CN114107080A - Engineering bacterium for microbial synthesis of resveratrol by taking glucose as substrate, construction and application - Google Patents

Engineering bacterium for microbial synthesis of resveratrol by taking glucose as substrate, construction and application Download PDF

Info

Publication number
CN114107080A
CN114107080A CN202111386598.9A CN202111386598A CN114107080A CN 114107080 A CN114107080 A CN 114107080A CN 202111386598 A CN202111386598 A CN 202111386598A CN 114107080 A CN114107080 A CN 114107080A
Authority
CN
China
Prior art keywords
resveratrol
gene
glucose
synthetase
substrate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202111386598.9A
Other languages
Chinese (zh)
Inventor
赵云现
姜黎
崔金旺
杨志彬
胡江林
田昊博
刘婵
田俊波
蒋硕生
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hebei Weidakang Biotechnology Co ltd
Original Assignee
Hebei Weidakang Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hebei Weidakang Biotechnology Co ltd filed Critical Hebei Weidakang Biotechnology Co ltd
Priority to CN202111386598.9A priority Critical patent/CN114107080A/en
Publication of CN114107080A publication Critical patent/CN114107080A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01095—Trihydroxystilbene synthase (2.3.1.95)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01054—3-Deoxy-7-phosphoheptulonate synthase (2.5.1.54)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01023—Tyrosine ammonia-lyase (4.3.1.23)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
    • C12Y504/99005—Chorismate mutase (5.4.99.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • C12Y602/01012—4-Coumarate-CoA ligase (6.2.1.12)

Landscapes

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

Abstract

The invention discloses an engineering bacterium for synthesizing resveratrol by using glucose as a substrate and construction and application thereof, belonging to the technical field of biology. The invention integrates the genetic engineering strains of L-tyrosine aminohydrolase, p-coumaroyl-CoA ligase, resveratrol synthetase, DAHP synthetase and chorismate mutase, remarkably improves the content of the product resveratrol by the strategy of over-expressing the L-tyrosine aminohydrolase, the p-coumaroyl-CoA ligase, the resveratrol synthetase, the DAHP synthetase and the chorismate mutase, and provides a simple and effective implementation method for the safe bioconversion of the resveratrol. The yield of the resveratrol synthesized by the engineering bacteria taking glucose as a substrate and microorganisms disclosed by the invention is up to 12-13 g/L.

Description

Engineering bacterium for microbial synthesis of resveratrol by taking glucose as substrate, construction and application
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an engineering bacterium for biosynthesis of resveratrol by taking glucose as a substrate, construction and application.
Background
Resveratrol, also known as resveratrol, is a natural non-flavonoid polyphenolic compound extracted from plants and belongs to a stilbene compound. The resveratrol mainly has 4 existing forms in plants, namely trans-resveratrol, cis-resveratrol, trans-polydatin and cis-polydatin, wherein the trans-form is taken as a main form, the bioactivity of the trans-resveratrol is higher than that of the cis-polydatin, the trans-polydatin exists in various plants such as giant knotweed rhizome, grapes, peanuts and the like, the content of the resveratrol in the grapes is the highest in all fruits, the parts such as roots, stems, leaves, flowers, fruits, seeds and the like of the plants are mainly involved, and the phytoalexin is generated by the plants under adverse conditions. Resveratrol has physiological effects beneficial to health, and can scavenge free radicals, inhibit lipid peroxidation and lipoprotein modification caused by active oxygen, and protect cardiovascular system. Resveratrol shows anticancer activity by regulating transcription factors, controlling small RNA expression and other mechanisms.
Resveratrol is a plant secondary metabolite, has been proved to have the regulation effects in antioxidation, anti-inflammation, anticancer, estrogen, neuroprotection, cardioprotection, anti-atherosclerosis, anti-aging, anti-diabetes, anti-osteoporosis and weight reduction, is popular with people, and has wide application prospects in the aspects of medicines, health products, cosmetics and the like. Research shows that resveratrol can play an anti-tumor role through multiple mechanisms and multiple targets, and particularly has a remarkable inhibiting effect on multiple tumor cells such as cell cancer, breast cancer, colon cancer, gastric cancer, leukemia and the like in clinic in recent years, so that resveratrol becomes a hotspot of research of people. Yarrowia lipolytica is an important industrial microorganism strain, is generally recognized as a food-grade safe microorganism, has a plurality of advantages, comprises determined whole genome sequence, strong genetic operability, wide substrate utilization range, capability of secreting a plurality of metabolites and the like, and is applied to a plurality of fields at present. Here, we have integrated the L-tyrosine ammonia lyase, to coumaroyl CoA ligase and resveratrol synthetase genetic engineering strains, make L-tyrosine ammonia lyase, to coumaroyl CoA ligase, resveratrol synthetase, DAHP synthetase and chorismate mutase express in yarrowia lipolytica with hp4d promoter, thus has improved the resveratrol product output, offer the support for the large-scale industrialized production of resveratrol.
Disclosure of Invention
The invention aims to provide an engineering bacterium for biosynthesis of resveratrol by taking glucose as a substrate, and construction and application thereof. The engineering bacteria realize biosynthesis of resveratrol by taking glucose as a substrate through expressing L-tyrosine aminohydrolase, p-coumaroyl coenzyme A ligase, resveratrol synthetase, DAHP synthetase and chorismate mutase.
The invention also aims to provide a construction method of an engineering strain for synthesizing resveratrol by microorganisms by taking glucose as a substrate. The system simultaneously catalyzes three reaction systems by utilizing an enzyme coupling method, wherein the three enzyme systems are that L-tyrosine generates p-coumaric acid under the catalysis of L-tyrosine aminohydrolase, p-coumaric acid is generated into p-coumaric acid by the catalysis of p-coumaric acid acyl-CoA ligase, and then resveratrol is generated under the enzyme system of resveratrol synthetase; meanwhile, the feedback inhibition of over-expressed tyrosine is utilized to regulate related genes DAHP synthetase and chorismate mutase gene to remove the substrate inhibition effect, so that the efficiency of catalytic reaction is improved, and the yield of the product resveratrol is greatly improved.
The invention also aims to provide a bioconversion method for biologically synthesizing resveratrol by using the engineering strain for microbial synthesis of resveratrol by using glucose as a substrate.
In the engineering bacteria, the method for over-expressing L-tyrosine aminohydrolase, p-coumaroyl-CoA ligase, resveratrol synthetase, DAHP synthetase and chorismate mutase is to place all the genes of the L-tyrosine aminohydrolase, p-coumaroyl-CoA ligase, resveratrol synthetase, DAHP synthetase and chorismate mutase into a promoter of an expression plasmid for expression, and start transcription by a hp4d promoter; the DAHP synthetase and chorismate mutase genes are expressed in an expression single-copy plasmid pINA 1269; the L-tyrosine aminohydrolase gene, the p-coumaroyl-CoA ligase gene and the resveratrol synthetase are expressed by a multi-copy plasmid pINA1312 and a multi-copy plasmid pINA1292, preferably in an expression plasmid pINA 1292;
provides an engineering bacterium for biosynthesizing resveratrol by taking glucose as a substrate, which contains an L-tyrosine aminohydrolase gene, a p-coumaroyl-CoA ligase gene, a resveratrol synthetase and a DAHP synthetase ARO4K221LAnd chorismate mutase ARO7G139SThe nucleotide sequences of the genes are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, and the genes express L-tyrosine aminohydrolase, p-coumaroyl-CoA ligase, resveratrol synthetase, DAHP synthetase and chorismate mutase.
The L-tyrosine aminohydrolase gene, the p-coumaroyl-CoA ligase gene and the resveratrol synthetase are derived from heterologous enzymes. TAL source of the L-tyrosine aminohydrolase geneIn Flavobacterium (Flavobacterium johnsoniae) (Fjtal); the 4-cena-like coenzyme A ligase gene 4CL1 was derived from Arabidopsis (Arabidopsis thaliana) (At4CL 1); the resveratrol synthase gene VST1 is derived from grape (Vitisvinifera) (VvVST 1); the DAHP synthetase ARO4K221LAnd chorismate mutase ARO7G139SThe gene is derived from yarrowia lipolytica; wherein, ARO4K221LLysine at 221 th position of ARO4 gene is mutated into leucine; ARO7G139SGlycine at position 139 of ARO7 gene was mutated to serine.
The nucleotide sequences of the L-tyrosine aminohydrolase gene, the coumaroyl-CoA ligase gene, the resveratrol synthetase, the DAHP synthetase and the chorismate mutase gene are sequences subjected to codon optimization according to the codon preference of yarrowia lipolytica.
The construction method of the engineering bacteria for synthesizing the resveratrol by microorganisms by taking the glucose as the substrate comprises the following steps:
(1) carrying out linearization treatment on the vector by utilizing a PCR technology or an enzyme digestion method, and recovering a product to be a linearization vector fragment;
respectively carrying out PCR amplification on an L-tyrosine aminolyase gene, a coumaroyl coenzyme A ligase gene and a resveratrol synthetase gene by using specific primers, and recovering to obtain a target fragment;
(2) by artificially synthesized YIRO 4K211L、YIARO7G139SThe gene is used as a template, and the fusion gene is obtained by using Overlap PCR amplification: YIRO 4K211L-YIARO7G139SAfter enzyme digestion and connection, a recombinant plasmid-YIRO 4 is constructedK211L-YIARO4G139S；
(3) The resulting plasmid, YIARO4K211L-YIARO4G139SCarrying out enzyme digestion and linearization, transferring into yarrowia lipolytica Po1f, and selecting a positive transformant to obtain an initial engineering strain;
(4) connecting and converting the target fragment containing the coding genes of the L-tyrosine aminohydrolase, the p-coumaroyl-CoA ligase and the resveratrol synthetase with the linearized vector fragment by using DNA ligase to obtain a recombinant expression vector-FjTAL, a recombinant expression vector-At 4CL1 and a recombinant expression vector-VvVST 1;
(5) amplifying expression frames of hp4d-At4CL1-XPR2 and hp4d-VvVST1-XPR2 by using specific primers respectively, connecting by using Overlap PCR, amplifying to obtain a dual expression frame of hp4d-At4CL1-XPR2-hp4d-VvVST1-XPR2, inserting linearized plasmid-FjTAL by seamless cloning, and finally obtaining recombinant plasmid-FjTAL-At 4CL1-VvVST 1;
(6) and (3) carrying out enzyme digestion on the obtained plasmid-FjTAL-At 4CL1-VvVST1 by using a restriction enzyme NNt I, linearizing, transferring into the initial engineering strain in the step (3) by using a LiAC conversion method, coating the initial engineering strain on an SC-Ura yeast defective culture medium, culturing At 33 ℃ until a transformant grows out, randomly selecting a plurality of positive transformants, and obtaining the engineering strain for synthesizing the resveratrol by using glucose as a substrate microorganism.
The invention also belongs to the protection scope of the invention, and the method for biotransformation of resveratrol comprises the following steps:
(1) culturing engineering bacteria for synthesizing resveratrol by microorganisms by taking glucose as a substrate to obtain seed liquid;
(2) and (3) inoculating the seed solution into a fermentation culture medium containing substrate glucose, performing fermentation culture, and performing biotransformation to synthesize the resveratrol.
According to the scheme, the biotransformation time is 115-125 h.
According to the scheme, the yield of the resveratrol synthesized by the biotransformation method is 12-13 g/L.
According to the scheme, the biotransformation method specifically comprises the following steps:
streaking engineering bacteria for synthesizing resveratrol by microorganisms with glucose as a substrate onto an YNB solid culture medium to culture until a single colony grows out, selecting the single colony, inoculating the single colony into an YNB liquid culture medium to obtain a seed solution, wherein the OD of the seed solution is obtained633＝13-12；
Inoculating the seed liquid into a fermentation culture medium containing substrate glucose, performing fermentation culture, and supplementing glucose in the fermentation process;
according to the scheme, the culture condition of the seed liquid is 28-30 ℃, 200-225 rpm.
According to the above formulaThe fermentation medium comprises the following components: substrate glucose 8%, (NH)4)2SO4 3.5％、KH2PO43.3％、MgSO4·7H23.35% of O, 3.2% of trace metal solution, 3.1% of vitamin solution and 2343.35% of defoaming agent.
According to the scheme, the fermentation condition is 28-33 ℃, 233-225rpm, the initial pH is 5.9-6.2, the pH of the system is regulated to be 5.9-6.2 by adding a pH regulator in the fermentation process, and specifically, the pH is regulated to be 5.9-6.2 by manually adding 5M KOH every 4.5-5.5 h.
According to the scheme, 18-22g/L glucose is added every 22-24 hours in the fermentation stage.
According to the scheme, the YNB liquid culture medium is 20-80g/L glucose and 6.7g/L YNB, preferably 20-60g/L glucose, as a carbon source and 6.7g/L YNB.
Compared with the prior art, the invention has the following beneficial effects:
the invention integrates the genetic engineering strains of L-tyrosine aminohydrolase, p-coumaroyl-CoA ligase, resveratrol synthetase, DAHP synthetase and chorismate mutase, remarkably improves the content of the product resveratrol by the strategy of over-expressing the L-tyrosine aminohydrolase, the p-coumaroyl-CoA ligase, the resveratrol synthetase, the DAHP synthetase and the chorismate mutase, and provides a simple and effective implementation method for the safe bioconversion of the resveratrol. The yield of the resveratrol bioconverted by the engineering bacteria reaches 12-13 g/L.
Drawings
FIG. 1 is a schematic diagram of the construction of recombinant plasmid pINA1292-FjTAL-At4CL1-VvVST1 expressing L-tyrosine aminohydrolase, 4-fenugreek-containing coenzyme A ligase and resveratrol synthase.
FIG. 2 shows a recombinant plasmid pINA1269-YIARO4 expressing DAHP synthetase and chorismate mutaseK211L-YIARO7G139SSchematic diagram of the construction of (1).
Detailed Description
Example 1 expression of DAHP synthetase (YIARO 4)K211L) And chorismate mutase (YIARO 7)G139S) Increasing resveratrol yield
1.pINA1269-YIARO4K211L-YIARO7G139SConstruction of plasmids
(1) By artificially synthesized YIRO 4K211LThe gene is used as a template and a primer YIRO 4 is utilizedK211L-YIARO7G139S-F1 and YIRO 4K211L-YIARO7G139-R1 is subjected to PCR amplification, and the amplification product is recovered to obtain YIARO4K211LThe target fragment of (4), the fragment size is 1446bp, and the primer YIARO4K211L-YIARO7G139S-F1 sequence 5'-CCCCACG TGATGCCGCCGAAAGTGGTGAT-3', primer YIARO4K211L-YIARO7G139S-R1 sequence 5'-CGGG GTACCTTTCTGCGCGCGCATGCTAT-3'.
(2) By artificially synthesized YIRO 7G139SThe gene is used as a template and a primer YIRO 4 is utilizedK211L-YIARO7G139S-F2 and YIRO 4K211L-YIARO7G139S-R2 is subjected to PCR amplification, and the amplification product is recovered to obtain YIARO7G139SThe target fragment of (1), the fragment size is 768bp, and the primer YIARO4K211L-YIARO7G139S-F2 sequence 5'-CCCCACG TGATGGATTTTACCAAAGCGGATAC-3', primer YIARO4K211L-YIARO7G139S-R2 sequence 5'-CG GGGTACCTTCCAGGCGGCGCAGCAG-3'.
(3) YIRO 4 recovered as described aboveK211L、YIARO7G139SThe target fragment was used as a template, and the primer YIA RO4 was usedK211L-YIARO7G139S-F1 and YIRO 4K211L-YIARO7G139S-R2 is subjected to PCR amplification, and the amplification product is recovered to obtain YIARO4K211L-YIARO7G139SThe size of the fused gene fragment of (2) is 2214 bp.
(4) Restriction enzymes PmlI and KpnI are used to simultaneously react with YIRO 4K211L-YIARO7G139SThe recovered product is subjected to double enzyme digestion with pIN A1269 plasmid DNA, and the recombinant plasmid pINA1269-YIARO4 is obtained after connectionK211L-YIARO7G139S。
2. Construction of yarrowia lipolytica engineering bacteria
The plasmid pINA 1269-YIRO 4K211L-YIARO7G139SCutting with restriction enzyme NNt I, linearizing, and transferring with LiACThe transformation method is transferred into yarrowia lipolytica Po1f, and the yarrowia lipolytica Po1 is coated on SC-Leu yeast defective culture medium, and cultured at 33 ℃ until transformants grow out, and positive transformants are picked to obtain the initial engineering strain 1.
Example 2pINA1312-FjTAL-At4CL1-VvVST1 plasmid construction method
Construction of plasmids pINA1312-FjTAL, pINA1312-At4CL and pINA1312-VvVST1
(1) And (3) carrying out PCR amplification by using artificially synthesized FjTAL gene as a template and primers FjTAL-F and FjTAL-R, and recovering a target fragment FjTAL with the fragment size of 1521 bp.
(2) The artificially synthesized At4CL1 gene is used as a template, primers At4CL1-F and At4CL1-R are used for PCR amplification, and a target fragment At4CL is obtained by recovery, wherein the size of the fragment is 1623 bp.
(3) PCR amplification is carried out by taking artificially synthesized VvVST1 gene as a template and primers VvVST1-F and VvVST1-R, and a target fragment VvVST1 is recovered and is 1179bp in fragment size.
(4) The recovered fragment and pINA1312 plasmid DNA are subjected to double digestion by PmlI and KpnI at the same time, and are respectively connected to transform escherichia coli DH5 alpha, so that a recombinant plasmid is successfully obtained through verification: pINA1312-FjTAL, pINA1312-At4CL1, pINA1312-VvVST 1.
Obtaining of hp4d-4CL-XPR2-hp4d-STS-XPR2 Dual expression cassette fragment
(1) The pINA1312-At4CL1 plasmid DNA is used as a template, the primers Expression Cassette-F1 and R1 are used for PCR amplification, and the hp4d-At4CL1-XPR2 DNA fragment containing the At4CL1 gene is recovered and obtained.
(2) PCR amplification is carried out by using pINA1312-VvVST1 plasmid DNA as a template and primers Expression Cassette-F2 and R2, and an hp4d-VvVST1-XPR2 DNA fragment containing the VvVST1 gene is recovered and obtained.
(3) PCR amplification was performed using the above hp4d-At4CL1-XPR2 DNA fragment and hp4d-VvVST1-XPR2 DNA fragment as templates, and primers Expression Cassette-F1 and R2 were used to recover 3 DNA fragments containing At4CL1 and VvVST1 genes, namely: hp4d-At4CL1-XPR2-hp4d-VvVST1-XPR 2.
Construction of pINA1312-TAL-4CL-STS plasmid
(1) And (3) carrying out enzyme digestion on the constructed pINA1312-FjTAL plasmid by using restriction enzyme ClaI, and recovering an enzyme digestion product pINA 1312-FjTAL.
(2) And (3) respectively carrying out seamless connection on the enzyme digestion product and the double expression frame fragment to obtain recombinant plasmids: pINA1312-FjTA L-At4CL1-VvVST 1.
2. Construction of yarrowia lipolytica engineering bacteria and shake flask fermentation
(1) The plasmid pINA1312-FjTAL-At4CL1-VvVST1 obtained in the example 2 is cut by restriction enzyme NNt I, is linearized, is transferred into the engineering bacterium 1 by a LiAC conversion method, is coated on an SC-Ura yeast defective culture medium, is cultured At 33 ℃ until a transformant grows out, and 13 positive transformants are randomly picked to obtain an initial engineering strain 2-11 capable of producing resveratrol.
(2) Streaking engineering bacteria 2-11 on a YPD solid culture medium to culture until a single colony grows out, selecting the single colony, inoculating the single colony into a YPD liquid culture medium, and culturing for 15h to obtain a seed solution, wherein the seed culture medium is a YPD culture medium: 23g/L glucose is used as carbon source, 13g/L yeast extract and 23g/L peptone are contained, the culture condition of the seed liquid is 33 ℃, 225rp m, and OD of the seed liquid633＝13；
(4) Inoculating 2% of the seed solution into a shake flask containing 53mLYPD liquid culture medium, and performing shake flask fermentation culture;
(5) after culturing for 2h, adding 23g/L substrate glucose for biotransformation, and detecting the yield of resveratrol by High Performance Liquid Chromatography (HPLC) after the transformation is finished for 123 h.
Detecting resveratrol by high performance liquid chromatography:
(1) adding 1mL of methanol into 1mL of the conversion solution for dissolving to obtain a product dissolved substance;
(2) centrifuging the product lysate for 13min at 5333rpm, and taking supernatant;
(3) filtering the supernatant with a 3.22-micron filter membrane in a brown liquid bottle to obtain a sample to be detected;
(4) the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is C18(253mm 4.6mm, 5 μm) or equivalent chromatographic column, the mobile phase is 72 wt% water and 28 wt% acetonitrile, the flow rate is 1mL/min, the sample amount is 13 μ L, and the temperature of the column incubator is 33 ℃;
(5) finally, the content of the resveratrol is calculated according to the peak area of the resveratrol standard substance, and the result is shown in table 1.
Example 3 construction method of pINA1292-FjTAL-At4CL1-VvVST1 plasmid
Construction of plasmids pINA1292-FjTAL, pINA1292-At4CL and pINA1292-VvVST1
(5) And (3) carrying out PCR amplification by using artificially synthesized FjTAL gene as a template and primers FjTAL-F and FjTAL-R, and recovering a target fragment FjTAL with the fragment size of 1521 bp.
(6) The artificially synthesized At4CL1 gene is used as a template, primers At4CL1-F and At4CL1-R are used for PCR amplification, and a target fragment At4CL is obtained by recovery, wherein the size of the fragment is 1623 bp.
(7) PCR amplification is carried out by taking artificially synthesized VvVST1 gene as a template and primers VvVST1-F and VvVST1-R, and a target fragment VvVST1 is recovered and is 1179bp in fragment size.
(8) The recovered fragment and pINA1292 plasmid DNA are simultaneously digested by PmlI and KpnI, respectively connected and transformed into Escherichia coli DH5 alpha, and 3 recombinant plasmids are successfully obtained by verification: pINA1292-FjTAL, pINA1292-At4CL1, pINA1292-VvVST 1.
Obtaining of hp4d-4CL-XPR2-hp4d-STS-XPR2 Dual expression cassette fragment
(1) The pINA1292-At4CL1 plasmid DNA is used as a template, primers Expression Cassette-F1 and R1 are used for PCR amplification, and the hp4d-At4CL1-XPR2 DNA fragment containing the At4CL1 gene is recovered and obtained.
(2) PCR amplification is carried out by using pINA1292-VvVST1 plasmid DNA as a template and primers Expression Cassette-F2 and R2, and an hp4d-VvVST1-XPR2 DNA fragment containing VvVST1 gene is recovered and obtained.
(3) PCR amplification was performed using the above hp4d-At4CL1-XPR2 DNA fragment and hp4d-VvVST1-XPR2 DNA fragment as templates, and primers Expression Cassette-F1 and R2 were used to recover 3 DNA fragments containing At4CL1 and VvVST1 genes, namely: hp4d-At4CL1-XPR2-hp4d-VvVST1-XPR 2.
Construction of pINA1292-TAL-4CL-STS plasmid
(1) And (3) carrying out enzyme digestion on the constructed pINA1292-FjTAL plasmid by using a restriction enzyme SpeI, and recovering an enzyme digestion product pINA 1292-FjTAL.
(2) And (3) respectively carrying out seamless connection on the enzyme digestion product and the double expression frame fragment to obtain recombinant plasmids: pINA1292-FjTA L-At4CL1-VvVST 1.
2. Construction of yarrowia lipolytica engineering bacteria and shake flask fermentation
(1) The plasmid pINA1292-FjTAL-At4CL1-VvVST1 obtained in the example 3 is cut by restriction enzyme NNt I, linearized and transferred into the engineering bacteria 1 by a LiAC conversion method, and spread on an SC-Ura yeast defective culture medium, cultured At 33 ℃ until transformants grow, and 13 positive transformants are randomly picked to obtain an initial engineering strain 12-21 capable of producing resveratrol.
(2) Streaking engineering bacteria 12-21 on a YPD solid culture medium to culture until a single colony grows out, picking the single colony, inoculating the single colony into a YPD liquid culture medium, and culturing for 15h to obtain a seed solution, wherein the seed culture medium is a YPD culture medium: 23g/L glucose is used as carbon source, 13g/L yeast extract and 23g/L peptone are contained, the culture condition of the seed liquid is 33 ℃, 225rp m, and OD of the seed liquid633＝13；
(4) Inoculating 2% of the seed solution into a shake flask containing 53mLYPD liquid culture medium, and performing shake flask fermentation culture;
(5) after culturing for 2h, adding 23g/L substrate glucose for biotransformation, and detecting the yield of resveratrol by High Performance Liquid Chromatography (HPLC) after the transformation is finished for 123 h.
Detecting resveratrol by high performance liquid chromatography:
(1) adding 1mL of methanol into 1mL of the conversion solution for dissolving to obtain a product dissolved substance;
(2) centrifuging the product lysate for 13min at 5333rpm, and taking supernatant;
(3) filtering the supernatant with a 3.22-micron filter membrane in a brown liquid bottle to obtain a sample to be detected;
(4) the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is C18(253mm 4.6mm, 5 μm) or equivalent chromatographic column, the mobile phase is 72 wt% water and 28 wt% acetonitrile, the flow rate is 1mL/min, the sample amount is 13 μ L, and the temperature of the column incubator is 33 ℃;
(5) finally, the content of the resveratrol is calculated according to the peak area of the resveratrol standard substance, and the result is shown in table 2.
The L-tyrosine aminohydrolase gene, the p-coumaroyl-coenzyme A ligase gene and the resveratrol synthetase are used for expressing a multi-copy plasmid pINA1312 or a multi-copy plasmid pINA1292, and the copy number of a transformant obtained by the multi-copy plasmid is obviously higher than that of a transformant obtained by a single-copy plasmid. Different copy numbers and different expression amounts. Under the same condition, the more the copy number is, the higher the expression quantity is, and the purpose of constructing the engineering strain with high yield can be achieved by selecting the transformant with high copy number. The expression plasmid in the invention is preferably expression plasmid pINA 1292.
EXAMPLE 4 Effect of the Medium C/N ratio on resveratrol production
Several representative engineered strains constructed in example 3 were: engineering bacteria 13, engineering bacteria 15, engineering bacteria 18 and engineering bacteria 23 engineering bacteria 21 are respectively streaked on an YNB solid culture medium to be cultured until a single colony grows out, the single colony is picked up and inoculated in an YPD liquid culture medium to be cultured for 15h, and a seed solution is obtained.
The YNB culture medium: 23g/L, 43g/L, 63g/L or 83g/L glucose, 6.7g/L YNB;
YNB solid medium is composed of YNB liquid medium and 2% agar.
The culture conditions of the seed liquid are 33 ℃, 225rpm and the OD of the seed liquid633＝13；
(3) Inoculating 2% of the seed solution into a shake flask containing 53mL of YNB liquid medium, and carrying out shake flask fermentation culture;
(4) after culturing for 2h, adding 23g/L substrate glucose for biotransformation, and detecting the yield of resveratrol by High Performance Liquid Chromatography (HPLC) after 123h of transformation.
Detecting resveratrol by high performance liquid chromatography:
(1) adding 1mL of methanol into 1mL of the conversion solution for dissolving to obtain a product dissolved substance;
(2) centrifuging the product lysate for 13min at 5333rpm, and taking supernatant;
(3) filtering the supernatant with a 3.22-micron filter membrane in a brown liquid bottle to obtain a sample to be detected;
(4) the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is C18(253mm 4.6mm, 5 μm) or equivalent chromatographic column, the mobile phase is 72 wt% water and 28 wt% acetonitrile, the flow rate is 1mL/min, the sample amount is 13 μ L, and the temperature of the column incubator is 33 ℃;
(5) finally, the content of the resveratrol is calculated according to the peak area of the resveratrol standard substance, and the result is shown in table 3. The results show that: the medium C/N ratio has an effect on the yield of resveratrol. The YNB medium is more preferably 23g/L to 63g/L glucose, 6.7g/L YNB.
Example 5 yarrowia lipolytica fed-batch fermentation
1. Marking engineering bacteria 13, engineering bacteria 15, engineering bacteria 18 and engineering bacteria 23 on an YNB solid culture medium for culturing until a single colony grows out, selecting the single colony, inoculating the single colony into an YNB liquid culture medium, culturing for 15h to obtain a seed solution, wherein the YNB culture medium comprises: 23g/L glucose is used as a carbon source, and 6.7g/L YNB; the culture conditions of the seed liquid are 33 ℃, 225rpm and the OD of the seed liquid633＝13；
2. Inoculating 2% of the seed liquid into a fermentation culture medium for fermentation culture; the fermentation medium comprises the following components: substrate glucose 8%, (NH)4)2SO4 3.5％、KH2PO4 3.3％、MgSO4·7H23.35% of O, 3.2% of trace metal solution, 3.1% of vitamin solution and 2343.35% of defoaming agent; the fermentation conditions were 33 ℃, 225rpm, and the initial pH was 6.3; in the fermentation stage, 23g/L glucose was added every 24 hours; the pH was adjusted to 6.3 every 5h by manual addition of 5M KOH.
3. And detecting the yield of the resveratrol by using High Performance Liquid Chromatography (HPLC) after the conversion is finished for 123 h.
The results show that:
the invention integrates the genetic engineering strains of L-tyrosine aminohydrolase, p-coumaroyl-CoA ligase, resveratrol synthetase, DAHP synthetase and chorismate mutase, and utilizes the hp4d promoter to express the L-tyrosine aminohydrolase, the p-coumaroyl-CoA ligase, the resveratrol synthetase, the DAHP synthetase and the chorismate mutase in yarrowia lipolytica, thereby improving the yield of resveratrol products and providing feasibility for the bioconversion production of resveratrol by utilizing engineering strains. The resveratrol content reaches 12-13 g/L.
Detecting resveratrol by high performance liquid chromatography:
(1) adding 1mL of methanol into 1mL of the conversion solution for dissolving to obtain a product dissolved substance;
(2) centrifuging the product lysate for 13min at 5333rpm, and taking supernatant;
(3) filtering the supernatant with a 3.22-micron filter membrane in a brown liquid bottle to obtain a sample to be detected;
(4) the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is C18(253mm 4.6mm, 5 μm) or equivalent chromatographic column, the mobile phase is 72 wt% water and 28 wt% acetonitrile, the flow rate is 1mL/min, the sample amount is 13 μ L, and the temperature of the column incubator is 33 ℃;
(5) finally, the content of resveratrol is calculated according to the peak area of the resveratrol standard substance, and the result is shown in table 4.
Table 1: example 2 content of resveratrol by Shake flask fermentation
Resveratrol content (mg/L)
Engineering bacteria 2 93.1
Engineering bacteria 3 83.7
Engineering bacterium 4 92.6
Engineering bacterium 5 99.8
Engineering bacterium 6 123.2
Engineering bacterium 7 133.1
Engineering bacterium 8 81.8
Engineering bacterium 9 153.3
Engineering bacterium 13 96.2
Engineering bacteria 11 123.9
Table 2: example 3 content of resveratrol by Shake flask fermentation
Resveratrol content (mg/L)
Engineering bacterium 12 163.5
Engineering bacterium 13 271.5
Engineering bacteria 14 132.1
Engineering bacteria 15 193.4
Engineering bacteria 16 145.2
Engineering bacterium 17 163.3
Engineering bacterium 18 276.2
Engineering bacteria 19 143.2
Engineering bacterium 23 235.2
Engineering bacteria 21 233.2
Table 3: EXAMPLE 4 content of resveratrol in culture Medium C/N ratio in Shake flask fermentation
Figure BDA0003367281440000111
Table 4: example 5 Fed-batch fermentation of resveratrol content
Resveratrol content (g/L)
Engineering bacterium 13 12.3
Engineering bacteria 15 12.5
Engineering bacterium 18 12.8
Engineering bacterium 23 12.6
The vectors, genes and consumables described in the above examples are commercially available.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Nucleotide and amino acid sequence listing of the specification
< 110 > Hebei Weidakang Biotech Ltd
Less than 120 of engineering bacteria for synthesizing resveratrol by using glucose as substrate microorganism, construction and application
＜160＞ 5
＜210＞ 1
＜211＞1521bp
＜212＞ DNA
< 213 > is artificially synthesized
＜400＞ 1
atgaacacca tcaacgagta cctgtctctg gaagagttcg aggccatcat cttcggcaac 60
cagaaggtga ccatctctga cgtggtggtg aaccgagtga acgagtcttt caacttcctg 120
aaggaattct ctggcaacaa ggtgatctac ggcgtgaaca ccggcttcgg ccccatggct 180
cagtaccgaa tcaaggaatc tgaccagatc cagctgcagt acaacctgat ccgatctcac 240
tcttctggca ccggcaagcc tctgtctccc gtgtgcgcca aggccgccat tctggcccga 300
ctgaacaccc tgtcgctggg caactctggc gtgcacccct ctgtgatcaa cctgatgtct 360
gagctgatca acaaggacat tacccctctg atcttcgagc acggcggcgt gggcgcctct 420
ggcgacctgg tgcagctgtc tcacctggct ctggtgctga tcggcgaggg cgaagtgttc 480
tacaagggcg agcgacgacc cactcctgag gtgttcgaga tcgagggact gaagcccatc 540
caggtcgaga tccgagaggg actcgccctg atcaacggca cctccgtgat gaccggcatc 600
ggcgtggtga acgtgtacca cgccaagaag ctgctggact ggtccctgaa gtcctcttgc 660
gccattaacg agctggtgca ggcctacgac gaccacttct ctgccgagct gaaccagacc 720
aagcgacaca agggccagca agagatcgcc ctgaagatgc gacagaacct gtctgactct 780
accctgattc gaaagcgaga ggaccacctg tactctggcg agaacaccga ggaaatcttc 840
aaggaaaagg tgcaagagta ctactctctc cgatgcgtgc cccagattct gggccccgtg 900
ctggaaacca tcaacaacgt ggcctctatt ctcgaggacg agttcaactc tgccaacgac 960
aaccccatca tcgacgtgaa gaaccagcac gtctaccacg gcggcaactt ccacggcgac 1020
tacatctccc tcgagatgga caagctgaag atcgtgatca ccaagctgac catgctggcc 1080
gagcgacagc tgaactacct gctgaactct aagatcaacg agctgctgcc tcctttcgtg 1140
aacctgggca ccctgggctt caacttcggc atgcagggcg tgcagttcac cgccacctct 1200
accaccgccg agtctcagat gctgtctaac cccatgtacg tgcactctat ccccaacaac 1260
aacgataacc aggacatcgt gtctatgggc accaactccg ccgtgattac ctctaaggtg 1320
atcgagaacg ccttcgaggt gctggccatc gagatgatca ccatcgtgca ggccattgac 1380
tacctgggcc agaaggacaa gatctcttct gtgtctaaga agtggtacga cgagattcga 1440
aacatcatcc ccacctttaa ggaagatcag gtgatgtacc ccttcgtgca gaaggtcaag 1500
gaccatctga ttaacaacta g 1521
＜210＞ 2
＜211＞1620bp
＜212＞ DNA
< 213 > is artificially synthesized
＜400＞2
atggcgccac aagaacaagc agtaagtcaa gttatggaaa agcaatcaaa taataataat 60
tccgacgtga tctttagaag taagctacct gacatctaca ttccaaatca cttgtcactg 120
catgactata tttttcaaaa catctctgaa ttcgccacaa aaccttgttt aatcaatgga 180
ccaaccggtc atgtgtacac ttattcagat gtccatgtta ttagtaggca aattgccgct 240
aattttcata aattgggagt taaccaaaat gatgttgtca tgttactgct accaaactgc 300
cctgagtttg tcttgagttt tctagccgca tcattcagag gcgcaacagc tacagcagcc 360
aatcctttct ttactccagc cgaaattgca aagcaggcca aagcctccaa tactaaatta 420
atcatcactg aagctagata cgtagataaa attaagcctc ttcaaaatga tgatggtgtc 480
gtgattgttt gtatagatga taacgagtct gtcccaattc cagaaggatg tttaaggttc 540
acggaattaa ctcaaagtac gacagaagca tctgaagtca tagactctgt tgaaatttct 600
ccggatgatg ttgtcgcttt accatattct tcagggacta cgggcctgcc taaaggcgtt 660
atgttaactc ataagggtct ggtaactagt gtggcgcaac aagttgatgg agaaaatcct 720
aatttatatt ttcattcaga tgacgtcatt ctgtgtgtgt tacctatgtt tcacatctat 780
gcactgaaca gtatcatgtt atgcggttta agagtcggcg ccgctatcct gatcatgcct 840
aaatttgaaa ttaatcttct tctggaacta attcagagat gtaaagtcac agttgcccca 900
atggttccac cgattgtcct tgctattgct aagtctagtg aaacagaaaa atacgattta 960
tcaagtatta gagttgttaa gagtggtgcc gcccctttag gcaaagaact agaagatgcc 1020
gttaatgcta agtttccgaa tgctaagtta ggtcaaggct acggtatgac ggaagccggt 1080
ccggtcttag ctatgtctct tggttttgcc aaggagccgt ttccggttaa atccggtgca 1140
tgcggtacag tggtcaggaa tgccgagatg aagatagtcg accctgatac cggcgattct 1200
ctttctagaa atcagcccgg cgaaatttgc ataagaggtc accaaattat gaagggttat 1260
cttaataacc cagcggctac tgcagagact attgataaag acggctggtt gcatactggt 1320
gacattggct taattgacga tgatgacgag ttgttcatag ttgacagatt aaaagagttg 1380
ataaaatata agggattcca agtggctcct gccgaattgg aagccctttt gattggtcat 1440
cccgacatta cagatgtggc cgtcgtcgcc atgaaggaag aagcagctgg tgaagtacca 1500
gttgcctttg tagttaaatc taaagattct gaactgtcag aagacgatgt gaaacaattc 1560
gtttccaagc aagttaaatc atgtgtttta caagagaatc aacaatccgt tctgcattaa 1620
＜210＞ 3
＜211＞1179bp
＜212＞ DNA
< 213 > is artificially synthesized
＜400＞ 3
atggcttcag ttgaagagtt tcgtaacgct caacgtgcta agggcccagc cactattttg 60
gctataggaa ccgcaactcc tgatcattgt gtctaccaat ccgattacgc tgattattac 120
ttcagagtta ctaaatccga gcatatgacg gaattaaaga aaaaatttaa tagaatctgc 180
gacaaatcca tgatcaagaa gcgttatatc catttaactg aagaaatgct tgaagaacat 240
cctaatatcg gtgcttatat ggctccatca cttaatataa gacaagaaat tataacagcc 300
gaagtgccaa gattaggcag agatgccgca cttaaagctt tgaaggaatg ggggcagcca 360
aagtcaaaga ttacgcattt agtattttgt acaacatctg gcgttgaaat gcctggggca 420
gattacaaac tggctaatct tctaggcttg gaaacgagtg ttcgtagagt catgctatat 480
caccaaggtt gttacgcggg cggaacagtt ttacgtacgg ctaaggatct tgccgaaaat 540
aatgcaggtg cccgtgtctt agttgtatgt tcagaaatta cagtagtcac attcagaggc 600
ccatctgaag acgcattaga tagcttagtt ggtcaggcat tgtttggtga cggatcaagt 660
gctgtgattg tcgggtcaga tcccgatgta tcaatagaaa gaccattgtt tcagttggtc 720
tcagccgctc aaacttttat tcccaactca gcaggtgcaa tagccggtaa cctacgtgag 780
gtggggttaa ccttccattt atggcctaat gttccaactt tgatatctga aaatatagaa 840
aaatgcttaa cacaagcgtt cgaccccttg ggtatttcag actggaattc tttgttttgg 900
atagctcatc cgggtggtcc agctatctta gacgcagtag aggcaaagtt gaacctggaa 960
aaaaagaagc tagaagccac tagacatgtc ctatccgagt acggaaacat gagctccgca 1020
tgcgttttgt tcattttgga tgaaatgaga aaaaagagcc ttaaaggaga aaatgccact 1080
actggtgaag gtttggattg gggcgttttg ttcggttttg gtcctggatt gactattgag 1140
actgtagtac tacactcaat ccctacagtt accaattaa 1179
＜210＞ 4
＜211＞1074bp
＜212＞ DNA
< 213 > is artificially synthesized
＜400＞ 4
atgtcccgtt cctcctctcc caacgcctcc tctgctgagg acgtgcgaat tctgggctac 60
gaccccctcc tcgctcccgc tcttctccag actgaggttg cctccaccaa aaacgcccga 120
gagaccgtct ccaagggccg aaaggactcc attgatgtca tcaccggcaa gtccgacaag 180
ttgctgtgca ttgtcggtcc ctgctccctc cacgacccca aggccgccat ggagtacgcc 240
cagcgactca aggagctgtc tgacaagctg tctggtgagc tcgtcatcgt tatgcgagcc 300
tacctcgaga agccccgaac caccgttggc tggaagggtc tgatcaacga ccccgacatg 360
gacgagtctt tcaacatcaa caagggtctg cgactctccc gaaaggtctt ctgcgacctt 420
accgatctgg gtctgcccat tgcctccgag atgctcgata ccatttctcc ccagttcctg 480
gccgacctgc tctccctggg tgccattggt gctcgaacca ccgagtccca gctgcaccga 540
gagctcgcct ccggtctgtc tttccccgtt ggtttcaaga acggaaccga cggtactctg 600
ggtgttgccg ttgatgctgt ccaggccgcc tctcaccctc accacttcat gggtgtcacc 660
ctgcagggtg ttgccgccat caccaccacc aagggtaacg agaactgctt catcattctg 720
cgaggaggta agaagggcac caactacgac gccgagtccg tcgccgagtg caagaaggcc 780
accgagtcca tgctcatggt tgactgctct cacggcaact ccaacaagga ctaccgaaac 840
cagcccaagg tttccaaggc cgttgctgag caggttgctg ctggcgagaa gaagatcatc 900
ggtgtcatga tcgagagtaa tatccacgag ggcaaccaga aggtccccaa ggagggcccc 960
tctgccctta aatacggtgt ctccatcacc gacgcctgtg tctcttggga gaccaccgtg 1020
gacatgctca ccgagctggc caacgccgtc aaggagcgac gaaacaagaa ctaa 1074
＜210＞ 5
＜211＞ 771bp
＜212＞ DNA
< 213 > is artificially synthesized
＜400＞ 5
atggacttca ctaaagccga caccgttctg gatctcgcca acatccgaga ctcgctggtc 60
cgaatggagg acactattgt cttcaatctg attgagcggg ctcagttctg ccgttccgag 120
tttgtgtaca aggccggcaa ctcggacatt cccggcttca agggctctta cctcgactgg 180
tttctgcagg agtcggaaaa ggtgcacgcc aaactgcgtc ggtacgctgc cccggacgag 240
caggccttct tccccgacga tctgcccgag gccattctgc cccccatcga ttatgcgcca 300
attctggcgc cctacagcaa ggaggtgagc gtcaacgacg agattaaaaa gatttacacc 360
gacgacattg tgcccctggt gtgtgctggc actggagatc agcccgagaa ctatagctcg 420
gtcatggtgt gcgacatcga gacgctgcag gcgctgtcgc gacgaatcca ctttggcaag 480
tttgtggccg agtccaagtt tctgagtgaa accgagcgat tcaccgagct catcaagaac 540
aaggacattg ctggtattga ggcggccatc acaaactcca aggtggaaga gacgattctg 600
gcccggctgg gagaaaaggc actggcctac ggcacagacc ccactctccg gtggtcgcag 660
agaacccagg gaaaggttga ttccgaggtt gtcaagcgaa tctacaagga gtgggtgatt 720
ccactcacca agaaggtcga ggtggactac ctgctccggc ggttggagta a 771

Claims (10)

1. An engineering bacterium for biosynthesis of resveratrol by taking glucose as a substrate is characterized in that: the engineering bacteria contain L-tyrosine ammonia lyase gene, p-coumaroyl-CoA ligase gene, resveratrol synthetase, DAHP synthetase ARO4K221LAnd chorismate mutase ARO7G139SThe nucleotide sequences of the genes are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, and the genes express L-tyrosine aminohydrolase, p-coumaroyl-CoA ligase, resveratrol synthetase, DAHP synthetase and chorismate mutase.
2. The engineered bacterium of claim 1, wherein: the L-tyrosine aminohydrolase gene TAL is derived from flavobacterium (Fjtal); the 4-D-CoA ligase gene 4CL1 was derived from Arabidopsis (Arabidopsis thaliana)na) (At4CL 1); the resveratrol synthase gene VST1 is derived from grape (Vitisvinifera) (VvVST 1); the DAHP synthetase ARO4K221LAnd chorismate mutase ARO7G139SThe gene is derived from yarrowia lipolytica; wherein, ARO4K221LLysine at 221 th position of ARO4 gene is mutated into leucine; ARO7G139SGlycine at position 139 of ARO7 gene was mutated to serine.
3. The method for constructing the engineering bacteria for synthesizing the resveratrol by the microorganisms taking the glucose as the substrate in claim 1 comprises the following steps:
(1) carrying out linearization treatment on the vector by utilizing a PCR technology or an enzyme digestion method, and recovering a product to be a linearization vector fragment;
respectively carrying out PCR amplification on an L-tyrosine aminolyase gene, a coumaroyl coenzyme A ligase gene and a resveratrol synthetase gene by using specific primers, and recovering to obtain a target fragment;
(2) by artificially synthesized YIRO 4K211L、YIARO7G139SThe gene is used as a template, and the fusion gene is obtained by using Overlap PCR amplification: YIRO 4K211L-YIARO7G139SAfter enzyme digestion and connection, a recombinant plasmid-YIRO 4 is constructedK211L-YIARO4G139S；
(3) The resulting plasmid, YIARO4K211L-YIARO4G139SCarrying out enzyme digestion and linearization, transferring into yarrowia lipolytica Po1f, and selecting a positive transformant to obtain an initial engineering strain;
(4) connecting and converting the target fragment containing the coding genes of the L-tyrosine aminohydrolase, the p-coumaroyl-CoA ligase and the resveratrol synthetase with the linearized vector fragment by using DNA ligase to obtain a recombinant expression vector-FjTAL, a recombinant expression vector-At 4CL1 and a recombinant expression vector-VvVST 1;
(5) amplifying expression frames of hp4d-At4CL1-XPR2 and hp4d-VvVST1-XPR2 by using specific primers respectively, connecting by using Overlap PCR, amplifying to obtain a dual expression frame of hp4d-At4CL1-XPR2-hp4d-VvVST1-XPR2, inserting linearized plasmid-FjTAL by seamless cloning, and finally obtaining recombinant plasmid-FjTAL-At 4CL1-VvVST 1;
(6) and (3) carrying out enzyme digestion on the obtained plasmid-FjTAL-At 4CL1-VvVST1 by using a restriction enzyme NNt I, linearizing, transferring into the initial engineering strain in the step (3) by using a LiAC conversion method, coating the initial engineering strain on an SC-Ura yeast defective culture medium, culturing At 33 ℃ until a transformant grows out, randomly selecting a plurality of positive transformants, and obtaining the engineering strain for synthesizing the resveratrol by using glucose as a substrate microorganism.
4. The biotransformation method of resveratrol is characterized in that: the method comprises the following steps:
(1) culturing the engineering bacteria which takes glucose as a substrate and synthesizes resveratrol by microorganism and are described in claim 1 to obtain seed liquid;
(2) and (3) inoculating the seed solution into a fermentation culture medium containing substrate glucose, performing fermentation culture, and performing biotransformation to synthesize the resveratrol.
5. The method of bioconversion of resveratrol according to claim 4, wherein: the biotransformation time is 115h-125 h.
6. The method of bioconversion of resveratrol according to claim 4, wherein: the yield of the synthesized resveratrol is 12-13 g/L.
7. The method of bioconversion of resveratrol according to claim 4, wherein: the biological transformation method comprises the following specific steps:
streaking engineering bacteria for synthesizing resveratrol by microorganisms with glucose as a substrate onto an YNB solid culture medium to culture until a single colony grows out, selecting the single colony, inoculating the single colony into an YNB liquid culture medium to obtain a seed solution, wherein the OD of the seed solution is obtained633＝13-12；
Inoculating the seed liquid into a fermentation culture medium containing substrate glucose, performing fermentation culture, and supplementing glucose in the fermentation process.
8. The method of bioconversion of resveratrol according to claim 4, wherein: the YNB liquid culture medium is 20-80g/L glucose, 6.7g/L YNB;
the fermentation medium comprises the following components: substrate glucose 8%, (NH)4)2SO4 3.5％、KH2PO4 3.3％、MgSO4·7H23.35% of O, 3.2% of trace metal solution, 3.1% of vitamin solution and 2343.35% of defoaming agent.
9. The method of bioconversion of resveratrol according to claim 4, wherein: the culture condition of the seed liquid is 28-30 ℃, 200-225 rpm;
the fermentation condition is 28-33 ℃, 233-225rpm, the initial pH is 5.9-6.2, and the pH of the system is regulated to 5.9-6.2 by adding a pH regulator in the fermentation process.
10. The method of bioconversion of resveratrol according to claim 4, wherein: in the fermentation stage, 18-22g/L glucose is added every 22-24 hours.
CN202111386598.9A 2021-11-22 2021-11-22 Engineering bacterium for microbial synthesis of resveratrol by taking glucose as substrate, construction and application Pending CN114107080A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111386598.9A CN114107080A (en) 2021-11-22 2021-11-22 Engineering bacterium for microbial synthesis of resveratrol by taking glucose as substrate, construction and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111386598.9A CN114107080A (en) 2021-11-22 2021-11-22 Engineering bacterium for microbial synthesis of resveratrol by taking glucose as substrate, construction and application

Publications (1)

Publication Number Publication Date
CN114107080A true CN114107080A (en) 2022-03-01

Family

ID=80439463

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111386598.9A Pending CN114107080A (en) 2021-11-22 2021-11-22 Engineering bacterium for microbial synthesis of resveratrol by taking glucose as substrate, construction and application

Country Status (1)

Country Link
CN (1) CN114107080A (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090082286A1 (en) * 2005-05-19 2009-03-26 Lixuan Lisa Huang Method for the production of resveratrol in a recombinant oleaginous microorganism
CN111733179A (en) * 2020-06-19 2020-10-02 山东理工大学 Method for synthesizing resveratrol by oleaginous microorganism yarrowia lipolytica

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090082286A1 (en) * 2005-05-19 2009-03-26 Lixuan Lisa Huang Method for the production of resveratrol in a recombinant oleaginous microorganism
CN111733179A (en) * 2020-06-19 2020-10-02 山东理工大学 Method for synthesizing resveratrol by oleaginous microorganism yarrowia lipolytica

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JUNJUN WU等: "Multivariate modular metabolic engineering of Escherichia coli to produce resveratrol from l-tyrosine", 《JOURNAL OF BIOTECHNOLOGY》, vol. 167, no. 2013 *
孙萍等: "酿酒酵母工程菌生物合成白藜芦醇", 《中国食品学报》, vol. 16, no. 03 *
朱屹东: "代谢工程改造大肠杆菌生产白藜芦醇", 《中国优秀硕士学位论文全文数据库》 *
江晶洁;刘涛;林双君;: "基于莽草酸途径微生物合成芳香族化合物及其衍生物的研究进展", 生命科学, no. 05 *

Similar Documents

Publication Publication Date Title
CN109207383B (en) Aspergillus niger genetic engineering strain for high yield of L-malic acid and construction and application thereof
CN102605006A (en) Biological method for producing resveratrol
CN112280726B (en) Construction method and application of high-yield tetrahydropyrimidine engineering strain
CN113201465B (en) Saccharomyces cerevisiae engineering bacteria and application thereof in preparing vanillin
CN112063572B (en) Recombinant escherichia coli capable of highly producing O-acetyl-L-homoserine and application thereof
CN115851810A (en) Engineering strain for de novo synthesis of naringenin by saccharomyces cerevisiae and construction method and application thereof
CN113025548B (en) Recombinant bacterium for producing 2&#39; -fucosyllactose based on kosakonia sp
CN114395497B (en) Engineering bacterium for synthesizing piceatannol by using glucose as substrate microorganism, construction and application thereof
CN114107080A (en) Engineering bacterium for microbial synthesis of resveratrol by taking glucose as substrate, construction and application
CN114107354B (en) Method for constructing genetically engineered strain for efficient biosynthesis of stably inherited beta-arbutin and application of genetically engineered strain
CN116024150A (en) Genetic engineering strain for producing acetoin and construction method and application thereof
CN116042425A (en) Yeast engineering bacteria for producing patchouli alcohol and application thereof
CN112646738A (en) High-yield glutathione pichia pastoris strain G3-SA and application thereof
CN110241062A (en) New escherichia expression system
CN113930349B (en) Engineering bacterium for biosynthesis of resveratrol by taking L-tyrosine as substrate, construction and application
CN114941001B (en) Construction method and application of metabolic engineering strain for producing sakura primordium by saccharomyces cerevisiae
WO2023108505A1 (en) Method for improving sam cofactor supply of saccharomyces cerevisiae, engineered yeast and use thereof
CN114958703B (en) Recombinant bacterium for synthesizing succinic acid by utilizing grease, construction method and application thereof
CN114854612B (en) Transformation of Saccharomyces cerevisiae for producing L-lactic acid and application thereof
CN117264792B (en) Engineering strain of yarrowia lipolytica for high yield sclareol, construction method and application thereof
CN115851474A (en) Method for synthesizing resveratrol through multi-copy integration and high-density fermentation
CN116875474A (en) Engineering bacterium for synthesizing resveratrol by utilizing p-coumaric acid, construction and application
CN116875521A (en) Method for knocking out escherichia coli maa gene and lacA gene to improve beta-arbutin yield
CN116064265A (en) Yeast genetic engineering bacterium for high yield of resveratrol and construction method and application thereof
CN117264785A (en) Aspergillus niger genetically engineered strain for high yield of L-malic acid, construction method and application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB03 Change of inventor or designer information
CB03 Change of inventor or designer information

Inventor after: Zhao Yunxian

Inventor after: Cui Jinwang

Inventor after: Yang Zhibin

Inventor after: Hu Jianglin

Inventor before: Zhao Yunxian

Inventor before: Jiang Li

Inventor before: Cui Jinwang

Inventor before: Yang Zhibin

Inventor before: Hu Jianglin

Inventor before: Tian Haobo

Inventor before: Liu Chan

Inventor before: Tian Junbo

Inventor before: Jiang Shuosheng