Yarrowia lipolytica engineering strain with high yield of resveratrol and construction and application thereof

Abstract

The application discloses a high-yield resveratrol Yarrowia lipolytica engineering strain and construction and application thereof, and belongs to the technical field of synthetic biology and metabolic engineering. The Yarrowia lipolytica engineering strain provided by the application can realize high-yield resveratrol by using glucose as a substrate, and the yield of resveratrol reaches 3.157 g / L in shake flask fermentation, and the yield of resveratrol is increased to 30.7 g / L in 5L scale batch feeding fermentation, which is the highest value reported at present.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and metabolic engineering technology, specifically relating to a high-yield resveratrol-producing Yersinia lipolytica engineered strain and its construction and application. Background Technology

[0002] Resveratrol (3,5,4'-trihydroxystilbene) is a natural polyphenol compound widely found in plants such as grapes, Japanese knotweed, and peanuts, possessing significant biological activity and pharmacological functions. It plays important roles in antioxidant and anti-inflammatory effects, cardiovascular protection, anti-aging, and neuroprotection, and has potential inhibitory effects on breast cancer and colon cancer. The natural synthesis of resveratrol relies on plant secondary metabolic pathways, resulting in extremely low concentrations in plants (e.g., only 0.1-14 mg / kg in grape skins). Extraction processes are complex, costly, and limited by planting cycles and geographical conditions. Chemical synthesis requires multiple steps (e.g., Wittig-Horner coupling), is difficult to construct chiral centers, produces numerous byproducts, and relies on toxic reagents, which does not align with the trend towards green chemistry. In vitro enzyme catalysis systems suffer from difficulties in coenzyme regeneration and poor enzyme stability, hindering large-scale production. Microbial cell factories based on synthetic biology offer a revolutionary solution for the efficient production of resveratrol.

[0003] Yarrowia lipolytica ( Yarrowia lipolytica *Yersinia lipophila* is an important industrial microbial species, widely recognized as a food-grade safe microorganism. *Yersinia lipophila* naturally possesses high-throughput malonyl-CoA and pentose phosphate pathways (PPP), metabolic traits that may be particularly relevant to the production of shikimic acid-derived compounds and plant natural products requiring aromatic amino acids and malonyl-CoA-derived building blocks. Therefore, *Yersinia lipophila* is an excellent host for resveratrol biosynthesis.

[0004] However, the current biosynthesis of resveratrol using Yeast Extract has technical problems such as low substrate conversion rate and low resveratrol yield. Summary of the Invention

[0005] The purpose of this invention is to provide a high-yield resveratrol-producing *Yarrowia lipolytica* engineered strain, its construction, and its application. This addresses the problems of low substrate conversion rate and low resveratrol yield in existing biosynthesis of resveratrol using *Yarrowia lipolytica*.

[0006] In a first aspect, the present invention provides a high-yield resveratrol-producing *Yersinia lipolytica* engineered strain, the *Yersinia lipolytica* engineered strain comprising an integrated 4-coumaric acid-CoA ligase gene. 4CL1(M4) Multiple copies of resveratrol synthase gene VST1(N3) transketolase 1 gene TKT Histidine phosphotransferase gene HIS5 With tyrosine aminolysin gene FjTAL Fusion genes HIS5-linker-FjTAL Glycolysis pathway hexokinase gene HXK 6-phosphofructokinase-1 gene PFC Phosphoglycerate kinase gene PGK And knocking out aromatic amino acid decarboxylase genes ARO10 It also integrates a branching acid mutase mutant gene that resists feedback inhibition. ARO7 fbr .

[0007] The *Yarrowia lipolyticis* engineered strain provided by this invention integrates the 4-coumaric acid-CoA ligase gene. 4CL1 (M4) Multiple copies of resveratrol synthase gene VST1(N3) transketolase 1 gene TKT Histidine phosphotransferase gene HIS5 With tyrosine aminolysin gene FjTAL Fusion genes HIS5-linker-FjTAL Glycolysis pathway hexokinase gene HXK 6-phosphofructokinase-1 gene PFC Phosphoglycerate kinase gene PGK And knocking out aromatic amino acid decarboxylase genes ARO10 It also integrates a branching acid mutase mutant gene that resists feedback inhibition. ARO7 fbr Afterwards, it is possible to achieve high yield of resveratrol using glucose as a substrate.

[0008] In some implementations, multiple copies of the resveratrol synthase gene are used. VST1(N3) It includes at least one of two copies, three copies, and four copies.

[0009] In some implementations, the transketolase 1 gene TKT Histidine phosphotransferase gene HIS5 All are endogenous genes of Yersinia lipophila (of which, gene TKT The gene number is YALI1_E07744g, gene HIS5 The nucleotide sequence is the sequence in NCBI accession number XM_503408.3 (tyrosine aminolysase gene). FjTAL The nucleotide sequence is shown in SEQ ID NO.1.

[0010] In some implementation schemes, fusion genes HIS5-linker-FjTAL middle linker The amino acid sequence is (GGGGS). n n is a positive integer greater than 1.

[0011] Understandably, linker The amino acid sequence can be conventionally selected according to the needs of use, as long as it enables efficient fusion of the target gene. For example, in this invention, the fusion gene... HIS5-linker-FjTAL middle linker The preferred amino acid sequence is (GGGGS). n , where n is a positive integer greater than 1, and more preferably GGGGS.

[0012] In some implementations, the hexokinase gene HXK 6-phosphofructokinase-1 gene PFC Phosphoglycerate kinase gene PGK All of these are endogenous genes from Yersinia lipolytica (their nucleotide sequences are the sequences in NCBI accession number XM_068282144.1, XM_502897.3, and positions 755-2666 in NCBI accession number M91598.1, respectively).

[0013] In some implementations, the cladoid mutase mutant gene ARO7 fbr The nucleotide sequence is shown in SEQ ID NO.2.

[0014] In a second aspect, the present invention provides a method for constructing an engineered *Yersinia lipolytica* strain as described in any of the above claims, comprising using a CRISPR / Cas9 localization and integration method, using *Yersinia lipolytica* jz104 as the starting strain, and integrating the 4-coumaric acid-CoA ligase gene. 4CL1(M4) Multiple copies of resveratrol synthase gene VST1(N3) transketolase 1 gene TKT Histidine phosphotransferase gene HIS5 With tyrosine aminolysin gene FjTAL Fusion genes HIS5- linker-FjTAL Glycolysis pathway hexokinase gene HXK 6-phosphofructokinase-1 gene PFC Phosphoglycerate kinase gene PGK And knocking out aromatic amino acid decarboxylase genes ARO10 It also integrates a branching acid mutase mutant gene that resists feedback inhibition. ARO7 fbr It was constructed.

[0015] It is understood that the gene integration in this invention can be performed using conventional methods from existing technologies, as long as the target gene can be integrated into the corresponding site of the starting strain, according to actual usage needs. For example, in this invention, CRISPR / Cas9 genome localization integration technology is used for gene integration. Specifically, a pylcas9-sgRNA plasmid and a plasmid expressing the target gene are constructed targeting the gene site to be integrated, and then co-transformed into Yersinia lipophila for gene integration.

[0016] In some embodiments, the construction method includes the following steps: 1) integrating the 4-coumaric acid-CoA ligase gene into the F17 site of the *Yarrowia lipolytica* strain jz104. 4CL1(M4) and resveratrol synthase gene VST1(N3) 2) Based on the strain obtained in step 1), overexpress the resveratrol synthase gene at sites D10 and E13, respectively. VST1(N3) 3) Based on the strain obtained in step 2), the upstream transketolase 1 gene is enhanced at the intC2 site. TKT 4) Based on the strain obtained in step 3), overexpress the histidine phosphotransferase gene at the F1 site. HIS5 With tyrosine aminolysin gene FjTAL Fusion genes HIS5-linker-FjTAL 5) Based on the strain obtained in step 4), enhance the hexokinase gene in the upstream glycolysis pathway at site A2. HXK 6-phosphofructokinase-1 gene PFC and phosphoglycerate kinase gene PGK 6) Based on the strain obtained in step 5), overexpress the tyrosine aminolysin gene at site B3. FjTAL 7) Based on the strain obtained in step 6), knock out the aromatic amino acid decarboxylase gene. ARO10 It also integrates a branching acid mutase mutant gene that resists feedback inhibition. ARO7 fbr .

[0017] In the construction method provided by this invention, in steps 1) and 2), the *Yarrowia lipolytica* strain jz104 (see Chinese patent document CN119752659A) is used as the starting strain, and the 4-coumaric acid-CoA ligase gene is used. 4CL1(M4) and resveratrol synthase gene VST1(N3) (See Chinese Patent Document CN116426492A) The resveratrol synthase gene was integrated into the F17 site of the starting strain, and then overexpressed at the D10 and E13 sites of the resulting strain. VST1(N3) To increase the resveratrol synthase gene VST1(N3)The copy number can be increased to further enhance the synthesis of resveratrol; a Yersinia lipolytica engineered strain that synthesizes resveratrol de novo using glucose as a substrate was obtained, and this Yersinia lipolytica engineered strain has a high yield when producing resveratrol by fermentation using glucose as a substrate.

[0018] In step 3), the upstream transketolase 1 gene is enhanced at the intC2 site of the obtained *Yarrowia lipolytica* engineered strain. TKT It can adjust carbon flux, balance the supply of upstream erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP), and thus further increase the yield of resveratrol.

[0019] In step 4), the yield of resveratrol is further increased by optimizing the tyrosine biosynthesis pathway.

[0020] In step 5), by strengthening the genes at key nodes of the glycolysis pathway, the glucose metabolic flux is increased, thereby further increasing the yield of resveratrol.

[0021] In step 6), the tyrosine aminolysin gene is overexpressed. FjTAL It can enhance the conversion efficiency of tyrosine to p-coumaric acid, thereby further increasing the yield of resveratrol.

[0022] In step 7), the cladodesmosome mutant gene is overexpressed. ARO7 fbr This can further increase the yield of resveratrol.

[0023] In a third aspect, the present invention provides the application of any of the above-mentioned engineered strains of Yersinia lipolytica in the high-yield production of resveratrol using glucose as a substrate.

[0024] In this invention, the resveratrol yield of the engineered strain of Yersinia lipolytica reached 3.157 g / L during shake-flask fermentation, and increased to 30.7 g / L during 5L fed-batch fermentation. This is the highest value reported to date for the Yersinia lipolytica system and has significant industrial application value.

[0025] The beneficial effects of this invention are: unlike the prior art, the *Yarrowia lipolyticis* engineered strain provided by this invention integrates the 4-coumaric acid-CoA ligase gene. 4CL1(M4) Multiple copies of resveratrol synthase gene VST1 (N3) transketolase 1 gene TKT Histidine phosphotransferase gene HIS5 With tyrosine aminolysin gene FjTAL Fusion genes HIS5-linker-FjTAL Glycolysis pathway hexokinase gene HXK6-phosphofructokinase-1 gene PFC Phosphoglycerate kinase gene PGK And knocking out aromatic amino acid decarboxylase genes ARO10 It also integrates a branching acid mutase mutant gene that resists feedback inhibition. ARO7 fbr Subsequently, it was found that high yields of resveratrol using glucose as a substrate were achieved. The resveratrol yield reached 3.157 g / L during shake-flask fermentation and increased to 30.7 g / L during 5L fed-batch fermentation. This is the highest value reported to date for the Yersinia lipolytica system and has significant industrial application value. Attached Figure Description

[0026] Figure 1 The metabolic pathway of the high-yield resveratrol-producing Yersinia lipolytica engineered strain constructed in this invention;

[0027] Figure 2 The results of resveratrol production of different high-resveratrol-producing Yersinia lipolytica engineered strains constructed in this invention;

[0028] Figure 3 This is the result of batch fermentation production of resveratrol using the engineered Yersinia lipophila strain JZ503 constructed in Example 7 of this invention. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0031] In this invention, the DNA ligase and other molecular biology reagents used were purchased from TaKaRa (https: / / www.takara.com.cn / ); the Uniclone One Step Seamless Cloning Kit was purchased from Beijing Jinsha Biotechnology Co., Ltd. (https: / / 400-688-7890.company.lookchem.cn / ); and other biochemical reagents were purchased from China Reagent Network (https: / / www.labgogo.com / ).

[0032] Example 1

[0033] In this embodiment, the 4-coumaric acid-CoA ligase gene obtained by the inventors' team through previous mutations is used. 4CL1(M4) and resveratrol synthase gene VST1(N3) (See Chinese Patent Document CN116426492A) This strain was integrated into the *Yersinia lipolytica* strain jz104, which produces p-coumaric acid (see Chinese Patent Document CN119752659A), to construct a *Yersinia lipolytica* strain that synthesizes resveratrol de novo using glucose as a substrate. The specific procedures are as follows:

[0034] artificial synthesis 4CL1(M4) Gene, VST1(N3) Gene sequences, and amplified separately. 4CL1(M4) Gene fragments and VST1(N3) Gene fragments were then used to construct a gene in E. coli DH5α using plasmid pUC19. 4CL1(M4) Gene expression cassette P TEF1 - 4CL1(M4) -T xpr2 and VST1(N3) Gene expression cassette P TEF1 - VST1(N3) -T xpr2 Using primers puc19-F17-F1 / F17-R1 (nucleotide sequences as shown in SEQ ID NO. 3-4) and F17-F2 / F17-puc19-R2 (nucleotide sequences as shown in SEQ ID NO. 5-6), fragments of the upstream and downstream homologous arms of the F17 site were amplified using the *Yarrowia lipolytica* genome as a template. CL1(M4) Gene expression cassette P TEF1 - 4CL1(M4) -T xpr2 and VST1(N3) Gene expression cassette P TEF1 - VST1(N3) -T xpr2 Using templates, amplify separately 4CL1(M4) Gene expression cassettes and VST1(N3) Gene expression cassettes, obtained 4CL1(M4)Gene expression cassettes and VST1(N3) Gene expression cassette fragments, connecting the upstream and downstream homologous arms of the F17 site with... CL(M4) Gene expression cassettes and VST1(N3) Gene expression cassette fragments were cloned and ligated in one step using plasmid pUC19 to construct plasmid pUC19-F17-P containing a homologous arm at the F17 site. TEF1 - 4CL1(M4) -T xpr2 -P TEF1 - VST1(N3) -T xpr2 Based on the sgRNA sequence targeting the F17 site, a tool plasmid pylcas9 was constructed to express Cas9 protein and sgRNA. Using pylcas9 as a template, the pYlCas9-sgRNA-F17 fragment containing Cas9 protein and located at the F17 site was amplified using sgRNA-F17-F / R primers (nucleotide sequences shown in SEQ ID NO. 7-8). Dpn After overnight digestion of the PCR product, it was recovered and constructed using T5 self-ligation to obtain the pYlCas9-sgRNA-F17 plasmid.

[0035] The pUC19-F17-P obtained above was transferred using a thermal transfer method involving lithium acetate / single-stranded DNA / PEG3350. TEF1 - 4CL1 (M4) -T xpr2 -P TEF1 - VST1(N3) -T xpr2 The pYlCas9-sgRNA-F17 plasmid was co-transformed into *Yersinia lipolytica* jz104 and plated on a leucine-deficient selection plate, YNB-leu. After 2-3 days, several transformants were randomly selected and cultured in the corresponding liquid YNB-leu medium at 30°C and 250 rpm for 48 hours. Genomic DNA was extracted and validated by PCR. Positive transformants were then selected to obtain the engineered *Yersinia lipolytica* strain jz200.

[0036] A single colony of the engineered *Yersinia lipolyticis* strain jz200 was picked and inoculated into a test tube containing 2 mL of YPD medium. The culture was incubated for 24 h in a shaker at 250 rpm and 30 °C. The primary seed culture was then inoculated into a 50 mL Erlenmeyer flask containing 10 mL of YPD medium and incubated for 24 h at an inoculation rate of 1% (v / v) to obtain the secondary seed culture. The secondary seed culture was then inoculated into a 250 mL Erlenmeyer flask containing 30 mL of YPD-40 medium at an inoculation rate of 5% (v / v). After fermentation under the same conditions for 96 h, the resveratrol yield was 580.3 mg / L. Figure 2 ).

[0037] Example 2

[0038] In this embodiment, the resveratrol synthase gene was added to the strain jz200 obtained in Example 1. VST1(N3) Copy number determination was performed to construct a *Yarrowia lipolytica* strain with higher resveratrol production. The specific steps are as follows:

[0039] Using primers puc19-D10-F1 / D10-R1 (nucleotide sequences as shown in SEQ ID NO. 9-10) and D10-F2 / D10-puc19-R2 (nucleotide sequences as shown in SEQ ID NO. 11-12), and with the *Yarrowia lipolytica* genome as a template, fragments of the upstream and downstream homologous arms of the D10 site were amplified, respectively, as described in Example 1. VST1(N3) Gene expression cassette P TEF1 - VST1(N3) -T xpr2 Using templates, amplification VST1(N3) Gene expression cassettes, obtained VST1(N3) Gene expression cassette fragment; combining the upstream and downstream homologous arms of the D10 site with... VST1(N3) Gene expression cassette fragments were cloned and ligated in one step using plasmid pUC19 to construct plasmid pUC19-D10-P containing a homologous arm at the D10 site. TEF1 - VST1(N3) -T xpr2 Using primers puc19-E13-F1 / E13-R1 (nucleotide sequence as shown in SEQ ID NO. 13-14) and E13-F2 / E13-puc19-R2 (nucleotide sequence as shown in SEQ ID NO. 15-16), and with the *Yarrowia lipolytica* genome as a template, fragments of the upstream and downstream homologous arms of the E13 site were amplified, respectively, as described in Example 1. VST1(N3) Gene expression cassette P TEF1 - VST1(N3) -T xpr2 Using templates, amplification VST1 (N3) Gene expression cassettes, obtained VST1(N3) Gene expression cassette fragments, including the upstream and downstream homologous arms of the E13 site, are combined with... VST1(N3) Gene expression cassette fragments were cloned and ligated in one step using plasmid pUC19 to construct plasmid pUC19-E13-P containing a homologous arm at the E13 site. TEF1 - VST1(N3) -T xpr2Based on the sgRNA sequences targeting the D10 and E13 sites, a tool plasmid pylcas9 was constructed to express Cas9 protein and sgRNA. Using pylcas9 as a template, primers sgRNA-D10-F / R (nucleotide sequences shown in SEQ ID NO. 17-18) and sgRNA-E13-F / R (nucleotide sequences shown in SEQ ID NO. 19-20) were used to amplify pYlCas9-sgRNA-D10 and pYlCas9-sgRNA-E13 fragments containing Cas9 protein and located at the D10 and E13 sites, respectively. Dpn After overnight digestion of the PCR product, it was recovered and constructed using T5 self-ligation to obtain the plasmids pYlCas9-sgRNA-D10 and pYlCas9-sgRNA-E13.

[0040] The pUC19-D10-P obtained above was transferred using a thermal transfer method involving lithium acetate / single-stranded DNA / PEG3350. TEF1 - VST1 (N3) -T xpr2 The pYlCas9-sgRNA-D10 plasmid was co-transformed into *Yersinia lipolytica* strain jz200 and plated on a leucine-deficient selection plate, YNB-leu. After 2-3 days, several transformants were randomly selected and cultured in the corresponding liquid YNB-leu medium at 30°C and 250 rpm for 48 hours. Genomic DNA was extracted and validated by PCR. Positive transformants were then selected to obtain *Yersinia lipolytica* strain jz202.

[0041] Furthermore, the pUC19-E13-P obtained above was transferred using a "lithium acetate / single-stranded DNA / PEG3350" thermal transfer method. TEF1 - VST1(N3) -T xpr2 The pYlCas9-sgRNA-E13 plasmid was co-transformed into *Yersinia lipolytica* strain jz202 and plated on a leucine-deficient selection plate, YNB-leu. After 2-3 days, several transformants were randomly selected and cultured in the corresponding liquid YNB-leu medium at 30°C and 250 rpm for 48 hours. Genomic DNA was extracted and validated by PCR. Positive transformants were then selected to obtain *Yersinia lipolytica* strain jz205.

[0042] A single colony of *Yersinia lipolyticis* strain JZ205 was picked and inoculated into a test tube containing 2 mL of YPD medium and cultured in a shaker at 250 rpm and 30 °C for 24 h. The primary seed culture was then inoculated into a 50 mL Erlenmeyer flask containing 10 mL of YPD medium and cultured for 24 h at an inoculation rate of 1% (v / v) to obtain a secondary seed culture. The secondary seed culture was then inoculated into a 250 mL Erlenmeyer flask containing 30 mL of YPD-40 medium at an inoculation rate of 5% (v / v). After fermentation under the same conditions for 96 h, the resveratrol yield was 941.8 mg / L. Figure 2 ).

[0043] Example 3

[0044] In this embodiment, the gene of *Yersinia lipophila* endogenous pentose phosphate pathway transketolase 1 was overexpressed on the strain jz205 obtained in Example 2. TKT To construct a *Yarrowia lipolytica* strain with higher resveratrol production, the carbon flux was adjusted to balance the supply of upstream erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP). The specific steps are as follows:

[0045] Using the genome of Yersinia lipophila as a template, the following was amplified: TKT The gene fragment (with the gene number YALI1_E07744g) was constructed in Escherichia coli DH5α using plasmid pUC19. TKT Gene expression cassette P TEF1 - TKT -T lip2 Using primers puc19-intC2-F1 / intC2-R1 (nucleotide sequences shown in SEQ ID NO. 21-22) and intC2-F2 / intC2-puc19-R2 (nucleotide sequences shown in SEQ ID NO. 23-24), the upstream and downstream homologous arms of intC2 were amplified using the *Yarrowia lipolytica* genome as a template; TKT Gene expression cassette P TEF1 - TKT -T lip2 Using templates, amplification TKT Gene expression cassettes will amplify TKT The gene expression cassette fragment and the upstream and downstream homologous arms of intC2 were cloned in one step using plasmid pUC19 to construct plasmid pUC19-intC2-P. TEF1 - TKT -T lip2Based on the sgRNA sequence targeting the intC2 site, a tool plasmid pylcas9 was constructed to express Cas9 protein and sgRNA. Using pylcas9 as a template, primers sgRNA-intC2-F / R (nucleotide sequences shown in SEQ ID NO. 25-26) were used to amplify the pYlCas9-sgRNA-intC2 fragment containing Cas9 protein and located at the intC2 site. Dpn After overnight digestion of the PCR product, it was recovered and constructed using T5 self-ligation to obtain the pYlCas9-sgRNA-intC2 plasmid.

[0046] The pUC19-intC2-P obtained above was transferred using a thermal transfer method involving lithium acetate / single-stranded DNA / PEG3350. TEF1 - TKT -T lip2 The plasmid pYlCas9-sgRNA-intC2 was co-transformed into *Yersinia lipolytica* strain jz205 and plated on a leucine-deficient selection plate, YNB-leu. After 2-3 days, several transformants were randomly selected and cultured in the corresponding liquid YNB-leu medium at 30°C and 250 rpm for 48 hours. Genomic DNA was extracted and validated by PCR. Positive transformants were then selected to obtain *Yersinia lipolytica* strain jz401.

[0047] A single colony of *Yersinia lipolyticis* strain JZ401 was picked and inoculated into a test tube containing 2 mL of YPD medium and cultured in a shaker at 250 rpm and 30 °C for 24 h. The primary seed culture was then inoculated into a 50 mL Erlenmeyer flask containing 10 mL of YPD medium and cultured for 24 h at an inoculation rate of 1% (v / v) to obtain a secondary seed culture. The secondary seed culture was then inoculated into a 250 mL Erlenmeyer flask containing 30 mL of YPD-40 medium at an inoculation rate of 5% (v / v). After fermentation under the same conditions for 96 h, the resveratrol yield was 881.4 mg / L. Figure 2 ).

[0048] Example 4

[0049] In this embodiment, the histidine phosphoaminotransferase gene was integrated into the strain jz401 obtained in Example 3. HIS5 With tyrosine aminolysin gene FjTAL Fusion genes HIS5- GGGGS -FjTAL To optimize the tyrosine biosynthesis pathway and construct a *Yarrowia lipolyticis* strain with higher resveratrol production, the specific procedures are as follows:

[0050] Using the genome of Yersinia lipophila as a template, the following was amplified: HIS5Gene fragment (its nucleotide sequence is the sequence in NCBI accession number: XM_503408.3); artificially synthesized FjTAL The gene (nucleotide sequence shown in SEQ ID NO.1) was used as a template to amplify the gene. FjTAL Gene fragment. Using primers pTEF1in-HIS5-F / HIS5-G4P-R (nucleotide sequence as shown in SEQ ID NO. 27-28), to... HIS5 Using a gene fragment as a template, amplification yields a gene with a linker. HIS5 Gene fragment; using G4P-FjTAL-F / Xpr2t-FjTAL-R (nucleotide sequence as shown in SEQ ID NO.29-30) primers, to FjTAL Using a gene fragment as a template, amplification yields a gene with a linker. FjTAL Gene fragments were constructed in Escherichia coli DH5α using plasmid pUC19. HIS5- GGGGS -FjTAL Gene expression cassette P TEF1in - HIS5- GGGGS -FjTAL -T xpr2 Using primers puc19-F1-F1 / F1-R1 (nucleotide sequences shown in SEQ ID NO. 31-32) and F1-F2 / F1-puc19-R2 (nucleotide sequences shown in SEQ ID NO. 33-34), the upstream and downstream homologous arms of F1 were amplified using the *Yarrowia lipolytica* genome as a template. HIS5- GGGGS -FjTAL Gene expression cassette P TEF1in - HIS5- GGGGS -FjTAL -T xpr2 Using templates, amplification HIS5- GGGGS -FjTAL Gene expression cassette fragments will amplify HIS5- GGGGS -FjTAL The gene expression cassette fragment, along with the upstream and downstream homologous arms of F1, was cloned in one step using plasmid pUC19 to construct plasmid pUC19-F1-P. TEF1in - HIS5- GGGGS -FjTAL -T xpr2Based on the sgRNA sequence targeting the F1 site, a tool plasmid pylcas9 expressing Cas9 protein and sgRNA was constructed. Using pylcas9 as a template, primers sgRNA-F1-F / R (nucleotide sequences shown in SEQ ID NO. 35-36) were used to amplify the pYlCas9-sgRNA-F1 fragment containing Cas9 protein and located at the F1 site. Dpn After overnight digestion of the PCR product, it was recovered and constructed using T5 self-ligation to obtain the pYlCas9-sgRNA-F1 plasmid.

[0051] The pUC19-F1-P obtained above was transferred using a thermal transfer method involving lithium acetate / single-stranded DNA / PEG3350. TEF1in - HIS5 - GGGGS -FjTAL -T xpr2 The pYlCas9-sgRNA-F1 plasmid was co-transformed into *Yersinia lipolytica* strain jz401 and plated on a leucine-deficient selection plate, YNB-leu. After 2-3 days, several transformants were randomly selected and cultured in the corresponding liquid YNB-leu medium at 30°C and 250 rpm for 48 hours. Genomic DNA was extracted and validated by PCR. Positive transformants were then selected to obtain *Yersinia lipolytica* strain jz403.

[0052] A single colony of *Yersinia lipolyticis* strain JZ403 was picked and inoculated into a test tube containing 2 mL of YPD medium and cultured in a shaker at 250 rpm and 30 °C for 24 h. The primary seed culture was then inoculated into a 50 mL Erlenmeyer flask containing 10 mL of YPD medium and cultured for 24 h at an inoculation rate of 1% (v / v) to obtain a secondary seed culture. The secondary seed culture was then inoculated into a 250 mL Erlenmeyer flask containing 30 mL of YPD-40 medium at an inoculation rate of 5% (v / v). After fermentation under the same conditions for 96 h, the resveratrol yield was 1820.2 mg / L. Figure 2 ).

[0053] Example 5

[0054] In this embodiment, the hexokinase gene was co-overexpressed based on strain jz403 obtained in Example 4. HXK 6-phosphofructokinase-1 gene PFC Phosphoglycerate kinase gene PGK To enhance key nodes in the glycolysis pathway and increase glucose metabolism flux, a *Yarrowia lipolyticis* strain with higher resveratrol production was constructed. The specific procedures are as follows:

[0055] Using the genome of Yersinia lipolyticis as a template, the following were amplified separately: HXK , PFC , PGK Gene fragments (their nucleotide sequences are from NCBI accession number XM_068282144.1, NCBI accession number XM_502897.3, and positions 755-2666 of NCBI accession number M91598.1, respectively). An expression cassette P was constructed in *E. coli* DH5α using plasmid pUC19. TEF1 - HXK -T lip2 P TEF1 - PFC -T xpr2 P TEF1 - PGK -T xpr2 Using primers puc19-A2-F1 / A2-R1 (nucleotide sequences shown in SEQ ID NO. 37-38) and A2-F2 / A2-puc19-R2 (nucleotide sequences shown in SEQ ID NO. 39-40), the upstream and downstream homologous arms of A2 were amplified using the *Yarrowia lipolytica* genome as a template. The amplification was performed using the aforementioned expression cassette P. TEF1 - HXK -T lip2 P TEF1 - PFC -T xpr2 P TEF1 - PGK -T xpr2 Using as a template, the amplified HXK , PFK , PGK The gene expression cassette fragment, along with the upstream and downstream homologous arms of A2, was cloned in one step using plasmid pUC19 to construct plasmid pUC19-A2- HXK - PFK - PGK Based on the sgRNA sequence targeting the A2 site, a tool plasmid pylcas9 was constructed to express Cas9 protein and sgRNA. Using pylcas9 as a template, primers sgRNA-A2-F / R (nucleotide sequences shown in SEQ ID NO. 41-42) were used to amplify the pYlCas9-sgRNA-A2 fragment containing Cas9 protein and located at the A2 site. Dpn After overnight digestion of the PCR product, it was recovered and constructed using T5 self-ligation to obtain the pYlCas9-sgRNA-A2 plasmid.

[0056] The pUC19-A2- obtained above was transferred using a thermal transfer method involving lithium acetate / single-stranded DNA / PEG3350. HXK - PFK - PGKThe pYlCas9-sgRNA-A2 plasmid was co-transformed into *Yersinia lipolytica* strain jz403 and plated on a leucine-deficient selection plate, YNB-leu. After 2-3 days, several transformants were randomly selected and cultured in the corresponding liquid YNB-leu medium at 30°C and 250 rpm for 48 hours. Genomic DNA was extracted and validated by PCR. Positive transformants were then selected to obtain *Yersinia lipolytica* strain jz405.

[0057] A single colony of *Yersinia lipolyticis* strain JZ405 was picked and inoculated into a test tube containing 2 mL of YPD medium and cultured in a shaker at 250 rpm and 30 °C for 24 h. The primary seed culture was then inoculated into a 50 mL Erlenmeyer flask containing 10 mL of YPD medium and cultured for 24 h at an inoculation rate of 1% (v / v) to obtain a secondary seed culture. The secondary seed culture was then inoculated into a 250 mL Erlenmeyer flask containing 3 mL of YPD-40 medium at an inoculation rate of 5% (v / v). After fermentation under the same conditions for 96 h, the resveratrol yield was 1951.6 mg / L. Figure 2 ).

[0058] Example 6

[0059] In this embodiment, the tyrosine ammonia-lyase gene was overexpressed based on strain jz405 obtained in Example 5. FjTAL To improve the efficiency of coumaric acid synthesis, a *Yarrowia lipolytica* strain with higher resveratrol production was constructed. The specific procedures are as follows:

[0060] Using the commercially available lipophilic yeast vector pINA1312 as a template, the following was amplified: HP4D Promoter fragment. To amplify HP4D The promoter fragment and the one in Example 4 FjTAL Gene fragments were cloned in one step using plasmid pUC19 to construct plasmid pUC19-P. HP4D - FjTAL -T xpt2 Using primers puc19-B3-F1 / B3-R1 (nucleotide sequences shown in SEQ ID NO. 43-44) and B3-F2 / B3-puc19-R2 (nucleotide sequences shown in SEQ ID NO. 45-46), the upstream and downstream homologous arms of B3 were amplified using the *Yarrowia lipolytica* genome as a template. The resulting plasmid pUC19-P... HP4D - FjTAL -T xpt2 Using P as a template, amplify HP4D - FjTAL Gene expression cassettes will amplify P HP4D - FjTALThe gene expression cassette fragment, along with the upstream and downstream homologous arms of B3, was cloned in one step using plasmid pUC19 to construct plasmid pUC19-B3-P. HP4D - FjTAL -T lip2 Based on the sgRNA sequence targeting the B3 site, a tool plasmid pylcas9 was constructed to express Cas9 protein and sgRNA. Using pylcas9 as a template, primers sgRNA-B3-F / R (nucleotide sequences shown in SEQ ID NO. 47-48) were used to amplify the pYlCas9-sgRNA-B3 fragment containing Cas9 protein and located at the B3 site. Dpn After overnight digestion of the PCR product, it was recovered and constructed using T5 self-ligation to obtain the pYlCas9-sgRNA-B3 plasmid.

[0061] The pUC19-B3-P obtained above was transferred using a thermal transfer method involving lithium acetate / single-stranded DNA / PEG3350. HP4D - FjTAL -T lip2 The pYlCas9-sgRNA-B3 plasmid was co-transformed into *Yersinia lipolytica* strain jz405 and plated on a leucine-deficient selection plate, YNB-leu. After 2-3 days, several transformants were randomly selected and cultured in the corresponding liquid YNB-leu medium at 30°C and 250 rpm for 48 hours. Genomic DNA was extracted and validated by PCR. Positive transformants were then selected to obtain *Yersinia lipolytica* strain jz501.

[0062] A single colony of *Yersinia lipolyticis* strain JZ501 was picked and inoculated into a test tube containing 2 mL of YPD medium and cultured in a shaker at 250 rpm and 30 °C for 24 h. The primary seed culture was then inoculated into a 50 mL Erlenmeyer flask containing 10 mL of YPD medium and cultured for 24 h at an inoculation rate of 1% (v / v) to obtain a secondary seed culture. The secondary seed culture was then inoculated into a 250 mL Erlenmeyer flask containing 30 mL of YPD-40 medium at an inoculation rate of 5% (v / v). After fermentation under the same conditions for 96 h, the resveratrol yield was 2531.8 mg / L. Figure 2 ).

[0063] Example 7

[0064] In this embodiment, the aromatic amino acid decarboxylase gene was knocked out based on the strain jz501 obtained in Example 6. ARO10 And integrates a branching acid mutase mutant gene that resists feedback inhibition. ARO7 fbr To increase tyrosine metabolic flux and obtain a *Yarrowia lipolyticis* strain with higher resveratrol production, the specific steps are as follows:

[0065] Using the genome of Yersinia lipophila as a template, the following was amplified: ARO7 Gene fragments were used to construct expression cassettes P in Escherichia coli DH5α using plasmid pUC19. TEF1 - ARO7 -T xpr2 Using ARO7 fbr -F / R (nucleotide sequence as shown in SEQ ID NO.49-50) primers, with pUC19-P TEF1 - ARO7 -T xpr2 Using the plasmid as a template, pUC19-P was amplified. TEF1 - ARO7 fbr - T xpr2 Fragment (of which, the cladositase mutant gene) ARO7 fbr The nucleotide sequence is shown in SEQ ID NO.2. Dpn After overnight digestion of the PCR product, it was recovered and constructed using T5 self-ligation to obtain pUC19-P. TEF1 - ARO7 fbr - T xpr2 Plasmids. Using the Yersinia lipolytica genome as a template, the aromatic amino acid decarboxylase gene was amplified using primers puc19-ARO10-F1 / ARO10-R1 (nucleotide sequence as shown in SEQ ID NO. 51-52) and ARO10-F2 / ARO10-puc19-R2 (nucleotide sequence as shown in SEQ ID NO. 53-54). ARO10 The upstream and downstream homologous arm fragments, with the above pUC19-P TEF1 - ARO7 fbr - T xpr2 Using plasmid as a template, amplification ARO7 fbr Gene expression cassettes will amplify ARO7 fbr The gene expression cassette fragment and the upstream and downstream homologous arms of ARO10 were cloned in one step using plasmid pUC19 to construct the plasmid pUC19-ΔARO10-P. TEF1 - ARO7 fbr -T xpr2 Using gRNA, the website chopchop (https: / / chopchop.cbu.uib.no / ) was used to design and position [the target audience / location]. ARO10The sgRNA site was used to construct the tool plasmid pylcas9, which expresses Cas9 protein and sgRNA. Using pylcas9 as a template, primers sgRNA-ARO10-F / R (nucleotide sequence shown in SEQ ID NO. 55-56) were used to amplify the Cas9 protein and its localized site. ARO10 pYlCas9-sgRNA at the site ARO10 Fragment, Dpn After overnight digestion of the PCR product, the fragment was recovered, and the vector fragment was constructed using T5 self-ligation to obtain pYlCas9-sgRNA- ARO10 Plasmid.

[0066] The pUC19-ΔARO10-P obtained above was transferred using a thermal transfer method involving lithium acetate / single-stranded DNA / PEG3350. TEF1 - ARO7 fbr -T xpr2 With pYlCas9-sgRNA- ARO10 The plasmid was co-transformed into *Yarrowia lipolytica* strain jz501 and plated on a leucine-deficient selection plate, YNB-leu. After 2-3 days, several transformants were randomly selected and cultured in the corresponding liquid YNB-leu medium at 30°C and 250 rpm for 48 hours. After genomic DNA extraction and PCR verification, positive transformants were selected to obtain *Yarrowia lipolytica* strain jz502.

[0067] A single colony of *Yersinia lipolyticis* strain JZ502 was picked and inoculated into a test tube containing 2 mL of YPD medium and cultured in a shaker at 250 rpm and 30 °C for 24 h. The primary seed culture was then inoculated into a 50 mL Erlenmeyer flask containing 10 mL of YPD medium and cultured for 24 h at an inoculation rate of 1% (v / v) to obtain a secondary seed culture. The secondary seed culture was then inoculated into a 250 mL Erlenmeyer flask containing 30 mL of YPD-40 medium at an inoculation rate of 5% (v / v). After fermentation under the same conditions for 96 h, the resveratrol yield was 3157 mg / L. Figure 2 ).

[0068] Example 8

[0069] In this embodiment, resveratrol is produced by batch feeding fermentation in a 5L fermenter, and the specific operation is as follows:

[0070] Single clones of strain JZ502 were picked from the plate and inoculated into test tubes containing 2 mL of YPD medium. The culture was incubated at 30°C and 220 rpm with shaking for 24 h. The culture was then transferred to a 500 mL Erlenmeyer flask containing 100 mL of YPD medium and incubated with shaking for another 24 h to obtain the seed culture for batch fermentation. All 100 mL of the seed culture was transferred to 2 L of fermentation medium, which consisted of: 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, 12 mL / L vitamins, and 10 mL / L trace metal salts. The trace metal salt solution contains: 5.75 g / L ZnSO4·7H2O, 0.32 g / L MnCl2, 0.32 g / L CuSO4, 0.47 g / L CoCl2, 0.48 g / L Na2MoO4, 2.9 g / L CaCl2·2H2O, 2.8 g / L FeSO4·7H2O, and 0.5 M EDTA. The vitamin solution contains: 0.05 g / L biotin, 1 g / L calcium pantothenate, 1 g / L nicotinic acid, 25 g / L inositol, 1 g / L thiamine hydrochloride, 1 g / L pyridoxal phosphate, and 0.2 g / L para-aminobenzoic acid.

[0071] The batch fermentation temperature was 30℃, and the pH was controlled to 5.5 using ammonia. The fed-batch medium was 800 g / L glucose. The culture medium was added continuously during fermentation, and the glucose concentration in the fermentation broth was controlled to be below 0.1 g / L. Product formation was monitored throughout the fermentation process. The final strain JZ502 (its metabolic pathway is as follows) Figure 1 (As shown) Fermentation produces 30.7 g / L of resveratrol, such as Figure 3 As shown, this represents the highest level of de novo synthesis of resveratrol reported to date.

[0072] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for constructing a high-yield resveratrol-producing engineered strain of *Yersinia lipolytica*, characterized in that, Includes the following steps: 1) The 4-coumaric acid-CoA ligase gene was integrated into the F17 site of the *Yarrowia lipolytica* strain jz104. 4CL1(M4) and resveratrol synthase gene VST1(N3) The 4-coumaric acid-CoA ligase gene 4CL1(M4) The nucleotide sequence of the resveratrol synthase gene is shown in SEQ ID NO.

57. VST1(N3) The nucleotide sequence is shown in SEQ ID NO. 58; 2) Based on the strain obtained in step 1), overexpress the resveratrol synthase gene at sites D10 and E13, respectively. VST1(N3) ; 3) Based on the strain obtained in step 2), the upstream transketolase 1 gene is enhanced at the intC2 site. TKT The transketolase 1 gene TKT The gene number is YALI1_E07744g; 4) Based on the strain obtained in step 3), overexpress the histidine phosphotransferase gene at the F1 site. HIS5 With tyrosine aminolysin gene FjTAL Fusion genes HIS5-linker-FjTAL The histidine phosphotransferase gene HIS5 The nucleotide sequence is the sequence in NCBI accession number: XM_503408.3, which is the tyrosine aminolysase gene. FjTAL The nucleotide sequence of the fusion gene is shown in SEQ ID NO.

1. HIS5-linker-FjTAL The amino acid sequence of the linker is (GGGGS)n, where n is a positive integer greater than 1; 5) Based on the strain obtained in step 4), the hexokinase gene in the upstream glycolysis pathway is enhanced at site A2. HXK 6-phosphofructokinase-1 gene PFK and phosphoglycerate kinase gene PGK The hexokinase gene HXK The nucleotide sequence is the sequence in NCBI accession number XM_068282144.1, which is the 6-phosphofructokinase-1 gene. PFK The nucleotide sequence is the sequence in NCBI accession number XM_502897.3, which is the phosphoglycerate kinase gene. PGK The nucleotide sequence is the sequence from position 755 to 2666 in NCBI accession number M91598.1; 6) Based on the strain obtained in step 5), overexpress the tyrosine aminolysin gene at site B3. FjTAL ; 7) Based on the strain obtained in step 6), knock out the aromatic amino acid decarboxylase gene. ARO10 It also integrates a branching acid mutase mutant gene that resists feedback inhibition. ARO7 fbr The cladoid mutase mutant gene ARO7 fbr The nucleotide sequence is shown in SEQ ID NO.

2.

2. The construction method according to claim 1, characterized in that, The transketolase 1 gene TKT The histidine phosphotransferase gene HIS5 All of these are endogenous genes from Yersinia lipolytica.

3. The construction method according to claim 1, characterized in that, The hexokinase gene HXK The 6-phosphofructokinase-1 gene PFK The phosphoglycerate kinase gene PGK All of these are endogenous genes from Yersinia lipolytica.

4. A high-yield resveratrol-producing engineered strain of *Yersinia lipolytica*, characterized in that, It is constructed by the construction method according to any one of claims 1-3.

5. The application of the engineered strain of Yersinia lipophila as described in claim 4 in the high-yield resveratrol production using glucose as a substrate.

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