Resveratrol production strain and construction method and application thereof

Abstract

This invention provides a resveratrol-producing strain, its construction method, and its application. The strain upregulates tyrA. fbr , tyrB, aroE, pntAB, ompF, aroG fbr The accA gene was knocked out, the carbon storage regulator csrA gene was eliminated, and the transcriptional level of the fabF gene was downregulated through the BBa_J23113 promoter, resulting in heterologous expression of the source gene on the genome. Rhodotorula glutinis TAL gene, Arabidopsis thaliana The 4CL gene, and derived from Vitis vinifera The strain carries the STS gene and also carries the high-copy plasmid PET-28a-WAL. The strain uses glucose as a carbon source to synthesize resveratrol efficiently and stably from scratch using fermentation, achieving high-efficiency production of resveratrol with low production cost.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and fermentation engineering technology, and in particular to a resveratrol-producing strain, its construction method, and its application. Background Technology

[0002] Resveratrol is a non-flavonoid polyphenolic compound with a stilbene structure, found in plants such as grapes, peanuts, mulberries, and Japanese knotweed. Its chemical name is 3,5,4-trihydroxy-catechol. Its chemical formula is C2. 14 H 12 O3, an antitoxin produced by plants, can combat diseases related to aging and oxidation, such as cancer, cardiovascular disease, and nervous system disorders. It has broad application prospects in the food, health product, and cosmetic industries.

[0003] Currently, the main methods for synthesizing resveratrol are plant extraction, chemical synthesis, enzymatic catalysis, and microbial fermentation. Plant extraction suffers from drawbacks such as long growth cycles, low yields, significant environmental impact, and high extraction costs. Chemical synthesis is characterized by high energy consumption, numerous byproducts, low yields, and high pollution. Enzymatic catalysis, using 4-coumaryl-CoA ligase and stilbene synthase as key enzymes, catalyzes the conversion of p-coumaric acid to resveratrol. This method is less environmentally polluting and has milder reaction conditions, but it requires the addition of p-coumaric acid as a substrate, increasing production costs. Microbial fermentation uses glucose or glycerol as the energy source for microbial growth, synthesizing resveratrol de novo. However, currently, microbial synthesis of resveratrol using glycerol as a substrate is costly, and using glucose as a carbon source results in long fermentation cycles, low sugar conversion efficiency, and low yields, hindering industrial production. Therefore, developing a genetically engineered bacterium that uses glucose as a substrate for inexpensive and efficient resveratrol production has significant practical value. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a resveratrol-producing strain.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned resveratrol-producing strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned resveratrol producing strain.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0008] A plasmid, PET-28a-WAL, has a nucleotide sequence as shown in SEQ ID NO.17 of the sequence listing.

[0009] The aforementioned plasmid PET-28a-WAL, derived from plasmid pET28a, carries plasmid elements such as a replication origin site, kanamycin resistance gene, T7 promoter, and terminator, and also carries elements derived from... Rhodotorula glutinis TAL gene, derived from Arabidopsis thaliana The 4CL gene and derived from Vitis vinifera The STS genes were all transcribed using the T7 promoter for enhanced transcription.

[0010] The above plasmids were constructed using ClonExpress® rapid cloning technology from Nanjing Novizan Biotechnology Co., Ltd.: cloning sites were selected, and the pET28a linear vector was amplified using reverse PCR. The pET28a linear vector was then ligated with the cloning fragments T7-TAL, 4CL, and STS using ClonExpress® recombinase to obtain the new plasmid PET-28a-WAL.

[0011] A resveratrol-producing strain, strain WAL13, is used to... E.coli W3110, as a chassis strain, upregulated tyrA. fbr ,tyrB,aroE,pntAB,ompF,aroG fbr accA gene ， Knocking out the csrA gene, a carbon storage regulator, downregulated the fabF gene, and heterologously expressed a gene derived from... Rhodotorula glutinis TAL gene, Arabidopsis thaliana The 4CL gene and derived from Vitis vinifera The STS gene also carries the aforementioned high-copy plasmid PET-28a-WAL.

[0012] Preferably, the above-mentioned resveratrol-producing strain, in order to E.coli W3110 was used as the chassis strain. The csrA gene was knocked out in the strain's genome, the fabF gene was downregulated using the BBa_J23113 promoter, and integrated into the yeeL gene locus in the genome; the tyrA gene was enhanced using the trc promoter. fbr The genes were enhanced using a trc promoter and integrated into the ycgH gene locus in the genome; the tyrB gene was enhanced using a trc promoter and integrated into the ylbE gene locus in the genome; the aroE gene was enhanced using a trc promoter and integrated into the ilvG gene locus in the genome; the pntAB gene was enhanced using a trc promoter and integrated into the ygaY gene locus in the genome; the ompF gene was enhanced using a trc promoter and integrated into the Yjgx gene locus in the genome; and the aroG gene was enhanced using a trc promoter. fbr The gene was enhanced using a TRC promoter and integrated into the mbhA gene locus in the genome; the accA gene was enhanced using a TRC promoter and integrated into the rph gene locus in the genome; the gene derived from...Rhodotorula glutinis The TAL gene was transcribed and integrated into the gapC gene locus in the genome; the trc promoter was used to enhance the expression of the gene derived from... Arabidopsis thaliana The 4CL gene was transcribed and integrated into the yeeP gene locus in the genome; the trc promoter was used to enhance the expression of the gene derived from... Vitis vinifera The STS gene is transcribed and integrated into the ycdN gene locus in the genome.

[0013] Preferably, the above-mentioned resveratrol-producing strain, wherein E.coli W3110 is E.coli W3110 ATCC 27325.

[0014] Preferably, in the above-mentioned resveratrol-producing strain, the nucleotide sequence of the csrA gene is shown in SEQ ID NO.1, and the nucleotide sequence of the fabF gene is shown in SEQ ID NO.2. fbr The nucleotide sequences of the following genes are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively. fbr The nucleotide sequences of the genes are shown in SEQ ID NO. 8, the accA gene in SEQ ID NO. 9, the TAL gene in SEQ ID NO. 10, the 4CL gene in SEQ ID NO. 11, the STS gene in SEQ ID NO. 12, the plasmid pET28a in SEQ ID NO. 13, the BBa_J23113 promoter in SEQ ID NO. 14, the trc promoter in SEQ ID NO. 15, the T7 promoter in SEQ ID NO. 16, and the PET-28a-WAL nucleotide sequence in SEQ ID NO. 17.

[0015] The above method for constructing resveratrol-producing strains involves starting with the strain... E.coli W3110 Based on this foundation, targeted modifications will be carried out, with the specific steps as follows:

[0016] (1) Modification of the chassis bacteria to open up and enhance the carbon pathway for resveratrol synthesis: tyrA was expressed by using the trc strong promoter. fbr tyrB, aroE, aroG fbrThe transcription levels of the TAL, 4CL, and STS genes were greatly increased.

[0017] (2) Increase carbon flux: Knock out the csrA gene, a carbon storage regulator, to enhance carbon source mobility;

[0018] (3) Increase reducing power and NADPH content: Overexpression of the pntAB gene by the trc promoter increases the transcription level, which increases the supply of NADPH, enhances reducing power, and is conducive to the efficient production of the final product;

[0019] (4) Optimize the malonyl-CoA supply system and improve the conversion rate: Overexpress the accA gene using the trc promoter to efficiently utilize carbon source to produce a large amount of malonyl-CoA, providing a large amount of precursors for the resveratrol synthesis pathway and accumulating the product;

[0020] (5) Weakening malonyl-CoA catabolism: The fabF gene is weakly expressed by the BBa_J23113 promoter to inhibit fatty acid synthesis, reduce malonyl-CoA catabolism, weaken resveratrol catabolism, and enable resveratrol to accumulate efficiently.

[0021] (6) Optimize the resveratrol transport system: Overexpress the ompF efflux protein gene using the trc promoter;

[0022] (7) Transformation of PET-28a-WAL plasmid system: Enhance resveratrol synthesis pathway.

[0023] The application of the above-mentioned resveratrol-producing strains in the fermentation production of resveratrol.

[0024] Preferably, in the application of the above-mentioned resveratrol-producing strain, the strain is fermented in a culture medium, which includes, but is not limited to, carbon sources, nitrogen sources, inorganic salts, vitamins, etc.; the fermentation conditions include fermentation temperature, fermentation pH, fermentation dissolved oxygen conditions, fermentation pressure, fermentation time, etc.

[0025] Preferably, the application of the above-mentioned resveratrol-producing strains follows these specific steps:

[0026] ① Slant culture: Inoculate the resveratrol producing strain onto a slant culture medium and incubate at 32-35℃ for 12-16 hours;

[0027] ② Shake flask seed culture: Take solid slant inoculum and inoculate it into shake flask culture medium for fermentation. The culture temperature is 32-35℃, the culture time is 12-20h, the shaking speed is 200-240r / min, and the pH is 6.4-6.7.

[0028] ③ Shake flask fermentation culture: The inoculum size is 15-20%, the culture temperature is 32-35℃, the pH is 6.4-6.7, the culture time is 24-36h, and the shaker speed is 200-240r / min.

[0029] Preferably, in the application of the above-mentioned resveratrol-producing strain, the slant culture medium used in step ① is the general-purpose LB solid medium.

[0030] Preferably, in the application of the above-mentioned resveratrol producing strain, the seed culture medium used in step ② is: glucose 30g / L, yeast 5g / L, peptone 3g / L, (NH4)2SO4 1g / L, KH2PO4·3H2O 2g / L, MgSO4·7H2O 1g / L, citric acid 3g / L, and glutamic acid 3g / L.

[0031] Preferably, in the application of the above-mentioned resveratrol-producing strain, the fermentation medium used in step ③ is: glucose 15g / L, yeast powder 4g / L, peptone 2g / L, (NH4)2SO4 1.5g / L, KH2PO4·3H2O 3.5g / L, MgSO4·7H2O 2g / L, glutamic acid 3g / L, citric acid 3g / L, FeSO4·7H2O 20mg / L, MnSO4·H2O 10mg / L.

[0032] Preferably, in the application of the above-mentioned resveratrol-producing strain, the fermentation medium used in step ③ is: glucose 15g / L, yeast powder 4g / L, peptone 2g / L, (NH4)2SO4 1.5g / L, KH2PO4·3H2O 3.5g / L, MgSO4·7H2O 2g / L, glutamic acid 3g / L, citric acid 3g / L, FeSO4·7H2O 20mg / L, MnSO4·H2O 10mg / L, pyridoxal phosphate 3mg / L, and betaine 1.2g / L.

[0033] All of the above-mentioned culture media can be prepared using standard methods.

[0034] Beneficial effects:

[0035] The above-mentioned resveratrol-producing strains exhibit good resveratrol synthesis ability, stable performance, high acid production efficiency, and upregulation of tyrA. fbr ,tyrB,aroE,pntAB,ompF,aroG fbr The accA gene was knocked out, the carbon storage regulator csrA gene was removed, and the fabF gene was downregulated through the BBa_J23113 promoter, resulting in heterologous expression of the gene derived from [a specific gene]. Rhodotorula glutinis TAL gene, Arabidopsis thaliana The 4CL gene, and derived from Vitis viniferaThe STS gene, along with the plasmid PET-28a-WAL, was overexpressed using the T7 promoter and heterologously expressed from [a specific gene / product]. Rhodotorula glutinis TAL gene, Arabidopsis thaliana The 4CL gene, and derived from Vitis vinifera The STS gene of this strain. The acetyl-CoA carboxylase of this strain is derived from the accA gene of E. coli W3110, which can efficiently produce malonyl-CoA as a raw material for resveratrol synthesis. The 3-oxoacyl-(acyl carrier protein) synthase II of this strain is derived from the fabF gene of E. coli W3110, which reduces carbon source loss by inhibiting fatty acid synthesis. The key enzymes of the resveratrol synthesis pathway are expressed using a highly stable plasmid system, opening up the synthesis pathway from tyrosine to resveratrol. This allows both the resveratrol and tyrosine metabolic pathways to function simultaneously in the engineered strain, resulting in efficient resveratrol accumulation. This effectively improves the efficiency of resveratrol synthesis, increases resveratrol production levels, and achieves high-efficiency resveratrol production, showing excellent industrial application prospects. Specifically:

[0036] By knocking out the carbon storage regulator csrA gene, the supply of carbon sources was either unblocked or increased, effectively reducing the catabolism of tyrosine and achieving efficient accumulation of the substrate tyrosine. Upregulation of the tyrB, aroE, and pntAB genes promoted the accumulation of substrate tyrosine. Targeted mutations in the tyrA and aroG genes were performed to remove the negative feedback inhibition of tyrosine, thereby achieving the goal of substrate tyrosine accumulation.

[0037] The three exogenous genes TAL, 4CL, and STS were upregulated, completely opening up the pathway from precursor tyrosine to resveratrol.

[0038] (3) By weakening the fabF gene and enhancing the transcription level of the accA gene, the fatty acid synthesis pathway was effectively inhibited, and the accumulation of malonyl-CoA, a raw material for resveratrol synthesis, was promoted. By increasing the transcription level of the ompF gene, the extracellular transport of 5-resveratrol was enhanced.

[0039] (4) By constructing the PET-28a-WAL plasmid, the T7 promoter on the plasmid is overexpressed from the TAL gene of Rhodotorula glutinis, the 4CL gene of Arabidopsis thaliana, and the STS gene of Vitisvinifera, thus opening up the synthesis pathway from tyrosine to resveratrol, enabling resveratrol to accumulate efficiently. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the structure of plasmid pET28a.

[0041] Figure 2 This is a schematic diagram of the structure of plasmid PET-28a-WAL.

[0042] Figure 3 This diagram illustrates the process of modifying the de novo synthesis pathway of resveratrol-producing genetically engineered bacteria. Detailed Implementation

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0044] Unless otherwise specified, the percentage sign "%" used in the examples refers to the mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0045] The starting strain used in the examples was wild-type. E.coli W3110ATCC 27325 (available through the ATCC cell bank), the corresponding promoter and gene are listed in the sequence listing. Primers used in the construction of the involved strains are listed in Table 1.

[0046] Table 1 Primers used in strain construction

[0047] Primer Name Sequence Number Primer Sequence (5'-3') pGRB-csrA-S SEQ ID NO.18 AGTCCTAGGTATAATACTAGTCTCAACTACCCACAGTTGTTGTTTTAGAGCTAGAA pGRB-csrA-A SEQ ID NO.19 TTCTAGCTCTAAAACAACAACTGTGGGTAGTTGAGACTAGTATTATACCTAGGACT csrA-US SEQ ID NO.20 AATGAACGGGAGTAAAGCG csrA-UA SEQ ID NO.21 ATTGGGGATGAGGTCACCGATCTACCAGCGTATCCAGG csrA-DS SEQ ID NO.22 CCTGGATACGCTGGTAGATCGGTGACCTCATCCCCAAT csrA-DA SEQ ID NO.23 TATGGTCGCTCAGCAGGTG pGRB-yeeL-S SEQ ID NO.24 AGTCCTAGGTATAATACTAGTTATGCGTCTGAACGACCGTGGTTTTAGAGCTAGAA pGRB-yeeL-A SEQ ID NO.25 TTCTAGCTCTAAAACCACGGTCGTTCAGACGCATAACTAGTATTATACCTAGGACT yeeL-US SEQ ID NO.26 TAAACTCGTCAGCGGCACAAC yeeL-UA SEQ ID NO.27 ATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTAGGCGTTTCTGTTGATTCTGAA yeeL-DS SEQ ID NO.28 TTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCGTGTCGGATTATCGTTCGA yesL-YES SEQ ID NO.29 GATTCAGGTTGCCATTTACGCCA fabF-S SEQ ID NO.30 CTAGCTCAGTCCTAGGGACTGTGCTAGCAGGAAACAGACCGTGTCTAAGCGTCGTGTGTAGT fabF-A SEQ ID NO.31 AACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTTCTTTGATCTTTAAAAAGATCTA pGRB-ycgH-S SEQ ID NO.32 ACCCAACCTTACGCAACCAGTTAA pGRB-ycgH-A SEQ ID NO.33 ATTGTTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCGCAGCATTG ycgH-US SEQ ID NO.34 ACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAACA ycgH-UA SEQ ID NO.35 GGGCGTAACTCAGCAGGCAG ycgH-DS SEQ ID NO.36 AGTCCTAGGTATACTAGTTATCGGCACTGACGCATTTCGTTTTAGAGCTAGAA ycgH-DA SEQ ID NO.37 TTCTAGCTCTAAAACGAAATGCGTCAGTGCCGATAACTAGTATTATACCTAGGACT <![CDATA[tyrA fbr -S]]> SEQ ID NO.38 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACAGGAAACAGACCATGGTTGCTGAATTGACCGC <![CDATA[tyrA fbr -A]]> SEQ ID NO.39 GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACTGGCGATTGTCATTCGC pGRB-ylbE-S SEQ ID NO.40 AGTCCTAGGTATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA pGRB-ylbE-A SEQ ID NO.41 TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTATTATACCTAGGACT ylbE-US SEQ ID NO.42 ACCCAACCTTACGCAACCAGTTAA ylbE-UA SEQ ID NO.43 ATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCGCAGCATTG ylbE-DS SEQ ID NO.44 ACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAACA ylbE-DA SEQ ID NO.45 GGGCGTAACTCAGCAGGCAG tyrB-S SEQ ID NO.46 GCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCGTGTTTCAAAAAGTTGACGCCTAC tyrB-A SEQ ID NO.47 CGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACATCACCGCAGCAAACG pGRB-ilvG-S SEQ ID NO.48 AGTCCTAGGTATAATACTAGTTATCGGCACTGACGCATTTCGTTTTAGAGCTAGAA pGRB-ilvG-A SEQ ID NO.49 TTCTAGCTCTAAAACGAAATGCGTCAGTGCCGATAACTAGTATTATACCTAGGACT ilvG-US SEQ ID NO.50 CCGAGGAGCAGACAATGAATAACAG ilvG-UA SEQ ID NO.51 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGGTGATGGCAACAACAGGG ilvG-DS SEQ ID NO.52 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCTATCTACGCGCCGTTGTTG ilvG-DA SEQ ID NO.53 GAAGGCGCTGGCTAACATGAGG aroE-S SEQ ID NO.54 ATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGAAACCTATGC aroE-A SEQ ID NO.55 CCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTCACGCGGACA pGRB-ygaY-S SEQ ID NO.56 AGTCCTAGGTATAATACTAGTCTCAACTACCCACAGTTGTTGTTTTAGAGCTAGAA pGRB-ygaY-A SEQ ID NO.57 TTCTAGCTCTAAAACAACAACTGTGGGTAGTTGAGACTAGTATTATACCTAGGACT ygaY-US SEQ ID NO.58 CCTACAAACCACATCGCACATT ygaY-UA SEQ ID NO.59 GCTAGCACAGTCCCTAGGACTGAGCTAGCTGTAAAACACCGAAGCAACCCAAAAGA ygaY-DS SEQ ID NO.60 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCTTGCCGCTCCACCTTTTA ygaY-DA SEQ ID NO.61 GGAGTAGGGCTTTCCATAGAGTGT pntAB-S SEQ ID NO.62 CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCGAATTGGCATACCAAGAG pntAB-A SEQ ID NO.63 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGAGCTTTCAGGATTGCATCCAC pGRB-yjgX-S SEQ ID NO.64 AGTCCTAGGTATAATACTAGTTCGCGACCACCGTAACTGGCGTTTTAGAGCTAGAA pGRB-yjgX-A SEQ ID NO.65 TTCTAGCTCTAAAACGCCAGTTACGGTGGTCGCGAACTAGTATTATACCTAGGAC yjgX-US SEQ ID NO.66 GGAAGTCAACGGGTTATGCGG yjgX-UA SEQ ID NO.67 TTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAATCACCACGAATACCAGAATCGC yjgX-DS SEQ ID NO.68 ACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACAGTGTCTTCCCTGAGCCG yjgX-DA SEQ ID NO.69 GGCGAAGGATACCATCAAGCTG OmpF-S SEQ ID NO.70 GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGATGAAGCGCAATATTCTGGC OmpF-A SEQ ID NO.71 CTGTGGGTATCGTTTACCAGTTCTAACAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCT pGRB-mbhA-S SEQ ID NO.72 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACACGGTGGCAGGTTTTGG pGRB-mbhA-A SEQ ID NO.73 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC mbhA-US SEQ ID NO.74 GCCAGCACGAACATAATCCC mbhA-UA SEQ ID NO.75 GGTCTGTTTCCTGCTAGCACTATACCTAGGACTGAGCTAGCCGTAAACACGGTGGCAGGTTTTGG mbhA-DS SEQ ID NO.76 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC mbhA-DA SEQ ID NO.77 CGGCGTAATCACAAACTGGC <![CDATA[aroG fbr -S]]> SEQ ID NO.78 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAATTATCAGAACGACGATTTACG <![CDATA[aroG fbr -A]]> SEQ ID NO.79 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACCCGCGACGCGCTTT pGRB-rph-S SEQ ID NO.80 AGTCCTAGGTATATACTAGTTGCGACGTGCTTCAGGCTGAGTTTTAGAGCTAGAA pGRB-rph-A SEQ ID NO.81 TTCTAGCTCTAAAACTCAGCCTGAAGCACGTCGCAACTAGTATTATACCTAGGACT rph-US SEQ ID NO.82 ATAGCGCAGGGTACATTCCACT rph-UA SEQ ID NO.83 TTGTTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCTTCTTCAATAGAGGCGGTACAC rph-DS SEQ ID NO.84 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTGCCGCAGAGACCGACGACATGAA rph-DA SEQ ID NO.85 ACAGCGGTTGTGGTGGCAAT accA-S SEQ ID NO.86 GAATTGTGAGCGGATAACAATTTCACAGGAAACAGACCATGAGTCTGAATTTCCTTGATTTTGAACAG accA-A SEQ ID NO.87 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACGCGTAACCGTAGCTCATC pGRB-gapC-S SEQ ID NO.88 AGTCCTAGGTATAATACTAGTAGGTCATGCGCAACGCGTGCGTTTTAGAGCTAGAA pGRB-gapC-A SEQ ID NO.89 TTCTAGCTCTAAAACGCACGCGTTGCGCATGACCTACTAGTATTATACCTAGGACT gapC-US SEQ ID NO.90 TGGGAAGAAACCACGAAACTCCA gapC-UA SEQ ID NO.91 TATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATGTTTCAGCAGGTAGGCGAGAA gapC-DS SEQ ID NO.92 TGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATAAAACGGTCGCCTGGTACG gapC-DA SEQ ID NO.93 TTATCCGCCGACATTGCTGC TAL-S SEQ ID NO.94 ATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGCGCCGCGCCCGACGAG TAL-A SEQ ID NO.95 CAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTATTTGTTAGGCTAACATTTTCAGCAGCAG pGRB-yeP-S SEQ ID NO.96 AGTCCTAGGTATAATACTAGTTCCCCTGATGACCCGACTGCGTTTTAGAGCTAGAA pGRB-andP-A SEQ ID NO.97 TTCTAGCTCTAAAACGCAGTCGGGTCATCAGGGGAACTAGTTATACCTAGGACT and P-US SEQ ID NO.98 GGTCAGGGTAACTTATCAGCG and P-UA SEQ ID NO.99 CCACACATTATACGAGCCGGATGATTAATTGTCAAATGGCAGGGCTCCGTTTT and P-DS SEQ ID NO.100 CGAAAGACTGGGCCTTTCGTTATCTCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGCAAATGAACTGGATTTTCTCTCTGAACCTGT and P-DA SEQ ID NO.101 ACGATGTCAGCAGCCAGCA 4CL-S SEQ ID NO.102 TATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGCGCCGCAAGAAAG 4CL-A SEQ ID NO.103 ACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGGCCGTTCGCCAGT pGRB-ycdN-S SEQ ID NO.104 AGTCCTAGGTATAATACTAGTGCGTGGAAATCATCATGGCTGTTTTAGAGCTAGAA pGRB-ycdN-A SEQ ID NO.105 TTCTAGCTCTAAAACAGCCATGATGATTTCCACGCACTAGTATTATACCTAGGACT ycdN-US SEQ ID NO.106 GATTTTGACGCCACCAACACC ycdN-UA SEQ ID NO.107 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAATCCACATCACACAATCCATC ycdN-DS SEQ ID NO.108 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAAGGGATTTTTGGCTATCAGG ycdN-DA SEQ ID NO.109 GTATTCGCCAGGCTGTAAATTC STS-S SEQ ID NO.110 GAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAATACAATTAATGAATATCTTAGTCTAGAAGA STS-A SEQ ID NO.111 ACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAAATAATCGGCACGCTGCG TAL-pet-S SEQ ID NO.112 TTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGCGCCGCGCCCGA TAL-pet-A SEQ ID NO.113 CCGCTTGTTCTTGCGGCGCCATTCTCCTTCTTAAAGTTAAACAAATTAGGCTAACATTTTCAGCAGCACG 4CL-pet-S SEQ ID NO.114 TTTGTTTAACTTTAAGAAGGAGAATGGCGCCGCAAGAAC 4CL-pet-A SEQ ID NO.115 [[ID=3j]]ATTGAGGCCATGGTATATCTCCTTTTACAGGCCGTTCGCCAGTTT STS-pet-S SEQ ID NO.116 / / 原内容此处有误,已修正为116 AAGGAGATATACCATGGCCTCAATAGAAGAATTTAGAAA STS-pet-A SEQ ID NO.117

[0048] like ACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTAGTTCGTCACGGTTGGTATGC As shown, a resveratrol-producing strain was constructed using the following four modules:

[0049] (1) Chassis bacteria modification: improve tyrA fbr Increase the transcriptional level of the tyrB gene, increase the transcriptional level of the aroE gene, and increase the transcriptional level of aroG. fbr The transcriptional level of genes was increased, the transcriptional level of TAL gene was increased, the transcriptional level of 4CL gene was increased, and the transcriptional level of STS gene was increased.

[0050] (2) Increase carbon flux: Knock out the carbon storage regulator csrA gene to enhance carbon source mobility.

[0051] (3) Increase reducing power and NADPH content: Overexpression of the pntAB gene by the trc promoter increases the transcription level, which increases the supply of NADPH, enhances reducing power, and is conducive to the efficient production of the final product;

[0052] (4) Optimize the malonyl-CoA supply system and improve the conversion rate: Overexpress the accA gene using the trc promoter to efficiently utilize carbon source to produce a large amount of malonyl-CoA, providing a large amount of precursors for the resveratrol synthesis pathway and accumulating the product;

[0053] (5) Weakening malonyl-CoA catabolism: By using the BBa_J23113 promoter to downregulate the fabF gene, fatty acid synthesis is inhibited, malonyl-CoA catabolism is reduced, and resveratrol catabolism is weakened, so that resveratrol can be accumulated efficiently.

[0054] (6) Optimize the resveratrol transport system: Overexpress the ompF efflux protein gene using the trc promoter;

[0055] (7) Transformation of PET-28a-WAL plasmid: Enhancement of resveratrol synthesis pathway (e.g.) Figure 3 As shown, it is specifically noted that, except for the plasmid elements specifically mentioned in this invention, other elements shown in the diagram can be replaced by any known equivalent effect element. The PET-28a-WAL plasmid is composed of plasmid pET28a (e.g., ...). Figure 2 Figure 1 (As shown) It was modified and carries plasmid elements such as the replication start site, kanamycin resistance gene, T7 promoter, and terminator, and also carries elements derived from... Rhodotorula glutinis TAL gene, Arabidopsis thaliana The 4CL gene and derived from Vitis vinifera The STS genes, all of which were transcribed using the T7 promoter for enhanced transcription;

[0056] The gene editing methods used in the above gene manipulations are referenced in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genomeediting. Metabolic Engineering, 2015, 31: 13-21.). Unless otherwise specified, all technical terms used in this invention are explained in this article. "Introduction" refers to the insertion of a foreign gene into the genome of an engineered bacterium after linking it with a promoter and terminator.

[0057] Example 1

[0058] This embodiment aims to illustrate the specific construction steps of strain WAL13. In particular, if there are similar gene manipulation methods in the embodiment, they will only be provided once and annotated, without further elaboration.

[0059] (1) Knockout of csrA gene: Using the E. coli W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using csrA-US, csrA-UA and csrA-DS, csrA-DA respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were obtained by overlapping PCR amplification using csrA-US and csrA-DA as primers. Using pGRB-csrA-S and pGRB-csrA-A as primers, gRNA fragments were annealed and ligated with the pGRB vector to obtain csrA-pGRB. E. coli W3110 electroporation competent cells were prepared, and the overlapping fragments and csrA-pGRB were electroporated into competent cells together. Positive transformants were screened to obtain strain WAL1.

[0060] (2) Downregulation of the fabF gene at the yeeL pseudogene locus using the BBa_J23113 promoter: E. coli W3110 Using the genome as a template, upstream and downstream homologous arms were amplified by PCR using yeeL-US, yeeL-UA, yeeL-DS, and yeeL-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were amplified by PCR using fabF-S and fabF-A primers. Annealing was then performed using pGRB-yeeL-S and pGRB-yeeL-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain yeeL-pGRB. Preparation... E. coli W3110 The overlapping fragment was electrotransformed into competent cells along with yeeL-pGRB, and positive transformants were obtained by screening to obtain strain WAL2.

[0061] (3) Use the trc promoter to control tyrA at the ycgH pseudogene site. fbr Gene overexpression: E. coli W3110 Using the genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using ycgH-US, ycgH-UA, ycgH-DS, and ycgH-DA, respectively. Then, using the upstream and downstream homologous arms as templates, tyrA... fbr -S、tyrA fbr Using primer A, overlapping fragments were amplified by overlapping PCR to obtain overlapping fragments; using pGRB-ycgH-S and pGRB-ycgH-A as primers, gRNA fragments were annealed to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain ycgH-pGRB; preparation E. coli W3110 The overlapping fragment was electroporated into competent cells along with ycgH-pGRB, and positive transformants were obtained by screening to obtain strain WAL3.

[0062] (4) Controlling tyrB gene overexpression at the ylbE pseudogene site using the trc promoter: E. coli Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using ylbE-US, ylbE-UA, ylbE-DS, and ylbE-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were obtained by PCR amplification using tyrB-S and tyrB-A primers. Annealing was then performed using pGRB-ylbE-S and pGRB-ylbE-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain ylbE-pGRB. Preparation... E. coli W3110 The overlapping fragments were electrotransformed into competent cells along with ylbE-pGRB, and positive transformants were screened to obtain strain WAL4.

[0063] (5) Controlling aroE gene overexpression at the ilvG pseudogene site using the trc promoter: E. coli Using the W3110 genome as a template, upstream and downstream homologous arms were amplified by PCR using ilvG-US, ilvG-UA, ilvG-DS, and ilvG-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were amplified by PCR using aroE-S and aroE-A primers. Annealing was then performed using pGRB-ilvG-S and pGRB-ilvG-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain ilvG-pGRB. Preparation... E. coli W3110 Competent cells were electrotransformed, and the overlapping fragment was electrotransformed together with ilvG-pGRB into competent cells. Positive transformants were then selected to obtain strain WAL5.

[0064] (6) Controlling pntAB gene overexpression at the ygaY pseudogene site using the trc promoter: E. coli W3110 Using the genome as a template, upstream and downstream homologous arms were amplified by PCR using ygaY-US, ygaY-UA, ygaY-DS, and ygaY-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were amplified by PCR using pntAB-S and pntAB-A primers. Annealing was then performed using pGRB-ygaY-S and pGRB-ygaY-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain ygaY-pGRB. Preparation... E. coli W3110 The overlapping fragment was electrotransformed into competent cells along with ygaY-pGRB, and positive transformants were obtained by screening to obtain strain WAL6.

[0065] (7) Controlling ompF gene overexpression at the Yjgx pseudogene site using the trc promoter: E. coli W3110 Using the genome as a template, upstream and downstream homologous arms were amplified by PCR using Yjgx-US, Yjgx-UA, Yjgx-DS, and Yjgx-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were amplified by overlapping PCR using ompF-S and ompF-A primers. Annealing was then performed using pGRB-Yjgx-S and pGRB-Yjgx-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain Yjgx-pGRB. Preparation... E. coli W3110 The overlapping fragment was electrotransformed into competent cells along with Yjgx-pGRB, and positive transformants were obtained by screening to obtain strain WAL7.

[0066] (8) Use the trc promoter to control the mbhA pseudogene site. aroG fbr Gene overexpression: E. coli W3110 Using the genome as a template, upstream and downstream homologous arms were obtained by PCR amplification of mbhA-US, mbhA-UA, mbhA-DS, and mbhA-DA, respectively. Then, using the upstream and downstream homologous arms as templates, aroG... fbr -S、aroG fbr Using primer A, overlapping fragments were amplified by overlapping PCR to obtain overlapping fragments; using pGRB-mbhA-S and pGRB-mbhA-A as primers, gRNA fragments were annealed to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain mbhA-pGRB; preparation E. coli W3110 electroporated competent cells, and the overlapping fragment and mbhA-pGRB were electroporated into competent cells together. Positive transformants were obtained by screening, and strain WAL8 was obtained.

[0067] (9) Controlling accA gene overexpression at the rph pseudogene site using the trc promoter: E. coli W3110 Using the genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using rph-US, rph-UA, rph-DS, and rph-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were obtained by overlapping PCR amplification using accA-S and accA-A primers. Annealing was performed using pGRB-rph-S and pGRB-rph-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain rph-pGRB. Preparation... E. coli W3110 electroporated competent cells, and the overlapping fragment was electroporated together with rph-pGRB into competent cells. Positive transformants were then screened to obtain strain WAL9.

[0068] (10) Controlling TAL gene overexpression using the trc promoter at the gapC pseudogene site: Rhodotorula glutinis Using the genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using gapC-US, gapC-UA, gapC-DS, and gapC-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were obtained by PCR amplification using TAL-S and TAL-A primers. Annealing was then performed using pGRB-gapC-S and pGRB-gapC-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain gapC-pGRB. Preparation... E. coli W3110 Competent cells were electrotransformed, and the overlapping fragments were electrotransformed together with gapC-pGRB into competent cells. Positive transformants were then screened to obtain strain WAL10.

[0069] (11) Controlling 4CL gene overexpression at the yeeP pseudogene site using the trc promoter: Arabidopsis thaliana Using the genome as a template, upstream and downstream homologous arms were amplified by PCR using yeeP-US, yeeP-UA, yeeP-DS, and yeeP-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were amplified by overlapping PCR using 4CL-S and 4CL-A primers. Annealing was then performed using pGRB-yeeP-S and pGRB-yeeP-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain yeeP-pGRB. Preparation... E. coli W3110 Competent cells were electrotransformed, and the overlapping fragment was electrotransformed together with yeeP-pGRB into competent cells. Positive transformants were then screened to obtain strain WAL11.

[0070] (12) Controlling STS gene overexpression at the ycdN pseudogene site using the trc promoter: Vitis vinifera Using the genome as a template, upstream and downstream homologous arms were amplified by PCR using ycdN-US, ycdN-UA, ycdN-DS, and ycdN-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were amplified by overlapping PCR using STS-S and STS-A primers. Annealing was then performed using pGRB-ycdN-S and pGRB-ycdN-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain ycdN-pGRB. Preparation... E. coli W3110 The overlapping fragment was electrotransformed into competent cells along with ycdN-pGRB, and positive transformants were obtained by screening to obtain strain WAL12.

[0071] (13) Transformation of plasmid PET-28a-WAL to obtain engineered bacteria WAL13: Transform the complete plasmid PET-28a-WAL into WAL12 competent cells by electrotransformation (or chemical transformation, etc.) to obtain engineered bacteria WAL13.

[0072] Example 2

[0073] This embodiment aims to illustrate the construction method of plasmid PET-28a-WAL in Example 1. The specific steps are as follows:

[0074] Using the Rhodotorula glutinis genome as a template, the TAL gene was amplified using TAL-pet-S and TAL-pet-A primers; using the Arabidopsis thaliana genome as a template, the 4CL gene was amplified using 4CL-pet-S and 4CL-pet-A primers; and using the Grapevine genome as a template, the STS gene was amplified using STS-pet-S and STS-pet-A primers. The pET28a plasmid linearized vector was amplified using reverse PCR. The gene fragments were ligated to the pET28a plasmid linearized vector using recombinase. The pET28a linearized vector was then ligated to the T7 promoter-TAL, 4CL, and STS-T7 terminator of the cloned fragment using ClonExpress® recombinase to obtain the new plasmid PET-28a-WAL, the nucleotide sequence of which is shown in SEQ ID NO.17 of the sequence listing.

[0075] Example 3

[0076] This embodiment aims to illustrate the application of engineered strain WAL13 in shake-flask fermentation. The specific steps are as follows:

[0077] ① Slant culture: Take the bacterial strain preserved in the -80℃ refrigerator and inoculate it on the slant culture medium. Incubate at 32-34℃ for 14 hours. The general LB solid medium is used as the slant culture medium.

[0078] ② Shake flask seed culture: Take solid slant inoculum and inoculate it into shake flask culture medium for fermentation. The culture temperature is 32-35℃, the culture time is 16h, the shaking speed is about 220r / min, and the pH is 6.4-6.7. The seed culture medium is glucose 30g / L, yeast 5g / L, peptone 3g / L, (NH4)2SO4 1g / L, KH2PO4·3H2O 2g / L, MgSO4·7H2O 1g / L, citric acid 3g / L, and glutamic acid 3g / L.

[0079] ③ Shake-flask fermentation culture: The inoculum size is 15-20%, the culture temperature is 32-35℃, the pH is 6.4-6.7, the culture time is 36h, and the shaker speed is about 220r / min; the fermentation medium used is: glucose 15g / L, yeast powder 4g / L, peptone 2g / L, (NH4)2SO4 1.5g / L, KH2PO4·3H2O 3.5g / L, MgSO4·7H2O 2g / L, glutamic acid 3g / L, citric acid 3g / L, FeSO4·7H2O 20mg / L, MnSO4·H2O 10mg / L.

[0080] wild type E. coli W3110 As a control group, after 36 days of fermentation verification, the wild type... E. coli W3110 Resveratrol failed to accumulate. However, the engineered strain WAL13 described in Example 1 accumulated 0.6 g / L of resveratrol, demonstrating the effectiveness of the strain.

[0081] Example 4

[0082] Using strain WAL13 as the production strain, this example aims to illustrate the effects of pyridoxal phosphate and betaine in resveratrol fermentation. Four control groups were set up based on the presence or absence of pyridoxal phosphate and betaine in a 60% glucose solution as the fermentation carbon source. This invention discloses data from four shake-flask fermentations over 36 hours, and the results are shown in Table 2:

[0083] Table 2 Effects of PLP and betaine on bacterial biomass and resveratrol yield

[0084] Group 1 Group 2 Group 3 Group 4 Pyridoxal phosphate dosage (mg / L) 0 0 3 3 Betaine addition amount (g / L) 0 1.2 0 1.2 <![CDATA[Cell biomass OD 600nm > 62 67.4 65.3 68.8 Resveratrol yield (g / L) 0.6 1.2 1.9 2.3

[0085] The results showed that the addition of either PLP or betaine to the fermentation carbon source could increase the yield of resveratrol. The yield of the target product was highest when both substances were added at the same time, accumulating to a total of 2.3 g / L.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A resveratrol-producing strain, characterized in that: by E. coli W3110 ATCC 27325 was used as the chassis strain, and tyrA was upregulated using the trc promoter. fbr ,tyrB,aroE,pntAB,ompF,aroG fbr accA gene ， The csrA gene, a carbon storage regulator, was knocked out, and the fabF gene was downregulated using the BBa_J23113 promoter, resulting in heterologous expression of a gene derived from [a specific gene]. Rhodotorula glutinis TAL gene, Arabidopsis thaliana The 4CL gene and derived from Vitis vinifera The STS gene was enhanced using the trc promoter and carried the PET-28a-WAL plasmid; the nucleotide sequence of the csrA gene is shown in SEQ ID NO.1 of the sequence listing, and the nucleotide sequence of the fabF gene is shown in SEQ ID NO.2 of the sequence listing; the tyrA gene... fbr The nucleotide sequences of the following genes are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively. fbr The nucleotide sequences of the genes are shown in SEQ ID NO. 8, the nucleotide sequences of the accA gene are shown in SEQ ID NO. 9, the nucleotide sequences of the TAL gene are shown in SEQ ID NO. 10, the nucleotide sequences of the 4CL gene are shown in SEQ ID NO. 11, the nucleotide sequences of the STS gene are shown in SEQ ID NO. 12, the nucleotide sequences of the BBa_J23113 promoter are shown in SEQ ID NO. 14, the nucleotide sequences of the trc promoter are shown in SEQ ID NO. 15, and the nucleotide sequences of the PET-28a-WAL plasmid are shown in SEQ ID NO.

17.

2. The resveratrol-producing strain according to claim 1, characterized in that: by E. coli W3110 ATCC27325 was used as the chassis strain. The csrA gene was knocked out in the strain's genome, the fabF gene was downregulated using the BBa_J23113 promoter, and integrated into the yeeL gene locus in the genome; the tyrA gene was enhanced using the trc promoter. fbr The genes were enhanced using a trc promoter and integrated into the ycgH gene locus in the genome; the tyrB gene was enhanced using a trc promoter and integrated into the ylbE gene locus in the genome; the aroE gene was enhanced using a trc promoter and integrated into the ilvG gene locus in the genome; the pntAB gene was enhanced using a trc promoter and integrated into the ygaY gene locus in the genome; the ompF gene was enhanced using a trc promoter and integrated into the Yjgx gene locus in the genome; and the aroG gene was enhanced using a trc promoter. fbr The gene was enhanced using a TRC promoter and integrated into the mbhA gene locus in the genome; the accA gene was enhanced using a TRC promoter and integrated into the rph gene locus in the genome; the gene derived from... Rhodotorula glutinis The TAL gene was transcribed and integrated into the gapC gene locus in the genome; the trc promoter was used to enhance the expression of the gene derived from... Arabidopsis thaliana The 4CL gene was transcribed and integrated into the yeeP gene locus in the genome; the trc promoter was used to enhance the expression of the gene derived from... Vitis vinifera The STS gene is transcribed and integrated into the ycdN gene locus in the genome.

3. The method for constructing the resveratrol-producing strain according to claim 1 or 2, characterized in that: In the originating strain E. coli The following are the specific steps for targeted modification based on the W3110 ATCC 27325: (1) Start the expression tyrA using the trc strong promoter respectively. fbr tyrB, aroE, aroG fbr TAL, 4CL, STS genes; (2) The carbon storage regulator csrA gene was knocked out; (3) Increase the transcription level of the pntAB gene by overexpressing it using the trc promoter; (4) Overexpressing the accA gene using the trc promoter; (5) The fabF gene was weakly expressed using the BBa_J23113 promoter; (6) Overexpression of the ompF efflux protein gene using the trc promoter; (7) Transformation of the PET-28a-WAL plasmid system.

4. The application of the resveratrol-producing strain according to claim 1 or 2 in the fermentation production of resveratrol.

5. The application according to claim 4, characterized in that: The specific steps are as follows: ① Slant culture: Inoculate the resveratrol producing strain onto a slant culture medium and incubate at 32-35℃ for 12-16 hours; ② Shake flask seed culture: Take solid slant inoculum and inoculate it into shake flask culture medium for fermentation. The culture temperature is 32-35℃, the culture time is 12-20h, the shaking speed is 200-240r / min, and the pH is 6.4-6.

7. ③ Shake flask fermentation culture: The inoculum size is 15-20%, the culture temperature is 32-35℃, the pH is 6.4-6.7, the culture time is 24-36h, and the shaker speed is 200-240r / min.

6. The application according to claim 5, characterized in that: The slant culture medium used in step ① is general-purpose LB solid medium; the seed culture medium used in step ② is: glucose 30g / L, yeast 5g / L, peptone 3g / L, (NH4)2SO4 1g / L, KH2PO4·3H2O 2g / L, MgSO4·7H2O 1g / L, citric acid 3g / L, glutamic acid 3g / L; the fermentation culture medium used in step ③ is: glucose 15g / L, yeast extract 4g / L, peptone 2g / L, (NH4)2SO4 1.5g / L, KH2PO4·3H2O 3.5g / L, MgSO4·7H2O 2g / L, glutamic acid 3g / L, citric acid 3g / L, FeSO4·7H2O 20mg / L, MnSO4·H2O 10mg / L.

7. The application according to claim 5, characterized in that: The fermentation medium used in step ③ is as follows: glucose 15g / L, yeast powder 4g / L, peptone 2g / L, (NH4)2SO4 1.5g / L, KH2PO4·3H2O 3.5g / L, MgSO4·7H2O 2g / L, glutamic acid 3g / L, citric acid 3g / L, FeSO4·7H2O 20mg / L, MnSO4·H2O 10mg / L, pyridoxal phosphate 3mg / L, and betaine 1.2g / L.

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