichia coli MG1655 strain was used as a template, and the DNA fragment obtained was amplified by PCR with CGAGCTCACATATGCAAGTATTACACGATCTCCG (SEQ ID NO: 29) and TCTAGAGCTATTTTGTTAGTGAAATAAAGG (SEQ ID NO: 30).
- the DNA fragment corresponding to the GAPDH promoter of about 110 bp was obtained by digestion.
- the obtained DNA fragment and the plasmid pBR322 (GenBank accession number J01749) were mixed with fragments obtained by digesting with restriction enzymes NdeI and PvuII, combined with ligase, and then Escherichia coli DH5 ⁇ strain competent cell (Toyo) Spinning Co., Ltd. DNA-903) was transformed to obtain a transformant that grew on an LB agar plate containing 50 ⁇ g / mL of ampicillin. The obtained colonies were cultured overnight at 37 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pBRgapP was recovered from the obtained cells.
- Escherichia coli O157 genomic DNA (SIGMA-ALDRICH: IRMM449) was used as a template, and GATCTAGACGGGAGAAGTCTTATGACGCAATCTCGATTTGCATG (SEQ ID NO: 31), and ATGGTACCTTAACCCAGTTGGCAGTG PCR method was obtained.
- the obtained DNA fragment was digested with the restriction enzyme XbaI to obtain an invertase gene fragment of about 1.4 kbp.
- the obtained DNA fragment and the previously prepared plasmid pBRgapP were digested with restriction enzymes XbaI and PshAI, mixed together using ligase, and then Escherichia coli DH5 ⁇ strain competent cell (Toyobo Co., Ltd.) Company transformant DNA-903) was transformed to obtain transformants that grew on LB agar plates containing ampicillin 50 ⁇ g / mL.
- the obtained colonies were cultured overnight at 37 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pGAP-cscA was recovered from the obtained cells.
- This plasmid pGAP-cscA was transformed into the MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp ⁇ fruR / GAPldhA genomic insert competent cell prepared in Example 7 and cultured at 37 ° C. overnight on ⁇ LBrth, Miller agar plate containing ⁇ fdr / ⁇ r A GAPldhA genome insertion strain / pGAP-cscA strain was obtained.
- the MG1655 ⁇ pfl ⁇ dld ⁇ GddhAdhAd / Aml / AdhdAgd strain / MG1655 ⁇ pfl ⁇ dldAGdA / GAPldhA genome-inserted strain was transformed into a competent cell and cultured at 37 ° C. overnight on an LB Broth, Miller agar plate containing ampicillin 50 ⁇ g / mL. A pGAP-cscA strain was obtained.
- Example 9 ⁇ Construction of Escherichia coli O157-derived invertase gene, Escherichia coli MG1655-derived fructose-1-phosphate kinase gene expression vector and the expression vector transformant>
- the amino acid sequence of the fructose-1-phosphate kinase of Escherichia coli MG1655 and the nucleotide sequence of the gene have already been reported. That is, a gene (fruK) encoding fructose-1-phosphate kinase is described in 2260387-2259449 of the Escherichia coli MG1655 strain genome sequence described in GenBank accession number U00096.
- fructose-1-phosphate kinase gene In order to obtain a fructose-1-phosphate kinase gene, using the genomic DNA of Escherichia coli MG1655 as a template, ATGGTACCGGAGAAAGTCTTATGGAGCAGACGTGTTCCTAC (SEQ ID NO: 33), and TCGGATCCTTATGCCTCTCCCTGCTGTCAG (SEQ ID NO: 34) were obtained by PCR. The obtained DNA fragment was digested with the restriction enzyme KpnI to obtain a fructose-1-phosphate kinase gene fragment of about 1.0 kbp.
- the resulting DNA fragment was mixed with the fragments obtained by digesting the plasmid pGAP-cscA prepared in Example 8 with restriction enzymes KpnI and EcoRV, and ligated using ligase, and then Escherichia coli DH5 ⁇ strain competent cell. (Toyobo Co., Ltd. Sakai DNA-903) was transformed to obtain a transformant that grows on an LB agar plate containing 50 ⁇ g / mL of ampicillin. The obtained colonies were cultured overnight at 37 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pGAP-cscA-fruK was recovered from the obtained cells.
- This plasmid pGAP-cscA-fruK was transformed into the MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genomic insert competent cell prepared in Example 6 and cultured overnight at 37 ° C. on LB Broth, Miller agar plates containing ampicillin 50 ⁇ g / mL. MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion strain / pGAP-cscA-fruK strain was obtained.
- Example 10 ⁇ MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp ⁇ fruR / GAPldhA genome insert strain / pGAP-cscA strain, MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insert strain / pGAP-cscA-fGAP strain / MG1655 ⁇ pDAP gene
- MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp ⁇ fruR / GAPldhA genome insertion strain / pGAP-cscAu strain (hereinafter referred to as “F disruption”) obtained in Example 8 was added to three 500 ml baffled Erlenmeyer flasks containing 25 ml of LB Broth and Miller culture medium (Difco244620).
- the medium composition was also the same as in Example 10, but sucrose was used after filter filtration sterilization.
- the D-lactic acid concentration in the culture solution after 48 hours of culture was 0 g / L.
- the concentrations of glucose and fructose in the culture were also 0 g / L. From this result, it was confirmed that deletion of the cscA gene failed to assimilate sucrose and produce lactic acid.
- Example 11 ⁇ D-lactic acid production from molasses by MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp ⁇ fruR / GAPldhA genome insertion strain / pGAP-cscA strain>
- the production of D-lactic acid from molasses of MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp ⁇ fruR / GAPldhA genome inserted strain / pGAP-cscA strain was examined.
- the same amount of pre-cultured flask contents (25 ml) as in Example 10 was inoculated into 475 g of the medium shown in Table 3 below.
- Example 12 ⁇ Construction of Bifidobacterium-derived ldh2 gene expression vector and the expression vector transformant MG1655 ⁇ pfl / pGAP-ldh2 strain>
- the amino acid sequence of L-lactate dehydrogenase of Bifidobacterium longum and the base sequence of the gene have already been reported. That is, a gene (ldh2) encoding L-lactate dehydrogenase is described in Bifidobacterium genome sequence 555-1517 described in GenBank accession number M33585.
- GAPDH glyceraldehyde 3-phosphate dehydrogenase
- the genomic DNA of Escherichia coli MG1655 strain was used as a template, and the DNA fragment obtained was amplified by PCR with CGAGCTCACATATGCAAGTATTACACGATCTCCG (SEQ ID NO: 29) and TCTAGAGCTATTTTGTTAGTGAAATAAAGG (SEQ ID NO: 30).
- the DNA fragment corresponding to the GAPDH promoter of about 110 bp was obtained by digestion.
- the obtained DNA fragment and the plasmid pBR322 (GenBank accession number J01749) were mixed with fragments obtained by digesting with restriction enzymes NdeI and PvuII, combined with ligase, and then Escherichia coli DH5 ⁇ strain competent cell (Toyo) Spinning Co., Ltd. DNA-903) was transformed to obtain a transformant that grew on an LB agar plate containing 50 ⁇ g / mL of ampicillin. The obtained colonies were cultured overnight at 37 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pBRgapP was recovered from the obtained cells.
- the obtained DNA fragment was digested with the restriction enzyme XbaI to obtain an L-lactate dehydrogenase gene fragment of about 1.0 kbp.
- the obtained DNA fragment and the previously prepared plasmid pBRgapP were digested with the restriction enzyme XbaI, mixed together using ligase, and then Escherichia coli DH5 ⁇ strain competent cell (Toyobo Co., Ltd. Sakai DNA) -903) to obtain transformants that grow on LB agar plates containing 50 ⁇ g / mL of ampicillin.
- the obtained colonies were cultured overnight at 37 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and plasmid pGAP-ldh2 was recovered from the obtained cells.
- This plasmid pGAP-ldh2 was transformed into a competent cell of the MG1655 strain (referred to as MG1655 ⁇ pfl strain) from which the pfl gene had been deleted in the same manner as in Example 2 using pTH ⁇ pfl prepared in Example 3, and 50 ⁇ g of ampicillin was used.
- MG1655 ⁇ pfl / pGAP-ldh2 strain was obtained by culturing overnight at 37 ° C. on LB Broth, Miller agar plate containing / mL.
- Example 13 ⁇ L-lactic acid production by MG1655 ⁇ pfl / pGAP-ldh2 strain>
- L-lactic acid production from glucose of the MG1655 ⁇ pfl / pGAP-ldh2 strain obtained in Example 12 was examined. 25 ml of the flask contents precultured in the same manner as that obtained in Example 10 was inoculated into 475 g of the medium shown in Table 4 below.
- L-lactic acid concentration in the culture solution after 18 hours of culture was 97.02 g / L. From this result, it was confirmed that L-lactic acid can be produced from glucose using L-lactic acid dehydrogenase derived from Bifidobacterium.
- Example 14 ⁇ Construction of MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion strain / pGAP-ldh2 strain>
- a transformant was prepared by introducing the pGAP-ldh2 plasmid prepared in Example 12 into the D-lactic acid producing strain constructed in Example 6. Specifically, it was performed as follows. Plasmid pGAP-ldh2 was transformed into a competent cell of MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion strain prepared in Example 6.
- MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion strain / pGAP-ldh2 strain was obtained by culturing overnight at 37 ° C. on an LB Broth, Miller agar plate containing 50 ⁇ g / mL of ampicillin.
- Example 15 ⁇ L-lactic acid production by MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome inserted strain / pGAP-ldh2 strain>
- L-lactic acid production from glucose of MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome inserted strain / pGAP-ldh2 strain obtained in Example 14 was examined. Cultivation was performed at atmospheric pressure for 18 hours with an aeration rate of 0.25 L / min, a stirring speed of 200 rpm, a culture temperature of 35 ° C., and a pH of 7.5 (adjusted with 24% NaOH).
- the concentration of L-lactic acid in the culture solution after 18 hours of culture was 116.84 g / L. From this result, it was confirmed that L-lactic acid can be produced using glucose as a raw material using an E. coli strain for D-lactic acid production. The production of L-lactic acid was confirmed by measuring the amount of L-lactic acid and the amount of D-lactic acid according to F-kit D- / L-lactic acid (Jay K. International product number 1112821).
- Example 16 ⁇ Construction of MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA / GAPldh2 genome insertion strain and MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA ⁇ fruR / GAPldh2 genome insertion strain>
- the ldhA gene in the Escherichia coli strain for production of D-lactic acid used in Example 6 (MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion strain) was replaced with the ldh2 gene, and the gene lddD, an enzyme that catalyzes the degradation of L-lactic acid, was destroyed.
- An E. coli strain for lactic acid production was constructed.
- the fruR gene was disrupted to construct an E. coli fruR disrupted strain for L-lactic acid production. Specifically, it was performed as follows.
- pTH ⁇ ldhA was transformed into a MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion strain competent cell, and an ldhA deletion strain was selected using the same method as in Example 2.
- the obtained strain was designated as MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion ⁇ ldhA strain.
- oligonucleotide primers CAACACCCAAGCTTTCGCG (SEQ ID NO: 40) and TGTTCTAGAAAGTTCTTTGAC (SEQ ID NO: 41) were synthesized based on the gene information of the region near the dld gene of E. coli MG1655 genomic DNA. Using these primers, PCR was carried out using the genomic DNA of E. coli MG1655 as a template, and the obtained DNA fragment was cleaved with restriction enzymes HindIII and XbaI.
- plasmid pTH18cs1 was cleaved with restriction enzymes HindIII and XbaI, mixed with the dld fragment, and ligated with ligase to construct a plasmid pTHLDD. Furthermore, pTHLDD was transformed into MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insert strain competent cells, and a dld-reverted strain was selected using the same method as in Example 2. The obtained strain was named as MG1655 ⁇ pfl ⁇ mdh ⁇ asp / GAPldhA genome insertion ⁇ ldhA strain.
- oligonucleotide primers GGTCTAGAGCAATGATTGACACGATTCCG SEQ ID NO: 48
- CGGAATTCCGCCTATTGTGTTAGGAATAAAAG SEQ ID NO: 49
- a plasmid obtained by cleaving pTH ⁇ ldhA obtained above with XbaI, the EcoRI-XbaI fragment of Bifidobacterium longum-derived ldh2 obtained above, and the EcoRI-XbaI fragment of the E. coli-derived GAPDH promoter were mixed, and ligase was used. After binding, Escherichia coli DH5 ⁇ strain competent cells (Toyobo Co., Ltd., DNA-903) were transformed to obtain transformants that grew on LB agar plates containing ampicillin 50 ⁇ g / mL. The obtained colony was cultured overnight at 37 ° C.
- the plasmid pTH ⁇ ldhA :: GAPLDH2 was recovered from the obtained bacterial cells.
- the obtained plasmid was transformed into the MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lld / GAPldhA genome insertion ⁇ ldhA strain, and the ldh2 genome insertion strain was selected by PCR amplification using the same method as in Example 2.
- the resulting strain was designated as MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA / GAPldh2 genome insertion strain.
- the plasmid pTH ⁇ fruR prepared in Example 7 was transformed into the MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA / GAPldh2 genome insertion strain, and the MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ hldDGldhGdAdD1 strain was obtained by disrupting the fruR gene in the same manner as in Example 2. This strain was designated as MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA ⁇ fruR / GAPldh2 genome insertion strain.
- Example 17 ⁇ Construction of MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA / GAPldh2 genomic insert / pGAP-cscA strain and MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA ⁇ fruR / GAPldh2 genomic insert / pGAP-cscA strain> Escherichia coli producing L-lactic acid from sucrose by introducing a sucrose hydrolase (invertase) gene expression vector into each of the L-lactic acid-producing E. coli strain and the L-lactic acid-producing E. coli fruR disrupted strain constructed in Example 16.
- a stock was built. Specifically, it was performed as follows.
- the plasmid pGAP-cscA constructed in Example 8 was transformed into the MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA / GAPldh2 genomic insertion strain and MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA ⁇ fruR / GAP1 lign cell strain B MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA / GAPldh2 genomic insert / pGAP-cscA strain and MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ ldhA ⁇ fruR / GAPldhAP genomic strain / GAPldh2APc strain were cultivated overnight at 37 ° C. on the plate.
- Example 18 ⁇ MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA / GAPldh2 genome insertion / pGAP-cscA-fruK strain construction> A sucrose hydrolase (invertase) and fructose-1-phosphate kinase gene expression vector were introduced into the E. coli strain for L-lactic acid production constructed in Example 16, and an L-lactic acid-producing Escherichia coli fruK-enhanced strain was constructed. Specifically, it was performed as follows.
- the plasmid pGAP-cscA-fruK constructed in Example 9 was transformed into a competent cell of the MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA / GAPldh2 genomic insert prepared in Example 16, and LB Broth, Miller ° C. agar plate containing ampicillin 50 ⁇ g / mL By culturing overnight, MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA / GAPldh2 genome insert / pGAP-cscA-fruK strain was obtained.
- Culturing was carried out under atmospheric pressure for 24 hours at an aeration rate of 0.25 L / min, a stirring speed of 350 rpm, a culture temperature of 35 ° C., and a pH of 7.5 (adjusted with 24% NaOH).
- the concentration of L-lactic acid in the culture medium after 24 hours of culture was 75.12 g / L for the MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA / GAPldh2 genome insert / pGAP-cscA strain (cscA), MG1655 ⁇ pfl ⁇ mdh ⁇ aspRhGrAPG strain was 83.79 g / L, and MG1655 ⁇ pfl ⁇ mdh ⁇ asp ⁇ lldD ⁇ ldhA / GAPldh2 genome insert / pGAP-cscA-fruK strain (fruK-enhanced strain) was 84.32 g / L.
- L-lactic acid can be produced from the molasses as a raw material using the lactic acid-producing Escherichia coli of the present invention. It was also clarified that the productivity of L-lactic acid is improved by disrupting the fruR gene of lactic acid-producing Escherichia coli. Furthermore, it has been clarified that the productivity of L-lactic acid is improved by strengthening the fruK gene of lactic acid-producing Escherichia coli.
- a DNA fragment was amplified by a PCR method in combination with SEQ ID NO: 29.
- the primer of SEQ ID NO: 29 has an NdeI recognition site on the 5 ′ end side
- the primer of SEQ ID NO: 50 has HindIII, PstI, SalI, BamHI, and SacI recognition sites in this order from the 5 ′ end side.
- the obtained DNA fragment was digested with restriction enzymes NdeI and HindIII to obtain a fragment encoding the GAPDH promoter of about 100 bp.
- the above DNA fragment was mixed with the Escherichia coli DH5 ⁇ competent cell (manufactured by Takara Bio Inc.) after mixing with the above-mentioned DNA fragment and the cloning vector pBR322 (GenBank accession number J01749) digested with NdeI and HindIII, and ligated with ligase.
- pBR322 GenBank accession number J01749
- the obtained transformants that grow on LB agar plates containing 50 ⁇ g / mL of ampicillin.
- the obtained colonies were cultured overnight at 30 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid was recovered from the obtained bacterial cells.
- This plasmid was named pGAP.
- the base sequence of the invertase gene (cscA) possessed by Escherichia coli O157 strain has already been reported. That is, it is described in Escherichia coli O157 strain genome sequence 3274383-3275816 described in GenBankBaccession number AE005174.
- primers having base sequences of GCGGATCCGCTGGTGGAATATAGACGCAATCTCGATTGC SEQ ID NO: 51
- GACGTCGAACTTAACCCAGTGCCAGATGC SEQ ID NO: 52
- the primer of SEQ ID NO: 51 has a BamHI recognition site and a 13-base GAPDH gene ribosome binding sequence in this order from the 5 ′ end.
- the primer of SEQ ID NO: 52 has a SalI recognition site on the 5 ′ end side.
- PCR is performed under normal conditions using the genomic DNA of Escherichia coli O157 strain (SIGMA-ALDRICH: IRMM449) as a template, and the resulting DNA fragment is digested with restriction enzymes BamHI and SalI. A 1.4 kbp invertase gene (cscA) fragment was obtained.
- This DNA fragment and the fragment obtained by digesting plasmid pGAP with restriction enzymes BamHI and SalI were mixed, ligated using ligase, and then transformed into Escherichia coli DH5 ⁇ competent cell (manufactured by Takara Bio Inc.). A transformant that grew on an LB agar plate containing 50 ⁇ g / mL of ampicillin was obtained. The obtained colonies were cultured overnight in an LB liquid medium containing 50 ⁇ g / mL of ampicillin at 30 ° C., and plasmid pGAP-cscA was recovered from the obtained cells to construct an invertase gene (cscA) expression vector.
- cscA invertase gene
- the base sequence of the sugar transporter glucose transport promoting protein gene (glf) possessed by Zymomonas mobilis has already been reported (GenBank accession number M60615).
- primers having the base sequences of CCTGTCGACGCTGGTGGAATATATGAGTTCTGAAAGTAGTCAGGG SEQ ID NO: 53
- CTACTGCAGCTACTTCTGGGAGCGCCCACA SEQ ID NO: 54
- the primer of SEQ ID NO: 53 has a SalI recognition site and a 13-base GAPDH gene ribosome binding sequence in this order from the 5 ′ end.
- the primer of SEQ ID NO: 54 has a PstI recognition site on the 5 ′ end side.
- PCR is performed under normal conditions using Zymomonas mobilis genomic DNA as a template, and the resulting DNA fragment is digested with the restriction enzymes SalI and PstI to promote glucose transport enzyme glucose transport at about 1.4 kbp.
- a protein gene (glf) fragment was obtained.
- This DNA fragment and the fragment obtained by digesting plasmid pGAP-cscA with restriction enzymes SalI and PstI were mixed, ligated with ligase, and transformed into Escherichia coli DH5 ⁇ competent cell (manufactured by Takara Bio Inc.).
- a transformant that grew on an LB agar plate containing 50 ⁇ g / mL of ampicillin was obtained.
- the obtained colonies were cultured overnight at 30 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pGAP-cscA-glf was recovered from the obtained cells to promote the invertase (cscA) gene and glucose transport.
- cscA invertase
- glf protein
- the concentration of D-lactic acid in the culture medium after 48 hours was 48.9 g / L for cscA, and 9.3 g / L for MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion strain / pGAP-cscA-glf strain. From these results, it can be seen that, as with cscA, the glucose transport enhancing protein gene (glf), which is a gene related to the sugar metabolism system, is used to enhance the uptake of sugar, and the effect of improving lactic acid productivity is not observed. I understood.
Abstract
Description
但しいずれの用途においても、原料たる乳酸には高い光学純度が要求されるのが事実である。
また、安価な糖原料であるスクロースから高選択的にD-乳酸を生産する大腸菌も知られている(Biotechnolohy Letters,Vol.27,pp.1891-1896(2005)参照)。しかし、スクロースからD-乳酸を生産する大腸菌は生産性が低く、また、スクロースの資化に非常に時間がかかり、工業化において課題となっていた。
L-乳酸については、グルコースを原料としてL-乳酸を高い選択性で高生産する大腸菌は知られているものの(特開2007-49993号公報)、スクロースからL-乳酸を生産する大腸菌はなかった。
[1] スクロース非PTS遺伝子群のうちスクロース加水分解酵素遺伝子を少なくとも含む1種以上の遺伝子(ただし、リプレッサー蛋白質(cscR)、スクロース加水分解酵素(cscA)、フルクトキナーゼ(cscK)及びスクロース透過酵素(cscB)の組み合わせと、スクロース加水分解酵素(cscA)、フルクトキナーゼ(cscK)及びスクロース透過酵素(cscB)の組み合わせとを除く)を有し、且つ、遺伝子組換えによる乳酸生産強化系を備えている乳酸生産大腸菌。
[2] 前記スクロース非PTS遺伝子群の中で、スクロース加水分解酵素遺伝子のみを有し、且つ遺伝子組み換えによる乳酸生産強化系を備えている[1]記載の乳酸生産大腸菌。
[3] 前記乳酸生産大腸菌が、フルクトースの代謝能力向上系を更に有する[1]又は[2]記載の乳酸生産大腸菌。
[4] 前記乳酸生産強化系が、ピルベートホルメートリアーゼ活性の不活化あるいは低減化を含む[1]~[3]のいずれかに記載の乳酸生産大腸菌。
[5] 前記乳酸生産強化系が、D-乳酸又はL-乳酸を生成するためのNADH依存性乳酸デヒドロゲナーゼ活性の増強を含む[1]~[4]のいずれかに記載の乳酸生産大腸菌。
[6] 前記乳酸生産強化系が、D-乳酸デヒドロゲナーゼ活性の増強と、該大腸菌が本来有しているFAD依存型D-乳酸デヒドロゲナーゼ活性の不活化あるいは低減化と、を含む[1]~[4]のいずれかに記載の乳酸生産大腸菌。
[7] 前記乳酸生産強化系が、L-乳酸デヒドロゲナーゼ活性の増強と、該大腸菌が本来有しているD-乳酸デヒドロゲナーゼ活性及びFMN依存性L-乳酸デヒドロゲナーゼ活性の少なくともどちらか一方の不活化あるいは低減化と、を含む[1]~[4]のいずれかに記載の乳酸生産大腸菌。
[8] 前記フルクトースの代謝能力向上系が、フルクトース代謝経路におけるリン酸化能力の強化またはフルクトース取り込み能力の強化である[3]~[7]のいずれかに記載の乳酸生産大腸菌。
[9] 前記フルクトース代謝経路におけるリン酸化能力の強化が、フルクトース-1-リン酸キナーゼ活性に由来する[8]に記載の乳酸生産大腸菌。
[10] 前記フルクトース代謝経路におけるフルクトース取り込み能力の強化が、該大腸菌が本来有しているFruR活性の不活化あるいは低減化に由来する[8]に記載の乳酸生産大腸菌。
[11] 前記スクロース加水分解酵素遺伝子が、エシェリヒア属細菌に由来する[1]~[10]のいずれかに記載の乳酸生産大腸菌。
[12] 前記スクロース加水分解酵素遺伝子が、エシェリヒア・コリO157細菌に由来する[1]~[10]のいずれかに記載の乳酸生産大腸菌。
[13] 前記フルクトース-1-リン酸キナーゼが、エシェリヒア属細菌に由来する[9]~[12]のいずれかに記載の乳酸生産大腸菌。
[14] 前記フルクトース-1-リン酸キナーゼがエシェリヒア・コリMG1655由来の蛋白質である[9]~[12]のいずれかに記載の乳酸生産大腸菌。
[15] 大腸菌K12由来株である[1]~[14]のいずれかに記載の乳酸生産大腸菌。
[16] [1]~[15]のいずれかに記載の乳酸生産大腸菌を用いて、スクロースを含む植物由来原料から乳酸を生産することを含む乳酸生産方法。
本発明の乳酸生産方法は、上記乳酸生産細菌を用いて、スクロースを含む植物由来原料から乳酸を生産することを含む乳酸生産方法である。
本発明は、スクロース非PTS遺伝子群を全てではなく不完全な状態で、即ち、スクロース加水分解酵素遺伝子を少なくとも含む1種以上の遺伝子を、乳酸生産大腸菌に導入することによって、スクロース由来フルクトースが高効率に資化されること、更には従来よりも著しく生産性が高まることを見出したものである。この結果、植物に由来し、安価で工業的に価値の高いスクロースから、効率よく短時間で乳酸を得ることができる。
一般に、大腸菌では通常グルコースの取り込みがフルクトースよりも優先され、グルコースの存在下ではフルクトースは充分に代謝されないことが知られている。また、糖代謝は生物にとって基本的な機能である。このため、フルクトース代謝経路のリン酸化活性又はフルクトース取り込み能力を強化することで、細菌の生育が阻害されず、またグルコースによる代謝抑制(カタボライトリプレッション)の影響を受けずに、効率よく乳酸の生産を行うことができたことは、驚くべきことである。
なかでも、乳酸を更に効率よく生産するという観点から、cscAをコードする遺伝子のみを有し、その他の遺伝子を含まないことが好ましい。
この酵素は、K12株等の大腸菌には本来保有されていない酵素であり、プロトン共輸送体、インベルターゼ、フルクトキナーゼ及びスクロース特異的リプレッサーを含む非PTS代謝経路の酵素の1つである(Canadian Journal of Microbiology, (1991) vol.45, pp418-422参照)。本発明においてこのCscAを付与することにより、特にcscAのみを付与することにより、菌体外におけるスクロースを細胞膜上でグルコース及びフルクトースに分解して細胞外へ放出し、グルコースPTS及びフルクトースPTSを介して細胞質内にリン酸化して取り込む。この結果、フルクトースを細菌におけるフルクトース代謝系へ供給して、解糖系を利用した資化を可能にすることができる。
本発明におけるフルクトースの代謝能力が向上しているとは、フルクトースの菌体内への取り込みが増加している状態を指す。フルクトースの代謝能力向上系とは、フルクトースの代謝能力を向上させるための構造を意味する。
なお、本発明において「宿主」とは、ひとつ以上の遺伝子の菌体外からの導入を受けた結果、本発明の乳酸生産大腸菌となる当該大腸菌を意味する。
本明細書中で示された数値範囲は、記載された数値をそれぞれ最小値及び最大値として含む範囲を示す。
なお、本発明における「遺伝子組み換えにより」との文言は、生来の遺伝子の塩基配列に対する別のDNAの挿入、あるいは遺伝子のある部分の置換、欠失又はこれらの組み合わせによって塩基配列上の変更が生じているものであれば全て包含し、例えば、突然変異が生じた結果得られたものであってもよい。
本発明における「低減化」とは、当該酵素又は転写因子であるFruRをコードする遺伝子の遺伝子組換えにより、それらの処理を行う前の状態よりも有意に当該酵素又はFruRの活性が低下している状態を指す。ここでいうFruRの活性とは、FruRによって制御される遺伝子の発現によって生じる蛋白質の量、又は蛋白質の機能を定量化したものを指す。
本発明において乳酸デヒドロゲナーゼ活性の増強とは、LdhA又はLdh2をコードする遺伝子の遺伝子組換えにより、それらの処理を行う前の状態よりも、有意にLdhA又はLdh2をコードする遺伝子より生産される酵素の活性が増加した状態を指す。
本発明におけるDld活性が不活化あるいは低減化されている、且つ/またはPfl活性が不活化あるいは低減化されている、且つ/またはLdhA活性が増強されていることを特徴とする微生物として、WO2005/033324号に記載のエシェリヒア・コリMT-10994(FERM BP-10058)株が例示できる。
本発明におけるプロモーターとはシグマ因子を有するRNAポリメラーゼが結合し、転写を開始する部位を意味する。例えばエシェリヒア・コリ由来のGAPDHプロモーターはGenBank accession number X02662の塩基配列情報において、塩基番号397-440に記されている。
エシェリヒア・コリMT-10994株は、ldhA遺伝子をゲノム上においてGAPDHプロモーターと機能的に連結することで発現させており、また遺伝子破壊によりPflB、Dldが不活化している。該菌株は、FERM BP-10058の寄託番号で、茨城県つくば市東1丁目1番1号中央第6の独立行政法人産業技術総合研究所 特許生物寄託センターに、特許手続上の微生物の寄託等の国際的承認に関するブタペスト条約に基いて、平成16年3月19日より寄託されている。
本発明におけるフルクトース取り込み能力の強化とは、FruRが制御する酵素活性が、FruRをコードする遺伝子の遺伝子組み換えにより、それらの処理を行う前の状態よりも、有意に当該酵素活性が低下している状態を指す。
本発明における酵素の活性は、既存の測定系のいずれによって測定された活性であってもよい。
また各種遺伝子を不活性化させるための手段としては、この目的で通常用いられる手段であれば特に制限されずに用いることができ、例えば相同組換え等による遺伝子破壊を挙げることができる。
なお、本発明に使用される培地としては、工業的生産に供する点を考慮すれば液体培地が好ましい。
上述した通気条件は培養初期から終了まで一貫して行う必要はなく、培養工程の一部で行うことでも好ましい結果を得ることができる。
本発明における培養物とは、上述した方法により生産された菌体、培養液、及びそれらの処理物を指す。
<エシェリヒア・コリMG1655株dld遺伝子欠失株の作製>
エシェリヒア・コリのゲノムDNAの全塩基配列は公知であり(GenBank accession number U00096)、エシェリヒア・コリのFAD依存性D-乳酸デヒドロゲナーゼをコードする遺伝子(以下dldと略すことがある)の塩基配列も報告されている(Genbank accession number M10038)。
なおエシェリヒア・コリMG1655株は細胞・微生物・遺伝子バンクであるアメリカンタイプカルチャーコレクションより入手することができる。
実施例1で得られたプラスミドpTHΔdldをMG1655株に30℃で形質転換し、クロラムフェニコール10μg/mlを含むLB寒天プレートに生育する形質転換体を得た。得られた形質転換体を寒天プレートに塗布し、30℃で一晩培養した。次にこれらの培養菌体が得られるようにクロラムフェニコール10μg/mlを含むLB寒天プレートに塗布し、42℃で生育するコロニーを得た。
さらにもう一度、42℃で生育するシングルコロニーを得る操作を繰り返し、相同組換えによりプラスミド全体が染色体に組込まれたクローンを選択した。本クローンがプラスミドを細胞質中に持たないことを確認した。
<エシェリヒア・コリMG1655pflB、dld遺伝子欠失株作製>
エシェリヒア・コリのゲノムDNAの全塩基配列は公知であり(GenBank accession number U00096)、エシェリヒア・コリのピルベートホルメートリアーゼをコードする遺伝子(pflB)の塩基配列も報告されている(Genbank accession number X08035)。pflB遺伝子の塩基配列近傍領域をクローニングするため、GCACGAAAGCTTTGATTACG(配列番号5)、TTATTGCATGCTTAGATTTGACTGAAATCG(配列番号6)TTATTGCATGCTTATTTACTGCGTACTTCG(配列番号7)AAGGCCTACGAAAAGCTGCAG(配列番号8)のオリゴヌクレオチドプライマーを4種合成した。
得られたクローンから、実施例2と同様の方法に従ってpfl遺伝子が破壊されたMG1655Δdld株を得、MG1655ΔpflΔdld株と命名した。
<エシェリヒア・コリMG1655ΔpflΔdldΔmdh株の作製>
エシェリヒア・コリのゲノムDNAの全塩基配列は公知であり(GenBank accession number U00096)、エシェリヒア・コリのmdh遺伝子の塩基配列も報告されている(Genbank accession number M24777)。mdh遺伝子(939bp)の塩基配列近傍領域をクローニングするため、AAAGGTACCAGAATACCTTCTGCTTTGCCC(配列番号9)、AAAGGATCCCCTAAACTCCTTATTATATTG(配列番号10)、AAAGGATCCAAACCGGAGCACAGACTCCGG(配列番号11)及びAAATCTAGAATCAGATCATCGTCGCCTTAC(配列番号12)のオリゴヌクレオチドプライマーを4種合成した。
<エシェリヒア・コリMG1655ΔpflΔdldΔmdhΔasp株の作製>
エシェリヒア・コリのゲノムDNAの全塩基配列は公知であり(GenBank accession number U00096)、エシェリヒア・コリのaspA遺伝子の塩基配列も報告されている(Genbank accession number X04066)。aspA遺伝子(1,482bp)の塩基配列近傍領域をクローニングするため、TTTTGAGCTCGATCAGGATTGCGTTGGTGG(配列番号13)、CGAACAGTAATCGTACAGGG(配列番号14)、TACGATTACTGTTCGGCATCGACCGAATACCCGAG(配列番号15)及びTTTTTCTAGACCTGGCACGCCTCTCTTCTC(配列番号16)に示すオリゴヌクレオチドプライマーを4種合成した。
<エシェリヒア・コリMG1655ΔpflΔdldΔmdhΔasp株のゲノム上ldhAプロモーターのGAPDHプロモーターへの置換>
エシェリヒア・コリのldhA遺伝子の塩基配列はすでに報告されている(GenBank accession number U36928)。グリセルデヒド3-リン酸デヒドロゲナーゼ(GAPDH)プロモーターを取得するためエシェリヒア・コリMG1655株のゲノムDNAをテンプレートに用いてAACGAATTCTCGCAATGATTGACACGATTC(配列番号17)、及びACAGAATTCGCTATTTGTTAGTGAATAAAAGG(配列番号18)によりPCR法で増幅し、得られたDNAフラグメントを制限酵素EcoRIで消化することで約100bpのGAPDHプロモーターをコードするフラグメントを得た。さらにD-乳酸デヒドロゲナーゼ遺伝子(ldhA)を取得するためにエシェリヒア・コリMG1655株のゲノムDNAをテンプレートに用いてGGAATTCCGGAGAAAGTCTTATGAAACT(配列番号19)、及びCCCAAGCTTTTAAACCAGTTCGTTCGGGC(配列番号20)によりPCR法で増幅し、得られたDNAフラグメントを制限酵素EcoRI及びHindIIIで消化することで約1.0kbpのD-乳酸デヒドロゲナーゼ(ldhA)遺伝子フラグメントを得た。上記の2つのDNAフラグメントとプラスミドpUC18を制限酵素EcoRI及びHindIIIで消化することで得られるフラグメントを混合し、リガーゼを用いて結合した後、エシェリヒア・コリDH5αコンピテントセル(東洋紡績株式会社 DNA-903)に形質転換し、アンピシリン50μg/mLを含むLB寒天プレートに生育する形質転換体を得た。得られたコロニーをアンピシリン50μg/mLを含むLB液体培地で30℃で一晩培養し、得られた菌体からプラスミドpGAP-ldhAを回収した。
<エシェリヒア・コリMG1655ΔpflΔdldΔmdhΔaspΔfruR/GAPldhAゲノム挿入株の作製>
エシェリヒア・コリのゲノムDNAの全塩基配列は公知であり(GenBank accession number U00096)、エシェリヒア・コリMG1655のfruR遺伝子の塩基配列は既に報告されている。すなわち、fruR遺伝子はGenBank accession number U00096に記載のエシェリヒア・コリMG1655株ゲノム配列の88028~89032に記載されている。
<エシェリヒア・コリO157由来スクロース加水分解酵素(インベルターゼ)遺伝子発現ベクターおよび該発現ベクター形質転換体の構築>
エシェリヒア・コリO157のインベルターゼのアミノ酸配列と遺伝子の塩基配列は既に報告されている。すなわち、インベルターゼをコードする遺伝子(cscA)はGenBank accession number AE005174に記載のエシェリヒア・コリO157株ゲノム配列の3274383~3275816に記載されている。上記遺伝子がコードする蛋白質のN末端側には、アミノ酸一文字表記でMTQSRLHAA(配列番号35)で表記される、疎水性が高くシグナルペプチダーゼで切断されるアミノ酸配列と同様な配列が存在する。上記の遺伝子を発現させるために必要なプロモーターの塩基配列として、GenBank accession number X02662の塩基配列情報において、397-440に記されているエシェリヒア・コリ由来のグリセルアルデヒド3-リン酸デヒドロゲナーゼ(以下GAPDHと呼ぶことがある)のプロモーター配列を使用することができる。
また実施例6で作成したMG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株コンピテントセルに形質転換し、アンピシリン50μg/mLを含むLB Broth,Miller寒天プレートで37℃一晩培養することにより、MG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-cscA株を得た。
<エシェリヒア・コリO157由来インベルターゼ遺伝子、エシェリヒア・コリMG1655由来フルクトース-1-リン酸キナーゼ遺伝子発現ベクターおよび該発現ベクター形質転換体の構築>
エシェリヒア・コリMG1655のフルクトース-1-リン酸キナーゼのアミノ酸配列と遺伝子の塩基配列は既に報告されている。すなわち、フルクトース-1-リン酸キナーゼをコードする遺伝子(fruK)はGenBank accession number U00096に記載のエシェリヒア・コリMG1655株ゲノム配列の2260387~2259449に記載されている。
<MG1655ΔpflΔdldΔmdhΔaspΔfruR/GAPldhAゲノム挿入株/pGAP-cscA株、MG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-cscA-fruK株、MG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-cscA株によるD-乳酸生産>
前培養としてLB Broth、Miller培養液(Difco244620)25mlを入れた500ml容バッフル付三角フラスコ3本に、実施例8で得られたMG1655ΔpflΔdldΔmdhΔaspΔfruR/GAPldhAゲノム挿入株/pGAP-cscA株(以下、「fruR破壊株」又は「ΔfruR株」)、MG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-cscA株(以下、「cscA株」)、実施例9で得られたMG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-cscA-fruK株(以下、「fruK強化株」又は「+fruK株」)を各々植菌し、一晩35℃、120rpmで攪拌培養を行った。その後、3台の1L容培養槽(ABLE社製培養装置BMJ-01)に、表1に示す培地475gを入れたものに、別々に上記フラスコ内容物全量を植菌した。
カラム:ULTRON PS-80H(信和化工社製)
溶離液:過塩素酸水溶液(pH2.1)
流速:1.0mL/min
検出器:UV検出器
測定波長:280nm
このとき、全ての株において培養開始時に投入されたスクロースは完全になくなっていた。また、fruK遺伝子を導入またはfruR遺伝子を破壊することによって、スクロースの分解によって得られるフルクトースが、遺伝子の導入または破壊のない株に比べて早く資化されることが明らかとなった。
<MG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pBRgapP株によるD-乳酸生産>
実施例10と同様に、MG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pBRgapP株のD-乳酸生産を調べた。本菌株は、導入したプラスミドにcscA遺伝子が含まれていないだけで、基本的にMG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-cscA株と同等である。培地組成も実施例10と同じであるが、スクロースはフィルターろ過滅菌をして用いた。48時間培養後の培養液中のD-乳酸濃度は、0g/Lだった。この時、培養液中のグルコースとフルクトースの濃度も0g/Lだった。
この結果より、cscA遺伝子を欠失するとスクロースを資化して乳酸を生産できないことが確認された。
<MG1655ΔpflΔdldΔmdhΔaspΔfruR/GAPldhAゲノム挿入株/pGAP-cscA株による廃糖蜜からのD-乳酸生産>
実施例10と同様に、MG1655ΔpflΔdldΔmdhΔaspΔfruR/GAPldhAゲノム挿入株/pGAP-cscA株の廃糖蜜からのD-乳酸生産を調べた。
下記の表3に示す培地475g入れたものに、実施例10で得られたものと同様の前培養したフラスコ内容物全量(25ml)を植菌した。
48時間培養後の培養液中のD-乳酸濃度は、96.47g/Lだった。この時、培養液中のグルコースとフルクトース、スクロースの濃度は0g/Lだった。
この結果より、本発明の乳酸生産大腸菌を用いて、廃糖蜜を原料として乳酸を生産できることが確認された。
<ビフィドバクテリウム由来ldh2遺伝子発現ベクターおよび該発現ベクター形質転換体MG1655Δpfl/pGAP-ldh2株の構築>
ビフィドバクテリウム・ロンガム(Bifidobacterium longum)のL-乳酸デヒドロゲナーゼのアミノ酸配列と遺伝子の塩基配列は既に報告されている。すなわち、L-乳酸デヒドロゲナーゼをコードする遺伝子(ldh2)はGenBank accession number M33585に記載のビフィドバクテリウムゲノム配列の555~1517に記載されている。
上記の遺伝子を発現させるために必要なプロモーターの塩基配列として、GenBank accession number X02662の塩基配列情報において、397-440に記されているエシェリヒア・コリ由来のグリセルアルデヒド3-リン酸デヒドロゲナーゼ(以下GAPDHと呼ぶことがある)のプロモーター配列を使用することができる。
<MG1655Δpfl/pGAP-ldh2株によるL-乳酸生産>
実施例10と同様に、実施例12で得られたMG1655Δpfl/pGAP-ldh2株のグルコースからのL-乳酸生産を調べた。
下記の表4に示す培地475g入れたものに、実施例10で得られたものと同様に前培養したフラスコ内容物25mlを植菌した。
18時間培養後の培養液中のL-乳酸濃度は、97.02g/Lだった。
この結果より、ビフィドバクテリウム由来のL-乳酸デヒドロゲナーゼを用いて、グルコースからL-乳酸を生産できることを確認した。
<MG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-ldh2株の構築>
実施例12で作成したpGAP-ldh2プラスミドを実施例6で構築したD-乳酸生産株に導入した形質転換体を作成した。具体的には以下のように行った。
プラスミドpGAP-ldh2を実施例6で作成したMG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株のコンピテントセルに形質転換した。アンピシリン50μg/mLを含むLB Broth,Miller寒天プレートで37℃一晩培養することにより、MG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-ldh2株を得た。
<MG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-ldh2株によるL-乳酸生産>
実施例13と同様に、実施例14で得られたMG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-ldh2株のグルコースからのL-乳酸生産を調べた。
培養は大気圧下、通気量0.25L/min、攪拌速度200rpm、培養温度35℃、pH7.5(24% NaOHで調整)で18時間行った。
18時間培養後の培養液中のL-乳酸濃度は、116.84g/Lだった。
この結果より、D-乳酸生産用大腸菌株を用いて、グルコースを原料としてL-乳酸を生産できることを確認した。L-乳酸が生産されたことは、F-キット D-/L-乳酸(ジェイ・ケイ・インターナショナル 製品番号1112821)に従い、L-乳酸量、及びD-乳酸量を測定することで確認した。
<MG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入株及びMG1655ΔpflΔmdhΔaspΔlldDΔldhAΔfruR/GAPldh2ゲノム挿入株の構築>
実施例6で用いたD-乳酸生産用大腸菌株(MG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株)のldhA遺伝子をldh2遺伝子に置き換え、更にL-乳酸の分解を触媒する酵素の遺伝子lldDを破壊して、L-乳酸生産用大腸菌株を構築した。更にfruR遺伝子を破壊し、L-乳酸生産用大腸菌fruR破壊株を構築した。具体的には、以下のようにして行った。
MG1655ゲノムDNAのldhA遺伝子近傍領域の遺伝子情報に基づいて、AAGGTACCACCAGAGCGTTCTCAAGC(配列番号21)、GCTCTAGATTCTCCAGTGATGTTGAATCAC(配列番号22)、GCTCTAGAGCATTCCTGACAGCAGAAGC(配列番号38)及びAACTGCAGTCGGCGTGTAGTAGTGAACC(配列番号39)のオリゴヌクレオチドプライマーを4種合成した。これらのプライマーを用い、実施例1と同様の手法で遺伝子破壊用プラスミドpTHΔldhAを構築した。更に、pTHΔldhAをMG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株コンピテントセルに形質転換し、実施例2と同様な手法を用いてldhA欠失株を選択した。得られた株をMG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入ΔldhA株と命名した。
大腸菌MG1655ゲノムDNAのdld遺伝子近傍領域の遺伝子情報に基づいて、CAACACCAAGCTTTCGCG(配列番号40)、TGTTCTAGAAAGTTCTTTGAC(配列番号41)のオリゴヌクレオチドプライマーを2種合成した。これらのプライマーを用い、大腸菌MG1655のゲノムDNAをテンプレートにしてPCRを実施し、得られたDNA断片を制限酵素HindIII、XbaIで切断した。さらにプラスミドpTH18cs1を制限酵素HindIII、XbaIで切断し、上記dld断片と混合した後、リガーゼで結合し、プラスミドpTHDLDを構築した。更に、pTHDLDをMG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株コンピテントセルに形質転換し、実施例2と同様な手法を用いてdld復帰株を選択した。得られた株をMG1655ΔpflΔmdhΔasp/GAPldhAゲノム挿入ΔldhA株と命名した。
MG1655株ゲノムDNAのlldD遺伝子近傍領域の遺伝子情報に基づいて、GGAAGCTTCAAATTGGCGTCTCTGATCT(配列番号42)、AAACCCGGGCCATCCATATAGTGGAACAGGAACGG(配列番号43)、GGGCTCGAGTGGCGATGACGCTGACTGG(配列番号44)及びCGTCTAGAACGGGTAAATCTGGTGGTGACCGTCACCCG(配列番号45)のオリゴヌクレオチドプライマーを4種合成した。これらのプライマーを用い、実施例1と同様の手法で遺伝子破壊用プラスミドpTHΔlldDを構築した。更に、pTHΔlldDをMG1655ΔpflΔmdhΔasp/GAPldhAゲノム挿入ΔldhA株コンピテントセルに形質転換し、実施例2と同様な手法を用いてlldD欠失株を選択した。得られた株をMG1655ΔpflΔmdhΔaspΔlldD/GAPldhAゲノム挿入ΔldhA株と命名した。
ビフィドバクテリウム・ロンガムのL-乳酸デヒドロゲナーゼのアミノ酸配列と遺伝子の塩基配列は既に報告されている。すなわち、L-乳酸デヒドロゲナーゼをコードする遺伝子(ldh2)はGenBank accession number M33585に記載のビフィドバクテリウムゲノム配列の555~1517に記載されている。
L-乳酸デヒドロゲナーゼをコードする遺伝子(ldh2)を取得する為にビフィドバクテリウム・ロンガム(ATCC15707)のゲノムDNAをテンプレートに用いてAAGAATTCCGGAGAAAGTCTTATGGCGGAAACTACCGTTAAGC(配列番号46)、CTGTCTAGATCAGAAGCCGAACTGGGCG(配列番号47)のオリゴヌクレオチドプライマーを2種合成した。これらのプライマーを用いてPCRを実施し、得られたDNA断片を制限酵素EcoRI、及びXbaIで切断した。
得られた株をMG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入株と命名した。
実施例7で作成したプラスミドpTHΔfruRをMG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入株に形質転換し、実施例2と同様の方法に従って、fruR遺伝子が破壊されたMG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入株を取得した。本株をMG1655ΔpflΔmdhΔaspΔlldDΔldhAΔfruR/GAPldh2ゲノム挿入株と命名した。
<MG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入/pGAP-cscA株及びMG1655ΔpflΔmdhΔaspΔlldDΔldhAΔfruR/GAPldh2ゲノム挿入/pGAP-cscA株の構築>
実施例16で構築したL-乳酸生産用大腸菌株とL-乳酸生産用大腸菌fruR破壊株のそれぞれに、スクロース加水分解酵素(インベルターゼ)遺伝子発現ベクターを導入し、スクロースからL-乳酸を生産する大腸菌株を構築した。具体的には以下のようにして行った。
<MG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入/pGAP-cscA-fruK株の構築>
実施例16で構築したL-乳酸生産用大腸菌株に、スクロース加水分解酵素(インベルターゼ)及びフルクトース-1-リン酸キナーゼ遺伝子発現ベクターを導入し、L-乳酸生産大腸菌fruK強化株を構築した。具体的には以下のようにして行った。
<MG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入/pGAP-cscA株及びMG1655ΔpflΔmdhΔaspΔlldDΔldhAΔfruR/GAPldh2ゲノム挿入/pGAP-cscA株MG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入/pGAP-cscA-fruK株によるL-乳酸生産>
MG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入/pGAP-cscA株及びMG1655ΔpflΔmdhΔaspΔlldDΔldhAΔfruR/GAPldh2ゲノム挿入/pGAP-cscA株MG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入/pGAP-cscA-fruK株の廃糖蜜からのL-乳酸生産を調べた。
前培養として、表5に示す前培養培地50mlを入れた500ml容バッフル付三角フラスコに、実施例17及び実施例18で得られたMG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入/pGAP-cscA株及びMG1655ΔpflΔmdhΔaspΔlldDΔldhAΔfruR/GAPldh2ゲノム挿入/pGAP-cscA株及びMG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入/pGAP-cscA-fruK株を各々植菌し、一晩35℃、120rpmで攪拌培養を行った。その後、下記の表6に示す培地475gを入れたものに、前培養したフラスコ内容物25mlを各々植菌し、実施例10と同様に培養実験を行った。
24時間培養後の培養液中のL-乳酸濃度は、MG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入/pGAP-cscA株(cscA)が75.12g/L、MG1655ΔpflΔmdhΔaspΔlldDΔldhAΔfruR/GAPldh2ゲノム挿入/pGAP-cscA株(fruR破壊株)が83.79g/L、MG1655ΔpflΔmdhΔaspΔlldDΔldhA/GAPldh2ゲノム挿入/pGAP-cscA-fruK株(fruK強化株)が84.32g/Lだった。
これにより、本発明の乳酸生産大腸菌を用いて、廃糖蜜を原料としてL-乳酸を生産できることが確認された。また、乳酸生産大腸菌のfruR遺伝子を破壊することによりL-乳酸の生産性が向上することが明らかになった。更に、乳酸生産大腸菌のfruK遺伝子を強化することによってもL-乳酸の生産性が向上することが明らかになった。
<エシェリヒア・コリO157由来インベルターゼ遺伝子、ザイモモナス菌由来グルコース輸送促進蛋白質(glf)遺伝子発現ベクターおよび該発現ベクター形質転換体の構築>
エシェリヒア・コリのGAPDH遺伝子の塩基配列はすでに報告されている。グリセルアルデヒド3-リン酸デヒドロゲナーゼ(GAPDH)プロモーターを取得するため、CCAAGCTTCTGCAGGTCGACGGATCCGAGCTCAGCTATTTGTTAGTGAATAAAAGG (配列番号50)の塩基配列を有するプライマーを合成した。エシェリヒア・コリMG1655株のゲノムDNAをテンプレートに用いて配列番号29との組み合わせによりPCR法でDNA断片を増幅した。配列番号29のプライマーはその5´末端側にNdeI認識部位を、配列番号50のプライマーはその5´末端側から順にHindIII、PstI、SalI、BamHI、SacI認識部位を有している。得られたDNAフラグメントを制限酵素NdeIとHindIIIで消化することで約100bpのGAPDHプロモーターをコードするフラグメントを得た。次に上記のDNAフラグメントと、NdeI、HindIIIで消化した大腸菌用クローニングベクターpBR322(GenBank accession number J01749)を混合し、リガーゼを用いて結合した後、エシェリヒア・コリDH5αコンピテントセル(タカラバイオ社製)に形質転換し、アンピシリン50μg/mLを含むLB寒天プレートに生育する形質転換体を得た。得られたコロニーを、アンピシリン50μg/mLを含むLB液体培地で30℃で一晩培養し、得られた菌体からプラスミドを回収した。このプラスミドをpGAPと命名した。
このプラスミドpGAP-cscA-glfを実施例6で作成したMG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株コンピテントセルに形質転換し、アンピシリン50μg/mLを含むLB Broth,Miller寒天プレートで37℃一晩培養することにより、MG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-cscA-glf株を得た。
前培養としてLB Broth、Miller培養液(Difco244620)3mlを入れた試験管に、上記記載のMG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-cscA-glf株を植菌し、30℃、200rpmで9時間攪拌培養を行った。
その後、10gのCaCO3(純正化学1級)を予め加え滅菌した100mlバッフル付き三角フラスコ4本に表7に示す培地20mlそれぞれ加えたものに、前培養液を各々100マイクロリットル植菌し、35℃、90rpm、培養時間48時間で攪拌培養を行った。コントロールとして実施例10に記載のcscAMG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-cscA株を用いて同様の培養を行い、培養終了後、実施例10に記載の方法により、得られた培養液中の乳酸濃度を定量した。
48時間後の培養液中のD-乳酸濃度は、cscAが48.9g/L、MG1655ΔpflΔdldΔmdhΔasp/GAPldhAゲノム挿入株/pGAP-cscA-glf株が9.3g/Lだった。
この結果より、cscAと同様に糖代謝系に関連する遺伝子であるグルコース輸送促進蛋白質遺伝子(glf)を利用して糖の取り込みを強化しても、乳酸生産性の向上効果はみられないことが分かった。
本明細書に記載された全ての文献、特許出願、および技術規格は、個々の文献、特許出願、および技術規格が参照により取り込まれることが具体的かつ個々に記された場合と同程度に、本明細書中に参照により取り込まれる。
Claims (16)
- スクロース非PTS遺伝子群のうちスクロース加水分解酵素遺伝子を少なくとも含む1種以上の遺伝子(ただし、リプレッサー蛋白質(cscR)、スクロース加水分解酵素(cscA)、フルクトキナーゼ(cscK)及びスクロース透過酵素(cscB)の組み合わせと、スクロース加水分解酵素(cscA)、フルクトキナーゼ(cscK)及びスクロース透過酵素(cscB)の組み合わせとを除く)を有し、且つ、遺伝子組換えによる乳酸生産強化系を備えている乳酸生産大腸菌。
- 前記スクロース非PTS遺伝子群の中で、スクロース加水分解酵素遺伝子のみを有し、且つ遺伝子組み換えによる乳酸生産強化系を備えている請求項1記載の乳酸生産大腸菌。
- 前記乳酸生産大腸菌が、フルクトースの代謝能力向上系を更に有する請求項1記載の乳酸生産大腸菌。
- 前記乳酸生産強化系が、ピルベートホルメートリアーゼ活性の不活化あるいは低減化を含む請求項1記載の乳酸生産大腸菌。
- 前記乳酸生産強化系が、D-乳酸又はL-乳酸を生成するためのNADH依存性乳酸デヒドロゲナーゼ活性の増強を含む請求項1記載の乳酸生産大腸菌。
- 前記乳酸生産強化系が、
D-乳酸デヒドロゲナーゼ活性の増強と、
該大腸菌が本来有しているFAD依存型D-乳酸デヒドロゲナーゼ活性の不活化あるいは低減化と、
を含む請求項1記載の乳酸生産大腸菌。 - 前記乳酸生産強化系が、
L-乳酸デヒドロゲナーゼ活性の増強と、
該大腸菌が本来有しているD-乳酸デヒドロゲナーゼ活性及びFMN依存性L-乳酸デヒドロゲナーゼ活性の少なくともどちらか一方の不活化あるいは低減化と、
を含む請求項1記載の乳酸生産大腸菌。 - 前記フルクトースの代謝能力向上系が、フルクトース代謝経路におけるリン酸化能力の強化またはフルクトース取り込み能力の強化である請求項3記載の乳酸生産大腸菌。
- 前記フルクトース代謝経路におけるリン酸化能力の強化が、フルクトース-1-リン酸キナーゼ活性に由来する請求項8に記載の乳酸生産大腸菌。
- 前記フルクトース代謝経路におけるフルクトース取り込み能力の強化が、該大腸菌が本来有しているFruR活性の不活化あるいは低減化に由来する請求項8に記載の乳酸生産大腸菌。
- 前記スクロース加水分解酵素遺伝子が、エシェリヒア属細菌に由来する請求項1~請求項10のいずれか一項に記載の乳酸生産大腸菌。
- [規則91に基づく訂正 10.12.2009]
前記スクロース加水分解酵素遺伝子が、エシェリヒア・コリO157細菌に由来する請求項1~請求項10のいずれか一項に記載の乳酸生産大腸菌。 - 前記フルクトース-1-リン酸キナーゼが、エシェリヒア属細菌に由来する請求項9記載の乳酸生産大腸菌。
- 前記フルクトース-1-リン酸キナーゼがエシェリヒア・コリMG1655由来の蛋白質である請求項9に記載の乳酸生産大腸菌。
- 大腸菌K12由来株である請求項1記載の乳酸生産大腸菌。
- 請求項1~請求項15のいずれか一項に記載の乳酸生産大腸菌を用いて、スクロースを含む植物由来原料から乳酸を生産することを含む乳酸生産方法。
Priority Applications (6)
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EP09814543.6A EP2336295B1 (en) | 2008-09-16 | 2009-09-11 | Method for producing lactic acid from plant-derived raw material, and lactic-acid-producing bacterium |
CA2737429A CA2737429C (en) | 2008-09-16 | 2009-09-11 | Method for producing lactic acid from plant-derived raw material, and lactic-acid-producing bacterium |
JP2010529744A JP5243546B2 (ja) | 2008-09-16 | 2009-09-11 | 植物由来原料から乳酸を生産する方法及び乳酸生産細菌 |
BRPI0919218-2A BRPI0919218A2 (pt) | 2008-09-16 | 2009-09-11 | Método para a produção de ácido lático a partir de máteria prima derivada de planta, e bactéria produtora de ácido lático |
US13/063,929 US8679800B2 (en) | 2008-09-16 | 2009-09-11 | Method for producing lactic acid from plant-derived raw material, and lactic-acid-producing bacterium |
CN200980135975.2A CN102333859B (zh) | 2008-09-16 | 2009-09-11 | 由来自植物的原料生产乳酸的方法及生产乳酸的细菌 |
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EP (1) | EP2336295B1 (ja) |
JP (1) | JP5243546B2 (ja) |
CN (1) | CN102333859B (ja) |
BR (1) | BRPI0919218A2 (ja) |
CA (1) | CA2737429C (ja) |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2012082720A2 (en) | 2010-12-13 | 2012-06-21 | Myriant Corporation | Method of producing succinic acid and other chemicals using sucrose-containing feedstock |
JP2013090600A (ja) * | 2011-10-26 | 2013-05-16 | Mitsui Eng & Shipbuild Co Ltd | 乳酸生産菌 |
WO2014017469A1 (ja) * | 2012-07-23 | 2014-01-30 | 三井化学株式会社 | D-乳酸の生産方法、ポリマーの生産方法およびポリマー |
KR101455360B1 (ko) * | 2011-11-01 | 2014-10-28 | 아주대학교산학협력단 | 수크로오스 대사 회로가 재구축된 대장균 |
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CN105026549B (zh) * | 2012-12-07 | 2021-03-16 | 韩国生命源株式会社 | 具有诱导il-12产生的能力的乳酸杆菌以及用于培养其的方法 |
CN105188749B (zh) * | 2012-12-21 | 2017-12-19 | 西雅图基因公司 | 抗ntb‑a抗体及相关组合物和方法 |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012082720A2 (en) | 2010-12-13 | 2012-06-21 | Myriant Corporation | Method of producing succinic acid and other chemicals using sucrose-containing feedstock |
US9845513B2 (en) | 2010-12-13 | 2017-12-19 | Myriant Corporation | Method of producing succinic acid and other chemicals using sucrose-containing feedstock |
JP2013090600A (ja) * | 2011-10-26 | 2013-05-16 | Mitsui Eng & Shipbuild Co Ltd | 乳酸生産菌 |
KR101455360B1 (ko) * | 2011-11-01 | 2014-10-28 | 아주대학교산학협력단 | 수크로오스 대사 회로가 재구축된 대장균 |
WO2014017469A1 (ja) * | 2012-07-23 | 2014-01-30 | 三井化学株式会社 | D-乳酸の生産方法、ポリマーの生産方法およびポリマー |
JPWO2014017469A1 (ja) * | 2012-07-23 | 2016-07-11 | 三井化学株式会社 | D−乳酸の生産方法およびポリマーの生産方法 |
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Publication number | Publication date |
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EP2336295A4 (en) | 2014-01-22 |
CN102333859B (zh) | 2015-04-22 |
US20110171704A1 (en) | 2011-07-14 |
JP5243546B2 (ja) | 2013-07-24 |
CN102333859A (zh) | 2012-01-25 |
CA2737429C (en) | 2014-10-28 |
CA2737429A1 (en) | 2010-03-25 |
TW201016849A (en) | 2010-05-01 |
US8679800B2 (en) | 2014-03-25 |
EP2336295A1 (en) | 2011-06-22 |
EP2336295B1 (en) | 2018-01-24 |
BRPI0919218A2 (pt) | 2015-08-11 |
JPWO2010032698A1 (ja) | 2012-02-09 |
TWI456055B (zh) | 2014-10-11 |
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