Biosynthesis method for improving yeast terpenoids

Abstract

The invention belongs to but not limited to the technical field of gene engineering, and discloses a method for improving the biosynthesis of yeast terpenoids and application of the method, and the method for improving the biosynthesis of the yeast terpenoids based on m7G methyltransferase complex genes TRM8-TRM82 comprises the following steps: overexpressing a TRM8 gene and a TRM82 gene; in a saccharomyces cerevisiae engineering strain genome for producing terpenoids, a promoter of a yeast endogenous TRM8 gene is replaced by a constitutive promoter PGK1p, and a promoter of a yeast endogenous TRM82 gene is replaced by a constitutive promoter TEF1p, so that the production efficiency of the saccharomyces cerevisiae terpenoids can be improved. The invention constructs a new wine brewing yeast engineering bacterium for producing the terpenoid lupeol as a representative compound, and the yield of the lupeol can be effectively improved. The new metabolic engineering method for improving the biosynthesis of the yeast terpenoids based on the TRM8-TRM82 has a remarkable effect.

Description

Technical Field

[0001] The invention belongs to but is not limited to the field of genetic engineering technology, and in particular relates to a method for improving the biosynthesis of yeast terpenoid compounds. Background Art

[0002] Terpenoids are a class of natural products with complex structures that are widely found in nature, with more than 50,000 representatives. Many of these compounds have been shown to have special physical and chemical properties and biological activities and can be used as drugs. For example, the triterpenoid lupeol is found in a variety of edible plants and Chinese herbal medicines. It has anti-inflammatory, anti-tumor, antioxidant, and wound healing effects, and has certain therapeutic effects on arthritis, diabetes, heart disease, nephrotoxicity and hepatotoxicity. In addition, triterpenes are also commonly used as spices, food additives, biofuels, etc., with great economic value and broad market prospects. At present, terpenoids are mainly obtained in two ways: one is to extract them from plants, but there are problems such as long plant growth cycle and low yield; the other is chemical synthesis, but the synthesis process is complicated, costly, and will cause environmental pollution. In contrast, the biosynthesis method is greener and more efficient.

[0003] In recent years, the construction of microbial cell factories to produce natural terpenoids has become an effective means. Among them, Saccharomyces cerevisiae has become an ideal cell factory for the efficient synthesis of a variety of terpenoids due to its advantages such as the convenience of genetic manipulation, the simplicity of the culture process, and the eukaryotic expression and modification system. The metabolic engineering method can not only increase the production of endogenous terpenoids in Saccharomyces cerevisiae, but also become an advantageous strain for the heterologous production of natural terpenoids. For example, by introducing heterologous α-amyrin synthase into yeast strains and increasing the supply of 2,3-oxidosqualene on this basis, the yield of α-amyrin can be increased to 11.97 mg / L; by introducing taxane synthesis-related genes into Saccharomyces cerevisiae, overexpressing key enzymes and transcription factors, inhibiting branch genes, expressing heterologous geranylgeranyl pyrophosphate synthase, and optimizing the codons of taxane synthase, the taxane yield in the engineered strain of Saccharomyces cerevisiae finally reached 8.7 mg / L; by introducing four non-natural metabolic enzymes, the carbon center metabolic flow of Saccharomyces cerevisiae was reconstructed, the ATP required for the generation of acetyl-CoA was reduced, and the metabolic flow in the strain was balanced, and finally the fermentation final yield of β-farnesene in the reactor of Saccharomyces cerevisiae reached 150 g / L. As the synthetic pathways of various natural products are resolved, more and more terpenoid compounds have obtained high-yield engineered strains through synthetic biology methods, providing a material basis for their large-scale preparation and industrialization.

[0004] At present, commonly used metabolic regulation methods include metabolic pathway construction and optimization, key enzyme mining and modification, cofactor regeneration engineering, cell compartmentalization engineering, and cell tolerance modification. However, when the yield reaches a certain level, the improvement effect of these methods becomes limited. Therefore, it is crucial to explore and develop new metabolic engineering strategies, and strategy optimization will bring broader development prospects for microbial cell factories. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a method based on m 7 A method for improving the biosynthesis of yeast terpenoids using the G methyltransferase complex gene TRM8-TRM82 and its application.

[0006] The present invention is implemented in this way: a method based on m 7 A method for improving the biosynthesis of yeast terpenoid compounds by using the G methyltransferase complex gene TRM8-TRM82, wherein the promoter of the yeast endogenous TRM8 gene is replaced with the constitutive promoter PGK1p, and the promoter of the yeast endogenous TRM82 gene is replaced with the constitutive promoter TEF1p in the genome of an engineered strain of Saccharomyces cerevisiae producing terpenoid compounds.

[0007] Another object of the present invention is to provide a method based on m 7 The invention relates to a method for improving the biosynthesis of terpenoids in yeast by using the G methyltransferase complex gene TRM8-TRM82 in an engineered strain of Saccharomyces cerevisiae that produces high terpenoids.

[0008] Another object of the present invention is to provide a method for constructing an engineered strain of Saccharomyces cerevisiae that produces a high yield of the terpenoid compound lupeol, comprising the following steps:

[0009] (1) Building Modules

[0010] The promoter GAL1p was connected to the promoter GAL10p to construct module I;

[0011] Module I, ERG8 gene and terminator CYC1t were connected to construct module II;

[0012] Connect the selection marker URA to module II to construct module III;

[0013] Module I, ERG12 gene and terminator ADH1t were connected to construct module IV;

[0014] Connect the selection marker HIS to module IV to construct module V;

[0015] Module I, ERG20 F96W / N127W The gene and terminator ADH1t are connected to form module VI;

[0016] Connecting the screening marker TRP to module VI to construct module VII;

[0017] Module Ⅷ was constructed by connecting module Ⅰ, EfMvaS gene and terminator ADH1t;

[0018] Module VIII, EfMvaE gene and terminator CYC1t were connected to construct module IX;

[0019] The promoter PGK1p, TRM8 gene and terminator ADH1 were connected to construct module X;

[0020] The selection marker URA, module X and promoter TEF1p were connected to construct module XI;

[0021] (2) Construction of expression vector

[0022] The vector pESC-LEU was digested with restriction endonuclease NotI;

[0023] The GgLUS1 gene was connected to the restriction vector through homologous recombination to construct the expression vector Pm01;

[0024] (3) Construction of yeast engineering strains

[0025] The above module III was introduced into the starting strain, integrated into the ERG9p site, and screened using yeast-deficient YNB-URA solid medium to construct strain MY01;

[0026] The above module V was introduced into strain MY01, integrated into the IDI1p site, and screened using yeast YNB-URA-HIS solid selection medium to construct strain MY02;

[0027] The above module VII was introduced into strain MY02, integrated into the ERG19p site, and screened using yeast YNB-URA-HIS-TRP solid selection medium to construct strain MY03;

[0028] The module IX was introduced into strain MY03 and integrated into the URA3 locus, and screened using yeast YPD-5FOA solid selection medium to construct strain MY04;

[0029] The module XI was introduced into strain MY04 and integrated into the TRM82p site, and screened using yeast YNB-URA-HIS-TRP solid selection medium to construct a new strain;

[0030] (4) Construction of a high-lupeol-producing yeast strain

[0031] The above expression vector Pm01 was introduced into strain MY06, and the strain MY07 capable of producing lupeol was constructed by screening using yeast YNB-URA-HIS-TRP-LEU solid selection medium.

[0032] Furthermore, the ERG8, ERG12, ERG19, IDI1, TRM8, and TRM82 genes were cloned from the genome of Saccharomyces cerevisiae CEN.PK2-1C, and their nucleotide sequences are shown in the sequence listing as SEQ ID NOs. 1 to 6;

[0033] The EfMvaS, EfMvaE, ERG20 F96W / N127W , GgLUS1 gene was obtained by gene synthesis of a biological company, and its nucleotide sequence is shown in the sequence list as SEQ ID NO.7-10;

[0034] The GAL1p, GAL10p, PGK1p and TEF1p are promoter sequences, which can be cloned from the genome of Saccharomyces cerevisiae CEN.PK2-1C, and their nucleotide sequences are shown in the sequence table as SEQ ID NO.11 to 14 respectively;

[0035] The ADH1t and CYC1t are terminator sequences, which can be cloned from the genome of Saccharomyces cerevisiae CEN.PK2-1C, and their nucleotide sequences are shown in the sequence table as SEQ ID NO.15-16 respectively;

[0036] The nucleotide sequences of the screening markers URA, HIS and TRP are shown in the sequence listing SEQ ID NOs. 17 to 19.

[0037] Another object of the present invention is to provide a method for increasing the yield of lupeol, a terpenoid compound in yeast, characterized in that the new yeast engineered strain with high lupeol yield is fermented in a shake flask to produce lupeol (C 30 H 50 O), whose structural formula is shown below:

[0038]

[0039] Furthermore, the method for increasing the yield of lupeol, a terpenoid compound of yeast, comprises the following steps:

[0040] (1) inoculating the new engineered yeast strain with high lupeol production onto a YNB-URA-HIS-TRP-LEU solid culture medium, culturing in a 30° C. incubator for 36 to 48 hours, picking a single clone colony and activating it in 6 mL of YNB-URA-HIS-TRP-LEU liquid culture medium, and culturing it at 30° C. and 220 rpm for 12 to 14 hours;

[0041] (2) Detect the OD value of the activated bacterial solution with a spectrophotometer and culture until the OD 600 When the initial OD is 2-3, take the corresponding amount of bacterial solution and inoculate it into a conical flask containing 50 mL of YNB-URA-HIS-TRP-LEU medium until the initial OD 600 The concentration of the mixture was 0.1, and the culture was shaken at 30°C and 220 rpm for 144 h.

[0042] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0043] The present invention is based on m 7 The method for improving the biosynthesis of terpenoid compounds by using the G methyltransferase complex gene TRM8-TRM82 can further improve the production efficiency of terpenoid compounds in Saccharomyces cerevisiae, and can effectively increase the production of lupeol by taking lupeol as a representative compound.

[0044] The technical solution of the present invention fills the technical gap in the industry at home and abroad: the technical solution of the present invention is the first to 7 G methylation modification was developed as a metabolic engineering optimization strategy for terpenoid biosynthesis, which can effectively increase the production of terpenoids in engineered strains of Saccharomyces cerevisiae. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a GC-MS detection result diagram of lupeol and lupeol standard in the engineered strain of Saccharomyces cerevisiae provided in the embodiment of the present invention.

[0046] Figure 2 This is the standard curve of the lupeol standard provided in the embodiment of the present invention.

[0047] Figure 3 This is a graph showing the production of lupeol in an engineered strain of Saccharomyces cerevisiae provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0049] The present invention only relates to a synthesis method, and the strains involved are existing products on sale.

[0050] The embodiment of the present invention provides a method based on m 7A method for improving the biosynthesis of yeast terpenoid compounds by using the G methyltransferase complex gene TRM8-TRM82, the method comprises overexpressing the TRM8 gene and the TRM82 gene; replacing the promoter of the endogenous TRM8 gene of the yeast with the constitutive promoter PGK1p, and replacing the promoter of the endogenous TRM82 gene of the yeast with the constitutive promoter TEF1p in the genome of an engineered strain of Saccharomyces cerevisiae producing terpenoid compounds.

[0051] The embodiment of the present invention provides a method based on m 7 The invention relates to a method for improving the biosynthesis of terpenoids in yeast by using the G methyltransferase complex gene TRM8-TRM82 in an engineered strain of Saccharomyces cerevisiae that produces high terpenoids.

[0052] Specifically, the embodiment of the present invention provides a new brewer's yeast engineered strain for producing terpenoid compounds represented by lupeol. The brewer's yeast engineered strain uses the brewer's yeast CEN.PK2-1C as the starting strain, firstly transforms the mevalonate pathway, introduces the EfMvaS gene and the EfMvaE gene from Enterococcus faecalis, and overexpresses ERG8, ERG9, ERG12, IDI1, ERG19, and ERG20. F96W / N127W Next, overexpression of m 7 The G methyltransferase complex gene TRM8-TRM82 genes further increased the synthesis of terpenoids, and finally the licorice-derived lupeol synthase GgLUS1 gene was introduced.

[0053] Furthermore, the new yeast engineered strain with high lupeol production was fermented in shake flasks to produce lupeol (C 30 H 50 O), whose structural formula is shown below:

[0054]

[0055] The embodiment of the present invention provides an application of a method for increasing the yield of lupeol, a terpenoid compound of yeast, comprising the following steps:

[0056] (1) The new engineered yeast strain with high lupeol production was inoculated onto a YNB-URA-HIS-TRP-LEU solid culture medium, cultured in a 30° C. incubator for 36 to 48 hours, and then a single clone was picked and activated in 6 mL of YNB-URA-HIS-TRP-LEU liquid culture medium, and cultured at 30° C. and 220 rpm for 12 to 14 hours.

[0057] (2) Detect the OD value of the activated bacterial solution with a spectrophotometer and culture until the OD 600When the initial OD is 2-3, take the corresponding amount of bacterial solution and inoculate it into a conical flask containing 50 mL of YNB-URA-HIS-TRP-LEU medium until the initial OD 600 The concentration of the mixture was 0.1, and the culture was shaken at 30°C and 220 rpm for 144 h.

[0058] An embodiment of the present invention provides a method based on m 7 The method for improving the biosynthesis of yeast terpenoid compounds by using the G methyltransferase complex gene TRM8-TRM82 is applied to the construction of an engineering strain of Saccharomyces cerevisiae with high lupeol production, but is not limited to the construction of an engineering strain of Saccharomyces cerevisiae with high lupeol production.

[0059] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0060] The reagents and culture medium involved in the embodiments of the present invention are as follows:

[0061] YNB medium: YNB yeast nitrogen base (without amino acids) 6.73 g / L, amino acid stock solution (lacking HIS / TRP / LEU / URA) (100×) 10 mL / L, glucose 20 g / L.

[0062] YPD medium: casein hydrolysate 20 g / L, yeast extract 10 g / L, glucose 20 g / L.

[0063] Amino acid stock solution (100×) (lacking HIS / TRP / LEU / URA): adenine sulfate 40 mg / L, arginine 20 mg / L, aspartic acid 100 mg / L, glutamate 100 mg / L, lysine 30 mg / L, phenylalanine 50 mg / L, serine 375 mg / L, threonine 200 mg / L, methionine 20 mg / L, tyrosine 30 mg / L, valine 150 mg / L.

[0064] Histidine (HIS) 0.2 g, tryptophan (TRP) 0.2 g, uracil (URA) 0.2 g, leucine (LEU) 0.6 g were respectively diluted to 100 mL with ultrapure water to prepare working concentrations of 20 mg / L histidine (HIS) stock solution (100×), 40 mg / L tryptophan (TRP) stock solution (100×), 20 mg / L uracil (URA) stock solution (100×), and 60 mg / L leucine (LEU) stock solution (100×).

[0065] YNB-URA medium: Add 1 mL each of the above LEU, HIS and TRP amino acid stock solutions (100×) to 50 mL of YNB nutrient-deficient medium. When preparing the solid medium, add 20 g / L of agar powder.

[0066] YNB-URA-HIS medium: Add 1 mL each of the above-mentioned LEU and TRP amino acid stock solutions (100×) to 50 mL of YNB nutrient-deficient medium. When preparing the solid medium, add 20 g / L of agar powder.

[0067] YNB-HIS-TRP-LEU medium: Add 1 mL of each of the above-mentioned URA amino acid stock solutions (100×) to 50 mL of YNB nutrient-deficient medium. When preparing the solid medium, add 20 g / L of agar powder.

[0068] YNB-URA-HIS-TRP medium: Add 1 mL of the above LEU amino acid stock solution (100×) to 50 mL of YNB nutrient-deficient medium. When preparing the solid medium, add 20 g / L of agar powder.

[0069] YNB-URA-HIS-TRP-LEU medium: Add 20 g / L agar powder to 50 mL of YNB nutrient-deficient medium when preparing solid culture medium.

[0070] TAE solution: 5g Tris, 0.74g Na2EDTA.2H2O, 1.15mL acetic acid, and finally add ddH2O to 1L.

[0071] NZY medium: casein hydrolysate 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, magnesium sulfate heptahydrate 2 g / L, 5 M sodium hydroxide solution to adjust the pH to 7.0.

[0072] NZY-Carb medium: Add 50 μL of 50 mg / mL carbenicillin solution to 50 mL of NZY medium.

[0073] Example 1: Cloning of yeast endogenous genes, promoters, terminators and selection marker genes and acquisition of exogenous genes

[0074] 1. Extraction of Yeast Genome

[0075] (1) The CEN.PK 2-1C monoclonal strain was selected and placed in 6 mL of YNB-deficient medium. The culture was shaken at 30°C and 220 rpm for 12 to 14 h in a shaker and cultured overnight.

[0076] (2) Transfer the yeast solution to a 1.5 mL centrifuge tube and centrifuge at 1000 rpm for 1 min. Discard the culture medium and collect the cells. Add liquid nitrogen to the collected cells and grind them thoroughly. Transfer the ground powder to a 1.5 mL centrifuge tube.

[0077] (3) The genomic DNA of Saccharomyces cerevisiae was extracted using a DNA extraction kit (DC104, Nanjing Novozymes Biotechnology Co., Ltd.). The specific operation process is shown in the instruction manual.

[0078] 2. Cloning of yeast endogenous genes, promoters and terminators

[0079] (1) Using the genome of Saccharomyces cerevisiae CEN.PK 2-1C obtained above as a template, the following yeast endogenous genes, promoters and terminator genes were cloned respectively (primer sequences are shown in Table 1):

[0080] Gene ERG8 (primers ERG8-F, ERG8-R), gene ERG12 (primers ERG12-F, ERG12-R), gene ERG20 (primers ERG20-F, ERG20-R), gene TRM8 (primers TRM8-F, TRM8-R), promoter GAL1p (primers GAL1p-F, GAL1p-R), promoter GAL10p (primers GAL10p-F, GAL10p-R), promoter PGK1p (primers PGK1 p-F, PGK1p-R), promoter TEF1p (primers TEF1p-F, TEF1p-R) (primers PGK1p-F, PGK1p-R) terminator ADH1t (primers ADH1t-FVG, ADH1t-R), terminator CYC1t (primers CYC1t-F, CYC1t-R), screening marker (primers URA-F, URA-R), screening marker (primers HIS-F, HIS-R), screening marker (primers TRP-F, TRP-R).

[0081] PCR reaction system: 2×Phanta Max Master Mix (P510, Nanjing Novozymes Biotech Co., Ltd.) 10 μL, 5' and 3' primers 0.5 μL each, ddH2O 8 μL, DNA template 1 μL, total reaction system 20 μL.

[0082] PCR amplification reaction conditions: 95°C for 1 min; 95°C for 15 sec, 58°C for 15 sec, 72°C for 5 sec / kb, 32 cycles; 72°C for 7 min.

[0083] Agarose gel electrophoresis detection: Take 20μL sample + 2μL 10×DNA Loading buffer (PO22-AA, Nanjing Novogene Biotech Co., Ltd.), mix and load, use Trans5K DNA Marker (BM141-01, Beijing Quanshijin Biotech Co., Ltd.) to analyze DNA bands, 120V constant voltage electrophoresis for 10-20min, save the electrophoresis image, and after confirming that the band size is correct, cut out the target band. (Preparation of agarose gel: 1×TAE buffer 30mL + 0.25g agarose, heat until the agarose is completely dissolved and then cool to below 60℃, add 0.2μL fluorescent nucleic acid staining reagent (GS101-01, Beijing Quanshijin Biotech Co., Ltd.), mix and pour the plate.

[0084] PCR product purification: The product was recovered according to the procedure of DNA gel recovery kit (DP209, Beijing Tiangen Biochemical Technology Co., Ltd.), and finally eluted with 35 μL ddH2O and stored at -20°C.

[0085] (2) The DNA fragment obtained above was connected to the pEASY-Blunt vector (CB111-01, Beijing Quanshijin Biotechnology Co., Ltd.), 4 μL of DNA fragment and 1 μL of pEASY-Blunt vector were taken, and after the system was prepared, they were placed on a float and placed in a 27°C water bath. After the reaction for 30 minutes, they were taken out. Then, competent Escherichia coli DH5α (DL1001, Shanghai Weidi Biotechnology Co., Ltd.) was transformed. The specific operation process is shown in the instruction manual.

[0086] (3) After overnight incubation at 37°C for 14 to 16 hours, single clones were selected for colony PCR.

[0087] PCR reaction system: 2×M5 HiPer Taq HiFi PCR mix (P515-03, Beijing PolyMei Biotechnology Co., Ltd.) 10 μL, 5' and 3' primers 0.3 μL each, ddH2O 10 μL.

[0088] PCR amplification conditions: 95°C for 3 min; 95°C for 15 sec, 58°C for 15 sec, 72°C for 30 s, 30 cycles; 72°C for 7 min.

[0089] After the PCR was completed, the PCR products were detected by agarose gel electrophoresis, and the positive strains were selected and cultured in 6 mL of NZY medium containing carbenicillin at 37°C for 12 to 14 h.

[0090] After the strain culture was completed, plasmids were extracted using a plasmid extraction kit (DP103, Tiangen Biochemical Technology (Beijing) Co., Ltd.). The detailed operation process is shown in the kit manual. After completion, the obtained plasmid was sequenced, and the next step was performed after the sequencing was correct.

[0091] 3. Synthesis of foreign genes

[0092] EfMvaS, EfMvaE, ERG20 F96W / N127W Genes such as GgLUS1 and GgLUS1 were commissioned to be synthesized by General Biotechnology (Anhui) Co., Ltd.

[0093] Table 1 Primer sequences used for cloning genes and expression elements in the present invention

[0094]

[0095]

[0096] Example 2: Construction of modules

[0097] The plasmid vector constructed and sequenced successfully was used for PCR. The PCR product was detected by agarose gel electrophoresis, and then the product was recovered using a DNA gel recovery kit. The specific operation process is shown in the instruction manual.

[0098] 1. Overlapping PCR Building Blocks

[0099] (1) First round PCR reaction system: 2×Phanta Max Master Mix 10 μL, 5' and 3' primers 0.5 μL each, ddH2O 8 μL, vector template 1 μL + 9 μL diluted 1 μL, total reaction system 20 μL.

[0100] The reaction conditions for one round of PCR were as follows: 95°C for 1 min; 95°C for 15 sec, 58°C for 15 sec, 72°C for 5 sec / kb, 32 cycles; 72°C for 7 min.

[0101] (2) After one round of PCR is completed, the PCR product is detected by agarose gel electrophoresis. After confirming that the bands are correct, the product is recovered using a DNA gel recovery kit. The specific operation process is shown in the instruction manual.

[0102] (3) Second-round PCR reaction system: 10 μL of 2×Phanta Max Master Mix, add appropriate μL of equal amounts of DNA fragments recovered from two gels, the total reaction system is 20 μL, and the remaining amount is made up with ddH2O.

[0103] PCR reaction conditions: 95°C for 1 min; 95°C for 15 sec, 60°C for 15 sec, 72°C for 5 sec / kb, 15 cycles; 72°C for 7 min.

[0104] (4) Take 1 μL of the second-round PCR reaction product as the template for the third-round overlapping PCR reaction

[0105] Three-round overlapping PCR reaction system: 2×Phanta Max Master Mix 10μL, 5' and 3' primers 0.5μL each, ddH2O 8μL, second-round product 1μL, total reaction system 20μL.

[0106] The reaction conditions of the second round of overlapping PCR were as follows: 95°C for 1 min; 95°C for 15 sec, 60°C for 15 sec, 72°C for 5 sec / kb, 32 cycles; 72°C for 7 min.

[0107] (3) After the three rounds of overlapping PCR are completed, the PCR products are detected by agarose gel electrophoresis. After confirming that the bands are correct, the products are recovered using a DNA gel recovery kit. The specific operation process is shown in the instruction manual.

[0108] (4) The DNA fragment obtained above was connected to the pEASY-Blunt vector and sequenced.

[0109] 2. Construction steps of modules I to XI

[0110] (1) The promoter GAL1p was connected to the promoter GAL10p to construct a GAL1p-GAL10p module, named module I; wherein the primers for cloning GAL1p in the first round of PCR were I-GAL1p-F1 and I-GAL1p-R1, and the primers for cloning GAL10p were I-GAL10p-F2 and I-GAL10p-R2; the primers for the second round of PCR were I-GAL1p-F1 and I-GAL10p-R2 (primer sequences are shown in Table 2).

[0111] (2) Module I, ERG8 gene and terminator ADH1t were connected to construct the I-ERG8-ADH1t module, named module II; the primers for cloning module I in the first round of PCR were II-I-F1 and II-I-R1, the primers for cloning ERG8 were II-ERG8-F2 and II-ERG8-R2; the primers for cloning ADH1t were II-ADH1t-F3 and II-ADH1t-R3, and the primers for the second round of PCR were II-I-F1 and II-ADH1t-R3 (primer sequences are shown in Table 2).

[0112] (3) Connect the screening marker URA to module II to construct the URA-II module, which is named module III; wherein the primers for cloning module URA in the first round of PCR are III-URA-F1 and III-URA-R1, the primers for cloning module II are III-II-F2 and III-II-R2, and the primers for the second round of PCR are III-F2 and III-R2 (primer sequences are shown in Table 2).

[0113] (4) Module I, ERG12 gene and terminator ADH1t were connected to construct the I-ERG12-ADH1t module, named module IV; wherein, the primers for cloning module I in the first round of PCR were IV-I-F1 and IV-I-R1, and the primers for cloning ERG12 were IV-ERG12-F2 and IV-ERG12-R2; the primers for cloning ADH1t were IV-ADH1t-F3 and IV-ADH1t-R3, and the primers for the second round of PCR were IV-I-F1 and IV-ADH1t-R3 (primer sequences are shown in Table 2).

[0114] (5) Connecting the screening marker HIS to module IV to construct an IV-HIS module, named module V; wherein the primers for cloning module HIS in the first round of PCR are V-HIS-F1 and V-HIS-R1, the primers for cloning module IV are V-IV-F2 and V-IV-R2, and the primers for the second round of PCR are V-F2 and V-R2 (primer sequences are shown in Table 2).

[0115] (6) Module I, ERG20 F96W / N127W The gene and terminator ADH1t were connected to construct an I-ERG20-ADH1t module, named module VI; wherein, the primers for cloning module I in the first round of PCR were VI-I-F1 and VI-I-R1, the primers for cloning ERG20 were VI-ERG20-F2 and VI-ERG20-R2; the primers for cloning ADH1t were VI-ADH1t-F3 and VI-ADH1t-R3, and the primers for the second round of PCR were VI-I-F1 and VI-ADH1t-R3 (primer sequences are shown in Table 2).

[0116] (7) Connecting the screening marker TRP to module VI to construct a VI-TRP module, named module VII; wherein the primers for cloning module TRP in the first round of PCR are VII-TRP-F1 and VII-TRP-R1, the primers for cloning module VI are VII-VI-F2 and VII-VI-R2, and the primers for the second round of PCR are VII-F2 and VII-R2 (primer sequences are shown in Table 2).

[0117] (8) Module I, EfMvaS gene and terminator ADH1t were connected to construct the I-EfMvaS-ADH1t module, named module VIII; the primers for cloning module I in the first round of PCR were VIII-I-F1 and VIII-I-R1, the primers for cloning EfMvaS were VIII-EfMvaS-F2 and VIII-EfMvaS-R2; the primers for cloning ADH1t were VIII-ADH1t-F3 and VIII-ADH1t-R3, and the primers for the second round of PCR were VIII-I-F1 and VIII-ADH1t-R3 (primer sequences are shown in Table 2).

[0118] (9) Module VIII, EfMvaE gene and terminator CYC1t were connected to construct the VIII-EfMvaS-CYC1t expression module, named module IX; the primers for cloning module VIII in the first round of PCR were IX-VIII-F1 and IX-VIII-R1, and the primers for cloning EfMvaE were IX-EfMvaE-F2 and IX-EfMvaE-R2; the primers for cloning CYC1t were IX-CYC1t-F3 and IX-CYC1t-R3, and the primers for the second round of PCR were IX-F2 and IX-R2 (primer sequences are shown in Table 2).

[0119] (10) The promoter PGK1p, TRM8 gene and terminator ADH1t were connected to construct a PGK1p-TRM8-ADH1t module, named module X; wherein the primers for cloning the module PGK1p in the first round of PCR were X-PGK1p-F1 and X-PGK1p-R1, and the primers for cloning TRM8 were X-TRM8-F2 and X-TRM8-R2; the primers for cloning ADH1t were X-ADH1t-F3 and X-ADH1t-R3, and the primers for the second round of PCR were X-PGK1p-F1 and X-ADH1t-R3 (primer sequences are shown in Table 2).

[0120] (11) The screening marker URA, module X and promoter TEF1p were connected to construct a TEF1p-X-URA module, named module XI; wherein the primers for cloning the module TEF1p in the first round of PCR were XI-TEF1p-F1 and XI-TEF1p--R1, and the primers for cloning the module X were XI-X-F2 and XI-X-R2; the primers for cloning URA were XI-URA-F3 and XI-URA-R3, and the primers for the second round of PCR were XI-F2 and XI-R2 (primer sequences are shown in Table 2).

[0121] (12) The successfully constructed modules I to XI were connected to the pEASY-Blunt vector and sequenced.

[0122] Example 3. Construction of expression vector Pm01

[0123] The vector pESC-LEU was cut with restriction endonucleases NotI and SacI; the GgLUS1 gene was connected to the cut vector through homologous recombination to construct the expression vector Pm01.

[0124] (1) PCR cloning of the GgLUS1 gene using primers GgLUS1-F and GgLUS1-R (primer sequences see Table 2).

[0125] PCR reaction system: 2×Phanta Max Master Mix 10μL, GgLUS1-F and GgLUS1-R primers 0.5μL each, ddH2O 8μL, vector template 1μL+9μL diluted to 1μL, total reaction system 20μL.

[0126] PCR reaction conditions: 95°C for 1 min; 95°C for 15 sec, 58°C for 15 sec, 72°C for 5 sec / kb, 32 cycles; 72°C for 7 min.

[0127] (2) Detect the PCR product by agarose gel electrophoresis. After confirming that the bands are correct, use a DNA gel recovery kit to recover the product. The specific operation process is shown in the instruction manual.

[0128] (3) The vector pESC-LEU was digested with restriction endonuclease FastDigest NotI (FD0593, Thermo Fisher Scientific (China) Co., Ltd.) and restriction endonuclease FastDigest SacI (FD1134, Thermo Fisher Scientific (China) Co., Ltd.).

[0129] Reaction system and reaction conditions: 1 μL 10×FastDigest buffer (B64, Thermo Fisher Scientific (China) Co., Ltd.), 0.5 μL endonuclease NotI, 0.5 μL endonuclease SacI, 1 μL vector, 7 μL ddH2O; after the system is prepared, react at 37°C for 1h 30min and at 85°C for 15min to inactivate the restriction endonuclease.

[0130] (4) Use the ClonExpress II One Step Cloning Kit (C112-01, Nanjing Novogene Biotechnology Co., Ltd.) to connect the target gene fragment GgLUS1 obtained in step (2) and the restriction vector pESC-LEU in step (3).

[0131] Reaction system and reaction conditions: 4 μL target gene fragment, 2 μL 5×CE II Buffer, 1 μL Express II, 2 μL ddH2O, 1 μL digested vector. After the system is prepared, place it at 37°C for 30 minutes.

[0132] (5) Take 35 μL of competent E. coli and thaw it on ice for 5 min. Add 3.5 μL of the recombinant system and place it on ice for 25 min. Heat shock at 42°C for 1 min and place it on ice for 2 min. Immediately add 200 μL of NZY medium and place it in a 37°C constant temperature shaker for 40 to 60 min. Centrifuge at 6000 rpm for 1 min, discard 100 μL of supernatant, mix the remaining supernatant and bacteria by pipetting, and evenly spread them on NZY-Carb solid culture medium. After incubation at 37°C for 14 to 18 h, select single clones for PCR verification.

[0133] (6) Pick a single clone with a toothpick and dip it into the corresponding PCR tube three times. Then insert the toothpick into a centrifuge tube containing 200 μL of the prepared NZY-Carb medium and culture it in a shaker at 37°C for 4 to 6 hours.

[0134] Colony PCR reaction system: 2×M5 HiPer plus Taq HiFi PCR mix (MF002-plus-01, Beijing PolyMei Biotechnology Co., Ltd.) 5 μL, 5' and 3' primers (primer sequences are shown in Table 1) 0.3 μL each, ddH2O 5 μL.

[0135] Colony PCR reaction conditions: 95℃5min; 95℃15sec, 58℃15sec, 72℃15sec / kb, 30 cycles; 72℃5min.

[0136] (7) Detect the PCR product by agarose gel electrophoresis, and select the corresponding bacterial solution for inoculation based on the electrophoresis results and the turbidity of the bacterial solution. Transfer the bacterial solution in the centrifuge tube into 6 mL of NZY-Carb liquid culture medium and culture at 37°C on a shaker for 12 to 14 hours.

[0137] (8) After the culture is completed, the plasmid is extracted using the Tiangen Plasmid Extraction Kit (DP103, Tiangen Biochemical Technology (Beijing) Co., Ltd.). The detailed operation process is shown in the kit manual. After the extraction is completed, sequencing is performed. After the sequencing is correct, the next step is performed.

[0138] Table 2 Primer sequences used to construct each module in the present invention

[0139]

[0140]

[0141]

[0142] Example 4: Construction of an engineered yeast strain producing lupeol

[0143] 1. Construction of the first new yeast engineering strain

[0144] (1) Module III was cloned from pEASY-Blunt-II-URA using primers III-URA-F1 and III-R2.

[0145] PCR reaction system: 2×Phanta Max Master Mix 25μL, III-URA-F1 and III-R2 primers 1μL each, vector 1μL, ddH2O 25μL. The PCR product was subjected to nucleic acid agarose gel electrophoresis. After the fragment size was confirmed by agarose gel electrophoresis, the PCR product was recovered using a DNA gel recovery kit.

[0146] PCR reaction conditions: 95°C for 1 min; 95°C for 15 sec, 58°C for 15 sec, 72°C for 5 sec / kb, 32 cycles; 72°C for 7 min.

[0147] (2) The recovered target PCR fragment was transformed into the Saccharomyces cerevisiae strain CEN.PK2-1C to replace the promoter sequence of the ERG9 gene in the chromosome of the yeast CEN.PK 2-1C. The strain was screened using a URA-deficient solid culture medium plate. After colony PCR verification, the Saccharomyces cerevisiae engineered strain MY01 was obtained.

[0148] Specific steps:

[0149] ①Pick the CEN.PK2-1C monoclonal strain into 6 mL of YNB defective medium, shake it in a shaker at 30°C and 220 rpm for 12 to 14 hours, and culture it overnight.

[0150] ② When OD 600 When the value is 0.6-0.8, take 1.5mL of the above bacterial solution and centrifuge at 9000rpm / min for 1min, discard the supernatant. Resuspend and wash with 1mL ddH2O, gently aspirate and beat, centrifuge at 9000rpm for 1min, and repeat the wash three times. When aspirating the supernatant for the third time, aspirate as much as possible and resuspend with 20μL ddH2O.

[0151] ③ Add 240μL PEG3350, 36μL lithium acetate solution, 5μL CarrierDNA and 50μL target fragment to the above bacterial solution, place it in a 42℃ water bath for 1h, and shake it up and down every 20min.

[0152] ④After the heat shock, centrifuge at 9000rpm for 1min, discard the supernatant, be careful not to aspirate the bacteria, add 500μL YPD with glucose, gently pipette to mix, and place in a yeast shaker at 30℃ and 220rpm for 3h.

[0153] ⑤ Centrifuge at 9000rpm for 1min, discard the supernatant, add 500μL ddH2O to resuspend and wash once, and plate as needed. Use YNB-URA solid medium for screening and culture at 30℃ for 48-60h.

[0154] ⑥ After colony PCR verification, the brewer's yeast engineering strain MY01 was obtained.

[0155] 2. Construction of the Second New Yeast Engineering Strain

[0156] (1) Clone module V from pEASY-Blunt-IV-HIS using primers V-HIS-F1 and V-R2. (The PCR reaction system and PCR reaction conditions are the same as those in Example 4, step 1. Construction of the first new yeast engineering strain.)

[0157] (2) The recovered target PCR fragment was transformed into the first new yeast engineering strain, the promoter sequence of the yeast IDI1 gene was replaced, and the strain was screened using a YNB-URA-HIS solid culture medium plate. After colony PCR verification, the cerevisiae engineering strain MY02 was obtained. (The specific steps are the same as those in Example 4, step 1. Construction of the first new yeast engineering strain.)

[0158] 3. Construction of the third new yeast engineering strain

[0159] (1) Module V was cloned from pEASY-Blunt-VI-TRP using primers VII-TRP-F1 and VII-R2. (The PCR reaction system and PCR reaction conditions are the same as those in Example 4, step 1. Construction of the first new yeast engineering strain.)

[0160] (2) The recovered target PCR fragment was transformed into the second new yeast engineering strain, the promoter sequence of the yeast ERG19 gene was replaced, and the strain was screened using a YNB-URA-HIS-TRP solid culture medium plate. After colony PCR verification, the cerevisiae engineering strain MY03 was obtained. (The specific steps are the same as those in Example 4, step 1. Construction of the first new yeast engineering strain.)

[0161] 4. Construction of the fourth new yeast engineering strain

[0162] (1) Clone module IX from pEASY-Blunt-VIII-EfMvaE-CYC1t using primers IX-VIII-F1 and IX-R2. (The PCR reaction system and PCR reaction conditions are the same as those in Example 4, step 1. Construction of the first new yeast engineering strain.)

[0163] (2) The recovered target PCR fragment was transformed into the third new yeast engineering strain, the yeast URA3 gene sequence was replaced, and the yeast YPD solid medium plate containing 5-fluoroorotic acid was used for screening. After colony PCR verification, the cerevisiae engineering strain MY04 was obtained. (The specific steps are the same as step 1. Construction of the first new yeast engineering strain in Example 4.)

[0164] 5. Construction of new yeast engineering strains

[0165] The expression vector Pm01 was introduced into strain MY04, and the strain MY05 capable of producing lupeol was constructed using solid selection medium of yeast YNB-HIS-TRP-LEU. (The specific steps are the same as step 1 of Example 4. Construction of the first new yeast engineering strain.)

[0166] 6. Construction of new yeast engineering strains

[0167] (1) Clone module IX from pEASY-Blunt-X-URA-TEF1t using primers XI-TEF1p-F1 and XI-R2. (The PCR reaction system and PCR reaction conditions are the same as those in Example 4, step 1. Construction of the first new yeast engineering strain.)

[0168] (2) The recovered target PCR fragment was transformed into the fourth new yeast engineering strain, the promoter sequence of the yeast TRM82 gene was replaced, and screening was performed using a solid culture medium plate containing YNB-URA-HIS-TRP. After colony PCR verification, the cerevisiae engineering strain MY05 was obtained. (The specific steps are the same as step 1. Construction of the first new yeast engineering strain in Example 4.)

[0169] 7. Construction of new yeast engineering strains

[0170] The expression vector Pm01 was introduced into strain MY06, and the strain MY07 with high lupeol production was constructed using solid selection medium of yeast YNB-URA-HIS-TRP-LEU. (The specific steps are the same as step 1 of Example 4. Construction of the first new yeast engineering strain.)

[0171] Example 5: Production of lupeol by shake flask fermentation of strains MY05 and MY07

[0172] (1) The yeast engineering strains MY05 and MY07 were inoculated onto YNB-HIS-TRP-LEU and YNB-URA-HIS-TRP-LEU solid culture media, respectively. After culturing in a 30° C. incubator for 36 to 48 h, single clones were picked and activated in 6 mL YNB-HIS-TRP-LEU and YNB-URA-HIS-TRP-LEU liquid culture media, respectively. The culture was shaken at 220 rpm at 30° C. for 12 to 14 h.

[0173] (2) Detect the OD value of the activated bacterial solution with a spectrophotometer and culture until the OD 600 When the initial OD value is 2 to 3, the corresponding amount of bacterial solution was inoculated into conical flasks containing 50 mL YNB-HIS-TRP-LEU and 50 mL YNB-URA-HIS-TRP-LEU culture medium respectively until the initial OD value is 2 to 3. 600 The concentration of the mixture was 0.1, and the culture was shaken at 30°C and 220 rpm for 144 h.

[0174] (3) Take out the bacterial solution, shake it evenly, pour it into a 50mL centrifuge tube, centrifuge it at 4℃ and 5000rpm for 5min, pour the supernatant of the bacterial solution back into the conical flask, and be careful to retain the precipitate. Add 10mL of alkaline lysis solution to the test tube containing the precipitate, vortex and shake to mix, and then place it in a 98℃ water bath and heat it for 15min, shaking it every three minutes. After the water bath heating is completed, cool it to room temperature

[0175] (4) Mix the lysed cells and supernatant and pour them back into the conical flask. Then add an equal volume of n-hexane. Ultrasonicate for 30 minutes. After the end, place the conical flask in a fume hood and let it stand for 48 hours.

[0176] (5) Pour the supernatant into a measuring cylinder, measure the supernatant volume with the measuring cylinder, take 1 / 10 of the supernatant, pour it into a rotary evaporation conical flask, and perform rotary evaporation. Stop evaporation when there is an appropriate amount of liquid at the bottom of the flask, and add an appropriate amount of n-hexane with a rubber-tipped dropper. Use a sterile syringe to take all the liquid in the rotary evaporation conical flask, add a filter, and inject it into a liquid phase vial for detection by GC-MS.

[0177] GC-MS detection conditions: HP-5ms capillary column; injection volume 1 μL, no split; injection port temperature 280°C, initial temperature 50°C maintained for 3 min, then increased to 70°C at a rate of 20°C / min and maintained for 1 min, then increased to 300°C at a rate of 15°C / min and maintained for 10 min, interface temperature 280°C; Electron Ionization ion source, energy intensity 70 eV; MS solvent delay set to 10 min, voltage multiplication mode turned on, gain factor set to 1.

[0178] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method based on m 7 A method for improving the biosynthesis of terpenoid compounds in yeast by using the G methyltransferase complex gene TRM8-TRM82, characterized in that: In the genome of an engineered yeast strain for producing terpenoid compounds, the promoter of the yeast endogenous TRM8 gene is replaced with the constitutive promoter PGK1p, and the promoter of the yeast endogenous TRM82 gene is replaced with the constitutive promoter TEF1p; the nucleotide sequence of the promoter PGK1p is shown in SEQ ID NO.13, and the nucleotide sequence of the promoter TEF1p is shown in SEQ ID NO.

14.

2. A method based on m as claimed in claim 1 7 The invention relates to a method for improving the biosynthesis of terpenoids in yeast by using the G methyltransferase complex gene TRM8-TRM82 in an engineered strain of Saccharomyces cerevisiae that produces high terpenoids.

3. A method for constructing an engineered strain of Saccharomyces cerevisiae that produces high yields of the terpenoid compound lupeol, characterized in that: The steps include: (1) Building Modules The promoter GAL1p was connected to the promoter GAL10p to construct module I; Module I, ERG8 gene and terminator CYC1t were connected to construct module II; Connect the selection marker URA to module II to construct module III; Module I, ERG12 gene and terminator ADH1t were connected to construct module IV; Connect the selection marker HIS to module IV to construct module V; Module I, ERG20F96W / N127W gene and terminator ADH1t were connected to construct module VI; Connecting the screening marker TRP to module VI to construct module VII; Module Ⅷ was constructed by connecting module Ⅰ, EfMvaS gene and terminator ADH1t; Module VIII, EfMvaE gene and terminator CYC1t were connected to construct module IX; The promoter PGK1p, TRM8 gene and terminator ADH1 were connected to construct module X; The selection marker URA, module X and promoter TEF1p were connected to construct module XI; (2) Construction of expression vector The vector pESC-LEU was digested with restriction endonuclease NotI; The GgLUS1 gene was connected to the restriction vector through homologous recombination to construct the expression vector Pm01; (3) Construction of yeast engineering strains The above module III was introduced into the starting strain, integrated into the ERG9p site, and screened using yeast-deficient YNB-URA solid medium to construct strain MY01; The above module V was introduced into strain MY01, integrated into the IDI1p site, and screened using yeast YNB-URA-HIS solid selection medium to construct strain MY02; The above module VII was introduced into strain MY02, integrated into the ERG19p site, and screened using yeast YNB-URA-HIS-TRP solid selection medium to construct strain MY03; The module IX was introduced into strain MY03 and integrated into the URA3 locus, and screened using yeast YPD-5FOA solid selection medium to construct strain MY04; The module XI was introduced into strain MY04 and integrated into the TRM82p site, and screened using yeast YNB-URA-HIS-TRP solid selection medium to construct a new strain; (4) Construction of a high-yield lupeol yeast strain The above expression vector Pm01 was introduced into strain MY06, and the strain MY07 capable of producing lupeol was constructed by screening using yeast YNB-URA-HIS-TRP-LEU solid selection medium.

4. The method for constructing an engineered strain of Saccharomyces cerevisiae for producing the terpenoid compound lupeol according to claim 1, characterized in that: The ERG8, ERG12, ERG19, IDI1, TRM8, and TRM82 genes are cloned from the genome of Saccharomyces cerevisiae CEN.PK2-1C, and their nucleotide sequences are shown in the sequence table as SEQ ID NO.1 to 6; The EfMvaS, EfMvaE, ERG20 F96W / N127W , GgLUS1 gene was obtained by gene synthesis of a biological company, and its nucleotide sequence is shown in the sequence list as SEQ ID NO.7-10; The GAL1p, GAL10p, PGK1p and TEF1p are promoter sequences, which can be cloned from the genome of Saccharomyces cerevisiae CEN.PK2-1C, and their nucleotide sequences are shown in the sequence table as SEQ ID NO.11 to 14 respectively; The ADH1t and CYC1t are terminator sequences, which can be cloned from the genome of Saccharomyces cerevisiae CEN.PK2-1C, and their nucleotide sequences are shown in the sequence table as SEQ ID NO.15-16 respectively; The nucleotide sequences of the screening markers URA, HIS and TRP are shown in the sequence listing SEQ ID NOs. 17 to 19.

5. Use of the method according to claim 1 in increasing the yield of lupeol, a terpenoid compound in yeast.

6. A method for increasing the yield of lupeol, a terpenoid compound of yeast, characterized in that: The new engineered yeast strain for high lupeol production according to claim 5 is fermented in a shake flask, and the product lupeol C 30 H 50 O, whose structural formula is shown below:

7. The method for increasing the yield of lupeol, a terpenoid compound in yeast according to claim 6, characterized in that: The steps include: (1) inoculating the new engineered yeast strain with high lupeol production onto a YNB-URA-HIS-TRP-LEU solid culture medium, culturing in a 30° C. incubator for 36 to 48 hours, picking a single clone colony and activating it in 6 mL of YNB-URA-HIS-TRP-LEU liquid culture medium, and culturing it at 30° C. and 220 rpm for 12 to 14 hours; (2) Detect the OD value of the activated bacterial solution with a spectrophotometer and culture until the OD 600 When the initial OD is 2-3, take the corresponding amount of bacterial solution and inoculate it into a conical flask containing 50 mL of YNB-URA-HIS-TRP-LEU medium until the initial OD 600 The concentration of the mixture was 0.1, and the culture was shaken at 30°C and 220 rpm for 144 h.

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