CN116478843A - Recombinant strain for synthesizing gastrodin, construction method thereof and method for synthesizing gastrodin by fermentation - Google Patents

Recombinant strain for synthesizing gastrodin, construction method thereof and method for synthesizing gastrodin by fermentation Download PDF

Info

Publication number
CN116478843A
CN116478843A CN202310371074.5A CN202310371074A CN116478843A CN 116478843 A CN116478843 A CN 116478843A CN 202310371074 A CN202310371074 A CN 202310371074A CN 116478843 A CN116478843 A CN 116478843A
Authority
CN
China
Prior art keywords
gene
seq
promoter
strain
nucleotide sequence
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
CN202310371074.5A
Other languages
Chinese (zh)
Inventor
顾洋
朱姜
林铭鑫
桑艾莉
刘桐糠
宋彦瑾
李昌凡
黄和
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.)
Nanjing Normal University
Original Assignee
Nanjing Normal University
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 Nanjing Normal University filed Critical Nanjing Normal University
Priority to CN202310371074.5A priority Critical patent/CN116478843A/en
Publication of CN116478843A publication Critical patent/CN116478843A/en
Pending legal-status Critical Current

Links

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
    • C12N15/63—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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • 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
    • 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
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04003—Adenylate kinase (2.7.4.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07009—UTP-glucose-1-phosphate uridylyltransferase (2.7.7.9), i.e. UDP-glucose-pyrophosphorylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/02—Phosphotransferases (phosphomutases) (5.4.2)
    • C12Y504/02002—Phosphoglucomutase (5.4.2.2)
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/34—Vector systems having a special element relevant for transcription being a transcription initiation element
    • 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
    • C12R2001/645—Fungi ; Processes using fungi

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (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)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to the field of genetic engineering, and discloses a recombinant strain for synthesizing gastrodin, a construction method thereof and a method for synthesizing gastrodin by fermentation. The recombinant strain is obtained by genetic modification of a starting strain, and compared with the starting strain, the recombinant strain is integrated and expressed with at least one of the genes described in the following (a) - (c): (a) From startMover P UGA2 A started phosphoglucomutase PGM1 gene; (b) From promoter P FBA1 A initiated glucose pyrophosphorylase UGP1 gene; (c) From promoter P TER A started adenylate kinase AK9 gene. The method for synthesizing gastrodin by fermentation comprises the following steps: inoculating the recombinant strain into a fermentation medium containing p-hydroxybenzyl alcohol for fermentation. The recombinant strain provided by the invention can effectively balance the metabolic flow of UDP-glucose synthesis and promote glycosylation reaction, so that the yield of gastrodin produced by converting p-hydroxybenzyl alcohol by the recombinant strain is improved.

Description

Recombinant strain for synthesizing gastrodin, construction method thereof and method for synthesizing gastrodin by fermentation
Technical Field
The invention relates to the field of genetic engineering, in particular to a recombinant strain for synthesizing gastrodin, a construction method of the recombinant strain for synthesizing gastrodin and a method for synthesizing gastrodin by fermentation.
Background
Gastrodin (also called 4-hydroxymethyl phenyl-beta-D-glucopyranoside) is a main bioactive component of traditional Chinese medicine rhizoma Gastrodiae, and has been widely used in two fields of medical drugs and health foods. The gastrodin can be used as a single therapeutic drug or can be used in combination, is mainly used for dilating blood vessels, enhancing vascular elasticity, improving cerebrovascular diseases and mental diseases caused by insufficient blood supply and senile dementia, has the effects of improving sleep, reducing blood pressure, enhancing immunity and the like, is a plurality of health care food types with the gastrodin as a main medicinal effect component, and is popular among the masses. At present, the demand of people for products with gastrodin as a main medicinal component is continuously increased, and the market prospect is wide along with the further expansion of the application range. However, for the industrial production of gastrodin, plant extraction and chemical synthesis methods are mainly relied on, which cannot be satisfied for the long-term, environment-friendly and sustainable development of modern industrial production. Therefore, development of a new green and sustainable production mode of gastrodin is needed, and the microbial synthesis method is the most main way for realizing green, safe and sustainable industrial production of gastrodin.
It has been found that microbial synthesis of gastrodin is formed by transfer of glucosyl groups to the 4-OH position of the substrate p-hydroxybenzyl alcohol by glycosyltransferase (UGT), wherein UDP-glucose (uridine diphosphate glucose) is the glycosyl donor and the supply of UDP-glucose precursor in yeast plays a vital role in the biosynthesis of glucoses. The metabolic flow of UDP-glucose synthesis is regulated and controlled to improve the yield of gastrodin produced by converting p-hydroxybenzyl alcohol by microorganisms, and the method has important significance for high-efficiency production of gastrodin, but no effective regulation and control method for the metabolic flow of UDP-glucose synthesis exists at present.
Disclosure of Invention
The invention aims to solve the problem that metabolic flow for UDP-glucose synthesis cannot be effectively regulated and controlled in the prior art, and provides a recombinant strain for synthesizing gastrodin, a construction method thereof and a method for synthesizing gastrodin by fermentation.
In order to achieve the above object, a first aspect of the present invention provides a recombinant strain for synthesizing gastrodin, which is obtained by genetic modification of a starting strain, wherein the recombinant strain is integrated and expressed with at least one of the genes described in the following (a) to (c) compared with the starting strain:
(a) From promoter P UGA2 A started phosphoglucomutase PGM1 gene;
(b) From promoter P FBA1 A initiated glucose pyrophosphorylase UGP1 gene;
(c) From promoter P TER A started adenylate kinase AK9 gene.
The second aspect of the present invention provides a method for constructing a recombinant strain for synthesizing gastrodin, the method comprising: genetically engineering a starting strain such that the starting strain is integrally expressed with at least one of the genes described in (a) - (c) below:
(a) From promoter P UGA2 A started phosphoglucomutase PGM1 gene;
(b) From promoter P FBA1 A initiated glucose pyrophosphorylase UGP1 gene;
(c) From promoter P TER A started adenylate kinase AK9 gene.
The third aspect of the invention provides the use of the recombinant strain described above or the method described above in the synthesis of gastrodin.
The fourth aspect of the invention provides a method for synthesizing gastrodin by fermentation, which comprises the following steps: inoculating the recombinant strain into a fermentation medium containing p-hydroxybenzyl alcohol for fermentation; alternatively, the recombinant strain is constructed as described above, and the resulting recombinant strain is inoculated into a fermentation medium containing p-hydroxybenzyl alcohol for fermentation.
Through the technical scheme, the recombinant strain for synthesizing gastrodin provided by the invention can utilize the promoter P UGA2 Regulation of phosphoglucomutase PGM1 gene expression using promoter P FBA1 Regulation of glucose pyrophosphorylase UGP1 gene expression, P TER Regulating and controlling the expression of an adenylate kinase AK9 gene, controlling the moderate expression of PGM1, UGP1 and AK9 genes, balancing the metabolic flow of UDP-glucose synthesis, promoting glycosylation reaction, thereby improving the yield of the gastrodin produced by transforming the p-hydroxybenzyl alcohol by the recombinant strain, obviously improving the yield of the gastrodin, laying a foundation for efficiently producing the gastrodin by metabolic engineering modification, and providing better potential choice for microbial fermentation production of the gastrodin; the construction method is simple, easy to operate and has good application prospect.
When the yarrowia lipolytica with the knock-out gene Ku70 and the glycosyltransferase Slysugt gene integrated and expressed at the Ku70 locus is taken as an original strain, the concentration of the cannabinoid in the fermentation liquor of the recombinant yarrowia lipolytica strain can reach 717.2mg/L, and the accumulation of the gastrodin is improved by 34 percent relative to the original strain.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
FIG. 1 is a recombinant plasmid pYL-P of example 1 UGA2 -PGM1-P FBA1 -UGP1-P TER -AK9 gene sequence structural diagram;
FIG. 2 is a recombinant yarrowia lipolytica po1 fk-Slysugt-P UGA2 -PGM1-P FBA1 -UGP1-P TER -AK9 (example 3) and starting strain Po1fΔku70-SlyUGT (comparative example 1) yields profile of gastrodin using p-hydroxybenzyl alcohol as substrate.
Detailed Description
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In a first aspect, the present invention provides a recombinant strain for synthesizing gastrodin, which is obtained by genetic modification of a starting strain, and is integrated and expressed with at least one of the genes described in (a) - (c) below compared with the starting strain:
(a) From promoter P UGA2 A started phosphoglucomutase PGM1 gene;
(b) From promoter P FBA1 A initiated glucose pyrophosphorylase UGP1 gene;
(c) From promoter P TER A started adenylate kinase AK9 gene.
The inventors of the present invention found during the course of the study that the promoter P was expressed integrally in the strain UGA2 Started phosphoglucomutase PGM1 and promoter P FBA1 Initiated glucose pyrophosphorylase UGP1 and promoter P TER Expression of the genes of the three started adenylate kinase AK9And on the basis, the combination of the strong constitutive promoter for over-expressing the target gene can further improve the yield of the gastrodin synthesized by the strain.
According to the present invention, preferably, the recombinant strain is integrally expressed with the genes described in (a), (b) and (c). The inventor finds that under the preferred embodiment, the strong and weak combination of promoters of three genes, namely PGM1 gene, UGP1 gene and AK9 gene, which are most suitable for UDP-glucose supply can be formed, thereby being beneficial to further optimizing the synthesis path of UTP-glucose to gastrodin, improving the yield of the gastrodin synthesized by the strain, and having important significance for improving the yield of the gastrodin produced by the conversion of the engineering strain into p-hydroxybenzyl alcohol.
According to the invention, promoter P UGA2 Promoter P FBA1 Promoter P TER Are all endogenous constitutions of yarrowia lipolytica. Preferably, the promoter P UGA2 The nucleotide sequence of (2) is shown as SEQ ID NO:1, said promoter P FBA1 The nucleotide sequence of (2) is shown as SEQ ID NO:2, said promoter P TER The nucleotide sequence of (2) is shown as SEQ ID NO: 3.
According to the present invention, preferably, the amino acid sequence encoded by the phosphoglucomutase PGM1 gene is as shown in SEQ ID NO:4, the amino acid sequence of the glucose pyrophosphorylase UGP1 gene is shown as SEQ ID NO:5, the amino acid sequence of the coding of the adenylate kinase AK9 gene is shown as SEQ ID NO: shown at 6. Further preferably, the nucleotide sequence of the phosphoglucomutase PGM1 gene is as shown in SEQ ID NO:7, the nucleotide sequence of the glucose pyrophosphorylase UGP1 gene is shown as SEQ ID NO:8, the nucleotide sequence of the adenylate kinase AK9 gene is shown as SEQ ID NO: shown at 9.
According to the invention, the starting strain can be any strain capable of synthesizing gastrodin by utilizing glucose. Preferably, the starting strain is yarrowia lipolytica (Yarrowia lipolytica). More preferably, the starting strain is yarrowia lipolytica which knocks out a gene Ku70 and integrates and expresses a glycosyltransferase Slysugt gene at a Ku70 site, wherein the nucleotide sequence of the gene Ku70 is shown in SEQ ID NO:10, the nucleotide sequence of the glycosyltransferase Slysugt gene is shown as SEQ ID NO: 11. The knock-out gene Ku70 in yarrowia lipolytica can be prepared by itself with reference to the methods disclosed in the prior art, for example, see documents Gao S, tong Y, zhu L, et al Iternate integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous. Beta. -carotene production [ J ]. Metabolic Engineering,2017:192, and after knock-out of the coding gene Ku70 responsible for non-homologous recombination on the basis of the original strain MYA2613 (purchased from American type culture Collection, ATCC), yarrowia lipolytica Yarrowia lipolytica Po1 f. DELTA.Ku70 is obtained and the expression cassette Ku70UP-SlyUGT-Ku70Down is integrated by homologous recombination to obtain yarrowia lipolytica Yarrowia lipolytica Po f. DELTA.Ku70-Slyt as the starting strain in the present invention.
In the present invention, the expression of the promoter P in the strain can be integrated by homologous recombination of a recombinant vector containing an expression cassette for the corresponding gene into the starting strain UGA2 The started phosphoglucomutase PGM1 gene, which is composed of a promoter P FBA1 Initiated glucose pyrophosphorylase UGP1 gene and promoter P TER At least one of the started adenylate kinase AK9 genes. In the present invention, integrated expression refers to a technique of integrating an exogenous gene into the genome of a target cell by homologous recombination so that expression of the exogenous gene occurs.
Further preferably, the recombinant strain is expressed integrally by promoter P UGA2 The started phosphoglucomutase PGM1 gene, which is composed of a promoter P FBA1 Initiated glucose pyrophosphorylase UGP1 gene and promoter P TER The three gene expression cassettes (P) of the started adenylate kinase AK9 gene UGA2 -PGM1-P FBA1 -UGP1-P TER AK 9). The inventors of the present invention found that the expression of the three gene expression cassette (P UGA2 -PGM1-P FBA1 -UGP1-P TER AK 9) can more significantly increase the yield of gastrodin in yarrowia lipolytica.
The second aspect of the invention provides a method for constructing a recombinant strain for synthesizing gastrodin, which comprises the following steps: genetically engineering a starting strain such that the starting strain is integrally expressed with at least one of the genes described in (a) - (c) below:
(a) From promoter P UGA2 A started phosphoglucomutase PGM1 gene;
(b) From promoter P FBA1 A initiated glucose pyrophosphorylase UGP1 gene;
(c) From promoter P TER A started adenylate kinase AK9 gene.
According to the present invention, preferably, the recombinant strain is integrally expressed with the genes described in (a), (b) and (c).
According to the present invention, preferably, the promoter P UGA2 The nucleotide sequence of (2) is shown as SEQ ID NO:1, said promoter P FBA1 The nucleotide sequence of (2) is shown as SEQ ID NO:2, said promoter P TER The nucleotide sequence of (2) is shown as SEQ ID NO:3 is shown in the figure;
the amino acid sequence of the phosphoglucomutase PGM1 gene is shown in SEQ ID NO:4, the amino acid sequence of the glucose pyrophosphorylase UGP1 gene is shown as SEQ ID NO:5, the amino acid sequence of the coding of the adenylate kinase AK9 gene is shown as SEQ ID NO:6 is shown in the figure;
preferably, the nucleotide sequence of the phosphoglucomutase PGM1 gene is shown in SEQ ID NO:7, the nucleotide sequence of the glucose pyrophosphorylase UGP1 gene is shown as SEQ ID NO:8, the nucleotide sequence of the adenylate kinase AK9 gene is shown as SEQ ID NO: shown at 9.
According to the invention, preferably, the starting strain is yarrowia lipolytica; further preferably, the starting strain is yarrowia lipolytica which knocks out a gene Ku70 and integrates and expresses a glycosyltransferase Slysugt gene at a Ku70 site, wherein the nucleotide sequence of the gene Ku70 is shown in SEQ ID NO:10, the nucleotide sequence of the glycosyltransferase Slysugt gene is shown as SEQ ID NO: 11.
In the invention, the homologous sequence fragments used for the integrated expression can be obtained by the following modes: the synthesis of the sequences of the fragments upstream and downstream of the target genes (e.g., the phosphoglucomutase UGP1 Gene ID: 2906446) in yarrowia lipolytica and Saccharomyces cerevisiae as homology arms is performed according to databases known in the art (e.g., genBank database, https:// www.ncbi.nlm.nih.gov/GenBank /); alternatively, the sequence of the upstream and downstream fragments of the target gene may be amplified from the genome of the starting strain (e.g., yarrowia lipolytica) as homology arms by PCR to obtain the initial homologous sequence fragment of the target gene, but the present invention is not limited thereto.
In the present invention, a recombinant vector capable of integrating an expression cassette expressing at least one of the genes (a) to (c) described above on the genome of a starting strain (e.g., yarrowia lipolytica), preferably a gene expression cassette (P) capable of integrating and expressing the three genes (a) to (c) described above on the genome of a starting strain, can be constructed first UGA2 -PGM1-P FBA1 -UGP1-P TER AK 9). Various methods are known in the art for constructing recombinant vectors for ligating a gene fragment of interest to a vector to prepare an integrated expression recombinant vector, such as, but not limited to, classical "restriction-ligation" methods, gateway cloning systems developed by Invitrogen, creator cloning systems developed by Clontech, unictor cloning systems developed by StephenElledge laboratories, and GoldenGate cloning based on type IIs restriction enzymes.
For example, recombinant vectors of the invention can be constructed using recombinant enzyme methods: amplifying by PCR method based on plasmid containing enzyme coding gene to obtain target enzyme coding gene; the sequence of the gene encoding the enzyme to be inserted, the promoter sequence, the screening gene expression cassette and the like are connected in series to obtain the recombinant vector, but the invention is not limited thereto.
The recombinant vector may then be introduced into an original strain (e.g., yarrowia lipolytica) using methods conventional in the art, such as, but not limited to, microinjection, gene gun, transformation (e.g., electrotransformation), infection, or transfection. Microinjection, gene gun, transformation, infection or transfection are all routine procedures in the art. For example, transformation refers to the treatment of cells by some known methods in molecular biology and genetic engineering, such that the treated cells are competent and thereby contacted with exogenous DNA, thereby allowing the exogenous DNA to enter the competent cells; common transformation methods include protoplast transformation, chemical transformation, and electroporation transformation.
After introduction of the recombinant vector into an original strain (e.g., yarrowia lipolytica), positive clones can be selected by a selection marker (e.g., resistance gene) and verified by genomic PCR or by sequencing of genomic DNA to obtain the recombinant strain of synthetic gastrodin.
According to the present invention, preferably, the process of integrating expression includes: first construct a promoter containing P UGA2 Expression cassette of started phosphoglucomutase PGM1 gene and promoter P FBA1 Expression cassette and promoter P of the UGP1 gene of the started glucose pyrophosphorylase TER Expression cassette (P) of the started adenylate kinase AK9 gene UGA2 -PGM1-P FBA1 -UGP1-P TER -AK 9) and then subjecting the three expression cassettes in the recombinant vector to homologous recombination to integrate and express them on the genome of the starting strain, to utilize the three gene expression cassettes (P UGA2 -PGM1-P FBA1 -UGP1-P TER -AK 9) replaces Ku70 in the starting strain.
The third aspect of the invention provides the use of the recombinant strain described above or the method described above in the synthesis of gastrodin.
The fourth aspect of the invention provides a method for synthesizing gastrodin by fermentation, which comprises the following steps: inoculating the recombinant strain into a fermentation medium containing p-hydroxybenzyl alcohol for fermentation; alternatively, the recombinant strain is constructed as described above, and the resulting recombinant strain is inoculated into a fermentation medium containing p-hydroxybenzyl alcohol for fermentation.
According to the invention, preferably, the recombinant strain is prepared into a seed solution, and then the seed solution is inoculated into a fermentation medium for fermentation to obtain a fermentation broth.
According to the present invention, preferably, the seed liquid preparation method includes: and (3) picking single colony of the recombinant strain, and inoculating the single colony into a seed culture medium for seed culture to obtain the seed solution. In the present invention, the single colony of the recombinant strain may be selected from the freshly prepared recombinant strain or the recombinant strain frozen at low temperature (e.g., recombinant strain for gastrodin synthesis frozen in a glycerol freezer in a-80 ℃ refrigerator). If the recombinant strains preserved in the glycerol tubes are inoculated, the strains in each glycerol tube are inoculated into 100mL of seed culture medium.
The method of seed culture is not particularly limited in the present invention, as long as the recombinant strain can be subjected to activated proliferation by the method; the parameters of temperature, pH, rotation speed, time and the like used for seed culture can be conventional settings in the art. Preferably, the seed culture conditions include: the temperature is 25-35 ℃, the rotating speed is 180-250rpm, and the time is 40-60h.
In the present invention, the seed medium is not particularly limited, and may be a seed medium conventionally used in the art. Preferably, when the starting strain is yarrowia lipolytica, the seed medium contains: glucose, peptone, yeast powder and p-hydroxybenzyl alcohol. Further preferably, the seed medium contains: 15-25g/L glucose, 15-25g/L peptone, 5-15g/L yeast powder and 0.5-1.5g/L p-hydroxybenzyl alcohol.
In the present invention, the method of fermentation is not particularly limited, and a method of synthesizing gastrodin by fermentation conventionally used in the art may be used, for example, by inoculating the seed solution into the fermentation medium (for example, into a shake flask or a fermenter containing the fermentation medium) and performing fermentation culture to obtain a fermentation liquid.
In the present invention, in order to increase the gastrodin yield, it is preferable that the seed liquid is inoculated in an amount of 0.5 to 2 parts by volume with respect to 100 parts by volume of the fermentation medium.
In the present invention, in order to increase the yield of gastrodin, preferably, the fermentation conditions include: the temperature is 25-35 ℃, the rotating speed is 180-250rpm, and the time is 80-160h.
According to the present invention, preferably, the fermentation medium further contains a carbon source and a nitrogen source; further preferably, the fermentation medium contains: 30-50g/L glucose, 10-30g/L peptone, 5-20g/L yeast powder and 0.5-1.5g/L p-hydroxybenzyl alcohol.
In the present invention, gastrodin in the obtained fermentation liquid can be separated by a known method, for example, the fermentation liquid is subjected to solid-liquid separation to obtain a fermentation liquid supernatant containing gastrodin and the like, thereby obtaining gastrodin in the cytoplasm of the cell.
In the invention, the gastrodin in the fermentation liquid or the gastrodin separated from the fermentation liquid can be detected by a known method. For example, the gastrodin content may be detected by gas chromatography and the like.
According to a particularly preferred embodiment of the present invention, the method for synthesizing gastrodin by fermenting recombinant strains comprises the following steps: selecting single colony of the recombinant strain, inoculating the single colony into a seed culture medium, and culturing the seed for 40-60 hours under the conditions that the temperature is 25-35 ℃ and the rotating speed is 180-250rpm to obtain seed liquid; inoculating the seed liquid into the fermentation culture medium according to the inoculum size of 0.5-2 vol% and fermenting and culturing for 80-160h under the conditions of the temperature of 25-35 ℃ and the rotating speed of 180-250rpm to obtain fermentation liquid; performing solid-liquid separation on the fermentation liquid to obtain fermentation thalli, crushing the fermentation thalli, and extracting to obtain gastrodin in cytoplasm of the thalli;
wherein the seed culture medium comprises: 15-25g/L of glucose, 15-25g/L of peptone, 5-15g/L of yeast powder and 0.5-1.5g/L of p-hydroxybenzyl alcohol;
the fermentation medium contains: 30-50g/L glucose, 15-25g/L peptone, 5-20g/L yeast powder and 0.5-1.5g/L p-hydroxybenzyl alcohol.
The present invention will be described in detail by the following examples, which are only for illustrating the present invention and are not intended to limit the scope of the present invention. In the examples which follow, unless otherwise indicated, the experimental methods used are conventional methods well known to those skilled in the art.
In the following examples, yarrowia lipolytica po1f is a derivative strain of yarrowia lipolytica ATCC 20460, offered directly by the university of Maryland Peng Xu professor, which is available from Taieaster Biotech company of Taiwan; the p-hydroxybenzyl alcohol standard is purchased from Shanghai Michelia Biochemical technology Co., ltd, product number H811051-25g; gastrodin standard is purchased from Shanghai Michelia Biochemical technology Co., ltd, and the product number is G828303-5G; unless otherwise specified, all reagents and materials used were commercially available, and all methods used were conventional.
1. Culture medium
Leucine-deficient plates: glucose 20g/L, ammonium sulfate 5g/L, an amino-free yeast nitrogen source YNB 1.7g/L, adenine hemisulfate 10mg/L, L-arginine 0.05g/L, L-aspartic acid 0.08g/L, L-histidine 0.02g/L, L-isoleucine 0.05g/L, L-lysine 0.05g/L, L-methionine 0.02g/L, L-phenylalanine 0.05g/L, L-threonine 0.1g/L, L-tryptophan 0.05g/L, L-tyrosine 0.05g/L, L-valine 0.14g/L, uracil 0.1g/L, agar 20g/L;
uracil-deficient plates: glucose 20g/L, ammonium sulfate 5g/L, an amino-free yeast nitrogen source YNB 1.7g/L, adenine hemisulfate 10mg/L, L-arginine 0.05g/L, L-aspartic acid 0.08g/L, L-histidine 0.02g/L, L-isoleucine 0.05g/L, L-lysine 0.05g/L, L-methionine 0.02g/L, L-phenylalanine 0.05g/L, L-threonine 0.1g/L, L-tryptophan 0.05g/L, L-tyrosine 0.05g/L, L-valine 0.14g/L, leucine 0.1g/L, and agar 20g/L;
YPD medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L;
YPD fermentation medium: yeast extract 10g/L, peptone 20g/L, glucose 40g/L;
seed culture medium: glucose 20g/L, peptone 20g/L, yeast powder 10g/L, and p-hydroxybenzyl alcohol 1g/L;
fermentation medium: 40g/L glucose, 20g/L peptone, 15g/L yeast powder and 1g/L p-hydroxybenzyl alcohol.
2. Detection method
The products gastrodin and p-hydroxybenzyl alcohol: all samples were subjected to the corresponding treatment before measurement, and after centrifugation at 12000rpm for 10min, 1mL of the supernatant was collected, and then the supernatant was subjected to a water-based filter (pore size: 0.22 μm) to remove impurities, and subjected to liquid phase detection.
The High Performance Liquid Chromatography (HPLC) detection parameters, chromatographic columns and detection conditions are as follows: shimadzu LC-40, UV-VIS detector, ZORBAX Eclipse Plus C column (4.6X100 mm,3.5 μm, agilent); the mobile phase is pure water (90%) and pure methanol (10%); the flow rate is 0.mL/min; an ultraviolet detector having a detection wavelength of 225nm; the sample injection amount is set to 10 mu L; the column temperature was set at 35 ℃. Preparing 5 gradient concentrations of gastrodin standard substance and p-hydroxybenzyl alcohol standard substance respectively, and detecting corresponding response values to obtain standard curve of concentration and light absorption value, and correlation coefficient R 2 The value of (2) should be greater than 0.99, and then determining the contents of gastrodin and p-hydroxybenzyl alcohol in the sample according to the respective standard curve.
Preparation example 1
(1) Connecting a Slysugt gene (the nucleotide sequence of which is shown as SEQ ID NO: 11) to an expression vector pYLXP ', carrying out enzyme tangential digestion on a plasmid pYLXP' -Slysugt by using AvrII and SalI, recovering DNA fragments after agarose gel electrophoresis, linearizing Purlox-Ku 70 (the nucleotide sequence of which is shown as SEQ ID NO: 12) by using SalI and Nhe I restriction endonucleases to serve as a vector, connecting the vector and the fragments by using T4 DNA ligase to obtain a recombinant plasmid Purlox-Ku 70-Slysugt, and carrying out sequencing verification on the recombinant plasmid Purlox-Ku 70-Slysugt to be used for genome integration;
(2) Culturing a po1f strain in 2mL of YPD culture medium to an exponential growth phase (16-24 h) by taking yarrowia lipolytica po1f as an original strain, collecting po1f thalli in 1mL of fermentation broth, centrifuging, discarding supernatant, adding 50 vol% PEG4000 solution containing 90 mu L of lithium acetate (2M), 5 mu L of single-stranded DNA (salmon sperm) and 5 mu L of Ku 70-Slysugt expression cassette (obtained by cutting recombinant plasmid Purlox-Ku 70-Slysugt obtained in the step (1) by AvrII and SalI) into the culture medium, mixing the culture medium evenly, incubating the culture medium at 39 ℃, coating uracil-deficient plates, and screening to obtain recombinant strain single colonies of uracil selectable markers URA (nucleotide sequence shown as SEQ ID NO: 13) and Ku 70-Sugt substitutional Ku70 genes (nucleotide sequence shown as SEQ ID NO: 10);
(3) And (3) selecting a single colony on the uracil-deficient plate in the step (2) for colony PCR verification, and using primer pairs Ku70_Chkup_F/TEF_R (the nucleotide sequences are shown as SEQ ID NO:14 and SEQ ID NO: 15) and Ku70_ChkDw_R/XPR_F (the nucleotide sequences are shown as SEQ ID NO:16 and SEQ ID NO: 17) to verify the upstream site and the downstream site of the integration of the Ku70-SlysugT, wherein the two sites are correct, and the upstream site and the downstream site of the integration of the Ku70-SlysugT expression frame are verified, and the two sites are correct, so that the Ku70-SlysugT expression frame is correctly verified to be integrated to the knockout site of the po1F thallus, and the obtained recombinant engineering bacterium is a yarrowia lipolytica strain Yarrowia lipolytica Po F Ku70-SugT which is subjected to Ku70 gene knockout and is integrated to express the glycosyltransferase encoding gene SlysugT at the Ku70 site.
EXAMPLE 1 construction of recombinant plasmid
Promoter replacement plasmid construction procedure with PYL-P FBA1 The following are examples: the plasmid pYLXP' is used as a template, a linearized vector fragment is obtained by amplifying a primer PYL01-F/PYL01-R (the nucleotide sequences are shown as SEQ ID NO:18 and SEQ ID NO: 19), and a promoter P with a homology arm is obtained by amplifying a primer FBA1-F/FBA1-R (the nucleotide sequences are shown as SEQ ID NO:20 and SEQ ID NO: 21) by using a yarrowia lipolytica genome as a template FBA1 Fragment (P) FBA1 The nucleotide sequence of (2) is shown as SEQ ID NO: 2) followed by the use ofInserting the target gene fragment into the amplified linearization vector fragment by the rapid cloning technology to obtain recombinant plasmid PYL-P FBA1 。
The same method is adopted to obtain the promoter P with homology arms through the amplification of the primer TER-F/TER-R (the nucleotide sequences are shown as SEQ ID NO:22 and SEQ ID NO: 23) TER Fragment (P) TER The nucleotide sequence of (2) is shown as SEQ ID NO: 3), and inserting the recombinant plasmid PYL-P into the amplified linearization vector to obtain a recombinant plasmid PYL-P TER The method comprises the steps of carrying out a first treatment on the surface of the The promoter P with homology arm is obtained by amplifying the primer UGA2-F/UGA2-R (the nucleotide sequence is shown as SEQ ID NO:24 and SEQ ID NO: 25) UGA2 Fragment (P) UGA2 The nucleotide sequence of (2) is shown as SEQ ID NO: 1) into the amplified linearized vectorObtaining recombinant plasmid PYL-P UGA2 。
Plasmid pYL-P is used in the construction process of single gene recombinant plasmid UGA2 PGM1 is for example: the PGM1 gene fragment (amino acid sequence shown in SEQ ID NO:4, nucleotide sequence shown in SEQ ID NO: 7) from Saccharomyces cerevisiae with optimized codon and synthesized by biological engineering (Shanghai) stock is used as template, the primer PGM1-F/PGM1-R (nucleotide sequences shown in SEQ ID NO:26 and SEQ ID NO: 27) is used to amplify the target gene fragment with homology arm, and Kpn I and Snab I restriction endonuclease is used to recombine PYL-P UGA2 Linearization of plasmid vectors using ClonInserting the target gene fragment into linearization vector by rapid cloning technology to obtain recombinant plasmid pYL-P UGA2 -PGM1。
Substitution of the plasmid vector with PYL-P by the same method as described above TER The target fragment is replaced by AK9 gene fragment (the amino acid sequence is shown as SEQ ID NO:6, the nucleotide sequence is shown as SEQ ID NO: 9), and recombinant plasmid pYL-P is constructed TER -AK9; substitution of plasmid vector with PYL-P FBA1 The target fragment is replaced by UGP1 gene fragment (the amino acid sequence is shown as SEQ ID NO:5, the nucleotide sequence is shown as SEQ ID NO: 8), and recombinant plasmid pYL-P is constructed FBA1 -UGP1。
The construction process of the polygene tandem recombinant plasmid comprises the following steps: plasmid pYL-P UGA2 PGM1 was linearized with Sal I and Nhe I restriction enzymes, and the large DNA fragment was recovered by agarose gel electrophoresis to obtain plasmid pYL-P TER AK9 is linearized by Sal I and Avr II restriction enzymes, DNA small fragments are recovered after agarose gel electrophoresis, larger fragments of the two recovered DNA fragments are used as vectors, and the larger fragments are connected by T4 DNA ligase to obtain recombinant plasmid pYL-P UGA2 -PGM1-P TER -AK9; plasmid pYL-P was then introduced UGA2 -PGM1-P TER AK9 was linearized with Sal I and Nhe I restriction enzymes, and the DNA fragment was recovered by agarose gel electrophoresis to obtain plasmid pYL-P FBA1 UGP1 is linear using Sal I and Avr II restriction enzymesAfter the transformation, DNA small fragments are recovered through agarose gel electrophoresis, larger fragments in the two recovered DNA fragments are used as vectors, and the T4 DNA ligase is used for connection to obtain recombinant plasmid pYL-P UGA2 -PGM1-P FBA1 -UGP1-P TER AK9 (structure see fig. 1).
Example 2
Culturing strain Yarrowia lipolytica Po fDeltaKu 70-Slysugt in 2mL YPD medium to exponential growth phase (16-24 h) with yarrowia lipolytica strain Yarrowia lipolytica Po fDeltaKu 70-Slysugt obtained in preparation example 1 as starting strain, collecting thallus in 1mL fermentation broth, centrifuging to remove supernatant, adding 50 vol% PEG4000 solution containing 90 μL, 5 μL lithium acetate (2M), 5 μL single-stranded DNA (salmon sperm) and 5 μL three-gene expression cassette P UGA2 -PGM1-P FBA1 -UGP1-P TER AK9 (recombinant plasmid pYL-P from example 1) UGA2 -PGM1-P FBA1 -UGP1-P TER -linearization products of AK9 digested with Not I restriction enzyme), mixing, incubating at 39deg.C for 1 hr, coating leucine-deficient plate, and screening to obtain endogenous constitutive promoter P of yarrowia lipolytica UGA2 The gene encoding the glucose mutase phosphate PGM1 and the endogenous constitutive promoter P of yarrowia lipolytica FBA1 The gene encoding the glucose pyrophosphorylase UGP1 and the constitutive promoter P endogenous to yarrowia lipolytica TER Three-gene expression frame P of started adenylate kinase AK9 coding gene UGA2 -PGM1-P FBA1 -UGP1-P TER Recombinant strain of AK9 Single colony Yarrowia lipolytica po fk-Slysugt-P UGA2 -PGM1-P FBA1 -UGP1-P TER -AK9。
Example 3
Gastrodin synthesis by recombinant strain fermentation
(1) The recombinant yarrowia lipolytica Yarrowia lipolytica po fk-Slysugt-P obtained in example 2 was picked up UGA2 -PGM1-P FBA1 -UGP1-P TER AK9, inoculating in seed culture medium, culturing at 30deg.C and 220rpm for 48 hr to obtain recombinant yarrowia lipolytica seed liquid;
(2) Inoculating the recombinant yarrowia lipolytica seed liquid obtained in the step (1) into a fermentation medium in an inoculum size of 1% by volume, and carrying out fermentation culture for 120 hours at a temperature of 30 ℃ and a rotating speed of 220rpm, and converting to produce gastrodin by taking p-hydroxybenzyl alcohol as a substrate to obtain a fermentation liquid; after the fermentation is finished, the content of the cannabinoid in the fermentation liquor is detected to be 717.2mg/L by a gastrodin measuring method, and the specific contents are shown in Table 1 and FIG. 2.
Comparative example 1
Gastrodin is synthesized by fermentation using P-hydroxybenzyl alcohol as substrate according to the method of example 3, except that the recombinant yarrowia lipolytica Yarrowia lipolytica po fk-Slysugt-P obtained in example 2 is UGA2 -PGM1-P FBA1 -UGP1-P TER AK9 was replaced with yarrowia lipolytica strain Yarrowia lipolytica Po fΔKu70-SlygGT obtained in preparation example 1.
After the fermentation is finished, the content of the cannabinoid in the fermentation liquor is detected to be 535.2mg/L by a gastrodin measuring method, and the content is specifically shown in Table 1 and FIG. 2.
Comparative example 2
Recombinant plasmid pYL-P was obtained as in example 1 FBA1 -AK9-P TER -PMG1-P TER UGP1, recombinant strain Yarrowia lipolytica po fk-SlysUGT-P was obtained as in example 2 FBA1 -AK9-P TER -PMG1-P TER UGP1, recombinant strain Yarrowia lipolytica po fk-SlysugT-P FBA1 -AK9-P TER -PMG1-P TER UGP1 is fermented to synthesize gastrodin by using p-hydroxybenzyl alcohol as a substrate according to the method of example 3.
After the fermentation is finished, the content of the cannabinoid in the fermentation liquor is detected to be 173.3mg/L by a gastrodin measuring method, and the specific content is shown in Table 1.
TABLE 1
Numbering device Gastrodin yield (mg/L)
Example 3 717.2
Comparative example 1 535.2
Comparative example 2 173.3
Based on the results of example 3 and comparative examples 1 and 2, it can be seen that the recombinant yarrowia lipolytica Yarrowia lipolytica po fk-Slysugt-P obtained in example 2 UGA2 -PGM1-P FBA1 -UGP1-P TER In the fermentation liquor obtained by fermenting AK9, the yield of gastrodin can be accumulated to 717.2mg/L, and is improved by 34% compared with the original strain Yarrowia lipolytica Po fDeltaKu 70-SlyUGT, so that the improvement of the intracellular yield of gastrodin in recombinant yarrowia lipolytica is realized; and compared with comparative example 2, the yield of gastrodin is improved by about 314%.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims (10)

1. A recombinant strain for synthesizing gastrodin, characterized in that the recombinant strain is obtained by genetic modification of a starting strain, and compared with the starting strain, the recombinant strain is integrated and expressed with at least one of the genes described in the following (a) - (c):
(a) From promoter P UGA2 A started phosphoglucomutase PGM1 gene;
(b) From promoter P FBA1 Initiated glucose pyrophosphorylase UGP1 gene；
(c) From promoter P TER A started adenylate kinase AK9 gene.
2. The recombinant strain of claim 1, wherein the recombinant strain is integrally expressed with the genes of (a), (b) and (c);
preferably, the promoter P UGA2 The nucleotide sequence of (2) is shown as SEQ ID NO:1, said promoter P FBA1 The nucleotide sequence of (2) is shown as SEQ ID NO:2, said promoter P TER The nucleotide sequence of (2) is shown as SEQ ID NO: 3.
3. The recombinant strain according to claim 1 or 2, wherein the phosphoglucomutase PGM1 gene encodes an amino acid sequence as set forth in SEQ ID NO:4, the amino acid sequence of the glucose pyrophosphorylase UGP1 gene is shown as SEQ ID NO:5, the amino acid sequence of the coding of the adenylate kinase AK9 gene is shown as SEQ ID NO:6 is shown in the figure;
preferably, the nucleotide sequence of the phosphoglucomutase PGM1 gene is shown in SEQ ID NO:7, the nucleotide sequence of the glucose pyrophosphorylase UGP1 gene is shown as SEQ ID NO:8, the nucleotide sequence of the adenylate kinase AK9 gene is shown as SEQ ID NO: shown at 9.
4. The recombinant strain according to claim 1 or 2, wherein the starting strain is yarrowia lipolytica;
preferably, the starting strain is yarrowia lipolytica with a knock-out gene Ku70 and integrated and expressed glycosyltransferase Slysugt gene at a Ku70 site, wherein the nucleotide sequence of the gene Ku70 is shown in SEQ ID NO:10, the nucleotide sequence of the glycosyltransferase Slysugt gene is shown as SEQ ID NO: 11.
5. A method of constructing a recombinant strain for the synthesis of gastrodin, the method comprising: genetically engineering a starting strain such that the starting strain is integrally expressed with at least one of the genes described in (a) - (c) below:
(a) From promoter P UGA2 A started phosphoglucomutase PGM1 gene;
(b) From promoter P FBA1 A initiated glucose pyrophosphorylase UGP1 gene;
(c) From promoter P TER A started adenylate kinase AK9 gene.
6. The method of claim 5, wherein the recombinant strain is integrally expressed with the genes of (a), (b) and (c);
preferably, the promoter P UGA2 The nucleotide sequence of (2) is shown as SEQ ID NO:1, said promoter P FBA1 The nucleotide sequence of (2) is shown as SEQ ID NO:2, said promoter P TER The nucleotide sequence of (2) is shown as SEQ ID NO:3 is shown in the figure;
the amino acid sequence of the phosphoglucomutase PGM1 gene is shown in SEQ ID NO:4, the amino acid sequence of the glucose pyrophosphorylase UGP1 gene is shown as SEQ ID NO:5, the amino acid sequence of the coding of the adenylate kinase AK9 gene is shown as SEQ ID NO:6 is shown in the figure;
preferably, the nucleotide sequence of the phosphoglucomutase PGM1 gene is shown in SEQ ID NO:7, the nucleotide sequence of the glucose pyrophosphorylase UGP1 gene is shown as SEQ ID NO:8, the nucleotide sequence of the adenylate kinase AK9 gene is shown as SEQ ID NO: shown at 9.
7. The method of claim 6, wherein the starting strain is yarrowia lipolytica;
preferably, the starting strain is yarrowia lipolytica with a knock-out gene Ku70 and integrated and expressed glycosyltransferase Slysugt gene at a Ku70 site, wherein the nucleotide sequence of the gene Ku70 is shown in SEQ ID NO:10, the nucleotide sequence of the glycosyltransferase Slysugt gene is shown as SEQ ID NO: 11;
preferably, the process of integrating expression comprises: first construct a promoter containing P UGA2 Started up phosphoglucoseExpression cassette and promoter P of glucose mutase PGM1 gene FBA1 Expression cassette and promoter P of the UGP1 gene of the started glucose pyrophosphorylase TER And (3) integrating and expressing three expression frames in the recombinant vector on the genome of the original strain through homologous recombination.
8. Use of a recombinant strain according to any one of claims 1 to 4 or a method according to any one of claims 5 to 7 in the synthesis of gastrodin.
9. A method for synthesizing gastrodin by fermentation, which is characterized by comprising the following steps: inoculating the recombinant strain of any one of claims 1 to 4 into a fermentation medium containing p-hydroxybenzyl alcohol for fermentation;
alternatively, a recombinant strain is constructed according to the method of any one of claims 5 to 7, and the resulting recombinant strain is inoculated into a fermentation medium containing p-hydroxybenzyl alcohol for fermentation.
10. The method of claim 9, wherein the fermentation medium further comprises a carbon source and a nitrogen source;
preferably, the fermentation medium contains: 30-50g/L of glucose, 10-30g/L of peptone, 5-20g/L of yeast powder and 0.5-1.5g/L of p-hydroxybenzyl alcohol;
the conditions of the fermentation include: the inoculation amount is 0.5-2 vol%, the temperature is 25-35 ℃, the rotating speed is 180-250rpm, and the time is 80-160h.
CN202310371074.5A 2023-04-07 2023-04-07 Recombinant strain for synthesizing gastrodin, construction method thereof and method for synthesizing gastrodin by fermentation Pending CN116478843A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310371074.5A CN116478843A (en) 2023-04-07 2023-04-07 Recombinant strain for synthesizing gastrodin, construction method thereof and method for synthesizing gastrodin by fermentation

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310371074.5A CN116478843A (en) 2023-04-07 2023-04-07 Recombinant strain for synthesizing gastrodin, construction method thereof and method for synthesizing gastrodin by fermentation

Publications (1)

Publication Number Publication Date
CN116478843A true CN116478843A (en) 2023-07-25

Family

ID=87220548

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310371074.5A Pending CN116478843A (en) 2023-04-07 2023-04-07 Recombinant strain for synthesizing gastrodin, construction method thereof and method for synthesizing gastrodin by fermentation

Country Status (1)

Country Link
CN (1) CN116478843A (en)

Similar Documents

Publication Publication Date Title
CN114480240B (en) Genetic engineering bacterium for producing fucosyllactose and production method thereof
CN110117601B (en) Grifola frondosa glucan synthase, encoding gene and application thereof
CN112708567B (en) Fructosyltransferase and high-yield strain thereof
CN113774075B (en) Escherichia coli genetically engineered bacterium and method for producing L-theanine by fermentation of escherichia coli genetically engineered bacterium
CN104131005A (en) High-ester-produced saccharomyces cerevisiae strain and method for seamlessly inserting promoter of high-ester-produced saccharomyces cerevisiae strain
US20210371843A1 (en) Genetically Engineered Bacteria, Its Construction Method And Its Application In Producing Nad+ Method
CN110804561B (en) Saccharomyces cerevisiae with high yield of C6-C10 ethyl ester and construction method and application thereof
CN106947705B (en) Gene recombination monascus purpureus M-piy strain with low-yield citrinin and high-yield monascus yellow pigment as well as preparation method and application thereof
CN109913380B (en) Recombinant yarrowia lipolytica for producing (-) -alpha-bisabolol and construction method and application thereof
CN101613707B (en) Method for producing glutathione by use of metabolic engineering bacteria
CN114507613A (en) Yeast engineering bacterium for producing alpha-santalene through fermentation and application thereof
CN112608936B (en) Promoter for regulating and controlling expression of exogenous gene of yeast, regulating and controlling method and application thereof
CN114107078A (en) High-yield valencene genetic engineering bacterium and construction method and application thereof
CN111154665B (en) Recombinant yarrowia lipolytica and construction method and application thereof
CN115838645B (en) Yeast strain for high production of orotic acid and application thereof
CN114317307B (en) Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield as well as construction method and application thereof
CN110551643A (en) Low-yield higher alcohol saccharomyces cerevisiae strain constructed by regulating proline metabolic pathway
CN116478843A (en) Recombinant strain for synthesizing gastrodin, construction method thereof and method for synthesizing gastrodin by fermentation
CN115948265A (en) Kluyveromyces marxianus haploid yeast and construction method and application thereof
CN114736918A (en) Recombinant escherichia coli for producing salidroside through integrated expression and application thereof
CN108913732B (en) Method for heterologous production of monacolin J and application
CN116478844A (en) Recombinant strain for synthesizing sakura extract, construction method thereof, and method for synthesizing sakura extract by fermentation and application
CN115058350B (en) Method for improving S-adenosylmethionine yield by introducing potassium ion transporter
CN112011473B (en) Genetic engineering bacterium with low-yield 2-phenethyl alcohol and application thereof
CN104004082A (en) Extraction method of recombinant human interleukin-2 fermentation inclusion body

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