Method for improving cordycepin synthesized by saccharomyces cerevisiae and saccharomyces cerevisiae engineering bacteria

Abstract

The invention relates to the technical field of microbial genetic engineering, in particular to a method for improving cordycepin synthesized by saccharomyces cerevisiae and saccharomyces cerevisiae engineering bacteria. According to the invention, codon-optimized 2 '-carbonyl-3'-deoxyadenosine reductase gene and 3 '-adenosine monophosphate phosphohydrolase gene from cordyceps militaris are expressed by using plasmids, and then the plasmids are transformed into host cells to obtain the saccharomyces cerevisiae engineering bacteria. And inoculating a seed solution obtained by seed culture of the activated saccharomyces cerevisiae engineering bacteria into a fermentation culture medium containing a metabolic effector, and carrying out fermentation culture to synthesize cordycepin. Metabolic effectors (Cu < 2 + >, Fe < 2 + >, Mg < 2 + >, Zn < 2 + >, citric acid, cysteine, aspartic acid, glycine, VB1 and tea polyphenol) are added into a fermentation culture medium strain, and the variety and concentration of the metabolic effectors are optimized, so that the yield and synthesis efficiency of cordycepin are remarkably improved. By using the combination of the strain obtained by the invention and the optimal effector, the yield of cordycepin in a fermentation tank reaches 2.9 g / L.

Description

Technical Field

[0001] The invention relates to the technical field of microbial genetic engineering, and in particular to a method for improving the synthesis of cordycepin by brewer's yeast and a brewer's yeast engineering bacterium. Background Art

[0002] Cordycepin, also known as 3'-deoxyadenosine, is a nucleoside compound with significant biological activity, which is widely found in medicinal fungi such as Cordyceps sinensis. Cordycepin has multiple pharmacological effects such as anti-tumor, anti-inflammatory, immunomodulatory, and antiviral, and has important application value in the fields of medicine, health care products, and cosmetics. In recent years, with the increase in people's demand for natural products, the market demand for cordycepin has continued to grow. However, the main source of cordycepin depends on the extraction of natural Cordyceps, which has problems such as scarce resources, high extraction cost, and low yield, making it difficult to meet market demand. Therefore, the production of cordycepin by microbial fermentation has become a potential alternative method. At present, the synthesis of cordycepin in Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae has been successfully achieved using relevant genetic engineering technologies. However, Saccharomyces cerevisiae is limited by factors such as differences in gene expression systems and complex regulatory networks, which makes it more difficult to redirect metabolic flux, resulting in the yield potential not being fully tapped. At the same time, current research lacks systematic research on the components of fermentation media, making it impossible to achieve a balance between energy-reducing power and the redistribution of metabolic flux, thus affecting the adaptability of microbial cell factories to the cordycepin synthesis pathway.

[0003] Based on this, the present invention provides an engineered strain of Saccharomyces cerevisiae and a method for improving the ability of Saccharomyces cerevisiae to synthesize cordycepin by optimizing a fermentation medium. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for improving the synthesis of cordycepin by brewer's yeast in view of the deficiencies of the prior art.

[0005] The technical problem that the present invention also solves is to provide an engineered yeast of saccharomyces cerevisiae for improving the synthesis of cordycepin by saccharomyces cerevisiae.

[0006] The final technical problem to be solved by the present invention is to provide a method for constructing the engineering bacteria of Saccharomyces cerevisiae.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for improving the synthesis of cordycepin by Saccharomyces cerevisiae, wherein the seed liquid of an engineered strain of Saccharomyces cerevisiae expressing a gene for an enzyme related to cordycepin synthesis is inoculated into a fermentation medium containing metabolic effectors for fermentation and cultivation to synthesize cordycepin.

[0009] The seed liquid of the brewer's yeast engineered bacteria is obtained by inoculating the activated brewer's yeast engineered bacteria into a primary seed culture medium (YPD liquid culture medium) for cultivation, and then inoculating the primary seed liquid into a secondary seed culture medium (YPD liquid culture medium) at an inoculation rate of 1-3% v / v for cultivation.

[0010] Specifically, the culture conditions are: 28-35° C., 100-500 rpm, and 18-24 hours.

[0011] Wherein, the inoculation amount is 5-15% v / v, and the preferred inoculation amount is 10% v / v.

[0012] The metabolic effectors include any one or a combination of metal ions, amino acids, and organic small molecules.

[0013] Preferably, the metabolic effector comprises a combination of metal ions and amino acids.

[0014] Specifically, the metal ions include Cu 2+ , Fe 2+ Mg 2+ and Zn 2+ any one or a combination of the amino acids including cysteine, aspartic acid and glycine; the organic small molecules including citric acid, VB 1 And any one or a combination of tea polyphenols.

[0015] Preferably, the metabolic effector comprises Cu 2+ , cysteine ​​and aspartic acid.

[0016] Specifically, in some embodiments of the present invention, the metabolic effector Cu 2+ The added concentration is 0.01 to 200 mM, preferably 0.05 to 50 mM, more preferably 0.1 mM;

[0017] Specifically, in some embodiments of the present invention, the metabolic effector Fe 2+ The added concentration is 0.01 to 200 mM, preferably 0.05 to 50 mM, and more preferably 1 mM.

[0018] Specifically, in some embodiments of the present invention, the metabolic effector Mg 2+ The added concentration is 0.01 to 200 mM, preferably 0.05 to 50 mM, and more preferably 1 mM.

[0019] Specifically, in some embodiments of the present invention, the metabolic effector Zn 2+The added concentration is 0.01 to 200 mM, preferably 0.05 to 50 mM, and more preferably 1 mM.

[0020] Specifically, in some embodiments of the present invention, the concentration of the metabolic effector citric acid added is 0.5 to 100 g / L, preferably 0.05 to 40 g / L, and more preferably 2 g / L;

[0021] Specifically, in some embodiments of the present invention, the added concentration of the metabolic effector cysteine ​​is 0.05 to 10 g / L, preferably 0.5 to 40 g / L, and more preferably 2 g / L;

[0022] Specifically, in some embodiments of the present invention, the added concentration of the metabolic effector aspartic acid is 0.05 to 50 g / L, preferably 0.5 to 20 g / L, and more preferably 2 g / L;

[0023] Specifically, in some embodiments of the present invention, the added concentration of the metabolic effector glycine is 0.1 to 200 g / L, preferably 1 to 80 g / L, and more preferably 4 g / L;

[0024] Specifically, in some embodiments of the present invention, the added concentration of the metabolic effector VB1 is 0.5 to 900 mg / L, preferably 5 to 50 mg / L, more preferably 50 mg / L;

[0025] Specifically, in some embodiments of the present invention, the added concentration of the metabolic effector tea polyphenols is 0.01-80 g / L, preferably 0.05-10 g / L, and more preferably 0.1 g / L.

[0026] The cordycepin synthesis-related enzyme genes are 2'-carbonyl-3'-deoxyadenosine reductase gene cns1 and 3'-adenosine monophosphate phosphohydrolase gene cns2.

[0027] Specifically, the 2'-carbonyl-3'-deoxyadenosine reductase gene cns1 and the 3'-adenosine monophosphate phosphohydrolase gene cns2 are derived from Cordyceps militaris, and their NCBI accession numbers are XP_006669647 and XP_006669648 respectively.

[0028] The fermentation medium further comprises: 0.1-100 g / L of carbon source, 10-50 g / L of nitrogen source and 0.1-15 g / L of adenine; preferably, the carbon source is glucose, and the nitrogen source is yeast extract and / or peptone.

[0029] Preferably, the fermentation medium has a formula of: 50 g / L glucose, 10 g / L yeast extract, 20 g / L tryptone, 1 g / L adenine, 2 g / L aspartic acid, 2 g / L cysteine, and 0.1 mM copper sulfate.

[0030] The fermentation culture conditions are as follows: temperature of 28-35°C, pH of 4-6, aeration rate of 2-4 vvm, initial stirring speed of 400-700 rpm. When the glucose concentration is lower than 0.5 g / L during fermentation, the DO value is controlled to be above 20%, and feed culture medium is added to maintain the glucose concentration at 0.5-2 g / L until the fermentation is completed.

[0031] Specifically, the feed medium has a formula of 300-800 g / L glucose, preferably 500 g / L glucose.

[0032] In a second aspect, the present invention provides an engineered yeast strain of Saccharomyces cerevisiae, which uses Saccharomyces cerevisiae as a host and expresses the 2'-carbonyl-3'-deoxyadenosine reductase gene cns1 and the 3'-adenosine monophosphate phosphohydrolase gene cns2 via a plasmid to construct the engineered yeast strain of Saccharomyces cerevisiae.

[0033] The nucleotide sequence of the 2'-carbonyl-3'-deoxyadenosine reductase gene cns1 is shown in SEQ ID NO.1; the nucleotide sequence of the 3'-adenosine monophosphate phosphohydrolase gene cns2 is shown in SEQ ID NO.2.

[0034] The Saccharomyces cerevisiae includes but is not limited to any one of S.cerevisiae BY4742, S.cerevisiae BY4741, S.cerevisiae W303-1A and S.cerevisiae CEN.PK2-1C; or, the Saccharomyces cerevisiae is a yeast whose genome lacks the triosephosphate isomerase TPI1 encoded by TPI1, including but not limited to S.cerevisiae CEN.PK530-1C.

[0035] Preferably, the Saccharomyces cerevisiae is any one of S. cerevisiae BY4742, S. cerevisiae BY4741, and S. cerevisiae CEN.PK530-1C; more preferably, the Saccharomyces cerevisiae is S. cerevisiae BY4742 or S. cerevisiae CEN.PK530-1C.

[0036] The plasmid includes but is not limited to any one of pG418, pYX212, pYES2, pESC-URA3, pESC-LEU2 and pESC-HIS; or, the plasmid carries a gene capable of complementing the function of TPI1, including but not limited to the CPOTud plasmid.

[0037] Preferably, the plasmid is any one of pG418, pESC-URA3, pESC-LEU2, pESC-HIS and CPOTud plasmids; more preferably, the plasmid is pG418, pESC-URA3 and CPOTud.

[0038] The 2'-carbonyl-3'-deoxyadenosine reductase gene cns1 and the 3'-adenosine monophosphate phosphohydrolase gene cns2 are expressed by plasmids in the form of cns1 expression frame and cns2 expression frame.

[0039] Specifically, the cns1 expression cassette consists of a promoter, cns1 and a terminator, and the cns2 expression cassette consists of a promoter, cns2 and a terminator.

[0040] Further, the promoter includes any one of PGK1, TEF1, GPD, TPI1, ADH1, TDH3, CYC1, HXT1, GAL1, GAL7, GAL10, CUP1 and MET25; preferably any one of TPI1, TDH3 and TEF1, and more preferably, the 2'-carbonyl-3'-deoxyadenosine reductase gene cns1 is driven to express by the promoter TPI1, and the 3'-adenosine monophosphate phosphohydrolase gene cns2 is driven to express by TEF1.

[0041] Furthermore, the terminator includes any one of ADH1, GAL2, PRM9, CPS1, GAT2, HIS5, IDP1, PDC6, AIP1, TIP1 and DIT1, preferably ADH1t and GAL2t.

[0042] In some embodiments of the present invention, the cns1 expression cassette consists of promoter TPI1, cns1 and terminator ADH1t, and the cns2 expression cassette consists of promoter TEF1, cns2 and terminator GAL2t.

[0043] In some embodiments of the present invention, the cns1 expression cassette and the cns2 expression cassette are connected back to back to form a cns expression cassette; wherein the back-to-back connection is composed of connecting the 3' end of the promoter TPI1 in the cns1 expression cassette to the cns1 gene, and connecting the 5' end to the 5' end of the promoter TEF1 in the cns2 expression cassette.

[0044] The construction method of the above-mentioned Saccharomyces cerevisiae engineering bacteria is as follows:

[0045] The genes of cordycepin (2'-carbonyl-3'-deoxyadenosine reductase gene cns1 and 3'-adenosine monophosphate phosphohydrolase gene cns2) were designed and synthesized, and the codons were optimized to adapt to the expression system of Saccharomyces cerevisiae; the expression cassettes of the relevant genes were connected to the linearized vector pG418 between the restriction endonuclease EcoRI and BamHI sites by one-step cloning technology, and the verified plasmids were electroporated into the Saccharomyces cerevisiae strain;

[0046] or,

[0047] The gene expression frame for synthesizing cordycepin (2'-carbonyl-3'-deoxyadenosine reductase gene cns1 and 3'-adenosine monophosphate phosphohydrolase gene cns2) was connected to the CPOTud linearized plasmid by a one-step cloning method, wherein the CPOTud linearized plasmid was obtained by PCR amplification, and the verified plasmid was electroporated into the Saccharomyces cerevisiae strain.

[0048] Beneficial effects:

[0049] (1) The present invention expresses the key enzyme gene in the cordycepin synthesis pathway via a plasmid in Saccharomyces cerevisiae, and the cordycepin yield reaches 0.87 g / L.

[0050] (2) The present invention improves the yield and efficiency of cordycepin synthesis by Saccharomyces cerevisiae by inoculating the seed liquid of the engineered yeast expressing the gene for the enzyme related to cordycepin synthesis into the fermentation medium added with different types of effectors and optimizing the types and concentrations of the effectors. By simultaneously adding 2 g / L aspartic acid, 2 g / L cysteine ​​and 0.1 mM CuSO 4 The yield of cordycepin was significantly improved, reaching 1.39 g / L in the shake flask and 2.9 g / L in the fermenter. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The present invention will be further described in detail below with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more clear.

[0052] Figure 1 This is the map of the recombinant plasmid pG418 (cns) in Example 1.

[0053] Figure 2 This is the map of the recombinant plasmid CPOTud (cns) in Example 1.

[0054] Figure 3 The cordycepin yield of the engineered yeast Saccharomyces cerevisiae SBG and SCC in Example 2.

[0055] Figure 4 The optimized effector Cu in Example 3 2+ Concentration of cordycepin production.

[0056] Figure 5 For the optimization of effector Fe in Example 4 2+ Concentration of cordycepin production.

[0057] Figure 6 Mg is the optimized effector in Example 5. 2+ Concentration of cordycepin production.

[0058] Figure 7 For the optimized effector Zn in Example 6 2+ Concentration of cordycepin production.

[0059] Figure 8 The cordycepin yield at the optimized effector citric acid concentration in Example 7.

[0060] Fig. 9 The cordycepin yield with optimized effector cysteine ​​concentration in Example 8.

[0061] Fig.10 This is the cordycepin yield when the effector aspartic acid concentration is optimized in Example 9.

[0062] Fig.11 The cordycepin yield at the optimized effector glycine concentration in Example 10.

[0063] Fig.12 Optimizing effector VB in Example 11 1 Concentration of cordycepin production.

[0064] Fig.13 This is the cordycepin yield when the concentration of effector tea polyphenols is optimized in Example 12.

[0065] Fig.14 This is a significance analysis of the effect of the optimal effector concentration on cordycepin production in Example 13.

[0066] Fig.15 Orthogonal experimental scheme and results.

[0067] Fig.16 The orthogonal experimental scheme in Example 14 and the yield of cordycepin obtained by the optimal combination using the orthogonal experiment.

[0068] Fig.17 The cordycepin yield in a 1 L fermenter using the fermentation medium without adding effectors in the engineered strain of Saccharomyces cerevisiae in Example 15.

[0069] Fig.18The cordycepin yield in a 1 L fermenter using the fermentation medium supplemented with effectors used in the engineered strain of Saccharomyces cerevisiae in Example 15. DETAILED DESCRIPTION

[0070] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

[0071] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0072] In the following examples, the pG418 plasmid vector was preserved in the laboratory, and the CPOTud plasmid vector was donated by Professor Liu Zihe of Beijing University of Chemical Technology; the Saccharomyces cerevisiae BY4742 was preserved in the laboratory, and the Saccharomyces cerevisiae CEN.PK530-1C was donated by Professor Huang Mingtao of South China University of Technology.

[0073] Example 1: Construction of pG418 (cns) expression plasmid and CPOTud (cns) expression plasmid

[0074] 1. Construction of cns1 and cns2 expression cassettes

[0075] According to the codon preference of Saccharomyces cerevisiae, the amino acid sequence as shown in XP_006669647 and the amino acid sequence as shown in XP_006669648 were codon optimized to obtain the nucleotide sequence of the cns1 gene as shown in SEQ ID NO.1 and the nucleotide sequence of the cns2 gene as shown in SEQ ID NO.2. The above process was completed by Suzhou Jinweizhi Biotechnology Co., Ltd., and the optimized cns1 gene sequence and cns2 gene sequence were loaded on the default vector.

[0076] The genome of Saccharomyces cerevisiae BY4742 was used as a template and TPI1p-F / TPI1p-R were used as primers to amplify TPI1p; the default vector was used as a template and PCR was performed using primers cns1-F / cns1-R to amplify the optimized cns1 gene sequence; the genome of Saccharomyces cerevisiae BY4742 was used as a template and ADH1t was amplified using primers ADH1t-F / ADH1t-R to obtain ADH1t; then, the primers TPI1p-F / ADH1t-R were used to overlap the fragments amplified above to obtain the cns1 expression cassette. The genome of Saccharomyces cerevisiae BY4742 strain was used as a template and TEF1p-F / TEF1p-R were used as primers to amplify TEF1p; the default vector was used as a template and primers cns2-F / cns2-R were used for PCR to amplify the optimized cns2 gene sequence; the genome of Saccharomyces cerevisiae BY4742 strain was used as a template and primers GAL2t-F / GAL2t-R were used to amplify GAL2t; then primers TEF1p-F / GAL2t-R were used to overlap the fragments amplified above to obtain the cns2 expression cassette. The primer sequences used above are shown in Table 1.

[0077] 2. Construction of pG418 (cns) expression plasmid

[0078] The empty vector pG418 was digested with restriction endonucleases EcoR I and BamH I, and then run on agarose gel electrophoresis at 37°C for 2 hours. After gel cutting, the pG418 linear vector was purified and recovered using a recovery kit. The pG418 linear vector was connected to the cns1 expression cassette using a one-step cloning enzyme. The system was as follows: 5×CE II Buffer 4μL Exnase II 2μL Target fragment 200ng Vector fragment 200ng ddH 2 O to 20μL. The ligated product was transformed into Trans1T1 competent medium by heat shock, cultured overnight, and the transformants were picked for verification and sequencing to obtain plasmid pG418 (cns1). The plasmid pG418 (cns1) was digested with restriction endonucleases SaC I and Sal I, reacted at 37℃ for 2h, and then run agarose gel electrophoresis. After gel cutting, the pG418 (cns1) linear vector was purified and recovered using a recovery kit. The pG418 (cns1) linearized vector was connected to the cns2 expression cassette using a one-step cloning enzyme. The system: 5×CE II Buffer 4μL Exnase II 2μL Target fragment 200ng Vector fragment 200ng ddH 2The ligated product was transformed into Trans1T1 competent medium by heat shock, cultured overnight at 37°C, and the transformants were picked for verification and sequencing to obtain plasmid pG418 (cns) (such as Figure 1 ).

[0079] 3. CPOTud (cns) expression plasmid

[0080] Use primers Ori-F-0105 / POT1-R-0105 containing homology arms (homologous to both ends of the CPOTud vector) for PCR amplification to obtain the CPOTud linear vector; use primers cns-F-0105 / cns-R-0105 for PCR amplification using pG418 (cns) plasmid as template to obtain cns1 and cns2 expression cassettes. The above primer sequences are shown in Table 1. Refer to the connection, transformation, and culture methods in step 2 to obtain the plasmid CPOTud (cns) (such as Figure 2 ).

[0081] Table 1 Primer sequences

[0082]

[0083]

[0084] Example 2: Construction and fermentation of engineered yeast Saccharomyces cerevisiae

[0085] 1. Construction of engineered Saccharomyces cerevisiae

[0086] Saccharomyces cerevisiae BY4742 and Saccharomyces cerevisiae CEN.PK530-1C were cultured in a 30°C incubator for 48 hours, and single colonies were picked and activated to prepare corresponding competent cells. The pG418 (cns) expression plasmid prepared in Example 1 was transformed into S.cerevisiae BY4742 competent cells by electroporation, and the CPOTud (cns) expression plasmid was transformed into S.cerevisiae CEN.PK530-1C competent cells. Transformants were screened using YPD solid medium containing 400 mg / L G418 and 100 mg / L Sh ble, and the transformants were verified by PCR to obtain Saccharomyces cerevisiae engineered bacteria SBG and SCC, respectively.

[0087] 2. Producing cordycepin by fermentation of engineered yeast Saccharomyces cerevisiae

[0088] After streaking the engineered yeast SBG and SCC, they were cultured in a 30°C incubator for 48 hours, and then single colonies were picked for activation. The activated recombinant strains SBG and SCC were inoculated into 5 ml YPD medium, respectively, and cultured at 30°C and 220 rpm for 18 to 24 hours to obtain seed liquid. The seed liquid was inoculated into the fermentation medium at an inoculation rate of 1% v / v, and cultured at 30°C and 220 rpm for 144 hours to obtain fermentation liquid, and the fermentation supernatant was collected by centrifugation at 13000 rpm for 5 minutes. The fermentation supernatant was filtered through a 0.22 μm membrane to remove impurities for subsequent detection and analysis.

[0089] The production of cordycepin in the fermentation broth supernatant was detected by high performance liquid chromatography, and the specific method was as follows: Agilent 1260 Infinity II high performance liquid chromatography with ultraviolet detector (VWD) was used to separate and quantitatively determine the cordycepin in the fermentation broth supernatant. The chromatographic column was ZORBAX SB-Aq, the mobile phase was 2% acetonitrile, 98% 0.3‰ trifluoroacetic acid, the flow rate was 0.8 mL / min, the column oven was set at 40°C, and the absorbance was 260 nm.

[0090] The fermentation medium has a formula of: 50 g / L glucose, 10 g / L yeast extract, 20 g / L tryptone, and 1 g / L adenine.

[0091] The results are as follows Figure 3 As shown, the cordycepin (COR) production of Saccharomyces cerevisiae engineered bacteria SBG and SCC were 0.16 g / L and 0.87 g / L, respectively. The cordycepin production of Saccharomyces cerevisiae engineered bacteria SCC was significantly higher than that of Saccharomyces cerevisiae engineered bacteria SBG, and Saccharomyces cerevisiae engineered bacteria SCC was subsequently used for fermentation optimization.

[0092] Example 3: Effector Cu 2+ Optimization of concentration

[0093] Different concentrations (0.1, 0.5, 1, 2, 5, 10 and 15 mM) of the effector CuSO were added to the fermentation medium of Example 2. 4 The other components remain unchanged. 4 The fermentation process and cordycepin detection method of the Saccharomyces cerevisiae engineering bacteria SCC are the same as those in Example 2.

[0094] The results are as follows Figure 4 As shown, when the effector CuSO 4 The yield of cordycepin was the highest when the concentration was 0.1mM.

[0095] Example 4: Effector Fe 2+ Optimization of concentration

[0096] Different concentrations (0.1, 0.5, 1, 2, 5, 10 and 15 mM) of the effector FeSO were added to the fermentation medium of Example 2. 4 The other components remain unchanged. 4 The fermentation process and cordycepin detection method of the Saccharomyces cerevisiae engineering bacteria SCC are the same as those in Example 2.

[0097] The results are as follows Figure 5 As shown, when the effector FeSO 4 When the concentration was 1 mM, the yield of cordycepin was the highest.

[0098] Example 5: Effector Mg 2+ Optimization of concentration

[0099] Different concentrations (0.1, 0.5, 1, 2, 5, 10 and 15 mM) of the effector MgSO were added to the fermentation medium of Example 2. 4 The other components remain unchanged. 4 The fermentation process and cordycepin detection method of the Saccharomyces cerevisiae engineering bacteria SCC are the same as those in Example 2.

[0100] The results are as follows Figure 6 As shown, when the effector MgSO 4 When the concentration was 1 mM, the yield of cordycepin was the highest.

[0101] Example 6: Effector Zn 2+ Optimization of concentration

[0102] Different concentrations (0.1, 0.5, 1, 2, 5, 10 and 15 mM) of the effector ZnSO were added to the fermentation medium of Example 2. 4 The other components remain unchanged. 4 The fermentation process and cordycepin detection method of the Saccharomyces cerevisiae engineering bacteria SCC are the same as those in Example 2.

[0103] The results are as follows Figure 7 As shown, when the effector ZnSO 4 When the concentration was 1 mM, the yield of cordycepin was the highest.

[0104] Example 7: Optimization of the concentration of effector citric acid

[0105] Different concentrations (0.5, 1, 2, 4, 5 and 10 g / L) of effector citric acid were added to the fermentation medium of Example 2, and the other components remained unchanged. The cordycepin production of the engineered yeast SCC at different citric acid concentrations was investigated. The fermentation process of the engineered yeast SCC and the cordycepin detection method were the same as those in Example 2.

[0106] The results are as follows Figure 8 As shown, when the concentration of effector citric acid was 2 g / L, the yield of cordycepin was the highest.

[0107] Example 8: Optimization of effector cysteine ​​concentration

[0108] Different concentrations (0.5, 1, 2, 4, 5 and 10 g / L) of effector cysteine ​​were added to the fermentation medium of Example 2, and the other components remained unchanged. The cordycepin production of the Saccharomyces cerevisiae engineered bacteria SCC was investigated under different cysteine ​​concentrations. The fermentation process of the Saccharomyces cerevisiae engineered bacteria SCC and the cordycepin detection method were the same as in Example 2.

[0109] The results are as follows Fig. 9 As shown, when the concentration of effector cysteine ​​was 2 g / L, the yield of cordycepin was the highest.

[0110] Example 9: Optimization of the concentration of effector aspartic acid

[0111] Different concentrations (0.5, 1, 2, 4 and 5 g / L) of effector aspartic acid were added to the fermentation medium of Example 2, and the other components remained unchanged. The cordycepin production of the Saccharomyces cerevisiae engineered bacteria SCC was investigated under different aspartic acid concentrations. The fermentation process of the Saccharomyces cerevisiae engineered bacteria SCC and the cordycepin detection method were the same as in Example 2.

[0112] The results are as follows Fig.10 As shown, when the concentration of effector aspartic acid was 2 g / L, the yield of cordycepin was the highest.

[0113] Example 10: Optimization of the concentration of the effector glycine

[0114] Different concentrations (1, 2, 4, 8, 16 and 20 g / L) of effector glycine were added to the fermentation medium of Example 2, and the other components remained unchanged. The cordycepin production of the Saccharomyces cerevisiae engineered bacteria SCC was investigated at different glycine concentrations. The fermentation process of the Saccharomyces cerevisiae engineered bacteria SCC and the cordycepin detection method were the same as in Example 2.

[0115] The results are as follows Fig.11 As shown, when the concentration of effector glycine was 4 g / L, the yield of cordycepin was the highest.

[0116] Example 11: Effector VB 1 Optimization of concentration

[0117] The effector VB was added to the fermentation medium of Example 2 at different concentrations (5, 10, 50, 100 and 200 mg / L). 1 , the other components remain unchanged. 1 The fermentation process and cordycepin detection method of the Saccharomyces cerevisiae engineering bacteria SCC are the same as those in Example 2.

[0118] The results are as follows Fig.12 As shown, when the effector VB 1 When the concentration is 50 mg / L, the yield of cordycepin is the highest.

[0119] Example 12: Optimization of the concentration of effector tea polyphenols

[0120] Different concentrations (0.1, 0.5, 1, 2, 4 and 8 g / L) of effector tea polyphenols were added to the fermentation medium of Example 2, and the other components remained unchanged. The cordycepin production of the engineered yeast SCC at different tea polyphenol concentrations was investigated. The fermentation process and cordycepin detection method of the engineered yeast SCC were the same as those in Example 2.

[0121] The results are as follows Fig.13 As shown, when the concentration of effector tea polyphenols was 0.1 g / L, the yield of cordycepin was the highest.

[0122] Example 13: Significance analysis of the effects of 10 effectors on cordycepin production

[0123] The effect of the optimal effector concentration on cordycepin production in Examples 3 to 12 was analyzed for significance. Specifically, statistical software (such as SPSS, GraphPad Prism or R language) was used for data analysis, and analysis of variance (ANOVA) was used to compare the differences in cordycepin production under different concentration gradients to determine whether the effector had a significant effect on cordycepin production. When p≤0.001, the difference was considered to be highly significant, indicating that the effector concentration had a very significant effect on cordycepin production. When p≤0.01, the difference was considered to be significant, indicating that the effector concentration had a significant effect on cordycepin production.

[0124] The results are as follows Fig.14 As shown in Figure 2, according to the significance analysis, the effectors with highly significant effects (p≤0.001) on cordycepin production are: Cu 2+ , glutamate, cysteine, aspartic acid and tea polyphenols; the effectors that have significant effects (p≤0.01) on cordycepin production are citric acid and glycine.

[0125] Example 14: Orthogonal experiment of effector

[0126] According to the significant analysis in Example 13, the top three effectors were selected: Cu 2+ , aspartic acid and cysteine, and a three-factor, three-level orthogonal experiment was designed. The orthogonal experiment factor level table is shown in Table 2, and a total of 9 orthogonal experiments were obtained. The fermentation process of the brewer's yeast engineering bacteria SCC and the cordycepin detection method in this embodiment are the same as those in Example 2.

[0127] Table 2 Orthogonal test factor level table

[0128]

[0129] Orthogonal experimental scheme and results are as follows Fig.15 As shown in the figure, the yields of No. 1 to 9 were 1.15 g / L, 0.90 g / L, 1.08 g / L, 1.05 g / L, 1.08 g / L, 1.12 g / L, 1.12 g / L, 1.28 g / L and 1.12 g / L, respectively. The order of influence of various factors on cordycepin yield was as follows: CuSO 4 >Aspartic acid>Cysteine ​​(according to the range R), the optimal combination is A 3 B 3 C 1 , that is, 2g / L aspartic acid, 2g / L cysteine ​​and 0.1mM Cu 2+ The optimal effector combination obtained by orthogonal experiment was used for fermentation. The results are as follows Fig.16 As shown, the cordycepin production reached 1.39 g / L.

[0130] Example 15: Production of cordycepin by fermentation in a 1L fermenter of engineered yeast Saccharomyces cerevisiae

[0131] After streaking the engineered yeast SCC in Example 3 and culturing it in a constant temperature incubator at 30° C. for 48 h, a single colony was picked for activation, and the activated bacteria were picked from the plate and inoculated into a 50 mL centrifuge tube containing 5 mL YPD liquid culture medium for culturing at a temperature of 30° C., a shaking speed of 220 rpm, and a culturing time of 18 to 24 h to obtain a primary seed solution; the primary seed solution was transferred to a 250 mL triangular flask containing 50 mL YPD liquid culture medium at an inoculation amount of 1% v / v, the culturing temperature was 30° C., the shaking speed was 220 rpm, and the culturing time was 18 to 24 h to obtain a secondary seed solution; the obtained secondary seed solution was inoculated into a 1 L fermenter containing 0.5 L fermentation medium at an inoculation amount of 10% v / v. The fermentation medium with and without effector addition was used respectively, wherein the fermentation medium with effector addition was: 50 g / L glucose, 10 g / L yeast extract, 20 g / L tryptone, 1 g / L adenine, 2 g / L aspartic acid, 2 g / L cysteine, 0.1 mM copper sulfate. The culture conditions were: the temperature was 30°C, the pH was maintained at 5.5 using 1M HCl and 1M ammonia water, the aeration rate was 3 vvm, the initial stirring speed was 500 rpm, after 12 hours of fermentation, the DO value was controlled to be above 20% by means of the association of DO (dissolved oxygen), stirring and aeration, and feed medium (500 g / L glucose) was added to maintain the glucose concentration at 0.5-2 g / L until the fermentation was completed after 144 hours.

[0132] The yield of cordycepin was determined to be 1.89 g / L ( Fig.17 ) Results: Using the fermentation medium with added effectors, the yield of cordycepin reached 2.9 g / L ( Fig.18 ), the cordycepin yield was 53.44% higher than that without adding effectors. The results show that the addition of effectors can significantly improve the synthesis efficiency of cordycepin.

[0133] The present invention provides a method for improving the synthesis of cordycepin by saccharomyces cerevisiae, a thought and method for engineering saccharomyces cerevisiae, and there are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for improving the synthesis of cordycepin by Saccharomyces cerevisiae, characterized in that: Inoculating the seed liquid of the engineered yeast Saccharomyces cerevisiae expressing enzymes related to cordycepin synthesis into a fermentation medium containing metabolic effectors to perform fermentation and cultivation to synthesize cordycepin; The metabolic effectors include any one or a combination of metal ions, amino acids, and organic small molecules.

2. The method according to claim 1, characterized in that The metal ions include Cu 2+ , Fe 2+ Mg 2+ and Zn 2+ The amino acids include any one or a combination of cysteine, aspartic acid and glycine; the organic small molecules include any one or a combination of citric acid, VB1 and tea polyphenols.

3. The method according to claim 2, characterized in that The metabolic effectors include Cu 2+ , cysteine ​​and aspartic acid.

4. The method according to claim 2, characterized in that: The Cu 2+ The added concentration is 0.01-200 mM; the Fe 2+ The added concentration is 0.01-200 mM; the Mg 2+ The added concentration is 0.01-200 mM; the Zn 2+ The added concentration is 0.01-200mM; the added concentration of cysteine ​​is 0.05-100g / L; the added concentration of aspartic acid is 0.05-50g / L; the added concentration of glycine is 0.1-200g / L; the added concentration of citric acid is 0.5-100g / L; the added concentration of VB1 is 0.5-900mg / L; the added concentration of the metabolic effector tea polyphenols is 0.01-80g / L.

5. The method according to claim 1, characterized in that The enzymes related to cordycepin synthesis are 2'-carbonyl-3'-deoxyadenosine reductase and 3'-adenosine monophosphate phosphohydrolase.

6. The method according to claim 1, characterized in that The fermentation medium comprises: 0.1-100 g / L of carbon source, 10-50 g / L of nitrogen source and 0.1-15 g / L of adenine; the fermentation culture conditions are: temperature of 28-35°C, pH of 4-6, aeration rate of 2-4 vvm, initial stirring speed of 400-700 rpm, when fermentation to a glucose concentration lower than 0.5 g / L, controlling the dissolved oxygen value to be above 20%, and adding feed medium to maintain the glucose concentration at 0.5-2 g / L until the fermentation ends; wherein the feed medium comprises 300-800 g / L of glucose.

7. An engineered strain of Saccharomyces cerevisiae, characterized in that: Using Saccharomyces cerevisiae as a host, the 2'-carbonyl-3'-deoxyadenosine reductase gene cns1 and the 3'-adenosine monophosphate phosphohydrolase gene cns2 are expressed by plasmids to construct the Saccharomyces cerevisiae engineered bacteria described in claim 1; wherein the nucleotide sequence of the 2'-carbonyl-3'-deoxyadenosine reductase gene cns1 is shown in SEQ ID NO.1, and the nucleotide sequence of the 3'-adenosine monophosphate phosphohydrolase gene cns2 is shown in SEQ ID NO.

2.

8. The engineered yeast of claim 7, characterized in that: The cerevisiae includes any one of S.cerevisiae BY4742, S.cerevisiae BY4741, S.cerevisiae W303-1A and S.cerevisiaeCEN.PK2-1C; the plasmid includes any one of pG418, pYX212, pYES2, pESC-URA3, pESC-LEU2 and pESC-HIS plasmids.

9. The engineered yeast of claim 7, characterized in that: The cerevisiae is a yeast lacking the phosphate triose isomerase TPI1 encoded by TPI1 in its genome, including S.cerevisiae CEN.PK530-1C; the plasmid carries a gene capable of complementing the function of TPI1, including a CPOTud plasmid.

10. The engineered yeast of claim 7, characterized in that: The 2'-carbonyl-3'-deoxyadenosine reductase gene cns1 and the 3'-adenosine monophosphate phosphohydrolase gene cns2 are expressed by plasmids in the form of a cns1 expression cassette and a cns2 expression cassette; wherein the cns1 expression cassette consists of a promoter, cns1 and a terminator, and the cns2 expression cassette consists of a promoter, cns2 and a terminator.

11. The engineered yeast of claim 10, characterized in that: The promoter includes any one of PGK1, TEF1, GPD, TPI1, ADH1, TDH3, CYC1, HXT1, GAL1, GAL7, GAL10, CUP1 and MET25; the terminator includes any one of ADH1, GAL2, PRM9, CPS1, GAT2, HIS5, IDP1, PDC6, AIP1, TIP1 and DIT1.

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