Genetically engineered bacterium, preparation method thereof and application of genetically engineered bacterium in synthesis of resveratrol

Abstract

The invention provides a genetically engineered bacterium, a preparation method of the genetically engineered bacterium and application of the genetically engineered bacterium in synthesis of resveratrol. The genetically engineered bacterium contains a p-coumaroyl coenzyme A ligase gene At4CL and a stilbene synthase gene PcSTS, wherein the p-coumaroyl coenzyme A ligase gene At4CL is derived from arabidopsis thaliana, and the stilbene synthase gene PcSTS is derived from Polygonum cuspidatum, and the p-coumaroyl coenzyme A ligase gene At4CL is derived from Polygonum cuspidatum. Wherein the genetically engineered bacteria are selected from escherichia coli. The method for catalytically synthesizing resveratrol by using the genetically engineered bacterium is short in fermentation period, high in yield and high in production intensity, has a relatively good application prospect, and lays a foundation for industrial production of resveratrol by using an escherichia coli microbial fermentation method.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a genetically engineered bacterium, a preparation method thereof and an application thereof in synthesizing resveratrol. Background Art

[0002] Resveratrol (3-4'-5-trihydroxystilbene), with the molecular formula C14H12O3, is a plant-derived non-flavonoid polyphenol compound that has received widespread attention due to the "French paradox". Resveratrol exists in more than 70 plants, including grapes, peanuts, and knotweed. It has multiple physiological activities such as anti-cancer, anti-oxidation, anti-inflammatory, anti-aging, and cardiovascular protection. It has great application value and market prospects in the fields of food, health products, nutritional supplements, and cosmetics.

[0003] At present, the main method of producing resveratrol is to extract it from knotweed or grapes, but it is limited by the low resveratrol content in plants, the complex extraction process and the low extraction efficiency, and the time and space constraints of plant growth. The chemical synthesis method has complicated steps, requires more organic reagents and has a low yield, which makes it difficult to apply on a large scale. Therefore, it is urgent to develop an efficient resveratrol production method to meet the growing market demand.

[0004] Microbial fermentation has gradually become an emerging production method due to its mild reaction conditions and green production process. With the development of synthetic biology and metabolic engineering, the economic and usability of microbial fermentation has been continuously improved, and it is an efficient production method for bulk chemicals and fine chemicals. Yield (titer) and production intensity (productivity) are two important indicators that have been reported to measure the level of microbial fermentation. Yield refers to the amount of product obtained per unit fermentation volume after the fermentation is completed. The unit is usually g / L. The higher the yield, the more the total yield of the target product obtained after the fermentation is completed, and the more conducive it is to collect the product; production intensity refers to the amount of product obtained per unit fermentation volume per unit time. The higher the production intensity, the more intensive the entire fermentation process, the stronger the capacity, and the higher the efficiency. In addition, fermentation stability, the types and prices of materials required for fermentation, and the complexity of the fermentation operation process are also key factors to consider in evaluating the fermentation process.

[0005] There are currently three main microbial systems for producing resveratrol, namely Yarrowia lipolytica, Saccharomyces cerevisiae and Escherichia coli. Yarrowia lipolytica can achieve high-yield synthesis of resveratrol due to its natural hydrophobic environment, high malonyl-CoA supply capacity and strong efflux system. However, the fermentation cycle is usually long, metabolic engineering is difficult, and as a typical dimorphic yeast, strict and complex fermentation processes are required to control it to maintain a yeast form with high production capacity, which increases the instability of industrial production. Saccharomyces cerevisiae is considered a safe strain, and resveratrol synthesis can be achieved by introducing heterologous pathways and strengthening the supply of endogenous precursors, but the overall fermentation yield and production intensity are low.

[0006] As a prokaryotic model organism, Escherichia coli has the advantages of clear genetic background, high genetic stability, simple genetic manipulation, simple nutrients required for fermentation, and easy fermentation process control. It is the preferred chassis for synthesizing high-value compounds. In addition, due to its short fermentation cycle, its production intensity is relatively high, which is conducive to improving the utilization rate of production equipment and thus reducing production costs. However, the highest yield of resveratrol synthesized from scratch by Escherichia coli reported so far is only 305 mg / L (Wu, Junjun, et al. "Efficient de novo synthesis of resveratrol by metabolically engineeredEscherichia coli." Journal of Industrial Microbiology and Biotechnology 44.7(2017): 1083-1095.).

[0007] In summary, although Yarrowia lipolytica can achieve efficient synthesis of resveratrol, it still faces problems such as long fermentation cycle and complex process control. The production of resveratrol using Saccharomyces cerevisiae faces the problem of low yield and production intensity. Therefore, breaking through the technical bottleneck of Escherichia coli's difficulty in synthesizing malonyl-CoA derivatives and achieving high-yield and high-production-intensity resveratrol synthesis is crucial to realize the industrial production of resveratrol on a diversified microbial chassis. Summary of the invention

[0008] The main purpose of the present invention is to provide a genetically engineered bacterium, a preparation method thereof and an application thereof in synthesizing resveratrol, so as to solve the problem of low yield of resveratrol produced by Escherichia coli in the prior art.

[0009] In order to achieve the above-mentioned object, according to the first aspect of the present invention, a genetically engineered bacterium is provided, which contains an exogenously introduced p-coumaroyl-CoA ligase gene At4CL from Arabidopsis thaliana and a stilbene synthase gene PcSTS from Polygonum cuspidatum; wherein the genetically engineered bacterium is selected from Escherichia coli.

[0010] Furthermore, the genetically engineered bacteria also contain an exogenously introduced amino acid ammonia lyase gene FjTAL derived from Flavobacterium johnsoniae.

[0011] Furthermore, the protein encoded by the p-coumaroyl-CoA ligase gene At4CL from Arabidopsis thaliana has an amino acid sequence shown in SEQ ID NO: 1;

[0012] The protein encoded by the stilbene synthase gene PcSTS from Polygonum cuspidatum has an amino acid sequence shown in SEQ ID NO: 5;

[0013] The protein encoded by the amino acid ammonia lyase gene FjTAL derived from Flavobacterium johnsoniae has the amino acid sequence shown in SEQ ID NO:7.

[0014] Furthermore, the genetically engineered bacteria do not contain any one or more of the following genes: pta or ackA.

[0015] Furthermore, the protein encoded by the pta gene has the amino acid sequence shown in SEQ ID NO:8; and the protein encoded by the ackA gene has the amino acid sequence shown in SEQ ID NO:9.

[0016] Furthermore, the genetically engineered bacteria mentioned above also contain any one or more of the following exogenously introduced genes: mutant aroG derived from Escherichia coli and mutant tyrA derived from Escherichia coli.

[0017] Further, the protein encoded by the mutant aroG gene from Escherichia coli has the amino acid sequence shown in SEQ ID NO: 11;

[0018] The protein encoded by the mutant tyrA gene derived from Escherichia coli has the amino acid sequence shown in SEQ ID NO:12.

[0019] Furthermore, the genetically engineered bacteria also contain the following exogenously introduced genes: accBC derived from Corynebacterium glutamicum and dtsR1 derived from Corynebacterium glutamicum.

[0020] Further, the protein encoded by the accBC gene from Corynebacterium glutamicum has the amino acid sequence shown in SEQ ID NO: 15;

[0021] The protein encoded by the dtsR1 gene derived from Corynebacterium glutamicum has the amino acid sequence shown in SEQ ID NO:16.

[0022] Furthermore, the genetically engineered bacteria also contain a fatty acid synthesis regulatory factor fadR, and the promoter of the fatty acid synthesis regulatory factor fadR is fliC.

[0023] Furthermore, the genetically engineered bacteria also overexpress any one or more of the following genes: glpF derived from Escherichia coli, glpK derived from Escherichia coli, glpD derived from Escherichia coli, or glpX derived from Escherichia coli.

[0024] Further, the protein encoded by the glpF gene has an amino acid sequence as shown in SEQ ID NO: 17;

[0025] The protein encoded by the glpK gene has the amino acid sequence shown in SEQ ID NO: 18;

[0026] The protein encoded by the glpD gene has the amino acid sequence shown in SEQ ID NO: 19;

[0027] The protein encoded by the glpX gene has the amino acid sequence shown in SEQ ID NO:10.

[0028] Furthermore, the genetically engineered bacteria do not contain the gene LamB and / or contain the gene ompF.

[0029] Further, the protein encoded by the LamB gene has the amino acid sequence shown in SEQ ID NO: 13;

[0030] The protein encoded by the ompF gene has the amino acid sequence shown in SEQ ID NO:14.

[0031] In order to achieve the above object, according to a second aspect of the present invention, a method for preparing a genetically engineered bacterium is provided, the method comprising: transferring a plasmid carrying a p-coumaroyl-CoA ligase gene At4CL and a stilbene synthase gene PcSTS into a starting engineering bacterium to obtain a genetically engineered bacterium;

[0032] The p-coumaroyl-CoA ligase gene At4CL is derived from Arabidopsis thaliana; the stilbene synthase gene PcSTS is derived from Polygonum cuspidatum; and the starting engineering bacteria is selected from Escherichia coli.

[0033] Furthermore, the preparation method further comprises: transferring a plasmid carrying the amino acid ammonia lyase gene FjTAL into the starting engineering bacteria or knocking in the amino acid ammonia lyase gene FjTAL into the starting engineering bacteria;

[0034] Among them, the amino acid ammonia lyase gene FjTAL comes from Flavobacterium johnsoniae.

[0035] Furthermore, the protein encoded by the p-coumaroyl-CoA ligase gene At4CL from Arabidopsis thaliana has an amino acid sequence shown in SEQ ID NO: 1;

[0036] The protein encoded by the stilbene synthase gene PcSTS from Polygonum cuspidatum has an amino acid sequence shown in SEQ ID NO: 5;

[0037] The protein encoded by the amino acid ammonia lyase gene FjTAL derived from Flavobacterium johnsoniae has the amino acid sequence shown in SEQ ID NO:7.

[0038] Furthermore, the preparation method further comprises: knocking out any one or more of the following genes in the starting engineering bacteria: pta or ackA.

[0039] Further, the protein encoded by the pta gene has an amino acid sequence as shown in SEQ ID NO: 8;

[0040] The protein encoded by the ackA gene has the amino acid sequence shown in SEQ ID NO:9.

[0041] Furthermore, the preparation method further comprises: knocking into the starting engineering bacteria any one or more of the following genes: a mutant aroG derived from Escherichia coli and a mutant tyrA derived from Escherichia coli.

[0042] Further, the protein encoded by the mutant aroG gene has an amino acid sequence as shown in SEQ ID NO: 11;

[0043] The protein encoded by the mutant tyrA gene has the amino acid sequence shown in SEQ ID NO:12.

[0044] Furthermore, the preparation method further comprises: knocking into the starting engineering bacteria the accBC gene derived from Corynebacterium glutamicum and the dtsR1 gene derived from Corynebacterium glutamicum.

[0045] Furthermore, the protein encoded by the accBC gene derived from Corynebacterium glutamicum has the amino acid sequence shown in SEQ ID NO: 15; and the protein encoded by the dtsR1 gene derived from Corynebacterium glutamicum has the amino acid sequence shown in SEQ ID NO: 16.

[0046] Furthermore, the preparation method further comprises: replacing the promoter of the fatty acid synthesis regulatory factor fadR in the starting engineering bacteria with the fliC promoter in situ.

[0047] Furthermore, the preparation method further comprises: knocking into the starting engineering bacteria any one or more of the following genes: glpF from Escherichia coli, glpK from Escherichia coli, glpD from Escherichia coli or glpX from Escherichia coli.

[0048] Further, the protein encoded by the glpF gene from Escherichia coli has the amino acid sequence shown in SEQ ID NO: 17;

[0049] The protein encoded by the glpK gene from Escherichia coli has the amino acid sequence shown in SEQ ID NO: 18;

[0050] The protein encoded by the glpD gene from Escherichia coli has the amino acid sequence shown in SEQ ID NO: 19;

[0051] The protein encoded by the glpX gene derived from Escherichia coli has the amino acid sequence shown in SEQ ID NO:10.

[0052] Furthermore, the preparation method further comprises: knocking out the LamB gene in the genetically engineered bacteria and / or knocking in the ompF gene into the genetically engineered bacteria.

[0053] Furthermore, the protein encoded by the LamB gene has the amino acid sequence shown in SEQ ID NO: 13; and the protein encoded by the ompF gene has the amino acid sequence shown in SEQ ID NO: 14.

[0054] To achieve the above object, according to the third aspect of the present invention, a method for synthesizing resveratrol is provided, which comprises: fermenting and synthesizing resveratrol in a fermentation medium using the above genetically engineered bacteria or the genetically engineered bacteria prepared by the above genetically engineered bacteria preparation method.

[0055] Furthermore, the fermentation is shake flask fermentation; the shake flask fermentation includes: inoculating the genetically engineered bacteria cultured overnight into the fermentation medium of the shake flask fermentation for expansion culture, culturing to OD600 of 0.8-1, adding an inducer to induce fermentation for 22-94 hours to obtain resveratrol.

[0056] Furthermore, the conditions for overnight culture are 30-37°C, 200-220rpm; the conditions for expanded culture are 30-37°C, 200-220rpm; the conditions for induced fermentation are 25-35°C, 200-220rpm; the inducer is IPTG; and the final concentration of the inducer is 0.1-1mM.

[0057] Further, the fermentation medium for shake flask fermentation is 20-30 g / L glycerol, 5-10 g / L yeast extract, 5-8 g / L disodium hydrogen phosphate, 2-5 g / L potassium dihydrogen phosphate, 0.3-0.8 g / L sodium chloride, 0.5-1.5 g / L ammonium chloride, 0.01-0.03 g / L calcium chloride, 0.2-0.5 g / L magnesium sulfate, 40-50 g / L MOPS and 50 mg / L-100 mg / L antibiotics and optionally 1-2 g / L tyrosine or optionally 1-2 mM p-coumaric acid or optionally 1-3 g / L sodium bicarbonate and 30-50 mg / L biotin and optionally 10%-20% tributyrin.

[0058] By applying the technical scheme of the present invention, the genetically engineered bacteria of the present invention contain exogenously introduced p-coumaroyl-CoA ligase gene At4CL and stilbene synthase gene PcSTS. Such genetically engineered bacteria can express p-coumaroyl-CoA ligase and stilbene synthase in vivo, thereby enabling the genetically engineered bacteria to catalyze the synthesis of resveratrol using p-coumaric acid as a precursor. The method for catalyzing the synthesis of resveratrol by the genetically engineered bacteria has a short fermentation cycle, high yield, high production intensity, good application prospects, and lays a foundation for the industrial production of resveratrol using the Escherichia coli microbial fermentation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0060] Figure 1 The metabolic pathway diagram of the biosynthesis of resveratrol in the present invention is shown.

[0061] Figure 2 The result diagram of the screening of sources of p-coumaroyl-CoA ligase and stilbene synthase according to Example 1 of the present invention is shown.

[0062] Figure 3The result diagram of the screening of amino acid ammonia lyase sources according to Example 2 of the present invention is shown.

[0063] Figure 4 The fermentation curve results of the engineered strain Res055 synthesizing resveratrol from tyrosine according to Example 3 of the present invention are shown, including (1) tyrosine curve, (2) p-coumaric acid curve, and (3) resveratrol curve.

[0064] Figure 5 A schematic diagram of tyrosine production during 96 h of shake flask fermentation of the engineered strains Res02 ​​and Res03 according to Example 4 of the present invention is shown.

[0065] Figure 6 A schematic diagram of the resveratrol synthesis curve of the engineered strains Res056 and Res058 according to Example 5 of the present invention is shown; wherein (1) represents the engineered strain Res056, and (2) represents the engineered strain Res058.

[0066] Figure 7 A schematic diagram of the resveratrol synthesis curve of the engineered strains Res178 and Res290 according to Example 6 of the present invention is shown; wherein (1) represents the engineered strain Res178, and (2) represents the engineered strain Res290.

[0067] Figure 8 The resveratrol yields of the engineered strains Res381, Res382, Res383 and Res384 according to Example 7 of the present invention after 72 h of shake flask fermentation are shown.

[0068] Fig. 9 The resveratrol yields of the engineered strains Res785 and Res787 according to Example 8 of the present invention after 72 h of shake flask fermentation are shown.

[0069] Fig.10 The graph shows the resveratrol yield of the engineered strain Res787 according to Example 9 of the present invention after fermentation for 72 h in a fermentation medium containing sodium bicarbonate and biotin. DETAILED DESCRIPTION

[0070] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0071] As mentioned in the background technology, resveratrol has multiple physiological activities such as anti-cancer, anti-oxidation, anti-inflammatory, anti-aging, cardiovascular protection, etc., and has great application value and market prospects in the fields of food, health products, nutritional supplements and cosmetics. In the prior art, Escherichia coli used to produce resveratrol has the defect of low yield.

[0072] The technical scheme of the present invention induces the genetic engineering bacteria containing exogenously introduced p-coumaryl-CoA ligase gene At4CL and stilbene synthase gene PcSTS to express p-coumaryl-CoA ligase and stilbene synthase, and uses p-coumaric acid as a precursor to catalyze the synthesis of resveratrol (the synthesis route of resveratrol of the present invention is shown in Figure 1 The resveratrol produced by the genetically engineered bacteria has high yield and high efficiency, has good application prospects, and lays a foundation for the industrial production of resveratrol using Escherichia coli microbial fermentation. On this basis, the inventors proposed a series of protection schemes of the present invention.

[0073] In a first typical embodiment of the present invention, a genetically engineered bacterium is provided, wherein the genetically engineered bacterium contains an exogenously introduced p-coumaroyl-CoA ligase gene At4CL from Arabidopsis thaliana and a stilbene synthase gene PcSTS from Polygonum cuspidatum; wherein the genetically engineered bacterium is selected from Escherichia coli.

[0074] The yield of resveratrol synthesized by coumaroyl-CoA ligase from different sources and stilbene synthase from different sources is different. The present invention combines At4CL from Arabidopsis thaliana, Pc4CL from Petroselinum crispum, Pp4CL from Pseudomonas protegens, AhSTS from Arachis hypogasa, PcSTS from Polygonum cuspidatum and VvSTS from Vitis vinifera, and finally screens out the optimal combination: At4CL from Arabidopsis thaliana and PcSTS from Polygonum cuspidatum. With p-coumaric acid as a precursor, the combination is used for fermentation for 48 hours, and the yield of resveratrol is 90.32 mg / L.

[0075] In a preferred embodiment of the present invention, the protein encoded by the p-coumaroyl-CoA ligase gene At4CL from Arabidopsis thaliana has the amino acid sequence shown in SEQ ID NO: 1; the protein encoded by the stilbene synthase gene PcSTS from Polygonum cuspidatum has the amino acid sequence shown in SEQ ID NO: 5.

[0076] In order to enable genetically engineered bacteria to efficiently synthesize p-coumaric acid in vivo, the present invention uses tyrosine as a precursor to screen amino acid ammonia lyases from different sources, and finally screens out amino acid ammonia lyases from Flavobacterium johnsoniae that can efficiently synthesize p-coumaric acid. The protein encoded by the amino acid ammonia lyase gene FjTAL from Flavobacterium johnsoniae has an amino acid sequence shown in SEQ ID NO: 7.

[0077] In a preferred embodiment of the present invention, the genetically engineered bacteria further contain an exogenously introduced amino acid ammonia lyase gene FjTAL from Flavobacterium johnsoniae. The genetically engineered bacteria of the present invention can achieve efficient synthesis of p-coumaric acid in vivo.

[0078] To achieve the de novo synthesis of resveratrol starting from a simple and renewable carbon source. In a preferred embodiment of the present invention, the genetically engineered bacteria do not contain any one or more of the following genes: pta or ackA. Among them, the knockout of pta (encoding protein is phosphate acetyltransferase) and ackA (encoding protein is acetate kinase) can avoid the formation of byproduct acetic acid. In a preferred embodiment of the present invention, the protein encoded by the pta gene has the amino acid sequence shown in SEQ ID NO: 8; the protein encoded by the ackA gene has the amino acid sequence shown in SEQ ID NO: 9.

[0079] Although the knockout of pta or ackA can enable more carbon flux to be used for tyrosine synthesis, when the genetically engineered bacteria do not contain key genes for tyrosine synthesis (for example, the genes encoding 3-deoxy-D-arabinoheptulose-7-phosphate synthase aroG / aroF, the gene encoding prephenate dehydrogenase tyrA, the gene encoding shikimate kinase aroK, etc.), the genetically engineered bacteria cannot efficiently synthesize tyrosine.

[0080] In a preferred embodiment of the present invention, the genetically engineered bacteria further contain any one or more of the following exogenously introduced genes: mutant aroG derived from Escherichia coli, mutant tyrA derived from Escherichia coli.

[0081] Among them, the knock-in of mutant aroG further strengthened the shikimate pathway, and the knock-in of mutant tyrA strengthened the terminal pathway of tyrosine synthesis.

[0082] It should be noted that the expression “the genetically engineered bacteria further contains any one or more of the following exogenously introduced genes: mutant aroG derived from Escherichia coli, mutant tyrA derived from Escherichia coli” here means that the genetically engineered bacteria, on the premise that it itself contains one copy of aroG and tyrA, further transfers one copy of mutant aroG derived from Escherichia coli and / or one copy of mutant tyrA derived from Escherichia coli into the above-mentioned genetically engineered bacteria.

[0083] In a preferred embodiment of the present invention, the protein encoded by the mutant aroG gene derived from Escherichia coli has the amino acid sequence shown in SEQ ID NO: 11; the protein encoded by the mutant tyrA gene derived from Escherichia coli has the amino acid sequence shown in SEQ ID NO: 12.

[0084] To enhance the metabolic flux from acetyl-CoA to malonyl-CoA, in a preferred embodiment of the present invention, the genetically engineered bacteria further contain the following exogenously introduced genes: accBC derived from Corynebacterium glutamicum and dtsR1 derived from Corynebacterium glutamicum.

[0085] It should be noted that the gene accBC encodes the α subunit protein of acetyl-CoA carboxylase, and the gene dtsR1 encodes the β subunit protein of acetyl-CoA carboxylase. When the gene accBC and the gene dtsR1 are introduced into the genetically engineered bacteria at the same time, the genetically engineered bacteria can play the role of carboxylating acetyl-CoA to synthesize malonyl-CoA.

[0086] In a preferred embodiment of the present invention, the protein encoded by the accBC gene derived from Corynebacterium glutamicum has the amino acid sequence shown in SEQ ID NO: 15; the protein encoded by the dtsR1 gene derived from Corynebacterium glutamicum has the amino acid sequence shown in SEQ ID NO: 16.

[0087] In order to avoid excessive consumption of malonyl-CoA in the fatty acid synthesis pathway and ensure normal cell growth, the natural promoter of the fatty acid synthesis regulatory factor fadR in the genetically engineered bacteria is replaced in situ using the growth-dependent promoter fliC, whose transcription level decreases during the stable period. In a preferred embodiment of the present invention, the genetically engineered bacteria also contain the fatty acid synthesis regulatory factor fadR, and the promoter of the fatty acid synthesis regulatory factor fadR is fliC.

[0088] Replacing the above promoter can normally express fadR during the growth period to ensure normal fatty acid synthesis and normal growth, while in the late growth exponential period and the stable growth period, the fadR transcription level is significantly downregulated, thereby inhibiting the synthesis of fatty acids so that more malonyl-CoA can be used to synthesize resveratrol. This will not cause premature inhibition of growth on the one hand, and is beneficial to the synthesis of resveratrol on the other.

[0089] According to literature reports, glucose has an inhibitory effect on the expression of 4CL and STS, the key genes for resveratrol synthesis. Therefore, the present invention selects glycerol as a carbon source to replace glucose. However, compared with glucose, Escherichia coli has a lower rate of glycerol utilization, which limits the speed of synthesizing high-value compounds using glycerol as a carbon source. Therefore, the glycerol utilization pathway is further strengthened to increase resveratrol production.

[0090] In a preferred embodiment of the present invention, the genetically engineered bacteria further overexpress any one or more of the following genes: glpF from Escherichia coli (the protein encoded is a water-glycerol channel protein), glpK from Escherichia coli (the protein encoded is a glycerol kinase), glpD from Escherichia coli (the protein encoded is glyceraldehyde-3-phosphate dehydrogenase) or glpX from Escherichia coli (the protein encoded is fructose-1,6-bisphosphatase).

[0091] It should be noted that the meaning of overexpression in the present invention refers to the copy number of the gene in the genetically engineered bacteria being ≥ 2. When the copy number of a gene in a genetically engineered bacteria increases, the effect of the gene in the genetically engineered bacteria may be enhanced. In a preferred embodiment of the present invention, the copy number of the above genes glpF, glpK, glpD and glpX in the above genetically engineered bacteria is 2.

[0092] In a preferred embodiment of the present invention, the protein encoded by the glpF gene has the amino acid sequence shown in SEQ ID NO: 17; the protein encoded by the glpK gene has the amino acid sequence shown in SEQ ID NO: 18; the protein encoded by the glpD gene has the amino acid sequence shown in SEQ ID NO: 19; and the protein encoded by the glpX gene has the amino acid sequence shown in SEQ ID NO: 10.

[0093] Resveratrol is a plant-derived polyphenolic substance that inhibits the growth of Escherichia coli to a certain extent. Therefore, in order to enhance the strain's tolerance to resveratrol and further increase resveratrol production, in a preferred embodiment of the present invention, the genetically engineered bacteria does not contain the gene LamB and / or contains an exogenously introduced gene ompF.

[0094] The protein encoded by the LamB gene has an amino acid sequence shown in SEQ ID NO: 13; and the protein encoded by the ompF gene has an amino acid sequence shown in SEQ ID NO: 14.

[0095] In a second typical embodiment of the present invention, a method for preparing a genetically engineered bacterium is provided, the method comprising: transferring a plasmid carrying a p-coumaroyl-CoA ligase gene At4CL and a stilbene synthase gene PcSTS into a starting engineered bacterium to obtain the genetically engineered bacterium.

[0096] The p-coumaryl-CoA ligase gene At4CL is derived from Arabidopsis thaliana; the stilbene synthase gene PcSTS is derived from Polygonum cuspidatum; and the starting engineering bacteria are selected from Escherichia coli. The genetically engineered bacteria prepared by the preparation method of the present invention can use p-coumaric acid as a substrate to efficiently synthesize resveratrol.

[0097] It should be noted that the p-coumaroyl-CoA ligase gene At4CL and the stilbene synthase gene PcSTS can be constructed on the same plasmid or on two plasmids respectively, and then the plasmids are transferred into genetically engineered bacteria.

[0098] In a preferred embodiment of the present invention, the preparation method further comprises: transferring a plasmid carrying the amino acid ammonia lyase gene FjTAL into the starting engineering bacteria or knocking in the amino acid ammonia lyase gene FjTAL into the starting engineering bacteria; wherein the amino acid ammonia lyase gene FjTAL is derived from Flavobacterium johnsoniae.

[0099] Among them, the protein encoded by the coumaroyl-CoA ligase gene At4CL from Arabidopsis thaliana has the amino acid sequence shown in SEQ ID NO: 1; the protein encoded by the stilbene synthase gene PcSTS from Polygonum cuspidatum has the amino acid sequence shown in SEQ ID NO: 5; the protein encoded by the amino acid ammonia lyase gene FjTAL from Flavobacterium johnsoniae has the amino acid sequence shown in SEQ ID NO: 7.

[0100] In a preferred embodiment of the present invention, the preparation method further comprises: knocking out any one or more of the following genes in the starting engineering bacteria: pta or ackA.

[0101] Preferably, the protein encoded by the pta gene has the amino acid sequence shown in SEQ ID NO:8; and the protein encoded by the ackA gene has the amino acid sequence shown in SEQ ID NO:9.

[0102] In a preferred embodiment of the present invention, the preparation method further comprises: knocking into the starting engineering bacteria any one or more of the following genes: mutant aroG derived from Escherichia coli and mutant tyrA derived from Escherichia coli.

[0103] Preferably, the protein encoded by the mutant aroG gene has the amino acid sequence shown in SEQ ID NO: 11; and the protein encoded by the mutant tyrA gene has the amino acid sequence shown in SEQ ID NO: 12.

[0104] In a preferred embodiment of the present invention, the preparation method further comprises: knocking in the accBC gene derived from Corynebacterium glutamicum and the dtsR1 gene derived from Corynebacterium glutamicum into the starting engineering bacteria.

[0105] Preferably, the protein encoded by the accBC gene derived from Corynebacterium glutamicum has the amino acid sequence shown in SEQ ID NO: 15; and the protein encoded by the dtsR1 gene derived from Corynebacterium glutamicum has the amino acid sequence shown in SEQ ID NO: 16.

[0106] In a preferred embodiment of the present invention, the preparation method further comprises: replacing the promoter of the fatty acid synthesis regulatory factor fadR in the starting engineering bacteria with the fliC promoter in situ.

[0107] In a preferred embodiment of the present invention, the preparation method further comprises: knocking into the starting engineering bacteria any one or more of the following genes: glpF from Escherichia coli, glpK from Escherichia coli, glpD from Escherichia coli or glpX from Escherichia coli.

[0108] Preferably, the protein encoded by the glpF gene has the amino acid sequence shown in SEQ ID NO: 17; the protein encoded by the glpK gene has the amino acid sequence shown in SEQ ID NO: 18; the protein encoded by the glpD gene has the amino acid sequence shown in SEQ ID NO: 19; and the protein encoded by the glpX gene has the amino acid sequence shown in SEQ ID NO: 10.

[0109] It should be noted that if the genetically engineered bacteria itself contains one copy of glpF, glpK, glpD and glpX, in order to achieve that the genetically engineered bacteria contain two copies of the above genes, it is only necessary to knock in one copy of the above genes into the genetically engineered bacteria.

[0110] In a preferred embodiment of the present invention, the preparation method further comprises: knocking out the LamB gene in the genetically engineered bacteria and / or knocking in the ompF gene into the genetically engineered bacteria.

[0111] Preferably, the protein encoded by the LamB gene has the amino acid sequence shown in SEQ ID NO: 13; and the protein encoded by the ompF gene has the amino acid sequence shown in SEQ ID NO: 14.

[0112] Chassis bacterial transformation is an important method to increase the yield of bacterial target metabolites, which involves the knock-in or knock-out of genes. The purpose of gene knockout is to reduce the accumulation of by-products in the metabolic pathway, while gene knock-in increases raw materials and power for the synthesis of the target product. Among them, knock-in gene refers to the direct integration of genes into the bacterial genome, and knock-out gene refers to the knock-out of genes on the bacterial genome. In a preferred embodiment of the present invention, gene knock-in or knock-out is performed using a gene editing method. Gene transfer into bacteria in the form of a plasmid and knock-in gene are different concepts. After the gene is transferred into bacteria in the form of a plasmid, the gene is not integrated into the bacterial genome.

[0113] It should be noted that there is no specific restriction on the order of knocking in and knocking out the above genes, and the timing of knocking in or knocking out the genes can be selected according to specific needs. The number of knock-ins of the same gene can also be selected according to actual needs. When multiple genes of the same kind are knocked into a genetically engineered bacterium, the effect of this gene in the genetically engineered bacterium will be enhanced.

[0114] In a third typical embodiment of the present invention, a method for synthesizing resveratrol is provided, wherein the genetically engineered bacteria or the genetically engineered bacteria prepared by the preparation method of the genetically engineered bacteria are fermented in a fermentation medium to synthesize the resveratrol. In a preferred embodiment of the present invention, the shake flask fermentation comprises: inoculating the genetically engineered bacteria cultured overnight into the fermentation medium of the shake flask fermentation for expansion culture, culturing to OD600 of 0.8-1, adding an inducer to induce fermentation for 22-94 h, and obtaining the resveratrol.

[0115] In a preferred embodiment of the present invention, the overnight culture conditions are 30-37°C, 200-220rpm; the expanded culture conditions are 30-37°C, 200-220rpm; the induced fermentation conditions are 25-35°C, 200-220rpm; the inducer is IPTG; the final concentration of the inducer is 0.1-1mM. Producing resveratrol under these conditions has the beneficial effect of high resveratrol yield.

[0116] In a preferred embodiment of the present invention, the fermentation medium for the shake flask fermentation is 20-30 g / L of glycerol, 5-10 g / L of yeast extract, 5-8 g / L of disodium hydrogen phosphate, 2-5 g / L of potassium dihydrogen phosphate, 0.3-0.8 g / L of sodium chloride, 0.5-1.5 g / L of ammonium chloride, 0.01-0.03 g / L of calcium chloride, 0.2-0.5 g / L of magnesium sulfate, 40-50 g / L of MOPS, 50 mg / L-100 mg / L of antibiotics, and optionally 1-2 g / L of tyrosine or optionally 1-2 mM p-coumaric acid or optionally 1-3 g / L of sodium bicarbonate and 30-50 mg / L of biotin and optionally 10%-20% of tributyrin.

[0117] It should be noted that when the genetically engineered bacteria cannot synthesize tyrosine by itself, tyrosine needs to be supplemented exogenously. Similarly, when the genetically engineered bacteria cannot synthesize p-coumaric acid by itself, p-coumaric acid needs to be supplemented exogenously. In some embodiments, in order to promote the supply of malonyl-CoA, sodium bicarbonate and biotin need to be supplemented exogenously. In some embodiments, tyrosine or p-coumaric acid or sodium bicarbonate and biotin are added together with the inducer during induction.

[0118] The type of antibiotic is determined by the resistance gene on the plasmid transferred into the genetically engineered bacteria. For example, when a plasmid is transferred into the genetically engineered bacteria, 50 mg / L-100 mg / L of the antibiotic corresponding to the antibiotic resistance gene contained in the plasmid is added to the fermentation medium. If the transferred plasmid is constructed with pACYC-Duet-1 as the starting plasmid, 50 mg / L-100 mg / L of chloramphenicol is added; if the transferred plasmid is constructed with pET22b as the starting plasmid, 50 mg / L-100 mg / L of ampicillin is added.

[0119] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed by the present invention.

[0120] Example 1: Screening of sources of coumaroyl-CoA ligase and stilbene synthase, key enzymes for resveratrol synthesis

[0121] The core of metabolic engineering to construct microbial cell factories is to maximize the carbon flux of the target metabolic pathway, thereby obtaining higher yields, conversion rates, and production intensity. In the same host, enzymes from different sources vary greatly in expression levels, catalytic activity, and compatibility between enzymes. Therefore, in order to achieve efficient synthesis of resveratrol, it is necessary to screen pathway enzymes from different sources for adaptation. 4-coumarate:CoA ligase (4CL) and stilbene synthase (STS) are the rate-limiting enzymes for the synthesis of resveratrol, so we first screened these two enzymes by adding p-coumaric acid as a substrate.

[0122] Three 4CLs were selected, namely At4CL from Arabidopsis thaliana, Pc4CL from Petroselinum crispum, and Pp4CL from Pseudomonas protegens.

[0123] In addition, three STSs were selected, namely AhSTS from peanut (Arachis hypogasa), PcSTS from Polygonum cuspidatum, and VvSTS from grape (Vitis vinifera). The three 4CLs and three STSs were combined in pairs, constructed on the pACYC-Duet-1 vector, and introduced into Escherichia coli BL21 (DE3), and a total of 9 engineered Escherichia coli strains were obtained as shown in Table 1. Among them, the amino acid sequences of the above 4CLs and STSs are shown in Table 1, and the genes of the above 6 proteins were optimized according to the codon usage preference of Escherichia coli.

[0124] The nine engineered strains were inoculated into LB liquid culture medium and cultured overnight at 37°C and 220 rpm to obtain seed solution. The seed solution was inoculated at 1% volume into fresh fermentation medium (20 g / L glycerol, 5 g / L yeast extract, 7 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 0.01 g / L calcium chloride, 0.3 g / L magnesium sulfate, 50 g / L MOPS, 50 mg / L chloramphenicol), cultured at 37°C and 220 rpm for 2 h, then induced by adding IPTG at a final concentration of 0.1 mM, and at the same time, adding p-coumaric acid at a final concentration of 2 mM as a precursor, and inducing fermentation culture at 30°C and 220 rpm for 48 h.

[0125] The resveratrol production was detected by high performance liquid chromatography. The conditions were: chromatographic column: xBridge BEH ShieldRP18 2.5um, mobile phase: H2O+0.1%TFA and ACN+0.1%TFA, flow rate: 1.0 ml / min, column temperature: 40°C, UV detector, detection wavelength: 220 nm, detection time: 15 min.

[0126] The fermentation results are as follows Figure 2 As shown, among these 9 engineered strains, the Res002 strain containing the combination of At4CL and PcSTS had the highest resveratrol production, which could reach 90.32 mg / L in 48 h. Therefore, At4CL and PcSTS were selected for further optimization of resveratrol synthesis.

[0127] Table 1 Screening strains from 4CL and STS sources

[0128]

[0129] The selected Res002 strain was inoculated into LB liquid medium, cultured overnight at 37°C and 220 rpm to obtain seed solution, and the seed solution was inoculated into fresh fermentation medium (glycerol 30 g / L, yeast extract 10 g / L, disodium hydrogen phosphate 5 g / L, potassium dihydrogen phosphate 5 g / L, sodium chloride 0.3 g / L, ammonium chloride 0.5 g / L, calcium chloride 0.03 g / L, magnesium sulfate 0.5 g / L, MOPS 40 g / L, chloramphenicol 100 mg / L) at a volume inoculation rate of 1%, cultured at 37°C and 220 rpm for 2 hours, and then induced by adding IPTG with a final concentration of 0.1 mM, and adding p-coumaric acid with a final concentration of 1.5 mM as a precursor, and induced fermentation culture at 30°C and 220 rpm for 48 hours. The experimental results showed that the resveratrol production of Res002 strain could reach 89.82 mg / L in 48 hours.

[0130] The Res002 strain screened above was inoculated into LB liquid medium, cultured overnight at 37°C and 220 rpm to obtain seed solution, and the seed solution was inoculated into fresh fermentation medium (glycerol 25 g / L, yeast extract 8 g / L, disodium hydrogen phosphate 8 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 0.8 g / L, ammonium chloride 1.5 g / L, calcium chloride 0.02 g / L, magnesium sulfate 0.2 g / L, MOPS 45 g / L, chloramphenicol 80 mg / L) at a volume inoculation rate of 1%, cultured at 37°C and 220 rpm for 2 h, and then induced by adding IPTG with a final concentration of 0.1 mM, and adding p-coumaric acid with a final concentration of 1 mM as a precursor, and induced fermentation culture at 30°C and 220 rpm for 48 h. The experimental results showed that the resveratrol production of the Res002 strain could reach 91.11 mg / L in 48 h.

[0131] The selected Res002 strain was inoculated into LB liquid medium, cultured overnight at 37°C and 220 rpm to obtain seed solution, and the seed solution was inoculated into fresh fermentation medium (glycerol 15 g / L, yeast extract 4 g / L, disodium hydrogen phosphate 4 g / L, potassium dihydrogen phosphate 1 g / L, sodium chloride 0.2 g / L, ammonium chloride 1.8 g / L, calcium chloride 0.04 g / L, magnesium sulfate 0.6 g / L, MOPS 60 g / L, chloramphenicol 120 mg / L) at a volume inoculation rate of 1%, cultured at 37°C and 220 rpm for 2 h, and then induced by adding IPTG with a final concentration of 0.1 mM, and adding p-coumaric acid with a final concentration of 0.5 mM as a precursor, and induced fermentation culture at 30°C and 220 rpm for 48 h. The experimental results showed that the resveratrol yield of Res002 strain could reach 78.32 mg / L in 48 h.

[0132] Example 2: Screening of amino acid ammonia lyase sources

[0133] p-Coumaric acid is a key precursor for the synthesis of resveratrol. Therefore, in order to ensure sufficient p-coumaric acid supply, we used tyrosine as an added precursor to screen 41 amino acid deaminase AADs stored in the laboratory. These 41 AADs were constructed in the pET22b vector and introduced into Escherichia coli BL21 (DE3), obtaining 41 strains of Escherichia coli as shown in Table 2. The 41 engineered strains were inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain seed solution. The seed solution was inoculated into fresh fermentation medium (glycerol 20 g / L, yeast extract 5 g / L, disodium hydrogen phosphate 7 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 0.5 g / L, ammonium chloride 1 g / L, calcium chloride 0.01 g / L, magnesium sulfate 0.3 g / L, MOPS 50 g / L, kanamycin 50 mg / L) at a volume inoculation rate of 1%. After culturing for 2 h at 37°C and 220 rpm, IPTG was added at a final concentration of 0.1 mM for induction. Tyrosine was added as a precursor at a final concentration of 1 g / L at 30°C and 220 rpm for 48 h for induction fermentation. The yield of p-coumaric acid was detected by high performance liquid chromatography. The conditions were: chromatographic column: xBridgeBEH Shield RP18 2.5um, mobile phase: H2O+0.1%TFA and ACN+0.1%TFA, flow rate: 1.0 ml / min, column temperature: 40°C, UV detector, detection wavelength: 220 nm, detection time: 15 min. The fermentation results are as follows Figure 3 As shown in the figure, among these 41 strains of E. coli, Res034 (containing AAD25) can convert 1 g / L of tyrosine into 900.11 mg / L p-coumaric acid in 48 h of shake flask fermentation, with the highest catalytic efficiency. Therefore, AAD25 was subsequently used to achieve the de novo synthesis and optimization of resveratrol. It should be noted that AAD25 is the amino acid ammonia lyase FjTAL (amino acid sequence is SEQ ID NO: 7) from Flavobacterium johnsoniae, so FjTAL is used instead of AAD25 for subsequent description.

[0134] Table 2 Screening strains for amino acid ammonia lyase sources

[0135]

[0136] The selected Res034 was inoculated into LB liquid medium and cultured overnight at 30°C and 200 rpm to obtain seed solution, which was inoculated into fresh fermentation medium (glycerol 30 g / L, yeast extract 10 g / L, disodium hydrogen phosphate 5 g / L, potassium dihydrogen phosphate 5 g / L, sodium chloride 0.3 g / L, ammonium chloride 0.5 g / L, calcium chloride 0.03 g / L, magnesium sulfate 0.5 g / L, MOPS 40 g / L, kanamycin 100 mg / L) at a volume inoculation rate of 1%, and cultured at 30°C and 200 rpm until OD600 was 0.8. Then, IPTG was added at a final concentration of 1 mM for induction, and tyrosine was added at a final concentration of 2 g / L as a precursor, and fermentation was induced at 25°C and 200 rpm for 48 h. The fermentation results showed that Res034 (containing AAD25) could convert 2 g / L tyrosine into 913.12 mg / L p-coumaric acid in 48 h of shake flask fermentation.

[0137] The selected Res034 was inoculated into LB liquid medium and cultured overnight at 35°C and 210 rpm to obtain seed solution, which was inoculated into fresh fermentation medium (glycerol 25 g / L, yeast extract 8 g / L, disodium hydrogen phosphate 8 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 0.8 g / L, ammonium chloride 1.5 g / L, calcium chloride 0.02 g / L, magnesium sulfate 0.2 g / L, MOPS 45 g / L, kanamycin 80 mg / L) at a volume inoculation rate of 1%, and cultured at 35°C and 210 rpm until OD600 was 1. Then, IPTG was added at a final concentration of 0.5 mM for induction, and tyrosine was added at a final concentration of 1.5 g / L as a precursor, and fermentation was induced at 35°C and 210 rpm for 48 h. The fermentation results showed that Res034 (containing AAD25) could convert 1.5 g / L tyrosine into 909.18 mg / L p-coumaric acid in 48 h of shake flask fermentation.

[0138] The screened Res034 was inoculated into LB liquid culture medium and cultured overnight at 28°C and 180 rpm to obtain seed solution. The seed solution was inoculated at 1% volume into fresh fermentation medium (15 g / L glycerol, 4 g / L yeast extract, 4 g / L disodium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 0.2 g / L sodium chloride, 1.8 g / L ammonium chloride, 0.04 g / L calcium chloride, 0.6 g / L magnesium sulfate, 60 g / L MOPS, and 120 mg / L kanamycin), and cultured at 28°C and 180 rpm until OD600 was 1. After induction, 0.05 mM IPTG was added at a final concentration. At the same time, 0.5 g / L tyrosine was added as a precursor at a final concentration, and fermentation was induced at 24°C and 180 rpm for 48 h. The fermentation results showed that Res034 (containing AAD25) could convert 0.5 g / L tyrosine into 423.08 mg / L p-coumaric acid in 48 h of shake flask fermentation.

[0139] Example 3: Synthesis of resveratrol from tyrosine

[0140] In order to synthesize resveratrol from tyrosine, the FjTAL screened in Example 2 was integrated into the genome of the W3110 chassis strain, and the resveratrol synthesis plasmid pACYC-At4CL-PcSTS was introduced to construct the engineered strain Res055. It should be noted that in the present invention, the integration of genes into the genome or the knockout of genes in the genome are both performed by gene editing methods.

[0141] To detect the ability of Res055 to synthesize resveratrol from tyrosine, it was inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain seed solution. The above seed solution was inoculated at a volume of 1% into fresh fermentation medium (20 g / L glycerol, 5 g / L yeast extract, 7 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 0.01 g / L calcium chloride, 0.3 g / L magnesium sulfate, 50 g / L MOPS, 50 mg / L chloramphenicol), and cultured at 37°C and 220 rpm for 2 h. After induction, a final concentration of 0.1 mM IPTG was added, and a final concentration of 3 mM tyrosine was added as a direct precursor. The fermentation was induced at 30°C and 220 rpm for 72 h, and the yields of tyrosine, p-coumaric acid and resveratrol were detected by high performance liquid chromatography.

[0142] The results are as follows Figure 4As shown in the figure, after 48 h of shake flask fermentation, tyrosine was completely consumed, and 285.01 mg / L p-coumaric acid and 130.74 mg / L resveratrol were produced. After fermentation for 72 h, the production of p-coumaric acid and resveratrol did not increase further. On the one hand, the production of target products caused by exogenous added precursors and endogenous synthetic precursors is different due to the different supply methods. On the other hand, the supply of malonyl-CoA is also another major factor limiting the production of resveratrol. Therefore, a resveratrol de novo synthesis strain was constructed in the subsequent step, and the supply of malonyl-CoA was increased at the same time.

[0143] Example 4: Construction of a high-yield tyrosine chassis strain

[0144] In order to achieve the de novo synthesis of resveratrol starting from a simple and renewable carbon source, we used Escherichia coli W3110 as the starting strain to construct a chassis strain with high tyrosine production. First, in order to avoid the formation of acetic acid as a byproduct, the key gene ackA-pta for acetic acid synthesis was knocked out to construct the engineered strain Res01. Among them, the protein encoded by the gene pta has the amino acid sequence shown in SEQ ID NO: 8, and the protein encoded by the gene ackA has the amino acid sequence shown in SEQ ID NO: 9.

[0145] In order to further strengthen the shikimate pathway, the 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene that is resistant to feedback inhibition was overexpressed. The specific method was as follows: using the W3110 genome as a template, the mutant aroG gene (the protein encoded by this gene has the amino acid sequence shown in SEQ ID NO: 11) was obtained by fragment cloning and overlap extension PCR technology, and it was integrated into Res01 to construct the engineered strain Res02; in order to strengthen the terminal pathway of tyrosine synthesis, the prephenatedehydrogenase gene that is resistant to feedback inhibition was overexpressed. The specific method was as follows: using the W3110 genome as a template, the mutant tyrA gene (the protein encoded by this gene has the amino acid sequence shown in SEQ ID NO: 12) was obtained by fragment cloning and overlap extension PCR technology, and it was integrated into Res02 ​​to construct the engineered strain Res03.

[0146] In order to evaluate the supply of tyrosine, the engineered strains Res02 ​​and Res03 were inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain seed solution. The seed solution was inoculated at 1% volume into fresh fermentation medium (20 g / L glycerol, 5 g / L yeast extract, 7 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 0.01 g / L calcium chloride, 0.3 g / L magnesium sulfate, 50 g / L MOPS), and cultured at 37°C and 220 rpm for 2 h. After induction, IPTG was added at a final concentration of 0.1 mM. The fermentation was induced at 30°C and 220 rpm for 96 h, and the tyrosine production was detected by high performance liquid chromatography. The conditions were: chromatographic column: xBridge BEH Shield RP18 2.5um, mobile phase: H2O+0.1%TFA and ACN+0.1%TFA, flow rate: 1.0 ml / min, column temperature: 40°C, UV detector, detection wavelength: 220 nm, detection time: 15 min.

[0147] The fermentation results are as follows Figure 5 As shown in the figure, after 96 h of shake flask fermentation, the tyrosine production of Res02 ​​and Res03 was 280 mg / L and 1642 mg / L, respectively. This indicates that genome integration of key genes is an effective way to boost metabolic pathways and increase target products, and can avoid the strain burden caused by plasmid overexpression, while leaving sufficient intracellular resources for the efficient expression optimization of key heterologous proteins for the synthesis of resveratrol.

[0148] Example 5: Construction of a de novo resveratrol synthesis strain

[0149] To achieve de novo synthesis of resveratrol, FjTAL was integrated into Res02 ​​and Res03, and the resveratrol synthesis plasmid pACYC-At4CL-PcSTS was introduced, respectively, to preliminarily obtain the resveratrol de novo synthesis strains Res056 and Res058.

[0150] To test the ability of Res056 and Res058 to synthesize resveratrol from scratch, they were inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain seed solution. The seed solution was inoculated at 1% volume into fresh fermentation medium (20 g / L glycerol, 5 g / L yeast extract, 7 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 0.01 g / L calcium chloride, 0.3 g / L magnesium sulfate, 50 g / L MOPS, 50 mg / L chloramphenicol), and cultured at 37°C and 220 rpm for 2 h. After induction, IPTG was added at a final concentration of 0.1 mM. The fermentation was induced at 30°C and 220 rpm for 72 h. The yields of tyrosine, p-coumaric acid and resveratrol were detected by high performance liquid chromatography.

[0151] The fermentation results are as follows Figure 6 As shown in Figure 2, Res056 and Res058 could synthesize 90.34 mg / L and 201.59 mg / L resveratrol, respectively, after 24 h of shake flask fermentation. When the fermentation was continued for 48 h, the resveratrol production increased by 80.2% and 63.4%, reaching 162.76 mg / L and 329.40 mg / L, respectively (e.g. Figure 6 ), but after 48 h, the resveratrol production no longer increased.

[0152] It can be seen from the p-coumaric acid yield curve that p-coumaric acid continues to accumulate as the fermentation time increases, especially at 48-72 h when resveratrol no longer accumulates, the p-coumaric acid accumulation rate increases significantly. It takes three malonyl-CoA molecules to synthesize one molecule of resveratrol, and because malonyl-CoA in E. coli is mainly used for fatty acid synthesis, it is only in sufficient supply during the logarithmic phase of E. coli growth. However, even if the supply is sufficient during the logarithmic phase, its content in the body is closely regulated by the overall metabolic pathway, which has always been a limiting bottleneck for the synthesis of stilbene compounds and flavonoids.

[0153] Therefore, it is speculated that after 48 h of fermentation, resveratrol no longer increased, while p-coumaric acid continued to increase, which may be due to insufficient supply of malonyl-CoA. Therefore, the supply of malonyl-CoA was further strengthened to increase resveratrol production.

[0154] Example 6: Enhancing the supply of malonyl-CoA to increase resveratrol production

[0155] In order to enhance the metabolic flux from acetyl-CoA to malonyl-CoA, the acetyl-CoA carboxylase subunit accBC gene from Corynebacterium glutamicum (the protein encoded by the gene has the amino acid sequence shown in SEQ ID NO: 15) and the acetyl-CoA carboxylase subunit dtsR1 from Corynebacterium glutamicum (the protein encoded by the gene has the amino acid sequence shown in SEQ ID NO: 16) were integrated into the genome of the engineering strain Res058 to construct the engineering strain Res178.

[0156] In order to avoid excessive consumption of malonyl-CoA in the fatty acid synthesis pathway while ensuring normal cell growth, the natural promoter of the fatty acid synthesis regulatory factor fadR in the engineered strain Res178 was replaced in situ using the growth-dependent promoter fliC, whose transcription level decreases during the stable period, to construct the engineered strain Res290.

[0157] Res178 and Res290 were inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain seed solution. The seed solution was inoculated at 1% volume into fresh fermentation medium (20 g / L glycerol, 5 g / L yeast extract, 7 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 0.01 g / L calcium chloride, 0.3 g / L magnesium sulfate, 50 g / L MOPS, 50 mg / L chloramphenicol), and cultured at 37°C and 220 rpm for 2 h. After induction, IPTG was added at a final concentration of 0.1 mM. At the same time, 10% tributyrin was added to improve the stability of resveratrol. The fermentation was induced at 30°C and 220 rpm for 72 h, and the resveratrol yield was detected by high performance liquid chromatography.

[0158] The fermentation results are as follows Figure 7As shown in the figure, the engineered strains Res178 and Res290 with enhanced malonyl-CoA supply can synthesize 743.92 mg / L and 832.44 mg / L resveratrol after 48 h of shake flask fermentation, which are 125.8% and 152.7% higher than the starting strain Res058. At the same time, it is worth noting that enhancing the supply of malonyl-CoA can extend the accumulation time of resveratrol to 72 h. Res178 and Res290 can synthesize 799.75 mg / L and 1034.84 mg / L resveratrol after 72 h of shake flask fermentation, respectively, which are 7.5% and 24.3% higher than the 48 h yield, respectively. This shows that enhancing the supply of malonyl-CoA can not only promote the synthesis of resveratrol, but also break the limitation of the growth cycle on the accumulation time of resveratrol to a certain extent, which is an effective means to increase the final yield of resveratrol.

[0159] Example 7: Enhancement of glycerol utilization pathway

[0160] Compared with glucose, Escherichia coli has a lower rate of glycerol utilization, which is the rate-limiting step in the synthesis of high-value compounds using glycerol as a carbon source. Therefore, we further strengthened the glycerol utilization pathway to increase resveratrol production. The glycerol utilization pathway involves four genes, glpF (the protein encoded by the gene has an amino acid sequence shown in SEQ ID NO: 17), glpK (the protein encoded by the gene has an amino acid sequence shown in SEQ ID NO: 18), glpD (the protein encoded by the gene has an amino acid sequence shown in SEQ ID NO: 19) and glpX (the protein encoded by the gene has an amino acid sequence shown in SEQ ID NO: 10). These four genes were integrated (using gene editing technology) into the Res290 genome to obtain Res381, Res382, Res383 and Res384. Res381, Res382, Res383 and Res384 were inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain seed solution. The seed solution was inoculated at 1% volume into fresh fermentation medium (20 g / L glycerol, 5 g / L yeast extract, 7 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 0.01 g / L calcium chloride, 0.3 g / L magnesium sulfate, 50 g / L MOPS, 50 mg / L chloramphenicol), and cultured at 37°C and 220 rpm for 2 h. After induction, IPTG was added at a final concentration of 0.1 mM. At the same time, 10% tributyrin was added to improve the stability of resveratrol. The fermentation was induced at 30°C and 220 rpm for 72 h, and the resveratrol yield was detected by high performance liquid chromatography.

[0161] The fermentation results are as follows Figure 8As shown, after 72 h of shake flask fermentation, Res381, Res382, Res383 and Res384 can synthesize 1806.50 mg / L, 1712.98 mg / L, 2251.48 mg / L and 2157.92 mg / L resveratrol, respectively, which are 74.6%, 65.5%, 117.6% and 108.5% higher than the starting strain, respectively. This shows that enhancing the glycerol utilization pathway can effectively drive the metabolic flux to resveratrol synthesis. Since the Res383 strain with integrated glpD has the highest resveratrol yield, this strain will be used for further optimization.

[0162] Example 8: Enhancing strain tolerance to resveratrol

[0163] Resveratrol is a plant-derived polyphenolic substance that inhibits the growth of Escherichia coli to a certain extent. Therefore, we enhanced the strain's tolerance to resveratrol to further increase resveratrol production. Taking Res383 as the starting strain, the lamB gene (the protein encoded by this gene has the amino acid sequence shown in SEQ ID NO: 13) was knocked out to construct the engineered strain Res785. Further, based on Res785, the outer membrane protein ompF gene (the protein encoded by this gene has the amino acid sequence shown in SEQ ID NO: 14) was integrated to construct the engineered strain Res787.

[0164] Res785 and Re787 were inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain seed solution. The seed solution was inoculated at 1% volume into fresh fermentation medium (20 g / L glycerol, 5 g / L yeast extract, 7 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 0.01 g / L calcium chloride, 0.3 g / L magnesium sulfate, 50 g / L MOPS, 50 mg / L chloramphenicol), and cultured at 37°C and 220 rpm for 2 h. After induction, IPTG was added at a final concentration of 0.1 mM, and 10% tributyrin was added to improve the stability of resveratrol. The fermentation was induced at 30°C and 220 rpm for 72 h, and the resveratrol yield was detected by high performance liquid chromatography.

[0165] The fermentation results are as follows Fig. 9 As shown, after 72 h of shake flask fermentation, the resveratrol production of Res785 and Res787 could reach 2649.04 mg / L and 3105.67 mg / L, respectively. This indicates that for resveratrol biosynthesis, which requires malonyl-CoA as a key precursor, maintaining cell activity and promoting cell growth are beneficial to increasing production.

[0166] The above Res787 was inoculated into LB liquid medium, cultured overnight at 37°C and 220 rpm to obtain seed solution, and the above seed solution was inoculated into fresh fermentation medium (glycerol 30 g / L, yeast extract 10 g / L, disodium hydrogen phosphate 5 g / L, potassium dihydrogen phosphate 5 g / L, sodium chloride 0.3 g / L, ammonium chloride 0.5 g / L, calcium chloride 0.03 g / L, magnesium sulfate 0.5 g / L, MOPS 40 g / L, chloramphenicol 100 mg / L) at a volume inoculation rate of 1%. After culturing for 2 h at 37°C and 220 rpm, IPTG was added at a final concentration of 0.1 mM for induction, and 20% tributyrin was added to improve the stability of resveratrol. The fermentation was induced at 30°C and 220 rpm for 72 h, and the resveratrol yield was detected by high performance liquid chromatography. The experimental results showed that the resveratrol yield reached 3104.27 mg / L after 72 h of shake flask fermentation.

[0167] The above Res787 was inoculated into LB liquid medium, cultured overnight at 37°C and 220 rpm to obtain seed solution, and the above seed solution was inoculated into fresh fermentation medium (glycerol 25 g / L, yeast extract 8 g / L, disodium hydrogen phosphate 8 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 0.8 g / L, ammonium chloride 1.5 g / L, calcium chloride 0.02 g / L, magnesium sulfate 0.2 g / L, MOPS 45 g / L, chloramphenicol 80 mg / L) at a volume inoculation rate of 1%. After culturing for 2 h at 37°C and 220 rpm, IPTG with a final concentration of 0.1 mM was added for induction, and 15% tributyrin was added to improve the stability of resveratrol. The fermentation was induced at 30°C and 220 rpm for 72 h, and the resveratrol yield was detected by high performance liquid chromatography. The experimental results showed that the resveratrol yield reached 3106.87 mg / L after 72 h of shake flask fermentation.

[0168] The above Res787 was inoculated into LB liquid medium, cultured overnight at 37°C and 220 rpm to obtain seed solution, and the above seed solution was inoculated into fresh fermentation medium (glycerol 18 g / L, yeast extract 4 g / L, disodium hydrogen phosphate 4 g / L, potassium dihydrogen phosphate 1 g / L, sodium chloride 0.2 g / L, ammonium chloride 1.8 g / L, calcium chloride 0.04 g / L, magnesium sulfate 0.6 g / L, MOPS 60 g / L, chloramphenicol 120 mg / L) at a volume inoculation rate of 1%. After culturing for 2 h at 37°C and 220 rpm, IPTG with a final concentration of 0.1 mM was added for induction, and 8% tributyrin was added to improve the stability of resveratrol. The fermentation was induced at 30°C and 220 rpm for 72 h, and the resveratrol yield was detected by high performance liquid chromatography. The experimental results showed that the resveratrol yield reached 2504.44 mg / L after 72 h of shake flask fermentation.

[0169] Example 9: Further strengthening the supply of malonyl-CoA to increase resveratrol production

[0170] In order to further enhance the supply of malonyl-CoA, sodium bicarbonate and biotin were added during the strain culture. The specific fermentation process was as follows: Res787 was inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain seed solution. The above seed solution was inoculated at a volume of 1% into fresh fermentation medium (30 g / L glycerol, 5 g / L yeast extract, 7 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 0.01 g / L calcium chloride, 0.3 g / L magnesium sulfate, 50 g / L MOPS, 50 mg / L chloramphenicol), and cultured at 37°C and 220 rpm for 2 h. After that, a final concentration of 0.1 mM IPTG, a final concentration of 2 g / L sodium bicarbonate, and a final concentration of 40 mg / L biotin were added. At the same time, 10% tributyrin was added to improve the stability of resveratrol. The fermentation was induced at 30°C and 220 rpm for 72 min. h, Resveratrol production was detected using high performance liquid chromatography.

[0171] The fermentation results are as follows Fig.10 As shown, after 72 h of shake flask fermentation, the resveratrol production of the engineered strain Res787 could reach 3971.03 mg / L, indicating that malonyl-CoA is the key rate-limiting precursor for the synthesis of resveratrol. Increasing the supply of malonyl-CoA is beneficial to increasing the resveratrol production, but further extending the fermentation time to 96 h did not further increase the resveratrol production.

[0172] Comparing the shake flask fermentation results of the present invention with those reported in the current literature (Table 3), it can be seen that the engineered strain for de novo synthesis of resveratrol of the present invention not only has the highest yield and production intensity reported so far, including Escherichia coli, Yarrowia lipolytica and Saccharomyces cerevisiae, but also has a short production cycle and does not require the addition of any pathway precursors during the entire fermentation process, thus having greater economic value and better industrial prospects.

[0173] Table 3

[0174]

[0175] Document 1: Wu, Junjun, et al. "Efficient de novo synthesis of resveratrol by metabolically engineered Escherichia coli." Journal of IndustrialMicrobiology and Biotechnology 44.7 (2017): 1083-1095.

[0176] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the present invention screens the sources of p-coumaroyl-CoA ligase and stilbene synthase for synthesizing resveratrol, optimizes the expression intensity of key genes in the resveratrol synthesis pathway, enhances the precursor supply, and knocks out the competitive pathway. A highly efficient de novo synthesis strain of resveratrol is constructed, which reduces the metabolic burden and improves the yield and conversion rate of resveratrol on the one hand, and on the other hand, does not need to add any antibiotics and any intermediate substances in the entire fermentation process, greatly reducing the production cost and simplifying the subsequent separation and purification steps. The engineered Escherichia coli provided by the present invention has high production intensity, simple fermentation process, good application prospects, and lays a foundation for the production of resveratrol by Escherichia coli microbial fermentation.

[0177] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria contain an exogenously introduced p-coumaroyl-CoA ligase gene At4CL from Arabidopsis thaliana and a stilbene synthase gene PcSTS from Polygonum cuspidatum; Wherein, the genetically engineered bacteria is selected from Escherichia coli; The genetically engineered bacteria does not contain any one or more of the following genes: pta or ackA.

2. The genetically engineered bacterium according to claim 1, characterized in that: The genetically engineered bacteria also contain an exogenously introduced amino acid ammonia lyase gene FjTAL derived from Flavobacterium johnsoniae.

3. The genetically engineered bacterium according to claim 2, characterized in that: The protein encoded by the p-coumaroyl-CoA ligase gene At4CL from Arabidopsis thaliana has an amino acid sequence shown in SEQ ID NO: 1; The protein encoded by the stilbene synthase gene PcSTS from Polygonum cuspidatum has an amino acid sequence shown in SEQ ID NO: 5; The protein encoded by the amino acid ammonia lyase gene FjTAL derived from Flavobacterium johnsoniae has an amino acid sequence shown in SEQ ID NO:

7.

4. The genetically engineered bacterium according to claim 3, characterized in that: The protein encoded by the pta gene has an amino acid sequence shown in SEQ ID NO: 8; The protein encoded by the ackA gene has an amino acid sequence shown in SEQ ID NO:

9.

5. The genetically engineered bacterium according to claim 4, characterized in that: The genetically engineered bacteria further contain any one or more of the following exogenously introduced genes: a mutant aroG derived from Escherichia coli or a mutant tyrA derived from Escherichia coli.

6. The genetically engineered bacterium according to claim 5, characterized in that: The protein encoded by the mutant aroG gene derived from Escherichia coli has an amino acid sequence shown in SEQ ID NO: 11; The protein encoded by the mutant tyrA gene derived from Escherichia coli has an amino acid sequence shown in SEQ ID NO:

12.

7. The genetically engineered bacterium according to claim 6, characterized in that: The genetically engineered bacteria also contain the following exogenously introduced genes: accBC derived from Corynebacterium glutamicum and dtsR1 derived from Corynebacterium glutamicum.

8. The genetically engineered bacterium according to claim 7, characterized in that: The protein encoded by the accBC gene derived from Corynebacterium glutamicum has an amino acid sequence shown in SEQ ID NO: 15; The protein encoded by the dtsR1 gene derived from Corynebacterium glutamicum has an amino acid sequence shown in SEQ ID NO:

16.

9. The genetically engineered bacterium according to claim 8, characterized in that: The genetically engineered bacteria also contain a fatty acid synthesis regulatory factor fadR, and the promoter of the fatty acid synthesis regulatory factor fadR is fliC.

10. The genetically engineered bacterium according to claim 9, characterized in that: The genetically engineered bacteria also overexpress any one or more of the following genes: glpF derived from Escherichia coli, glpK derived from Escherichia coli, glpD derived from Escherichia coli or glpX derived from Escherichia coli.

11. The genetically engineered bacterium according to claim 10, characterized in that: The protein encoded by the glpF gene has an amino acid sequence shown in SEQ ID NO: 17; The protein encoded by the glpK gene has an amino acid sequence shown in SEQ ID NO: 18; The protein encoded by the glpD gene has an amino acid sequence shown in SEQ ID NO: 19; The protein encoded by the glpX gene has an amino acid sequence shown in SEQ ID NO:

10.

12. The genetically engineered bacterium according to claim 11, characterized in that: The genetically engineered bacteria do not contain the gene LamB and / or contain the gene ompF.

13. The genetically engineered bacterium according to claim 12, characterized in that: The protein encoded by the LamB gene has an amino acid sequence shown in SEQ ID NO: 13; The protein encoded by the ompF gene has an amino acid sequence shown in SEQ ID NO:

14.

14. A method for preparing a genetically engineered bacterium, characterized in that: The preparation method comprises: transferring a plasmid carrying a p-coumaroyl-CoA ligase gene At4CL and a stilbene synthase gene PcSTS into a starting engineering bacterium to obtain the genetically engineered bacterium; Among them, the p-coumaroyl-CoA ligase gene At4CL comes from Arabidopsis thaliana; the stilbene synthase gene PcSTS comes from Polygonum cuspidatum; The starting engineering bacteria is selected from Escherichia coli; The preparation method further comprises: knocking out any one or more of the following genes in the starting engineering bacteria: pta or ackA.

15. The preparation method according to claim 14, characterized in that: The preparation method further comprises: transferring a plasmid carrying an amino acid ammonia lyase gene FjTAL into the starting engineering bacteria or knocking in the amino acid ammonia lyase gene FjTAL into the starting engineering bacteria; Among them, the amino acid ammonia lyase gene FjTAL comes from Flavobacterium johnsoniae.

16. The preparation method according to claim 15, characterized in that: The protein encoded by the p-coumaroyl-CoA ligase gene At4CL from Arabidopsis thaliana has an amino acid sequence shown in SEQ ID NO: 1; The protein encoded by the stilbene synthase gene PcSTS from Polygonum cuspidatum has an amino acid sequence shown in SEQ ID NO: 5; The protein encoded by the amino acid ammonia lyase gene FjTAL derived from Flavobacterium johnsoniae has an amino acid sequence shown in SEQ ID NO:

7.

17. The preparation method according to claim 16, characterized in that: The protein encoded by the pta gene has an amino acid sequence shown in SEQ ID NO: 8; The protein encoded by the ackA gene has an amino acid sequence shown in SEQ ID NO:

9.

18. The preparation method according to claim 17, characterized in that: The preparation method further comprises: knocking into the starting engineering bacteria any one or more of the following genes: mutant aroG derived from Escherichia coli and mutant tyrA derived from Escherichia coli.

19. The preparation method according to claim 18, characterized in that: The protein encoded by the mutant aroG gene has an amino acid sequence shown in SEQ ID NO: 11; The protein encoded by the mutant tyrA gene has an amino acid sequence shown in SEQ ID NO:

12.

20. The preparation method according to claim 19, characterized in that: The preparation method further comprises: knocking in the accBC gene derived from Corynebacterium glutamicum and the dtsR1 gene derived from Corynebacterium glutamicum into the starting engineering bacteria.

21. The preparation method according to claim 20, characterized in that: The protein encoded by the accBC gene derived from Corynebacterium glutamicum has an amino acid sequence shown in SEQ ID NO: 15; and the protein encoded by the dtsR1 gene derived from Corynebacterium glutamicum has an amino acid sequence shown in SEQ ID NO:

16.

22. The preparation method according to claim 21, characterized in that: The preparation method further comprises: replacing the promoter of the fatty acid synthesis regulatory factor fadR in the starting engineering bacteria with the fliC promoter in situ.

23. The preparation method according to claim 22, characterized in that: The preparation method further comprises: knocking into the starting engineering bacteria any one or more of the following genes: glpF derived from Escherichia coli, glpK derived from Escherichia coli, glpD derived from Escherichia coli or glpX derived from Escherichia coli.

24. The preparation method according to claim 23, characterized in that: The protein encoded by the glpF gene from Escherichia coli has an amino acid sequence shown in SEQ ID NO: 17; The protein encoded by the glpK gene from Escherichia coli has an amino acid sequence shown in SEQ ID NO: 18; The protein encoded by the glpD gene from Escherichia coli has an amino acid sequence shown in SEQ ID NO: 19; The protein encoded by the glpX gene derived from Escherichia coli has an amino acid sequence shown in SEQ ID NO:

10.

25. The preparation method according to claim 24, characterized in that: The preparation method further comprises: knocking out the LamB gene in the genetically engineered bacteria and / or knocking in the ompF gene into the genetically engineered bacteria.

26. The preparation method according to claim 25, characterized in that: The protein encoded by the LamB gene has an amino acid sequence shown in SEQ ID NO: 13; The protein encoded by the ompF gene has an amino acid sequence shown in SEQ ID NO:

14.

27. A method for synthesizing resveratrol, characterized in that: The genetically engineered bacteria prepared by the method for preparing the genetically engineered bacteria according to any one of claims 1 to 13 or any one of claims 14 to 26 are used to ferment and synthesize the resveratrol in a fermentation medium.

28. The method according to claim 27, characterized in that The fermentation is shake flask fermentation; the shake flask fermentation comprises: inoculating the genetically engineered bacteria cultured overnight into the fermentation medium of the shake flask fermentation for expansion culture, culturing until the OD600 is 0.8-1, adding an inducer to induce fermentation for 22-94 hours, and obtaining the resveratrol.

29. The method according to claim 28, characterized in that The conditions for overnight culture are 30-37°C, 200-220rpm; the conditions for expanded culture are 30-37°C, 200-220rpm; the conditions for induced fermentation are 25-35°C, 200-220rpm; the inducer is IPTG; and the final concentration of the inducer is 0.1-1mM.

30. The method according to claim 28, characterized in that The fermentation medium of the shake flask fermentation includes 20-30 g / L glycerol, 5-10 g / L yeast extract, 5-8 g / L disodium hydrogen phosphate, 2-5 g / L potassium dihydrogen phosphate, 0.3-0.8 g / L sodium chloride, 0.5-1.5 g / L ammonium chloride, 0.01-0.03 g / L calcium chloride, 0.2-0.5 g / L magnesium sulfate, 40-50 g / L MOPS and 50 mg / L-100 mg / L antibiotics and optionally 1-2 g / L tyrosine or optionally 1-2 mM p-coumaric acid or optionally 1-3 g / L sodium bicarbonate and 30-50 mg / L biotin and optionally 10%-20% tributyrin.

Images