WO2023208146A1 - Procédé et support pour la biosynthèse d'ergothionéine - Google Patents

Procédé et support pour la biosynthèse d'ergothionéine Download PDF

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WO2023208146A1
WO2023208146A1 PCT/CN2023/091345 CN2023091345W WO2023208146A1 WO 2023208146 A1 WO2023208146 A1 WO 2023208146A1 CN 2023091345 W CN2023091345 W CN 2023091345W WO 2023208146 A1 WO2023208146 A1 WO 2023208146A1
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seq
dependent
sequence
amino acid
methyltransferase
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常莹莹
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武汉合生科技有限公司
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • 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
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/045—Actinoplanes

Definitions

  • the present application belongs to the technical field of ergothioneine biosynthesis, and specifically relates to a method and carrier for biosynthesizing ergothioneine.
  • Ergothioneine is a sulfur-containing non-protein amino acid derived from histidine. It is present in the liver and other organs or blood of animals including humans. It is considered to maintain cellular redox. and bioenergy homeostasis. It is also recommended as one of the top ten vitamins for longevity. At the same time, as a natural antioxidant, it has good application prospects in the fields of food, medicine, cosmetics and other fields. It is a high value-added vitamin. New fermentation products.
  • EGT can be naturally synthesized by mushrooms, cyanobacteria, mycobacteria and red bread mold, but cannot be synthesized by animals and plants.
  • the biosynthesis of EGT requires only two enzymes, Egt1 and Egt2, while in Mycobacterium smegmatis, it requires five steps of enzyme-catalyzed reactions.
  • These enzymes are encoded by the gene cluster egtABCDE.
  • egtABCDE the gene cluster composed of egtABCDE only exists in the phylum Actinobacteria, and most strains belonging to the suborder Frankella and Corynebacteria contain EgtE homologous proteins and belong to the suborder Micromonospora and the suborder Propionibacteria.
  • the purpose of this application is to provide a method for biosynthesizing ergothioneine, as well as the carriers required in the biosynthesis process.
  • the first aspect of the present application provides a nucleic acid construct, which includes encoding glutamylcysteine synthetase, Fe2 + -dependent oxidase, glutamine amidotransferase, histidine methyltransferase and phosphoric acid At least one polynucleotide sequence of a pyridoxal-dependent carbon-sulfur lyase, wherein the glutamylcysteine synthetase, Fe 2+ -dependent oxidase, glutamine amidotransferase, histamine Acid methyltransferase and pyridoxal phosphate-dependent carbon-sulfur lyase are from A. swimming actinomycetes.
  • the second aspect of the present application provides a method for biosynthesizing ergothioneine, which includes expressing glutamylcysteine synthase, Fe 2+ -dependent oxidase, glutamine amidotransferase, histidine methyl Ergothioneine is synthesized by a recombinant bacterium of base transferase and pyridoxal phosphate-dependent carbon-sulfur lyase, wherein the recombinant bacterium is obtained by introducing the nucleic acid construct provided in the first aspect of the application into a host cell.
  • the third aspect of the present application provides a recombinant bacterium for biosynthesizing ergothioneine, which can express glutamylcysteine synthase, Fe 2+ -dependent oxidase, glutamine amidotransferase, histamine Acid methyltransferase and pyridoxal phosphate-dependent carbon-sulfur lyase, wherein the recombinant bacterium is obtained by introducing the nucleic acid construct provided in the first aspect of the application into a host cell.
  • the fourth aspect of this application provides an enzyme for ergothioneine biosynthesis, which has the same characteristics as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO. 5 or the amino acid sequence shown in SEQ ID NO.6 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity Amino acid sequence.
  • the fifth aspect of this application provides a polynucleotide molecule, which contains SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO. .12 A nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity sequence.
  • the sixth aspect of the present application provides a kind of mobile actinomycetes, which is Actinoplanes sp. HS, and its strain preservation number is CCTCC No:M 2022390.
  • the mobile actinomycetes can express The amino acid sequences are glutamylcysteine synthetase, Fe 2+ -dependent oxidase, and glutamine shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4 respectively. Amide amidotransferase and histidine methyltransferase, and a pyridoxal phosphate-dependent carbon-sulfur lyase whose amino acid sequence is shown in SEQ ID NO. 5 and SEQ ID NO. 6.
  • the seventh aspect of the present application provides the nucleic acid construct of the first aspect of the present application, the recombinant bacterium of the third aspect of the present application, the enzyme of the fourth aspect of the present application, the polynucleotide molecule of the fifth aspect of the present application, or the sixth aspect of the present application.
  • the present application provides a method comprising encoding glutamylcysteine synthase, Fe 2+ -dependent oxidase, glutamine amidotransferase, histidine methyltransferase and pyridoxine phosphate encoding a mobile actinomycete.
  • a nucleic acid construct of a polynucleotide sequence of an aldehyde-dependent carbon-sulfur lyase by introducing the nucleic acid construct into a host cell, the host cell recombinantly expresses glutamylcysteine synthetase, Fe2 + -dependent oxidase, glutamine amidotransferase, histidine methyltransferase and pyridoxal phosphate-dependent carbon-sulfur lyase, thereby achieving efficient synthesis of ergothioneine in the host cell.
  • the yield of biosynthesized ergothioneine can be significantly increased.
  • Figure 1A is the precise molecular weight extracted ion chromatogram of the EGT product in the EGT standard substance and the Actinobacteria HS fermentation broth of Example 1 of the present application;
  • Figure 1B is the first-level accurate molecular weight mass spectrum of the EGT product in the EGT standard and the EGT product in the HS fermentation broth of Example 1 of the present application;
  • Figure 1C shows the secondary fragment mass spectrum of the EGT standard
  • Figure 1D is a secondary fragment mass spectrum of the EGT product in the fermentation broth of Actinobacteria HS in Example 1 of the present application;
  • Figure 2 shows the structural schematic diagram of plasmids pCIL001A, pCIL001, pCIL003 and pCIL008;
  • Figure 3A is the precise molecular weight extracted ion chromatogram of the intermediate HER-Cys-Sul in the fermentation broth of YC300 strain and YC301 strain in Example 2 of the present application;
  • Figure 3B is the first-level accurate molecular weight mass spectrum of the intermediate HER-Cys-Sul in the fermentation broth of YC300 strain and YC301 strain in Example 2 of the present application;
  • Figure 4 is the precise molecular weight extracted ion chromatogram of the intermediate HER-Cys-Sul in the fermentation broth of strains YC301, YC303 and YC308 in Example 2 of the application;
  • Figure 5 is a histogram of ergothioneine production in the fermentation broth of strains YC303 and YC308 in Example 2 of the present application;
  • Figure 6A and Figure 6B are schematic structural diagrams of plasmids pCIL013 and pCIL014 respectively;
  • Figure 7 is a bar chart of ergothioneine production of the mobile actinomycetes HS and recombinant strains YC313 and YC314 of the present application.
  • the terms "about” and “similar to” mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which error range may depend in part on how the value is measured or determined, or Depends on the limitations of the measurement system.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single- or double-stranded form.
  • nucleic acid or “polynucleotide” also includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and behave in a manner similar to naturally occurring nucleosides. Acid is metabolized in a similar manner (see, U.S. Patent No.
  • Construct refers to any recombinant polynucleotide molecule (e.g., plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, linear or circular single- or double-stranded DNA or RNA polynucleotide molecule) that can Derived from any source, capable of integrating into the genome or replicating autonomously, it can be operably linked to one or more polynucleotide molecules.
  • a construct generally includes a polynucleotide molecule of the present application operably linked to transcription initiation regulatory sequences that direct the transcription of the polynucleotide molecule of the present application in a host cell.
  • Heterologous promoters or endogenous promoters may be used to direct expression of the nucleic acids of the present application.
  • Vector refers to any recombinant nucleic acid construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell).
  • the vector may contain a bacterial resistance gene for growth in bacteria and a promoter for expression of a protein of interest in an organism.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, vectors with an origin of replication functioning in the host cell).
  • Other vectors can be introduced into a host cell, integrated into the host cell's genome, and thus replicated with the host genome.
  • certain preferred vectors are capable of directing the expression of foreign genes to which they are linked.
  • Plasmid generally refers to a circular double-stranded DNA circle into which additional DNA segments (foreign genes) can be ligated, but may also include linear double-stranded molecules, such as those derived from polymerase chains. Amplification by reaction (PCR) or treatment of circular plasmid with restriction enzymes yields linear double-stranded molecules.
  • Plasmid vectors include vector backbone (i.e. empty vector) and expression framework.
  • expression framework refers to a sequence with the potential to encode a protein.
  • host cell refers to a cell, such as a microorganism, that can introduce a gene of interest and provide conditions for cloning and/or expression of the gene of interest.
  • mutant bacteria refers to genetically engineered bacteria (such as bacteria, yeasts, actinomycetes, etc.), which means that their cells have introduced Foreign gene fragments, among which one way of transformation includes the modification of the bacterial genome after the introduction of new DNA fragments, and the other way includes the introduction of artificially constructed or modified plasmids into the bacterial body, thereby making the bacterial genome Gain the ability to express the gene of interest.
  • genetically engineered bacteria such as bacteria, yeasts, actinomycetes, etc.
  • the first aspect of the present application provides a nucleic acid construct, which includes encoding glutamylcysteine synthetase, Fe2 + -dependent oxidase, glutamine amidotransferase, histidine methyltransferase and phosphoric acid At least one polynucleotide sequence of a pyridoxal-dependent carbon-sulfur lyase, wherein the glutamylcysteine synthetase, Fe 2+ -dependent oxidase, glutamine amidotransferase, histamine Acid methyltransferase and pyridoxal phosphate-dependent carbon-sulfur lyase are from A. swimming actinomycetes.
  • enzymes encoding glutamylcysteine synthase, Fe2 + -dependent oxidase, glutamine amidotransferase, histidine methyltransferase and pyridoxal phosphate-dependent carbon-sulfur lyase are At least one of the polynucleotide sequences is referred to as the gene of interest.
  • Doral-dependent carbon-sulfur lyase in which histidine methyltransferase transfers three methyl groups of S-adenosylmethionine (SAM) to L-histidine, catalyzing the synthesis of L-histidine Betaine (HER), glutamylcysteine synthase connects glutamate to cysteine to synthesize ⁇ -glutamylcysteine ( ⁇ -Glu-Cys), Fe 2+ -dependent oxidase Catalyzes the formation of CS bond between ⁇ -Glu-Cys and HER, synthesizes Hersienyl- ⁇ -glutamyl cysteine sulfoxide (HER- ⁇ -Glu-Cys-Sul), and then transfers glutamine amide
  • the enzyme removes the L-glutamic acid moiety in HER- ⁇ -Glu-Cys-Sul to generate acetyl cysteine sulfoxide (HER-Cys-Sul), which
  • the nucleic acid construct of the present application contains genes encoding glutamylcysteine synthetase, Fe2 + -dependent oxidase, glutamine amidotransferase, histidine methyltransferase and pyridoxal phosphate-dependent carbon-sulfur enzyme.
  • At least one polynucleotide sequence of a lytic enzyme which can be used to introduce polynucleotide sequences encoding these enzymes into a host cell, so that the host cell obtains expression of glutamylcysteine synthetase, Fe2 + -dependent
  • the ability of oxidases, glutamine amidotransferase, histidine methyltransferase, and pyridoxal phosphate-dependent carbon-sulfur lyase in turn, can be used to biosynthesize ergothioneine.
  • the nucleic acid construct comprises encoding glutamylcysteine synthetase, Fe2 + -dependent oxidase, glutamine amidotransferase, histidine methyltransferase, and pyridoxal phosphate Polynucleotide sequence of a dependent carbon-sulfur lyase.
  • the glutamylcysteine synthetase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% similarity to the amino acid sequence shown in SEQ ID NO.
  • the Fe 2+ -dependent oxidase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Amino acid sequences with 99% or 100% sequence identity;
  • the glutamine aminotransferase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence shown in SEQ ID NO. % or 100% sequence identity of the amino acid sequence;
  • the histidine methyltransferase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Amino acid sequences with 99% or 100% sequence identity;
  • the pyridoxal phosphate-dependent carbon-sulfur lyase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, An amino acid sequence that is 98%, 99% or 100% sequence identical; or has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to the amino acid sequence shown in SEQ ID NO.6 , 97%, 98%, 99% or 100% sequence identity of the amino acid sequence.
  • the glutamylcysteine synthetase (BC03_Glimmer_04016) has the amino acid sequence shown below:
  • the Fe2 + -dependent oxidase (BC03_Glimmer_04015) has the amino acid sequence shown below:
  • the glutamine aminotransferase (BC03_Glimmer_04014) has the amino acid sequence shown below:
  • the histidine methyltransferase (BC03_Glimmer_04013) has the amino acid sequence shown below:
  • the pyridoxal phosphate-dependent carbon-sulfur lyase has the amino acid sequence shown below:
  • the above two carbon-sulfur lyases can be introduced into the host cell alone to participate in the biosynthesis of ergothioneine, or they can be introduced into the host cell together to participate in the biosynthesis of ergothioneine.
  • the polynucleotide sequence encoding glutamylcysteine synthetase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity;
  • the polynucleotide sequence encoding Fe 2+ -dependent oxidase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% similarity with the nucleotide sequence shown in SEQ ID NO.8 , 97%, 98%, 99% or 100% sequence identity;
  • the polynucleotide sequence encoding glutamine aminotransferase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity;
  • the polynucleotide sequence encoding histidine methyltransferase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% similarity with the nucleotide sequence shown in SEQ ID NO.10 , 97%, 98%, 99% or 100% sequence identity;
  • the polynucleotide sequence encoding a pyridoxal phosphate-dependent carbon-sulfur lyase has at least 90%, 91%, 92%, and 93% similarity with the nucleotide sequence shown in SEQ ID NO.11 or SEQ ID NO.12 , 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • nucleotide sequence encoding glutamylcysteine synthetase includes:
  • Nucleotide sequences encoding Fe2 + -dependent oxidases include:
  • Nucleotide sequences encoding glutamine amidotransferase include:
  • Nucleotide sequences encoding histidine methyltransferase include:
  • the nucleotide sequence encoding a pyridoxal phosphate-dependent carbon-sulfur lyase includes:
  • codon optimization in order to express the enzyme of the present application in different host cells, those skilled in the art can perform codon optimization on the target gene of the present application according to the codon preference of different host cells to obtain the enzyme in different host cells. Faster or greater expression of the target protein.
  • the method of codon optimization is a conventional method in this field. For example, DNAWorks (Hoover and Lubkowski 2002), Codon Optimizer (Fuglsang 2003) and other websites can be used for codon optimization design. This application is not limited here.
  • the nucleic acid construct is a plasmid vector, and the encoding glutamylcysteine synthetase, Fe2 + -dependent oxidase, glutamine amidotransferase, histidine methyltransferase and the polynucleotide of the pyridoxal phosphate-dependent carbon-sulfur lyase are located in the expression framework of the plasmid vector.
  • the target genes can be independently connected to different plasmid vectors, or the target genes can also be partially or completely connected to the same plasmid vector.
  • the plasmid vector also contains a promoter that regulates the expression of the target gene.
  • a promoter that regulates the expression of the target gene.
  • This application does not limit the type, quantity and position of the promoter, as long as it can achieve the purpose of this application, for example, A promoter can be set upstream of each target gene fragment, or multiple target genes can share a promoter.
  • the promoter can be a promoter commonly used in this field, such as ermE* promoter, kasOp promoter, hrdB promoter, etc.
  • the plasmid vector is a prokaryotic expression vector.
  • Those skilled in the art can specifically select the starting plasmid of the plasmid vector according to needs, such as host cell type, enzyme cleavage site, etc., and this application is not limited here.
  • the plasmid vector is an integrating plasmid, which can integrate the target gene of the present application into the genome of the host cell, so that the obtained recombinant bacteria can stably express the target gene.
  • the starting plasmid of the plasmid vector is pSET152 plasmid.
  • the attP site can be used to quickly integrate the target gene carried into the host cell genome, which is conducive to the rapid acquisition of recombinant strains.
  • the structure of the plasmid vector is as shown in Figure 6A.
  • the starting plasmid of the plasmid vector is pSET152, encoding genes for glutamylcysteine synthetase, Fe2 + -dependent oxidase, glutamine amidotransferase and histidine methyltransferase Under the control of the same promoter, the gene encoding the pyridoxal phosphate-dependent carbon-sulfur lyase BC03_Glimmer_04046 is under the control of another promoter.
  • the plasmid vector also contains attP, oriT, acc(3)IV, and ori components, which can be used for site integration, vector replication, and resistance screening.
  • the structure of the plasmid vector is as shown in Figure 6B.
  • the starting plasmid of the plasmid vector is pSET152, encoding genes for glutamylcysteine synthetase, Fe2 + -dependent oxidase, glutamine amidotransferase and histidine methyltransferase Under the control of the same promoter, the coding gene BC03_Glimmer_04917 for pyridoxal phosphate-dependent carbon-sulfur lyase is under the control of another promoter; the plasmid vector also contains attP, oriT, acc(3)IV, and ori components .
  • the second aspect of the present application provides a method for biosynthesizing ergothioneine, which includes expressing glutamylcysteine synthase, Fe 2+ -dependent oxidase, glutamine amidotransferase, histidine methyl Ergothioneine is synthesized by a recombinant bacterium of base transferase and pyridoxal phosphate-dependent carbon-sulfur lyase, wherein the recombinant bacterium is obtained by introducing the nucleic acid construct provided in the first aspect of the application into a host cell.
  • the present application adopts a method comprising encoding glutamylcysteine synthase, Fe2 + -dependent oxidase, glutamine amidotransferase, histidine methyltransferase and pyridoxal phosphate-dependent carbon-sulfur lyase.
  • At least one nucleic acid construct of a polynucleotide sequence is introduced into a host cell, thereby obtaining a nucleic acid construct capable of expressing glutamylcysteine synthase, Fe 2+ -dependent oxidase, glutamine amidotransferase, and histamine
  • a recombinant strain of acid methyltransferase and pyridoxal phosphate-dependent carbon-sulfur lyase is used to biosynthesize ergothioneine.
  • the nucleic acid constructs described in the present application may include proteins encoding glutamylcysteine synthetase, Fe2 + -dependent oxidase, glutamine amidotransferase, histidine methyltransferase and pyridoxal phosphate-dependent One or more or all of the polynucleotide sequences of carbon-sulfur lyase, when the nucleic acid construct includes encoding glutamylcysteine synthetase, Fe 2+ -dependent oxidase, glutamine amidotransferase When one or more of the polynucleotide sequences of histidine methyltransferase and pyridoxal phosphate-dependent carbon-sulfur lyase are used, the host cell can be introduced into the host cell containing polynucleotide sequences encoding different enzymes.
  • Nucleic acid construct thereby enabling the simultaneous expression of glutamylcysteine synthetase, Fe 2+ -dependent oxidase, glutamine amidotransferase, histidine methyltransferase and pyridoxine phosphate in the host cell Aldehyde-dependent carbon-sulfur lyase.
  • the present application adopts a method comprising encoding glutamylcysteine synthase, Fe2 + -dependent oxidase, glutamine amidotransferase, histidine methyltransferase and pyridoxal phosphate-dependent carbon-sulfur lyase.
  • the nucleic acid construct of the polynucleotide sequence is introduced into the host cell by known methods to obtain recombinant bacteria.
  • Those skilled in the art can specifically select a method for introducing the nucleic acid construct into the host cell according to needs, such as the type of host cell, the type of nucleic acid construct, etc., which is not limited in this application.
  • the calcium transfer method can be used for Escherichia coli
  • the lithium acetate transformation method can be used for yeast
  • the conjugative transfer method, protoplast transformation method, etc. can be used for swimming actinomycetes.
  • the enzyme encoding glutamylcysteine synthetase, Fe2 + -dependent oxidase, glutamine amidotransferase, histidine methyltransferase, and pyridoxal phosphate-dependent carbon-sulfur enzyme
  • the polynucleotide sequence of the lytic enzyme is integrated into the genome of the host cell. Integrating the target gene into the genome of the host cell is conducive to the stable expression of the target gene by the recombinant bacteria.
  • the polynucleotide sequence of the enzyme can be independently integrated into the host cell genome at a site that does not interfere with the normal physiological metabolism of the host cell, and the recombinant bacteria of the present application can be obtained.
  • attP in the nucleic acid construct can be obtained.
  • site, the target gene of the present application is inserted into the attB site of the mobile actinomycete, thereby obtaining the recombinant bacterium of the present application.
  • the host cell is selected from at least one of Escherichia coli, Saccharomyces cerevisiae, or Actinobacteria tumefaciens.
  • the swimming actinomycetes is swimming actinomycetes HS, and its deposit number is CCTCC No: M 2022390.
  • the third aspect of the present application provides a recombinant bacterium for biosynthesizing ergothioneine, which can express glutamylcysteine synthase, Fe 2+ -dependent oxidase, glutamine amidotransferase, histamine Acid methyltransferase and pyridoxal phosphate-dependent carbon-sulfur lyase, wherein the recombinant bacterium is obtained by introducing the nucleic acid construct provided in the first aspect of the application into a host cell.
  • the enzyme encoding glutamylcysteine synthetase, Fe2 + -dependent oxidase, glutamine amidotransferase, histidine methyltransferase, and pyridoxal phosphate-dependent carbon-sulfur enzyme
  • the polynucleotide sequence of the lytic enzyme is integrated into the genome of the host cell.
  • the host cell is selected from at least one of Escherichia coli, Saccharomyces cerevisiae, or Actinobacteria tumefaciens.
  • the host cell is Actinobacterium HS, and its deposit number is CCTCC No: M 2022390.
  • actinomycetes when used as host cells, they do not produce endotoxins, so the fermentation products have higher biological safety compared to E. coli as host cells; in addition, actinomycetes can use crops, For example, soybean cake powder, as a nitrogen source, can reduce production costs and realize resource reuse.
  • the recombinant bacterium is obtained by introducing the nucleic acid construct into the mobile actinomycete HS, wherein the nucleic acid construct is a plasmid vector, and the starting plasmid of the plasmid vector is pSET152 plasmid.
  • the structure of the plasmid vector is shown in Figure 6A or Figure 6B.
  • the fourth aspect of this application provides an enzyme for ergothioneine biosynthesis, which has the same characteristics as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO. 5 or the amino acid sequence shown in SEQ ID NO.6 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity Amino acid sequence.
  • the fifth aspect of this application provides a polynucleotide molecule encoding the enzyme provided in the fourth aspect of this application, which includes SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO. 10.
  • the nucleotide sequence shown in SEQ ID NO.11 or SEQ ID NO.12 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% sequence identity to the nucleotide sequence.
  • the sixth aspect of the present application provides a kind of mobile actinomycetes, which is Actinoplanes sp. HS, and its strain preservation number is CCTCC No:M 2022390.
  • the mobile actinomycetes can express The amino acid sequences are glutamylcysteine synthetase, Fe 2+ -dependent oxidase, glutamylcysteine synthetase, and glutamylcysteine synthetase shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4 respectively.
  • the mobile actinomycetes HS of the present application can naturally and efficiently synthesize ergothioneine.
  • the seventh aspect of the present application provides the nucleic acid construct of the first aspect of the present application, the recombinant bacterium of the third aspect of the present application, the enzyme of the fourth aspect of the present application, the polynucleotide molecule of the fifth aspect of the present application, or the sixth aspect of the present application.
  • the enzyme composition and its application of the present application will be described below through specific examples.
  • the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present invention.
  • the plasmids involved in the following examples are all plasmids well known to those skilled in the art. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
  • Step 1 Fermentation of Mobile Actinobacteria HS
  • a strain with high ergothioneine production was obtained through laboratory screening. Its 16s rRNA sequence is 5'--3' (SEQ ID NO.38), which belongs to the genus Swim Actinomyces, so it is named Swim Actinomyces HS (preserved No. CCTCC No:M 2022390).
  • seed culture medium 1% glucose, 4% soybean cake powder, 1% glycerol, 1% soluble starch, 0.2% CaCO 3 , pH 7.0
  • Step 2 Identification of ergothioneine products in fermentation broth
  • Step 3 Quantitative analysis of ergothioneine products in fermentation broth
  • the ergothioneine in the fermentation broth was quantitatively detected using high-performance liquid chromatography-ultraviolet-visible spectrophotometric detector (HPLC-UV).
  • the yield of ergothioneine in the fermentation broth of the swimming actinomycetes HS of the present application is 43 mg/L, indicating that the swimming actinomycetes HS of the present application can naturally and efficiently synthesize ergothioneine.
  • Amplification primers are shown in Table 1.
  • Step 1 Use pETDuet-1 plasmid as the vector, use NcoI/EcoRI double enzyme digestion to obtain the fragmented vector, use the genome of the Actinobacteria HS strain extracted with a small amount of phenol chloroform method as the template, and use Primer 1/Primer 2 as the template.
  • the fragment BC03_Glimmer_04016 was obtained by primer amplification, and the fragment BC03_Glimmer_04015 was amplified using Primer3/Primer 4 as primers.
  • the above fragmentation vector and the two amplified fragments were carried out using the multi-fragment one-step rapid cloning kit (Yisheng Biotechnology Co., Ltd., China). Assemble and obtain pCIL001A plasmid.
  • the schematic structural diagram of pCIL001A plasmid is pCIL001A in Figure 2.
  • 04016 and 04015 represent the amplified fragments BC03_Glimmer_04016 and BC03_Glimmer_04015 respectively.
  • the other parts of the plasmid vector are derived from the vector backbone part of the pETDuet-1 plasmid.
  • pCIL001 plasmid The schematic structural diagram of pCIL001 plasmid is shown in pCIL001 in Figure 2. Among them, 04014 and 04013 represent the amplified fragments BC03_Glimmer_04014 and BC03_Glimmer_04013 respectively. The other parts of the plasmid vector are the same as the pCIL001A plasmid.
  • Step 2 Obtaining and fermentation of heterologous expression strains in BL21(DE3)
  • pCIL001 and pETDuet-1 were transformed into the BL21 (DE3) competent state respectively through the calcium transfer method to obtain strains YC301 and YC300.
  • YC301 is a heterologous expression strain
  • YC300 is a control strain.
  • Single colonies of strains YC300 and YC301 were isolated from LA plates containing 100 ⁇ g/mL ampicillin. The single colonies were picked out and placed in 10 mL of LB liquid culture medium containing 100 ⁇ g/mL ampicillin. They were cultured overnight at 37°C and 220 rpm.
  • Step 3 Detection of intermediate HER-Cys-Sul in fermentation broth
  • the results show that the control strain YC300 cannot synthesize the intermediate HER-Cys-Sul, while the strain YC301 that heterologously expressed BC03_Glimmer_04016, BC03_Glimmer_04015, BC03_Glimmer_04014 and BC03_Glimmer_04013 can produce HER-Cys-Sul, thus confirming that the actinomycetes HS
  • the proteins (enzymes) encoded by the genes BC03_Glimmer_04016, BC03_Glimmer_04015, BC03_Glimmer_04014, and BC03_Glimmer_04013 can synthesize the ergothioneine precursor HER-Cys-Sul.
  • the HS genome of Actinomycete niger was used as a template to amplify, and the relevant genes were constructed into the vector pBBR1MCS-2.
  • the constructed plasmid was then transferred into strain YC301, followed by shake flask fermentation and product measurement.
  • pBBR1MCS-2 plasmid Use pBBR1MCS-2 plasmid as the vector, use Primer 9/Primer 10 as primers to amplify the vector fragment, and then use NdeI/SacI double enzyme digestion to obtain the fragmented vector fragment, using the genome of the Actinobacterium HS strain as the template.
  • Primer 13/Primer 14 is the primer amplified to obtain the fragment BC03_Glimmer_04917.
  • the above fragmented vector and amplified fragment were assembled using a multi-fragment one-step rapid cloning kit (Yisheng Biotechnology Co., Ltd., China) to obtain the pCIL008 plasmid.
  • BC03_Glimmer_04917 represents the amplified fragment BC03_Glimmer_04917, and the rest are from the vector backbone part of the pBBR1MCS-2 plasmid.
  • Amplification primers are shown in Table 2.
  • Step 2 Obtaining and fermentation of heterologous expression strains in YC301
  • YC303 and YC308 strains were isolated from single colonies on LA plates containing 100 ⁇ g/mL ampicillin and 50 ⁇ g/mL kanamycin, and their single colonies were picked out and placed in 10 mL of LA plates containing 100 ⁇ g/mL ampicillin and 50 ⁇ g/mL kanamycin.
  • LB liquid medium cultivate overnight at 37°C and 220 rpm, and transfer it to a fermentation medium containing 100 ⁇ g/mL ampicillin and 50 ⁇ g/mL kanamycin (same as the fermentation medium of the YC301 strain) at a transfer volume of 1%.
  • Step 3 Detection of ergothioneine in fermentation broth
  • the fermentation broth processing and product detection methods are the same as step 3 of 2.1 in Example 2.
  • Example 3 Obtaining a high-yielding strain of ergothioneine
  • Step 1 Overexpression plasmid construction
  • pSET152 plasmid use XbaI/Eco32I double enzyme digestion to obtain the fragmented vector fragment, denoted as 152-XE.
  • the amplified fragments ermE*(2) and BC03_Glimmer_04046 were used using Prim er 19/ Primer 22 primer was used for OE-PCR to obtain the spliced fragment ermE*-BC03_Glimmer_04046, recorded as E-46.
  • the above fragments 152-XE, K16-13 and E-46 were used with the multi-fragment one-step rapid cloning kit (Yisheng Biotechnology Co., Ltd. , China) was assembled to obtain the pCIL013 plasmid.
  • the schematic diagram of the plasmid structure is shown in Figure 6A.
  • 04016, 04015, 04014, 04013, and 04046 represent gene fragments BC03_Glimmer_04016, BC03_Glimmer_04015, BC03_Glimmer_04014, BC03_Glimmer_04013, and BC03_ respectively.
  • Glimmer_04046, other components in the plasmid are all from pSET152 plasmid.
  • the promoter fragment ermE*(3) was amplified.
  • the fragment BC03_Glimmer_04917 was amplified.
  • the above amplified fragments ermE*(3) and BC03_Glimmer_04917 were used for OE-PCR using Primer 19/Primer 25 primers to obtain the spliced fragment ermE*-BC03_Glimmer_04917, recorded as E-17.
  • BC03_Glimmer_04014, BC03_Glimmer_04013 and BC03_Glimmer_04917, other components in the plasmid are all from the pSET152 plasmid.
  • the primer sequences are shown in Table 3.
  • Step 2 Obtain recombinant strain
  • pCIL013 and pCIL014 plasmids were transformed into the strain ET12567-pUZ8002 (Chang et al., Overproduction of gentamicin B in industrial strain Micromonospora echinospora CCTCC M 2018898 by cloning of the missing genes genR and genS, 2019).
  • pick Single clone was cultured overnight at 37°C in LB liquid medium containing 50 ⁇ g/mL apramycin, 50 ⁇ g/mL kanamycin, and 25 ⁇ g/mL chloramphenicol. The overnight culture broth was transferred at a volume ratio of 1/100.
  • LB liquid medium containing 50 ⁇ g/mL apramycin, 50 ⁇ g/mL kanamycin, and 25 ⁇ g/mL chloramphenicol, and culture at 37°C until the OD value is 0.6 to 0.8. Centrifuge to collect the cells and equal volumes. Wash the bacterial cells three times with anti-LB liquid medium, and then resuspend them in 100 ⁇ L anti-LB liquid medium to obtain an E. coli suspension, which is used as the donor bacteria.
  • mL apramycin SFM plate take an appropriate amount of the grown colonies into 50 ⁇ L dd H 2 O, boil in boiling water for 5 minutes and then incubate on ice for 5 minutes, repeat three times, centrifuge briefly and take the supernatant for PCR verification to verify the correct naming of the mutant strain. for YC313 and YC314.
  • Step 3 Shake flask fermentation of the recombinant strain and product detection
  • step 2 of Example 1 the fermentation broth of strains Actinomycete HS, YC313 and YC314 is processed.
  • the ergothioneine in the fermentation broth of Actinomyces HS, YC313 and YC314 strains is quantitatively detected using HPLC-UV.
  • the ergothioneine production in the fermentation broth of Actinobacterium HS (marked as Actinoplanes sp.HS in the figure), YC313 and YC314 strains is shown in Figure 7.
  • the ergothioneine production of YC313 and YC314 strains reached 125mg/L and 108mg/L respectively.
  • L which is 2.9 times and 2.5 times the ergothioneine production (43 mg/L) of the starting strain Actinobacterium swimming HS.
  • Step 4 Fermentation of recombinant strain in 50L fermenter and product detection
  • the glycerol bacteria of YC313 Take the glycerol bacteria of YC313, prepare the seed liquid with the same process as in step 3 of Example 3, and transfer it into a 15L seed tank according to the transfer amount of 1 ⁇ (the culture medium is the same as the above seed culture medium), and the stirring speed is 150 rpm.
  • Aeration rate 1 0.5 ⁇ 1 (m 3 /m 3 ⁇ min), after culturing for 48 hours at 28°C, transfer it to a 50L fermenter according to a transfer volume of 20% (medium composition: 5% maltose, 2% glucose, 3 % soybean cake powder, 0.3% yeast powder, 0.25% CaCl 2 , 0.25% CaCO 3 , pH 6.5 ⁇ 7.0), stirring speed 150 rpm, ventilation volume 1: 0.8 ⁇ 1.2 (m 3 /m 3 ⁇ min) , cultured at 28°C for 80h to obtain fermentation liquid.
  • step 3 of Example 3 the fermentation broth of strain YC313 was processed and detected.
  • YC313 strain was fermented in a 50L fermenter for 80 hours, and the ergothioneine production reached 323 mg/L.

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Abstract

L'invention concerne un procédé et un support pour la biosynthèse d'ergothionéine. Le procédé comprend : l'introduction dans une cellule hôte d'au moins une construction d'acide nucléique d'une séquence polynucléotidique comprenant : une glutamylcystéine synthétase de codage, une oxydase dépendante de Fe2+, de la glutamine amidotransférase, de l'histidine méthyltransférase et de la carbone-soufre lyase dépendante de phosphate de pyridoxal ; ce qui permet d'obtenir des bactéries recombinantes capables d'exprimer la glutamylcystéine synthétase, l'oxydase dépendante de Fe2+ , la glutamine amidotransférase, l'histidine méthyltransférase et la carbone-soufre lyase dépendante de phosphate de pyridoxal, et d'utiliser lesdites bactéries recombinantes pour la biosynthèse d'ergothionéine.
PCT/CN2023/091345 2022-04-27 2023-04-27 Procédé et support pour la biosynthèse d'ergothionéine WO2023208146A1 (fr)

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