Method for producing acetyl-coa derivatives

Abstract

To enhance the productivity of acetyl-CoA derivatives in the production of acetyl-CoA derivatives through the cultivation of hydrogen-oxidizing bacteria using hydrogen, oxygen, and carbon dioxide.SOLUTION: Hydrogen-oxidizing bacteria transformed with an exogenous phosphoketolase gene are cultivated in the presence of hydrogen, oxygen, and carbon dioxide to produce acetyl-CoA derivatives. The hydrogen-oxidizing bacteria may be those classified under the genus Cupriavidus. The acetyl-CoA derivatives may be citric acid, 3-hydroxybutyric acid, or polyhydroxyalkanoic acid.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing an acetyl-CoA derivative by culturing hydrogen-oxidizing bacteria. [Background technology]

[0002] In recent years, growing awareness of global environmental issues has accelerated the movement away from fossil fuel resources. In this context, advances in biotechnology and digital technology have attracted attention to the development of "biomachining" technology, which applies synthetic biology to increase the substance production capabilities of microorganisms and other organisms, or to give them the ability to produce new target substances. In addition, the importance of carbon dioxide resource technology has been recognized as one of the measures against global warming, and specifically, development of technology to convert carbon dioxide, hydrogen, water, etc. into fuels and chemicals is being promoted both domestically and internationally.

[0003] Hydrogen-oxidizing bacteria can fix carbon dioxide using energy obtained by oxidizing hydrogen without relying on light energy, and can convert the fixed carbon dioxide into useful substances. For this reason, the production of useful substances using hydrogen-oxidizing bacteria that does not rely on fossil resources, carbohydrates, oils, etc. is attracting attention.

[0004] One example of a useful substance that can be produced by hydrogen-oxidizing bacteria is polyhydroxyalkanoic acid (hereinafter referred to as PHA). PHA is a biodegradable polymeric material that can be used as an alternative to plastics, and social implementation is already underway. PHA is biodegradable in seawater and has attracted attention as a material that can solve the marine pollution caused by waste plastic.

[0005] It is known that hydrogen-oxidizing bacteria such as Cupriavidus necator accumulate polyhydroxyalkanoic acid (hereinafter, also referred to as PHA) by being cultured in a mixed gas containing hydrogen, oxygen, and carbon dioxide. There are reports of genetic engineering modifications to improve PHA productivity in the gas culture of C. necator. For example, Non-Patent Document 1 reports that overexpression of a group of genes that regulate the expression level of the Calvin-Benson-Bassham operon in the C. necator H16 strain was performed, improving the bacterial yield and PHA content. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Kim S.,et al.,Microb. Cell Fact.,21:231(2022) Summary of the Invention [Problem to be solved by the invention]

[0007] As described above, production technologies for useful substances such as PHAs by culturing hydrogen-oxidizing bacteria using hydrogen, oxygen, and carbon dioxide have been reported. However, the methods developed so far are still insufficient and the production costs are high, so there is a strong need to improve productivity.

[0008] In view of the above-mentioned current situation, the present invention aims to improve the productivity of acetyl-CoA derivatives in the production of acetyl-CoA derivatives by culturing hydrogen-oxidizing bacteria using hydrogen, oxygen, and carbon dioxide. [Means for solving the problem]

[0009] As a result of intensive research to solve the above-mentioned problems, the inventors discovered that the productivity of acetyl-CoA derivatives by hydrogen-oxidizing bacteria can be improved by culturing hydrogen-oxidizing bacteria transformed with an exogenous phosphoketolase gene in the presence of hydrogen, oxygen, and carbon dioxide, and thus completed the present invention.

[0010] That is, the present invention relates to a method for producing an acetyl-CoA derivative, which comprises a step of culturing a hydrogen-oxidizing bacterium transformed with an exogenous phosphoketolase gene in the presence of hydrogen, oxygen, and carbon dioxide. Effect of the Invention

[0011] According to the present invention, in the production of acetyl-CoA derivatives by culturing hydrogen-oxidizing bacteria using hydrogen, oxygen, and carbon dioxide, the productivity of acetyl-CoA derivatives can be improved. According to the present invention, the productivity of acetyl-CoA derivatives per unit volume of solution can be improved, the production efficiency can be improved, and the production cost can be reduced. [Brief description of the drawings]

[0012] [Figure 1] Graph showing the change over time in optical density (600 nm) of each culture solution in Example 1 and Comparative Example 1 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, an embodiment of the present invention will be described, however, the present invention is not limited to the following embodiment. The present embodiment relates to a method for producing an acetyl-CoA derivative, which includes a step of culturing a hydrogen-oxidizing bacterium transformed with an exogenous phosphoketolase gene in the presence of hydrogen, oxygen, and carbon dioxide.

[0014] (Acetyl CoA derivative) The acetyl-CoA derivative in the present application is a general term for compounds that are produced through a pathway in which acetyl-CoA, one of the compounds that constitute the basis of central metabolism, is an important intermediate, and that are located downstream of acetyl-CoA when the Calvin-Benson-Bassham cycle is considered as the upstream in the metabolic pathway of hydrogen-oxidizing bacteria. Specific examples of acetyl-CoA derivatives include, but are not limited to, citric acid, 3-hydroxybutyric acid, and PHA. In the method for producing an acetyl-CoA derivative in this embodiment, the acetyl-CoA derivative to be produced may be one type or two or more types.

[0015] (Hydrogen-oxidizing bacteria) Hydrogen-oxidizing bacteria are chemosynthetic bacteria that can grow by oxidizing hydrogen molecules, utilizing the energy produced by the reaction to fix carbon dioxide and produce acetyl-CoA via the Calvin-Benson-Bassham cycle.

[0016] The transformed hydrogen-oxidizing bacteria in this embodiment are hydrogen-oxidizing bacteria capable of producing acetyl-CoA derivatives and are transformed with an exogenous gene encoding phosphoketolase.

[0017] The host of the transformed hydrogen-oxidizing bacteria in this embodiment is not particularly limited as long as it is a hydrogen-oxidizing bacterium, and examples thereof include bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, Alcaligenes, Pseudomonas, Bacillus, Hydrogenobacter, Streptomyces, Mycobacterium, etc. From the viewpoint of safety and productivity, bacteria belonging to the genera Ralstonia, Cupriavidus, and Wautersia are preferred, hydrogen-oxidizing bacteria belonging to the genus Cupriavidus are more preferred, and Cupriavidus necator is particularly preferred.

[0018] The host of the transformed hydrogen-oxidizing bacteria in this embodiment may be a wild-type strain, a mutant strain obtained by artificially mutating a wild-type strain so as to be suitable for the acetyl-CoA derivative to be produced, or a transformed strain into which an exogenous gene has been introduced by genetic engineering techniques, and can be appropriately selected.

[0019] For example, when the acetyl-CoA derivative to be produced is PHA, the host of the transformed hydrogen-oxidizing bacterium in this embodiment may be a wild-type strain that inherently has a PHA synthase gene, or a mutant strain obtained by artificially mutating such a wild-type strain, or a transformed strain into which an exogenous PHA synthase gene has been introduced by genetic engineering techniques.

[0020] The method for introducing a foreign gene is not particularly limited, and may include a method for directly inserting or replacing a gene on a host chromosome, a method for directly inserting or replacing a gene on a megaplasmid possessed by the host, or a method for arranging a gene on a vector such as a plasmid, phage, or phagemid and introducing the gene, or two or more of these methods may be used in combination. Considering the stability of the introduced gene, a method for directly inserting or replacing a gene on a host chromosome or a megaplasmid possessed by the host is preferred, and a method for directly inserting or replacing a gene on a host chromosome is more preferred.

[0021] When the acetyl-CoA derivative to be produced is a PHA, the type of PHA is not particularly limited as long as it is a PHA that can be produced by hydrogen-oxidizing bacteria, but is preferably a homopolymer of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, a copolymer of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, a copolymer of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and another hydroxyalkanoic acid (e.g., 2-hydroxyalkanoic acids, 4-hydroxyalkanoic acids, 5-hydroxyalkanoic acids, 6-hydroxyalkanoic acids, etc. having 4 to 16 carbon atoms), and a copolymer of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and another hydroxyalkanoic acid. Particularly preferred are a homopolymer of 3-hydroxyalkanoic acid having 4 carbon atoms and a copolymer containing 3-hydroxyalkanoic acid having 4 carbon atoms. Examples of PHAs include, but are not limited to, P(3HB), a homopolymer of 3-hydroxybutyric acid (abbreviation: 3HB), P(3HB-co-3HV), a copolymer of 3HB and 3-hydroxyvaleric acid (abbreviation: 3HV), P(3HB-co-3HH) (abbreviation: PHBH), P(3HB-co-4HB), a copolymer of 3HB and 4-hydroxybutyric acid (abbreviation: 4HB), and PHAs containing lactic acid (abbreviation: LA) as a component, such as P(LA-co-3HB), a copolymer of 3HB and LA. Among these, PHBH is preferred from the viewpoint of its wide range of application as a polymer. The type of PHA produced can be appropriately selected depending on the purpose, the type of PHA synthase gene possessed by the hydrogen-oxidizing bacteria used or introduced separately, the type of metabolic system gene involved in the synthesis, the culture conditions, and the like.

[0022] (phosphoketolase gene) The transformed hydrogen-oxidizing bacteria according to this embodiment are transformed with an exogenous phosphoketolase gene. According to this embodiment, the productivity of acetyl-CoA derivatives can be improved by introducing an exogenous phosphoketolase gene into the hydrogen-oxidizing bacteria to newly secure or strengthen a pathway for synthesizing acetyl-CoA from the carbon dioxide fixation pathway (Calvin-Benson-Bassham cycle). In particular, in the case of hydrogen-oxidizing bacteria that do not inherently have a phosphoketolase gene, such as Capriavidus necator, a new pathway for synthesizing acetyl-CoA from the carbon dioxide fixation pathway can be secured by introducing an exogenous phosphoketolase gene.

[0023] In addition, the foreign phosphoketolase gene means that the biological species from which the phosphoketolase gene is derived is different from the biological species of the host of the transformed hydrogen-oxidizing bacterium. In addition, the number of phosphoketolase genes to be introduced may be one or more.

[0024] Phosphoketolase refers to an enzyme that catalyzes the reaction of xylulose-5-phosphate as a substrate to produce glyceraldehyde-3-phosphate and acetyl phosphate, an enzyme that catalyzes the reaction of fructose-6-phosphate as a substrate to produce erythrose-4-phosphate and acetyl phosphate, or an enzyme that catalyzes the reaction of sedoheptulose-7-phosphate as a substrate to produce ribose-5-phosphate and acetyl phosphate. Acetyl CoA and acetyl CoA derivatives can be produced from the acetyl phosphate produced by these enzymatic reactions.

[0025] The origin of the phosphoketolase gene is not particularly limited, and examples thereof include microorganisms belonging to the genus Pseudomonas, microorganisms belonging to the genus Ideonella, microorganisms belonging to the genus Afipia, and microorganisms belonging to the genus Achromobacter.

[0026] Specific examples of phosphoketolase genes derived from the genus Pseudomonas include genes encoding the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. Specific examples of phosphoketolase genes derived from the genus Ideonella include genes encoding the amino acid sequence set forth in SEQ ID NO: 4. Specific examples of phosphoketolase genes derived from the genus Aphidopia include genes encoding the amino acid sequence set forth in SEQ ID NO: 5. Specific examples of phosphoketolase genes derived from the genus Achromobacter include genes encoding the amino acid sequence set forth in SEQ ID NO: 6.

[0027] The phosphoketolase gene to be introduced into the transformed hydrogen-oxidizing bacterium according to this embodiment is preferably a gene encoding a phosphoketolase containing an amino acid sequence showing 90 to 100% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6. The sequence identity is preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more.

[0028] An amino acid sequence having less than 100% sequence identity to an amino acid sequence shown in any one of SEQ ID NOs: 1 to 6 is preferably an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted or added in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6.

[0029] Such amino acid substitution, deletion, insertion or addition is preferably a conservative mutation that maintains the normal function of the protein, and more preferably a conservative substitution. Examples of conservative substitution include substitution between aromatic amino acids, substitution between hydrophobic amino acids, substitution between polar amino acids, substitution between basic amino acids, and substitution between amino acids having a hydroxyl group. The amino acid substitution, deletion, insertion or addition may be an artificial mutation, or a natural mutation due to individual differences in the organism from which the gene originates, differences in species, etc.

[0030] The method for introducing a target gene into a host is not particularly limited, and may include directly inserting or replacing the target gene on a chromosome of the host, directly inserting or replacing the target gene on a megaplasmid carried by the host, or placing the target gene on a vector such as a plasmid, phage, or phagemid and introducing it, and two or more of these methods may be used in combination.

[0031] Considering the stability of the introduced gene, a method in which the gene of interest is directly inserted or replaced onto the host chromosome or onto a megaplasmid carried by the host is preferred, and a method in which the gene of interest is directly inserted or replaced onto the host chromosome is more preferred.

[0032] In order to ensure the expression of the introduced gene, it is preferable to introduce the target gene so that it is located downstream of a "gene expression regulatory sequence" that the host originally has, or to introduce the target gene so that it is located downstream of an exogenous "gene expression regulatory sequence." In this application, the "gene expression regulatory sequence" is a DNA sequence that includes a base sequence (e.g., a promoter sequence) that controls the transcription amount of the gene, and / or a base sequence (e.g., a Shine-Dalgarno sequence) that controls the translation amount of messenger RNA transcribed from the gene. As the "gene expression regulatory sequence," any base sequence that exists in nature can be used, or an artificially constructed or modified base sequence can be used.

[0033] Examples of promoter sequences and Shine-Dalgarno sequences contained in the "gene expression regulatory sequence" include, but are not limited to, the base sequences shown in any of SEQ ID NOs: 7 to 12, or base sequences containing parts of these base sequences.

[0034] Substitution, deletion, insertion and / or addition of at least a portion of genomic DNA can be performed by methods well known to those skilled in the art, including a method utilizing a transposon and the mechanism of homologous recombination (Ohman et al., J. Bacteriol., 162:1068-1074 (1985)) and a method based on site-specific integration caused by the mechanism of homologous recombination and loss by a second step of homologous recombination (Noti et al., Methods Enzymol., 154:197-217 (1987)). Alternatively, a method can be used in which the sacB gene from Bacillus subtilis is coexisted and a hydrogen-oxidizing bacteria strain in which the gene has been eliminated by a second-stage homologous recombination is easily isolated as a sucrose-resistant strain (Schweizer, Mol. Microbiol., 6:1195-1204 (1992); Lenz et al., J. Bacteriol., 176:4385-4393 (1994)). As another method, genome editing technology using the CRISPR / Cas9 system to modify target DNA (Y. Wang et al., ACS Synth Biol. 2016, 5(7):721-732) can also be used. In the CRISPR / Cas9 system, guide RNA (gRNA) has a sequence that can bind to a part of the base sequence of the genomic DNA to be modified, and plays a role in carrying Cas9 to the target.

[0035] The method for introducing a vector into a cell is not particularly limited, and examples thereof include the calcium chloride method, electroporation, polyethylene glycol method, and spheroplast method.

[0036] (Production of acetyl-CoA derivatives by gas culture) Culturing the transformed hydrogen-oxidizing bacteria in the presence of hydrogen, oxygen, and carbon dioxide enables the production of acetyl-CoA derivatives using carbon dioxide as a carbon source. The transformed hydrogen-oxidizing bacteria may be cultured in a medium in the presence of a mixed gas containing hydrogen, oxygen, and carbon dioxide. The medium composition, gas addition method, culture scale, aeration and stirring conditions, culture temperature, culture time, and the like are not particularly limited. The gas to be aerated may be a gas in which appropriate components are mixed in advance, or each gas may be aerated separately to obtain an appropriate composition in the culture tank. It is preferable to aerate the gas into the culture tank continuously or intermittently.

[0037] The composition of the gas to be aerated is not particularly limited as long as the transformed hydrogen-oxidizing bacteria can produce an acetyl-CoA derivative, but the hydrogen gas concentration as a ratio in the culture gas phase is preferably 1 vol% or more, more preferably 10 vol% or more, even more preferably 30 vol% or more, and particularly preferably 60 vol% or more, from the viewpoint of utilization efficiency by the hydrogen-oxidizing bacteria. The oxygen gas concentration is preferably 0.1 to 80 vol%, more preferably 0.1 to 50 vol%, even more preferably 0.1 to 10 vol%, and particularly preferably 0.1 to 5 vol%, from the viewpoint of utilization efficiency by the hydrogen-oxidizing bacteria and toxicity due to high concentration oxygen. The carbon dioxide gas concentration is preferably 0.1 to 80 vol%, more preferably 1 to 50 vol%, even more preferably 2 to 30 vol%, and particularly preferably 5 to 20 vol%, from the viewpoint of utilization efficiency by the hydrogen-oxidizing bacteria and toxicity due to high concentration carbon dioxide.

[0038] In the production of the acetyl-CoA derivative in this embodiment, it is preferable to culture the hydrogen-oxidizing bacteria in a medium containing a nitrogen source, inorganic salts, and other organic nutrient sources in the presence of the mixed gas. Examples of the nitrogen source include, but are not limited to, ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, yeast extract, and the like. Examples of the inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride, and the like. Examples of the other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C, and the like.

[0039] (Recovery of acetyl-CoA derivatives) The transformed hydrogen-oxidizing bacteria may be cultured for an appropriate period of time to produce an acetyl-CoA derivative, and the acetyl-CoA derivative may then be collected from the medium by any method without particular limitation.

[0040] For example, when the acetyl-CoA derivative produced is PHA, the PHA can be collected from the cells by the following method. After the culture is completed, the cells are separated from the culture liquid by a centrifuge or the like, washed with distilled water, methanol, or the like, and dried. PHA is extracted from the dried cells using an organic solvent such as chloroform. Cell components are removed from the solution containing PHA by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA. Furthermore, the supernatant is removed by filtration or centrifugation, and the PHA can be collected by drying.

[0041] As another example, the bacterial cells are separated from the culture medium using a centrifuge or the like, and the bacterial cells are washed with distilled water, methanol, etc. Then, the washed sample is mixed with a sodium lauryl sulfate (SDS) solution, the cell membrane is disrupted by ultrasonication, the bacterial components and PHA are separated using a centrifuge or the like, and the PHA can be recovered by drying the PHA.

[0042] The following items enumerate preferred aspects of the present disclosure, but the present invention is not limited to the following items. [Item 1] A method for producing an acetyl-CoA derivative, comprising the step of culturing a hydrogen-oxidizing bacterium transformed with an exogenous phosphoketolase gene in the presence of hydrogen, oxygen, and carbon dioxide. [Item 2] Item 2. The method for producing an acetyl-CoA derivative according to Item 1, wherein the hydrogen gas concentration in the culture gas phase is 1 vol% or more. [Item 3] 3. The method for producing an acetyl-CoA derivative according to item 1 or 2, wherein the carbon dioxide gas concentration in the culture gas phase is 0.1 vol% or more. [Item 4] 4. The method for producing an acetyl-CoA derivative according to any one of items 1 to 3, wherein the hydrogen-oxidizing bacteria belongs to the genus Capriavidus. [Item 5] 5. The method for producing an acetyl-CoA derivative according to Item 4, wherein the hydrogen-oxidizing bacterium is Capriavidus necator. [Item 6] 6. The method for producing an acetyl-CoA derivative according to any one of items 1 to 5, wherein the phosphoketolase gene is derived from the genus Pseudomonas, Ideonella, Aphidipia, or Achromobacter. [Item 7] 7. The method for producing an acetyl-CoA derivative according to Item 6, wherein the phosphoketolase gene is a gene encoding a phosphoketolase comprising an amino acid sequence showing 90 to 100% sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 1 to 6. [Item 8] 8. The method according to any one of items 1 to 7, wherein the acetyl-CoA derivative is at least one selected from the group consisting of citric acid, 3-hydroxybutyric acid, and polyhydroxyalkanoic acid. [Item 9] Item 9. The method according to item 8, wherein the acetyl-CoA derivative is a polyhydroxyalkanoic acid. EXAMPLES

[0043] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples. The overall genetic manipulation can be carried out, for example, as described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Enzymes, cloning hosts, and the like used in the genetic manipulation can be purchased from commercial suppliers and used according to their instructions. There are no particular limitations on the enzymes used, so long as they can be used in genetic manipulation.

[0044] (Microbial strain preparation example 1) Preparation of hydrogen oxidizing bacteria strain 1 transformed with phosphoketolase gene (hereinafter sometimes referred to as "PK strain 1") First, a plasmid for introducing the phosphoketolase gene was prepared as follows. A DNA fragment (SEQ ID NO: 13) containing the nucleotide sequence upstream of the A0006 structural gene of Cupriavidus necator H16 strain and a DNA fragment (SEQ ID NO: 14) containing the nucleotide sequence downstream of the A0006 structural gene of Cupriavidus necator H16 strain were obtained by PCR using the genomic DNA of Cupriavidus necator H16 strain as a template. These DNA fragments were simultaneously ligated to the EcoRI site of plasmid pK18mobsacB (National Institute of Genetics, Shizuoka, Japan) using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain plasmid pK18-A0006Del.

[0045] Next, a DNA fragment (SEQ ID NO: 15) containing the base sequence of a gene encoding a phosphoketolase having a trc promoter sequence and the amino acid sequence shown in SEQ ID NO: 1 was obtained by PCR using synthetic oligo DNA. Furthermore, PCR was performed using PCR primers with the base sequences shown in SEQ ID NO: 16 and SEQ ID NO: 17, and the plasmid pK18-A0006Del as a template to amplify the DNA fragment. These obtained DNA fragments were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to prepare a plasmid pK18-PaXFPK for introducing the phosphoketolase gene.

[0046] Next, the PK strain 1 was prepared as follows using the plasmid pK18-PaXFPK for introducing the phosphoketolase gene. The Escherichia coli S17-1 strain (ATCC47055) was transformed with the plasmid pK18-PaXFPK for introducing the phosphoketolase gene, and the resulting transformed microorganism was mixed and cultured with Cupriavidus necator H16 strain on LB agar medium for conjugation transfer. The resulting culture solution was cultured on MBM medium (beef extract 1% w / v, peptone 1% w / v, yeast extract 0.2% w / v, Na 2 Cl 0.01% w / v, 10 μg / mL gentamicin and 200 μg / mL kanamycin) containing 10 μg / mL gentamicin. 2 HPO 4 12H 2 O 0.9% w / v, KH 2 PO 4 The strains were then plated on agar medium (0.15% w / v, 1.5% w / v agar) and the strains that grew on the agar medium were selected to obtain a strain in which the plasmid had been integrated into the chromosome of Cupriavidus necator H16 strain. The strains were then cultured in MBM medium containing 10 μg / mL gentamicin and 200 μg / mL kanamycin, and then diluted and spread on MBM medium containing 5% sucrose and 10 μg / mL gentamicin. The strains that grew were obtained as strains in which the plasmid had been lost. Furthermore, one strain in which the phosphoketolase gene had been introduced into the chromosome was isolated by PCR and DNA sequencer analysis and named PK strain 1.

[0047] (Comparative Example 1) Production of acetyl-CoA derivatives by gas culture using Cupriavidus necator H16 strain A culture study was carried out using Cupriavidus necator H16 strain under the following conditions.

[0048] (Culture medium) The composition of the medium was 1.1 w / v% Na 2 HPO 4 12H 2 O, 0.19w / v%KH 2 PO 4 , 1.0w / v%(NH 4 ) 2 SO 4 , 0.1w / v% MgSO 4 7H 2 0, 0.895 w / v% Tricine, 0.1 v / v% trace metal salt solution (1.62 w / v% FeCl in 0.1 N hydrochloric acid) 3 6H 2 O, 1.03w / v% CaCl 2 2H 2 O, 0.022w / v% CoCl 2 6H 2 O, 0.016w / v% CuSO 4 5H 2 O, 0.012w / v% NiCl 2 6H 2 2O was dissolved in the solution) and the pH was adjusted to 7.1 with NaOH.

[0049] (Analysis of Acetyl CoA Derivatives) Low molecular weight acetyl-CoA derivatives were analyzed as follows: First, 1 mL of culture medium was suspended in 3 mL of washing solution (40 v / v % ethanol, 0.8 w / v % NaCl), centrifuged at 4°C and 6000 × g for 5 minutes, and the supernatant was removed. The cells were then suspended in an extract solution (37.38 μM L-methionine sulfone, piperazine-1, and 37.38 μM 4-bis(2-ethanesulfonic acid) dissolved in methanol) to extract intracellular metabolites, which were then analyzed by capillary electrophoresis-mass spectrometry (CE-MS) (CE; Agilent G7100, MS; Agilent G6224AA LC / MSD TOF; Agilent Technologies, Palo Alto, CA, USA) as described previously (Hasunuma, T.; Matsuda M.; Kondo, A. Improved sugar-free succinate production by Synechocystis sp. PCC 6803 following identification of the limiting steps in glycogen catabolism. Metab. Eng. Commun. 2016, 3, 130-141. DOI: 10.1016 / j.meteno.2016.04.003).

[0050] (Method of measuring PHA production volume) The amount of PHA produced was measured according to the method described in a previous publication (J. Biosci. Bioeng., 124, 250-254 (2017)) as follows. First, 20 mL of culture medium was collected, and the supernatant was removed by centrifugation to obtain wet cells. Next, the obtained wet cells were suspended in a 1% SDS solution, disrupted by ultrasonic treatment, and then the PHA was collected by centrifugation. The obtained PHA was washed with distilled water, freeze-dried, and weighed to measure the amount of PHA produced.

[0051] (Gas Culture) Gas culture was carried out as follows. First, Cupriavidus necator H16 strain was inoculated into 30 mL of the above medium in a 300 mL baffled Erlenmeyer flask so that the optical density at 600 nm was 0.2. Then, the medium was stirred using a magnetic stirrer and H 2 :O 2 :CO 2 The mixture was cultured at 30°C for 54 hours while aerating (70 mL / min) the gases mixed at a volume ratio of 8:1:1 to obtain a preculture solution.

[0052] Next, the preculture solution was inoculated into 70 mL of the above medium in a separate 300 mL baffled Erlenmeyer flask so that the optical density at 600 nm was 0.2. After that, the medium was stirred using a magnetic stirrer and heated at 120°C for 1 hour. 2 :O 2 :CO 2 The bacteria were cultured at 30°C for 64 hours while aerating (70mL / min) a mixture of gases at a volume ratio of 8:1:1, and PHA production was carried out. During the culture, the culture solution was sampled at appropriate times and the optical density (600nm) was measured. After 64 hours of culture, the amount of low molecular weight acetyl-CoA derivatives and PHA produced were measured using the method described above.

[0053] (Example 1) Production of acetyl-CoA derivatives by gas culture using PK strain 1 Under the same conditions as in Comparative Example 1, a culture study was carried out using PK strain 1 obtained in Microorganism Production Example 1, and the amounts of low molecular weight acetyl-CoA derivatives and PHA produced were measured.

[0054] The amounts of citric acid, 3-hydroxybutyric acid, and PHA produced by each strain are shown in Table 1. The amounts of citric acid and 3-hydroxybutyric acid are shown per cell weight (RCW) (subtracting PHA from the bacterial cell weight), and the amount of PHA is shown per culture medium. FIG. 1 shows the change in optical density (600 nm) of each strain during gas culture.

[0055] [Table 1]

[0056] From Table 1, it can be seen that the production amounts of 3-hydroxybutyric acid, citric acid, and PHA were each improved in Example 1 compared to Comparative Example 1. This clearly shows that the introduction of an exogenous phosphoketolase gene into the hydrogen oxidizing bacteria improved the productivity of acetyl-CoA derivatives by culturing the hydrogen oxidizing bacteria using hydrogen, oxygen, and carbon dioxide.

[0057] Furthermore, from FIG. 1, it can be seen that the optical density of the culture medium in Example 1 was higher than that in Comparative Example 1, and the growth rate of the hydrogen oxidizing bacteria was improved by introducing an exogenous phosphoketolase gene into the hydrogen oxidizing bacteria.

Claims

1. A method for producing an acetyl-CoA derivative, comprising culturing a hydrogen-oxidizing bacterium transformed with an exogenous phosphoketolase gene in the presence of hydrogen, oxygen, and carbon dioxide.

2. The method for producing an acetyl-CoA derivative according to claim 1, wherein the hydrogen gas concentration in the culture gas phase is 1 vol% or more.

3. 3. The method for producing an acetyl-CoA derivative according to claim 1 or 2, wherein the carbon dioxide gas concentration in the culture gas phase is 0.1 vol% or more.

4. The method for producing an acetyl-CoA derivative according to claim 1 or 2, wherein the hydrogen-oxidizing bacteria belong to the genus Capriavidus.

5. The method for producing an acetyl-CoA derivative according to claim 4, wherein the hydrogen-oxidizing bacterium is Capriavidus necator.

6. The method for producing an acetyl-CoA derivative according to claim 1 or 2, wherein the phosphoketolase gene is derived from the genus Pseudomonas, Ideonella, Aphidipia, or Achromobacter.

7. The method for producing an acetyl-CoA derivative according to claim 6, wherein the phosphoketolase gene is a gene encoding a phosphoketolase comprising an amino acid sequence showing 90 to 100% sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 1 to 6.

8. The method according to claim 1 or 2, wherein the acetyl-CoA derivative is at least one selected from the group consisting of citric acid, 3-hydroxybutyric acid, and polyhydroxyalkanoic acid.

9. The method according to claim 8, wherein the acetyl-CoA derivative is a polyhydroxyalkanoic acid.

Images