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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/44—Polycarboxylic acids

Definitions

• the present invention relates to genetically modified microorganisms that produce high amounts of 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid, and to a method for producing 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid using the genetically modified microorganisms.
• 3-Hydroxyadipic acid (IUPAC name: 3-hydroxyhexandioic acid), ⁇ -hydromuconic acid (IUPAC name: (E)-hex-2-enedioic acid), and adipic acid (IUPAC name: hexanedioic acid) are dicarboxylic acids with six carbon atoms. These can be polymerized with polyhydric alcohols to make polyesters, and with polyamines to make polyamides. Compounds that have been lactamized by adding ammonia to the ends of these compounds can also be used as polyamide raw materials.
• Patent Document 1 which is a document related to the production of 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid using microorganisms, describes a method for producing 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid using a polypeptide that exhibits excellent activity in catalyzing the reduction reaction from 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, and describes that the biosynthetic pathway for these substances goes through an enzymatic reaction that reduces 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA.
• Patent Document 2 describes a method for producing 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid using a polypeptide that exhibits excellent activity in catalyzing the reduction reaction from 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA and a genetically modified microorganism that lacks the function of pyruvate kinase.
• Patent Document 3 describes a method for producing 3-hydroxyadipic acid and/or ⁇ -hydromuconic acid using a genetically modified microorganism in which the reaction for producing acetyl-CoA from pyruvate is enhanced and the function of pyruvate kinase and/or phosphotransferase enzymes is reduced.
• blocking the acetic acid production pathway to reduce the by-production of acetic acid also reduces the productivity of the target substance.
• a method for blocking the acetic acid production pathway a method of inactivating or deleting a gene encoding an enzyme that catalyzes the acetic acid production reaction can be mentioned.
• the main enzymes that catalyze the acetic acid production reaction are phosphate acetyltransferase (EC 2.3.1.8), which catalyzes the reaction of producing acetyl phosphate and CoA from acetyl-CoA and phosphate, acetate kinase (EC 2.7.2.1), which catalyzes the reaction of producing acetic acid from acetyl phosphate, and pyruvate dehydrogenase (EC 1.2.5.1), which catalyzes the reaction of producing acetic acid and carbon dioxide from pyruvic acid.
• phosphate acetyltransferase EC 2.3.1.8
• acetate kinase EC 2.7.2.1
• pyruvate dehydrogenase EC 1.2.5.1
• Non-Patent Document 1 it is known that in Escherichia coli, which is widely used industrially, the growth of the microorganism is reduced by the deletion of ackA, pta, and poxB, which are genes encoding the enzymes that catalyze the acetic acid production reaction, and the productivity of the target substance is also reduced accordingly.
• the present invention aims to provide a genetically modified microorganism capable of producing 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid, which improves the productivity of 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid while reducing the by-production of acetic acid by modifying the metabolic pathway of the microorganism without blocking the acetic acid production pathway.
• the present inventors discovered that in a microorganism capable of producing 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid, by strengthening the glyoxylic acid cycle and decreasing the reaction producing lactic acid or ethanol, the productivity of 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid is improved and the productivity of acetic acid is decreased, leading to the completion of the present invention.
• the present invention comprises the following (1) to (16).
• the enhancement of the glyoxylic acid cycle is a reduction in the function of isocitrate lyase repressor, an enhancement in the function of malate synthase, and/or an enhancement in the function of isocitrate dehydrogenase kinase/phosphatase.
• the enhancement of the glyoxylic acid cycle is any one of the following (i) to (iii).
• the genetically modified microorganism according to (6) wherein the gene encoding the alcohol dehydrogenase is the adhE gene.
• the genetically modified microorganism according to any one of (1) to (7) further comprising an enhanced reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA.
• the genetically modified microorganism according to (1) which maintains a reaction pathway for producing acetate from acetyl-CoA or pyruvate.
• a method for producing 3-hydroxyadipic acid, ⁇ -hydromuconic acid and/or adipic acid comprising a step of culturing the genetically modified microorganism according to any one of (1) to (9).
• a method for producing adipic acid comprising the steps of producing 3-hydroxyadipic acid and/or ⁇ -hydromuconic acid by the method according to (10) above, and reacting the acid and the acid with hydrogen in the presence of a hydrogenation catalyst (hydrogenation step).
• a method for producing a polyamide comprising: a step of producing adipic acid by the method according to (10) or (11); and a step of polycondensing adipic acid and a diamine.
• the diamine is a diamine containing 1,4-butanediamine, 1,5-pentanediamine or hexamethylenediamine.
• the genetically modified microorganism of the present invention can increase the productivity of 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid, and can also decrease the productivity of acetic acid.
• the production ratio of 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid relative to acetic acid can be increased.
• 3-hydroxyadipyl acid may be abbreviated as “3HA”, ⁇ -hydromuconic acid as “HMA”, and adipic acid as “ADA”.
• 3-oxoadipyl-CoA may be abbreviated as “3OA-CoA”, 3-hydroxyadipyl-CoA as “3HA-CoA”, 2,3-dehydroadipyl-CoA as “HMA-CoA”, and adipyl-CoA as "ADA-CoA”.
• the enzyme that catalyzes the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA may be referred to as "3-oxoadipyl-CoA reductase”.
• Isocitrate lyase repressor may be referred to as "IclR”, malate synthase as “AceB”, and isocitrate dehydrogenase kinase/phosphatase as "AceK”.
• Nucleic acids that code for functional polypeptides may be referred to as genes.
• the genetically modified microorganism of the present invention can biosynthesize 3HA, HMA and/or ADA using acetyl-CoA and succinyl-CoA as intermediates, as shown in the metabolic pathway below.
• the metabolic pathway from glucose to acetyl-CoA is known as the glycolysis pathway, while the metabolic pathway from glucose to succinyl-CoA is known as the TCA cycle, the glyoxylate cycle, and the supplementary pathway that produces oxaloacetate from PEP, as well as the reductive TCA cycle.
• reaction A shows the reaction of producing 3-oxoadipyl-CoA from acetyl-CoA and succinyl-CoA.
• reaction B shows the reaction of producing 3-hydroxyadipyl-CoA by reducing 3-oxoadipyl-CoA.
• reaction C shows the reaction of producing 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA.
• reaction D shows the reaction of producing adipyl-CoA from 2,3-dehydroadipyl-CoA.
• Reaction E shows the reaction of producing 3-hydroxyadipyl-CoA from 3-hydroxyadipyl-CoA.
• Reaction F shows the reaction of producing ⁇ -hydromuconic acid from 2,3-dehydroadipyl-CoA.
• Reaction G shows a reaction for producing adipic acid from adipyl-CoA. Enzymes that catalyze each reaction in the following metabolic pathways, as well as a method for creating a microorganism capable of producing 3HA, HMA and/or ADA using the following metabolic pathways, are described in detail in WO2019/107516.
• the present invention is characterized by reducing the function of isocitrate lyase repressor, enhancing the function of malate synthase, and/or enhancing the function of isocitrate dehydrogenase kinase/phosphatase in a microorganism capable of producing 3HA, HMA, and/or ADA.
• Isocitrate lyase repressor is a protein that has the activity of suppressing the expression of genes encoding isocitrate lyase, malate synthase, and isocitrate dehydrogenase kinase/phosphatase.
• IclR NCBI-Protein ID: NP_418442, SEQ ID NO: 1 derived from Escherichia coli str. K-12 substr. MG1655 strain
• a specific example of a gene encoding IclR is the nucleic acid sequence of SEQ ID NO: 2.
• Isocitrate lyase is an enzyme (EC 4.1.3.1) that reversibly catalyzes the reaction that produces succinic acid and glyoxylic acid from isocitrate.
• a specific example of isocitrate lyase is AceA (NCBI-Protein ID: NP_418439) derived from Escherichia coli str. K-12 substr. MG1655 strain.
• Malate synthase is an enzyme (EC 2.3.3.9) that catalyzes the reaction of producing malic acid and CoA from glyoxylic acid and acetyl-CoA.
• a specific example of malate synthase is AceB (NCBI-Protein ID: NP_418438, SEQ ID NO: 35) derived from Escherichia coli str. K-12 substr. MG1655 strain, and a specific example of a gene encoding AceB is the nucleic acid sequence of SEQ ID NO: 36.
• Isocitrate dehydrogenase kinase/phosphatase is an enzyme (EC 2.7.11.5) that catalyzes the phosphorylation of isocitrate dehydrogenase, an enzyme that catalyzes the reaction of producing ⁇ -ketoglutarate and carbon dioxide from isocitrate in the TCA cycle. It is known that the catalytic activity of isocitrate dehydrogenase decreases upon phosphorylation.
• a specific example of isocitrate dehydrogenase kinase/phosphatase is AceK (NCBI-Protein ID: NP_418440, SEQ ID NO: 37) derived from Escherichia coli str. K-12 substr. MG1655 strain, and a specific example of a gene encoding AceK is the nucleic acid gene of SEQ ID NO: 38.
• the aceA gene which codes for isocitrate lyase
• the aceB gene which codes for malate synthase
• the aceK gene which codes for isocitrate dehydrogenase kinase/phosphatase
• isocitrate lyase, malate synthase, and isocitrate dehydrogenase kinase/phosphatase catalyze reactions that form the glyoxylate cycle, which bypasses part of the TCA cycle
• the aceBAK operon is also known as the glyoxylate bypass operon.
• the protein encoded by the IclR gene is an isocitrate lyase repressor that functions to suppress the expression of the aceBAK operon. It is believed that by reducing the function of the isocitrate lyase repressor, the expression of the aceBAK operon is induced, and the expression of isocitrate lyase, malate synthase, and isocitrate dehydrogenase kinase/phosphatase is enhanced.
• FadR (NCBI-Protein ID: NP_415705) and ArcA (NCBI-Protein ID: NP_418818) have also been reported as proteins other than the isocitrate lyase repressor that function to control the expression of the aceBAK operon (J. Bacteriol. 1996, 178(15), 4704-4709.; BMC Microbiol. 2011, 11:70.).
• the method for enhancing the glyoxylate cycle is not particularly limited as long as it is a method for reducing the function of the isocitrate lyase repressor or a method for enhancing the function of malate synthase and/or isocitrate dehydrogenase kinase/phosphatase.
• Methods for reducing the function of the isocitrate lyase repressor include, for example, deletion, loss-of-function mutation, or suppression of expression of the gene encoding the enzyme, with deletion or suppression of expression of the gene encoding the enzyme being preferred.
• the iclR gene is preferred as the isocitrate lyase repressor gene whose function is reduced.
• Methods for enhancing the function of malate synthase and/or isocitrate dehydrogenase kinase/phosphatase include, for example, enhancing the catalytic activity of the enzyme itself or increasing the expression level of the enzyme, with increasing the expression level of the enzyme being preferred.
• the aceB gene is preferred as the malate synthase gene whose function is enhanced, and the aceK gene is preferred as the isocitrate dehydrogenase kinase/phosphatase gene.
• the method of suppressing the expression of the iclR gene is not particularly limited, but examples thereof include a method of inhibiting transcription by introducing a mutation into the nucleic acid sequence of a promoter region present upstream of the gene on the microbial genome and reducing promoter activity, a method of inhibiting translation by introducing a base sequence that forms a secondary structure such as a hairpin into the nucleic acid sequence of mRNA obtained by transcription of the gene region, and a method of inhibiting translation by expressing an RNA nucleic acid that is a complementary strand of a part of the gene and forming a complex with the nucleic acid molecule of the gene (J. Microbiol. Methods 2018, 154, 25-32.; Nat. Biotechnol.
• RNA nucleic acid that is a complementary strand of a part of the gene and inhibits translation by expressing an RNA nucleic acid that is a complementary strand of the gene and forming a complex with the nucleic acid molecule of the gene is preferred.
• the catalytic activity of the enzyme itself can be enhanced, for example, by introducing a mutation that leads to the deletion, substitution, and/or insertion of a portion of amino acids in a polypeptide having the catalytic activity of the enzyme, and expressing an enzyme that exhibits higher catalytic activity than before the mutation is introduced.
• the method for increasing the expression amount of malate synthase and/or isocitrate dehydrogenase kinase/phosphatase is not particularly limited, and examples include a method of introducing a gene encoding the enzyme from outside the host microorganism into the host microorganism, increasing the copy number of the gene, and modifying a promoter region or a ribosome binding sequence upstream of the coding region of the gene.
• the method for introducing a gene encoding the enzyme from outside the host microorganism into the host microorganism or increasing the copy number of the gene may be a method of artificially introducing the gene originally possessed by the host gene, or a method of introducing an exogenous gene.
• the method for increasing the expression amount of the enzyme may be performed alone or in combination with the above-mentioned methods.
• JP-A-2008-513023 discloses a method for high production of succinic acid, fumaric acid, malic acid, oxaloacetic acid, or glyoxylic acid produced using the TCA cycle by reducing the activity of the protein encoded by the iclR gene to reduce the function of the isocitrate lyase repressor, or by overexpressing the aceB gene or aceK gene or increasing the activity of the protein encoded by these genes to enhance the function of malate synthase or isocitrate dehydrogenase kinase/phosphatase.
• the intermediate acetyl-CoA is essential for the production of 3HA, HMA and/or ADA by microorganisms capable of producing 3HA, HMA and/or ADA, and WO2022/102635 discloses that the productivity of 3HA and/or HMA can be improved by enhancing the reaction that produces acetyl-CoA.
• the reaction catalyzed by malate synthase in the glyoxylate cycle consumes acetyl-CoA and competes with the production reactions of 3HA, HMA, and ADA.
• the main by-products obtained by culturing microorganisms capable of producing 3HA, HMA and/or ADA include lactic acid and ethanol in addition to acetic acid, but the present invention is characterized by reducing the lactic acid or ethanol production reaction in microorganisms capable of producing 3HA, HMA and/or ADA.
• Lactic acid is produced by the reduction of pyruvate.
• a specific example of an enzyme that catalyzes the reduction of pyruvate to lactate is lactate dehydrogenase (EC 1.1.1.27, 1.1.1.28).
• a specific example of lactate dehydrogenase is LdhA (NCBI-Protein ID: NP_415898, SEQ ID NO: 3) derived from Escherichia coli str. K-12 substr. MG1655 strain, and a specific example of a gene encoding LdhA is the nucleic acid sequence of SEQ ID NO: 4.
• Ethanol is produced by the reduction of acetyl-CoA.
• An example of an enzyme that catalyzes the reduction of acetyl-CoA to ethanol is alcohol dehydrogenase (EC 1.2.1.10, 1.1.1.1).
• An example of an alcohol dehydrogenase is AdhE (NCBI-Protein ID: NP_415757, SEQ ID NO: 5) derived from Escherichia coli str. K-12 substr. MG1655 strain, and an example of a gene encoding AdhE is the nucleic acid sequence of SEQ ID NO: 6.
• the method for reducing the lactic acid or ethanol production reaction is not particularly limited, but examples thereof include a method for reducing the function of the above-mentioned lactate dehydrogenase or alcohol dehydrogenase.
• the method for reducing the function of lactate dehydrogenase or alcohol dehydrogenase is not particularly limited, but examples thereof include deletion, loss-of-function mutation, or expression inhibition of the gene encoding the enzyme, with deletion or expression inhibition of the gene encoding the enzyme being preferred.
• the ldhA gene is preferred as the gene encoding lactate dehydrogenase whose function is reduced, and the adhE gene is preferred as the gene encoding alcohol dehydrogenase whose function is reduced.
• the method of gene deletion or loss-of-function mutation is not particularly limited, but can be, for example, gene mutation treatment using a gene mutation agent or ultraviolet light irradiation, deletion of part or all of a nucleic acid sequence using site-specific mutagenesis, introduction of a frameshift mutation into a nucleic acid sequence, insertion of a stop codon into a base sequence, etc.
• function can be lost by removing all or part of a nucleic acid sequence or replacing it with another nucleic acid sequence using recombinant gene technology. Of these, the method of deleting part or all of a nucleic acid sequence is preferred.
• Enhancement of the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA can be achieved, for example, by increasing the expression level of the enzyme that catalyzes this reaction.
• Methods for increasing the expression level include, for example, introducing the enzyme gene from outside the host microorganism into the host microorganism, increasing the copy number of the gene, and modifying the promoter region or ribosome binding sequence upstream of the coding region of the gene. These methods may be performed alone or in combination, but it is preferable to introduce the enzyme gene from outside the host microorganism into the host microorganism by the method described in WO2019/107516.
• 3-oxoadipyl-CoA reductase that catalyzes the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA
• enzymes classified as 3-hydroxyacyl-CoA dehydrogenases in EC 1.1.1.35 enzymes classified as 3-hydroxybutyryl-CoA dehydrogenases in EC 1.1.1.157, and enzymes that have 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and even more preferably 99% or more sequence identity to any of the amino acid sequences of SEQ ID NOs: 1 to 6 and 213 described in WO2019/107516 and have 3-oxoadipyl-CoA reductase activity.
• PaaH derived from Pseudomonas putida KT2440 strain (NCBI-Protein ID: NP_745425.1), Escherichia coli str. K-12 substr.
• polypeptides include PaaH derived from Acinetobacter baylyi strain ADP1 (NCBI Protein ID: NP_415913.1), DcaH derived from Acinetobacter baylyi strain ADP1 (NCBI Protein ID: CAG68533.1), PaaH derived from Serratia plymuthica strain NBRC102599 (NCBI Protein ID: WP_063197120), and a polypeptide derived from Serratia marcescens strain ATCC13880 (NCBI Protein ID: KFD11732.1).
• amino acid sequences of the polypeptides of SEQ ID NOs: 1 to 6 and 213 described in WO2019/107516 and the nucleotide sequences encoding them are shown in SEQ ID NOs: 51 to 56 and 57, and SEQ ID NOs: 17, 58 to 62 and 63, respectively.
• sequence identity means the percentage of identical amino acids or bases in the total overlapping amino acid sequence (including the amino acid at the translation initiation point) or base sequence (including the initiation codon) in the optimal alignment when two amino acid sequences or base sequences are aligned with or without introducing gaps, and is calculated by formula (1).
• the shorter sequence length to be compared is 400 amino acids or more, and sequence identity is not defined when it is less than 400 amino acids. Sequence identity can be easily examined using BLAST (Basic Local Alignment Search Tool), an algorithm commonly used in this field.
• microorganisms are examples of microorganisms that have the inherent ability to produce 3-hydroxyadipic acid:
• the genus Escherichia such as Escherichia fergusonii and Escherichia coli.
• Serratia grimesii Serratia ficaria, Serratia fonticoia, Serratia odorifera, Serratia plymuthica, Serratia Genus Serratia, such as Entomophila or Serratia nematodiphila.
• Pseudomonas genus such as Pseudomonas chlororaphis, Pseudomonas putida, Pseudomonas azotoformans, and Pseudomonas chlororaphis subsp. aureofaciens.
• Hafnia such as Hafnia alvei.
• Bacillus genus such as Bacillus badius, Bacillus megaterium, and Bacillus roseus.
• Streptomyces genus such as Streptomyces vinaceus, Streptomyces karnatakensis, Streptomyces olivaceus.
• Cupriavidus genus such as Cupriavidus metallidurans, Cupriavidus necator, Cupriavidus oxalaticus.
• the genus Acinetobacter such as Acinetobacter baylyi and Acinetobacter radiores Istens.
• the genus Alcaligenes such as Alcaligenes faecalis.
• the genus Nocardioides such as Nocardioides albus.
• the genus Brevibacterium such as Brevibacterium iodinum.
• the genus Delftia such as Delftia acidovorans.
• the genus Shimwellia such as Shimwellia blattae.
• the genus Aerobacter such as Aerobacter cloacae.
• Rhizobium genus such as Rhizobium radiobacter.
• microorganisms that inherently have the ability to produce 3-hydroxyadipic acid in the present invention, microorganisms that do not metabolize sugars via the phosphoketolase pathway, such as those belonging to the genera Escherichia, Serratia, Hafnia, Corynebacterium, Brevibacterium, Shimwellia, and Aerobacter, are preferred, and microorganisms belonging to the genus Escherichia or Serratia are more preferably used.
• the genus Escherichia such as Escherichia fergusonii and Escherichia coli.
• Serratia grimesii Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia Genus Serratia, such as Entomophila or Serratia nematodiphila.
• Pseudomonas genus such as Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas azotoformans, and Pseudomonas chlororaphis subsp. aureofaciens.
• the genus Hafnia such as Hafnia alvei.
• the genus Bacillus such as Bacillus badius.
• Cupriavidus genus such as Cupriavidus metallidurans, Cupriavidus numazuensis, Cupriavidus oxalaticus.
• the genus Acinetobacter such as Acinetobacter baylyi and Acinetobacter radioresistens.
• the genus Alcaligenes such as Alcaligenes faecalis.
• the genus Delftia such as Delftia acidovorans.
• the genus Shimwellia such as Shimwellia blattae.
• the genetically modified microorganism of the present invention does not inherently have the ability to produce 3-hydroxyadipic acid, this ability can be imparted by introducing into the microorganism an appropriate combination of nucleic acids encoding enzymes that catalyze reactions A, B, and E. If the genetically modified microorganism of the present invention does not inherently have the ability to produce ⁇ -hydromuconic acid, this ability can be imparted by introducing into the microorganism an appropriate combination of nucleic acids encoding enzymes that catalyze reactions A, B, C, and F.
• the genetically modified microorganism of the present invention does not inherently have the ability to produce adipic acid, this ability can be imparted by introducing into the microorganism an appropriate combination of nucleic acids encoding enzymes that catalyze reactions A, B, C, D, and G.
• microorganisms that can be used as hosts for obtaining genetically modified microorganisms in the present invention are not particularly limited as long as they can be genetically modified, and may or may not be capable of producing 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid.
• suitable microorganisms include Escherichia, Serratia, Hafnia, Psuedomonas, Corynebacterium, Bacillus, Streptomyces, Cupriavidus, Acinetobacter, Alcaligen, and the like.
• Microorganisms belonging to the genera Escherichia, Brevibacterium, Delftia, Shimwellia, Aerobacter, Rhizobium, Thermobifida, Clostridium, Schizosaccharomyces, Kluyveromyces, Pichia and Candida are preferred, microorganisms belonging to the genera Escherichia, Serratia, Hafnia and Pseudomonas are more preferred, and microorganisms belonging to the genus Escherichia or Serratia are particularly preferred.
• the genetically modified microorganism of the present invention preferably maintains a reaction pathway for producing acetate from acetyl-CoA or pyruvate. This can be achieved, for example, by possessing a gene encoding any of the enzymes that catalyze the acetate production reaction and maintaining the function of the enzyme.
• Examples of enzymes that catalyze the acetate production reaction include phosphate acetyltransferase (EC 2.3.1.8), which catalyzes the reaction of producing acetyl phosphate and CoA from acetyl-CoA and phosphate, acetate kinase (EC 2.7.2.1), which catalyzes the reaction of producing acetate from acetyl phosphate, and pyruvate dehydrogenase (EC 1.2.5.1), which catalyzes the reaction of producing acetate and carbon dioxide from pyruvate.
• phosphate acetyltransferase EC 2.3.1.8
• acetate kinase EC 2.7.2.1
• pyruvate dehydrogenase EC 1.2.5.1
• the method for introducing a gene to create the genetically modified microorganism of the present invention is not particularly limited, and may involve incorporating the gene into an expression vector capable of autonomously replicating within the microorganism and then introducing the vector into the host microorganism, or incorporating the gene into the genome of the microorganism.
• Gene introduction and enhanced expression may also be combined.
• the expression vector or the nucleic acid to be incorporated into the genome is preferably composed of a promoter, a ribosome binding sequence, the gene or nucleic acid fragment to be expressed, and a transcription termination sequence. It may also contain a gene that controls the promoter activity.
• the promoter used in the present invention is not particularly limited as long as it can express a gene in a host microorganism, but examples include the gap promoter, trp promoter, lac promoter, tac promoter, T7 promoter, and lambda promoter (PL or PR).
• an expression vector When an expression vector is used in the present invention to introduce a gene or enhance gene expression, it is not particularly limited as long as it is capable of autonomously replicating in the microorganism, but examples include pBBR1MCS vector, pBR322 vector, pMW vector, pET vector, pRSF vector, pCDF vector, pACYC vector, and derivatives of the above vectors.
• the gene is introduced using site-specific homologous recombination.
• site-specific homologous recombination There are no particular limitations on the method of site-specific homologous recombination, but examples include a method using ⁇ Red recombinase and FLP recombinase (Proc. Natl. Acad. Sci. USA. 2000, 97(12), 6640-6645.) and a method using ⁇ Red recombinase and the sacB gene (Biosci. Biotechnol. Biochem. 2007, 71(12), 2905-2911.).
• the method for introducing an expression vector or a nucleic acid for genome integration is not particularly limited as long as it is a method for introducing a nucleic acid into a microorganism, but examples include the calcium ion method (J. Mol. Biol. 1970, 53, 159-162.) and the electroporation method (J. Bacteriol. 1988, 170, 2796-2801.).
• the production ratio of 3HA to acetic acid is calculated according to formula (2).
• the production ratio of HMA to acetic acid or the production ratio of ADA to acetic acid is calculated by replacing 3HA in formula (2) with HMA or ADA.
• Production ratio of 3HA to acetic acid 3HA production (g/L) / acetic acid production (g/L) ... formula (2).
• the genetically modified microorganism of the present invention is cultured in a medium, preferably a liquid medium, that contains a carbon source that can be utilized by ordinary microorganisms as a fermentation raw material.
• a medium that contains an appropriate amount of a nitrogen source, inorganic salts, and organic trace nutrients such as amino acids and vitamins as necessary is used.
• Either a natural medium or a synthetic medium can be used as long as it contains the above nutrient sources.
• the fermentation raw material is a raw material that can be metabolized by the genetically modified microorganism.
• “Metabolism” refers to the conversion of a certain chemical compound that the microorganism has taken in from outside the cell or that is produced from another chemical compound inside the cell into another chemical compound through an enzymatic reaction.
• Sugars can be preferably used as the carbon source. Specific examples of sugars include monosaccharides such as glucose, fructose, galactose, mannose, xylose, arabinose, etc., disaccharides such as sucrose that are formed by combining these monosaccharides, polysaccharides, and starch saccharification liquid, molasses, cellulose-containing biomass saccharification liquid, etc. that contain these.
• the above carbon sources may be used alone or in combination, but it is particularly preferable to culture in a medium containing glucose.
• concentration of the carbon source in the medium is not particularly limited and can be set appropriately depending on the type of carbon source, etc.
• the preferred concentration of glucose is 5 to 300 g/L.
• the nitrogen source used in culturing the genetically modified microorganism may be, for example, ammonia gas, ammonia water, ammonium salts, urea, nitrates, or other supplementary organic nitrogen sources, such as oil cakes, soybean hydrolysate, casein hydrolysate, other amino acids, vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermentation bacteria and their hydrolysates.
• the concentration of the nitrogen source in the medium is not particularly limited, but is preferably 0.1 to 50 g/L.
• Inorganic salts that can be used in the culture of the genetically modified microorganism include, for example, phosphates, magnesium salts, calcium salts, iron salts, and manganese salts.
• the culture conditions for the genetically modified microorganisms to produce 3-hydroxyadipic acid, ⁇ -hydromuconic acid, and/or adipic acid are set by appropriately adjusting or selecting the medium with the above-mentioned component composition, culture temperature, stirring speed, pH, aeration amount, inoculation amount, etc. depending on the type of the genetically modified microorganism and external conditions, etc.
• pH range during cultivation there are no particular limitations on the pH range during cultivation as long as the genetically modified microorganism can grow, but a pH of 5 to 8 is preferred, and a pH of 5.5 to 7.0 is more preferred.
• antifoaming agents such as mineral oil, silicone oil, and surfactants can be added to the medium as appropriate.
• the produced product can be recovered.
• the produced product can be recovered, for example isolated, by stopping the culture when the accumulated amount has increased appropriately, and collecting the fermentation product from the culture in accordance with a general method. Specifically, the bacterial cells can be separated by centrifugation, filtration, etc., and then the product can be isolated from the culture by column chromatography, ion exchange chromatography, activated carbon treatment, crystallization, membrane separation, distillation, etc.
• examples of the method include, but are not limited to, a method of adding an acid component to the salt of the product and recovering the precipitate; a method of increasing the concentration of the product by removing water from the culture by concentrating the culture using a reverse osmosis membrane or an evaporator, etc., and then precipitating crystals of the product and/or the salt of the product by cooling crystallization or adiabatic crystallization, and obtaining crystals of the product and/or the salt of the product by centrifugation or filtration, and a method of adding alcohol to the culture to convert the product into an ester, recovering the ester of the product by distillation, and obtaining the product by hydrolysis.
• These recovery methods can be appropriately selected and optimized depending on the physical properties of the product.
• Adipic acid can be produced by reacting (hydrogenating) the ⁇ -hydromuconic acid obtained in the present invention with hydrogen in the presence of a hydrogenation catalyst.
• a method for producing adipic acid from a 3-hydroxyadipic acid fermentation liquid is described in detail in WO2021/060335.
• 3-hydroxyadipic acid-3,6-lactone can be prepared by dissolving 3-hydroxyadipic acid in water and adjusting the pH to 4 or less.
• a method for preparing 3-hydroxyadipic acid-3,6-lactone from 3-hydroxyadipic acid is described in detail in WO2022/191314.
• Adipic acid can be produced by reacting (hydrogenating) 3-hydroxyadipic acid-3,6-lactone with hydrogen in an aqueous solvent in the presence of a hydrogenation catalyst.
• the hydrogenation catalyst preferably contains a transition metal element, specifically, preferably contains one or more elements selected from the group consisting of palladium, platinum, ruthenium, rhodium, rhenium, nickel, cobalt, iron, iridium, osmium, copper and chromium, and more preferably contains one or more elements selected from the group consisting of palladium, platinum, nickel, cobalt, iron, copper and chromium.
• the hydrogenation catalyst is preferably used supported on a carrier from the viewpoints of saving the amount of metal used and increasing the active surface area of the catalyst.
• the hydrogenation catalyst can be supported on a carrier by known methods such as impregnation, deposition/precipitation, and gas-phase support.
• carriers include carbon, polymers, metal oxides, metal sulfides, zeolites, clays, heteropolyacids, solid phosphoric acid, and hydroxyapatite.
• Hydrogen to be reacted with ⁇ -hydromuconic acid and/or 3-hydroxyadipic acid-3,6-lactone may be added to the reactor all at once or gradually.
• the partial pressure of hydrogen is preferably 0.1 MPa or more and 10 MPa or less (gauge pressure) at room temperature, more preferably 0.3 MPa or more and 5 MPa or less (gauge pressure), and even more preferably 0.5 MPa or more and 3 MPa or less (gauge pressure).
• the reaction may be carried out in any of the following reactors: a batch tank reactor, a semi-batch tank reactor, a continuous tank reactor, a continuous tubular reactor, or a trickle-bed tubular reactor.
• a solid hydrogenation catalyst When used, the reaction may be carried out in any of the following systems: a suspended bed, a fixed bed, a moving bed, or a fluidized bed.
• the hydrogenation reaction temperature is not particularly limited, but if it is too low, the reaction rate will be slow, and if the reaction temperature is too high, the energy consumption will be high, which is undesirable. From this perspective, the reaction temperature is preferably 100 to 350°C, more preferably 120 to 300°C, even more preferably 130 to 280°C, even more preferably 140 to 250°C, even more preferably 150 to 230°C, and even more preferably 160 to 220°C.
• the atmosphere in the reactor may contain inert gases such as nitrogen, helium, and argon, but the oxygen concentration is preferably 5% by volume or less, as this can lead to deterioration of the hydrogenation catalyst and the generation of explosive gas.
• the amount of ammonia relative to the ⁇ -hydromuconic acid and/or 3-hydroxyadipic acid-3,6-lactone raw material is preferably 5% by weight or less, more preferably 3% by weight or less, and even more preferably 0% by weight (i.e., reaction in the absence of ammonia).
• the hydrogenation of ⁇ -hydromuconic acid and/or 3-hydroxyadipic acid-3,6-lactone is preferably carried out in the presence of a solvent.
• Solvents that can be used for hydrogenation include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, pentane, hexane, cyclohexane, heptane, octane, decane, dimethyl ether, diethyl ether, 1,2-dimethoxyethane, diglyme, tetrahydrofuran, dioxane, methyl acetate, ethyl acetate, n-propyl acetate, n-butyl acetate, ⁇ -butyrolactone, N-methylpyrrolidone, dimethyl sulfoxide, and aqueous solvents. A mixture of two or more of these may also be used, but from the standpoint of economical and environmental considerations, it is preferable to use an aqueous solvent.
• the aqueous solvent means water or a mixed solvent that is mainly water mixed with a water-miscible organic solvent.
• "Mainly water” means that the proportion of water in the mixed solvent is more than 50% by volume, preferably 70% by volume or more, and more preferably 90% by volume or more.
• Water-miscible organic solvents include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, 1,2-dimethoxyethane, diglyme, tetrahydrofuran, dioxane, ⁇ -butyrolactone, N-methylpyrrolidone, dimethylsulfoxide, dimethylformamide, dimethylacetamide, and acetone.
• the pH of the aqueous solvent is not particularly limited, but taking into consideration the prevention of catalyst deterioration, the prevention of by-product formation, and corrosiveness to the reaction equipment, a pH of 2 to 13 is preferable, a pH of 3 to 11 is more preferable, and a pH of 4 to 10 is even more preferable.
• the carboxylic acid, carboxylate, and carboxylate ester of ⁇ -hydromuconic acid and/or 3-hydroxyadipic acid-3,6-lactone produce the corresponding adipic acid, adipate, and adipate ester, respectively.
• a solvent containing a primary or secondary alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, or isobutanol is used as the hydrogenation solvent
• a mixture of adipic acid, adipate, adipic acid monoester, and adipic acid diester is obtained after the reaction.
• the carboxylic acid, carboxylate, and carboxylate ester of adipic acid and mixtures thereof are collectively referred to as "adipic acid.”
• the carboxylic acid of adipic acid obtained in the present invention can be further converted to an adipic acid ester by subjecting it to an esterification reaction.
• the esterification method is not particularly limited, but examples include dehydration condensation of a carboxylic acid and an alcohol using an acid catalyst or a condensing agent, and a method using an alkylating agent such as diazomethane or an alkyl halide.
• the adipic acid obtained in this invention can be separated and purified by conventional unit operations such as centrifugation, filtration, membrane filtration, distillation, extraction, crystallization, and drying.
• Adiponitrile can be produced from the adipic acid obtained in the present invention by known methods (e.g., JP-B-61-24555). Hexamethylenediamine can be produced by hydrogenating the obtained adiponitrile by known methods (e.g., JP-T-2000-508305).
• Polyamides can be produced by polycondensing the adipic acid obtained in the present invention with diamines using known methods (see, for example, "Polyamide Resin Handbook," edited by Fukumoto Osamu, published by Nikkan Kogyo Publishing Co., Ltd. (January 1998)). Specifically, by using 1,4-diaminobutane, 1,5-pentanediamine, or hexamethylenediamine as the diamine, polyamide 46, polyamide 56, or polyamide 66, respectively, can be produced.
• Polyamide can be processed by known methods (e.g., WO 2019/208427) to produce polyamide fibers.
• the polyamide fibers thus obtained can be used for clothing applications such as innerwear, sportswear, and casual wear, and for industrial material applications such as airbags and tire cords.
• Polyamides can also be molded by known methods (e.g., WO 2021/006257) to produce polyamide molded products.
• the polyamide molded products thus obtained can be used for automobile parts, electrical parts, electronic parts, building materials, various containers, daily necessities, household goods, and sanitary products, etc.
• Reference Example 1 Preparation of a plasmid expressing an enzyme that catalyzes the reaction of producing 3OA-CoA and coenzyme A from acetyl-CoA and succinyl-CoA (reaction A), the reaction of producing 3HA-CoA from 3OA-CoA (reaction B), the reaction of producing 3-hydroxyadipic acid from 3HA-CoA (reaction E), the reaction of producing ⁇ -hydromuconic acid from HMA-CoA (reaction F), and the reaction of producing adipic acid from ADA-CoA (reaction G)
• Vector pBBR1MCS-2 (ME Kovach, (1995), Gene 166:175-176), which can autonomously replicate in Escherichia coli, was cleaved with XhoI to obtain pBBR1MCS-2/XhoI.
• pBBR1MCS-2::Pgap the plasmid whose base sequence was confirmed by a conventional method was named pBBR1MCS-2::Pgap. Subsequently, pBBR1MCS-2::Pgap was cleaved with ScaI to obtain pBBR1MCS-2::Pgap/ScaI.
• primers were designed (SEQ ID NOs: 11 and 12) for PCR amplification of the full length of the acyltransferase gene pcaF (NCBI-Gene ID: 1041755, SEQ ID NO: 10) using the genomic DNA of Pseudomonas putida KT2440 strain as a template, and PCR reaction was carried out according to a conventional method.
• the obtained fragment and pBBR1MCS-2::Pgap/ScaI were ligated using "In-Fusion HD Cloning Kit" and introduced into Escherichia coli strain DH5 ⁇ .
• pBBR1MCS-2::AT The plasmid was extracted from the obtained recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was named pBBR1MCS-2::AT. Subsequently, pBBR1MCS-2::AT was cleaved with HpaI to obtain pBBR1MCS-2::AT/HpaI.
• primers were designed (SEQ ID NOs: 15 and 16) for PCR amplification of a continuous sequence including the full length of CoA transferase genes pcaI and pcaJ (NCBI-GeneID: 1046613, 1046612, SEQ ID NOs: 13 and 14) using the genomic DNA of Pseudomonas putida KT2440 strain as a template, and PCR reaction was carried out according to a conventional method.
• the obtained fragment and pBBR1MCS-2::AT/HpaI were ligated using "In-Fusion HD Cloning Kit" and introduced into Escherichia coli strain DH5 ⁇ .
• the plasmid was extracted from the obtained recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was named pBBR1MCS-2::ATCT.
• pBBR1MCS-2::ATCT was cleaved with ScaI to obtain pBBR1MCS-2::ATCT/ScaI.
• primers were designed (SEQ ID NOs: 18 and 19) to amplify the nucleic acid described in SEQ ID NO: 17 using the genomic DNA of Serratia marcescens ATCC13880 strain as a template, and a PCR reaction was performed according to a conventional method.
• the obtained fragment and pBBR1MCS-2::ATCT/ScaI were ligated using "In-Fusion HD Cloning Kit” (manufactured by Takara Bio Inc.) and introduced into Escherichia coli strain DH5 ⁇ .
• the plasmid was extracted from the obtained recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was named pBBR1MCS-2::ATCTOR.
• primers for PCR amplification of the nucleic acid fragment for gene inhibition were designed (SEQ ID NO: 21, 22), and a PCR reaction was performed according to a conventional method.
• the obtained fragment and pCDF-1b/XbaI, BamHI were ligated using "In-Fusion HD Cloning Kit” (manufactured by Takara Bio Inc.) and introduced into Escherichia coli strain DH5 ⁇ .
• the plasmid was extracted from the obtained recombinant strain, and the base sequence was confirmed by a conventional method.
• the plasmid was named pCDF-1b::temp.
• Reference Example 3 Preparation of a Plasmid for Suppressing the Expression of the iclR Gene pCDF-1b::temp was cleaved with NcoI and further subjected to dephosphorylation treatment with Bacterial Alkaline Phosphatase to obtain pCDF-1b::temp/NcoI,BAP.
• a nucleic acid fragment for suppressing the expression of the iclR gene into the vector a nucleic acid fragment (SEQ ID NO:23) containing a 24-base complementary strand including the start codon of the iclR gene was genetically synthesized (manufactured by Azenta Co., Ltd.).
• the fragment and pCDF-1b::temp/NcoI,BAP were ligated using "In-Fusion HD Cloning Kit” (manufactured by Takara Bio Inc.) and introduced into Escherichia coli strain DH5 ⁇ .
• the plasmid was extracted from the obtained recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was named pCDF-1b::iclRi.
• Reference Example 4 Preparation of a Plasmid for Inhibiting Expression of the fadR Gene
• a nucleic acid fragment for inhibiting expression of the fadR gene into the pCDF-1b::temp vector, a nucleic acid fragment (SEQ ID NO: 24) containing a 24-base complementary strand including the start codon of the fadR gene was genetically synthesized (manufactured by Azenta Co., Ltd.).
• This fragment was ligated to pCDF-1b::temp/NcoI,BAP prepared by the method described in Reference Example 3 using "In-Fusion HD Cloning Kit” (manufactured by Takara Bio Inc.), and introduced into Escherichia coli strain DH5 ⁇ .
• the plasmid was extracted from the resulting recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was named pCDF-1b::fadRi.
• a loopful of the strain was inoculated into 5 mL of LB medium (Bacto tryptone (Difco Laboratories) 10 g/L, Bacto yeast extract (Difco Laboratories) 5 g/L, sodium chloride 5 g/L) containing kanamycin 25 ⁇ g/mL and streptomycin 50 ⁇ g/mL adjusted to pH 7, and cultured at 30°C and 120 min-1 for 24 hours with shaking.
• LB medium Bacto yeast extract (Difco Laboratories) 5 g/L, sodium chloride 5 g/L
• 0.05 mL of the culture solution was added to 5 mL of medium I (glucose 10 g/L, ammonium sulfate 1 g/L, potassium phosphate 50 mM, magnesium sulfate 0.025 g/L, ferrous sulfate 0.0625 mg/L, manganese sulfate 2.7 mg/L, calcium chloride 0.33 mg/L, sodium chloride 1.25 g/L, Bacto tryptone 2.5 g/L, Bacto yeast extract 1.25 g/L) containing 25 ⁇ g/mL kanamycin and 50 ⁇ g/mL streptomycin adjusted to pH 6.5 in a test tube, and cultured with shaking at 30°C and 120 min-1 for 24 hours.
• medium I glucose 10 g/L, ammonium sulfate 1 g/L, potassium phosphate 50 mM, magnesium sulfate 0.025 g/L, ferrous sulfate 0.0625 mg/L,
• the supernatant obtained by centrifuging the cells from the culture medium was treated with a Millex-GV membrane (0.22 ⁇ m, PVDF, Merck), and the permeate was analyzed by HPLC and LC-MS/MS. Quantitative analysis of the acetic acid and 3HA accumulated in the culture supernatant was performed, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.
• the pKD46 plasmid required for expressing ⁇ Red recombinase was introduced into the MG1655 strain by electroporation. After the introduction, the strain was cultured at 30° C. on LB agar medium containing 50 ⁇ g/mL ampicillin to obtain the E. coli MG1655/pKD46 strain.
• the nucleic acid fragment includes 500 b of the upstream region of the ldhA gene, the sacB gene, the kanamycin resistance gene, and 500 b of the downstream region of the ldhA gene on the genome of the E. coli MG1655 strain (SEQ ID NO: 25).
• Primers were designed (SEQ ID NOs: 26 and 27) to PCR amplify the nucleic acid fragment obtained by gene synthesis, and PCR reaction was performed according to a conventional method.
• the obtained fragment was purified by electrophoresis using agarose gel and a nucleic acid column purification kit, and used to create a strain lacking the ldhA gene.
• the nucleic acid fragment for deleting the ldhA gene was introduced into the E. coli MG1655/pKD46 strain by electroporation. After introduction, the strain was cultured at 30°C on LB agar medium containing 25 ⁇ g/mL kanamycin. The resulting recombinant strain is the E.
• coli MG1655/ldhA-sacB-kan strain in which the full length of the ldhA gene sequence has been replaced with a sequence (sacB-kan sequence) containing the full length of the sacB gene and the kanamycin resistance gene.
• a nucleic acid fragment for removing the sacB-kan sequence was synthesized by gene synthesis (Azenta Co., Ltd.).
• the fragment includes 500b of the upstream region and 500b of the downstream region of the ldhA gene on the genome of the E. coli MG1655 strain (SEQ ID NO: 28).
• a PCR reaction was carried out according to a conventional method using primers of SEQ ID NO: 26 and 27.
• the obtained fragment was purified by electrophoresis using agarose gel and a nucleic acid column purification kit, and used for removing the sacB-kan sequence.
• the nucleic acid fragment for removing the sacB-kan sequence was introduced into the E. coli MG1655/ldhA-sacB-kan strain by electroporation. After introduction, the strain was cultured at 30°C on LB agar medium containing 50 g/L sucrose. The colonies obtained were cultured at 30°C on LB agar medium and LB agar medium containing 25 ⁇ g/mL kanamycin, and strains without kanamycin resistance were selected. The strain obtained is an E. coli MG1655/ldhA gene deletion strain, in which the ldhA gene on the genome has been deleted.
• the pBBR1MCS-2::ATCTOR plasmid was introduced into the E. coli MG1655/ldhA gene deletion strain by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin.
• the pCDF-1b::temp plasmid was introduced into the resulting strain by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin and 50 ⁇ g/mL streptomycin.
• the pKD46 plasmid required for expressing ⁇ Red recombinase was introduced into the W3110 strain by electroporation. After the introduction, the strain was cultured at 30° C. on LB agar medium containing 50 ⁇ g/mL ampicillin to obtain the E. coli W3110/pKD46 strain.
• the nucleic acid fragment includes 500 b of the upstream region of the adhE gene, the sacB gene, the kanamycin resistance gene, and 500 b of the downstream region of the adhE gene on the genome of the E. coli MGW3110 strain (SEQ ID NO: 29).
• Primers were designed (SEQ ID NOs: 30 and 31) to PCR amplify the nucleic acid fragment obtained by gene synthesis, and a PCR reaction was performed according to a conventional method.
• the obtained fragment was purified by electrophoresis using agarose gel and a nucleic acid column purification kit, and used to create an adhE gene deletion strain.
• the nucleic acid fragment for deleting the adhE gene was introduced into the E. coli W3110/pKD46 strain by electroporation. After introduction, the strain was cultured at 30°C on LB agar medium containing 25 ⁇ g/mL kanamycin. The resulting recombinant strain is the E. coli W3110/adhE-sacB-kan strain, in which the full length of the adhE gene sequence has been replaced with a sequence (sacB-kan sequence) containing the full length of the sacB gene and the kanamycin resistance gene.
• a nucleic acid fragment for removing the sacB-kan sequence was synthesized by gene synthesis (Azenta Co., Ltd.).
• the fragment includes 500b of the upstream region and 500b of the downstream region of the adhE gene on the genome of the E. coli W3110 strain (SEQ ID NO: 32).
• a PCR reaction was carried out according to a conventional method using primers of SEQ ID NOs: 30 and 31.
• the obtained fragment was purified by electrophoresis using agarose gel and a nucleic acid column purification kit, and used for removing the sacB-kan sequence.
• the nucleic acid fragment for removing the sacB-kan sequence was introduced into the E.
• coli W3110/adhE-sacB-kan strain by electroporation. After introduction, the strain was cultured at 30°C on LB agar medium containing 50 g/L sucrose. The colonies obtained were cultured at 30°C on LB agar medium and LB agar medium containing 25 ⁇ g/mL kanamycin, and a strain lacking kanamycin resistance was selected. The resulting strain was an E. coli W3110/adhE gene deletion strain, in which the adhE gene on the genome was deleted.
• the pBBR1MCS-2::ATCTOR plasmid was introduced into the E. coli W3110/adhE gene deletion strain by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin.
• the pCDF-1b::temp plasmid was introduced into the resulting strain by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin and 50 ⁇ g/mL streptomycin.
• Example 1 Preparation and culture of ldhA gene deleted Escherichia coli str. K-12 substr. MG1655 strain into which pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::iclRi plasmid have been introduced
• the pCDF-1b::iclRi plasmid for suppressing the expression of the iclR gene was introduced by electroporation into the ldhA gene deleted Escherichia coli str.
• Example 2 Preparation and culture of adhE gene-deleted Escherichia coli str. K-12 substr. W3110 strain into which pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::iclRi plasmid have been introduced
• the pCDF-1b::iclRi plasmid for suppressing the expression of the iclR gene was introduced by electroporation into the adhE gene-deleted Escherichia coli str.
• Example 3 Preparation and culture of Escherichia coli str. K-12 substr. MG1655 strain lacking pykA, pykF and ldhA genes introduced with pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::iclRi plasmid
• pBBR1MCS-2 ::ATCTOR plasmid
• pCDF-1b :iclRi plasmid
• an Escherichia coli str. K-12 substr. MG1655 strain lacking the full length of the pykF and pykA genes (SEQ ID NOs: 33 and 34) encoding pyruvate kinase and the ldhA gene was prepared.
• the deletion method of the pykF and pykA genes was performed according to the method described in WO2020/230718.
• the ldhA gene was deleted using the method described in Comparative Example 2.
• the pBBR1MCS-2::ATCTOR plasmid was introduced into the pykF, pykA, and ldhA gene deletion strain by electroporation. After the introduction, the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin.
• the pCDF-1b::iclRi plasmid for suppressing the expression of the iclR gene was introduced into the obtained strain by electroporation. After the introduction, the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin and 50 ⁇ g/mL streptomycin.
• Example 3 it was shown that the production ratio of 3HA to acetate was significantly improved by deleting the ldhA gene in a genetically modified microorganism in which the pyruvate kinase function was deleted as described in detail in WO2020/230718 and further suppressing the expression of the iclR gene.
• Reference Example 7 Preparation of a plasmid for expressing an enzyme that catalyzes the reaction (reaction C) that produces HMA-CoA from 3HA-CoA
• reaction C an enzyme that catalyzes the reaction
• reaction C an enzyme that catalyzes the reaction
• pMW119 capable of autonomous replication in E. coli was cleaved with SacI to obtain pMW119/SacI.
• primers were designed (SEQ ID NOs: 39 and 40) for PCR amplification of the upstream region 200b (SEQ ID NO: 7) of gapA (NCBI Gene ID: NC_000913.3) using the genomic DNA of Escherichia coli K-12 MG1655 as a template, and a PCR reaction was carried out according to a conventional method.
• the obtained fragment and pMW119/SacI were ligated using "In-Fusion HD Cloning Kit” (manufactured by Takara Bio Inc.) and introduced into E. coli strain DH5 ⁇ .
• the plasmid was extracted from the resulting recombinant E.
• pMW119::Pgap the plasmid whose base sequence was confirmed by a conventional method was named pMW119::Pgap. Then, pMW119::Pgap was cleaved with SphI to obtain pMW119::Pgap/SphI.
• primers were designed (SEQ ID NOs: 42 and 43) for PCR amplification of the full length of the enoyl-CoA hydratase gene paaF (NCBI Gene ID: 1046932, SEQ ID NO: 41) using the genomic DNA of Pseudomonas putida KT2440 strain as a template, and a PCR reaction was carried out according to a conventional method.
• the obtained fragment and pMW119::Pgap/SphI were ligated using "In-Fusion HD Cloning Kit” (manufactured by Takara Bio Inc.) and introduced into Escherichia coli strain DH5 ⁇ .
• the plasmid was extracted from the obtained recombinant strain and the base sequence was confirmed by a conventional method.
• the obtained plasmid was named pMW119::EH.
• Reference Example 8 Preparation of a plasmid for expressing an enzyme that catalyzes the reaction (reaction C) of producing HMA-CoA from 3HA-CoA and the reaction (reaction D) of producing ADA-CoA from HMA-CoA pMW119::EH was cleaved with HindIII to obtain pMW119::EH/HindIII.
• primers were designed (SEQ ID NOs: 45 and 46) for PCR amplification of the full length of dcaA (NCBI-Protein ID: AAL09094.1, SEQ ID NO: 44) derived from Acinetobacter baylyi ADP1 strain, and a PCR reaction was carried out according to a conventional method.
• the obtained fragment and pMW119::EH/HindIII were ligated using "In-Fusion HD Cloning Kit” (manufactured by Takara Bio Inc.) and introduced into Escherichia coli strain DH5 ⁇ .
• the plasmid was extracted from the obtained recombinant strain, and the nucleotide sequence was confirmed by a standard method.
• the plasmid was named pMW119::EHER.
• the obtained fragment and pMW119::Pgap/KpnI were ligated using "In-Fusion HD Cloning Kit” (manufactured by Takara Bio Inc.) and introduced into Escherichia coli strain DH5 ⁇ .
• the plasmid was extracted from the obtained recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was named pMW119::aceB.
• Reference Example 10 Preparation of a Plasmid for Expressing aceK Gene pMW119::Pgap was cleaved with KpnI to obtain pMW119::Pgap/KpnI.
• primers for PCR amplification of the full length of aceK SEQ ID NO: 38
• SEQ ID NOs: 49, 50 primers for PCR amplification of the full length of aceK (SEQ ID NOs: 49, 50)
• the obtained fragment and pMW119::Pgap/KpnI were ligated using "In-Fusion HD Cloning Kit” (manufactured by Takara Bio Inc.) and introduced into Escherichia coli strain DH5 ⁇ .
• the plasmid was extracted from the obtained recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was named pMW119::aceK.
• Reference Example 11 Preparation and culture of Escherichia coli str. K-12 substr. MG1655 strain into which pBBR1MCS-2::ATCTOR plasmid, pCDF-1b::temp plasmid and pMW119::EH plasmid have been introduced
• the pMW119::EH plasmid was introduced by electroporation into the Escherichia coli str. K-12 substr. MG1655 strain into which the pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::temp plasmid prepared in Reference Example 5 had been introduced.
• the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin and 100 ⁇ g/mL ampicillin.
• a loopful of the strain was inoculated into 5 mL of LB medium (Bacto tryptone (Difco Laboratories) 10 g/L, Bacto yeast extract (Difco Laboratories) 5 g/L, sodium chloride 5 g/L) containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin, and 100 ⁇ g/mL ampicillin adjusted to pH 7, and cultured at 30°C and 120 min-1 for 24 hours with shaking.
• LB medium Bacto yeast extract (Difco Laboratories) 10 g/L, Bacto yeast extract (Difco Laboratories) 5 g/L, sodium chloride 5 g/L
• 0.05 mL of the culture solution was added to 5 mL of medium I (glucose 10 g/L, ammonium sulfate 1 g/L, potassium phosphate 50 mM, magnesium sulfate 0.025 g/L, ferrous sulfate 0.0625 mg/L, manganese sulfate 2.7 mg/L, calcium chloride 0.33 mg/L, sodium chloride 1.25 g/L, Bacto tryptone 2.5 g/L, Bacto yeast extract 1.25 g/L) containing kanamycin 25 ⁇ g/mL, streptomycin 50 ⁇ g/mL, and ampicillin 100 ⁇ g/mL adjusted to pH 6.5 in a test tube, and cultured with shaking at 30°C and 120 min-1 for 24 hours.
• medium I glucose 10 g/L, ammonium sulfate 1 g/L, potassium phosphate 50 mM, magnesium sulfate 0.025 g/L, ferrous
• the supernatant obtained by centrifuging the cells from the culture medium was treated with a Millex-GV membrane (0.22 ⁇ m, PVDF, Merck), and the permeate was analyzed by HPLC and LC-MS/MS. Quantitative analysis of the acetic acid and HMA accumulated in the culture supernatant was performed, and the production ratio of HMA to acetic acid calculated by replacing 3HA in formula (2) with HMA is shown in Table 2.
• Example 4 Preparation and Cultivation of ldhA Gene-Deleted Escherichia coli str. K-12 substr. MG1655 Strain Introduced with pBBR1MCS-2::ATCTOR Plasmid, pCDF-1b::iclRi Plasmid, and pMW119::EH Plasmid The pMW119::EH plasmid was introduced by electroporation into the ldhA gene-deleted Escherichia coli str. K-12 substr. MG1655 strain into which the pBBR1MCS-2::ATCTOR plasmid and the pCDF-1b::iclRi plasmid prepared in Example 1 had been introduced. After the introduction, the strain was cultured at 37° C. on LB agar medium containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin, and 100 ⁇ g/mL ampicillin.
• Reference Example 12 Preparation and culture of Escherichia coli str. K-12 substr. W3110 strain into which pBBR1MCS-2::ATCTOR plasmid, pCDF-1b::temp plasmid and pMW119::EH plasmid have been introduced The pMW119::EH plasmid was introduced by electroporation into the Escherichia coli str. K-12 substr. W3110 strain into which the pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::temp plasmid prepared in Reference Example 6 had been introduced. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin and 100 ⁇ g/mL ampicillin.
• Comparative Example 8 Preparation and Cultivation of adhE Gene-Deleted Escherichia coli str. K-12 substr.
• W3110 strain in which the pBBR1MCS-2::ATCTOR plasmid and the pCDF-1b::temp plasmid prepared in Comparative Example 3 were introduced was introduced with the pMW119::EH plasmid by electroporation. After the introduction, the strain was cultured at 37° C. on LB agar medium containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin, and 100 ⁇ g/mL ampicillin.
• the strain was cultured at 37°C on an LB agar medium containing 25 ⁇ g/mL kanamycin and 50 ⁇ g/mL streptomycin.
• the pMW119::EH plasmid was introduced into the obtained strain by electroporation.
• the strain was cultured at 37° C. on LB agar medium containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin, and 100 ⁇ g/mL ampicillin.
• Example 5 Preparation and Cultivation of adhE Gene-Deleted Escherichia coli str. K-12 substr.
• the pMW119::EH plasmid was introduced by electroporation into the adhE gene-deleted Escherichia coli str. K-12 substr.
• Reference Example 13 Preparation and culture of Escherichia coli str. K-12 substr. MG1655 strain into which pBBR1MCS-2::ATCTOR plasmid, pCDF-1b::temp plasmid and pMW119::EHER plasmid have been introduced
• the pMW119::EHER plasmid was introduced by electroporation into the Escherichia coli str. K-12 substr. MG1655 strain into which the pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::temp plasmid prepared in Reference Example 5 had been introduced.
• the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin and 100 ⁇ g/mL ampicillin.
• a loopful of the strain was inoculated into 5 mL of LB medium (Bacto tryptone (Difco Laboratories) 10 g/L, Bacto yeast extract (Difco Laboratories) 5 g/L, sodium chloride 5 g/L) containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin, and 100 ⁇ g/mL ampicillin adjusted to pH 7, and cultured at 30°C and 120 min-1 for 24 hours with shaking.
• LB medium Bacto yeast extract (Difco Laboratories) 10 g/L, Bacto yeast extract (Difco Laboratories) 5 g/L, sodium chloride 5 g/L
• 0.05 mL of the culture solution was added to 5 mL of medium I (glucose 10 g/L, ammonium sulfate 1 g/L, potassium phosphate 50 mM, magnesium sulfate 0.025 g/L, ferrous sulfate 0.0625 mg/L, manganese sulfate 2.7 mg/L, calcium chloride 0.33 mg/L, sodium chloride 1.25 g/L, Bacto tryptone 2.5 g/L, Bacto yeast extract 1.25 g/L) containing kanamycin 25 ⁇ g/mL, streptomycin 50 ⁇ g/mL, and ampicillin 100 ⁇ g/mL adjusted to pH 6.5 in a test tube, and cultured with shaking at 30°C and 120 min-1 for 24 hours.
• medium I glucose 10 g/L, ammonium sulfate 1 g/L, potassium phosphate 50 mM, magnesium sulfate 0.025 g/L, ferrous
• the supernatant obtained by centrifuging the cells from the culture medium was treated with a Millex-GV membrane (0.22 ⁇ m, PVDF, Merck), and the permeate was analyzed by HPLC and LC-MS/MS. Quantitative analysis of the acetic acid and ADA accumulated in the culture supernatant was performed, and the production ratio of ADA to acetic acid calculated by replacing 3HA in formula (2) with ADA is shown in Table 3.
• Comparative Example 10 Preparation and culture of ldhA gene-deleted Escherichia coli str. K-12 substr. MG1655 strain into which pBBR1MCS-2::ATCTOR plasmid, pCDF-1b::temp plasmid, and pMW119::EHER plasmid have been introduced.
• the pMW119::EHER plasmid was introduced by electroporation into the ldhA gene-deleted Escherichia coli str. K-12 substr. MG1655 strain into which the pBBR1MCS-2::ATCTOR plasmid and the pCDF-1b::temp plasmid prepared in Comparative Example 2 had been introduced.
• the strain was cultured at 37° C. on LB agar medium containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin, and 100 ⁇ g/mL ampicillin.
• Example 6 Preparation and Cultivation of ldhA Gene-Deleted Escherichia coli str. K-12 substr. MG1655 Strain Introduced with pBBR1MCS-2::ATCTOR Plasmid, pCDF-1b::iclRi Plasmid, and pMW119::EHER Plasmid The pMW119::EHER plasmid was introduced by electroporation into the ldhA gene-deleted Escherichia coli str. K-12 substr. MG1655 strain into which the pBBR1MCS-2::ATCTOR plasmid and the pCDF-1b::iclRi plasmid prepared in Example 1 had been introduced. After the introduction, the strain was cultured at 37° C. on LB agar medium containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin, and 100 ⁇ g/mL ampicillin.
• Reference Example 14 Preparation and culture of Escherichia coli str. K-12 substr. W3110 strain into which pBBR1MCS-2::ATCTOR plasmid, pCDF-1b::temp plasmid and pMW119::EHER plasmid have been introduced The pMW119::EHER plasmid was introduced by electroporation into the Escherichia coli str. K-12 substr. W3110 strain into which the pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::temp plasmid prepared in Reference Example 6 had been introduced. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin and 100 ⁇ g/mL ampicillin.
• Comparative Example 12 Preparation and Cultivation of adhE Gene-Deleted Escherichia coli str. K-12 substr.
• W3110 strain into which the pBBR1MCS-2::ATCTOR plasmid and the pCDF-1b::temp plasmid prepared in Comparative Example 3 had been introduced was introduced with the pMW119::EHER plasmid by electroporation. After the introduction, the strain was cultured at 37° C. on LB agar medium containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin, and 100 ⁇ g/mL ampicillin.
• the strain was cultured at 37°C on an LB agar medium containing 25 ⁇ g/mL kanamycin and 50 ⁇ g/mL streptomycin.
• the pMW119::EHER plasmid was introduced by electroporation into the obtained strain.
• the strain was cultured at 37° C. on LB agar medium containing 25 ⁇ g/mL kanamycin, 50 ⁇ g/mL streptomycin, and 100 ⁇ g/mL ampicillin.
• Example 7 Preparation and Cultivation of adhE Gene-Deleted Escherichia coli str. K-12 substr.
• the pMW119::EHER plasmid was introduced by electroporation into the adhE gene-deleted Escherichia coli str. K-12 substr.
• Reference Example 15 Preparation and culture of Escherichia coli str. K-12 substr. MG1655 strain into which pBBR1MCS-2::ATCTOR plasmid and pMW119::Pgap plasmid have been introduced The pMW119::Pgap plasmid was introduced by electroporation into the Escherichia coli str. K-12 substr. MG1655 strain into which the pBBR1MCS-2::ATCTOR plasmid prepared in Reference Example 5 had been introduced. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin and 100 ⁇ g/mL ampicillin.
• a loopful of the strain was inoculated into 5 mL of LB medium (Bacto tryptone (Difco Laboratories) 10 g/L, Bacto yeast extract (Difco Laboratories) 5 g/L, sodium chloride 5 g/L) containing 25 ⁇ g/mL kanamycin and 100 ⁇ g/mL ampicillin adjusted to pH 7, and cultured at 30°C and 120 min-1 for 24 hours with shaking.
• LB medium Bacto yeast extract (Difco Laboratories) 5 g/L, sodium chloride 5 g/L
• 0.05 mL of the culture solution was added to 5 mL of medium I (glucose 10 g/L, ammonium sulfate 1 g/L, potassium phosphate 50 mM, magnesium sulfate 0.025 g/L, ferrous sulfate 0.0625 mg/L, manganese sulfate 2.7 mg/L, calcium chloride 0.33 mg/L, sodium chloride 1.25 g/L, Bacto tryptone 2.5 g/L, Bacto yeast extract 1.25 g/L) containing kanamycin 25 ⁇ g/mL and ampicillin 100 ⁇ g/mL adjusted to pH 6.5 in a test tube, and cultured with shaking at 30°C and 120 min-1 for 24 hours.
• medium I glucose 10 g/L, ammonium sulfate 1 g/L, potassium phosphate 50 mM, magnesium sulfate 0.025 g/L, ferrous sulfate 0.0625 mg/L, manganes
• the supernatant obtained by centrifuging the cells from the culture medium was treated with a Millex-GV membrane (0.22 ⁇ m, PVDF, Merck), and the permeate was analyzed by HPLC and LC-MS/MS. Quantitative analysis of the acetic acid and 3HA accumulated in the culture supernatant was performed, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 4.
• Comparative Example 14 Preparation and culture of ldhA gene deleted Escherichia coli str. K-12 substr. MG1655 strain into which pBBR1MCS-2::ATCTOR plasmid and pMW119::Pgap plasmid were introduced The pMW119::Pgap plasmid was introduced by electroporation into the ldhA gene deleted Escherichia coli str. K-12 substr. MG1655 strain into which the pBBR1MCS-2::ATCTOR plasmid prepared in Comparative Example 2 was introduced. After introduction, the strain was cultured at 37 ° C. on LB agar medium containing 25 ⁇ g / mL kanamycin and 100 ⁇ g / mL ampicillin.
• Example 8 Preparation and culture of ldhA gene deleted Escherichia coli str. K-12 substr. MG1655 strain into which pBBR1MCS-2::ATCTOR plasmid and pMW119::aceB plasmid were introduced The pMW119::aceB plasmid was introduced by electroporation into the ldhA gene deleted Escherichia coli str. K-12 substr. MG1655 strain into which the pBBR1MCS-2::ATCTOR plasmid prepared in Comparative Example 2 was introduced. After introduction, the strain was cultured at 37 ° C. on LB agar medium containing 25 ⁇ g / mL kanamycin and 100 ⁇ g / mL ampicillin.
• Example 9 Preparation and culture of ldhA gene deleted Escherichia coli str. K-12 substr. MG1655 strain into which pBBR1MCS-2::ATCTOR plasmid and pMW119::aceK plasmid were introduced The pMW119::aceK plasmid was introduced by electroporation into the ldhA gene deleted Escherichia coli str. K-12 substr. MG1655 strain into which the pBBR1MCS-2::ATCTOR plasmid prepared in Comparative Example 2 was introduced. After introduction, the strain was cultured at 37 ° C. on LB agar medium containing 25 ⁇ g / mL kanamycin and 100 ⁇ g / mL ampicillin.
• Reference Example 16 Preparation and culture of Escherichia coli str. K-12 substr. W3110 strain into which pBBR1MCS-2::ATCTOR plasmid and pMW119::Pgap plasmid have been introduced The pMW119::Pgap plasmid was introduced by electroporation into the Escherichia coli str. K-12 substr. W3110 strain into which the pBBR1MCS-2::ATCTOR plasmid prepared in Reference Example 6 had been introduced. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin and 100 ⁇ g/mL ampicillin.
• Comparative Example 17 Preparation and culture of adhE gene-deleted Escherichia coli str. K-12 substr. W3110 strain into which pBBR1MCS-2::ATCTOR plasmid and pMW119::Pgap plasmid were introduced The pMW119::Pgap plasmid was introduced by electroporation into the adhE gene-deleted Escherichia coli str. K-12 substr. W3110 strain into which the pBBR1MCS-2::ATCTOR plasmid prepared in Comparative Example 3 was introduced. After introduction, the strain was cultured at 37 ° C. on LB agar medium containing 25 ⁇ g / mL kanamycin and 100 ⁇ g / mL ampicillin.
• Comparative Example 18 Preparation and culture of Escherichia coli str. K-12 substr. W3110 strain into which pBBR1MCS-2::ATCTOR plasmid and pMW119::aceB plasmid have been introduced The pMW119::aceB plasmid was introduced by electroporation into the Escherichia coli str. K-12 substr. W3110 strain into which the pBBR1MCS-2::ATCTOR plasmid prepared in Reference Example 6 had been introduced. After introduction, the strain was cultured at 37 ° C. on LB agar medium containing 25 ⁇ g / mL kanamycin and 100 ⁇ g / mL ampicillin.
• Example 10 Preparation and culture of adhE gene-deleted Escherichia coli str. K-12 substr. W3110 strain into which pBBR1MCS-2::ATCTOR plasmid and pMW119::aceB plasmid were introduced The pMW119::aceB plasmid was introduced by electroporation into the adhE gene-deleted Escherichia coli str. K-12 substr. W3110 strain into which the pBBR1MCS-2::ATCTOR plasmid prepared in Comparative Example 3 was introduced. After introduction, the strain was cultured at 37 ° C. on LB agar medium containing 25 ⁇ g / mL kanamycin and 100 ⁇ g / mL ampicillin.
• Comparative Example 19 Preparation and culture of Escherichia coli str. K-12 substr. W3110 strain into which pBBR1MCS-2::ATCTOR plasmid and pMW119::aceK plasmid have been introduced The pMW119::aceK plasmid was introduced by electroporation into the Escherichia coli str. K-12 substr. W3110 strain into which the pBBR1MCS-2::ATCTOR plasmid prepared in Reference Example 6 had been introduced. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 ⁇ g/mL kanamycin and 100 ⁇ g/mL ampicillin.
• Example 11 Preparation and culture of adhE gene deleted Escherichia coli str. K-12 substr. W3110 strain into which pBBR1MCS-2::ATCTOR plasmid and pMW119::aceK plasmid were introduced The pMW119::aceK plasmid was introduced by electroporation into the adhE gene deleted Escherichia coli str. K-12 substr. W3110 strain into which the pBBR1MCS-2::ATCTOR plasmid prepared in Comparative Example 3 was introduced. After introduction, the strain was cultured at 37 ° C. on LB agar medium containing 25 ⁇ g / mL kanamycin and 100 ⁇ g / mL ampicillin.
• Reference Example 15 and Examples 8 and 9 show that in an E. coli strain in which the ldhA gene has been deleted and the expression level of the aceB gene or the aceK gene has been increased, the production ratio of 3HA to acetic acid is improved.
• the results of Reference Example 16 and Examples 10 and 11 show that in an E. coli strain in which the adhE gene has been deleted and the expression level of the aceB gene or the aceK gene has been increased, the production ratio of 3HA to acetic acid is improved.

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