Synthesis of resveratrol by p-coumaric acid and construction and application of engineering bacteria

Abstract

The application discloses a kind of resveratrol engineering bacteria synthesized by p-coumaric acid, construction and application, belong to biotechnology field.The application is by expressing the coding gene of 4-coumaroyl coenzyme A ligase mutant and / or the coding gene of resveratrol synthase mutant and the coding gene of acetyl-coa carboxylase and / or the coding gene of acetyl-coa synthase, on the basis of significantly improving the catalytic activity of 4-coumaroyl coenzyme A ligase, resveratrol synthase respectively, by the enhanced expression of acetyl-coa, malonyl-coa, the obtained Yarrowia lipolytica engineering bacteria can efficiently utilize p-coumaric acid to produce resveratrol, the yield reaches 2.4g / L in flask fermentation, substrate conversion rate reaches 96.3% in 5L fermenter batch feeding fermentation, and the yield of resveratrol reaches 47.8g / L.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an engineered bacterium that synthesizes resveratrol using p-coumaric acid, its construction, and its application. Background Technology

[0002] Resveratrol is a natural plant polyphenol compound with anti-inflammatory, antibacterial, anticancer, and anti-aging bioactivities, showing great potential in health products, food, and cosmetics. Currently, resveratrol production mainly relies on chemical synthesis and plant extraction. Chemical synthesis is hampered by safety concerns such as severe pollution and toxic byproduct residues; plant extraction, primarily from plants like peanuts and grapes, is cumbersome, yields low product rates, and consumes significant natural resources. Therefore, considering both environmental and economic benefits, microbial production of resveratrol is a highly promising alternative.

[0003] Currently, with advancements in microbial metabolic engineering and synthetic biology, efficient heterologous synthetic pathways can be designed and constructed in hosts such as *Escherichia coli* and *Saccharomyces cerevisiae*. Combined with large-scale fermentation by engineered strains, this enables the industrial production of rare plant-derived natural products such as resveratrol. This invention aims to provide an engineered strain capable of efficiently utilizing p-coumaric acid to synthesize resveratrol and its applications, thus supporting the large-scale industrial production of resveratrol. Summary of the Invention

[0004] This invention provides the construction and application of a recombinant genetically engineered bacterium that synthesizes resveratrol using p-coumaric acid, which can be used to efficiently synthesize resveratrol using p-coumaric acid as a substrate.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a recombinant genetically engineered bacterium, wherein the recombinant genetically engineered bacterium contains an expression vector comprising the coding gene of a 4-coumaroyl-CoA ligase mutant and / or the coding gene of a resveratrol synthase mutant, as well as the coding gene of acetyl-CoA carboxylase and / or the coding gene of acetyl-CoA synthase, or the genome of the bacterium containing the coding gene of a 4-coumaroyl-CoA ligase mutant and / or the coding gene of a resveratrol synthase mutant, as well as the coding gene of acetyl-CoA carboxylase and / or the coding gene of acetyl-CoA synthase. The two genes, namely the coding gene of the 4-coumaroyl-CoA ligase mutant and / or the coding gene of the resveratrol synthase mutant, can be expressed individually or fused together to form a fusion protein.

[0007] According to the above scheme, the recombinant genetically engineered bacteria is *Yarrowia lipophila*.

[0008] According to the above scheme, the acetyl-CoA carboxylase and acetyl-CoA synthase are derived from endogenous enzymes of Yersinia lipolytica.

[0009] According to the above scheme, the coding gene sequence of the acetyl-CoA carboxylase is shown in SEQ ID NO.9; the coding sequence of the acetyl-CoA synthase is shown in SEQ ID NO.10.

[0010] The 4-coumaroyl-CoA ligase mutant provided by this invention has an amino acid sequence derived from the sequence shown in SEQ ID NO:1 by mutation at one or more amino acid residue sites selected from the following group: 260, 328, 330, 333, 338, 351, 353, 363, 364, 378, 381, 442, 451, 452, 453, 457, 460, 492, and 498, that is, one or more of these sites are mutated to any amino acid other than the original amino acid;

[0011] Preferably, the mutation modes at each site in the 4-coumaroyl-CoA ligase mutant are: Y260F, S328V, A330S, L333M, E338R, G351S, G353C, L363A, A364S, S378P, C381V, I442V, L451F, F452D, I453V, L457M, L460M, K492P, and E498Q.

[0012] The resveratrol synthase mutant provided by this invention has an amino acid sequence derived from the sequence shown in SEQ ID NO:2, with mutations occurring at one or more amino acid residue sites selected from the following group: positions 51, 54, 57, 59, 61, 62, 203, 204, 205, 208, 252, 254, 265, 266, 267, 268, 269, 270, 273, 276, 307, 312, 313, and 315.

[0013] Preferably, the mutation modes at each site in the resveratrol synthase mutant are: E51D, K54E, N57K, I59M, D61Q, K62S, E203Q, D204T, A205H, S208C, G252C, I254V, F265C, H266D, L267M, W268L, P269K, N270D, T273G, S276D, P307H, A312Q, V313I, A315S.

[0014] The 4-coumaroyl-CoA ligase mutant and resveratrol synthase mutant in this invention can be obtained by plasmid amplification and mutagenesis.

[0015] In this invention, the term "expression vector" refers to bacterial plasmids, yeast plasmids, or other vectors well known in the art. Those skilled in the art can use well-known methods to construct expression vectors, such as enzyme digestion and ligation, and seamless cloning.

[0016] The present invention can transform host cells with the coding genes of the above-mentioned 4-coumaroyl-CoA ligase mutant and / or resveratrol synthase mutant, as well as the coding genes of acetyl-CoA carboxylase and / or acetyl-CoA synthase, and the expression vector, so that the host cells can express the proteins of 4-coumaroyl-CoA ligase mutant and / or resveratrol synthase mutant, as well as the proteins of acetyl-CoA carboxylase and / or acetyl-CoA synthase.

[0017] The host cell described in this invention includes a host cell containing the above-mentioned expression vector or a genome that integrates the coding sequences of the 4-coumaroyl-CoA ligase mutant and / or resveratrol synthase mutant of this invention as well as acetyl-CoA carboxylase and / or acetyl-CoA synthase.

[0018] The host cells or strains of the present invention can efficiently express 4-coumaroyl-CoA ligase and / or resveratrol synthase mutant enzymes with high catalytic performance, as well as enhance the expression of acetyl-CoA and malonyl-CoA.

[0019] The host cell of the present invention can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell. Specifically, the host cell can be a bacterium or a yeast; preferably, the host cell is *Escherichia coli*, *Saccharomyces cerevisiae*, *Pichia pastoris*, *Hansenula polymorpha*, or *Yarrowia lipolytica*; more preferably, the host cell is *Yarrowia lipolytica*.

[0020] Secondly, this invention provides a method for obtaining recombinant genetically engineered bacteria: An expression vector containing the coding gene for a 4-coumaroyl-CoA ligase mutant and / or the coding gene for a resveratrol synthase mutant, as well as the coding gene for acetyl-CoA carboxylase and / or the coding gene for acetyl-CoA synthase, is transferred into a host cell. When the recombinant genetically engineered bacteria contain both the coding gene for the 4-coumaroyl-CoA ligase mutant and the coding gene for the resveratrol synthase mutant, the two genes can be expressed individually or fused to construct a fusion protein. For example, the 4-coumaroyl-CoA ligase mutant and the resveratrol synthase mutant can be fused together using a linker peptide to construct a fusion protein. Preferably, the linker peptide is GPGPGPGP or GPGPGPGPGPGPGPGP.

[0021] According to the above scheme, the host cell is Yeast lipolytica.

[0022] Thirdly, the present invention provides the application of the above-mentioned recombinant genetically engineered bacteria in the production of resveratrol.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] This invention significantly improves the substrate conversion rate and final yield of resveratrol by expressing the coding genes of 4-coumaroyl-CoA ligase mutant and / or resveratrol synthase mutant, as well as the coding genes of acetyl-CoA carboxylase and / or acetyl-CoA synthase, based on the significantly enhanced catalytic activities of 4-coumaroyl-CoA ligase and resveratrol synthase, and by enhancing the expression of acetyl-CoA and malonyl-CoA. The engineered bacteria achieve a resveratrol yield of up to 47.8 g / L and a substrate conversion rate of 96.3% through biotransformation. Attached Figure Description

[0025] Figure 1 The figure shows the results of whole-cell catalytic production of resveratrol by the 4CL1 mutant strain.

[0026] Figure 2 The image shows the results of whole-cell catalytic production of resveratrol by the VST1 mutant strain.

[0027] Figure 3 This is a diagram of the pINA1312-4CL1(M4)-4GP-VST1(N3) plasmid vector.

[0028] Figure 4 Figure showing the results of fermentation of resveratrol using engineered Yeast strains.

[0029] Figure 5 Figure showing the results of resveratrol production by a strain expressing acetyl-CoA carboxylase.

[0030] Figure 6 Figure showing the results of resveratrol production by a strain expressing acetyl-CoA synthase.

[0031] Figure 7 Figure showing the results of fed-batch fermentation of engineered Yeast strains to produce resveratrol. Detailed Implementation

[0032] Part 1: Construction of a high-resveratrol-producing *Yersinia lipolytica* strain without enhanced intracellular acetyl-CoA and malonyl-CoA supply Example 1 Construction of a high-efficiency mutant of 4-coumaryl-CoA ligase (4CL1)

[0033] (1) Using the pRSFDuet-1 vector as a template, PCR amplification was performed using primers P1-F and P1-R. The primer sequences are shown in Table 1. The pRSFDuet-1 vector is a commercially available vector purchased from Novagen. After recovery of the PCR product, the linearized vector pRSFDuet-1 was obtained, and the size of the linearized vector fragment was 3471 bp.

[0034] (2) Using the artificially synthesized 4CL1 gene with optimized codons as a template, PCR amplification was performed using primers 4CL1-F and 4CL1-R. The primer sequences are shown in Table 1. The amplification product was recovered to obtain the target fragment of the 4CL1 gene, which has a size of 1716 bp. The nucleotide sequence of the 4CL1 gene is shown in SEQ ID NO.3.

[0035] (3) Using the codon-optimized artificially synthesized VST1 gene as a template, PCR amplification was performed using primers VST1-F and VST1-R. The primer sequences are shown in Table 1. The amplification product was recovered to obtain the target fragment of the VST1 gene, which has a size of 1209 bp. The nucleotide sequence of the VST1 gene is shown in SEQ ID NO.4.

[0036] (4) Using the pRSFDuet-1 vector as a template, PCR amplification was performed using primers P2-F and P2-R. The primer sequences are shown in Table 1. After recovery of the PCR product, the target fragment T7pro was obtained, with a fragment size of 171 bp.

[0037] (5) The above-mentioned 4CL1, VST1, T7pro target fragments and linearized vector pRSFDuet-1 were ligated using the ClonExpress II one-step cloning kit to obtain the recombinant plasmid pRSFDuet-4CL1-VST1. The correctness was verified by sequencing, and the recombinant plasmid was successfully constructed.

[0038] (6) The plasmid pRSFDuet-4CL1-VST1 was transformed into the Escherichia coli expression host strain BL21(DE3) by electroporation and plated onto LB solid medium containing kanamycin. The LB plates were incubated at 37°C until transformants grew. Positive transformants were picked to obtain wild-type engineered bacteria (WT).

[0039] (7) Using the constructed pRSFDuet-4CL1-VST1 plasmid as a template, primers were designed to amplify and induced the plasmid, resulting in a linearized plasmid vector with base mutations. This vector was then transformed into Escherichia coli BL21(DE3), and after in vivo repair and circularization, a plasmid with base mutations was obtained. Specifically, PCR amplification was performed using primers 4CL1-M1-F and 4CL1-M1-R. The primer sequences are shown in Table 1, and the mutant sequence was obtained. The mutation mode is: Y260F / S328V / A330S / L333M / E338R. The resulting plasmid expressing the mutant was named pRSFDuet1-4CL1(M1)-VST1, and the resulting mutant engineered bacteria was named M1.

[0040] (8) Using the constructed pRSFDuet1-4CL1(M1)-VST1 plasmid as a template, PCR amplification was performed using primers 4CL1-M2-F and 4CL1-M2-R. The primer sequences are shown in Table 1. The mutant sequence was obtained. The mutation mode was: L363A / A364S / S378P / C381V. The resulting plasmid expressing the mutant was named pRSFDuet1-4CL1(M2)-VST1, and the resulting mutant engineered bacteria was named M2.

[0041] (9) Following step 8, using the constructed pRSFDuet1-4CL1(M2)-VST1 plasmid as a template, PCR amplification was performed using primers 4CL1-M3-F and 4CL1-M3-R. The mutation pattern was: G351S / G353C / L451F / F452D / I453V / L457M / L460M. The mutant plasmid pRSFDuet1-4CL1(M3)-VST1 and the mutant engineered bacterium M3 were obtained.

[0042] (10) Following step 8, using the constructed pRSFDuet1-4CL1(M3)-VST1 plasmid as a template, PCR amplification was performed using primers 4CL1-M4-F and 4CL1-M4-R. The mutation mode was: I442V / K492P / E498Q. The mutant plasmid pRSFDuet1-4CL1(M4)-VST1 and the mutant engineered bacterium M4 were obtained. The amino acid sequence of 4CL1(M4) is shown in SEQ ID NO.5;

[0043] (11) Wild-type and mutant engineered bacteria were cultured overnight in a seed culture medium containing 50 μg / mL kanamycin to obtain a seed solution. The seed culture medium (mass percentage) consisted of 1% tryptone, 1% sodium chloride and 0.5% yeast extract. The culture conditions for the seed solution were 37°C and 220 rpm.

[0044] (12) The above seed culture was inoculated at 2% into a protein expression medium (mass percentage) containing 1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 0.231% KH2PO4 and 1.254% K2HPO4. The culture conditions were 37℃ and 220 rpm. After culturing for 3 h, IPTG was added to a final concentration of 0.5 mM for induction expression. The induction conditions were 25℃ and 220 rpm for 16 h.

[0045] (13) After protein induction expression was completed, the bacterial cells were collected by centrifugation at 4000 rpm for 20 min at room temperature, washed with sterile 0.9% physiological saline, and centrifuged again; the bacterial cells were resuspended in a certain volume of transformation buffer until the OD of the resuspended solution was reached. 600 The value was 10. 10 mL of the system was used for whole-cell catalysis. The reaction conditions were 30℃, 150 rpm, and 3 h. The composition of the conversion solution was: 10 g / L glucose, 10 g / L glycerol, 6 g / L Na2HPO4, 0.5 g / L NaCl, 3 g / L KH2PO4, 1 g / L NH4Cl, 246.5 mg / L MgSO4·7H2O, 14.7 mg / L CaCl2·2H2O, 27.8 mg / L FeSO4·7H2O, and 200 mg / L p-coumaric acid;

[0046] (14) After the reaction is complete, take 0.5 mL of the reaction solution and mix it thoroughly with 1 mL of methanol. Centrifuge at 12000 rpm for 2 min and filter it into a liquid phase bottle using a 0.22 μm filter membrane for high performance liquid chromatography detection.

[0047] (15) High performance liquid chromatography for the detection of resveratrol: The chromatographic column is a C18 (250mm*4.6mm, 5μm) or equivalent column, the mobile phase is acetonitrile and 1% acetic acid solution (gradient elution conditions: 5% acetonitrile for 5min, 5-50% acetonitrile for 15min, 50% acetonitrile for 5min, 50-5% acetonitrile for 2min), the flow rate is 1mL / min, the injection volume is 10μL, the column temperature is 25℃, and resveratrol is detected at a wavelength of 305nm. The content is determined by external standard method.

[0048] (16) The results are as follows Figure 1 As shown, compared with the wild-type (WT) strain, the 4CL1 mutant engineered strain has a 249.6% higher resveratrol production capacity, reaching a yield of 81.1 mg / L;

[0049] The aforementioned single mutants of 4-coumaroyl-CoA ligases are not limited to any of the mutations at positions 260, 328, 330, 333, 338, 351, 353, 363, 364, 378, 381, 442, 451, 452, 453, 457, 460, 492, and 498; mutations at any of these positions can enhance enzyme activity. The aforementioned multiple mutants of 4-coumaroyl-CoA ligases are not limited to mutants M1, M2, M3, M4, or the multiple mutants of 4-coumaroyl-CoA ligases listed in Table 3. Multiple mutant engineered strains of 4-coumaroyl-CoA ligases constructed based on the aforementioned sites can also enhance catalytic activity.

[0050] Table 1. Primer sequences for constructing the 4CL1 mutant

[0051]

[0052] Example 2 Construction of a high-efficiency mutant of resveratrol synthase (VST1)

[0053] Using the constructed pRSFDuet-4CL1(M4)-VST1 plasmid as a template, primers were designed for plasmid amplification and mutagenesis to obtain a linearized plasmid vector with base mutations. This vector was then transformed into E. coli BL21(DE3), and after in vivo repair and circularization, the plasmid with base mutations was obtained.

[0054] (1) Using the constructed pRSFDuet1-4CL1(M4)-VST1 plasmid as a template, PCR amplification was performed using primers VST1-N1-F and VST1-N1-R. The primer sequences are shown in Table 2, and the mutant sequence was obtained. The mutation mode is: E51D / K54E / N57K / I59M / D61Q / K62S / P307H / A312Q / V313I / A315S. The resulting expression mutant plasmid was named pRSFDuet1-4CL1-VST1(N1), and the resulting mutant engineered bacteria was named N1;

[0055] (2) Using the constructed pRSFDuet1-4CL1-VST1(N1) plasmid as a template, PCR amplification was performed using primers VST1-N2-F and VST1-N2-R. The primer sequences are shown in Table 2, and the mutant sequence was obtained. The mutation mode is: G252C / I254V / F265C / H266D / L267M / W268L / P269K. The resulting expression mutant plasmid was named pRSFDuet1-4CL1-VST1(N2), and the resulting mutant engineered bacteria was named N2;

[0056] (3) Following step 2, using the constructed pRSFDuet1-4CL1-VST1(N2) plasmid as a template, PCR amplification was performed using primers VST1-N3-F and VST1-N3-R. The mutation mode was: E203Q / D204T / A205H / S208C / N270D / T273G / S276D. The mutant plasmid pRSFDuet1-4CL1-VST1(N3) and the mutant engineered bacterium N3 were obtained. The amino acid sequence of VST1(N3) is shown in SEQ ID NO.6;

[0057] (4) The activity of the VST1 mutant engineered bacteria was detected using whole-cell catalysis. The specific method is described in Example 1 (steps 11-15). The results are as follows: Figure 2 As shown, compared with the control strain M4, the VST1 mutant engineered strain had a 117.4% higher resveratrol production capacity, with a maximum yield of 176.3 mg / L.

[0058] The aforementioned resveratrol synthase single mutants are not limited to any of the mutations at positions 51, 54, 57, 59, 61, 62, 203, 204, 205, 208, 252, 254, 265, 266, 267, 268, 269, 270, 273, 276, 307, 312, 313, and 315, all of which can enhance enzyme activity. The resveratrol synthase multiple mutants are also not limited to mutants N1, N2, N3, and the resveratrol synthase multiple mutants listed in Table 3; multiple resveratrol synthase mutant engineered bacteria constructed based on the aforementioned sites can also enhance catalytic activity.

[0059] Table 2. Primer sequences for constructing VST1 mutants

[0060]

[0061] Table 3. Resveratrol yield of other mutant strains of 4CL1 and VST1

[0062]

[0063] Example 3. Co-expression of 4CL1 (M4) and VST1 (N3) genes in Yersinia lipolytica.

[0064] 1) Construction of pINA1312-4CL1(M4)-VST1(N3) integrative plasmid

[0065] In Examples 1 and 2, the nucleotide codon preference of the 4-coumaroyl-CoA ligase mutant 4CL1(M4) gene and the resveratrol synthase mutant VST1(N3) gene is Escherichia coli. Considering codon adaptability, the nucleotides of the 4CL1(M4) gene described in Example 1 were optimized with codons from Yersinia lipolytica to obtain the 4CL1(M4) nucleotide sequence, as shown in SEQ ID NO. 7; the nucleotides of the VST1(N3) mutant described in Example 2 were optimized with codons from Yersinia lipolytica to obtain the VST1(N3) nucleotide sequence, as shown in SEQ ID NO. 8. The two optimized genes were then synthesized by Wuhan Jinkairui Biotechnology Co., Ltd.

[0066] A) Using hp4d-4CL1-F / xpr2-4CL1-R as primers, the 4CL1(M4) nucleotide fragment carrying 20 bp homologous sequences of the pINA1312 vector backbone at both ends was amplified using the 4CL1(M4) nucleotide optimized according to the Yersinia lipolyticis codon as a template. Similarly, using xpr2-F / hp4d-R as primers, the pINA1312-vector vector backbone fragment was amplified using the pINA1312 plasmid as a template. The two fragments were then ligated into a vector using the ClonExpress II one-step cloning kit to construct the pINA1312-4CL1(M4) plasmid.

[0067] B) Similarly, using hp4d-VST1-F / xpr2-VST1-R as primers, and the VST1(N3) nucleotides optimized according to the Yersinia lipophila codon as templates, the VST1(N3) fragment carrying 20 bp homologous sequences of the pINA1312 vector backbone at both ends was amplified; the pINA1312-vector vector backbone fragment and the VST1(N3) fragment were ligated into a vector using the ClonExpress II one-step cloning kit to construct the pINA1312-VST1(N3) plasmid.

[0068] C) Using Xpr-1312-yz-up / xpr2-Zeta-R as primers and pINA1312-VST1(N3) plasmid as template, an operon fragment containing hp4d-VST1-xpr2 was amplified, with 20 bp homologous sequences at each end of the vector backbone fragment. Simultaneously, using Zeta-F1 / Xpr2-R1 as primers and pINA1312-4CL1(M4) plasmid as template, a backbone fragment containing the full length of pINA1312-4CL1(M4) plasmid was amplified. The fragments were then ligated using the ClonExpress II one-step cloning kit to construct the pINA1312-4CL1(M4)-VST1(N3) plasmid. The primers used are shown in the table below:

[0069]

[0070] 2) Transformation of Yersinia lipolytica with genes 4CL1 (M4) and VST1 (N3)

[0071] The transformation of the *Yeast lipolyticis* strain was performed primarily according to the instructions of the Zymo Frozen-EZ Yeast Transformation kit II, which are briefly described below:

[0072] Linearization of the transformation fragment vector: The pINA1312-4CL1(M4)-VST1(N3) plasmid constructed in Example 3(1) was digested with NEB restriction endonucleases. The digestion system is shown in the table below:

[0073]

[0074] The prepared enzyme digestion system was placed in a 37℃ water bath for 4 h to obtain linearized plasmid fragments, which were then reserved for later use.

[0075] Preparation of competent cells: Pick a single po1f colony from the resuscitation plate and incubate it overnight at 30°C with shaking at 250 rpm. When the OD value of the bacterial culture reaches 1.0, centrifuge the cells at 500 g for 4 min, discard the supernatant, add 1 ml of Solution 1 to resuspend the cells, centrifuge again and discard the supernatant; add 100 μl of Solution 2 to resuspend the cells. The resulting competent cells of Yersinia lipophila can be directly used for transformation.

[0076] Transformation of competent cells: Take 50 μL of competent cells and mix them with the linearized plasmid fragments mentioned above. Add 500 μL of Solution 3 and mix thoroughly. Incubate in a 30°C water bath for 45 min. During incubation, gently tap or vortex at low speed 2-3 times to mix thoroughly. Spread the incubation solution onto the corresponding SD defect plates and place the plates in a 30°C incubator for static culture for 2-4 days to obtain transformants. The correct transformant is named VST1-01.

[0077]

[0078] 3) VST1-01 strain transformed with p-coumaric acid to produce resveratrol

[0079] The VST1-01 strain obtained in Example 3 (2) was selected for shake-flask fermentation of resveratrol strain. Single clones were selected into test tubes containing 3 ml of YPD liquid medium (20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose) and cultured overnight at 30°C and 220 rpm. After overnight culture, the culture was transferred to 50 ml of YPD liquid medium (250 ml Erlenmeyer flask) and cultured for 24 h. 3 g / L of p-coumaric acid was added, and fermentation was carried out for 72 h. The fermentation broth was taken for liquid chromatography analysis. The liquid chromatography method was as shown in Example 1. The fermentation results are shown in the table below:

[0080]

[0081] Example 4: Fusion expression of 4CL1 (M4) and VST1 (N3) to increase resveratrol yield

[0082] Protein fusion expression is often used to reduce the consumption of potentially unstable intermediates, thereby increasing the yield of the final product. Resveratrol yield was increased by using rigid peptides of (GP)*4 (GPGPGPGP) and (GP)*8 (GPGPGPGPGPGPGPGP). The specific construction method is shown below:

[0083] Using 4GP-4CL1-R / 4GP-VST1-F as primers and pINA1312-4CL1(M4)-VST1(N3) as a template, an operon fragment containing hp4d-4CL1-VST1-Xpr was amplified. This fragment carries a rigid peptide of (GP)*4. Subsequently, the vector was self-ligated using the ClonExpress II one-step cloning kit to construct the vector plasmid pINA1312-4CL1(M4)-GP4-VST1(N3). The plasmid vector is as follows. Figure 3 As shown; using 8GP-4CL1-R / 8GP-VST1-F as primers and pINA1312-4CL1(M4)-VST1(N3) as a template, an operon fragment containing hp4d-4CL1-VST1-Xpr was amplified. This fragment carries a rigid peptide of (GP)*8. Subsequently, the vector was self-ligated using the ClonExpress II one-step cloning kit to construct the vector plasmid pINA1312-4CL1(M4)-GP8-VST1(N3). The primer sequences used are shown in the table below:

[0084]

[0085] The constructed pINA1312-4CL1(M4)-GP4-VST1(N3) and pINA1312-4CL1(M4)-GP8-VST1(N3) plasmids were transformed into po1f strain according to the yeast transformation method in Example 3(2), and the new strains were constructed as shown below:

[0086]

[0087] The newly constructed strain was fermented according to the fermentation method of Example 3 (3), and the fermentation yield of resveratrol is shown in the table below:

[0088]

[0089] Example 5. Production of resveratrol by fed-batch fermentation of strain VST1-11

[0090] VST1-11 single clones were picked from the plate and inoculated into test tubes containing 2 ml of YPD medium. The culture was carried out 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 cultured with shaking for another 24 h to obtain the seed culture for batch fermentation. All 100 ml of the seed culture was transferred to 3 L of fermentation medium, the composition of which was: 60 g / L glucose, 15 g / L (NH4)2SO4, 8 g / L KH2PO4, 6.15 g / L MgSO4, 12 ml / L vitamins, 10 ml / L trace metal salts, 0.5 g / L leucine, and 10 g / L p-coumaric acid. The trace metal salt solution contained the following components: 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 contained: 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.

[0091] Supplemented culture medium: 700 g / L glucose, 120 g / L p-coumaric acid, 10 ml / L trace metal salts, 10 ml / L vitamins, supplemented with 680 mL.

[0092] The batch fermentation temperature was 30℃, and the pH was controlled to 6.0 using NaOH. The glucose concentration was controlled to 30 g / L in batches, and the product formation was monitored during the fermentation process.

[0093] Ultimately, strain VST1-11 produced a maximum of 35.8 g / L resveratrol, achieving a conversion rate of 92.3%. A schematic diagram of the fermentation process is shown below. Figure 4 As shown.

[0094] Part Two: Construction of a high-yield resveratrol-producing *Yersinia lipolytica* strain to enhance intracellular acetyl-CoA and malonyl-CoA supply.

[0095] By expressing endogenous acetyl-CoA carboxylase and acetyl-CoA synthase genes, the intracellular supply of acetyl-CoA and malonyl-CoA was enhanced, and a high-yield resveratrol-producing *Yarrowia lipolytica* strain was constructed. Taking strain VST1-11 as an example, the specific construction method is as follows:

[0096] Example 6: Expression of acetyl-CoA carboxylase (ACC) to increase resveratrol production

[0097] 1. Construction of pINA1269-ACC plasmid vector

[0098] (1) Total RNA was extracted from *Yeast Inonotus lipolyticus* Po1f using a rapid total RNA extraction kit (purchased from Sangon Biotech Co., Ltd.) according to the instructions. Using the extracted total RNA as a template, cDNA was synthesized using a one-step reverse transcription kit (purchased from TransGen Biotech Co., Ltd.). Using the synthesized cDNA as a template, PCR amplification was performed using primers ACC-F and ACC-R. The primer sequences are shown in Table 1. The amplified product was recovered to obtain the target fragment of the ACC gene, with a fragment size of 6801 bp. The nucleotide sequence of the ACC gene is shown in SEQ ID NO. 9.

[0099] (2) Using plasmid vector pINA1269 as a template, PCR amplification was performed using primers P1-F and P1-R. The primer sequences are shown in Table 1. After recovery of the PCR product, the linearized vector pINA1269 was obtained, and the linearized vector fragment size was 7279 bp.

[0100] (3) The above-mentioned ACC target fragment and linearized vector pINA1269 were ligated using the ClonExpress II one-step cloning kit to obtain the recombinant plasmid pINA1269-ACC. The sequence was verified to be correct, and the recombinant plasmid was successfully constructed.

[0101] Table 1 Primer sequences for constructing ACC plasmids

[0102]

[0103] 2. Construction and shake-flask fermentation of engineered *Yarrowia lipolyticis* strain

[0104] (1) The obtained plasmid pINA1269-ACC was digested with the restriction endonuclease Not I, linearized, and then transformed into the engineered Yersinia lipophila strain VST1-11 using the LiAC transformation method, and then plated onto SC. On Leu yeast auxotrophic medium, the culture was carried out at 30℃ until transformants grew. Five positive transformants were selected to obtain engineered strains R1, R2, R3, R4 and R5 expressing acetyl-CoA carboxylase, respectively.

[0105] (2) Engineered strains R1, R2, R3, R4, R5 and control strain VST1-11 were streaked onto YPD culture medium and cultured until single colonies grew. Single colonies were picked and inoculated into seed culture medium and cultured for 15 hours to obtain seed culture. The seed culture medium was YPD medium: 20 g / L glucose as the carbon source, containing 10 g / L yeast extract and 20 g / L peptone. The culture conditions for the seed culture were 30℃ and 220 rpm. The OD of the seed culture was... 600 =10;

[0106] (3) Inoculate 2% of the seed culture into a shake flask containing 50 mL of YPD medium and carry out shake flask fermentation culture;

[0107] (4) After culturing for 24 hours, 5 g / L of substrate was added to biotransform coumaric acid and 3 g / L sodium acetate. After 72 hours of transformation, the yield of resveratrol was detected by high performance liquid chromatography (HPLC) of the fermentation broth.

[0108] (5) High performance liquid chromatography (HPLC) for the detection of resveratrol: The chromatographic column was a C18 (250 mm * 4.6 mm, 5 μm) or an equivalent column. The mobile phase was acetonitrile and 1% acetic acid solution (gradient elution conditions: 5% acetonitrile for 5 min, 5-50% acetonitrile for 15 min, 50% acetonitrile for 5 min, 50-5% acetonitrile for 2 min), the flow rate was 1 mL / min, the injection volume was 10 μL, the column temperature was 25℃, and resveratrol was detected at a wavelength of 305 nm. The content was determined by external standard method.

[0109] (6) The results are as follows Figure 5 As shown, compared with the starting strain VST1-11, the engineered strain expressing acetyl-CoA carboxylase showed a significant increase in resveratrol production. Taking the positive transformant R4 of the engineered strain expressing acetyl-CoA carboxylase as an example, its resveratrol production capacity increased by 40.3% compared with the starting strain VST1-11, with a maximum yield of 1.8 g / L.

[0110] Example 7: Expression of acetyl-CoA synthase (ACS) to increase resveratrol production

[0111] 1. Construction of pINA1269-ACC-ACS plasmid vector

[0112] (1) Using the cDNA synthesized in Example 6 as a template, PCR amplification was performed using primers ACS-F and ACS-R. The primer sequences are shown in Table 2. The amplification product was recovered to obtain the target fragment of the ACS gene, which was 1974 bp in size. The nucleotide sequence of the ACS gene is shown in SEQ ID NO. 10.

[0113] (2) Using the plasmid vector pINA1269-ACC as a template, PCR amplification was performed using primers Hp4d-F and Hp4d-R, and XPR2t-F and XPR2t-R, respectively. The primer sequences are shown in Table 2. After the PCR products were recovered, the target fragments Hp4d and XPR2t were obtained, with sizes of 612 bp and 569 bp, respectively.

[0114] (3) Using the target fragments ACS, Hp4d and XPR2t as templates, Overlap PCR was performed using primers Hp4d-F and XPR2t-R. After the PCR product was recovered, the target fragment Hp4d-ACS-XPR2t was obtained. The size of the fragment was 3135bp.

[0115] (4) Using the plasmid vector pINA1269-ACC as a template, PCR amplification was performed using primers P2-F and P2-R. The primer sequences are shown in Table 2. After the PCR product was recovered, the linearized vector pINA1269-ACC was obtained. The linearized vector fragment size was 14060 bp.

[0116] (5) The above Hp4d-ACS-XPR2t target fragment and linearized vector pINA1269-ACC were ligated using the ClonExpress II one-step cloning kit to obtain the recombinant plasmid pINA1269-ACC-ACS. The recombinant plasmid was successfully constructed and verified by sequencing.

[0117] Table 2 Primer sequences for constructing ACS plasmids

[0118]

[0119] 2. Construction and shake-flask fermentation of engineered *Yarrowia lipolyticis* strain

[0120] (1) The obtained plasmid pINA1269-ACC-ACS was digested with restriction endonuclease Not I, linearized, and then transformed into the engineered Yersinia lipophila strain VST1-11 using the LiAC transformation method, and then plated onto SC. On Leu yeast auxotrophic medium, the culture was carried out at 30℃ until transformants grew. Five positive transformants were selected to obtain engineered strains R6, R7, R8, R9 and R10 expressing acetyl-CoA synthase, respectively.

[0121] (2) Engineered strains R6, R7, R8, R9, R10 and control strain R4 were streaked onto YPD culture medium and cultured until single colonies grew. Single colonies were picked and inoculated into seed culture medium and cultured for 15 hours to obtain seed culture. The seed culture medium was YPD medium: 20 g / L glucose as the carbon source, containing 10 g / L yeast extract and 20 g / L peptone. The culture conditions for the seed culture were 30℃ and 220 rpm. The OD of the seed culture was... 600 =10;

[0122] (3) Inoculate 2% of the seed culture into a shake flask containing 50 mL of YPD medium and carry out shake flask fermentation culture;

[0123] (4) After culturing for 24 hours, 5 g / L of substrate was added to biotransform coumaric acid and 3 g / L sodium acetate. After 72 hours of transformation, the yield of resveratrol was detected by high performance liquid chromatography (HPLC) of the fermentation broth.

[0124] (5) High performance liquid chromatography (HPLC) for the detection of resveratrol: The chromatographic column was a C18 (250 mm * 4.6 mm, 5 μm) or an equivalent column. The mobile phase was acetonitrile and 1% acetic acid solution (gradient elution conditions: 5% acetonitrile for 5 min, 5-50% acetonitrile for 15 min, 50% acetonitrile for 5 min, 50-5% acetonitrile for 2 min), the flow rate was 1 mL / min, the injection volume was 10 μL, the column temperature was 25℃, and resveratrol was detected at a wavelength of 305 nm. The content was determined by external standard method.

[0125] The results are as follows Figure 6 As shown, compared with the control strain R4, the engineered strain expressing acetyl-CoA synthase showed a significant increase in resveratrol production. Taking the positive transformant R7 of the engineered strain expressing acetyl-CoA synthase as an example, the resveratrol production capacity increased by 30.3%, with the highest yield reaching 2.4 g / L.

[0126] Example 8: High-yield resveratrol production through fed-batch fermentation

[0127] (1) Pick R7 single clones from the plate and inoculate them into test tubes containing 2 mL of YPD medium and culture at 30°C and 220 rpm for 24 h with shaking.

[0128] (2) Transfer to a 500 mL Erlenmeyer flask containing 100 mL of YPD medium, shake and culture for 24 h to obtain the seed culture required for batch fermentation;

[0129] (3) Transfer 100 mL of seed culture to an initial volume of 2 L of fermentation medium. The fermentation medium consists of: 60 g / L glucose, 15 g / L (NH4)2SO4, 8 g / L KH2PO4, 6.15 g / L MgSO4, 12 mL / L vitamin solution, 10 mL / L trace metal salt solution, 0.5 g / L leucine, 3 g / L sodium acetate, and 15 g / L p-coumaric acid. The trace metal salt solution consists of: 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 niacin, 25 g / L inositol, 1 g / L thiamine hydrochloride, 1 g / L pyridoxal phosphate, and 0.2 g / L para-aminobenzoic acid.

[0130] (4) Supplemented culture medium: 700 g / L glucose, 120 g / L p-coumaric acid, 20 g / L sodium acetate, 10 mL / L trace metal salt solution, 10 mL / L vitamin solution, supplemented 910 mL.

[0131] (5) The batch fermentation temperature is 30℃, and the pH is controlled to 6.0 using NaOH. The glucose concentration is controlled to 30g / L during the batch feeding. The product formation is detected during the fermentation process.

[0132] The results are as follows Figure 7 As shown, the R7 strain produced a resveratrol yield of up to 47.8 g / L after 168 h of fermentation, with a conversion rate of 96.3%.

Claims

1. A recombinant genetically engineered bacteria, characterized in that: The recombinant genetically engineered bacteria are Yarrowia lipolytica engineered bacteria, and the recombinant genetically engineered bacteria comprise an expression vector of a coding gene of a 4-coumaric acid coenzyme A ligase mutant and a coding gene of a resveratrol synthase mutant and an acetyl coenzyme A carboxylase, or an expression vector of a coding gene of a 4-coumaric acid coenzyme A ligase mutant and a coding gene of a resveratrol synthase mutant and coding genes of an acetyl coenzyme A carboxylase and an acetyl coenzyme A synthetase, or the genome of the recombinant genetically engineered bacteria is integrated with a coding gene of a 4-coumaric acid coenzyme A ligase mutant, a coding gene of a resveratrol synthase mutant and a coding gene of an acetyl coenzyme A carboxylase, or integrated with a coding gene of a 4-coumaric acid coenzyme A ligase mutant, a coding gene of a resveratrol synthase mutant and coding genes of an acetyl coenzyme A carboxylase and an acetyl coenzyme A synthetase, the amino acid sequence of the 4-coumaric acid coenzyme A ligase mutant is as shown in SEQ ID NO. 5, the amino acid sequence of the resveratrol synthase mutant is as shown in SEQ ID NO. 6, the coding gene sequence of the acetyl coenzyme A carboxylase is as shown in SEQ ID NO. 9, and the coding sequence of the acetyl coenzyme A synthetase is as shown in SEQ ID NO.

10.

2. The method for obtaining the recombinant genetically engineered bacteria according to claim 1, characterized in that: The expression vector comprising a coding gene of a 4-coumaric acid coenzyme A ligase mutant, a coding gene of a resveratrol synthase mutant and a coding gene of an acetyl coenzyme A carboxylase, or the expression vector comprising a coding gene of a 4-coumaric acid coenzyme A ligase mutant, a coding gene of a resveratrol synthase mutant and coding genes of an acetyl coenzyme A carboxylase and an acetyl coenzyme A synthetase is introduced into a host cell to obtain the recombinant genetically engineered bacteria, wherein the coding gene of the 4-coumaric acid coenzyme A ligase mutant and the coding gene of the resveratrol synthase mutant are expressed separately or are expressed as a fusion protein by fusion expression.

3. The recombinant genetically engineered bacteria of claim 1 are used for producing resveratrol from p-coumaric acid as a substrate.

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