WO2023198006A1 - 一种s-乳酰谷胱甘肽的制备方法 - Google Patents

一种s-乳酰谷胱甘肽的制备方法 Download PDF

Info

Publication number
WO2023198006A1
WO2023198006A1 PCT/CN2023/087385 CN2023087385W WO2023198006A1 WO 2023198006 A1 WO2023198006 A1 WO 2023198006A1 CN 2023087385 W CN2023087385 W CN 2023087385W WO 2023198006 A1 WO2023198006 A1 WO 2023198006A1
Authority
WO
WIPO (PCT)
Prior art keywords
glutathione
gene
escherichia coli
gshf
seq
Prior art date
Application number
PCT/CN2023/087385
Other languages
English (en)
French (fr)
Inventor
王宇辰
张科春
Original Assignee
元素驱动(杭州)生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 元素驱动(杭州)生物科技有限公司 filed Critical 元素驱动(杭州)生物科技有限公司
Publication of WO2023198006A1 publication Critical patent/WO2023198006A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • 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
    • 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.)
    • 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
    • C12N9/93—Ligases (6)
    • 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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y404/00—Carbon-sulfur lyases (4.4)
    • C12Y404/01—Carbon-sulfur lyases (4.4.1)
    • C12Y404/01005—Lactoylglutathione lyase (4.4.1.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
    • C12Y603/02003—Glutathione synthase (6.3.2.3)
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention relates to the technical fields of genetic engineering and fermentation engineering, and specifically relates to a method for preparing S-lactoylglutathione.
  • Glutathione is a biologically active non-protein thiol compound widely present in living organisms. Glutathione is the main antioxidant in the body. It can resist the damaging effects of oxidants on sulfhydryl groups and protect proteins and enzymes containing sulfhydryl groups in cell membranes.
  • glutathione The physiological functions of glutathione are mainly manifested in six aspects: (1) As the most abundant antioxidant in cells, it protects DNA, proteins and other biomolecules against oxidative damage; (2) Through glutathione-S-transfer Enzymes complete the detoxification of foreign substances; (3) participate in the absorption and transport of amino acids; (4) participate in cell signaling and maintain normal life activities of cells; (5) participate in the reduction of methemoglobin and promote the absorption of iron; (6) ) regulates lymphocyte function and exerts antiviral effects. Therefore, reduced glutathione is widely used in medicine, food, cosmetics and other fields.
  • S-lactoylglutathione is generated from glutathione and methylglyoxal through glyoxalase catalysis and is part of the methylglyoxal detoxification pathway.
  • methylglyoxal and glutathione first generate S-lactylglutathione under the action of glyoxalase, and then S-lactylglutathione Under the action of hydrolase, glutathione is hydrolyzed back to produce lactic acid, and the detoxification of methylglyoxal is finally completed through lactic acid.
  • the generation of S-lactylglutathione during the above detoxification process can be regarded as using the lactyl group to protect the sulfhydryl group of glutathione that is easily oxidized, thereby obtaining a more stable glutathione derivative that is less likely to be oxidized.
  • the technical problem solved by the present invention is to provide a preparation method of S-lactoylglutathione with low production cost and suitable for industrial production.
  • the second technical problem solved by the present invention is to improve the conversion rate during the production process and increase the output of S-lactoylglutathione.
  • the invention provides a method for preparing S-lactyl glutathione, which uses glutamic acid, glycine, cysteine and methylglyoxal as raw materials, in the presence of glutathione synthase and glyoxalase. Catalytically converted into S-lactylglutathione.
  • glutamic acid, glycine, cysteine and methylglyoxal are used as substrates, and a recombinant microorganism containing a glutathione synthase encoding gene and a glyoxalase encoding gene is added for fermentation, Overexpression of recombinant microorganisms produces the glutathione synthetase and glyoxalase.
  • the glutathione synthase encoding gene is selected from any one or more of gshF, gshA, and gshB, preferably gshF; further preferably, the nucleotide sequence of gshF is such as SEQ ID NO :1 shown;
  • the glyoxalase encoding gene includes gloA; preferably, the nucleotide sequence of gloA is shown in SEQ ID NO: 2.
  • the preparation method includes constructing the recombinant microorganism by genetic engineering methods, including plasmid expression or genome integration.
  • the recombinant microorganism is constructed by plasmid expression
  • the construction method is: obtain the glutathione synthase encoding gene and the glyoxalase encoding gene through PCR amplification, jointly connect the obtained genes to a plasmid vector containing an IPTG inducible promoter and transform into a competent state In the cells, the recombinant vector is obtained after sequencing; the recombinant vector is transformed into the recipient microorganism to obtain the recombinant microorganism;
  • the plasmid vector is selected from any one or both of pZAlac and pZElac.
  • the pZE-gshF-gloA construction method is: amplify the gshF gene and gloA gene by PCR, connect the gshF gene and gloA gene together to the pZElac vector containing the IPTG inducible promoter and transform into competent cells.
  • plasmid pZE-gshF_gloA was obtained after sequencing;
  • the gshF gene and the gloA gene are respectively amplified by PCR using the genome of E. coli MG1655 as a template;
  • the competent cell is E. coli dh5a.
  • the recipient microorganism is selected from one or more of Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces.
  • nucleotide sequence of gloC is shown in SEQ ID NO: 6;
  • nucleotide sequence of yeiG is shown in SEQ ID NO:7;
  • the recipient microorganism is Escherichia coli MG1655 ⁇ gloB, Escherichia coli MG1655 ⁇ gloC, Escherichia coli MG1655 ⁇ yeiG, Escherichia coli MG1655 ⁇ gloB ⁇ gloC, Escherichia coli MG1655 ⁇ gloC ⁇ yeiG, Escherichia coli MG1655 ⁇ gloB ⁇ yeiG, Escherichia coli MG1655 ⁇ gloB ⁇ gloC ⁇ yeiG.
  • the recipient microorganism contains a gene expressing cysteine hydrolase or glutathione hydrolase, it is necessary to further knock out the expression of cysteine hydrolase or glutathione hydrolase on the recipient microorganism.
  • the gene expressing cysteine hydrolase is tnaA; further preferably, the nucleotide sequence of tnaA is shown in SEQ ID NO: 3;
  • the gene expressing glutathione hydrolase is ggt; further preferably, the nucleotide sequence of ggt is as shown in SEQ ID NO: 4;
  • the recipient microorganism is E. coli MG1655 ⁇ tnaA, E. coli MG1655 ⁇ ggt or E. coli MG1655 ⁇ tnaA ⁇ ggt.
  • the recipient microorganism is E. coli MG1655 ⁇ tnaA ⁇ ggt ⁇ gloB ⁇ gloC ⁇ yeiG.
  • step 3 Use the ⁇ tnaA single-deficient bacterium obtained in step 1) as the recipient bacterium, and add the phage obtained in step 2) in sequence for transfection to obtain E. coli MG1655 ⁇ tnaA ⁇ ggt ⁇ gloB ⁇ gloC ⁇ yeiG.
  • the fermentation temperature is 20-90°C.
  • the molar concentration ratio of glutamic acid, glycine, cysteine, and methylglyoxal ranges from 8 to 12:8 to 12:6 to 10:1 to 4.
  • glutamic acid, glycine, cysteine and recombinant microorganisms are first added, and the fermentation culture is preferably performed for 1 to 4 hours to accumulate glutathione, and then methylglyoxal is added to continue the fermentation; preferably Preferably, during fermentation, the concentration of methylglyoxal in the fermentation tank is maintained at 0.2 to 4mM using a slow fed method.
  • the gene expressing S-lactylglutathione hydrolase in the recombinant microorganism is knocked out;
  • the gene expressing S-lactylglutathione hydrolase is, for example, gloB, gloC, or yeiG;
  • nucleotide sequence of gloB is shown in SEQ ID NO:5;
  • nucleotide sequence of gloC is shown in SEQ ID NO: 6;
  • nucleotide sequence of yeiG is shown in SEQ ID NO:7;
  • the recombinant microorganism is Escherichia coli MG1655 ⁇ gloB, Escherichia coli MG1655 ⁇ gloC, Escherichia coli MG1655 ⁇ yeiG, Escherichia coli MG1655 ⁇ gloB ⁇ gloC, Escherichia coli MG1655 ⁇ gloC ⁇ yeiG, Escherichia coli MG1655 ⁇ gloB ⁇ yeiG, Escherichia coli MG1655 ⁇ gloB ⁇ gloC ⁇ yeiG.
  • the present invention also provides a recombinant microorganism for preparing S-lactylglutathione, which overexpresses endogenous or exogenous glutathione synthetase encoding genes and glyoxalase encoding genes;
  • the glutathione synthase encoding gene is selected from any one or more of gshF, gshA, and gshB, preferably gshF; further preferably, the nucleotide sequence of gshF is as shown in SEQ ID NO: 1 Show;
  • the glyoxalase encoding gene includes gloA; preferably, the nucleotide sequence of gloA is shown in SEQ ID NO: 2;
  • the recipient microorganism is selected from one of Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces Or several; further preferably, the recipient microorganism is selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, One or more of Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris.
  • the present invention also provides recombinant DNA or biological materials for preparing S-lactoylglutathione, which is characterized in that the recombinant DNA or biological materials contain glutathione synthase encoding genes and glyoxalase encoding genes;
  • the glutathione synthase encoding gene is selected from any one or more of gshF, gshA, and gshB, preferably gshF; further preferably, the nucleotide sequence of gshF is as shown in SEQ ID NO: 1 Show;
  • the glyoxalase encoding gene includes gloA; preferably, the nucleotide sequence of gloA is shown in SEQ ID NO: 2;
  • the biological material is an expression cassette, transposon, plasmid vector, phage vector or viral vector.
  • the present invention also provides the use of the above recombinant microorganisms, recombinant DNA or biological materials in the preparation of S-lactyl glutathione.
  • the preparation method of S-lactoylglutathione of the present invention has a simple preparation process and convenient operation. Compared with directly using glutathione as a raw material, the raw material cost is significantly reduced, and the product The conversion rate and output are high and suitable for industrial mass production.
  • Figure 1 is a schematic diagram of the production results of S-lactoylglutathione under different conditions in Example 1.
  • Figure 2 is a schematic diagram of gene knockout in recipient bacteria.
  • gene synthesis means produced using recombinant DNA technology or obtained using synthetic DNA or amino acid sequence technology available and well known in the art.
  • Coding refers to the inherent property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA that serves as a template for the synthesis of other polymers and macromolecules used in biological processes. Molecules have either a defined sequence of nucleotides (ie, rRNA, tRNA, and mRNA) or a defined sequence of amino acids and biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produce a protein in a cell or other biological system.
  • Both the coding strand where the nucleotide sequence is equivalent to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, which serves as a template for transcribing a gene or cDNA, may be said to encode the protein or other product of that gene or cDNA.
  • endogenous refers to any substance from or produced within an organism, cell, tissue or system.
  • exogenous refers to any substance introduced from or produced outside an organism, cell, tissue or system.
  • expression is defined as the transcription and/or translation of a specific nucleotide sequence driven by its promoter.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.
  • the phrase nucleotide sequence encoding a protein or RNA may also include introns, to the extent that the nucleotide sequence encoding the protein may in some versions include intron(s).
  • the term "vector” is a composition of matter that includes an isolated nucleic acid and that can be used to deliver the isolated nucleic acid into the interior of a cell.
  • the transferred nucleic acid is typically ligated, eg, inserted, into a vector nucleic acid molecule.
  • the vector may contain sequences that direct autonomous replication in the cell or may contain sequences sufficient to allow integration into the host cell DNA.
  • Many vectors are known in the art, including but not limited to plasmids, phagemids, artificial chromosomes, bacterial phages, and animal viruses.
  • the term “vector” includes autonomously replicating plasmids or viruses.
  • DNA polymerase Phanta Max Super-Fidelity DNA Polymerase and ligase-independent single fragment rapid cloning kit used in examples of the present invention One Step was purchased from Nanjing Novozan Biotechnology Co., Ltd. company.
  • the recombinant vectors used in some examples are constructed as follows:
  • the gloA gene fragment was amplified by PCR using the genome of E. coli MG1655 as the template, and the gshF gene fragment was amplified by PCR using the genome of Streptococcus thermophilus as the template, and they were jointly ligated using the ligase-independent single fragment rapid cloning kit. into the vector pZElac containing an IPTG-inducible promoter, then transformed into BW25113 competent cells, coated with kanamycin sulfate-resistant plates and cultured overnight, and positive clones were selected for sequencing verification.
  • the correct recombinant vector was named pZE-gshF_gloA.
  • the nucleotide sequence of gshF is shown in SEQ ID NO:1.
  • the nucleotide sequence of gloA is shown in SEQ ID NO:2.
  • the receptor bacteria used in some examples are constructed as follows:
  • step 2) Cultivate the four monodeficient strains of ⁇ ggt, ⁇ gloB, ⁇ gloC and ⁇ yeiG with the kanamycin sulfate resistance gene prepared by the homologous recombination method in step 1) at 37°C overnight, and then transfer them to In LB medium with 5 mmol/L CaCl2 and 0.1% glucose, culture at 37°C for 1 hour, then add wild-type P1 phage and continue to culture for 1-3 hours. Add a few drops of chloroform and then incubate for 3 to 8 minutes.
  • step 1) Use the ⁇ tnaA single-deficient bacterium obtained in step 1) as the recipient bacterium, and add the phage obtained in step 2) in sequence for transfection to obtain E. coli MG1655 ⁇ tnaA ⁇ ggt ⁇ gloB ⁇ gloC ⁇ yeiG.
  • ⁇ tnaA monodeficient bacteria as the recipient bacteria, transform the pCP20 plasmid, express the flippase recombinase gene, promote the homologous recombination of the FRT site itself, and knock out the kanamycin sulfate resistance gene.
  • pCP20 is a temperature-sensitive plasmid, and the plasmid can be cleared by changing the environmental temperature.
  • the ⁇ tnaA ⁇ ggt ⁇ gloB ⁇ gloC ⁇ yeiG strain containing the kanamycin sulfate resistance gene was obtained by repeated knockout and transfection processes.
  • the ⁇ tnaA ⁇ ggt ⁇ gloB ⁇ gloC ⁇ yeiG target strain was obtained by knocking out the kanamycin sulfate resistance gene again with pcp20. During this process, the order of transfection is not restricted.
  • gloB, gloC, and yeiG are all S-lactylglutathione hydrolases, and knocking out tnaA and ggt can further increase product production.
  • the conception principle is shown in Figure 2.
  • the nucleotide sequence of ggt is shown in SEQ ID NO:4.
  • the nucleotide sequence of gloB is shown in SEQ ID NO:5.
  • the nucleotide sequence of gloC is shown in SEQ ID NO:6.
  • the nucleotide sequence of yeiG is shown in SEQ ID NO:7.
  • the recombinant plasmid pZE-gshF_gloA was electroporated into E. coli MG1655 ⁇ tnaA ⁇ ggt ⁇ gloB ⁇ gloC ⁇ yeiG, and the ampicillin resistance gene was used as a screening marker to select positive clones.
  • the transformants were inoculated into 2 mL LB medium and cultured for 12 hours.
  • the obtained activated bacterial strain was inoculated into M9 medium containing 1/1000 ampicillin and 20 g/L glucose at an inoculation amount of 1%, and incubated at 30°C at 240 rpm.
  • E. coli MG1655 ⁇ tnaA ⁇ ggt ⁇ gloB ⁇ gloC ⁇ yeiG was used for direct fermentation, sampling was carried out under the same conditions for inoculation, fermentation and fermentation broth was collected.
  • the glutathione concentration was detected by high performance liquid chromatography to be 0.20mM, and S-lactoylglutathione was not detected.
  • the recombinant vector containing only the gshF gene was transformed into E. coli MG1655 ⁇ tnaA ⁇ ggt ⁇ gloB ⁇ gloC ⁇ yeiG, and the same conditions were sampled for inoculation, fermentation, and fermentation broth was collected.
  • the glutathione concentration was detected by high-performance liquid chromatography to be 1.7mM, S -Lactylglutathione was not detected.
  • MG1655 represents Comparative Example 1
  • MG123 represents Comparative Example 2
  • YC1123 represents Comparative Example 3
  • YC2123+MG methylglyoxal
  • the ordinate in the figure is measured as the total content of glutathione and S-lactoylglutathione. Except for the data marked in the figure of YC2123+MG (methylglyoxal) at 12 hours, which contains 38% SLG (S- Except lactylglutathione), no SLG was detected in the other groups.
  • This example is the synthesis of S-lactoylglutathione in a fermentation tank.
  • the recombinant plasmid pZE-gshF_gloA was electroporated into E. coli MG1655 ⁇ tnaA ⁇ ggt ⁇ gloB ⁇ gloC ⁇ yeiG, and the ampicillin resistance gene was used as a screening marker to select positive clones. Inoculate a single colony of the above recombinant bacteria into 50 mL of LB liquid culture medium containing 100 ⁇ g/mL ampicillin, culture it at 37°C and 220 rpm for 14 hours, then insert the bacterial strain into M9 culture medium containing ampicillin and 50 g/L glucose, and place it in a 1L fermentation tank.
  • pZE-gloA was electroporated into E. coli MG1655 ⁇ tnaA ⁇ ggt and MG1655 ⁇ tnaA ⁇ ggt ⁇ gloB ⁇ gloC ⁇ yeiG, and the ampicillin resistance gene was used as a screening marker to select positive clones.
  • the transformants were inoculated into 2 mL LB medium and cultured for 12 h.
  • the obtained activated strains were inserted into 2XYT medium containing 1/1000 ampicillin at 1% inoculum volume and placed in a 150 ml Erlenmeyer flask at 30°C and 240 rpm. (Liquid volume 15mL) culture to OD 0.4-0.6, add IPTG to a final concentration of 0.2mM.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明属于基因工程和发酵工程技术领域，具体涉及一种S-乳酰谷胱甘肽的制备方法，其以谷氨酸、甘氨酸、半胱氨酸以及甲基乙二醛为原料，谷氨酸、甘氨酸、半胱氨酸以及甲基乙二醛在谷胱甘肽合成酶及乙二醛酶的催化下转化为S-乳酰谷胱甘肽。本发明采用成本较低的原料进行发酵，操作简易，转化率高，制备得到的S-乳酰谷胱甘肽产量高，适合批量化工业生产。

Description

一种S-乳酰谷胱甘肽的制备方法
本申请要求于2022年4月12日提交中国专利局、申请号为202210381545.6、发明名称为“一种S-乳酰谷胱甘肽的制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及基因工程及发酵工程技术领域，具体涉及一种S-乳酰谷胱甘肽的制备方法。
背景技术
谷胱甘肽(Glutathione)，是一种广泛存在于生物体内的生物活性非蛋白硫醇化合物。谷胱甘肽是体内的主要抗氧化剂，能抵抗氧化剂对巯基的破坏作用，保护细胞膜中含巯基的蛋白质和酶。谷胱甘肽的生理功能主要表现在6个方面：(1)作为胞内最丰富的抗氧化剂，保护DNA、蛋白质和其他生物分子抵抗氧化损伤；(2)通过谷胱甘肽-S-转移酶完成对外源物质的解毒作用；(3)参与氨基酸的吸收与转运；(4)参与细胞信号传导，维持细胞正常生命活动；(5)参与高铁血红蛋白的还原作用以及促进铁的吸收；(6)调节淋巴细胞功能，发挥抗病毒作用。因此，还原型谷胱甘肽被广泛应用在在医药、食品、化妆品等领域，但由于谷胱甘肽结构中巯基的不稳定(易被氧化)，使其在储存、运输、应用时条件要求苛刻，限制了谷胱甘肽的进一步应用。因此相对更稳定且可以被代谢分解回谷胱甘肽的谷胱甘肽衍生物，被认为可作为未来谷胱甘肽的供应替代。
S-乳酰谷胱甘肽(S-lactoylglutathione)是由谷胱甘肽和甲基乙二醛通过乙二醛酸酶催化生成的，是甲基乙二醛解毒途径的一部分。在甲基乙二醛解毒途径中，甲基乙二醛和谷胱甘肽先在乙二醛酸酶的作用下生成S-乳酰谷胱甘肽，再在S-乳酰谷胱甘肽水解酶的作用下，水解回谷胱甘肽并生成乳酸，通过乳酸最终完成了甲基乙二醛的解毒。上述解毒过程中生成S-乳酰谷胱甘肽，可视为利用了乳酰基将谷胱甘肽易被氧化的巯基保护起来，得到不易被氧化更稳定的谷胱甘肽衍生物。
而目前S-乳酰谷胱甘肽合成的研发较少，文献集中于体外酶催化谷胱甘肽和醛直接合成，该制备方法酶提取过程复杂，原料中谷胱甘肽价格昂贵，高昂的成本使S-乳酰谷胱甘肽难以 工业化大量生产。
发明内容
本发明所解决的技术问题在于提供一种生产成本低、适合工业化生产的S-乳酰谷胱甘肽的制备方法。
本发明所述解决的技术问题之二在于提高生产过程中的转化率，提高S-乳酰谷胱甘肽的产量。
本发明提供一种S-乳酰谷胱甘肽的制备方法，以谷氨酸、甘氨酸、半胱氨酸以及甲基乙二醛为原料，在谷胱甘肽合成酶及乙二醛酶的催化下转化为S-乳酰谷胱甘肽。
在一些实施方案中，以谷氨酸、甘氨酸、半胱氨酸以及甲基乙二醛为底物，加入含有谷胱甘肽合成酶编码基因和乙二醛酶编码基因的重组微生物进行发酵，重组微生物过表达产生所述谷胱甘肽合成酶及乙二醛酶。
在一些实施方案中，所述谷胱甘肽合成酶编码基因选自gshF、gshA、gshB中的任一种或几种，优选为gshF；进一步优选的，gshF的核苷酸序列如SEQ ID NO:1所示；
在一些实施方案中，所述乙二醛酶编码基因包括gloA；优选的，gloA的核苷酸序列如SEQ ID NO:2所示。
在一些实施方案中，所述制备方法包括通过基因工程方法构建所述重组微生物，所述基因工程方法包括质粒表达或基因组整合。
在一些实施方案中，重组微生物通过质粒表达的方法进行构建；
优选地，构建方法为：通过PCR扩增获得谷胱甘肽合成酶编码基因和乙二醛酶编码基因，将获得的基因共同连接至含有IPTG诱导型启动子的质粒载体上并转化至感受态细胞中，测序后获得重组载体；将重组载体转化至受体微生物中即得到重组微生物；
优选地，质粒载体选自pZAlac、pZElac中的任一种或两种。
在一些实施方案中，重组载体为pZE-gshF-gloA，
优选地，所述pZE-gshF-gloA构建方法为：通过PCR扩增得gshF基因和gloA基因，将gshF基因和gloA基因共同连接至含有IPTG诱导型启动子的pZElac载体上并转化至感受态细胞中，测序后得到质粒pZE-gshF_gloA；
优选地，以大肠杆菌MG1655的基因组为模板分别通过PCR扩增得gshF基因和gloA基因；
优选地，感受态细胞为大肠杆菌E.coli dh5a。
在一些实施方案中，所述受体微生物选自大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌中的一种或几种。
在一些实施方案中，受体微生物选自大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、巨大芽孢杆菌(Bacillus megaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Candida utilis)或毕赤酵母(Pichia pastoris)中的一种或几种。
在一些实施方案中，若受体微生物含有表达S-乳酰谷胱甘肽水解酶的基因，需敲除受体微生物上表达S-乳酰谷胱甘肽水解酶的基因；表达S-乳酰谷胱甘肽水解酶的基因例如为gloB、gloC、yeiG；
优选的，gloB的核苷酸序列如SEQ ID NO:5所示；
优选的，gloC的核苷酸序列如SEQ ID NO:6所示；
优选的，yeiG的核苷酸序列如SEQ ID NO:7所示；
优选的，所述受体微生物为大肠杆菌MG1655ΔgloB、大肠杆菌MG1655ΔgloC、大肠杆菌MG1655ΔyeiG、大肠杆菌MG1655ΔgloBΔgloC、大肠杆菌MG1655ΔgloCΔyeiG、大肠杆菌MG1655ΔgloBΔyeiG、大肠杆菌MG1655ΔgloBΔgloCΔyeiG。
在一些实施方案中，若受体微生物含有表达半胱氨酸水解酶或谷胱甘肽水解酶的基因，需进一步敲除受体微生物上表达半胱氨酸水解酶或谷胱甘肽水解酶的基因；
优选的，表达半胱氨酸水解酶的基因为tnaA；进一步优选的，tnaA的核苷酸序列如SEQ ID NO:3所示；
优选的，表达谷胱甘肽水解酶的基因为ggt；进一步优选的，ggt的核苷酸序列如SEQ ID NO:4所示；
优选的，所述受体微生物为大肠杆菌MG1655ΔtnaA、大肠杆菌MG1655Δggt或大肠杆菌MG1655ΔtnaAΔggt。
在一些实施方案中，所述受体微生物为大肠杆菌MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG。
在一些实施方案中，所述大肠杆菌MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG的构建方法包括如下步骤：
1)用同源重组法对野生型大肠杆菌MG1655菌株的tnaA、ggt、gloB、gloC、yeiG基因进行单独敲除获得五株单缺菌；
2)将步骤1)中得到的Δggt、ΔgloB、ΔgloC和ΔyeiG四株单缺菌分别加入野生型P1噬菌体进行培养，得到分别含有ggt、gloB、gloC和yeiG敲除性状的大肠杆菌基因片段的噬菌体P1vir ggt、P1vir gloB、P1vir gloC和P1vir yeiG；
3)将步骤1)得到的ΔtnaA单缺菌作为受体菌，依次加入步骤2)中得到的噬菌体进行转染，得到大肠杆菌MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG。
在一些实施方案中，发酵时，发酵温度为20～90℃。
在一些实施方案中，所述谷氨酸、甘氨酸、半胱氨酸、甲基乙二醛的摩尔浓度比例范围为8～12:8～12:6～10:1～4。
在一些实施方案中，发酵时，先加入谷氨酸、甘氨酸、半胱氨酸以及重组微生物，发酵培养优选1～4h使谷胱甘肽积累，然后再加入甲基乙二醛继续发酵；优选地，发酵时，采用缓慢流加的方式使发酵罐中所述甲基乙二醛浓度维持为0.2～4mM。
本发明还提供一种S-乳酰谷胱甘肽的制备方法，以谷胱甘肽和甲基乙二醛为原料，在过表达乙二醛酸酶的重组微生物或其粗酶液催化下转化为S-乳酰谷胱甘肽；
优选的，所述重组微生物中表达S-乳酰谷胱甘肽水解酶的基因被敲除；所述表达S-乳酰谷胱甘肽水解酶的基因例如为gloB、gloC、yeiG；
优选的，gloB的核苷酸序列如SEQ ID NO:5所示；
优选的，gloC的核苷酸序列如SEQ ID NO:6所示；
优选的，yeiG的核苷酸序列如SEQ ID NO:7所示；
优选的，所述重组微生物为大肠杆菌MG1655ΔgloB、大肠杆菌MG1655ΔgloC、大肠杆菌MG1655ΔyeiG、大肠杆菌MG1655ΔgloBΔgloC、大肠杆菌MG1655ΔgloCΔyeiG、大肠杆菌MG1655ΔgloBΔyeiG、大肠杆菌MG1655ΔgloBΔgloCΔyeiG。
本发明还提供制备S-乳酰谷胱甘肽的重组微生物，所述重组微生物过表达内源或外源谷胱甘肽合成酶编码基因及乙二醛酶编码基因；
优选的，所述谷胱甘肽合成酶编码基因选自gshF、gshA、gshB中的任一种或几种，优选为gshF；进一步优选的，gshF的核苷酸序列如SEQ ID NO:1所示；
优选的，所述乙二醛酶编码基因包括gloA；优选的，gloA的核苷酸序列如SEQ ID NO:2所示；
优选的，所述受体微生物选自大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌中的一种 或几种；进一步优选地，所述受体微生物选自大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、巨大芽孢杆菌(Bacillus megaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Candida utilis)或毕赤酵母(Pichia pastoris)中的一种或几种。
本发明还提供用于制备S-乳酰谷胱甘肽的重组DNA或生物材料，其特征在于，所述重组DNA或生物材料含有谷胱甘肽合成酶编码基因及乙二醛酶编码基因；
优选的，所述谷胱甘肽合成酶编码基因选自gshF、gshA、gshB中的任一种或几种，优选为gshF；进一步优选的，gshF的核苷酸序列如SEQ ID NO:1所示；
优选的，所述乙二醛酶编码基因包括gloA；优选的，gloA的核苷酸序列如SEQ ID NO:2所示；
优选的，所述生物材料为表达盒、转座子、质粒载体、噬菌体载体或病毒载体。
本发明还提供上述重组微生物、重组DNA或生物材料在制备S-乳酰谷胱甘肽中的应用。
有益效果：本发明所述的S-乳酰谷胱甘肽的制备方法，制备过程简易，操作便利，相较于直接以谷胱甘肽作为原料而言，原料成本得到显著降低，且产物的转化率及产量高，适合工业化大量生产。
附图说明
图1为实施例1中不同条件下S-乳酰谷胱甘肽的生成结果示意图。
图2为受体菌基因敲除原理图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例进一步阐述本发明。
在本公开中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了 更好地理解本公开，下面提供相关术语的定义和解释。
也应理解本文使用的术语仅是为了描述具体实施方式的目的，并不意欲是限制性的。
本文中使用冠词“一”和“所述”来指代冠词的语法宾语中的一个或多于一个。
替代方案(例如，“或”)的使用应当被理解为意指替代方案中的一个、两个或其任何组合。术语“和/或”应当被理解为意指替代方案中的一个或两个。
如本文所用，术语“基因合成”，指利用重组DNA技术产生或利用本领域可用和公知的合成DNA或氨基酸序列技术获得。
“编码”指的是多核苷酸诸如基因、cDNA或mRNA中核苷酸的特异性序列用作模板合成在生物学过程中的其他多聚体和大分子的固有性质，所述多聚体和大分子具有核苷酸(即，rRNA、tRNA和mRNA)的限定序列或氨基酸的限定序列中的任一个和由其产生的生物学性质。因此，如果相应于那个基因的mRNA的转录和翻译在细胞或其他生物学系统中产生蛋白质，则基因编码蛋白质。核苷酸序列等同mRNA序列并通常提供在序列表中的编码链，和用作转录基因或cDNA的模板的非编码链两者，都可被称为编码那个基因或cDNA的蛋白质或其他产物。
如本文所用，术语“内源的”指的是来自有机体、细胞、组织或系统的或在有机体、细胞、组织或系统内产生的任何物质。
如本文所用，术语“外源的”指的是任何从有机体、细胞、组织或系统引入的或在有机体、细胞、组织或系统外产生的物质。
如本文所用，术语“表达”被定义为由它的启动子驱动的特定核苷酸序列的转录和/或翻译。
除非另有规定，“编码氨基酸序列的多核苷酸序列”包括为彼此简并版本并编码相同的氨基酸序列的所有的核苷酸序列。短语编码蛋白质或RNA的核苷酸序列也可包括内含子，其程度为编码该蛋白质的核苷酸序列可在某些版本中包含内含子(一个或多个)。
如本文所用的，术语“载体”为物质组合物，其包括分离的核酸，并且其可用于传递分离的核酸至细胞内部。转移的核酸通常连接到例如插入到载体核酸分子中。载体可以包含引导细胞中的自主复制的序列或可以包含足以允许整合到宿主细胞DNA中的序列。很多载体在本领域中是已知的，包含但不限于质粒、噬菌粒、人工染色体、细菌噬菌体以及动物病毒。因此，术语“载体”包括自主复制的质粒或病毒。
本发明实例所用的DNA聚合酶Phanta Max Super-Fidelity DNA Polymerase、非连接酶依赖型单片段快速克隆试剂盒One Step购自南京诺唯赞生物科技股份有限 公司。
一些实施例用到的重组载体构建如下：
根据NCBI公布的大肠杆菌MG1655的基因组及嗜热链球菌的基因组分别设计引物：引物与模板结合部分用大写字母表示且Tm值为58～62℃，同源臂部分用英文字母小写表示且均为20bp：
gshF_F：ttaaagaggagaaaggtaccATGACATTAAACCAACTTCTTCAAAAACTGG
gshF_R：ttaatttctcctgtcgacTTAAGTTTGACCAGCCACTATTTCTGG
gloA_F：gtcgacaggagaaattaactATGCGTCTTCTTCATACCATGCTG：
gloA_R：ttgatgcctctagaaagcttTTAGTTGCCCAGACCGCG
以大肠杆菌MG1655的基因组为模板通过PCR扩增得gloA基因片段，以嗜热链球菌的基因组为模板通过PCR扩增得gshF基因片段，并通过非连接酶依赖型单片段快速克隆试剂盒共同连接到含有IPTG诱导型启动子的载体pZElac上，然后转化至BW25113感受态细胞，涂布硫酸卡那霉素抗性平板过夜培养，挑阳性克隆进行测序验证，正确的重组载体命名为pZE-gshF_gloA。
其中：
gshF的核苷酸序列如SEQ ID NO:1所示。
gloA的核苷酸序列如SEQ ID NO:2所示。
一些实施例用到的受体菌构建如下：
大肠杆菌MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG菌株构建：
1)用同源重组法对野生型大肠杆菌MG1655菌株的tnaA、ggt、gloB、gloC、yeiG基因进行单独敲除获得五株单缺菌；制备得到的五株单缺菌均带有抗硫酸卡那霉素抗性基因；
2)将步骤1)中通过同源重组法制备的带有抗硫酸卡那霉素抗性基因的Δggt、ΔgloB、ΔgloC和ΔyeiG四株单缺菌均于37℃过夜培养，然后转接于含5mmol/L CaCl2和0.1％葡萄糖的LB培养基中，37℃培养1h，然后加入野生型P1噬菌体继续培养1-3h。加几滴氯仿后再培养3～8min分钟，离心取上清，即得到分别含有ggt、gloB、gloC和yeiG敲除性状的大肠杆菌基因片段的噬菌体P1vir ggt、P1vir gloB、P1vir gloC和P1vir yeiG；
3)将步骤1)得到的ΔtnaA单缺菌作为受体菌，依次加入步骤2)中得到的噬菌体进行转染，得到大肠杆菌MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG。具体为：将ΔtnaA单缺菌作为受体菌，转化pCP20质粒，表达翻转酶重组酶基因，促进FRT位点自身的同源重组，敲除抗硫酸卡那霉素抗性基因。pCP20为温敏型质粒，通过改变环境温度可以实现质粒的清除。 过夜培养ΔtnaA受体菌，取1.5mL菌液10000g离心2分钟后，用0.75mL的P1盐溶液(溶剂为水，溶质为10mM CaCl2和5mM MgSO4)重悬ΔtnaA受体菌，将100μL噬菌体P1vir ggt与100μLΔtnaA受体菌悬浮液混合，37℃孵育30min，然后加入1mL的LB培养基和200μL的1mol/L柠檬酸钠，37℃继续培养1h，离心收集菌体，用100μL的LB培养基重悬后，涂布含卡那霉素的LB平板(卡那霉素的浓度为50μg/ml)上，37℃培养过夜后，挑选克隆进行PCR扩增鉴定，阳性克隆即为含抗硫酸卡那霉素抗性基因的ΔtnaAΔggt。将含抗硫酸卡那霉素抗性基因的ΔtnaAΔggt作为受体菌，利用pcp20敲除抗硫酸卡那霉素抗性基因后转染P1vir gloB获得ΔtnaAΔggtΔgloB。以此类推，通过重复敲除转染过程获得含抗硫酸卡那霉素抗性基因的ΔtnaAΔggtΔgloBΔgloCΔyeiG菌株，pcp20再敲除一次抗硫酸卡那霉素抗性基因后获得ΔtnaAΔggtΔgloBΔgloCΔyeiG目标菌株。在此过程中，转染先后顺序不受限制。
其中gloB、gloC、yeiG均为S-乳酰谷胱甘肽水解酶，而tnaA、ggt敲除后可进一步提高产物产量。构思原理如图2所示。
tnaA的核苷酸序列如SEQ ID NO:3所示。
ggt的核苷酸序列如SEQ ID NO:4所示。
gloB的核苷酸序列如SEQ ID NO:5所示。
gloC的核苷酸序列如SEQ ID NO:6所示。
yeiG的核苷酸序列如SEQ ID NO:7所示。
实施例1
本实施例为摇瓶中S-乳酰谷胱甘肽的合成。
将重组质粒pZE-gshF_gloA电转至大肠杆菌MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG中，使用氨苄青霉素(Ampicillin)抗性基因作为筛选标志，挑阳性克隆。将转化子接种到2mL LB培养基中培养12h，获得的活化菌种以1％接种量接入含有千分之一氨苄青霉素和20g/L的葡萄糖的M9培养基中，在30℃转速240rpm下于150ml三角瓶中(装液量15mL，三角瓶含0.5g CaCO3)培养至OD 0.4-0.6，加入IPTG至终浓度0.2mM，同时一次性加入10mM谷氨酸、10mM甘氨酸和8mM半胱氨酸(其中半胱氨酸比例较低可减少对大肠杆菌的毒性，且有利于降低成本的同时不减少产量)。而由于甲基乙二醛对大肠杆菌有一定毒性，选择在37℃继续培养发酵2h有一定谷胱甘肽积累后，再加入甲基乙二醛至3mM，继续持续发酵12h后，收集发酵液，利用高效液相色谱检测得谷胱甘肽浓度为1.3mM，S-乳酰谷胱甘肽的浓度 为0.8mM，谷胱甘肽的转化率为38.1％。转化率＝S-乳酰谷胱甘肽/(S-乳酰谷胱甘肽终产量+谷胱甘肽终产量)
作为对照实施例1，采用大肠杆菌MG1655的野生菌直接进行发酵，采样同样条件进行接种、发酵并收集发酵液，利用高效液相色谱检测得谷胱甘肽浓度为0.08mM，S-乳酰谷胱甘肽未检出。
作为对照实施例2，采用大肠杆菌MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG直接进行发酵，采样同样条件进行接种、发酵并收集发酵液，利用高效液相色谱检测得谷胱甘肽浓度为0.20mM，S-乳酰谷胱甘肽未检出。
作为对照实施例3，将仅含有gshF基因的重组载体转化入大肠杆菌MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG后，采样同样条件进行接种、发酵并收集发酵液，利用高效液相色谱检测得谷胱甘肽浓度为1.7mM，S-乳酰谷胱甘肽未检出。
数据结果参见图1所示。
图中：MG1655表示对照实施例1，MG123表示对照实施例2，YC1123表示对照实施例3，YC2123+MG(甲基乙二醛)表示实施例1。图中纵坐标测为谷胱甘肽和S-乳酰谷胱甘肽的总含量，除图中标注的YC2123+MG(甲基乙二醛)在12h时的数据含有38％SLG(S-乳酰谷胱甘肽)外，其余组均未检出SLG。
由此说明，本发明的技术方案可以有效提高S-乳酰谷胱甘肽的产量和产率。
实施例2
本实施例为发酵罐中S-乳酰谷胱甘肽的合成。
将重组质粒pZE-gshF_gloA电转至大肠杆菌MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG中，使用氨苄青霉素(Ampicillin)抗性基因作为筛选标志，挑阳性克隆。将上述重组菌单菌落分别接种50mL含有100μg/mL氨苄青霉素的LB液体培养基，37℃，220rpm培养14h后将菌种接入含有氨苄青霉素和50g/L葡萄糖的M9培养基，1L发酵罐中(装液量500mL)，转速600rpm，发酵培养6h，加入IPTG至终浓度0.1mM，同时一次性加入谷氨酸至25mM、甘氨酸至25mM和半胱氨酸至17mM，实验中发现一次性添加3mM甲基乙二醛对大肠杆菌生长有影响，因此发酵时选择通过缓慢流加控制甲基乙二醛浓度维持在1mM，继续持续发酵24h，收集发酵液，利用高效液相色谱检测谷胱甘肽浓度为2.1g/L和S-乳酰谷胱甘肽浓度约2.8g/L，谷胱甘肽的转化率为57.1％。
实施例3
本实施例以受体菌粗酶液催化合成S-乳酰谷胱甘肽对比水解酶的敲除效果
将pZE-gloA电转至大肠杆菌MG1655ΔtnaAΔggt和MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG，使用氨苄青霉素(Ampicillin)抗性基因作为筛选标志，挑阳性克隆。将转化子接种到2mL LB培养基中培养12h，获得的活化菌种以1％接种量接入含有千分之一氨苄青霉素2XYT培养基中，在30℃转速240rpm下于150ml三角瓶中(装液量15mL)培养至OD 0.4-0.6，加入IPTG至终浓度0.2mM。在16℃转速240rpm下培养20h后18000g离心，弃上清后用10mM磷酸钾缓冲液(pH 7.0)洗两次，离心后沉淀物用5mL 10mM磷酸钾缓冲液(pH 7.0)重悬。重悬液0℃超声处理5min，18000g离心40min后取上清做为粗酶液。
1mL反应体系中，甲基乙二醛和谷胱甘肽摩尔比1:1，加入缓冲液和200uL粗酶液，37℃水浴反应。以10mM甲基乙二醛和10mM谷胱甘肽为例，2h后取样检测，MG1655ΔtnaAΔggt和MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG两组谷胱甘肽均消耗完，分别生成9.79mM和9.91mM的S-乳酰谷胱甘肽。再经过12h后取样检测，S-乳酰谷胱甘肽的浓度为8.52mM和9.67mM，说明水解酶基因gloB、gloC、yeiG的敲除减少了S-乳酰谷胱甘肽的水解。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。







Claims (19)

  1. 一种S-乳酰谷胱甘肽的制备方法，其特征在于，以谷氨酸、甘氨酸、半胱氨酸以及甲基乙二醛为原料，在谷胱甘肽合成酶及乙二醛酶的催化下转化为S-乳酰谷胱甘肽。
  2. 根据权利要求1所述的S-乳酰谷胱甘肽的制备方法，其特征在于，以谷氨酸、甘氨酸、半胱氨酸以及甲基乙二醛为底物，加入含有谷胱甘肽合成酶编码基因和乙二醛酶编码基因的重组微生物进行发酵，重组微生物过表达产生所述谷胱甘肽合成酶及乙二醛酶。
  3. 根据权利要求2所述的S-乳酰谷胱甘肽的制备方法，其特征在于，
    所述谷胱甘肽合成酶编码基因选自gshF、gshA、gshB中的任一种或几种，优选为gshF；进一步优选的，gshF的核苷酸序列如SEQ ID NO:1所示；
    和/或，所述乙二醛酶编码基因包括gloA；优选的，gloA的核苷酸序列如SEQ ID NO:2所示。
  4. 根据权利要求2或3所述的S-乳酰谷胱甘肽的制备方法，其特征在于，所述制备方法包括通过基因工程方法构建所述重组微生物，所述基因工程方法包括质粒表达或基因组整合；
  5. 根据权利要求4所述的S-乳酰谷胱甘肽的制备方法，其特征在于，重组微生物通过质粒表达的方法进行构建；
    优选地，构建方法为：通过PCR扩增获得谷胱甘肽合成酶编码基因和乙二醛酶编码基因，将获得的基因共同连接至含有IPTG诱导型启动子的质粒载体上并转化至感受态细胞中，测序后获得重组载体；将重组载体转化至受体微生物中即得到重组微生物；
    优选地，质粒载体选自pZAlac、pZElac中的任一种或两种。
  6. 根据权利要求5所述的S-乳酰谷胱甘肽的制备方法，其特征在于，
    重组载体为pZE-gshF-gloA，
    优选地，所述pZE-gshF-gloA构建方法为：通过PCR扩增得gshF基因和gloA基因，将gshF基因和gloA基因共同连接至含有IPTG诱导型启动子的pZElac载体上并转化至感受态细胞中，测序后得到质粒pZE-gshF_gloA；
    优选地，以大肠杆菌MG1655的基因组为模板分别通过PCR扩增得gshF基因和gloA基因；优选地，感受态细胞为大肠杆菌E.coli dh5a。
  7. 根据权利要求5所述的S-乳酰谷胱甘肽的制备方法，其特征在于，所述受体微生物选自大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌中的一种或几种。
  8. 根据权利要求5或7所述的S-乳酰谷胱甘肽的制备方法，其特征在于，受体微生物选自大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、巨大芽孢杆菌 (Bacillus megaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Candida utilis)或毕赤酵母(Pichia pastoris)中的一种或几种。
  9. 根据权利要求5-8任一项所述的S-乳酰谷胱甘肽的制备方法，其特征在于，若受体微生物含有表达S-乳酰谷胱甘肽水解酶的基因，需敲除受体微生物上表达S-乳酰谷胱甘肽水解酶的基因；表达S-乳酰谷胱甘肽水解酶的基因例如为gloB、gloC、yeiG；
    优选的，gloB的核苷酸序列如SEQ ID NO:5所示；
    优选的，gloC的核苷酸序列如SEQ ID NO:6所示；
    优选的，yeiG的核苷酸序列如SEQ ID NO:7所示；
    优选的，所述受体微生物为大肠杆菌MG1655ΔgloB、大肠杆菌MG1655ΔgloC、大肠杆菌MG1655ΔyeiG、大肠杆菌MG1655ΔgloBΔgloC、大肠杆菌MG1655ΔgloCΔyeiG、大肠杆菌MG1655ΔgloBΔyeiG、大肠杆菌MG1655ΔgloBΔgloCΔyeiG。
  10. 根据权利要求5-9任一项所述的S-乳酰谷胱甘肽的制备方法，其特征在于，若受体微生物含有表达半胱氨酸水解酶或谷胱甘肽水解酶的基因，需进一步敲除受体微生物上表达半胱氨酸水解酶或谷胱甘肽水解酶的基因；
    优选的，表达半胱氨酸水解酶的基因为tnaA；进一步优选的，tnaA的核苷酸序列如SEQ ID NO:3所示；
    优选的，表达谷胱甘肽水解酶的基因为ggt；进一步优选的，ggt的核苷酸序列如SEQ ID NO:4所示；
    优选的，所述受体微生物为大肠杆菌MG1655ΔtnaA、大肠杆菌MG1655Δggt或大肠杆菌MG1655ΔtnaAΔggt。
  11. 根据权利要求5-10任一项所述的S-乳酰谷胱甘肽的制备方法，其特征在于，所述受体微生物为大肠杆菌MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG。
  12. 根据权利要求11所述的S-乳酰谷胱甘肽的制备方法，其特征在于，所述大肠杆菌MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG的构建方法包括如下步骤：
    1)用同源重组法对野生型大肠杆菌MG1655菌株的tnaA、ggt、gloB、gloC、yeiG基因进行单独敲除获得五株单缺菌；
    2)将步骤1)中得到的Δggt、ΔgloB、ΔgloC和ΔyeiG四株单缺菌分别加入野生型P1噬菌体进行培养，得到分别含有ggt、gloB、gloC和yeiG敲除性状的大肠杆菌基因片段的噬菌体P1vir ggt、P1vir gloB、P1vir gloC和P1vir yeiG；
    3)将步骤1)得到的ΔtnaA单缺菌作为受体菌，依次加入步骤2)中得到的噬菌体进行转染，得到大肠杆菌MG1655ΔtnaAΔggtΔgloBΔgloCΔyeiG。
  13. 根据权利要求2-12任一项所述的S-乳酰谷胱甘肽的制备方法，其特征在于，发酵时，发酵温度为20～90℃。
  14. 根据权利要求1-13任一项所述的S-乳酰谷胱甘肽的制备方法，其特征在于，
    所述谷氨酸、甘氨酸、半胱氨酸、甲基乙二醛的摩尔浓度比例范围为8～12:8～12:6～10:1～4。
  15. 根据权利要求1所述的S-乳酰谷胱甘肽的制备方法，其特征在于，发酵时，先加入谷氨酸、甘氨酸、半胱氨酸以及重组微生物，发酵培养优选1～4h使谷胱甘肽积累，然后再加入甲基乙二醛继续发酵；优选地，发酵时，采用缓慢流加的方式使发酵罐中所述甲基乙二醛浓度维持为0.2～4mM。
  16. 一种S-乳酰谷胱甘肽的制备方法，其特征在于，以谷胱甘肽和甲基乙二醛为原料，在过表达乙二醛酸酶的重组微生物或其粗酶液催化下转化为S-乳酰谷胱甘肽；
    优选的，所述重组微生物中表达S-乳酰谷胱甘肽水解酶的基因被敲除；所述表达S-乳酰谷胱甘肽水解酶的基因例如为gloB、gloC、yeiG；
    优选的，gloB的核苷酸序列如SEQ ID NO:5所示；
    优选的，gloC的核苷酸序列如SEQ ID NO:6所示；
    优选的，yeiG的核苷酸序列如SEQ ID NO:7所示；
    优选的，所述重组微生物为大肠杆菌MG1655ΔgloB、大肠杆菌MG1655ΔgloC、大肠杆菌MG1655ΔyeiG、大肠杆菌MG1655ΔgloBΔgloC、大肠杆菌MG1655ΔgloCΔyeiG、大肠杆菌MG1655ΔgloBΔyeiG、大肠杆菌MG1655ΔgloBΔgloCΔyeiG。
  17. 制备S-乳酰谷胱甘肽的重组微生物，所述重组微生物过表达内源或外源谷胱甘肽合成酶编码基因及乙二醛酶编码基因；
    优选的，所述谷胱甘肽合成酶编码基因选自gshF、gshA、gshB中的任一种或几种，优选为gshF；进一步优选的，gshF的核苷酸序列如SEQ ID NO:1所示；
    优选的，所述乙二醛酶编码基因包括gloA；优选的，gloA的核苷酸序列如SEQ ID NO:2所示；优选的，所述受体微生物选自大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌中的一种或几种；进一步优选地，所述受体微生物选自大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、巨大芽孢杆菌(Bacillus megaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Candida utilis)或毕赤酵母(Pichia  pastoris)中的一种或几种。
  18. 用于制备S-乳酰谷胱甘肽的重组DNA或生物材料，其特征在于，所述重组DNA或生物材料含有谷胱甘肽合成酶编码基因及乙二醛酶编码基因；
    优选的，所述谷胱甘肽合成酶编码基因选自gshF、gshA、gshB中的任一种或几种，优选为gshF；进一步优选的，gshF的核苷酸序列如SEQ ID NO:1所示；
    优选的，所述乙二醛酶编码基因包括gloA；优选的，gloA的核苷酸序列如SEQ ID NO:2所示；优选的，所述生物材料为表达盒、转座子、质粒载体、噬菌体载体或病毒载体。
  19. 权利要求17所述的重组微生物、权利要求18所述的重组DNA或生物材料在制备S-乳酰谷胱甘肽中的应用。
PCT/CN2023/087385 2022-04-12 2023-04-10 一种s-乳酰谷胱甘肽的制备方法 WO2023198006A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210381545.6 2022-04-12
CN202210381545.6A CN116949119A (zh) 2022-04-12 2022-04-12 一种s-乳酰谷胱甘肽的制备方法

Publications (1)

Publication Number Publication Date
WO2023198006A1 true WO2023198006A1 (zh) 2023-10-19

Family

ID=88329039

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/087385 WO2023198006A1 (zh) 2022-04-12 2023-04-10 一种s-乳酰谷胱甘肽的制备方法

Country Status (2)

Country Link
CN (1) CN116949119A (zh)
WO (1) WO2023198006A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6349073A (ja) * 1986-08-15 1988-03-01 Takara Shuzo Co Ltd 大型酵母の造成方法
JPS63267727A (ja) * 1987-04-23 1988-11-04 Nonogawa Shoji:Kk Ｓ−ラクトイルグルタチオンおよび／またはその塩を有効成分とする医薬
CN1760359A (zh) * 2004-10-17 2006-04-19 肖强 一种生物工程法生产谷胱甘肽的方法
CN102586369A (zh) * 2011-01-10 2012-07-18 华东理工大学 一种利用重组大肠杆菌发酵生产谷胱甘肽的方法
CN105238797A (zh) * 2015-10-23 2016-01-13 山东金城生物药业有限公司 一种无乳链球菌的gshF基因的突变基因及其应用
CN112301050A (zh) * 2019-07-26 2021-02-02 浙江大学 一种构建高产谷胱甘肽重组菌株的方法及应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6349073A (ja) * 1986-08-15 1988-03-01 Takara Shuzo Co Ltd 大型酵母の造成方法
JPS63267727A (ja) * 1987-04-23 1988-11-04 Nonogawa Shoji:Kk Ｓ−ラクトイルグルタチオンおよび／またはその塩を有効成分とする医薬
CN1760359A (zh) * 2004-10-17 2006-04-19 肖强 一种生物工程法生产谷胱甘肽的方法
CN102586369A (zh) * 2011-01-10 2012-07-18 华东理工大学 一种利用重组大肠杆菌发酵生产谷胱甘肽的方法
CN105238797A (zh) * 2015-10-23 2016-01-13 山东金城生物药业有限公司 一种无乳链球菌的gshF基因的突变基因及其应用
CN112301050A (zh) * 2019-07-26 2021-02-02 浙江大学 一种构建高产谷胱甘肽重组菌株的方法及应用

Also Published As

Publication number Publication date
CN116949119A (zh) 2023-10-27

Similar Documents

Publication Publication Date Title
JP5486029B2 (ja) 遺伝子増幅によるリジン産生の増加
KR101511639B1 (ko) 재조합 미생물 및 이의 사용 방법
JP2017523787A (ja) フィードバック抵抗性アセトヒドロキシ酸シンターゼ変異体及びそれを用いたｌ−バリンの生産方法
CN110195036B (zh) 一种高产乙酰氨基葡萄糖的重组谷氨酸棒杆菌及其应用
CN114381415B (zh) 高产pha的基因重组菌及其构建方法
US10508279B2 (en) Recombinant Escherichia coli for high efficiency production of fructosylated chondroitin and method for making thereof
CN112522223A (zh) 一种用于l-肌氨酸生产的基因工程菌及构建方法与应用
CN113604521A (zh) 一种增强n-乙酰基-5-羟基色胺产量的方法
RU2546239C1 (ru) РЕКОМБИНАНТНЫЙ ШТАММ Escherichia coli, ОБЛАДАЮЩИЙ КОНСТИТУТИВНОЙ АСПАРТАЗНОЙ АКТИВНОСТЬЮ И СПОСОБ СИНТЕЗА L-АСПАРАГИНОВОЙ КИСЛОТЫ С ИСПОЛЬЗОВАНИЕМ ЭТОГО ШТАММА В КАЧЕСТВЕ БИОКАТАЛИЗАТОРА
CN107287144B (zh) 一种代谢改造的枯草芽孢杆菌生物转化细胞及其制备方法与应用
CN110305855B (zh) 天麻GeCPR基因及其应用
WO2023198006A1 (zh) 一种s-乳酰谷胱甘肽的制备方法
CN106148336B (zh) 新型的l-苏氨酸-诱导型启动子及其应用
Lee et al. Mass production of thermostable D‐hydantoinase by batch culture of recombinant Escherichia coli with a constitutive expression system
US9222110B2 (en) Microorganism and method for lactic acid production
CN109097315B (zh) 一种高产脂肽的基因工程菌及其构建方法和应用
CN110904102A (zh) 一种用于重组蛋白质表达的启动子
CN116376995B (zh) 一种利用苏氨酸制备甘氨酸、乙酰辅酶a及乙酰辅酶a衍生物的方法
WO2002055716A1 (en) Antibiotics-independent vector for constant high-expression and method for gene expression using the same
CN110331121B (zh) 一种高产脂肽的重组菌及其应用
CN116376989B (zh) 一种制备酮酸的方法及该方法在制备氨基酸或氨基酸衍生物中的应用
EP3978515A1 (en) Genetically modified methylobacillus bacteria having improving properties
KR101123070B1 (ko) 세포내 에너지 농도가 높은 세포 및 그의 용도
CN115851657A (zh) 一种促进精氨酸合成的乙酰谷氨酸激酶
CN115960807A (zh) 一种厌氧高产l-精氨酸工程菌及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23787650

Country of ref document: EP

Kind code of ref document: A1