WO2024236787A1 - 物質合成を実施する方法、およびｃｏ２を吸収し、コハク酸を製造するための方法 - Google Patents

物質合成を実施する方法、およびｃｏ２を吸収し、コハク酸を製造するための方法 Download PDF

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WO2024236787A1
WO2024236787A1 PCT/JP2023/018494 JP2023018494W WO2024236787A1 WO 2024236787 A1 WO2024236787 A1 WO 2024236787A1 JP 2023018494 W JP2023018494 W JP 2023018494W WO 2024236787 A1 WO2024236787 A1 WO 2024236787A1
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gene encoding
acid
gene
culturing
organism
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French (fr)
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麻衣子 田邉
美和 佐藤
康一 渡辺
敦史 望月
崇 岡田
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Hitachi Ltd
Kyoto University NUC
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Kyoto University NUC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to a method for performing material synthesis and a method for absorbing CO2 and producing succinic acid.
  • a non-patent document (Scientific Reports 5:17321) describes the co-expression of the bicA gene and genes encoding sodium ion-dependent bicarbonate transporters (pck and sbtA) in E. coli.
  • pck and sbtA sodium ion-dependent bicarbonate transporters
  • the present invention aims to provide a method for performing material synthesis.
  • One embodiment of the present invention is a method for synthesizing a substance by modifying a gene and culturing the modified organism to form a metabolic pathway in which a non-genetically modified CO2 excretion site is converted to absorption.
  • the genes are a gene encoding a sodium ion-dependent bicarbonate transporter and a gene encoding phosphoenolpyruvate carboxykinase, and the method includes culturing a microorganism in which each of the genes is forcibly expressed, and the substance may be succinic acid.
  • the metabolic pathway in which the CO2 excretion site is converted to absorption may cause the following reaction in the TCA cycle: (1) Succinyl CoA + CO 2 ⁇ ⁇ -ketoglutaric acid (2) ⁇ -ketoglutaric acid + CO 2 ⁇ D-isocitric acid.
  • Another embodiment of the present invention is a method for absorbing CO2 , comprising culturing a microorganism in which a gene encoding a sodium ion-dependent bicarbonate transporter and a gene encoding phosphoenolpyruvate carboxykinase are forcibly expressed.
  • a further embodiment of the present invention is a method for producing succinic acid, comprising the step of culturing a microorganism in which the bicA gene and the pckA gene are overexpressed.
  • a further embodiment of the present invention is a method for inhibiting CO2 emission, comprising culturing a microorganism in which the bicA and pckA genes are overexpressed.
  • the bicA gene may be expressed at 10 copies or more per cell, or at least 100- fold over background, and the pckA gene may be expressed at 10-fold over vector alone.
  • the microorganism may be Escherichia coli.
  • the bicA gene may be derived from Synechococcus, and the pckA gene may be derived from Escherichia coli.
  • FIG. 1 shows (A) the base sequence of the coding region of the bicA gene in one embodiment of the present invention, and (B) the amino acid sequence of the protein encoded thereby.
  • FIG. 1 shows (A) the base sequence of the coding region of the pckA gene in one embodiment of the present invention, and (B) the amino acid sequence of the protein encoded thereby.
  • This is a diagram showing the sequences of the expression vectors expressing the bicA gene and the pckA gene used in one embodiment of the present invention. The continuous sequences are shown in three parts, (A) to (C). The sequences in (A) and (C) are derived from the pET vector, and the sequence in (B) is the inserted sequence.
  • FIG. 1 shows the results of examining the expression level of a gene inserted into an expression vector in one example of the present invention.
  • FIG. 1 shows the results of investigating the expression levels of elements of the TCA cycle in one example of the present invention.
  • FIG. 1 is a diagram showing a stoichiometric matrix calculated assuming fluxes for normal rotation of the TCA cycle in one embodiment of the present invention.
  • FIG. 1 shows the results of verification performed by decomposing each reaction in which the synthesis amount increases and is consistent with the experimental results in a stoichiometric matrix calculated assuming a reverse rotation flux of the TCA cycle in one embodiment of the present invention.
  • One embodiment of the present invention is a method for synthesizing substances by genetically modifying and culturing the modified organism to form a metabolic pathway that converts non-genetically modified CO2 emission sites into absorption.
  • succinic acid can be synthesized and the following reaction can occur in the TCA cycle of the organism: (1) Succinyl CoA + CO 2 ⁇ ⁇ -ketoglutaric acid (2) ⁇ -ketoglutaric acid + CO 2 ⁇ D-isocitric acid This allows the reaction of the TCA cycle to run in the opposite direction to the normal direction.
  • a gene encoding a sodium ion-dependent bicarbonate transporter and a gene encoding phosphoenolpyruvate carboxykinase are inserted into an expression vector, which is then introduced into a suitable host for forced expression therein.
  • the gene encoding the sodium ion-dependent bicarbonate transporter may be, for example, the bicA gene, and in particular the Synechococcus bicA gene (Ordered Locus Names: SYNPCC7002_A2371).
  • the nucleotide sequence of the coding region of the Synechococcus bicA gene (A) and the amino acid sequence of the protein it encodes (B) are shown in Figure 1.
  • the gene encoding phosphoenolpyruvate carboxykinase may be, for example, the pckA gene, and in particular the E. coli pckA gene (Ordered Locus Names: J7MG59_ECOLX).
  • the nucleotide sequence of the coding region of the E. coli pckA gene (A) and the amino acid sequence of the protein it encodes (B) are shown in Figure 2.
  • the origin of the gene encoding the sodium ion-dependent bicarbonate transporter used is not particularly limited, but is preferably derived from Cyanobacterium, and more preferably from Synechococcus.
  • the origin of the gene encoding phosphoenolpyruvate carboxykinase used is also not particularly limited, but is preferably derived from Enterobacter, and more preferably from E. coli.
  • the host microorganism for forced expression is preferably a unicellular organism, more preferably a bacterium, and most preferably Escherichia coli (E. coli).
  • the gene encoding the sodium ion-dependent bicarbonate transporter is preferably expressed at a level 10-fold higher than the expression level of the endogenous pckA gene when only the vector used for forced expression of the gene is introduced into the microorganism.
  • the gene encoding phosphoenolpyruvate carboxykinase is preferably expressed at a level of 106 or more copies per cell, or at a level 100-fold higher than the background. Examples of such expression vectors include pET vectors and pUC vectors.
  • This recombinant microorganism can absorb CO2 and produce succinic acid.
  • the recombinant microorganism can be allowed to live and preferably grow in the liquid.
  • the recombinant microorganism can be grown in a medium suitable for growth, and after a given period of incubation, the microorganism can be harvested and succinic acid can be separated by a method routine to those skilled in the art.
  • the resulting transformants were inoculated into 50 mL of LB + ampicillin medium and grown overnight (usually for more than 16 hours) before being harvested.
  • mRNA was extracted using RNAprotect Bacteria Reagent (Qiagen) and purified using RNeasy Mini Kits (Qiagen). The resulting mRNA was reverse transcribed using Random Hexamers and SuperScript III RT (200 U/ul).
  • the expression level of the obtained cDNA was examined by real-time PCR. Using Premix Ex Taq (Perfect Real Time) (Takara Bio) and the primers and probes having the following sequences, amplification and melting curve analysis were performed using a QuantStudio 12 K Flex real-time PCR system (Thermo Fisher Scientific, US) to calculate the expression level. The amplification conditions were denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds. The results are shown in Figure 4. As negative controls, the same operation was performed without the addition of DNA (NC), and transformation was performed only with the nul pET vector without an insert sequence (WT).
  • NC DNA
  • WT insert sequence
  • bicA gene Fw: TTTTTTTGCGGCCCTCTTC (SEQ ID NO: 1) Rev: GGGCCGGTCGGTTCAC (SEQ ID NO: 2) Probe: ACCCCGACGCTGAT (SEQ ID NO: 3)
  • pckA gene Fw: GCCGATCAAAACCCAGTATCACT (SEQ ID NO: 4) Rev: TCAGTACCGGCCAGTTTGG (SEQ ID NO:5)
  • Probe: CCTCTCTGGCTTCAC SEQ ID NO: 6
  • the expression levels were 104 - fold or more above background for the bicA gene, and 10-fold or more above that of the vector alone for the pckA gene.
  • WT vector-introduced strain
  • ENG recombinant gene-introduced strain
  • SOC medium with 100 mg/mL Amp added
  • ENG recombinant gene-introduced strain
  • the strains were diluted to OD600 of 0.9 with SOC medium and cultured overnight in a desiccator under 12CO2 ( 1 atm) or 13CO2 (1 atm).
  • the OD600 after culture was 0.7 for WT cultured under 12CO2 ( 1 atm ), 1.32 for ENG, 1.23 for WT cultured under 13CO2 (1 atm), and 0.75 for ENG.
  • the cells were collected by centrifugation and adjusted to OD 600 of 1.0. 10 mL of the mixture was frozen, suspended in 6.4 mM NH 4 HCO 3 aqueous solution, and then ultrasonically disrupted. After centrifugation, the supernatant was subjected to ultrafiltration (3 kDa filter), and 20 ⁇ L of the filtrate was measured by LC/MS. LC (liquid chromatography) and MS (mass spectrometry) were performed under the following conditions. L.C.
  • Structural sensitivity analysis is a technique that suggests enzymes that affect the amount of a substance on a metabolic network based on the structure of the metabolic network (A. Mochizuki and B. Fiedler, J. Theor. Biol. 367, 189(2015)).
  • Using this method we constructed a stoichiometry matrix from the chemical reaction network of the central metabolic pathway of E. coli (T. Okada and A. Mochizuki, Phys. Rev. Lett. 117, 048101(2016)), and interpreted the simulation results based on the experimental results described in (3).
  • the present invention provides a method for carrying out novel material synthesis.

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PCT/JP2023/018494 2023-05-17 2023-05-17 物質合成を実施する方法、およびｃｏ２を吸収し、コハク酸を製造するための方法 Ceased WO2024236787A1 (ja)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130130339A1 (en) * 2010-07-31 2013-05-23 Myriant Corporation Fermentation process for the production of organic acids
JP2022530475A (ja) * 2019-04-24 2022-06-29 ジェノマティカ インコーポレイティド 遺伝子操作された微生物及びアルデヒド脱水素酵素活性の改善方法
WO2023068295A1 (ja) * 2021-10-21 2023-04-27 伊藤忠商事株式会社 バイオプロセス、微生物を培養する方法及び標的物質を製造する方法並びにバイオプロセス装置

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130130339A1 (en) * 2010-07-31 2013-05-23 Myriant Corporation Fermentation process for the production of organic acids
JP2022530475A (ja) * 2019-04-24 2022-06-29 ジェノマティカ インコーポレイティド 遺伝子操作された微生物及びアルデヒド脱水素酵素活性の改善方法
WO2023068295A1 (ja) * 2021-10-21 2023-04-27 伊藤忠商事株式会社 バイオプロセス、微生物を培養する方法及び標的物質を製造する方法並びにバイオプロセス装置

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE UniProt 22 February 2023 (2023-02-22), "Bicarbonate transporter BicA", XP093241165, Database accession no. Q14SY0 *
DATABASE UniProt 3 May 2023 (2023-05-03), "Phosphoenolpyruvate carboxykinase", XP093241166, Database accession no. V0ANF8 *
MOCHIZUKI ATSUSHI; FIEDLER BERNOLD: "Sensitivity of chemical reaction networks: A structural approach. 1. Examples and the carbon metabolic network", JOURNAL OF THEORETICAL BIOLOGY, vol. 367, 13 November 2014 (2014-11-13), GB , pages 189 - 202, XP029134008, ISSN: 0022-5193, DOI: 10.1016/j.jtbi.2014.10.025 *
ZHU LI-WEN, ZHANG LEI, WEI LI-NA, LI HONG-MEI, YUAN ZHAN-PENG, CHEN TAO, TANG YA-LING, LIANG XIN-HUA, TANG YA-JIE: "Collaborative regulation of CO2 transport and fixation during succinate production in Escherichia coli", SCIENTIFIC REPORTS, vol. 5, no. 1, 1 January 2015 (2015-01-01), US , pages 1 - 12, XP093240852, ISSN: 2045-2322, DOI: 10.1038/srep17321 *

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