CN110628657A - Saccharomyces cerevisiae engineering bacterium for synthesizing resveratrol as well as preparation method and application thereof - Google Patents
Saccharomyces cerevisiae engineering bacterium for synthesizing resveratrol as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a saccharomyces cerevisiae gene engineering bacterium which can simultaneously and independently express a grape source 4-coumarate coenzyme A ligase (4CL) and a stilbene synthase (STS) and is used for biosynthesis of resveratrol. The invention also provides a construction method of the 4CL and STS double-gene transformed saccharomyces cerevisiae engineering bacteria, a biosynthesis method of resveratrol, and application of the saccharomyces cerevisiae engineering bacteria and fermentation products thereof. The saccharomyces cerevisiae engineering bacteria can catalyze and synthesize resveratrol by taking grape juice as a substrate through independently and stably expressing two enzymes of 4CL and STS, and remarkably improve the synthesis efficiency of the resveratrol. The saccharomyces cerevisiae engineering bacteria and the fermentation product thereof can be widely applied to the preparation of food, medicine or cosmetics. The saccharomyces cerevisiae is non-toxic and harmless, has high safety, and the thalli and fermentation products can be used for preparing products without strict separation and purification. Therefore, the method for synthesizing the resveratrol by utilizing the genetic engineering saccharomyces cerevisiae has the advantages of low production cost and high safety.
Description
Technical Field
The invention belongs to the field of synthetic biology and metabolic engineering, and particularly relates to a saccharomyces cerevisiae engineering bacterium for biosynthesis of resveratrol, and a preparation method and application thereof.
Background
Resveratrol (Resveratrol) is a non-flavonoid polyphenolic compound with the chemical name of 3,4,5-trihydroxystilbene (3,4,5-trihydroxystilbene), and is mainly present in plants such as grapes, peanuts, mulberry, giant knotweed and the like. A large number of researches show that the resveratrol has multiple biological effects of oxidation resistance, inflammation resistance, cancer resistance, cardiovascular protection and the like. Resveratrol is commonly added as a raw material or additive to foods, pharmaceuticals and cosmetics to improve product efficacy.
At present, the production method of resveratrol mainly comprises a plant extraction method and a chemical synthesis method. The content of resveratrol in general plants is low, and the problems of low yield, complex process, long time period and the like exist in the process of directly extracting resveratrol from natural plants, so that the market demand is difficult to meet. The chemical synthesis method has the defects of long synthesis route and high energy consumption, is easy to cause environmental pollution and is not suitable for large-scale production.
The microbial synthesis method is a development trend of resveratrol production. The microbial synthesis method not only has the advantages of simple and convenient operation and less natural resource damage, but also can overcome the problem of environmental pollution existing in the chemical synthesis method. However, no microorganism capable of efficiently synthesizing resveratrol has been found at present. Generally, special strains are screened or genetic engineering bacteria are constructed to improve the synthesis efficiency of the resveratrol. For example, researchers screen a strain of saccharomyces bouhegeri, and the synthesis efficiency of resveratrol is improved by adding a precursor substance into a culture medium. However, the Dermatopteris yeast itself lacks the high-efficiency resveratrol synthesis related enzyme gene, and several gene-mutated yeast strains have very limited capability for improving the synthesis efficiency of resveratrol.
More resveratrol biosynthesis methods are implemented by constructing genetically engineered bacteria. For example, patent 1 (application No. 201510396741.0) discloses a fusion gene for biosynthesis of resveratrol, an expression vector and a preparation method thereof, wherein an arabidopsis thaliana-derived p-coumaroyl-coenzyme A ligase (4CL) gene and a peanut-derived resveratrol synthase RS gene are fused to obtain a 4CL fusion gene, the RS fusion gene is further transformed into escherichia coli, and coumaric acid is used as a substrate for fermentation to obtain resveratrol. Patent 2 (application No. 201210036763.2) discloses a method for biologically producing resveratrol, which comprises the steps of connecting 4 genes required for synthesizing resveratrol, namely phenylalanine hydroxylase gene, tyrosine ammonia lyase gene, 4-coumaroyl-CoA ligase gene and resveratrol synthase, in series with pEC-XK99E vector plasmid, and further transforming Escherichia coli and corynebacterium glutamicum to express and realize the synthesis of resveratrol. Patent 3 (application number: 201810178242.8) discloses a construction method and application of a high-yield resveratrol escherichia coli engineering bacterium, wherein a 4CL fusion gene obtained by fusing an STS gene from a grape source and a 4CL gene from arabidopsis thaliana is adopted, the fusion gene is further transformed into escherichia coli, and 4-coumaric acid is used as a substrate for fermentation to obtain resveratrol. Patent 4 (application number: 201510107705.8) discloses a genetically engineered bacterium for synthesizing resveratrol and a construction method thereof, wherein 4 genes such as tyrosine deaminase gene tal of rhodotorula glutinis, 4-coumaric acid-coenzyme A ligase gene 4cl of parsley, stilbene synthase gene sts of grape and the like are fused, escherichia coli E.coli BW25113 which limits tyrR and trpED of glucose to synthesize tyrosine is transformed and knocked out to obtain the genetically engineered bacterium, and the resveratrol is biosynthesized by taking glucose as a substrate. Patent 5 (application number: 201310723683.9) provides a method for biosynthesizing pterocarpus by utilizing O-methyltransferase, which is characterized in that p-coumaroyl-CoA ligase gene 4CL of arabidopsis thaliana and STS gene of grape source are fused to obtain 4CL, STS fusion gene is obtained, escherichia coli or yeast is transformed to obtain genetic engineering bacteria, p-coumaric acid is used as a substrate, fermentation is firstly carried out to obtain resveratrol, and then the genetic synthesis is carried out by transferring the engineering bacteria to resveratrol O-methyltransferase (ROMT) to obtain the pterocarpus. Patent 6 (application number: CN201710009219.1) discloses a method for preparing resveratrol dimer by using pichia pastoris, which comprises constructing an expression vector containing 4-hydroxystilbene peroxidase gene, transforming pichia pastoris to obtain pichia pastoris expressing 4-hydroxystilbene peroxidase, and synthesizing resveratrol dimer by using resveratrol as a raw material.
The above method has the following disadvantages: (1) the resveratrol biosynthesis by utilizing genetic engineering escherichia coli has the safety problem. Because escherichia coli belongs to conditional pathogenic bacteria and contains endotoxin, the resveratrol product extracted from escherichia coli fermentation liquor has the risk of endotoxin residue, and the cost for extracting and purifying the resveratrol is higher; (2) some patent genetic engineering bacteria carry 4 related enzyme genes synthesized by resveratrol, and the preparation method of the engineering bacteria is too complicated. And excessive exogenous genes are simultaneously expressed in bacteria, which is a serious metabolic burden on host bacteria. Excessive foreign proteins not only influence the proliferation and growth of host bacteria, but also influence the synthesis of resveratrol; (3) in the above-mentioned patents 3 and 5, the 4CL and STS genes used are fusion genes 4CL:: STS fusion genes. The active structures of two enzymes in the fusion protein produced by expression are easy to interfere with each other, and the activity of the two enzymes and the synthesis efficiency of resveratrol are influenced. (4) In the above patent 5, 4CL: (STS fusion protein) is expressed by using Escherichia coli or yeast engineering bacteria, the final purpose is to obtain pterocarpus santalinus, and resveratrol is only one intermediate product. The pterocarpus santalinus can be obtained by co-transfecting yeast with a 4CL: (STS) fusion gene and a resveratrol O-methyltransferase (ROMT) gene expression vector. In fact, co-transfection of yeast with two vectors is technically difficult to achieve. Therefore, the preferable scheme in the patent is to utilize ROMT genetic engineering bacteria and add resveratrol as a raw material to synthesize pterocarpus santalinus. (5) The 4-coumaroyl-coenzyme A ligase (4CL) genes adopted in the patents are not grape-derived 4CL genes, raw materials and metabolic intermediates in grape juice cannot be utilized to synthesize resveratrol, only expensive raw materials such as p-coumaric acid, phenylalanine or tyrosine can be added, and the synthesis cost is increased. (6) Patent 6 discloses the synthesis of resveratrol dimer by using genetic engineering pichia pastoris, wherein resveratrol is required to be used as a raw material, and cannot be directly synthesized.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a saccharomyces cerevisiae gene engineering bacterium capable of simultaneously expressing 4-coumarate-coenzyme A ligase (4CL) and stilbene synthase (STS) of a grape source, a preparation method of the saccharomyces cerevisiae gene engineering bacterium, and a technical method for biologically synthesizing resveratrol by using the saccharomyces cerevisiae gene engineering bacterium and taking grape juice as a substrate.
In order to achieve the purpose, the invention adopts the technical scheme that:
the first purpose of the invention is to provide a saccharomyces cerevisiae engineering bacterium for synthesizing resveratrol, which is a genetically engineered saccharomyces cerevisiae capable of simultaneously expressing grape-derived 4-coumarate-coenzyme A ligase (4CL) and stilbene synthase (STS).
Preferably, the 4CL and STS genes are respectively controlled by a Gap promoter, and can stably and independently express the two enzymes of the 4CL and the STS at the same time.
Preferably, the upstream of the 4CL gene contains a his8 tag gene sequence, and the STS gene introduces a Flag tag gene sequence.
Preferably, the nucleotide sequence of the 4-coumarate-CoA ligase (4CL) is shown as SEQ ID NO. 1; the nucleotide sequence of stilbene synthase (STS) is shown in SEQ ID NO. 2.
Preferably, the saccharomyces cerevisiae is saccharomyces cerevisiae INVSc 1.
The invention also provides application of the saccharomyces cerevisiae engineering bacteria for synthesizing resveratrol in biosynthesis of resveratrol.
The invention provides a biosynthesis method of resveratrol, which comprises the following steps:
(1) firstly, according to the gene sequences of 4CL and STS of grapes, codons are optimized to be suitable for high-efficiency expression of saccharomyces cerevisiae;
(2) introducing a his8 tag gene sequence upstream of the 4CL gene; introducing a Gap promoter sequence at the upstream of the STS gene and introducing a Flag tag gene sequence at the downstream of the STS;
(3) inserting his8-4CL gene into the downstream of a Gap promoter of a yeast expression vector pTH847 to construct a recombinant expression vector pGAP-4CL and convert escherichia coli, screening positive clones, and performing DNA sequencing identification; inserting the Gap-STS-Flag gene into a pGAP-4CL vector to construct a recombinant expression vector pGAP-4CL-STS, converting saccharomyces cerevisiae, screening positive clones, and performing DNA sequencing identification to obtain a saccharomyces cerevisiae engineering bacterium converted from pGAP-4 CL-STS;
(4) and when the proliferation density OD600 of the saccharomyces cerevisiae engineering bacteria converted by pGAP-4CL-STS reaches 0.8-1.2, adding fresh grape juice into the fermentation liquor, wherein the volume ratio of the fermentation liquor to the fresh grape juice is 1:5-10, and continuously culturing for 24-72 hours to produce the synthetic resveratrol.
(5) The concentration of the biosynthetic resveratrol was determined by HPLC.
(6) Separating the thalli of the saccharomyces cerevisiae engineering bacteria, collecting fermentation liquor, and separating and purifying resveratrol or yeast protein and polysaccharide from thalli lysate and fermentation liquor.
Preferably, the saccharomyces cerevisiae engineering bacteria in the step (3) are transformed bacteria of 4-coumarate-coa ligase (4CL) and stilbene synthase (STS) double genes.
Preferably, the yeast expression vector in step (3) is pTH847, which contains a Gap promoter and URA3 selection marker gene, and the recombinant yeast can be screened by using histidine-deficient culture medium.
Preferably, the sequencing in step (3) identifies the primers used as shown in SEQ ID NO: 4, respectively.
Preferably, the saccharomyces cerevisiae is saccharomyces cerevisiae INVSc 1.
The invention also provides the application of the resveratrol prepared by the resveratrol biosynthesis method, the saccharomyces cerevisiae engineering bacteria or the fermentation product thereof in food, medicine or cosmetics.
Compared with the prior art, the invention has the following advantages:
1. the invention takes the saccharomyces cerevisiae as the genetic engineering bacteria and the grape juice as the fermentation substrate to synthesize the resveratrol, and has low production cost and high safety. The invention uses high-efficiency promoter to respectively control the expression of grape-derived 4CL and STS genes, the expression efficiency is high, and the synthesis efficiency of resveratrol is naturally greatly improved. The grape juice contains compounds and intermediate metabolites required by the biosynthesis of resveratrol, so that the saccharomyces cerevisiae gene engineering bacteria can directly utilize grape juice for fermentation, and use cheap grape juice as a raw material, thereby reducing the production cost of the synthesis of resveratrol.
2. The method has high efficiency of synthesizing resveratrol by transforming saccharomyces cerevisiae engineering bacteria by using the 4CL and STS double genes. The 4CL and STS genes are both derived from grape genome and are two key enzymes for biosynthesis of resveratrol in grape fruits. The 4CL and STS double-gene transformed saccharomyces cerevisiae engineering bacteria can efficiently express two enzymes of 4CL and STS, compounds and intermediate metabolites in fresh grape juice can be used for catalyzing and synthesizing resveratrol, and the biosynthesis efficiency of resveratrol is remarkably improved.
3. Compared with the prior method for synthesizing resveratrol by using 4CL fusion gene engineering bacteria, the invention respectively expresses two genes of 4CL and STS, can avoid the mutual interference between the active structural domains of the two enzymes, and ensures the activity of the two enzymes of 4CL and STS and the synthesis efficiency of resveratrol.
4. The saccharomyces cerevisiae adopted by the invention is edible strain and has high safety. The thalli and metabolites thereof of the saccharomyces cerevisiae contain rich substances such as protein, B vitamins, amino acids, polysaccharide, glucan and the like, have good economic value, and can obtain byproducts with economic value while utilizing saccharomyces cerevisiae engineering bacteria to biologically synthesize resveratrol.
Drawings
FIG. 1 is a schematic diagram of the construction of an expression vector of the present invention (grape-derived 4CL and pGAP-STS genes are synthesized respectively and inserted into BamHI, MluI and XbaI restriction sites of a yeast expression vector pTH in sequence to obtain a pGAP-4CL-STS recombinant vector).
FIG. 2 shows the electrophoresis of recombinant yeast expression vector pGAP-4CL-STS (after inserting two gene fragments of 4CL and STS into empty vector pTH, the size of the vector is increased, after digestion with SacI, the recombinant vector pGAP-4CL-STS can cut out a fragment of about 3.0kb to meet the expected size).
FIG. 3 shows the sequencing and identification map of recombinant yeast expression vector pGAP-4CL-STS (the extracted recombinant expression vector is sequenced by using a universal upstream sequencing primer (SEQ ID NO: 3), and the result shows that the sequence after the corresponding enzyme cutting site is completely consistent with the expected 4CL and GAP-STS sequences, which indicates that the sequencing and identification are correct).
FIG. 4 is a High Performance Liquid Chromatography (HPLC) identification peak diagram of resveratrol biosynthesized by 4CL and STS (4CL-STS) double-gene transformed Saccharomyces cerevisiae engineering bacteria (HPLC detection results of Saccharomyces cerevisiae engineering bacteria fermentation liquor show that a peak diagram of resveratrol appears in 10.7 minutes, the position of the peak diagram is the same as that of the fermentation liquor of 4CL:: STS fusion gene Saccharomyces cerevisiae under the same condition, but the peak of the fermentation liquor of 4CL and STS double-gene engineering bacteria (4CL-STS D) is obviously larger than that of 4CL: (4CL:: STS F)).
FIG. 5 is a comparison graph of resveratrol contents generated by 4CL and STS double-gene transformed Saccharomyces cerevisiae engineering bacteria (4CL-STS D) and 4CL:: STS fusion gene yeast engineering bacteria (4CL:: STS F) and empty carrier yeast engineering bacteria (Vector) under the same conditions (HPLC detection result of engineering bacteria fermentation liquor shows that resveratrol synthesized by the 4CL-STS D engineering bacteria is significantly higher than resveratrol synthesized by the 4CL-STS engineering bacteria and Vector engineering bacteria).
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.
Example 14 preparation, screening and identification of engineered Saccharomyces cerevisiae with CL and STS double-Gene transformation
1. Synthesis and recombination of grape-derived 4CL and STS genes
(1) According to the 4CL and STS gene sequences of the grape, the codon is optimized to be suitable for high-efficiency expression of the saccharomyces cerevisiae, and cDNA sequences of the 4CL and STS genes are respectively designed.
(2) To facilitate detection of 4CL gene expression, a His8 tag gene sequence is introduced at the upstream of the 4CL gene, BamHI and MluI restriction endonuclease site sequences are added at the 5 'end and 3' end of a fusion gene (Gap-His8-4CL) respectively, and the fusion gene is handed to a gene synthesis company to synthesize His8-4CL fusion gene whole-gene double-stranded DNA, the nucleotide sequence of which is shown as SEQ ID NO: 1, is convenient to insert into pTH847 vector downstream of the Gap promoter for stable and high-efficiency expression.
(3) In order to improve the expression efficiency of STS gene, a Gap promoter sequence is introduced at the upstream of the STS gene, a Flag tag gene sequence is introduced at the downstream of the STS, MluI and XbaI restriction endonuclease site sequences are respectively added at the 5 'end and the 3' end of a fusion gene (Gap-STS-Flag), and the fusion gene is handed over to a gene synthesis company to synthesize pGAP-STS-Flag fusion gene whole-gene double-stranded DNA, the nucleotide sequence of which is shown as SEQ ID NO: 2, respectively.
2. Construction of recombinant 4CL and STS double-gene saccharomyces cerevisiae expression vector
(1) The His8-4CL fusion gene and the yeast expression vector pTH847 are cut by BamHI endonuclease and MluI endonuclease respectively, and the His8-4CL gene fragment and the vector fragment are separated by agarose electrophoresis.
(2) Cutting His8-4CL fusion gene fragment and pTH847 vector fragment (Addgene biological resource center source), and recovering and purifying His8-4CL fusion gene and vector DNA fragment by using gel DNA recovery kit. Mixing the fusion gene and the vector DNA fragment according to the proportion of 3:1, adding a connecting buffer solution and T4 DNA ligase, and connecting for 6-12 hours at 16 ℃ to connect the fusion gene and the vector DNA fragment into a recombinant expression vector pGAP-His8-4 CL.
(3) Using CaCl2The chemical transformation method is used for transforming the Escherichia coli. 100-500 mu g of recombinant expression vector pGAP-His8-4CL ligation product is taken and added into 100 mu l of Top10 escherichia coli competent cell suspension, the recombinant expression vector pGAP-His8-4CL is transformed into Top10 escherichia coli according to the conventional operation, and LB selection culture medium containing 100 mu g/ml is used for screening gene transformation bacteria.
(4) Selecting a single colony for amplification culture, extracting plasmids by using a conventional plasmid extraction kit, sending the plasmids to a DNA sequencing company, and carrying out sequencing identification on pGAP-His8-4CL transformed escherichia coli by using an expression vector universal sequencing primer (SEQ ID NO: 4).
(5) Selecting a recombinant expression vector pGAP-His8-4CL with correct sequencing identification to transform escherichia coli for amplification culture, and extracting and purifying the recombinant plasmid by a conventional method.
(6) And constructing a yeast recombinant expression vector pGAP-4 CL-STS. The method is the same as above. Respectively using MluI and XbaI endonucleases to enzyme-cut the Pgap-STS-Flag fusion gene and the recombinant yeast expression vector pGAP-4CL, and separating and purifying the Pgap-STS-Flag gene fragment and the vector fragment by agarose electrophoresis.
(7) The Pgap-STS-Flag fusion gene and pGAP-His8-4CL vector DNA fragment were ligated in the order of 3:1, adding a ligation buffer and T4 DNA ligase, and ligating at 16 ℃ for 6-12 hours to obtain a recombinant expression vector pGAP-4CL-STS (pGAP-His 8-4CL-Pgap-STS-Flag, as shown in FIG. 1).
(8) pGAP-4CL-STS transformed saccharomyces cerevisiae is obtained by transformation by an electroporation method. Adding 1-3 μ g of recombinant expression vector pGAP-4CL-STS into 100 μ l of competent cell suspension of Saccharomyces cerevisiae INVSC1, uniformly mixing, transferring into a 2mm wide electric shock cup, placing the electric shock cup in an electric shock groove, and setting electric shock conditions: 2.5kV, a capacitance of 25 muF and a resistance of 200 omega.
(9) And (4) culturing and screening the pGAP-4CL-STS transformed saccharomyces cerevisiae colonies. After the electric shock is finished, 200-400 mu L of basic culture medium lacking histidine is added to suspend and wash the bacteria, the bacteria are coated on a selective culture medium agarose plate lacking histidine, and the agarose plate is placed in an anaerobic box at the temperature of 30 ℃ to be cultured for 48 to 72 hours. Single colonies were picked and expanded in 5mL histidine-deficient minimal medium for 48 h.
(10) Meanwhile, 4CL: (STS) fusion gene expression vector pGAP-4CL: (STS) is constructed and converted to obtain 4CL: (STS) fusion gene saccharomyces cerevisiae engineering bacteria as control engineering bacteria. The methods of gene recombination, electrotransformation and engineering bacteria screening are the same as the steps (1) to (9). The main difference is that 4CL and STS gene are connected into 4CL:: STS fusion gene, 4CL:: His8 label sequence is introduced into 5 ' upstream of STS fusion gene, BamHI and XbaI restriction endonuclease site sequences are added into 5 ' end and 3 ' end respectively, and then synthesized into His8-4CL-STS fusion gene whole gene double-stranded DNA (SEQ ID NO: 3) by gene synthesis company. His8-4CL-STS is cloned to corresponding close site of yeast expression vector pTH847 to obtain recombinant 4CL:: STS fusion gene yeast expression vector pGAP-4CL:: STS, and Saccharomyces cerevisiae INVSC1 is transformed to obtain pGAP-4CL:: STS fusion gene transformed Saccharomyces cerevisiae engineering bacteria.
(11) Sequencing identification of pGAP-4CL-STS transformed Saccharomyces cerevisiae (the yeast expression vector pTH847 has a Gap promoter sequence thereon, and His8-4CL gene is inserted into the downstream of the Gap promoter and is controlled by the Gap promoter, therefore, SEQ ID NO: 1 is a His8-4CL sequence, and SEQ ID NO: 2 is a Gap-STS-Flag gene sequence). Collecting 5-10ml bacterial liquid bacteria, adding 10mg/ml lysozyme for digestion at 37 ℃ for 30min, and extracting plasmids by using a conventional plasmid extraction kit. The extracted plasmid was sent to DNA sequencing company for DNA sequencing identification using universal sequencing primer for expression vector (SEQ ID NO: 4). Identification results 4CL-STS and 4CL:, the nucleotide sequence of STS fusion gene is completely consistent with the designed expected sequence.
(12) Selecting pGAP-4CL-STS with correct sequencing identification to convert Saccharomyces cerevisiae, culturing with selective culture medium, adding glycerol to final concentration of 25%, subpackaging, and storing as original strain in a refrigerator at-20 deg.C.
3. Biosynthesis of resveratrol
(1) Recovering and culturing the saccharomyces cerevisiae engineering bacteria INVSC1 transformed by pGAP-4CL-STS, and carrying out amplification culture by using a general yeast culture medium YPD at 30 ℃. Meanwhile, the empty vector pTH847 transformed yeast was recovered as a negative control.
(2) When the proliferation density OD600 of the saccharomyces cerevisiae engineering bacteria reaches 0.8, adding fresh grape juice according to the ratio of 1:5, and continuing to culture for 24 hours.
4. HPLC method for measuring concentration of biosynthetic resveratrol
(1) Preparation of fermentation sample: and (3) putting 1mL of the bacterial liquid into a centrifuge tube, centrifuging at 5000rpm for 10min to collect the yeast, discarding the supernatant, adding 1mL of acetone to resuspend and mix, and ultrasonically crushing the yeast on ice by using an ultrasonic crusher.
(2) And centrifuging the yeast lysate subjected to ultrasonic crushing at 10000rpm for 10 min. And (3) taking the supernatant, performing rotary evaporation to dryness, and adding 1mL of methanol for dissolving to obtain a sample to be detected.
(3) Detecting resveratrol by High Performance Liquid Chromatography (HPLC): an Agilent 1220Infinity HPLC instrument is adopted, and the chromatographic conditions are as follows: column ODS C18 (4.6mm 250mm, 5 μm); the column temperature is 30 ℃; wavelengths of 308nm (trans-resveratrol and trans-polydatin) and 285nm (cis-resveratrol and cis-polydatin); the mobile phase A is ultrapure water: acetic acid 94: 6, the mobile phase B is acetonitrile; the flow rate is 0.5 mL/min; the detection wavelengths are 285nm and 308 nm; the column temperature is 30 ℃; the amount of the sample was 10. mu.L. The detection spectrum of the resveratrol standard substance (100mg/L) was used as a reference to draw a standard curve, and the concentration of resveratrol was calculated, with the result shown in FIG. 4.
(4) Through 3 times of repeated experiments, the average concentration of resveratrol in the 4CL-STS double-gene transformed saccharomyces cerevisiae lysate is 234.68 mg/L; 4CL, the average concentration of resveratrol in the saccharomyces cerevisiae lysate transformed by the STS fusion gene is 89.21 mg/L; the average concentration of resveratrol in the pTH847 empty vector-transformed Saccharomyces cerevisiae lysate was 22.86mg/L (FIG. 5).
Example 2
The only difference between this example and example 1 is that in step 3(2) of resveratrol biosynthesis, when the proliferation density OD600 of the saccharomyces cerevisiae engineering bacteria reaches 1.0, fresh grape juice is added according to a ratio of 1:8, and the culture is continued for 36 hours.
Example 3
The only difference between this example and example 1 is that in step 3(2) of resveratrol biosynthesis, when the proliferation density OD600 of the engineered saccharomyces cerevisiae strain reaches 1.2, fresh grape juice is added according to a ratio of 1:10, and the culture is continued for 72 hours.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> Guangzhou Lanxing bioengineering Co., Ltd
<120> saccharomyces cerevisiae engineering bacteria for synthesizing resveratrol and preparation method and application thereof
<130> 9.19
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1737
<212> DNA
<213> Artificial Synthesis
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Claims (9)
1. The saccharomyces cerevisiae engineering bacteria for synthesizing resveratrol is characterized by being genetic engineering saccharomyces cerevisiae capable of simultaneously and independently expressing grape-derived 4-coumaric acid coenzyme A ligase (4CL) and stilbene synthase (STS).
2. The engineered saccharomyces cerevisiae for synthesizing resveratrol according to claim 1, wherein the 4CL and STS genes are respectively regulated by a Gap promoter, and can stably and independently express two enzymes, namely 4CL and STS.
3. The engineered saccharomyces cerevisiae strain for synthesizing resveratrol according to claim 1 or 2, wherein the upstream of the 4CL gene comprises a his8 tag gene sequence; and a Flag tag gene sequence is introduced into the downstream of the STS gene.
4. The engineered saccharomyces cerevisiae strain for synthesizing resveratrol according to claim 1, wherein the nucleotide sequence of 4-coumarate-coa ligase (4CL) is shown as SEQ ID No. 1; the nucleotide sequence of stilbene synthase (STS) is shown in SEQ ID NO. 2.
5. The engineered strain of saccharomyces cerevisiae for synthesizing resveratrol according to claim 1, wherein the saccharomyces cerevisiae is saccharomyces cerevisiae INVSc 1.
6. The saccharomyces cerevisiae engineering bacteria for synthesizing resveratrol according to any one of claims 1-5 is used for biosynthesis of resveratrol.
7. A biosynthesis method of resveratrol is characterized by comprising the following steps:
(1) firstly, according to the gene sequences of 4CL and STS of grapes, codons are optimized to be suitable for high-efficiency expression of saccharomyces cerevisiae;
(2) introducing a his8 tag gene sequence upstream of the 4CL gene; introducing a Gap promoter sequence at the upstream of the STS gene and introducing a Flag tag gene sequence at the downstream of the STS;
(3) inserting his8-4CL gene into the downstream of a Gap promoter of a yeast expression vector pTH847 to construct a recombinant expression vector pGAP-4CL and convert escherichia coli, screening positive clones, and performing DNA sequencing identification; inserting the Gap-STS-Flag gene into a pGAP-4CL vector to construct a recombinant expression vector pGAP-4CL-STS, converting saccharomyces cerevisiae, screening positive clones, and performing DNA sequencing identification to obtain a saccharomyces cerevisiae engineering bacterium converted from pGAP-4 CL-STS;
(4) and when the proliferation density OD600 of the saccharomyces cerevisiae engineering bacteria converted by pGAP-4CL-STS reaches 0.8-1.2, adding fresh grape juice into the fermentation liquor, wherein the volume ratio of the fermentation liquor to the fresh grape juice is 1:5-10, and continuously culturing for 24-72 hours to produce the synthetic resveratrol.
(5) The concentration of the biosynthetic resveratrol was determined by HPLC.
(6) Separating the thalli of the saccharomyces cerevisiae engineering bacteria, collecting fermentation liquor, and separating and purifying resveratrol or yeast protein and polysaccharide from thalli lysate and fermentation liquor.
8. The method for the biological production of resveratrol according to claim 7, wherein the engineered Saccharomyces cerevisiae in step (3) is a transformant with 4-coumarate-CoA ligase (4CL) and stilbene synthase (STS) double genes.
9. The resveratrol-engineered saccharomyces cerevisiae bacteria prepared by the resveratrol biosynthesis method according to claim 7 or 8, or fermentation products thereof are used in food, medicine or cosmetics.
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