WO2024099089A1 - 一种生产假尿苷的基因工程菌株及其构建方法与应用 - Google Patents

一种生产假尿苷的基因工程菌株及其构建方法与应用 Download PDF

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WO2024099089A1
WO2024099089A1 PCT/CN2023/126720 CN2023126720W WO2024099089A1 WO 2024099089 A1 WO2024099089 A1 WO 2024099089A1 CN 2023126720 W CN2023126720 W CN 2023126720W WO 2024099089 A1 WO2024099089 A1 WO 2024099089A1
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gene
pseudouridine
escherichia coli
fragment
geneid
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French (fr)
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范晓光
王倩倩
王昭颖
黄馨禾
屈琰
徐庆阳
陈宁
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天津科技大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the technical field of genetic engineering, and relates to the breeding of industrial microorganisms, in particular to a genetic engineering strain for producing pseudouridine, and a construction method and application thereof.
  • Pseudouridine also known as 5-BD-nucleofuracil, has a molecular formula of C 9 H 12 N 2 O 6 and a molecular weight of 244.201.
  • Pseudouridine is a pyrimidine nucleoside and the 5-ribose isomer of uridine. The ribose is not connected to the N1 of uracil, but to the C5 of the pyrimidine ring.
  • Pseudouridine is the most abundant modified nucleoside on RNA and is also known as the "fifth nucleoside" of RNA. It has important application value in mRNA COVID-19 vaccines.
  • pseudouridine as a metabolite of RNA, has the characteristic of being excreted only from the kidneys. It can be used in clinical medicine to monitor the occurrence and development of tumors and the diagnosis and treatment of kidney diseases. Pseudouridine has very important application value. With the continuous expansion of the market demand for pseudouridine, it is urgent to develop a cheap pseudouridine production process that can be applied on a large scale.
  • pseudouridine The production methods of pseudouridine include chemical synthesis, chemical enzyme method and microbial fermentation method.
  • Li Jingjian et al. used 2,3,5-tribenzyloxy-D-ribonic acid-1,4-lactone as a raw material to carry out addition reaction with 2,4-dialkoxy-5-bromopyrimidine, and then carried out reduction reaction with triethylsilane and boron trifluoride ether in sequence, and finally carried out deprotection reaction with boron trichloride to obtain pseudouridine (ZL202210798651.4); Jin Feng et al.
  • pseudouridine ZL202210473532.1
  • the production method of pseudouridine mainly relies on chemical synthesis, there are problems such as long synthesis steps, high production cost, low yield, etc. in the chemical synthesis process, and dangerous reagents such as flammable and explosive are used.
  • Xiao Cong and others invented a chemical enzyme method to synthesize pseudouridine, using a 5'-pseudouridine monophosphoglycosidase mutant to catalyze 5-phosphoribose and uracil to synthesize 5'-pseudouridine monophosphate, and then chemically dephosphorylated and separated to obtain pseudouridine (ZL202111581013.9).
  • this method also has the disadvantages of high raw material cost, complex production process, and low conversion rate.
  • the microbial fermentation method has the advantages of being environmentally friendly, low cost, simple process, and sustainable production.
  • the use of metabolic engineering and synthetic biology to breed strains for nucleoside product production has been a hot topic in recent years.
  • the present invention mainly uses metabolic engineering technology to systematically and comprehensively optimize the modules of the uridine acid synthesis pathway, pseudouridine synthesis pathway, transport system and pseudouridine branch metabolic pathway to improve the pseudouridine fermentation performance of the strain.
  • the purpose of the present invention is to overcome the problems existing in the prior art and provide a genetically engineered strain for producing pseudouridine and a construction method and application thereof.
  • a genetically engineered strain for producing pseudouridine wherein the genetically engineered strain heterologously overexpresses a pyrimidine nucleoside operon, overexpresses pseudouridylate synthetase, overexpresses ribokinase, overexpresses ribonucleoside hydrolase, overexpresses uracil permease, and does not express pseudouridine transporter and pseudouridine kinase.
  • pyrimidine nucleoside operon is an operon encoded by the pyrBCAKDFE gene of Bacillus subtilis, and the nucleotide sequence is SEQ ID NO.1;
  • the pseudouridylate synthetase is a protein encoded by the Escherichia coli yeiN gene (NCBI-GeneID: 946699), a protein encoded by the Thermotoga maritima TM1464 gene (NCBI-GeneID: 896983), or a protein encoded by the Agrobacterium tumefaciens AWN88_14600 gene (NCBI-GeneID: 29364903);
  • the ribokinase is a protein encoded by the Escherichia coli rbsK gene (NCBI-GeneID: 948260) or a protein encoded by the Bacillus subtilis rbsK gene (NCBI-GeneID: 936844);
  • the ribonucleoside hydrolase is a protein encoded by the Escherichia coli rihA gene (NCBI-GeneID: 945503) or the Escherichia coli rihB gene (NCBI-GeneID: 946646) or the Escherichia coli rihC gene (NCBI-GeneID: 944796);
  • the uracil permease is a protein encoded by the Escherichia coli uraA gene (NCBI-GeneID: 946978);
  • the pseudouridine transporter is a protein encoded by the Escherichia coli psuT gene (NCBI-GeneID: 946671);
  • the pseudouridine kinase is a protein encoded by the Escherichia coli yeiI gene (NCBI-GeneID: 946640) and the Escherichia coli yeiC gene (NCBI-GeneID: 946664).
  • the gene of the pyrimidine nucleoside operon is connected to a promoter P trc ;
  • the gene of the pseudouridylate synthetase is connected to the promoter P trc ; and/or
  • the gene of the ribokinase is connected to the promoter P trc ; and/or
  • the gene of the ribonucleoside hydrolase is connected to the promoter P trc ; and/or
  • the gene of the uracil permease is connected to the promoter P trc ;
  • nucleotide sequence of the promoter P trc is shown as SEQ ID NO.2.
  • the starting strain used in the construction of the genetically engineered strain is Escherichia coli.
  • Escherichia coli is E. coli MG1655.
  • the method for constructing the genetically engineered strain as described above comprises the following steps:
  • the pyrimidine nucleoside operon gene pyrBCAKDFE from Bacillus subtilis is overexpressed, the endogenous pseudouridylate synthetase gene yeiN of Escherichia coli or the pseudouridylate synthetase gene TM1464 of Thermotoga maritima or the pseudouridylate synthetase gene AWN88_14600 of Agrobacterium tumefaciens is overexpressed, the endogenous ribokinase gene rbsK of Escherichia coli or the ribokinase gene rbsK of Bacillus subtilis is overexpressed, the endogenous ribonucleoside hydrolase gene rihA or rihB or rihC of Escherichia coli is overexpressed, the endogenous uracil permease gene uraA of Escherichia coli is overexpressed, the pseudouridine
  • the genetically engineered bacteria overexpresses a pyrimidine nucleoside operon, overexpresses pseudouridylate synthetase, overexpresses ribokinase, overexpresses ribonucleoside hydrolase, overexpresses uracil permease, and does not express pseudouridine transporter and pseudouridine kinase.
  • the method comprises the following steps:
  • the pyrimidine nucleoside operon gene pyrBCAKDFE from Bacillus subtilis was overexpressed, the endogenous pseudouridylate synthetase gene yeiN of Escherichia coli was overexpressed, the endogenous ribokinase gene rbsK of Escherichia coli was overexpressed, the endogenous ribonucleoside hydrolase gene rihB of Escherichia coli was overexpressed, the endogenous uracil permease gene uraA of Escherichia coli was overexpressed, the pseudouridine transporter gene psuT was knocked out or inactivated, and the pseudouridine kinase genes yeiI and yeiC were knocked out or inactivated.
  • the method comprises the following steps:
  • a method for producing pseudouridine by fermentation using the genetically engineered strain as described above comprises the following steps:
  • the suitable conditions include a culture temperature of 37°C, a pH of 7.0 ⁇ 0.5, dissolved oxygen of 25%-35%, and a culture medium composition of:
  • the genetically engineered strain of the present invention overexpresses pyrimidine nucleoside operons, overexpresses pseudouridylate synthetase, overexpresses ribokinase, overexpresses ribonucleoside hydrolase, overexpresses uracil permease, and does not express pseudouridine transporter and pseudouridine kinase.
  • the present invention mainly uses metabolic engineering technology to systematically and comprehensively optimize the modules of the uridylate synthesis pathway, pseudouridine synthesis pathway, transport system and pseudouridine branch metabolic pathway, thereby improving the pseudouridine fermentation performance of the strain.
  • the pseudouridine yield of the genetically engineered strain is high, the production performance is stable, and it can be used for pseudouridine production.
  • the present invention uses a rational metabolic engineering method to obtain an engineered strain with a clear genetic background, no plasmid, using cheap carbon sources such as glucose as substrates, and efficiently synthesizing pseudouridine from scratch.
  • the highest yield of pseudouridine in the current prior art is obtained by an engineered bacterium carrying a plasmid, with a yield of 7.2g/L and a fermentation cycle of 72-96h (ZL202011637153.9).
  • the strain of the present invention has a pseudouridine yield of 20g/L after fermentation on a 5L tank for 48h.
  • the fermentation yield is the highest level reported so far, and the fermentation cycle is significantly shortened compared with existing reports.
  • the present invention uses metabolic engineering technology to systematically and comprehensively optimize the modules of uridine acid synthesis pathway, pseudouridine synthesis pathway, transport system and pseudouridine branch metabolic pathway.
  • the overall metabolic transformation strategy (see Figure 1) has not been reported. Since all operations are completed on the genome, the resulting engineered strain can produce pseudouridine more stably and efficiently, and has good prospects for industrial application.
  • FIG1 is a diagram showing the metabolic transformation strategy of pseudouridine engineering strains in the present invention.
  • FIG2 is an electrophoretic diagram showing the construction and verification of the segmented integration fragments of the pyrimidine nucleoside operon pyrBCAKDFE in the present invention. including:
  • FIG2A is an electrophoresis diagram of the construction and verification of the pyr1 integration fragment in the present invention; wherein: M: 1kb DNA Marker; 1: upstream homology arm; 2: pyr1 fragment; 3: downstream homology arm; 4: overlapping fragment; 5: original bacteria PCR fragment; 6: positive single colony PCR identification fragment;
  • FIG2B is an electrophoresis diagram of the construction and verification of the pyr2 integration fragment in the present invention; wherein: M: 1kb DNA Marker; 1: pyr2 upstream fragment-pyr2 fragment; 2: downstream homology arm; 3: overlapping fragment; 4: original bacteria PCR fragment; 5: positive single colony PCR identification fragment;
  • FIG2C is an electrophoresis diagram of the construction and verification of the pyr3 integration fragment in the present invention.
  • M 1kb DNA Marker
  • 1 pyr3 upstream fragment-pyr3 fragment
  • 2 downstream homology arm
  • 3 overlapping fragment
  • 4 original bacteria PCR fragment
  • 5 positive single colony PCR identification fragment
  • Figure 3 is an electrophoresis diagram of the construction and verification of the yeiN integration fragment in the present invention; wherein: M: 1kb DNA Marker; 1: upstream homology arm; 2: target gene; 3: downstream homology arm; 4: overlapping fragment; 5: original bacteria PCR fragment; 6: positive single colony PCR identification fragment;
  • Figure 4 is an electrophoresis diagram of the construction and verification of the rbsK integration fragment in the present invention; wherein: M: 1kb DNA Marker; 1: upstream homology arm; 2: target gene; 3: downstream homology arm; 4: overlapping fragment; 5: original bacteria PCR fragment; 6: positive single colony PCR identification of fragments;
  • Figure 5 is an electrophoresis diagram of the construction and verification of the rihB integration fragment in the present invention; wherein: M: 1kb DNA Marker; 1: upstream homology arm; 2: target gene; 3: downstream homology arm; 4: overlapping fragment; 5: original bacteria PCR fragment; 6: positive single colony PCR identification fragment;
  • Figure 6 is an electrophoresis diagram of the construction and verification of the uraA integration fragment in the present invention; wherein: M: 1kb DNA Marker; 1: upstream homology arm; 2: target gene; 3: downstream homology arm; 4: overlapping fragment; 5: original bacteria PCR fragment; 6: positive single colony PCR identification fragment;
  • Figure 7 is an electrophoresis diagram of the construction and verification of the psuT gene knockout fragment in the present invention; wherein: M: 1kb DNA Marker; 1: upstream homology arm; 2: downstream homology arm; 3: overlapping fragment; 4: original bacteria PCR fragment; 5: positive single colony PCR identification fragment;
  • Figure 8 is an electrophoresis diagram of the construction and verification of the yeiI gene knockout fragment in the present invention; wherein: M: 1kb DNA Marker; 1: upstream homology arm; 2: downstream homology arm; 3: overlapping fragment; 4: original bacteria PCR fragment; 5: positive single colony PCR identification fragment;
  • Figure 9 is an electrophoresis diagram of the construction and verification of the yeiC gene knockout fragment in the present invention.
  • M 1kb DNA Marker
  • 1 upstream homology arm
  • 2 downstream homology arm
  • 3 overlapping fragment
  • 4 original bacteria PCR fragment
  • 5 positive single colony PCR identification fragment
  • FIG10 is a diagram showing the shake flask fermentation results of Example 2 of the present invention.
  • FIG11 is a fermentation process curve diagram of a 5L fermenter in Example 3 of the present invention.
  • FIG12 is a high performance liquid chromatogram of the pseudouridine standard product of the present invention, wherein the peak at 4.3 min is pseudouridine;
  • FIG. 13 is a high performance liquid chromatogram of the fermentation broth of the present invention, wherein the peak at 4.3 min is pseudouridine.
  • the raw materials used in the present invention are all conventional commercially available products.
  • the methods used in the present invention are all conventional methods in the art.
  • the quality of each substance used in the present invention is the conventional quality.
  • a genetically engineered strain for producing pseudouridine wherein the genetically engineered strain heterologously overexpresses a pyrimidine nucleoside operon, overexpresses pseudouridylate synthetase, overexpresses ribokinase, overexpresses ribonucleoside hydrolase, overexpresses uracil permease, and does not express pseudouridine transporter and pseudouridine kinase.
  • the pyrimidine nucleoside operon is an operon encoded by the pyrBCAKDFE gene of Bacillus subtilis, and the nucleotide sequence is SEQ ID NO.1;
  • the pseudouridylate synthetase is a protein encoded by the Escherichia coli yeiN gene (NCBI-GeneID: 946699), a protein encoded by the Thermotoga maritima TM1464 gene (NCBI-GeneID: 896983), or a protein encoded by the Agrobacterium tumefaciens AWN88_14600 gene (NCBI-GeneID: 29364903);
  • the ribokinase is a protein encoded by the Escherichia coli rbsK gene (NCBI-GeneID: 948260) or a protein encoded by the Bacillus subtilis rbsK gene (NCBI-GeneID: 936844);
  • the ribonucleoside hydrolase is a protein encoded by the Escherichia coli rihA gene (NCBI-GeneID: 945503) or the Escherichia coli rihB gene (NCBI-GeneID: 946646) or the Escherichia coli rihC gene (NCBI-GeneID: 944796);
  • the uracil permease is a protein encoded by the Escherichia coli uraA gene (NCBI-GeneID: 946978);
  • the pseudouridine transporter is a protein encoded by the Escherichia coli psuT gene (NCBI-GeneID: 946671);
  • the pseudouridine kinase is a protein encoded by the Escherichia coli yeiI gene (NCBI-GeneID: 946640) and the Escherichia coli yeiC gene (NCBI-GeneID: 946664).
  • the gene of the pyrimidine nucleoside operon is connected to a promoter P trc ; and/or
  • the gene of the pseudouridylate synthetase is connected to the promoter P trc ; and/or
  • the gene of the ribokinase is connected to the promoter P trc ; and/or
  • the gene of the ribonucleoside hydrolase is connected to the promoter P trc ; and/or
  • the gene of the uracil permease is connected to the promoter P trc ;
  • nucleotide sequence of the promoter P trc is shown as SEQ ID NO.2.
  • the starting strain used in the construction of the genetically engineered strain is Escherichia coli.
  • the Escherichia coli is E. coli MG1655.
  • the method for constructing the genetically engineered strain as described above comprises the following steps:
  • the pyrimidine nucleoside operon gene pyrBCAKDFE from Bacillus subtilis is overexpressed, the endogenous pseudouridylate synthetase gene yeiN of Escherichia coli or the pseudouridylate synthetase gene TM1464 of Thermotoga maritima or the pseudouridylate synthetase gene AWN88_14600 of Agrobacterium tumefaciens is overexpressed, the endogenous ribokinase gene rbsK of Escherichia coli or the ribokinase gene rbsK of Bacillus subtilis is overexpressed, the endogenous ribonucleoside hydrolase gene rihA or rihB or rihC of Escherichia coli is overexpressed, the endogenous uracil permease gene uraA of Escherichia coli is overexpressed, the pseudouridine
  • the genetically engineered bacteria overexpresses a pyrimidine nucleoside operon, overexpresses pseudouridylate synthetase, overexpresses ribokinase, overexpresses ribonucleoside hydrolase, overexpresses uracil permease, and does not express pseudouridine transporter and pseudouridine kinase.
  • the method comprises the following steps:
  • the pyrimidine nucleoside operon gene pyrBCAKDFE from Bacillus subtilis was overexpressed, the endogenous pseudouridylate synthetase gene yeiN of Escherichia coli was overexpressed, the endogenous ribokinase gene rbsK of Escherichia coli was overexpressed, the endogenous ribonucleoside hydrolase gene rihB of Escherichia coli was overexpressed, the endogenous uracil permease gene uraA of Escherichia coli was overexpressed, the pseudouridine transporter gene psuT was knocked out or inactivated, and the pseudouridine kinase genes yeiI and yeiC were knocked out or inactivated.
  • the method comprises the following steps:
  • a method for producing pseudouridine by fermentation using the genetically engineered strain as described above comprises the following steps:
  • the suitable conditions include a culture temperature of 37°C, a pH of 7.0 ⁇ 0.5, dissolved oxygen of 25%-35%, and a culture medium composition of:
  • a genetically engineered strain of Escherichia coli heterologously overexpresses a pyrimidine nucleoside operon, overexpresses pseudouridylate synthetase, overexpresses ribokinase, overexpresses ribonucleoside hydrolase, overexpresses uracil permease, and does not express pseudouridine transporter and pseudouridine kinase.
  • nucleotide sequence of the pyrimidine nucleoside operon gene pyrBCAKDFE is as shown in SEQ ID NO.1.
  • NCBI-GeneID of the pseudouridylate synthetase gene yeiN is: 946699.
  • NCBI-GeneID of the ribokinase gene rbsK is: 948260.
  • NCBI-GeneID of the ribonucleoside hydrolase gene rihB is: 946646.
  • the NCBI-GeneID of the uracil permease gene uraA is: 946978.
  • the NCBI-GeneID of the pseudouridine transporter gene psuT is: 946671.
  • NCBI-GeneID of the pseudouridine kinase gene yeiI is: 946640.
  • NCBI-GeneID of the pseudouridine kinase gene yeiC is: 946664.
  • the gene may not be expressed by conventional means in the art, for example, the gene may be inactivated or knocked out by conventional means in the art.
  • the non-expression means that the amount of the gene expression product is significantly lower than the original level, for example, significantly reduced by 50%, 60%, 70%, 80%, 90%, 100%.
  • the above-mentioned gene can be overexpressed by conventional means in the art, for example, increasing the copy number of the gene or connecting the gene to a strong promoter.
  • the overexpression means that the amount of the gene expression product is significantly higher than the original level.
  • the pyrimidine nucleoside operon gene pyrBCAKDFE is connected to the promoter P trc ; and/or the pseudouridylate synthetase gene yeiN is connected to the promoter P trc ; and/or the ribokinase gene rbsK is connected to the promoter P trc ; and/or the ribonucleoside hydrolase gene rihB is connected to the promoter P trc ; and/or the uracil permease gene uraA is connected to the promoter P trc ; preferably, the nucleotide sequence of the promoter is shown in SEQ ID NO.2.
  • the starting strain used to construct the genetically engineered Escherichia coli strain can be any Escherichia coli. According to a preferred embodiment of the present invention, the starting strain is E. coli MG1655.
  • the method for constructing the genetically engineered strain of Escherichia coli as described above comprises: overexpressing the pyrimidine nucleoside operon gene pyrBCAKDFE derived from Bacillus subtilis in the starting strain Escherichia coli, overexpressing the endogenous pseudouridylate synthetase gene yeiN of Escherichia coli, overexpressing the endogenous ribokinase gene rbsK of Escherichia coli, overexpressing the endogenous ribonucleoside hydrolase gene rihB of Escherichia coli, overexpressing the endogenous uracil permease gene uraA of Escherichia coli, knocking out or inactivating the pseudouridine transporter gene psuT, and knocking out or inactivating the pseudouridine kinase genes yeiI and yeiC.
  • the method comprises:
  • the fusion fragment Ptrc-pyrBCAKDFE of the pyrimidine nucleoside operon of B. subtilis A260 and the promoter Ptrc was integrated into the yghX pseudogene site; this step enhanced the de novo synthesis flux of pyrimidine nucleoside and relieved the feedback inhibition of uridine monophosphate on carbamoyl phosphate synthetase;
  • the fusion fragment Ptrc -yeiN of the endogenous pseudouridylate synthetase gene yeiN and the promoter Ptrc was integrated into the ygay pseudogene site to strengthen the conversion of 5-phosphate ribose and uracil to 5'-pseudouridine monophosphate;
  • the fusion fragment Ptrc -rbsK of the endogenous ribokinase gene rbsK and the promoter Ptrc was integrated into the mbhA pseudogene site to strengthen the conversion of ribose to 5-phosphate ribose;
  • the fusion fragment Ptrc-rihB of the endogenous ribonucleoside hydrolase gene rihB and the promoter Ptrc was integrated into the yjiT pseudogene site to strengthen the conversion of uridine to uracil; 5'-pseudouridine monophosphate generates pseudouridine through
  • the fusion fragment Ptrc -uraA which is a fusion of the endogenous uracil permease gene uraA of Escherichia coli and the promoter Ptrc , was integrated into the ycgh pseudogene site to enhance the transport of uracil into the cell; the pseudouridine transporter gene psuT was knocked out to weaken the transport of pseudouridine into the cell.
  • the use of the genetically engineered strain of Escherichia coli as described above in high production of pseudouridine comprises: culturing the genetically engineered strain under suitable conditions and collecting pseudouridine from the culture.
  • the suitable conditions refer to a culture temperature of 37°C, maintaining a pH of about 7.0, a dissolved oxygen content of 25%-35%, and a culture medium composed of: 20-25 g/L glucose, 4-8 g/L yeast powder, 5-10 g/L peptone, 1-5 g/L sodium citrate , 2-4 g /L KH2PO4 , 1.2-2.0 g/L MgSO4 ⁇ 7H2O, 20-30 mg/ L FeSO4 ⁇ 7H2O, 10-20 mg/L MnSO4 ⁇ 7H2O, 2-4 mg/L each of VB1 , VB3 , VB5 , VB12 , and VBH , and the rest water , pH 7.0-7.2.
  • the gene editing methods involved in the embodiments of the present invention are carried out with reference to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic engineering, 2015, 31: 13-21.), and other specific operating methods such as molecular biology and genetic engineering involved can be implemented according to technical manuals, textbooks or literature reports that are easily available to personnel in this field.
  • Example 1 Construction of pseudouridine engineering strain E. coli psu8
  • the present invention adopts a CRISPR/Cas9-mediated gene editing method, which can be carried out with reference to the literature (Metabolic Engineering, 2015, 31: 13-21.).
  • CRISPR/Cas9 is a precise and efficient new gene targeting modification technology.
  • the two plasmids used in this method are pGRB and pREDCas9.
  • the pREDCas9 plasmid is a temperature-sensitive plasmid, carrying a gRNA plasmid elimination system, a lambda phage Red recombination system and a Cas9 protein expression system, with spectinomycin resistance (working concentration: 100 mg/L), and an optimal culture temperature of 32°C;
  • the pGRB plasmid, with pUC18 as the backbone contains the promoter J23100, a gRNA-Cas9 binding region sequence and a terminator sequence, and has ampicillin resistance (working concentration: 100 mg/L), and an optimal culture temperature of 37°C.
  • the pyrimidine nucleoside operon pyrBCAKDFE (including eight genes, pyrB, pyrC, pyrAA, pyrAB, pyrK, pyrD, pyrF, and pyrE) of Bacillus subtilis (B. subtilis A260) totaling 9492 bp was divided into three segments, pyr1, pyr2, and pyr3, and sequentially integrated into the yghX gene site of Escherichia coli in this embodiment, and the transcriptional expression of the exogenous operon was initiated by the promoter P trc to construct the strain E. coli psu1-3. Specifically comprising:
  • upstream homology arm primers UP-yghX-S, UP-yghX-A
  • downstream homology arm primers DN-yghX-S1, DN-yghX-A
  • primers (pyr1-S, pyr1-A) were designed according to pyr1 (positions 1-3260 of the nucleotide sequence shown in SEQ ID NO.1), and the pyr1 gene fragment was amplified by PCR, and the promoter P trc was designed in the downstream primer of the upstream homology arm and the upstream primer of the pyr1 gene.
  • the above fragments were fused by overlapping PCR to obtain the integrated fragment of the pyr1 gene (upstream homology arm-P trc -pyr1-downstream homology arm), and the DNA fragment containing the target sequence of the target gene was obtained by annealing primers gRNA-yghX-S and gRNA-yghX-A, and the recombinant pGRB-yghX was obtained after recombination with the linearized pGRB vector.
  • the recombinant fragment and plasmid pGRB-yghX were electrotransferred to the competent cells of MG1655 containing the pREDCas9 plasmid, and the plasmid was eliminated after screening the positive strain to obtain the strain E. coli psu1-1.
  • the electrophoresis diagram of the construction of the P trc -pyr1 integrated fragment and the PCR verification of the positive strain is shown in Figure 2A. Among them, the length of the upstream homology arm is 658bp, the length of the pyr1 gene fragment is 3363bp, the length of the downstream homology arm is 604bp, and the total length of the integrated fragment is 4560bp.
  • the length of the PCR amplification fragment for positive bacteria should be 4560bp, and the length of the PCR amplification fragment for original bacteria should be 1765bp.
  • primers (pyr2-S, pyr2-A) were designed based on pyr2 and its upstream sequence (nucleotide sequence shown in SEQ ID NO.1, position 2448-6469), and PCR amplification of the pyr2 fragment was performed; using the E. coli MG1655 genome as a template, PCR amplification of the downstream homology arm fragment of the yghX gene was performed based on the downstream homology arm primers (DN-yghX-S2, DN-yghX-A). The above fragments were fused by overlapping PCR to obtain the entire pyr2 gene.
  • the integrated fragment (pyr2-downstream homology arm) was synthesized, and a DNA fragment containing the target sequence of the target gene was obtained by annealing primers gRNA-S1 and gRNA-A1, and the recombinant pGRB-pyr2 was obtained after recombination with the linearized pGRB vector.
  • the recombinant fragment and plasmid pGRB-pyr2 were electrotransferred to the competent cells of E.coli psu1-1 containing the pREDCas9 plasmid, and the plasmid was eliminated after screening the positive strain to obtain the strain E.coli psu1-2.
  • the electrophoresis diagram of the construction of the pyr2 integrated fragment and the PCR verification of the positive strain is shown in Figure 2B.
  • the length of the pyr2 gene fragment and its upstream homology arm is 4065bp
  • the length of the downstream homology arm is 604bp
  • the total length of the integrated fragment is 4646bp.
  • the length of the PCR amplification fragment of the positive bacteria should be 4646bp
  • the length of the PCR amplification fragment of the original bacteria should be 1437bp.
  • primers (pyr3-S, pyr3-A) were designed according to pyr3 and its upstream sequence (nucleotide sequence shown in SEQ ID NO.1, position (5671-9492)), and PCR amplification of the pyr3 fragment was performed; using the E. coli MG1655 genome as a template, according to the downstream homology arm primers of its yghX gene (DN-yghX-S3, DN-yghX-A), PCR amplification of its downstream homology arm fragment.
  • DN-yghX-S3, DN-yghX-A downstream homology arm primers of its yghX gene
  • the above fragments were fused by overlapping PCR to obtain the integrated fragment of the pyr3 gene (pyr3-downstream homology arm), and the DNA fragment containing the target sequence of the target gene was obtained by annealing primers gRNA-S2 and gRNA-A2.
  • the recombinant pGRB-pyr3 was obtained after recombination with the linearized pGRB vector.
  • the recombinant fragment and plasmid pGRB-pyr3 were electroporated into competent cells of E. coli psu1-2 containing the pREDCas9 plasmid, and the plasmid was eliminated after screening the positive strain to obtain the strain E. coli psu1-3.
  • the electrophoresis diagram of the construction of the pyr3 integration fragment and the PCR verification of the positive strain is shown in Figure 2C.
  • the length of the pyr3 gene fragment and its upstream homologous arm is 3885bp
  • the length of the downstream homologous arm is 622bp
  • the total length of the integration fragment is 4468bp.
  • the length of the PCR amplification fragment of the positive bacteria should be 4468bp
  • the length of the PCR amplification fragment of the original bacteria should be 1426bp.
  • upstream homologous arm primers UP-ygaY-S, UP-ygaY-A
  • downstream homologous arm primers DN-ygaY-S, DN-ygaY-A
  • primers yeiN-S, yeiN-A
  • primers were designed according to its yeiN gene (NCBI-GeneID: 946699) to amplify the yeiN gene fragment
  • the promoter P trc was designed in the downstream primer of the upstream homologous arm and the upstream primer of the yeiN gene
  • the terminator T trc was designed in the upstream primer of the downstream homologous arm and the downstream primer of the yeiN gene.
  • the above fragments were fused by overlapping PCR to obtain the integration fragment of the yeiN gene (upstream homology arm-P trc -yeiN-T trc -downstream homology arm), and the DNA fragment containing the target sequence of the target gene was obtained by annealing primers gRNA-ygaY-S and gRNA-ygaY-A, and recombined with the linearized pGRB vector to obtain the recombinant pGRB-ygaY.
  • the recombinant fragment and plasmid pGRB-ygaY were electrotransferred to the competent cells of E.
  • the electrophoresis diagram of the construction of the yeiN integration fragment and the PCR verification of the positive strain is shown in Figure 3.
  • the length of the upstream homology arm is 629bp
  • the length of the yeiN gene fragment is 1063bp
  • the length of the downstream homology arm is 549bp
  • the total length of the integration fragment is 2152bp.
  • the length of the PCR amplification fragment for positive bacteria should be 1642bp
  • the length of the PCR amplification fragment for original bacteria should be 2083bp.
  • upstream homologous arm primers (UP-mbhA-S, UP-mbhA-A) and downstream homologous arm primers (DN-mbhA-S, DN-mbhA-A) were designed according to the upstream and downstream sequences of its mbhA gene, and PCR was used to amplify the upstream and downstream homologous arm fragments; primers (rbsK-S, rbsK-A) were designed according to its rbsK gene (NCBI-GeneID: 948260) to amplify the rbsK gene fragment, the promoter P trc was designed in the downstream primer of the upstream homologous arm and the upstream primer of the rbsK gene, and the terminator T trc was designed in the upstream primer of the downstream homologous arm and the downstream primer of the rbsK gene.
  • the above fragments were fused by overlapping PCR to obtain the integrated fragment of the rbsK gene (upstream homology arm-P trc -rbsK-T trc -downstream homology arm), and the DNA fragment containing the target sequence of the target gene was obtained by annealing primers gRNA-mbhA-S and gRNA-mbhA-A, which was recombined with the linearized pGRB vector to obtain the recombinant pGRB-mbhA.
  • the recombinant fragment and plasmid pGRB-mbhA were electroporated into competent cells of E.coli psu2 containing the pREDCas9 plasmid, and the positive strains were screened and then the plasmid was eliminated to obtain the strain E.coli psu3.
  • the electrophoresis diagram of the construction of the rbsK integrated fragment and the PCR verification of the positive strains is shown in Figure 4. Among them, the length of the upstream homology arm is 692bp, the length of the rbsK gene fragment is 1053bp, the length of the downstream homology arm is 749bp, and the total length of the integrated fragment is 2413bp. During PCR verification, the length of the PCR amplified fragment of the positive bacteria should be 2413bp, the length of the PCR amplified fragment of the original bacteria should be 1837bp.
  • upstream homologous arm primers UP-yjit-S, UP-yjit-A
  • downstream homologous arm primers DN-yjit-S, DN-yjit-A
  • primers rihB-S, rihB-A
  • rihB gene NCBI-GeneID: 946646
  • the above fragments were fused by overlapping PCR to obtain the integration fragment of the rihB gene (upstream homology arm-P trc -rihB-T trc -downstream homology arm), and the DNA fragment containing the target sequence of the target gene was obtained by annealing primers gRNA-yjit-S and gRNA-yjit-A, and the recombinant pGRB-yjit was obtained after recombination with the linearized pGRB vector.
  • the recombinant fragment and plasmid pGRB-yjit were electrotransferred to the competent cells of E.coli psu3 containing the pREDCas9 plasmid, and the plasmid was eliminated after screening the positive strain to obtain the strain E.coli psu4.
  • the electrophoresis diagram of the construction of the rihB integration fragment and the PCR verification of the positive strain is shown in Figure 5.
  • the length of the upstream homology arm is 380bp
  • the length of the rihB gene fragment is 1065bp
  • the length of the downstream homology arm is 532bp
  • the total length of the integration fragment is 1889bp.
  • the length of the PCR amplification fragment for positive bacteria should be 1396bp
  • the length of the PCR amplification fragment for original bacteria should be 1873bp.
  • upstream homologous arm primers (UP-ycgh-S, UP-ycgh-A) and downstream homologous arm primers (DN-ycgh-S, DN-ycgh-A) were designed according to the upstream and downstream sequences of its ycgh gene, and PCR was used to amplify the upstream and downstream homologous arm fragments; primers (uraA-S, uraA-A) were designed according to its uraA gene (NCBI-GeneID: 946978) to amplify the uraA gene fragment, the promoter P trc was designed in the downstream primer of the upstream homologous arm and the upstream primer of the uraA gene, and the terminator T trc was designed in the upstream primer of the downstream homologous arm and the downstream primer of the uraA gene.
  • the above fragments were fused by overlapping PCR to obtain the integration fragment of the uraA gene (upstream homology arm-P trc -uraA-T trc -downstream homology arm), and the DNA fragment containing the target sequence of the target gene was obtained by annealing primers gRNA-ycgh-S and gRNA-ycgh-A, and the recombinant pGRB-ycgh was obtained after recombination with the linearized pGRB vector.
  • the recombinant fragment and plasmid pGRB-ycgh were electrotransferred to the competent cells of E.
  • the electrophoresis diagram of the construction of the uraA integration fragment and the PCR verification of the positive strain is shown in Figure 6.
  • the length of the upstream homology arm is 580bp
  • the length of the uraA gene fragment is 1413bp
  • the length of the downstream homology arm is 630bp
  • the total length of the integration fragment is 2535bp.
  • the length of the PCR amplification fragment for positive bacteria should be 2535bp
  • the length of the PCR amplification fragment for original bacteria should be 1409bp.
  • upstream homology arm primers UP-psuT-S, UP-psuT-A
  • downstream homology arm primers DN-psuT-S, DN-psuT-A
  • upstream homology arm primers UP-psuT-S, UP-psuT-A
  • DN-psuT-S, DN-psuT-A downstream homology arm primers
  • the DNA fragment containing the target sequence of the target gene was obtained by annealing primers gRNA-psuT-S and gRNA-psuT-A, and recombined with the linearized pGRB vector to obtain the recombinant pGRB-psuT.
  • the recombinant fragment and plasmid pGRB-psuT were electroporated into the competent cells of E. coli psu5 containing the pREDCas9 plasmid, and the positive strains were screened and the plasmid was eliminated to obtain the strain E. coli psu6.
  • the electrophoresis diagram of the construction of the psuT knockout fragment and the PCR verification of the positive strain is shown in Figure 7.
  • the length of the upstream homology arm is 303bp
  • the length of the downstream homology arm is 513bp
  • the total length of the overlapping fragment is 775bp.
  • the length of the PCR amplification fragment of the positive bacteria should be 775bp
  • the length of the PCR amplification fragment of the original bacteria should be 1970bp.
  • upstream homology arm primers UP-yeiI-S, UP-yeiI-A
  • downstream homology arm primers DN-yeiI-S, DN-yeiI-A
  • NCBI-GeneID 946640
  • PCR amplification of its upstream and downstream homology arm fragments was performed.
  • the above fragments were fused by overlapping PCR to obtain the knockout fragment of the yeiI gene (upstream homology arm-downstream homology arm).
  • the primers gRNA-yeiI-S and gRNA-yeiI-A were annealed to obtain the knockout fragment containing
  • the DNA fragment of the target sequence of the target gene was recombined with the linearized pGRB vector to obtain the recombinant pGRB-yeiI.
  • the recombinant fragment and plasmid pGRB-yeiI were electrotransferred to the competent cells of E.coli psu6 containing the pREDCas9 plasmid, and the plasmid was eliminated after screening the positive strain to obtain the strain E.coli psu7.
  • the electrophoresis diagram of the construction of the yeiI knockout fragment and the PCR verification of the positive strain is shown in Figure 8.
  • the length of the upstream homology arm is 617bp
  • the length of the downstream homology arm is 314bp
  • the total length of the overlapping fragment is 885bp.
  • the length of the PCR amplification fragment of the positive bacteria should be 885bp
  • the length of the PCR amplification fragment of the original bacteria should be 2372bp.
  • upstream homology arm primers UP-yeiC-S, UP-yeiC-A
  • downstream homology arm primers DN-yeiC-S, DN-yeiC-A
  • upstream homology arm primers UP-yeiC-S, UP-yeiC-A
  • DN-yeiC-S, DN-yeiC-A downstream homology arm primers
  • the DNA fragment containing the target sequence of the target gene was obtained by annealing the primers gRNA-yeiC-S and gRNA-yeiC-A, and the recombinant pGRB-yeiC was obtained after recombination with the linearized pGRB vector.
  • the recombinant fragment and plasmid pGRB-yeiC were electroporated into the competent cells of E. coli psu7 containing the pREDCas9 plasmid, and the positive strains were screened and the plasmid was eliminated to obtain the strain E. coli psu8.
  • the electrophoresis diagram of the construction of the psuT knockout fragment and the PCR verification of the positive strain is shown in Figure 9.
  • the length of the upstream homology arm is 734 bp
  • the length of the downstream homology arm is 860 bp
  • the total length of the overlapping fragment is 1547 bp.
  • the length of the PCR amplification fragment of the positive bacteria should be 1547 bp
  • the length of the PCR amplification fragment of the original bacteria should be 2408 bp.
  • the shake flask culture method is as follows:
  • Slant activation culture Take the -80°C stored strain and inoculate it on the activated slant, culture it at 37°C for 12h, and then subculture it once;
  • Seed culture Use an inoculation loop to scrape a ring of slant seeds and inoculate it into a 500mL Erlenmeyer flask containing 30mL of seed culture medium, seal it with nine layers of gauze, and culture it at 37°C, 200rmp for 7-10h;
  • Fermentation culture inoculate 10-15% of the volume of the seed culture solution into a 500mL conical flask containing fermentation medium (final volume is 30ml), seal with nine layers of gauze, and culture at 37°C, 200r/min with shaking.
  • the pH is maintained at 7.0-7.2 by adding ammonia water; 60% (m/v) glucose solution is added to maintain the fermentation; the fermentation period is 36h.
  • Slant culture medium glucose 1-5g/L, peptone 5-10g/L, beef extract 5-10g/L, yeast powder 1-5g/L, sodium chloride 1-2.5g/L, agar 25-30g/L, and the rest is water, pH 7.0-7.2.
  • Seed culture medium glucose 20-25g/L, yeast powder 4-8g/L, KH 2 PO 4 1-2g/L, MgSO 4 ⁇ 7H 2 O 0.1-0.5g/L, FeSO 4 ⁇ 7H 2 O 2-5mg/L, MnSO 4 ⁇ 7H 2 O 2-5mg/L, VB1 , VB3 , VB5 , VB12 , VBH each 1-2mg/L, the rest is water, pH 7.0-7.2.
  • Fermentation medium glucose 20-25g/L, yeast powder 4-8g/L, KH2PO4 2-4g / L, MgSO4 ⁇ 7H2O 0.1-0.5g/ L , FeSO4 ⁇ 7H2O 20-30mg/L, MnSO4 ⁇ 7H2O 10-20mg/L, VB1 , VB3 , VB5 , VB12 , VBH each 2-4mg/L, the rest is water , pH 7.0-7.2 .
  • the fermentation tank culture method is as follows:
  • Slant activation culture Take the -80°C stored strain and inoculate it on the activated slant, culture it at 37°C for 12h, transfer it to the eggplant-shaped bottle and continue to culture it for 12-16h;
  • Seed culture Take an appropriate amount of sterile water in an eggplant-shaped bottle, inoculate the bacterial suspension into the seed culture medium, stabilize the pH at around 7.0, keep the temperature constant at 37°C, and keep the dissolved oxygen between 25-35%, and culture until the cell dry weight reaches 5-6g/L;
  • Fermentation culture add fresh fermentation medium at an inoculation rate of 15-20% and start fermentation. During the fermentation process, control the pH to be stable at about 7.0, the temperature to be maintained at 37°C, and the dissolved oxygen to be between 25-35%. When the glucose in the medium is consumed, add 80% (m/v) glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-2 g/L.
  • Slant culture medium glucose 1-5g/L, peptone 5-10g/L, beef extract 5-10g/L, yeast powder 1-5g/L, sodium chloride 1-2.5g/L, agar 25-30g/L, and the rest is water, pH 7.0-7.2.
  • Seed culture medium glucose 20-25g/L, yeast powder 4-8g/L, peptone 1-5g/L, KH2PO4 1-4g /L, MgSO4 ⁇ 7H2O 1.2-2.0g/L, FeSO4 ⁇ 7H2O 20-30mg/L, MnSO4 ⁇ 7H2O 10-20mg/L, VB1 , VB3 , VB5 , VB12 , VBH each 2-4mg/L, the rest is water , pH 7.0-7.2 .
  • Fermentation medium glucose 20-25g/L, yeast powder 4-8g/L, peptone 5-10g/L, sodium citrate 1-5g/ L , KH2PO4 2-4g /L, MgSO4 ⁇ 7H2O 1.2-2.0g /L, FeSO4 ⁇ 7H2O 20-30mg/L, MnSO4 ⁇ 7H2O 10-20mg/L, VB1, VB3, VB5 , VB12 , VBH each 2-4mg / L , the rest is water, pH 7.0-7.2.
  • the highest yield of pseudouridine in the prior art is obtained by a plasmid-carrying engineered bacterium, with a yield of 7.2 g/L and a fermentation cycle of 72-96 h (ZL202011637153.9).
  • the pseudouridine genetic engineering bacteria and fermentation method of the present invention have a pseudouridine yield of up to 20 g/L and a fermentation cycle of 48 h, which are superior to the yield and fermentation cycle of pseudouridine in the prior art.
  • Bacillus subtilis (B. subtilis A260)

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Abstract

提供了一种生产假尿苷的基因工程菌株及其在发酵生产假尿苷中的应用。该菌株异源过表达嘧啶核苷操纵子，过表达假尿苷酸合成酶，过表达核糖激酶，过表达核糖核苷水解酶，过表达尿嘧啶渗透酶，不表达假尿苷转运蛋白和假尿苷激酶。该菌株能够提高假尿苷的产量。

Description

一种生产假尿苷的基因工程菌株及其构建方法与应用 技术领域
本发明属于基因工程技术领域，涉及工业微生物的育种，尤其是一种生产假尿苷的基因工程菌株及其构建方法与应用。
背景技术
假尿苷又称5-B-D-核呋喃基尿嘧啶，分子式为C9H12N2O6，分子量为244.201。假尿苷是一种嘧啶核苷，是尿苷的5位核糖异构体，核糖不与尿嘧啶N1相连，而与嘧啶环的C5连接。假尿苷是RNA上最丰富的修饰核苷，又被称为RNA的“第五种核苷”，在mRNA新冠疫苗中具有重要的应用价值。将假尿苷及其类似物替换尿苷加入mRNA，可以解决mRNA药物容易被免疫系统识别而被清除、产生免疫副反应的问题。同时，假尿苷作为一种RNA的代谢产物，具有仅从肾脏排泄的特点，在临床医学上可用于监测肿瘤的发生、发展及肾病的诊治等。假尿苷具有十分重要的应用价值，随着假尿苷市场需求量的不断扩大，迫切需要开发廉价的可大规模应用的假尿苷生产工艺。
假尿苷的生产方法有化学合成法、化学酶法、微生物发酵法。而目前，关于假尿苷的制备方面的报道颇少。李静简等人以2，3，5-三苄氧基-D-核糖酸-1，4-内酯为原料与2，4-二烷氧基-5-溴嘧啶进行加成反应，而后依次与三乙基硅烷、三氟化硼乙醚进行还原反应，最后用三氯化硼进行脱保护反应得到假尿苷(ZL202210798651.4)；金峰等人以D-核糖和尿嘧啶为起始反应物，在有机溶剂中加入乙酰溴与D-核糖反应以及在有机溶剂中使用强路易斯碱后加入(Boc)2O与尿嘧啶反应，两者发生缩合反应后使用三氟乙酸脱去保护基，而后加入乙酸酐进行手性拆分，最后脱去保护基得到假尿苷(ZL202210473532.1)。虽说假尿苷的生产方法主要依赖于化学方法合成，但是化学合成过程中存在合成步骤长、生产成本高、收率低等问题，并且使用易燃易爆等危险试剂。肖聪等人发明了一种化学酶法来合成假尿苷，使用5’-假尿苷单磷酸糖苷酶突变体催化5-磷酸核糖与尿嘧啶合成5’-假尿苷单磷酸，再经化学去磷酸化、分离后得到假尿苷(ZL202111581013.9)。但是该方法也存在原料成本高、生产工艺复杂、转化率低等缺点。与上述方法相比，微生物发酵法具有环境友好、成本低廉、工艺简单、生产可持续等优势。陈伟等人筛选得到了一种链霉菌，可以通过有氧发酵产生1.36g/L的假尿苷(ZA202011421953.7)。进一步，将来自于链霉菌Streptomyces sp.ID38640中的假尿苷合成基因pumH、pumJ和pumD基因克隆至大肠杆菌体内，转化得到可产7.2g/L假尿苷的工程菌(ZL202011637153.9)。但是该工程菌携带质粒、生长负担重、产量较低且发酵周期长。
利用代谢工程和合成生物技术选育核苷类产品生产菌株是近年来研究的热点。本发明从大肠杆菌基因组水平出发，主要通过代谢工程技术手段，对尿苷酸合成途径、假尿苷合成途径、转运系统及假尿苷分支代谢途径各模块进行系统全面的组合优化，提高菌株的假尿苷发酵性能。
通过检索，尚未发现与本发明专利申请相关的专利公开文献。
发明内容
本发明的目的在于克服现有技术上存在的问题，提供一种生产假尿苷的基因工程菌株及其构建方法与应用。
本发明解决技术问题所采用的技术方案是：
一种生产假尿苷的基因工程菌株，所述基因工程菌异源过表达嘧啶核苷操纵子，过表达假尿苷酸合成酶，过表达核糖激酶，过表达核糖核苷水解酶，过表达尿嘧啶渗透酶，不表达假尿苷转运蛋白和假尿苷激酶。
进一步地，所述嘧啶核苷操纵子为枯草芽孢杆菌pyrBCAKDFE基因编码的操纵子，核苷酸序列为SEQ ID NO.1；
所述假尿苷酸合成酶为大肠杆菌yeiN基因(NCBI-GeneID:946699)编码的蛋白或海栖热袍菌TM1464基因(NCBI-GeneID:896983)编码的蛋白或根癌农杆菌AWN88_14600基因(NCBI-GeneID:29364903)编码的蛋白；
所述核糖激酶为大肠杆菌rbsK基因(NCBI-GeneID:948260)编码的蛋白或枯草芽孢杆菌rbsK基因(NCBI-GeneID:936844)编码的蛋白；
所述核糖核苷水解酶为大肠杆菌rihA基因(NCBI-GeneID:945503)或大肠杆菌rihB基因(NCBI-GeneID:946646)或大肠杆菌rihC基因(NCBI-GeneID:944796)编码的蛋白；
所述尿嘧啶渗透酶为大肠杆菌uraA基因(NCBI-GeneID:946978)编码的蛋白；
所述假尿苷转运蛋白为大肠杆菌psuT基因(NCBI-GeneID:946671)编码的蛋白；
所述假尿苷激酶为大肠杆菌yeiI基因(NCBI-GeneID:946640)和大肠杆菌yeiC基因(NCBI-GeneID:946664)编码的蛋白。
进一步地，所述嘧啶核苷操纵子的基因连接有启动子Ptrc；和/或
所述假尿苷酸合成酶的基因连接有启动子Ptrc；和/或
所述核糖激酶的基因连接有启动子Ptrc；和/或
所述核糖核苷水解酶的基因连接有启动子Ptrc；和/或
所述尿嘧啶渗透酶的基因连接有启动子Ptrc；
其中，所述启动子Ptrc的核苷酸序列如SEQ ID NO.2所示。
进一步地，所述基因工程菌株在构建时使用的出发菌株为大肠杆菌。
进一步地，所述大肠杆菌为E.coli MG1655。
如上所述的基因工程菌株的构建方法，所述方法包括如下步骤：
在出发菌株中过表达枯草芽孢杆菌来源的嘧啶核苷操纵子基因pyrBCAKDFE，过表达大肠杆菌内源的假尿苷酸合成酶基因yeiN或海栖热袍菌的假尿苷酸合成酶基因TM1464或根癌农杆菌的假尿苷酸合成酶基因AWN88_14600，过表达大肠杆菌内源的核糖激酶基因rbsK或枯草芽孢杆菌的核糖激酶基因rbsK，过表达大肠杆菌内源的核糖核苷水解酶基因rihA或rihB或rihC，过表达大肠杆菌内源的尿嘧啶渗透酶基因uraA，敲除或失活假尿苷转运蛋白基因psuT，敲除或失活假尿苷激酶基因yeiI和yeiC。所述基因工程菌过表达嘧啶核苷操纵子，过表达假尿苷酸合成酶，过表达核糖激酶，过表达核糖核苷水解酶，过表达尿嘧啶渗透酶，不表达假尿苷转运蛋白和假尿苷激酶。
进一步地，所述方法包括如下步骤：
在出发菌株中过表达枯草芽孢杆菌来源的嘧啶核苷操纵子基因pyrBCAKDFE，过表达大肠杆菌内源的假尿苷酸合成酶基因yeiN，过表达大肠杆菌内源的核糖激酶基因rbsK，过表达大肠杆菌内源的核糖核苷水解酶基因rihB，过表达大肠杆菌内源的尿嘧啶渗透酶基因uraA，敲除或失活假尿苷转运蛋白基因psuT，敲除或失活假尿苷激酶基因yeiI和yeiC。
进一步地，所述方法包括如下步骤：
(1)从大肠杆菌E.coli MG1655基因组出发，将核苷酸序列如SEQ ID NO.1所示的嘧啶核苷操纵子pyrBCAKDFE与启动子Ptrc的融合片段Ptrc-pyrBCAKDFE整合在yghX假基因位点；
(2)将大肠杆菌内源的假尿苷酸合成酶基因yeiN与启动子Ptrc的融合片段Ptrc-yeiN整合在ygay假基因位点；
(3)将大肠杆菌内源的核糖激酶基因rbsK与启动子Ptrc的融合片段Ptrc-rbsK整合在mbhA假基因位点；
(4)将大肠杆菌内源的核糖核苷水解酶基因rihB与启动子Ptrc的融合片段Ptrc-rihB整合在yjiT假基因位点；
(5)将大肠杆菌内源的尿嘧啶渗透酶基因uraA与启动子Ptrc的融合片段Ptrc-uraA整合在ycgh假基因位点；
(6)将假尿苷转运蛋白基因psuT敲除或失活；
(7)将假尿苷激酶基因yeiI和yeiC敲除或失活。
如上述的基因工程菌株在发酵生产假尿苷中的应用。
利用如上所述的基因工程菌株在发酵生产假尿苷的方法，所述方法包括如下步骤：
在适宜条件培养所述基因工程菌株，并从其培养物中收集假尿苷，得到假尿苷；
其中，所述适宜条件是指培养温度37℃，维持pH在7.0±0.5，溶氧在25％-35％之间，并且培养基组成为：
葡萄糖20-25g/L，酵母粉4-8g/L，蛋白胨5-10g/L，柠檬酸钠1-5g/L，KH2PO4 2-4g/L，MgSO4·7H2O 1.2-2.0g/L，FeSO4·7H2O 20-30mg/L，MnSO4·7H2O 10-20mg/L，VB1、VB3、VB5、VB12、VH各2-4mg/L，其余为水，pH 7.0-7.2。
本发明取得的有益效果是：
1、本发明基因工程菌株过表达嘧啶核苷操纵子，过表达假尿苷酸合成酶，过表达核糖激酶，过表达核糖核苷水解酶，过表达尿嘧啶渗透酶，不表达假尿苷转运蛋白和假尿苷激酶。本发明从大肠杆菌基因组水平出发，主要通过代谢工程技术手段，对尿苷酸合成途径、假尿苷合成途径、转运系统及假尿苷分支代谢途径各模块进行系统全面的组合优化，提高菌株的假尿苷发酵性能。本基因工程菌株的假尿苷产量高、生产性能稳定、能用于假尿苷生产。
2、本发明利用理性代谢工程改造的方法获得了具有清晰遗传背景、不含质粒、以葡萄糖等廉价碳源为底物并且从头高效合成假尿苷的工程菌株。目前现有技术中假尿苷的产量最高的是通过一株携带质粒的工程菌获得的，产量为7.2g/L，发酵周期为72-96h(ZL202011637153.9)。从生产表型来看，本发明所述菌株在5L罐上发酵48h后假尿苷产量达到了20g/L，发酵产量是现有报道的最高水平，且发酵周期与现有报道相比明显缩短。
3、从构建策略来看，本发明通过代谢工程技术手段，分模块对尿苷酸合成途径、假尿苷合成途径、转运系统及假尿苷分支代谢途径各模块进行了系统全面的组合优化，整体的代谢改造策略(见图1)未见相关报道。由于所有操作均在基因组上完成，最终所得工程菌株可更加稳定高效生产假尿苷，具有良好的工业化应用前景。
附图说明
图1为本发明中假尿苷工程菌株的代谢改造策略图；
图2为本发明中嘧啶核苷操纵子pyrBCAKDFE分段整合片段构建及验证电泳图；包括：
图2A为本发明中pyr1整合片段构建及验证电泳图；其中：M:1kb DNA Marker；1:上游同源臂；2:pyr1片段；3:下游同源臂；4:重叠片段；5:原菌PCR片段；6:阳性单菌落PCR鉴定片段；
图2B为本发明中pyr2整合片段构建及验证电泳图；其中：M:1kb DNA Marker；1:pyr2上游片段-pyr2片段；2:下游同源臂；3:重叠片段；4:原菌PCR片段；5:阳性单菌落PCR鉴定片段；
图2C为本发明中pyr3整合片段构建及验证电泳图；其中：M:1kb DNA Marker；1:pyr3上游片段-pyr3片段；2:下游同源臂；3:重叠片段；4:原菌PCR片段；5:阳性单菌落PCR鉴定片段；
图3为本发明中yeiN整合片段构建及验证电泳图；其中：M:1kb DNA Marker；1:上游同源臂；2:目的基因；3:下游同源臂；4:重叠片段；5:原菌PCR片段；6:阳性单菌落PCR鉴定片段；
图4为本发明中rbsK整合片段构建及验证电泳图；其中：M:1kb DNA Marker；1:上游同源臂；2:目的基因；3:下游同源臂；4:重叠片段；5:原菌PCR片段；6:阳性单菌落 PCR鉴定片段；
图5为本发明中rihB整合片段构建及验证电泳图；其中：M:1kb DNA Marker；1:上游同源臂；2:目的基因；3:下游同源臂；4:重叠片段；5:原菌PCR片段；6:阳性单菌落PCR鉴定片段；
图6为本发明中uraA整合片段构建及验证电泳图；其中：M:1kb DNA Marker；1:上游同源臂；2:目的基因；3:下游同源臂；4:重叠片段；5:原菌PCR片段；6:阳性单菌落PCR鉴定片段；
图7为本发明中psuT基因敲除片段构建及验证电泳图；其中：M:1kb DNA Marker；1:上游同源臂；2:下游同源臂；3:重叠片段；4:原菌PCR片段；5:阳性单菌落PCR鉴定片段；
图8为本发明中yeiI基因敲除片段构建及验证电泳图；其中：M:1kb DNA Marker；1:上游同源臂；2:下游同源臂；3:重叠片段；4:原菌PCR片段；5:阳性单菌落PCR鉴定片段；
图9为本发明中yeiC基因敲除片段构建及验证电泳图；其中：M:1kb DNA Marker；1:上游同源臂；2:下游同源臂；3:重叠片段；4:原菌PCR片段；5:阳性单菌落PCR鉴定片段；
图10为本发明中实施例2的摇瓶发酵结果图；
图11为本发明中实施例3的5L发酵罐发酵过程曲线图；
图12为本发明中假尿苷标品的高效液相色谱图，其中4.3min中是假尿苷的峰；
图13为本发明中发酵液的高效液相色谱图，其中4.3min中是假尿苷的峰。
具体实施方式
为更好理解本发明，下面结合实施例对本发明做进一步地详细说明，但是本发明要求保护的范围并不局限于实施例所表示的范围。
本发明中所使用的的原料，如无特殊说明，均为常规市售产品，本发明中所使用的方法，如无特殊说明，均为本领域常规方法，本发明所使用的各物质质量均为常规使用质量。
一种生产假尿苷的基因工程菌株，所述基因工程菌异源过表达嘧啶核苷操纵子，过表达假尿苷酸合成酶，过表达核糖激酶，过表达核糖核苷水解酶，过表达尿嘧啶渗透酶，不表达假尿苷转运蛋白和假尿苷激酶。
较优地，所述嘧啶核苷操纵子为枯草芽孢杆菌pyrBCAKDFE基因编码的操纵子，核苷酸序列为SEQ ID NO.1；
所述假尿苷酸合成酶为大肠杆菌yeiN基因(NCBI-GeneID:946699)编码的蛋白或海栖热袍菌TM1464基因(NCBI-GeneID:896983)编码的蛋白或根癌农杆菌AWN88_14600基因(NCBI-GeneID:29364903)编码的蛋白；
所述核糖激酶为大肠杆菌rbsK基因(NCBI-GeneID:948260)编码的蛋白或枯草芽孢杆菌rbsK基因(NCBI-GeneID:936844)编码的蛋白；
所述核糖核苷水解酶为大肠杆菌rihA基因(NCBI-GeneID:945503)或大肠杆菌rihB基因(NCBI-GeneID:946646)或大肠杆菌rihC基因(NCBI-GeneID:944796)编码的蛋白；
所述尿嘧啶渗透酶为大肠杆菌uraA基因(NCBI-GeneID:946978)编码的蛋白；
所述假尿苷转运蛋白为大肠杆菌psuT基因(NCBI-GeneID:946671)编码的蛋白；
所述假尿苷激酶为大肠杆菌yeiI基因(NCBI-GeneID:946640)和大肠杆菌yeiC基因(NCBI-GeneID:946664)编码的蛋白。
较优地，所述嘧啶核苷操纵子的基因连接有启动子Ptrc；和/或
所述假尿苷酸合成酶的基因连接有启动子Ptrc；和/或
所述核糖激酶的基因连接有启动子Ptrc；和/或
所述核糖核苷水解酶的基因连接有启动子Ptrc；和/或
所述尿嘧啶渗透酶的基因连接有启动子Ptrc；
其中，所述启动子Ptrc的核苷酸序列如SEQ ID NO.2所示。
较优地，所述基因工程菌株在构建时使用的出发菌株为大肠杆菌。
较优地，所述大肠杆菌为E.coli MG1655。
如上所述的基因工程菌株的构建方法，所述方法包括如下步骤：
在出发菌株中过表达枯草芽孢杆菌来源的嘧啶核苷操纵子基因pyrBCAKDFE，过表达大肠杆菌内源的假尿苷酸合成酶基因yeiN或海栖热袍菌的假尿苷酸合成酶基因TM1464或根癌农杆菌的假尿苷酸合成酶基因AWN88_14600，过表达大肠杆菌内源的核糖激酶基因rbsK或枯草芽孢杆菌的核糖激酶基因rbsK，过表达大肠杆菌内源的核糖核苷水解酶基因rihA或rihB或rihC，过表达大肠杆菌内源的尿嘧啶渗透酶基因uraA，敲除或失活假尿苷转运蛋白基因psuT，敲除或失活假尿苷激酶基因yeiI和yeiC。所述基因工程菌过表达嘧啶核苷操纵子，过表达假尿苷酸合成酶，过表达核糖激酶，过表达核糖核苷水解酶，过表达尿嘧啶渗透酶，不表达假尿苷转运蛋白和假尿苷激酶。
较优地，所述方法包括如下步骤：
在出发菌株中过表达枯草芽孢杆菌来源的嘧啶核苷操纵子基因pyrBCAKDFE，过表达大肠杆菌内源的假尿苷酸合成酶基因yeiN，过表达大肠杆菌内源的核糖激酶基因rbsK，过表达大肠杆菌内源的核糖核苷水解酶基因rihB，过表达大肠杆菌内源的尿嘧啶渗透酶基因uraA，敲除或失活假尿苷转运蛋白基因psuT，敲除或失活假尿苷激酶基因yeiI和yeiC。
较优地，所述方法包括如下步骤：
(1)从大肠杆菌E.coli MG1655基因组出发，将核苷酸序列如SEQ ID NO.1所示的嘧啶核苷操纵子pyrBCAKDFE与启动子Ptrc的融合片段Ptrc-pyrBCAKDFE整合在yghX假基因位点；
(2)将大肠杆菌内源的假尿苷酸合成酶基因yeiN与启动子Ptrc的融合片段Ptrc-yeiN整合在ygay假基因位点；
(3)将大肠杆菌内源的核糖激酶基因rbsK与启动子Ptrc的融合片段Ptrc-rbsK整合在mbhA假基因位点；
(4)将大肠杆菌内源的核糖核苷水解酶基因rihB与启动子Ptrc的融合片段Ptrc-rihB整合在yjiT假基因位点；
(5)将大肠杆菌内源的尿嘧啶渗透酶基因uraA与启动子Ptrc的融合片段Ptrc-uraA整合在ycgh假基因位点；
(6)将假尿苷转运蛋白基因psuT敲除或失活；
(7)将假尿苷激酶基因yeiI和yeiC敲除或失活。
如上述的基因工程菌株在发酵生产假尿苷中的应用。
利用如上所述的基因工程菌株在发酵生产假尿苷的方法，所述方法包括如下步骤：
在适宜条件培养所述基因工程菌株，并从其培养物中收集假尿苷，得到假尿苷；
其中，所述适宜条件是指培养温度37℃，维持pH在7.0±0.5，溶氧在25％-35％之间，并且培养基组成为：
葡萄糖20-25g/L，酵母粉4-8g/L，蛋白胨5-10g/L，柠檬酸钠1-5g/L，KH2PO4 2-4g/L，MgSO4·7H2O 1.2-2.0g/L，FeSO4·7H2O 20-30mg/L，MnSO4·7H2O 10-20mg/L，VB1、VB3、VB5、VB12、VH各2-4mg/L，其余为水，pH 7.0-7.2。
具体地，相关的制备及检测如下：
一种大肠杆菌基因工程菌株，该基因工程菌异源过表达嘧啶核苷操纵子，过表达假尿苷酸合成酶，过表达核糖激酶，过表达核糖核苷水解酶，过表达尿嘧啶渗透酶，不表达假尿苷转运蛋白和假尿苷激酶。
优选的，所述嘧啶核苷操纵子基因pyrBCAKDFE的核苷酸序列如SEQ ID NO.1所示。
优选的，所述假尿苷酸合成酶基因yeiN的NCBI-GeneID:946699。
优选的，所述核糖激酶基因rbsK的NCBI-GeneID:948260。
优选的，所述核糖核苷水解酶基因rihB的NCBI-GeneID:946646。
优选的，所述尿嘧啶渗透酶基因uraA的NCBI-GeneID:946978。
优选的，所述假尿苷转运蛋白基因psuT的NCBI-GeneID:946671。
优选的，所述假尿苷激酶基因yeiI的NCBI-GeneID:946640。
优选的，所述假尿苷激酶基因yeiC的NCBI-GeneID:946664。
根据本发明，不表达上述基因的方式可以采用本领域常规手段，例如，通过本领域常规手段使该基因失活或者将该基因敲除。
根据本发明，所述不表达是指该基因表达产物的量显著低于原有水平，例如，显著降低了50％、60％、70％、80％、90％、100％。
根据本发明，过表达上述基因的方式可以采取本领域常规手段，例如，增加基因的拷贝数或者使该基因连接强启动子。
根据本发明，所述过表达是指该基因表达产物的量显著高于原有水平。
根据本发明一种优选的实施方式，所述嘧啶核苷操纵子基因pyrBCAKDFE连接有启动子Ptrc；和/或所述假尿苷酸合成酶基因yeiN连接有启动子Ptrc；和/或所述核糖激酶基因rbsK连接有启动子Ptrc；和/或核糖核苷水解酶基因rihB连接有启动子Ptrc；和/或尿嘧啶渗透酶基因uraA连接有启动子Ptrc；优选的，所述启动子的核苷酸序列如SEQ ID NO.2所示。
根据本发明，用于构建所述大肠杆菌基因工程菌株的出发菌株可以是任意的大肠杆菌，根据本发明一种优选的实施方式，所述出发菌株是E.coli MG1655。
如上所述的大肠杆菌基因工程菌株的构建方法，包括:在出发菌株大肠杆菌中过表达枯草芽孢杆菌来源的嘧啶核苷操纵子基因pyrBCAKDFE，过表达大肠杆菌内源的假尿苷酸合成酶基因yeiN，过表达大肠杆菌内源的核糖激酶基因rbsK，过表达大肠杆菌内源的核糖核苷水解酶基因rihB，过表达大肠杆菌内源的尿嘧啶渗透酶基因uraA，敲除或失活假尿苷转运蛋白基因psuT，敲除或失活假尿苷激酶基因yeiI和yeiC。
上面已经详细介绍了上述各基因的选择、启动子的选择、出发菌株的选择等，此处不再赘述。
一种具体的实施方式，该方法包括：
(1)增强尿苷酸合成途径
从菌株E.coli MG1655基因组出发，将B.subtilis A260的嘧啶核苷操纵子pyrBCAKDFE与启动子Ptrc的融合片段Ptrc-pyrBCAKDFE整合在yghX假基因位点；该步骤强化了嘧啶核苷从头合成通量，解除了尿苷酸对氨甲酰磷酸合成酶的反馈抑制；
(2)增强假尿苷合成途径
将大肠杆菌内源的假尿苷酸合成酶基因yeiN与启动子Ptrc的融合片段Ptrc-yeiN整合在ygay假基因位点，强化5-磷酸核糖与尿嘧啶向5’-假尿苷单磷酸的转化；将大肠杆菌内源的核糖激酶基因rbsK与启动子Ptrc的融合片段Ptrc-rbsK整合在mbhA假基因位点，强化核糖到5-磷酸核糖的转化；将大肠杆菌内源的核糖核苷水解酶基因rihB与启动子Ptrc的融合片段Ptrc-rihB整合在yjiT假基因位点，强化尿苷到尿嘧啶的转化；5’-假尿苷单磷酸通过胞内的核苷酸酶的作用生成假尿苷，该步骤促进了假尿苷的合成和分泌；
(3)修饰转运系统
将大肠杆菌内源的尿嘧啶渗透酶基因uraA与启动子Ptrc的融合片段Ptrc-uraA整合在ycgh假基因位点，强化尿嘧啶向胞内的转运；敲除假尿苷转运蛋白基因psuT，减弱了假尿苷向胞内的转运。
(4)减弱假尿苷分支代谢途径
敲除假尿苷激酶基因yeiI和yeiC，减弱了假尿苷向5’-假尿苷单磷酸的转化。
本发明上述构建过程的原理可参考图1所示。
如上所述的大肠杆菌基因工程菌株在高产假尿苷中的应用，包括：在适宜条件培养所述基因工程菌株，并从其培养物中收集假尿苷。
一种优选的实施方式，所述适宜条件是指培养温度37℃，维持pH在7.0左右，溶氧在25％-35％之间，并且培养基组成为：葡萄糖20-25g/L，酵母粉4-8g/L，蛋白胨5-10g/L，柠檬酸钠1-5g/L，KH2PO4 2-4g/L，MgSO4·7H2O 1.2-2.0g/L，FeSO4·7H2O 20-30mg/L，MnSO4·7H2O 10-20mg/L，VB1、VB3、VB5、VB12、VH各2-4mg/L，其余为水，pH7.0-7.2。
以下将通过具体的实施例对本发明进行更详细描述。以下实施例中：
如无特别说明，本发明实施例中所涉及的基因编辑方法参照文献(Li Y,Lin Z,Huang C,et al.Metabolic engineering of Escherichia coli using CRISPR-Cas9meditated genome editing.Metabolic engineering,2015,31:13-21.)进行，其他所涉及的分子生物学、基因工程等具体操作手段根据本领域人员容易获得的技术手册、教科书或文献报道均可以实现。
实施例1：假尿苷工程菌株E.coli psu8的构建
1基因编辑方法
本发明中采用CRISPR/Cas9介导的基因编辑方法，可参照文献(Metabolic Engineering,2015,31:13-21.)进行。CRISPR/Cas9是一种精确、高效的新型基因靶向修饰技术，该方法所用的两种质粒分别为pGRB与pREDCas9。pREDCas9质粒为温敏型质粒，携带gRNA质粒的消除系统，λ噬菌体的Red重组系统及Cas9蛋白表达系统，具有奇霉素抗性(工作浓度：100mg/L)，适培温度32℃；pGRB质粒，以pUC18为骨架，包含启动子J23100，gRNA-Cas9结合区域序列和终止子序列，具有氨苄青霉素抗性(工作浓度：100mg/L)，适培温度37℃。
2增强尿苷酸合成途径
将枯草芽孢杆菌(B.subtilis A260)的嘧啶核苷操纵子pyrBCAKDFE(包含pyrB、pyrC、pyrAA、pyrAB、pyrK、pyrD、pyrF、pyrE八个基因)共9492bp，在本实施例中分pyr1、pyr2、pyr3三段依次整合至大肠杆菌yghX基因位点，并由启动子Ptrc启动该外源操纵子的转录表达，构建了菌株E.coli psu1-3。具体包括：
2.1 Ptrc-pyr1的整合
以E.coli MG1655基因组为模板，根据其yghX基因的上下游序列设计上游同源臂引物(UP-yghX-S、UP-yghX-A)和下游同源臂引物(DN-yghX-S1、DN-yghX-A)，PCR扩增其上下游同源臂片段；以B.subtilis A260基因组为模板，根据pyr1(SEQ ID NO.1所示核苷酸序列的第1-3260位)设计引物(pyr1-S、pyr1-A)，PCR扩增pyr1基因片段，启动子Ptrc则设计在上游同源臂的下游引物和pyr1基因的上游引物中。上述片段通过重叠PCR的方法融合获得pyr1基因的整合片段(上游同源臂-Ptrc-pyr1-下游同源臂)，通过引物gRNA-yghX-S和gRNA-yghX-A的退火制得含目的基因靶序列的DNA片段，与线性化的pGRB载体重组后获得重组的pGRB-yghX。将该重组片段和质粒pGRB-yghX电转至含有pREDCas9质粒的MG1655的感受态细胞，筛选阳性菌株后再将质粒消除获得菌株E.coli psu1-1。Ptrc-pyr1整合片段的构建和阳性菌株的PCR验证的电泳图见附图2A。其中，上游同源臂的长度为658bp，pyr1基因片段长度为3363bp，下游同源臂的长度为604bp，整合片段的总长为4560bp。PCR验证时，阳性菌PCR扩增片段长度应为4560bp，原菌PCR扩增片段长度应为1765bp。
2.2 Ptrc-pyr2的整合
以B.subtilis A260基因组为模板，根据pyr2及其上游序列(SEQ ID NO.1所示核苷酸序列的第2448-6469位)设计引物(pyr2-S、pyr2-A)，PCR扩增pyr2片段；以E.coli MG1655基因组为模板，根据其yghX基因的下游同源臂引物(DN-yghX-S2、DN-yghX-A)，PCR扩增其下游同源臂片段。上述片段通过重叠PCR的方法融合获得pyr2基因的整 合片段(pyr2-下游同源臂)，通过引物gRNA-S1和gRNA-A1的退火制得含目的基因靶序列的DNA片段，与线性化的pGRB载体重组后获得重组的pGRB-pyr2。将该重组片段和质粒pGRB-pyr2电转至含有pREDCas9质粒的E.coli psu1-1的感受态细胞，筛选阳性菌株后再将质粒消除获得菌株E.coli psu1-2。pyr2整合片段的构建和阳性菌株的PCR验证的电泳图见附图2B。其中pyr2基因片段及其上游同源臂的长度为4065bp，下游同源臂的长度为604bp，整合片段的总长为4646bp。PCR验证时，阳性菌PCR扩增片段长度应为4646bp，原菌PCR扩增片段长度应为1437bp。
2.3 Ptrc-pyr3的整合
以B.subtilis A260基因组为模板，根据pyr3及其上游序列(SEQ ID NO.1所示核苷酸序列的第(5671-9492位)设计引物(pyr3-S、pyr3-A)，PCR扩增pyr3片段；以E.coli MG1655基因组为模板，根据其yghX基因的下游同源臂引物(DN-yghX-S3、DN-yghX-A),PCR扩增其下游同源臂片段。上述片段通过重叠PCR的方法融合获得pyr3基因的整合片段(pyr3-下游同源臂)，通过引物gRNA-S2和gRNA-A2的退火制得含目的基因靶序列的DNA片段，与线性化的pGRB载体重组后获得重组的pGRB-pyr3。将该重组片段和质粒pGRB-pyr3电转至含有pREDCas9质粒的E.coli psu1-2的感受态细胞，筛选阳性菌株后再将质粒消除获得菌株E.coli psu1-3。pyr3整合片段的构建和阳性菌株的PCR验证的电泳图见附图2C。其中pyr3基因片段及其上游同源臂的长度为3885bp，下游同源臂的长度为622bp，整合片段的总长为4468bp。PCR验证时，阳性菌PCR扩增片段长度应为4468bp，原菌PCR扩增片段长度应为1426bp。
3增强假尿苷合成途径
3.1 yeiN基因的整合
以E.coli MG1655基因组为模板，根据其ygaY基因的上下游序列设计上游同源臂引物(UP-ygaY-S、UP-ygaY-A)和下游同源臂引物(DN-ygaY-S、DN-ygaY-A)，PCR扩增其上下游同源臂片段；根据其yeiN基因(NCBI-GeneID:946699)设计引物(yeiN-S、yeiN-A)，扩增yeiN基因片段，启动子Ptrc则设计在上游同源臂的下游引物和yeiN基因的上游引物中，终止子Ttrc则设计在下游同源臂的上游引物和yeiN基因的下游引物中。上述片段通过重叠PCR的方法融合获得yeiN基因的整合片段(上游同源臂-Ptrc-yeiN-Ttrc-下游同源臂)，通过引物gRNA-ygaY-S和gRNA-ygaY-A的退火制得含目的基因靶序列的DNA片段，与线性化的pGRB载体重组后获得重组的pGRB-ygaY。将该重组片段和质粒pGRB-ygaY电转至含有pREDCas9质粒的E.coli psu1-3的感受态细胞，筛选阳性菌株后再将质粒消除获得菌株E.coli psu2。yeiN整合片段的构建和阳性菌株的PCR验证的电泳图见附图3。其中，上游同源臂的长度为629bp，yeiN基因片段长度为1063bp，下游同源臂的长度为549bp，整合片段的总长为2152bp。PCR验证时，阳性菌PCR扩增片段长度应为1642bp，原菌PCR扩增片段长度应为2083bp。
3.2 rbsK基因的整合
以E.coli MG1655基因组为模板，根据其mbhA基因的上下游序列设计上游同源臂引物(UP-mbhA-S、UP-mbhA-A)和下游同源臂引物(DN-mbhA-S、DN-mbhA-A)，PCR扩增其上下游同源臂片段；根据其rbsK基因(NCBI-GeneID:948260)设计引物(rbsK-S、rbsK-A)，扩增rbsK基因片段，启动子Ptrc则设计在上游同源臂的下游引物和rbsK基因的上游引物中，终止子Ttrc则设计在下游同源臂的上游引物和rbsK基因的下游引物中。上述片段通过重叠PCR的方法融合获得rbsK基因的整合片段(上游同源臂-Ptrc-rbsK-Ttrc-下游同源臂)，通过引物gRNA-mbhA-S和gRNA-mbhA-A的退火制得含目的基因靶序列的DNA片段，与线性化的pGRB载体重组后获得重组的pGRB-mbhA。将该重组片段和质粒pGRB-mbhA电转至含有pREDCas9质粒的E.coli psu2的感受态细胞，筛选阳性菌株后再将质粒消除获得菌株E.coli psu3。rbsK整合片段的构建和阳性菌株的PCR验证的电泳图见附图4。其中，上游同源臂的长度为692bp，rbsK基因片段长度为1053bp，下游同源臂的长度为749bp，整合片段的总长为2413bp。PCR验证时，阳性菌PCR扩增片段长度应为 2413bp，原菌PCR扩增片段长度应为1837bp。
3.3 rihB基因的整合
以E.coli MG1655基因组为模板，根据其yjit基因的上下游序列设计上游同源臂引物(UP-yjit-S、UP-yjit-A)和下游同源臂引物(DN-yjit-S、DN-yjit-A)，PCR扩增其上下游同源臂片段；根据其rihB基因(NCBI-GeneID:946646)设计引物(rihB-S、rihB-A)，扩增rihB基因片段，启动子Ptrc则设计在上游同源臂的下游引物和rihB基因的上游引物中，终止子Ttrc则设计在下游同源臂的上游引物和rihB基因的下游引物中。上述片段通过重叠PCR的方法融合获得rihB基因的整合片段(上游同源臂-Ptrc-rihB-Ttrc-下游同源臂)，通过引物gRNA-yjit-S和gRNA-yjit-A的退火制得含目的基因靶序列的DNA片段，与线性化的pGRB载体重组后获得重组的pGRB-yjit。将该重组片段和质粒pGRB-yjit电转至含有pREDCas9质粒的E.coli psu3的感受态细胞，筛选阳性菌株后再将质粒消除获得菌株E.coli psu4。rihB整合片段的构建和阳性菌株的PCR验证的电泳图见附图5。其中，上游同源臂的长度为380bp，rihB基因片段长度为1065bp，下游同源臂的长度为532bp，整合片段的总长为1889bp。PCR验证时，阳性菌PCR扩增片段长度应为1396bp，原菌PCR扩增片段长度应为1873bp。
4修饰转运系统
4.1 uraA基因的整合
以E.coli MG1655基因组为模板，根据其ycgh基因的上下游序列设计上游同源臂引物(UP-ycgh-S、UP-ycgh-A)和下游同源臂引物(DN-ycgh-S、DN-ycgh-A)，PCR扩增其上下游同源臂片段；根据其uraA基因(NCBI-GeneID:946978)设计引物(uraA-S、uraA-A)，扩增uraA基因片段，启动子Ptrc则设计在上游同源臂的下游引物和uraA基因的上游引物中，终止子Ttrc则设计在下游同源臂的上游引物和uraA基因的下游引物中。上述片段通过重叠PCR的方法融合获得uraA基因的整合片段(上游同源臂-Ptrc-uraA-Ttrc-下游同源臂)，通过引物gRNA-ycgh-S和gRNA-ycgh-A的退火制得含目的基因靶序列的DNA片段，与线性化的pGRB载体重组后获得重组的pGRB-ycgh。将该重组片段和质粒pGRB-ycgh电转至含有pREDCas9质粒的E.coli psu4的感受态细胞，筛选阳性菌株后再将质粒消除获得菌株E.coli psu5。uraA整合片段的构建和阳性菌株的PCR验证的电泳图见附图6。其中，上游同源臂的长度为580bp，uraA基因片段长度为1413bp，下游同源臂的长度为630bp，整合片段的总长为2535bp。PCR验证时，阳性菌PCR扩增片段长度应为2535bp，原菌PCR扩增片段长度应为1409bp。
4.2 psuT基因的敲除
以E.coli MG1655基因组为模板，根据其psuT基因(NCBI-GeneID:946671)的上下游序列设计上游同源臂引物(UP-psuT-S、UP-psuT-A)和下游同源臂引物(DN-psuT-S、DN-psuT-A),PCR扩增其上下游同源臂片段。上述片段通过重叠PCR的方法融合获得psuT基因的敲除片段(上游同源臂-下游同源臂)。通过引物gRNA-psuT-S和gRNA-psuT-A的退火制得含目的基因靶序列的DNA片段，与线性化的pGRB载体重组后获得重组的pGRB-psuT。将该重组片段和质粒pGRB-psuT电转至含有pREDCas9质粒的E.coli psu5的感受态细胞，筛选阳性菌株后再将质粒消除获得菌株E.coli psu6。psuT敲除片段的构建和阳性菌株的PCR验证的电泳图见附图7。其中，上游同源臂的长度为303bp，下游同源臂的长度为513bp，重叠片段的总长为775bp。PCR验证时，阳性菌PCR扩增片段长度应为775bp，原菌PCR扩增片段长度应为1970bp。
5减弱假尿苷分支代谢途径
5.1 yeiI基因的敲除
以E.coli MG1655基因组为模板，根据其yeiI基因(NCBI-GeneID:946640)的上下游序列设计上游同源臂引物(UP-yeiI-S、UP-yeiI-A)和下游同源臂引物(DN-yeiI-S、DN-yeiI-A),PCR扩增其上下游同源臂片段。上述片段通过重叠PCR的方法融合获得yeiI基因的敲除片段(上游同源臂-下游同源臂)。通过引物gRNA-yeiI-S和gRNA-yeiI-A的退火制得含 目的基因靶序列的DNA片段，与线性化的pGRB载体重组后获得重组的pGRB-yeiI。将该重组片段和质粒pGRB-yeiI电转至含有pREDCas9质粒的E.coli psu6的感受态细胞，筛选阳性菌株后再将质粒消除获得菌株E.coli psu7。yeiI敲除片段的构建和阳性菌株的PCR验证的电泳图见附图8。其中，上游同源臂的长度为617bp，下游同源臂的长度为314bp，重叠片段的总长为885bp。PCR验证时，阳性菌PCR扩增片段长度应为885bp，原菌PCR扩增片段长度应为2372bp。
5.2 yeiC基因的敲除
以E.coli MG1655基因组为模板，根据其yeiC基因(NCBI-GeneID:946664)的上下游序列设计上游同源臂引物(UP-yeiC-S、UP-yeiC-A)和下游同源臂引物(DN-yeiC-S、DN-yeiC-A),PCR扩增其上下游同源臂片段。上述片段通过重叠PCR的方法融合获得yeiC基因的敲除片段(上游同源臂-下游同源臂)。通过引物gRNA-yeiC-S和gRNA-yeiC-A的退火制得含目的基因靶序列的DNA片段，与线性化的pGRB载体重组后获得重组的pGRB-yeiC。将该重组片段和质粒pGRB-yeiC电转至含有pREDCas9质粒的E.coli psu7的感受态细胞，筛选阳性菌株后再将质粒消除获得菌株E.coli psu8。psuT敲除片段的构建和阳性菌株的PCR验证的电泳图见附图9。其中，上游同源臂的长度为734bp，下游同源臂的长度为860bp，重叠片段的总长为1547bp。PCR验证时，阳性菌PCR扩增片段长度应为1547bp，原菌PCR扩增片段长度应为2408bp。
6菌株构建过程中用到的引物
上述构建过程所涉及的引物见下表：



实施例2：假尿苷工程菌株摇瓶发酵实验
摇瓶培养方法如下：
斜面活化培养：取-80℃保藏菌种划线接种于活化斜面，37℃培养12h，再传代一次；
种子培养：用接种环刮取一环斜面种子接种于装有30mL种子培养基的500mL三角瓶中，九层纱布封口，37℃，200rmp培养7-10h；
发酵培养：按种子培养液体积10-15％的接种量接种到装有发酵培养基的500mL三角瓶中(终体积为30ml)，九层纱布封口，37℃，200r/min振荡培养，发酵过程中通过补加氨水维持pH在7.0-7.2；补加60％(m/v)葡萄糖溶液维持发酵进行；发酵周期36h。
斜面培养基：葡萄糖1-5g/L，蛋白胨5-10g/L，牛肉膏5-10g/L，酵母粉1-5g/L，氯化钠1-2.5g/L，琼脂25-30g/L，其余为水，pH 7.0-7.2。
种子培养基：葡萄糖20-25g/L，酵母粉4-8g/L，KH2PO4 1-2g/L，MgSO4·7H2O 0.1-0.5g/L，FeSO4·7H2O 2-5mg/L，MnSO4·7H2O 2-5mg/L，VB1、VB3、VB5、VB12、VH各1-2mg/L，其余为水，pH 7.0-7.2。
发酵培养基：葡萄糖20-25g/L，酵母粉4-8g/L，KH2PO4 2-4g/L，MgSO4·7H2O 0.1-0.5g/L，FeSO4·7H2O 20-30mg/L，MnSO4·7H2O 10-20mg/L，VB1、VB3、VB5、VB12、VH各2-4mg/L，其余为水，pH 7.0-7.2。
摇瓶发酵结果：如图10所示，对菌株psu4、psu5、psu6、psu7、psu8进行摇瓶发酵，结果显示五个菌株的OD600值均无明显变化，但假尿苷产率均得到不同程度的提升。菌株psu8的假尿苷产率最高，达到4g/L。
实施例3：假尿苷工程菌株5L罐发酵实验
发酵罐培养方法如下：
斜面活化培养：取-80℃保藏菌种划线接种于活化斜面，37℃培养12h，转接茄形瓶继续培养12-16h；
种子培养：取适量无菌水于茄形瓶中，将菌悬液接入种子培养基中，pH稳定在7.0左右，温度恒定在37℃，溶氧在25-35％之间，培养至细胞干重达到5-6g/L；
发酵培养：按照15-20％接种量接入新鲜的发酵培养基，开始发酵，发酵过程中控制pH稳定在7.0左右，温度维持在37℃，溶氧在25-35％之间；当培养基中的葡萄糖消耗完之后，流加80％(m/v)的葡萄糖溶液，维持发酵培养基中的葡萄糖浓度在0.1-2g/L。
斜面培养基：葡萄糖1-5g/L，蛋白胨5-10g/L，牛肉膏5-10g/L，酵母粉1-5g/L，氯化钠1-2.5g/L，琼脂25-30g/L，其余为水，pH 7.0-7.2。
种子培养基：葡萄糖20-25g/L，酵母粉4-8g/L，蛋白胨1-5g/L，KH2PO4 1-4g/L，MgSO4·7H2O 1.2-2.0g/L，FeSO4·7H2O 20-30mg/L，MnSO4·7H2O 10-20mg/L，VB1、VB3、VB5、VB12、VH各2-4mg/L，其余为水，pH 7.0-7.2。
发酵培养基：葡萄糖20-25g/L，酵母粉4-8g/L，蛋白胨5-10g/L，柠檬酸钠1-5g/L，KH2PO4 2-4g/L，MgSO4·7H2O 1.2-2.0g/L，FeSO4·7H2O 20-30mg/L，MnSO4·7H2O 10-20mg/L，VB1、VB3、VB5、VB12、VH各2-4mg/L，其余为水，pH 7.0-7.2。
5L发酵罐分批补料发酵结果：如图11所示，发酵初期菌株生长迅速，OD600值达到最高后逐渐降低，48h的假尿苷产量达到20g/L。假尿苷标品的液相检测结果如图12所示，发酵液的液相检测结果如图13所示。
目前现有技术中假尿苷的产量最高的是通过一株携带质粒的工程菌获得的，产量为7.2g/L，发酵周期为72-96h(ZL202011637153.9)。本发明假尿苷的基因工程菌及其发酵方法的假尿苷产量高达20g/L，发酵周期为48h，均优于现有技术中的假尿苷的产量和发酵周期。
相关序列如下：
1
9495
DNA
枯草芽孢杆菌(B.subtilis A260)
1



2
74
DNA
人工序列
2
尽管为说明目的公开了本发明的实施例，但是本领域的技术人员可以理解：在不脱离本发明及所附权利要求的精神和范围内，各种替换、变化和修改都是可能的，因此，本发明的范围不局限于实施例所公开的内容。

Claims (10)

  1. 一种生产假尿苷的基因工程菌株，其特征在于：所述基因工程菌异源过表达嘧啶核苷操纵子，过表达假尿苷酸合成酶，过表达核糖激酶，过表达核糖核苷水解酶，过表达尿嘧啶渗透酶，不表达假尿苷转运蛋白和假尿苷激酶。
  2. 根据权利要求1所述的基因工程菌，其特征在于：所述嘧啶核苷操纵子为枯草芽孢杆菌pyrBCAKDFE基因编码的操纵子，核苷酸序列为SEQ ID NO.1；
    所述假尿苷酸合成酶为大肠杆菌yeiN基因(NCBI-GeneID:946699)编码的蛋白或海栖热袍菌TM1464基因(NCBI-GeneID:896983)编码的蛋白或根癌农杆菌AWN88_14600基因(NCBI-GeneID:29364903)编码的蛋白；
    所述核糖激酶为大肠杆菌rbsK基因(NCBI-GeneID:948260)编码的蛋白或枯草芽孢杆菌rbsK基因(NCBI-GeneID:936844)编码的蛋白；
    所述核糖核苷水解酶为大肠杆菌rihA基因(NCBI-GeneID:945503)或大肠杆菌rihB基因(NCBI-GeneID:946646)或大肠杆菌rihC基因(NCBI-GeneID:944796)编码的蛋白；
    所述尿嘧啶渗透酶为大肠杆菌uraA基因(NCBI-GeneID:946978)编码的蛋白；
    所述假尿苷转运蛋白为大肠杆菌psuT基因(NCBI-GeneID:946671)编码的蛋白；
    所述假尿苷激酶为大肠杆菌yeiI基因(NCBI-GeneID:946640)和大肠杆菌yeiC基因(NCBI-GeneID:946664)编码的蛋白。
  3. 根据权利要求1或2所述的基因工程菌株，其特征在于：所述嘧啶核苷操纵子的基因连接有启动子Ptrc；和/或
    所述假尿苷酸合成酶的基因连接有启动子Ptrc；和/或
    所述核糖激酶的基因连接有启动子Ptrc；和/或
    所述核糖核苷水解酶的基因连接有启动子Ptrc；和/或
    所述尿嘧啶渗透酶的基因连接有启动子Ptrc；
    其中，所述启动子Ptrc的核苷酸序列如SEQ ID NO.2所示。
  4. 根据权利要求1或2所述的基因工程菌株，其特征在于：所述基因工程菌株在构建时使用的出发菌株为大肠杆菌。
  5. 根据权利要求4所述的基因工程菌株，其特征在于：所述大肠杆菌为E.coli MG1655。
  6. 如权利要求1至5任一所述的基因工程菌株的构建方法，其特征在于：所述方法包括如下步骤：
    在出发菌株中过表达枯草芽孢杆菌来源的嘧啶核苷操纵子基因pyrBCAKDFE，过表达大肠杆菌内源的假尿苷酸合成酶基因yeiN或海栖热袍菌的假尿苷酸合成酶基因TM1464或根癌农杆菌的假尿苷酸合成酶基因AWN88_14600，过表达大肠杆菌内源的核糖激酶基因rbsK或枯草芽孢杆菌的核糖激酶基因rbsK，过表达大肠杆菌内源的核糖核苷水解酶基因rihA或rihB或rihC，过表达大肠杆菌内源的尿嘧啶渗透酶基因uraA，敲除或失活假尿苷转运蛋白基因psuT，敲除或失活假尿苷激酶基因yeiI和yeiC。所述基因工程菌过表达嘧啶核苷操纵子，过表达假尿苷酸合成酶，过表达核糖激酶，过表达核糖核苷水解酶，过表达尿嘧啶渗透酶，不表达假尿苷转运蛋白和假尿苷激酶。
  7. 根据权利要求6所述的构建方法，其特征在于：所述方法包括如下步骤：
    在出发菌株中过表达枯草芽孢杆菌来源的嘧啶核苷操纵子基因pyrBCAKDFE，过表达大肠杆菌内源的假尿苷酸合成酶基因yeiN，过表达大肠杆菌内源的核糖激酶基因rbsK，过表达大肠杆菌内源的核糖核苷水解酶基因rihB，过表达大肠杆菌内源的尿嘧啶渗透酶基因uraA，敲除或失活假尿苷转运蛋白基因psuT，敲除或失活假尿苷激酶基因yeiI和yeiC。
  8. 根据权利要求7所述的构建方法，其特征在于：所述方法包括如下步骤：
    (1)从大肠杆菌E.coli MG1655基因组出发，将核苷酸序列如SEQ ID NO.1所示的嘧啶核苷操纵子pyrBCAKDFE与启动子Ptrc的融合片段Ptrc-pyrBCAKDFE整合在yghX假基因位点；
    (2)将大肠杆菌内源的假尿苷酸合成酶基因yeiN与启动子Ptrc的融合片段Ptrc-yeiN整合在ygay假基因位点；
    (3)将大肠杆菌内源的核糖激酶基因rbsK与启动子Ptrc的融合片段Ptrc-rbsK整合在mbhA假基因位点；
    (4)将大肠杆菌内源的核糖核苷水解酶基因rihB与启动子Ptrc的融合片段Ptrc-rihB整合在yjiT假基因位点；
    (5)将大肠杆菌内源的尿嘧啶渗透酶基因uraA与启动子Ptrc的融合片段Ptrc-uraA整合在ycgh假基因位点；
    (6)将假尿苷转运蛋白基因psuT敲除或失活；
    (7)将假尿苷激酶基因yeiI和yeiC敲除或失活。
  9. 如权利要求1至5任一所述的基因工程菌株在发酵生产假尿苷中的应用。
  10. 利用如权利要求1至5任一所述的基因工程菌株在发酵生产假尿苷的方法，其特征在于：所述方法包括如下步骤：
    在适宜条件培养所述基因工程菌株，并从其培养物中收集假尿苷，得到假尿苷；
    其中，所述适宜条件是指培养温度37℃，维持pH在7.0±0.5，溶氧在25％-35％之间，并且培养基组成为：
    葡萄糖20-25g/L，酵母粉4-8g/L，蛋白胨5-10g/L，柠檬酸钠1-5g/L，KH2PO42-4g/L，MgSO4·7H2O 1.2-2.0g/L，FeSO4·7H2O 20-30mg/L，MnSO4·7H2O 10-20mg/L，VB1、VB3、VB5、VB12、VH各2-4mg/L，其余为水，pH 7.0-7.2。
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