Engineering bacterium for de novo biosynthesis of cycloastragenol as well as construction method and application of engineering bacterium

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an engineering bacterium for de novo biosynthesis of cycloastragenol as well as a construction method and application of the engineering bacterium. A mevalonic acid (MVA) pathway is reconstructed in a saccharomyces cerevisiae strain peroxidase body by utilizing a cell region compartment strategy, a key enzyme gene of the MVA pathway is over-expressed by using a strong promoter, and the cycloastragenol is obtained. The key enzyme genes are used as expression elements of cycloastragenol, are introduced into the chassis saccharomyces cerevisiae through a CRISPR-Cas9 technology, and are integrated on a saccharomyces cerevisiae genome through homologous recombination, so that a saccharomyces cerevisiae engineering bacterium CycZ23 and an engineering bacterium CAG-1 for efficiently synthesizing cycloastragenol are constructed, and de novo synthesis of plant triterpenoid natural products is realized; the highest titer of the generated cycloastragenol is 236 mg / L; the synthesis method is economical, efficient, green, environmentally friendly and easy to operate, and has important application value.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to an engineering bacterium for de novo biosynthesis of cycloastragenol. Background Art

[0002] Cycloastragenol (CAG), a tetracyclic triterpenoid extracted from the traditional medicinal plant Astragalus membranaceus, has shown significant anti-aging, immunomodulatory, and telomerase-activating activities and has attracted considerable attention in recent years in the fields of anti-aging drugs, functional foods, and cosmetics. However, its natural source relies on plant extraction, which presents both resource and efficiency bottlenecks. Astragalus membranaceus has a long growth cycle (typically 3-5 years), and the cycloastragenol content in its roots is extremely low (typically <0.01% dry weight), resulting in high raw material costs and difficulty in meeting scale-up requirements. Traditional extraction requires multiple purification steps, including organic solvent extraction and column chromatography, which are energy-intensive and yield-poor, while also generating large amounts of organic wastewater and posing significant environmental risks. While chemical synthesis can avoid plant dependence, its industrial application is hampered by the complex structure of cycloastragenol (containing multiple chiral centers and a tetracyclic skeleton), the lengthy chemical synthesis steps, and the difficulty in controlling stereoselectivity, making industrial feasibility low. Therefore, there is an urgent need to develop sustainable and efficient methods for producing cycloastragenol, a natural plant ingredient.

[0003] In recent years, synthetic biology has developed rapidly, and the use of microbial cell factories to synthesize rare active ingredients has become a major green synthetic route. Compared with plant extraction and chemical synthesis, microbial cell factories have a shorter growth cycle and can use a variety of simple sugar raw materials to mass-produce a variety of complex plant active natural products. It is a green and economical biomanufacturing model. Patent 202310293488.0 provides a method for preparing cycloastragenol. By preparing a mixed solution of astragaloside IV, xylosidase and GSM inoculant, cycloastragenol is prepared through a conversion reaction. Although it can achieve efficient conversion of astragaloside IV, it relies on the natural plant extract astragaloside IV as a raw material and is still limited by the source of raw materials. Patent 202111608350.2 discloses a biocatalytic preparation method for cycloastragenol. By adding a composite enzyme during the processing of cycloastragenol, high-purity cycloastragenol is extracted and purified, with high conversion rate and high product purity. It also relies on the hydrolysis of astragaloside IV.

[0004] Therefore, synthetic biology technology is used to construct a brewer's yeast cell factory for the de novo synthesis of cycloastragenol, and direct biosynthesis of cycloastragenol is achieved through genetic engineering. This not only gets rid of the dependence on natural plant raw materials, but also can further improve the yield and purity of cycloastragenol by optimizing fermentation conditions and metabolic pathways, which will effectively solve the problem of limited sources of plant natural products such as cycloastragenol. Summary of the Invention

[0005] To solve the above problems, the present invention provides an engineering bacterium for de novo biosynthesis of cycloastragenol, a construction method and application thereof, and the engineering bacterium can produce cycloastragenol by microbial fermentation.

[0006] The technical solution of the present invention is achieved as follows:

[0007] The present invention utilizes a cell compartmentalization strategy to reconstruct the mevalonate (MVA) pathway in the peroxisome of a Saccharomyces cerevisiae strain, and uses a strong promoter to overexpress key enzyme genes of the MVA pathway: acetoacetyl CoA thiolase (ERG10), HMG-CoA synthase (3-hydroxy-3-methylglutaryl-CoA synthase, ERG13), truncated expression of HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase, tHMG1), mevalonate kinase (ERG12), phosphomevalonate kinase (ERG8), 5-pyrophosphomevalonate decarboxylase (ERG19), isoprenyl pyrophosphate isomerase (IDI1), etc. to enhance the synthesis of terpenoid precursors; and express ERG20 F96C The content of triterpenoid precursors was increased by strategies such as mutants, overexpression of farnesyl diphosphate farnesyltransferase (ERG9), and squalene epoxidase (ERG1); heterologous cycloastragenol synthase (CAS), cytochrome P450 enzyme (CYP450s), oxoglutarate / iron-dependent dioxygenase 1, etc. were introduced, and these key enzyme genes were used as expression elements of cycloastragenol. They were introduced into the chassis brewer's yeast through CRISPR-Cas9 technology and integrated into the brewer's yeast genome by homologous recombination to construct a brewer's yeast strain that efficiently synthesizes cycloastragenol, realizing the de novo synthesis of the plant triterpenoid natural product cycloastragenol.

[0008] Based on this, the present application provides a method for constructing an engineered bacterium for de novo biosynthesis of cycloastragenol, comprising the following steps:

[0009] (1) Constructing expression cassette G1 containing ERG10 gene, expression cassette G2 containing IDI1 gene, and expression cassette G3 containing ERG20 F96C , an expression cassette G3 comprising the ERG1 gene, an expression cassette G4 comprising the tHMG1 gene, an expression cassette G5 comprising the AmCAS1 gene, an expression cassette G6 comprising the ERG13, ERG12, ERG19, ERG9 and ERG8 genes, an expression cassette G7 comprising the ERG1 and AmCAS1 genes, an expression cassette G8 comprising the ACS2 gene, an expression cassette G9 comprising the AmOGD1, AmCYP88D25, AmCYP88D7 and AmCYP71D756 genes, and an expression cassette G10 comprising the ERG1, ERG9, EGFP and AmCAS1 genes;

[0010] (2) The expression cassette G1 was transferred into the Saccharomyces cerevisiae strain to construct the CycZ01 strain, the expression cassette G2 was transferred into the CycZ01 strain to construct the CycZ02 strain, the expression cassette G3 was transferred into the CycZ02 strain to construct the CycZ03 strain, the expression cassette G4 was transferred into the CycZ03 strain to construct the CycZ04 strain, the expression cassette G5 was transferred into the CycZ04 strain to construct the CycZ05 strain, the expression cassette G6 was transferred into the CycZ05 strain to construct the CycZ07 strain, the expression cassette G7 was transferred into the CycZ07 strain to construct the CycZ12 strain, the expression cassette G8 was transferred into the CycZ12 strain to construct the CycZ20 strain, the expression cassette G9 was transferred into the CycZ20 strain to construct the engineered strain CycZ23, and the expression cassette G10 was transferred into the strain CycZ23 to construct the engineered strain CAG-1.

[0011] Preferably, the above-mentioned genes encode acetoacetyl CoA thiolase (ERG10), HMG-CoA synthase (3-hydroxy-3-methylglutaryl-CoA synthase, ERG13), truncated expression of HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase, tHMG1), mevalonate kinase (ERG12), phosphomevalonate kinase (ERG8), 5-pyrophosphomevalonate decarboxylase (ERG19), isoprene pyrophosphate isomerase (IDI1), ERG20 F96C The mutant, farnesyl diphosphate farnesyltransferase (ERG9), squalene epoxidase (ERG1), and acetyl-CoA synthetase (ACS2) genes are all derived from Saccharomyces cerevisiae.

[0012] Preferably, the above-mentioned gene encoding cycloartenol synthase CAS is derived from the AmCAS1 gene of Astragalus membranaceus, and the codon-optimized AmCAS1 gene has an artificially synthesized sequence as shown in SEQ ID No. 1; the genes encoding cytochrome P450 enzymes (CYP450s) are derived from the AmCYP88D25, AmCYP88D7, and AmCYP71D756 genes of Astragalus membranaceus, and the codon-optimized AmCYP88D25, AmCYP88D7, and AmCYP71D756 genes have artificially synthesized sequences as shown in SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively; the gene encoding oxoglutarate / iron-dependent dioxygenase 1 is derived from the AmOGD1 gene of Astragalus membranaceus, and the codon-optimized AmOGD1 gene has an artificially synthesized sequence as shown in SEQ ID No. 5; the codon-optimized EGFP gene has an artificially synthesized sequence as shown in SEQ ID No. 6.

[0013] Preferably, the promoter of the expression cassette in step (1) is the promoter P of Saccharomyces cerevisiae. TEF1 、P PGK1 、P TDH3 、PENO2 、P INO1 and P TPI1 Any one of the terminator T of Saccharomyces cerevisiae CYC1、 T ADH1 、T HSP26 、T TPS1 and T CPS1 Any one of .

[0014] Preferably, the integration site of the expression cassette in the above step (1) is any one of the X-2 site, X-3 site, X-4 site, XI-1 site, XI-2 site, XII-1 site, XII-5 site, XII-4 site, XI-5 site and delta site of Saccharomyces cerevisiae.

[0015] Preferably, the Saccharomyces cerevisiae strain in the above step (2) is Saccharomyces cerevisiae IMX581; the expression cassette is transferred into the Saccharomyces cerevisiae cells by CRISPR-Cas9 technology and integrated into the Saccharomyces cerevisiae genome by homologous recombination.

[0016] The acetoacetyl CoA thiolase expression cassette, isoprene pyrophosphate isomerase expression cassette, ERG20 F96C The mutant co-expression squalene epoxidase expression cassette, the truncated expression HMG-CoA reductase expression cassette, the squalene epoxidase co-expression cycloartenol synthase expression cassette, the acetyl-CoA synthetase 2 expression cassette, the HMG-CoA synthase, the mevalonate kinase, the farnesyl diphosphate farnesyltransferase, the 5-pyrophosphomevalonate decarboxylase co-expression phosphomevalonate kinase expression cassette, the cycloartenol synthase expression cassette, the acetyl-CoA synthetase expression cassette, and the cytochrome CYP450 enzyme co-expression oxoglutarate / iron-dependent dioxygenase 1 expression cassette are introduced into the chassis cerevisiae cells and recombined into the cerevisiae genome.

[0017] Preferably, the recombinant plasmid used for the above-mentioned transfer is any one of pMEL10-X-2, pMEL10-X-3, pMEL10-X-4, pMEL10-XI-1, pMEL10-XI-2, pMEL10-XII-1, pMEL10-XII-5, pMEL10-XII-4, pMEL10-XI-5 and pMEL10-delta.

[0018] In the second aspect, the engineered bacteria constructed by the above construction method are the engineered strain CycZ23 or the engineered strain CAG-1.

[0019] In a third aspect, the application of the above-mentioned engineered bacteria in the biosynthesis of cycloastragenol comprises the following steps: inoculating the seed liquid of the engineered strain CycZ23 or the engineered strain CAG-1 into YPD fermentation medium, fermenting at 28-30°C and 200-220 rpm in a shaker for 120-144 hours, crushing the cells after the fermentation, and extracting with ethyl acetate to obtain cycloastragenol.

[0020] Preferably, the OD value of the seed solution is 0.06-0.1.

[0021] The YPD fermentation medium has a formula of 22 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, 8 g / L ammonium sulfate, 3 g / L KH2PO4, 1.5 g / L MgSO4·7H2O, 10 mL / L trace element mixture, 2 mg / L thiamine, and a pH of 6.0.

[0022] The present invention has the following beneficial effects:

[0023] The present invention provides a method for constructing an engineered bacterium for de novo biosynthesis of cycloastragenol, wherein the mevalonate (MVA) pathway is reconstructed in the peroxisome of a Saccharomyces cerevisiae strain, and a strong promoter is used to overexpress key enzyme genes of the MVA pathway: acetoacetyl CoA thiolase (ERG10), HMG-CoA synthase (ERG13), truncated expression of HMG-CoA reductase (tHMG1), mevalonate kinase (ERG12), phosphomevalonate kinase (ERG8), 5-pyrophosphomevalonate decarboxylase (ERG19), isoprenyl pyrophosphate isomerase (IDI1), etc., to enhance the synthesis of terpenoid precursors; and ERG20 is expressed. F96C The researchers used strategies such as mutants, overexpression of farnesyl diphosphate farnesyltransferase (ERG9), and squalene epoxidase (ERG1) to increase the content of triterpenoid precursors. Furthermore, they introduced heterologous cycloartenol synthase, cytochrome P450 enzymes, and oxoglutarate / iron-dependent dioxygenase 1. Key enzyme genes, serving as expression elements for cycloastragenol, were introduced into chassis Saccharomyces cerevisiae cells via CRISPR-Cas9 technology and integrated into the Saccharomyces cerevisiae genome by homologous recombination. This led to the construction of engineered Saccharomyces cerevisiae strains CycZ23 and CAG-1, which efficiently synthesize cycloastragenol, achieving de novo synthesis of plant triterpenoid natural products. The maximum titer of cycloastragenol produced was 236 mg / L. The synthesis method of the present invention is economical, efficient, environmentally friendly, and simple to operate, possessing significant application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 Diagram of the synthesis pathway of cycloastragenol in Saccharomyces cerevisiae.

[0026] Figure 2 Figure 3 is a diagram of the expression cassette structure of the overexpressed gene.

[0027] Figure 3 This is the HPLC chromatogram of cycloastragenol synthesized by genetically engineered Saccharomyces cerevisiae.

[0028] Figure 4 HRMS mass spectrum of cycloastragenol synthesized by genetically engineered Saccharomyces cerevisiae.

[0029] Figure 5 This is a statistical analysis chart of the synthesis of cycloastragenol by genetically engineered Saccharomyces cerevisiae. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0032] The present invention utilizes a cell compartmentalization strategy to reconstruct the mevalonate (MVA) pathway in the peroxisome of a Saccharomyces cerevisiae strain, and uses a strong promoter to overexpress key MVA pathway enzyme genes, such as acetoacetyl-CoA thiolase (ERG10), HMG-CoA synthase (3-hydroxy-3-methylglutaryl-CoA synthase, ERG13), truncated expression of HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase, tHMG1), mevalonate kinase (ERG12), phosphomevalonate kinase (ERG8), 5-pyrophosphomevalonate decarboxylase (ERG19), and isoprenyl pyrophosphate isomerase (IDI1), to enhance the synthesis of terpenoid precursors; and to express ERG20 F96CThe content of triterpenoid precursors was increased by strategies such as mutants, overexpression of farnesyl diphosphate farnesyltransferase (ERG9) and squalene epoxidase (ERG1); and heterologous cycloartenol synthase, cytochrome P450 enzyme, oxoglutarate / iron-dependent dioxygenase 1, etc. were introduced. These key enzyme genes were used as expression elements of cycloastragenol to construct genetically engineered yeast Saccharomyces cerevisiae to achieve heterologous synthesis of cycloastragenol. The synthetic pathway of cycloastragenol is shown in Figure 1 .

[0033] 1. Experimental instruments:

[0034] Clean bench, electronic balance, centrifuge, constant temperature shaker, constant temperature incubator, water bath, cell disruptor, PCR instrument, gel imager, nucleic acid quantifier.

[0035] 2. Strains and vectors:

[0036] The Saccharomyces cerevisiae strain IMX581 and the recombinant plasmids pMEL10-X-2, pMEL10-X-3, pMEL10-X-4, pMEL10-XI-1, pMEL10-XI-2, pMEL10-XII-1, pMEL10-XII-5, pMEL10-XII-4, and pMEL10-XI-5 were all preserved in the laboratory.

[0037] Saccharomyces cerevisiae strain IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3); strain IMX581 is a strain in which the Cas9 gene is inserted into the yeast mutant strain CEN.PK 113-5D (purchased from the EUROSCARF strain bank (Frankfurt, Germany)) to establish the CRISPR / Cas9 gene editing system.

[0038] 3. Test kit:

[0039] High-fidelity enzymes, plasmid extraction kits, and gel recovery kits were purchased from Nanjing Novozymes Biotechnology Co., Ltd., recombinant enzymes were purchased from Anhui Tolo Biotechnology Co., Ltd., and yeast transformation kits were purchased from Coolaber Technology Co., Ltd.

[0040] 4. Culture medium and reagent configuration:

[0041] (1) YPD medium: 10 g / L yeast extract powder, 20 g / L peptone, 22 g / L glucose. For solid culture medium, 20 g / L agar powder should be added before sterilizing the liquid culture medium. The volume should be adjusted to 1 L with ddH2O and sterilized at high temperature.

[0042] (2) YNB medium: 6.7 g / L amino-free yeast nitrogen source; 22 g / L glucose; 0.77 g / L amino acid-deficient medium (URA); solid medium should be supplemented with 20 g / L agar powder before sterilizing the liquid medium, and the volume should be adjusted to 1 L with ddH2O, and sterilized at high temperature.

[0043] (3) 50×TAE agarose gel electrophoresis buffer: Tris 121 g, glacial acetic acid 28.6 mL, 0.5 mol / L EDTA (pH 8.0) 50 mL, distilled water to 500 mL, stored at room temperature;

[0044] (4) 0.2M LiAc + 1% SDS: Take 1.02g lithium acetate and 0.5g sodium dodecyl sulfate (SDS), dilute to 50mL with ddH2O, stir evenly with a magnetic stirrer, and sterilize at high temperature for 20min.

[0045] (5) LB medium preparation: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, dilute to 1 L with ddH2O; solid culture medium needs to be supplemented with 20 g / L agar powder before sterilization and sterilized at 121°C for 20 min.

[0046] Example 1: Construction of gene expression cassette

[0047] 1. Amplification of genetic elements

[0048] The AmCAS1 gene encoding cycloartenol synthase CAS is derived from Astragalus membranaceus, and the codon-optimized AmCAS1 gene and the artificially synthesized sequence are shown in SEQ ID No. 1; the AmCYP88D25, AmCYP88D7, and AmCYP71D756 genes encoding cytochrome P450 enzymes (CYP450s) are derived from Astragalus membranaceus, and the codon-optimized AmCYP88D25, AmCYP88D7, and AmCYP71D756 genes and the artificially synthesized sequences are shown in SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively; the AmOGD1 gene encoding oxoglutarate / iron-dependent dioxygenase 1 is derived from Astragalus membranaceus, and the codon-optimized AmOGD1 gene and the artificially synthesized sequence are shown in SEQ ID No. 5.

[0049] Using the genome of IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3) as a template, primers were designed to amplify the genes encoding acetoacetyl-CoA thiolase (ERG10), HMG-CoA synthase (3-hydroxy-3-methylglutaryl-CoA synthase, ERG13), truncated HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase, tHMG1), mevalonate kinase (ERG12), phosphomevalonate kinase (ERG8), 5-pyrophosphomevalonate decarboxylase (ERG19), isoprenyl pyrophosphate isomerase (IDI1), and ERG20. F96C mutants, farnesyl diphosphate farnesyltransferase (ERG9), squalene epoxidase (ERG1), acetyl-CoA synthetase (ACS2) and other genes.

[0050] Using the IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3) genome as a template, primers were designed to amplify the promoter P TEF1 、P PGK1 、P TDH3 、P ENO2 、P INO1 、P TPI1 and terminator T CYC1、 T ADH1 、T HSP26 、T TPS1 、T CPS1 .

[0051] Using the IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3) genome as a template, primers were designed to amplify the upstream and downstream homology arms of chromosome X-2, X-3, X-4, XI-1, XI-2, XII-1, XII-5, XII-4, XI-5 and delta sites.

[0052] 2. Construction of gene expression cassette: The structure of gene expression cassette is as follows Figure 2 As shown, the steps are as follows:

[0053] (1) Construction of gene expression cassette G1

[0054] The gene expression cassette G1 is composed of the upstream homology arms, gene expression elements, and downstream homology arms at the X-2 locus on chromosome 1 in IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3). Using the IMX581 yeast genome as a template, the upstream and downstream homology arms X-2 / UP and X-2 / DW of the expression cassette G1 were amplified using primers X-2 / UP-F / R and X-2 / DW-F / R. The promoter P of the expression cassette G1 was amplified using the IMX581 yeast genome as a template using primers TDH3p-F and TDH3p-R. TDH3 Using the IMX581 yeast genome as a template, CYC1t-F and CYC1t-R as primers, amplify the terminator T of the expression cassette G1. CYC1 The ERG10 gene was amplified using the IMX581 yeast genome as a template and ERG10-F and ERG10-R as primers (Table 1). The FastPure Gel DNA Extraction Mini Kit (purchased from Nanjing Novozyme Technology Co., Ltd.) was used to purify and recover the gene fragments. PrimeSTAR Max DNA polymerase (Bao Ri Yi Biotechnology (Beijing) Co., Ltd.) was used to splice the gene fragments, and the spliced gene expression cassette G1 (X-2 / UP-P TDH3 -ERG10-T CYC1 -DW).

[0055] Table 1 Primer sequences used to construct expression cassette G1

[0056]

[0057] (2) Construction of gene expression cassette G2

[0058] The gene expression cassette G2 is composed of the upstream homology arms, gene expression elements, and downstream homology arms at the X-3 locus on chromosome 1 in IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3). Using the IMX581 yeast genome as a template, the upstream and downstream homology arms X-3 / UP and X-3 / DW of the expression cassette G2 were amplified using primers X-3 / UP-F / R and X-3 / DW-F / R. The promoter P of the expression cassette G2 was amplified using the IMX581 yeast genome as a template using primers ENO2p-F and ENO2p-R. ENO2 Using the IMX581 yeast genome as a template, ADH1t-F and ADH1t-R as primers were used to amplify the terminator T of the expression cassette G2. ADH1The IDI1 gene was amplified using the IMX581 yeast genome as a template and primers IDI1-F and IDI1-R (Table 2). The Fast PureGel DNA Extraction Mini Kit (purchased from Nanjing Novozyme Technology Co., Ltd.) was used to purify and recover the gene fragments. PrimeSTAR Max DNA polymerase (Baori Biotechnology (Beijing) Co., Ltd.) was used to splice the gene fragments, and the spliced gene expression cassette G2 (X-3 / UP-P) was recovered by agarose gel electrophoresis. ENO2 -IDI1-T ADH1 -DW).

[0059] Table 2 Primer sequences used to construct expression cassette G2

[0060]

[0061] (3) Construction of gene expression cassette G3

[0062] The gene expression cassette G3 is composed of the upstream homology arms, gene expression elements, and downstream homology arms at the X-4 locus on chromosome 1 in IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3). Using the IMX581 yeast genome as a template, the upstream and downstream homology arms X-4 / UP and X-4 / DW of the expression cassette G3 were amplified using primers X-4 / UP-F / R and X-4 / DW-F / R. The promoter P of the expression cassette G3 was amplified using the IMX581 yeast genome as a template using primers TDH3p-F, TDH3p-R, ENO2p-F, and ENO2p-R. TDH3 、P ENO2 Using the IMX581 yeast genome as a template, CYC1t-F, CYC1t-R, ADH1t-F, and ADH1t-R as primers, amplify the terminator T of the expression cassette G3. CYC1 、T ADH1 Using the IMX581 yeast genome as a template, ERG20-1-F, ERG20-1-R, ERG20-2-F, ERG20-2-R, ERG1-F, ERG1-R were used as primers to amplify ERG20 and ERG1 genes (Table 3). Each gene fragment was purified and recovered using FastPure Gel DNA Extraction Mini Kit (purchased from Nanjing Novozyme Technology Co., Ltd.). Each gene fragment was spliced using PrimeSTAR Max DNA polymerase (Bao Ri Yi Biotechnology (Beijing) Co., Ltd.), and the spliced gene expression cassette G3 (X-4 / UP-P TDH3 -ERG20 F96C -T CYC1 -PENO2 -ERG1-T ADH1 -DW).

[0063] Table 3 Primer sequences used to construct expression cassette G3

[0064]

[0065]

[0066] (4) Construction of gene expression cassette G4

[0067] The gene expression cassette G4 is composed of the upstream homology arms, gene expression elements, and downstream homology arms at the XI-2 locus on chromosome 1 in IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3). Using the IMX581 yeast genome as a template, the upstream and downstream homology arms XI-2 / UP and XI-2 / DW of the expression cassette G4 were amplified using primers XI-2 / UP-F / R and XI-2 / DW-F / R. The promoter P of the expression cassette G4 was amplified using the IMX581 yeast genome as a template using primers pTEF1-F and pTEF1-R. TEF1 Using the IMX581 yeast genome as a template, CYC1t-F and CYC1t-R as primers, amplify the terminator T of the expression cassette G4. CYC1 The tHMG1 gene was amplified using the IMX581 yeast genome as a template and tHMG1-F and tHMG1-R as primers (Table 4). The FastPure Gel DNA Extraction Mini Kit (purchased from Nanjing Novozyme Technology Co., Ltd.) was used to purify and recover the gene fragments. PrimeSTAR Max DNA polymerase (Baori Biotechnology (Beijing) Co., Ltd.) was used to splice the gene fragments, and the spliced gene expression cassette G4 (XI-2 / UP-P) was recovered by agarose gel electrophoresis. TEF1 -tHMG1-T CYC1 -DW).

[0068] Table 4 Primer sequences used to construct expression cassette G4

[0069]

[0070] (5) Construction of gene expression cassette G5

[0071] The gene expression cassette G5 is composed of the upstream homology arms, gene expression elements, and downstream homology arms at the XI-1 locus on the chromosome of IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3). Using the IMX581 yeast genome as a template, the upstream and downstream homology arms XI-1 / UP and XI-1 / DW of the expression cassette G5 were amplified using primers XI-1 / UP-F / R and XI-1 / DW-F / R; the promoter P of the expression cassette G5 was amplified using the IMX581 yeast genome as a template using primers pTEF1-F and pTEF1-R. TEF1 Using the IMX581 yeast genome as a template, CYC1t-F and CYC1t-R as primers, amplify the terminator T of the expression cassette G5. CYC1 ; Using IMX581 yeast genome as template, using ERG13-F, ERG13-R, ERG12-F, ERG12-R, ERG19-F, ERG19-R, ERG9-F, ERG9-R, ERG8-F, ERG8-R as primer amplification ERG13, ERG12, ERG19, ERG9, ERG8 gene; With reference to AmCAS1 (OM913798) gene in Astragalus membranaceus in NCBI, artificial sequence (SEQ ID No.1) was synthesized after codon optimization, using CAS1-F, CAS1-R as primer amplification AmCAS1 gene (Table 5). Use Fast Pure Gel DNA Extraction Mini Kit (purchased from Nanjing Noviyon Technology Co., Ltd.) to purify and reclaim each gene fragment. Use PrimeSTAR Max DNA polymerase (Bao Riyi Biotechnology (Beijing) Co., Ltd.) to carry out the splicing of each gene fragment, and agarose gel electrophoresis gel reclaims the spliced gene expression cassette G5 (XI-1 / UP-P TEF1 -AmCAS1-T CYC1 -DW).

[0072] Table 5 Primer sequences used to construct expression cassette G5

[0073]

[0074] (6) Construction of gene expression cassette G6

[0075] The gene expression cassette G6 is composed of upstream homology arms, gene expression elements, and downstream homology arms at the chromosome XII-1 site in IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3). Using the IMX581 yeast genome as a template, the upstream and downstream homology arms XII-1 / UP and XII-1 / DW of the expression cassette G6 were amplified using primers XII-1 / UP-F / R and XII-1 / DW-F / R. The promoter P of the expression cassette G6 was amplified using the IMX581 yeast genome as a template using primers TDH3p-F, TDH3p-R, pENO2-F, pENO2-R, pTEF1-F, pTEF1-R, pTPI1-F, pTPI1-R, INO1p-F, and INO1p-R. TDH3 、P ENO2 、P TEF1 、P TPI1 、P INO1 Using the IMX581 yeast genome as a template, CYC1t-F, CYC1t-R, tHSP26-F, tHSP26-R, ADH1t-F, ADH1t-R, TPS1t-F, TPS1t-R, CPS1t-F, and CPS1t-R were used as primers to amplify the terminator T of the expression cassette G6. CYC1 、T HSP26 、T ADH1 、T TPS1 、T CPS1 ; Using IMX581 yeast genome as template, ERG13-F, ERG13-R, ERG12-F, ERG12-R, ERG19-F, ERG19-R, ERG9-F, ERG9-R, ERG8-F, ERG8-R were primers for amplification of ERG13, ERG12, ERG19, ERG9, and ERG8 genes (Table 6). Each gene fragment was purified and recovered using Fast Pure Gel DNA Extraction Mini Kit (purchased from Nanjing NovaZan Technology Co., Ltd.). Each gene fragment was spliced using PrimeSTAR Max DNA polymerase (Bao Ri Yi Biotechnology (Beijing) Co., Ltd.), and the spliced gene expression cassette G6 (XII-1 / UP-P TDH3 -ERG13-T CYC1 -T HSP26 -ERG12-P ENO2 -P TEF1 -ERG19-T ADH1 -T TPS1 -ERG9-P TPI1 -P INO1 -ERG8-T CPS1 -DW).

[0076] Table 6 Primer sequences used to construct expression cassette G6

[0077]

[0078] (7) Construction of gene expression cassette G7

[0079] The gene expression cassette G7 is composed of the upstream homology arms, gene expression elements, and downstream homology arms at the chromosome XII-5 site in IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3). Using the IMX581 yeast genome as a template, the upstream and downstream homology arms XII-5 / UP and XII-5 / DW of the expression cassette G7 were amplified using primers XII-5 / UP-F / R and XII-5 / DW-F / R. The promoter P of the expression cassette G7 was amplified using the IMX581 yeast genome as a template using primers pTPI1-F, pTPI1-R, PGK1p-F, and PGK1p-R. TPI1 、P PGK1 Using the IMX581 yeast genome as a template, tHSP26-F, tHSP26-R, ADH1t-F, and ADH1t-R as primers were used to amplify the terminator T of the expression cassette G7. HSP26 、T ADH1 ; With IMX581 yeast genome as template, with ERG1-F, ERG1-R as primer amplification ERG1 gene; With reference to AmCAS1 gene (OM913798) in Astragalus membranaceus in NCBI, after codon optimization, synthesize artificial sequence (SEQ ID No.1), with AmCAS1-F, AmCAS1-R as primer amplification AmCAS1 gene (Table 7). Use Fast Pure Gel DNA Extraction Mini Kit (purchased from Nanjing Noviyon Technology Co., Ltd.) to purify and reclaim each gene fragment. Use PrimeSTAR Max DNA polymerase (Bao Riyi Biotechnology (Beijing) Co., Ltd.) to carry out the splicing of each gene fragment, agarose gel electrophoresis gel reclaims the spliced gene expression cassette G7 (XII-5 / UP-T HSP26 -ERG1-P TPI1 -P PGK1 -AmCAS1-T ADH1 -DW).

[0080] Table 7 Primer sequences used to construct expression cassette G7

[0081]

[0082] (8) Construction of gene expression cassette G8

[0083] The gene expression cassette G8 is composed of upstream homology arms, gene expression elements, and downstream homology arms at the XII-4 locus on chromosome IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3). Using the IMX581 yeast genome as a template, the upstream and downstream homology arms XII-4 / UP and XII-4 / DW of the expression cassette G8 were amplified using primers XII-4 / UP-F / R and XII-4 / DW-F / R. The promoter P of the expression cassette G8 was amplified using the IMX581 yeast genome as a template using primers TDH3p-F and TDH3p-R. TDH3 Using the IMX581 yeast genome as a template, tHSP26-F and tHSP26-R as primers were used to amplify the terminator T of the expression cassette G8. HSP26 ; Using the IMX581 yeast genome as a template, ACS2-F and ACS2-R were used as primers to amplify the ACS2 gene (Table 8). FastPure Gel DNA Extraction Mini Kit (purchased from Nanjing Novozyme Technology Co., Ltd.) was used to purify and recover each gene fragment. PrimeSTAR Max DNA polymerase (Bao Ri Yi Biotechnology (Beijing) Co., Ltd.) was used to splice each gene fragment, and agarose gel electrophoresis was used to recover the spliced gene expression cassette G8 (XII-4 / UP-P TDH3 -ACS2-T HSP26 -DW).

[0084] Table 8 Primer sequences used to construct expression cassette G8

[0085]

[0086] (9) Construction of gene expression cassette G9

[0087] The gene expression cassette G9 is composed of upstream homology arms, gene expression elements, and downstream homology arms at the XI-5 locus on chromosome IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3). Using the IMX581 yeast genome as a template, the upstream and downstream homology arms XI-5 / UP and XI-5 / DW of the expression cassette G9 were amplified using primers XI-5 / UP-F / R and XI-5 / DW-F / R. The promoter P of the expression cassette G9 was amplified using the IMX581 yeast genome as a template using primers PGK1p-F, PGK1p-R, TDH3p-F, TDH3p-R, pTPI1-F, pTPI1-R, pTEF1-F, and pTEF1-R. PGK1 、P TDH3 、P TPI1 、P TEF1Using the IMX581 yeast genome as a template, ADH1t-F, ADH1t-R, tHSP26-F, tHSP26-R, CPS1t-F, CPS1t-R, TPS1t-F, TPS1t-R were used as primers to amplify the terminator T of the expression cassette G9. ADH1 、T HSP26 、T CPS1 、T TPS1 With reference to the AmOGD1 (OQ365044), AmCYP88D25 (OQ365041), AmCYP88D7 (OQ365042), and AmCYP71D756 (OQ365043) genes in Astragalus membranaceus in NCBI, artificial sequences (shown as SEQ ID No.5, SEQ ID No.2, SEQ ID No.3, and SEQ ID No.4, respectively) were synthesized after codon optimization. AmOGD1-F / R, AmCYP88D25-F / R, AmCYP88D7-F / R, and AmCYP71D756-F / R were used as primers to amplify the AmOGD1, AmCYP88D25, AmCYP88D7, and AmCYP71D756 genes (Table 9). Each gene fragment was purified and recovered using Fast Pure Gel DNA Extraction Mini Kit (purchased from Nanjing Novigene Technology Co., Ltd.). PrimeSTARMax DNA polymerase (Bao Ri Yi Biotechnology (Beijing) Co., Ltd.) was used to splice the gene fragments, and the spliced gene expression cassette G9 (XI-5 / UP-P PGK1 -AmOGD1-T ADH1 -T HSP26 -AmCYP88D25-P TDH3 -P TPI1 -AmCYP88D7-T CPS1 -T TPS1 -AmCYP71D756-P TEF1 -DW).

[0088] Table 9 Primer sequences used to construct expression cassette G9

[0089]

[0090]

[0091] Example 2: Construction of genetically engineered yeast Saccharomyces cerevisiae

[0092] 1. Construction of genetically engineered Saccharomyces cerevisiae strain CycZ01: Coolaber's Super Yeast Transformation Kit was used to transfer the laboratory-stored recombinant plasmid pMEL10-X-2 and gene expression cassette G1 into the IMX581 strain for homologous recombination to construct the CycZ01 strain.

[0093] The specific experimental steps are as follows:

[0094] (1) The IMX581 strain was inoculated into YPD liquid medium and cultured overnight to prepare competent cells. The transformation premix was prepared according to the instructions of Coolaber's Super Yeast Transformation Kit. The recombinant plasmid pMEL10-X-2 and gene expression cassette G1 were transformed into IMX581 competent cells. The cells were heat-shocked at 30°C and 42°C for 30 min respectively, centrifuged and the supernatant was discarded. The cells were resuspended in sterile H2O and plated on YNB plates to wait for the growth of transformants.

[0095] (2) Select the positive clones on the YNB plate and transfer them to YNB liquid medium. The positive clones identified by PCR were transferred to YPD liquid medium and cultured overnight for 3-5 generations. The bacterial liquid was streaked onto YPD plates and cultured in a 30℃ incubator. Single colonies were streaked onto YNB and YPD plates in turn. After culture in a 30℃ incubator, colonies that grew on the YPD plate but not on the YNB plate were picked and transferred to YPD liquid medium. After culture in a 30℃ shaker, the strain was named CycZ01.

[0096] 2. Construction of genetically engineered yeast strain CycZ02

[0097] The recombinant plasmid pMEL10-X-3 and gene expression cassette G2 stored in the laboratory were transferred into the CycZ01 strain using Coolaber's Super Yeast Transformation Kit, and homologous recombination was performed to construct the CycZ02 strain.

[0098] The specific experimental steps are as follows:

[0099] (1) The CycZ01 strain was inoculated into YPD liquid medium and cultured overnight to prepare competent cells. The transformation premix was prepared according to the instructions of Coolaber's Super Yeast Transformation Kit. The recombinant plasmid pMEL10-X-3 and gene expression cassette G2 were transformed into CycZ01 competent cells. The cells were heat-shocked at 30°C and 42°C for 30 min respectively, centrifuged and the supernatant was discarded. The cells were resuspended in sterile H2O and plated on YNB plates to wait for the growth of transformants.

[0100] (2) Select positive clones on the YNB plate and place them in YNB liquid medium. The positive clones identified by PCR were transferred to YPD liquid medium and cultured overnight for 3-5 generations. The bacterial liquid was streaked onto YPD plates and cultured in a 30°C incubator. Single colonies were streaked onto YNB and YPD plates in turn. After culture in a 30°C incubator, colonies that grew on the YPD plate but not on the YNB plate were picked and placed in YPD liquid medium. After culture in a 30°C shaker, the strain was preserved and named CycZ02.

[0101] 3. Construction of genetically engineered yeast strain CycZ03

[0102] The recombinant plasmid pMEL10-X-4 and gene expression cassette G3 stored in the laboratory were transferred into the CycZ02 strain using Coolaber's Super Yeast Transformation Kit, and homologous recombination was performed to construct the CycZ03 strain.

[0103] The specific experimental steps are as follows:

[0104] (1) The CycZ02 strain was inoculated into YPD liquid medium and cultured overnight to prepare competent cells. The transformation premix was prepared according to the instructions of Coolaber's Super Yeast Transformation Kit. The recombinant plasmid pMEL10-X-4 and gene expression cassette G3 were transformed into CycZ02 competent cells. The cells were heat-shocked at 30°C and 42°C for 30 min respectively, centrifuged and the supernatant was discarded. The cells were resuspended in sterile H2O and plated on YNB plates to wait for the growth of transformants.

[0105] (2) Select positive clones on the YNB plate and place them in YNB liquid medium. The positive clones identified by PCR were transferred to YPD liquid medium and cultured overnight for 3-5 generations. The bacterial liquid was streaked onto YPD plates and cultured in a 30°C incubator. Single colonies were streaked onto YNB and YPD plates in turn. After culture in a 30°C incubator, colonies that grew on the YPD plate but not on the YNB plate were picked and placed in YPD liquid medium. After culture in a 30°C shaker, the strain was preserved and named CycZ03.

[0106] 4. Construction of genetically engineered yeast strain CycZ04

[0107] The recombinant plasmid pMEL10-XI-2 and gene expression cassette G4 stored in the laboratory were transferred into the CycZ03 strain using Coolaber's Super Yeast Transformation Kit, and homologous recombination was performed to construct the CycZ04 strain.

[0108] The specific experimental steps are as follows:

[0109] (1) The CycZ03 strain was inoculated into YPD liquid medium and cultured overnight to prepare competent cells. The transformation premix was prepared according to the instructions of Coolaber's Super Yeast Transformation Kit. The recombinant plasmid pMEL10-XI-2 and gene expression cassette G4 were transformed into CycZ03 competent cells. The cells were heat-shocked at 30°C and 42°C for 30 min respectively, centrifuged and the supernatant was discarded. The cells were resuspended in sterile H2O and plated on YNB plates to wait for the growth of transformants.

[0110] (2) Select the positive clones on the YNB plate and transfer them to YNB liquid medium. The positive clones identified by PCR were transferred to YPD liquid medium and cultured overnight for 3-5 generations. The bacterial liquid was streaked onto YPD plates and cultured in a 30℃ incubator. Single colonies were streaked onto YNB and YPD plates in turn. After culture in a 30℃ incubator, colonies that grew on the YPD plate but not on the YNB plate were picked and transferred to YPD liquid medium. After culture at 30℃ in a shaker, the strain was preserved and named CycZ04.

[0111] 5. Construction of genetically engineered yeast strain CycZ05

[0112] The recombinant plasmid pMEL10-XI-1 and gene expression cassette G5 stored in the laboratory were transferred into the CycZ04 strain using Coolaber's Super Yeast Transformation Kit, and homologous recombination was performed to construct the CycZ05 strain.

[0113] The specific experimental steps are as follows:

[0114] (1) The CycZ04 strain was inoculated into YPD liquid medium and cultured overnight to prepare competent cells. The transformation premix was prepared according to the instructions of Coolaber's Super Yeast Transformation Kit. The recombinant plasmid pMEL10-XI-1 and gene expression cassette G5 were transformed into CycZ04 competent cells. The cells were heat-shocked at 30°C and 42°C for 30 min respectively, centrifuged and the supernatant was discarded. The cells were resuspended in sterile H2O and plated on YNB plates to wait for the growth of transformants.

[0115] (2) Select positive clones on the YNB plate and place them in YNB liquid medium. The positive clones identified by PCR were transferred to YPD liquid medium and cultured overnight for 3-5 generations. The bacterial liquid was streaked onto YPD plates and cultured in a 30°C incubator. Single colonies were streaked onto YNB and YPD plates in turn. After culture in a 30°C incubator, colonies that grew on the YPD plate but not on the YNB plate were picked and placed in YPD liquid medium. After culture in a 30°C shaker, the strain was preserved and named CycZ05.

[0116] 6. Construction of genetically engineered yeast strain CycZ07

[0117] The recombinant plasmid pMEL10-XII-1 and gene expression cassette G6 stored in the laboratory were transferred into the CycZ05 strain using Coolaber's Super Yeast Transformation Kit, and homologous recombination was performed to construct the CycZ07 strain.

[0118] The specific experimental steps are as follows:

[0119] (1) The CycZ05 strain was inoculated into YPD liquid medium and cultured overnight to prepare competent cells. The transformation premix was prepared according to the instructions of Coolaber's Super Yeast Transformation Kit. The recombinant plasmid pMEL10-XII-1 and gene expression cassette G6 were transformed into CycZ05 competent cells. The cells were heat-shocked at 30°C and 42°C for 30 min respectively, centrifuged and the supernatant was discarded. The cells were resuspended in sterile H2O and plated on YNB plates to wait for the growth of transformants.

[0120] (2) Select positive clones on the YNB plate and place them in YNB liquid medium. The positive clones identified by PCR were transferred to YPD liquid medium and cultured overnight for 3-5 generations. The bacterial liquid was streaked onto YPD plates and cultured in a 30°C incubator. Single colonies were streaked onto YNB and YPD plates in turn. After culture in a 30°C incubator, colonies that grew on the YPD plate but not on the YNB plate were picked and placed in YPD liquid medium. After culture in a 30°C shaker, the strain was preserved and named CycZ07.

[0121] 7. Construction of genetically engineered yeast strain CycZ12

[0122] The recombinant plasmid pMEL10-XII-5 and gene expression cassette G7 stored in the laboratory were transferred into the CycZ07 strain using Coolaber's Super Yeast Transformation Kit, and homologous recombination was performed to construct the CycZ12 strain.

[0123] The specific experimental steps are as follows:

[0124] (1) The CycZ07 strain was inoculated into YPD liquid medium and cultured overnight to prepare competent cells. The transformation premix was prepared according to the instructions of Coolaber's Super Yeast Transformation Kit. The recombinant plasmid pMEL10-XII-5 and gene expression cassette G7 were transformed into CycZ07 competent cells. The cells were heat-shocked at 30°C and 42°C for 30 min respectively, centrifuged and the supernatant was discarded. The cells were resuspended in sterile H2O and plated on YNB plates to wait for the growth of transformants.

[0125] (2) Select the positive clones on the YNB plate and transfer them to YNB liquid medium. The positive clones identified by PCR were transferred to YPD liquid medium and cultured overnight for 3-5 generations. The bacterial liquid was streaked on YPD plates and cultured in a 30℃ incubator. Single colonies were streaked on YNB and YPD plates in turn. After culture in a 30℃ incubator, colonies that grew on the YPD plate but not on the YNB plate were picked and transferred to YPD liquid medium. After culture in a 30℃ shaker, the strain was preserved and named CycZ12.

[0126] 8. Construction of genetically engineered yeast strain CycZ20

[0127] The recombinant plasmid pMEL10-XII-4 and gene expression cassette G8 stored in the laboratory were transferred into the CycZ12 strain using Coolaber's Super Yeast Transformation Kit, and homologous recombination was performed to construct the CycZ20 strain.

[0128] The specific experimental steps are as follows:

[0129] (1) The CycZ12 strain was inoculated into YPD liquid medium and cultured overnight to prepare competent cells. The transformation premix was prepared according to the instructions of Coolaber's Super Yeast Transformation Kit. The recombinant plasmid pMEL10-XII-4 and gene expression cassette G8 were transformed into CycZ12 competent cells. The cells were heat-shocked at 30°C and 42°C for 30 min respectively, centrifuged and the supernatant was discarded. The cells were resuspended in sterile H2O and plated on YNB plates to wait for the growth of transformants.

[0130] (2) Select the positive clones on the YNB plate and transfer them to YNB liquid medium. The positive clones identified by PCR were transferred to YPD liquid medium and cultured overnight for 3-5 generations. The bacterial liquid was streaked onto YPD plates and cultured in a 30℃ incubator. Single colonies were streaked onto YNB and YPD plates in turn. After culture in a 30℃ incubator, colonies that grew on the YPD plate but not on the YNB plate were picked and transferred to YPD liquid medium. After culture in a 30℃ shaker, the strain was named CycZ20.

[0131] 9. Construction of genetically engineered yeast strain CycZ23

[0132] The recombinant plasmid pMEL10-XI-5 and gene expression cassette G9 stored in the laboratory were transferred into the CycZ20 strain using Coolaber's Super Yeast Transformation Kit, and homologous recombination was performed to construct the CycZ23 strain.

[0133] The specific experimental steps are as follows:

[0134] (1) The CycZ20 strain was inoculated into YPD liquid medium and cultured overnight to prepare competent cells. The transformation premix was prepared according to the instructions of Coolaber's Super Yeast Transformation Kit. The recombinant plasmid pMEL10-XI-5 and gene expression cassette G9 were transformed into CycZ20 competent cells. The cells were heat-shocked at 30°C and 42°C for 30 min respectively, centrifuged and the supernatant was discarded. The cells were resuspended in sterile H2O and plated on YNB plates to wait for the growth of transformants.

[0135] (2) Select the positive clones on the YNB plate and transfer them to YNB liquid medium. The positive clones identified by PCR were transferred to YPD liquid medium and cultured overnight for 3-5 generations. The bacterial liquid was streaked onto YPD plates and cultured in a 30℃ incubator. Single colonies were streaked onto YNB and YPD plates in turn. After culture in a 30℃ incubator, colonies that grew on the YPD plate but not on the YNB plate were picked and transferred to YPD liquid medium. After culture in a 30℃ shaker, the strain was preserved and named CycZ23.

[0136] Example 3: Optimization of engineered yeast strains for de novo synthesis of cycloastragenol

[0137] The Saccharomyces cerevisiae delta locus is a highly repetitive region of the genome containing multiple repeat sequences. Inserting multiple copies of a gene into the delta locus can increase its expression. In metabolic engineering of Saccharomyces cerevisiae, multiple copies of a gene can be integrated into the delta locus to increase its expression, thereby increasing the content of the target metabolite.

[0138] 1. Design and construction of sgRNA vector targeting delta site

[0139] Using the pMEL10 plasmid as a template, primers 1-F / XR and XF / 1-R (Table 10) were used for amplification. The two fragments were purified and recovered using the FastPure GelDNA Extraction Mini Kit and recombined using 2xEzmax-SingleCloneMix Plus. The recombinant plasmid (pMEL10-delta) was transformed into E. coli TOP10 competent cells and plated onto LB plates containing kanamycin resistance. The cells were cultured in a 37°C incubator for 12-14 hours. A single colony was picked and plated onto LB liquid medium containing kanamycin resistance. After sequencing verification, the pMEL10-delta recombinant plasmid was extracted using a plasmid extraction kit for subsequent transformation.

[0140] 2. Construction of delta site expression cassette G10

[0141] The delta gene expression cassette G10 is composed of upstream homology arms, gene expression elements, and downstream homology arms at the delta locus on the chromosome of IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2HIS3). Using the IMX581 yeast genome as a template, delta1-F / delta1-R primers were used to amplify the upstream and downstream homology arms delta1 and delta2 of the expression cassette G10. Using the IMX581 yeast genome as a template, TDH3p-F, TDH3p-R, PGK1p-F, PGK1p-R, pTPI1-F, pTPI1-R, pTEF1-F, and pTEF1-R primers were used to amplify the promoter P of the expression cassette G10. TDH3 、P PGK1 、P TPI1 、P TEF1 Using the IMX581 yeast genome as a template, CYC1t-F, CYC1t-R, ADH1t-F, ADH1t-R, tHSP26-F, tHSP26-R, tGRE3-F, and tGRE3-R were used as primers to amplify the terminator T of the expression cassette G10. CYC1 、T ADH1 、T HSP 26 、T GRE3; With IMX581 yeast genome as template, with ERG1-F / R, ERG9-F / R as primer amplification expression cassette G10 ERG1, ERG9 gene; With reference to EGFP amino acid sequence (AAK08507.1) in NCBI, after codon optimization, artificially synthesize this sequence (SEQ ID No.6), with artificially synthesized sequence as template, EGFP-F / R is primer amplification EGFP gene; With reference to AmCAS1 gene in Astragalus membranaceus in NCBI, with AmCAS1-F / R as primer amplification AmCAS1 gene (table 10). Use Fast Pure Gel DNA Extraction Mini Kit (purchased from Nanjing Noviyan Technology Co., Ltd.) to purify and reclaim each gene fragment. Use PrimeSTAR Max DNA polymerase (Bao Ri Yi Biotechnology (Beijing) Co., Ltd.) to carry out the splicing of each gene fragment, agarose gel electrophoresis gel reclaims the spliced gene expression cassette G10 (delta1-P T DH3 -EGFP-T CYC1 -T HSP26 -ERG1-P TPI1 -P GPK1 -AmCAS1-T ADH1 -T GRE3 -ERG9-P TEF1 -delta2).

[0142] Table 10 Primer sequences used for delta sites

[0143]

[0144]

[0145] 3. Transformation of expression cassette G10 and fluorescence intensity detection

[0146] The recombinant plasmid pMEL10-delta and gene expression cassette G10 were transferred into the CycZ23 strain using Coolaber's Super Yeast Transformation Kit, and homologous recombination was performed to construct the CAG-1 strain.

[0147] The specific experimental steps are as follows:

[0148] (1) The CycZ23 strain was inoculated into YPD liquid medium and cultured overnight to prepare competent cells. The transformation premix was prepared according to the instructions of Coolaber's Super Yeast Transformation Kit. The recombinant plasmid pMEL10-delta and gene expression cassette G10 were transformed into CycZ23 competent cells. The cells were heat-shocked at 30°C and 42°C for 30 min respectively, centrifuged and the supernatant was discarded. The cells were resuspended in sterile H2O and plated on YNB plates to wait for the growth of transformants.

[0149] (2) All positive clones on the YNB plate were selected and cultured in YNB liquid medium overnight. After PCR verification, 1 mL of bacterial solution was transferred to a 2 mL sterile EP tube, centrifuged at 5000 rpm for 3 min, and the supernatant was discarded; the bacteria were resuspended in 2 mL ddH2O, mixed, and 200 μL of bacterial solution was transferred to a transparent 96-well plate using a microplate reader to measure its OD 600 At the same time, 200 μL of bacterial solution was taken into a black 96-well plate and its fluorescence intensity was measured using a microplate reader. After detection, the expression level of fluorescent protein = fluorescence signal / OD 600 The fluorescent protein expression intensity of the strain was calculated and the strain with high fluorescence intensity was named CAG-1.

[0150] (3) Transfer the CAG-1 bacterial suspension to YPD liquid medium and culture overnight for 3-5 generations. Streak the bacterial suspension onto a YPD plate and culture in a 30°C incubator. Pick a single colony and streak it onto a YNB plate and then a YPD plate. After culture in a 30°C incubator, pick a colony that grows on the YPD plate but not on the YNB plate and transfer it to YPD liquid medium. Culture in a shaking incubator at 30°C and preserve the seed.

[0151] Example 4: Application of genetically engineered yeast Saccharomyces cerevisiae in the production of cycloastragenol

[0152] 1. Fermentation and product extraction of genetically engineered Saccharomyces cerevisiae CAG-1

[0153] The initial chassis strain IMX581 and the yeast engineered strain CAG-1 constructed in Example 3 were fermented to synthesize cycloastragenol.

[0154] The specific method is as follows:

[0155] Fermentation medium formula: glucose 30 g / L, ammonium sulfate 8 g / L, yeast extract 10 g / L, KH2PO4 3 g / L, MgSO4·7H2O 1.5 g / L, trace element mixture 10 mL / L, thiamine 2 mg / L, adjust pH to 6.0, and sterilize at 121°C for 20 min.

[0156] The chassis strain IMX581 and the engineered strain CAG-1 were cultured overnight in YPD liquid medium at 30°C, 220 rpm in a shaker to obtain a fermentation seed broth. A certain amount of IMX581 and CAG-1 seed broth was added to 100 mL of YPD fermentation medium to an initial OD of 0.06-0.1 and fermented for 120 hours at 30°C, 220 rpm in a shaker. After fermentation, the broth was transferred to a 50 mL centrifuge tube, and the cells were disrupted by sonication. The broth was extracted three times with equal volumes of ethyl acetate. The combined extracts were concentrated and reconstituted in 200 μL of methanol.

[0157] 2. Qualitative and quantitative analysis of cycloastragenol

[0158] A series of cycloastragenol standard solutions (purchased from Merck KGaA, Darmstadt, Germany) were prepared, and the above extracts were filtered through a 0.22 μm filter membrane, and qualitative and quantitative analyses were performed using HPLC-UV.

[0159] The specific conditions are as follows:

[0160] Chromatographic column: C18 column; mobile phase: 45% A (water-0.1% formic acid), 55% B (acetonitrile); flow rate: 1 mL / min; column temperature: 30°C, detection wavelength: 203 nm, injection volume: 10 μL.

[0161] Figure 3 This is the HPLC chromatogram of cycloastragenol synthesized by genetically engineered yeast Saccharomyces cerevisiae. Figure 3 It can be seen that compared with the cycloastragenol standard, the target compound produced a characteristic peak in the CycZ23 chromatogram, and its retention time was consistent with that of the standard.

[0162] Figure 4 HRMS mass spectrum of cycloastragenol synthesized by genetically engineered yeast Saccharomyces cerevisiae. Figure 4 It can be seen that HRMS analysis showed that the characteristic ion peak of the target substance was detected at m / z 513.35, and the error between its accurate mass and the theoretical value was within the range of ±5ppm, indicating that the target compound was successfully detected.

[0163] Specific implementation method According to Example 4, laboratory shake flask fermentation was performed, and carbon source was added during the fermentation process to control the accumulation of ethanol. Figure 5 Statistical analysis of the synthesis of cycloastragenol by genetically engineered yeast Saccharomyces cerevisiae. Figure 5 It can be seen that after 120 hours of fermentation, the titer of cycloastragenol produced by the CAG-1 strain was 236 mg / L.

[0164] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing an engineered bacterium for de novo biosynthesis of cycloastragenol, characterized in that: The steps are: (1) Construct expression cassette G1 containing ERG10 gene, expression cassette G2 containing IDI1 gene, and expression cassette G3 containing ERG20 gene. F96C , an expression cassette G3 comprising the ERG1 gene, an expression cassette G4 comprising the tHMG1 gene, an expression cassette G5 comprising the AmCAS1 gene, an expression cassette G6 comprising the ERG13, ERG12, ERG19, ERG9 and ERG8 genes, an expression cassette G7 comprising the ERG1 and AmCAS1 genes, an expression cassette G8 comprising the ACS2 gene, an expression cassette G9 comprising the AmOGD1, AmCYP88D25, AmCYP88D7 and AmCYP71D756 genes, and an expression cassette G10 comprising the ERG1, ERG9, EGFP and AmCAS1 genes; (2) The expression cassette G1 was transferred into the Saccharomyces cerevisiae strain to construct the CycZ01 strain, the expression cassette G2 was transferred into the CycZ01 strain to construct the CycZ02 strain, the expression cassette G3 was transferred into the CycZ02 strain to construct the CycZ03 strain, the expression cassette G4 was transferred into the CycZ03 strain to construct the CycZ04 strain, the expression cassette G5 was transferred into the CycZ04 strain to construct the CycZ05 strain, the expression cassette G6 was transferred into the CycZ05 strain to construct the CycZ07 strain, the expression cassette G7 was transferred into the CycZ07 strain to construct the CycZ12 strain, the expression cassette G8 was transferred into the CycZ12 strain to construct the CycZ20 strain, the expression cassette G9 was transferred into the CycZ20 strain to construct the engineered strain CycZ23, and the expression cassette G10 was transferred into the strain CycZ23 to construct the engineered strain CAG-1.

2. The method for constructing an engineered bacterium for de novo biosynthesis of cycloastragenol according to claim 1, characterized in that: The ERG10 gene, IDI1 gene, ERG20 F96C , ERG1 gene, tHMG1 gene, ERG13 gene, ERG12 gene, ERG19 gene, ERG9 gene, ERG8 gene and ACS2 gene are all derived from Saccharomyces cerevisiae; AmCAS1 gene, AmOGD1 gene, AmCYP88D25 gene, AmCYP88D7 gene and AmCYP71D756 gene are all derived from Astragalus membranaceus.

3. The method for constructing an engineered bacterium for de novo biosynthesis of cycloastragenol according to claim 2, characterized in that: The promoter of the expression cassette in step (1) is P TEF1 、P PGK1 、P TDH3 、P ENO2 、P INO1 and P TPI1 Any one of the following; the terminator is T CYC1 、T ADH1 、T HSP26 、T TPS1 and T CPS1 Any one of .

4. The method for constructing an engineered bacterium for de novo biosynthesis of cycloastragenol according to claim 3, characterized in that: The integration site of the expression cassette in step (1) is any one of the X-2 site, X-3 site, X-4 site, XI-1 site, XI-2 site, XII-1 site, XII-5 site, XII-4 site, XI-5 site and delta site of Saccharomyces cerevisiae.

5. The method for constructing an engineered bacterium for de novo biosynthesis of cycloastragenol according to claim 4, characterized in that: The Saccharomyces cerevisiae strain in step (2) is Saccharomyces cerevisiae IMX581; the expression cassette is transferred into the Saccharomyces cerevisiae cells by CRISPR-Cas9 technology and integrated into the Saccharomyces cerevisiae genome by homologous recombination.

6. The method for constructing an engineered bacterium for de novo biosynthesis of cycloastragenol according to claim 5, characterized in that: The recombinant plasmid used in step (2) is any one of pMEL10-X-2, pMEL10-X-3, pMEL10-X-4, pMEL10-XI-1, pMEL10-XI-2, pMEL10-XII-1, pMEL10-XII-5, pMEL10-XII-4, pMEL10-XI-5 and pMEL10-delta.

7. The engineered bacteria constructed by the construction method according to any one of claims 1 to 6, characterized in that: The engineered bacteria is the engineered strain CycZ23 or the engineered strain CAG-1.

8. Use of the engineered bacteria according to claim 7 in the biosynthesis of cycloastragenol.

9. The use according to claim 8, characterized in that The method comprises the following steps: inoculating the seed liquid of the engineered strain CycZ23 or the engineered strain CAG-1 into a YPD fermentation medium, fermenting the culture medium at 28-30° C. and 200-220 rpm in a shaking incubator for 120-144 hours, crushing the cells after the fermentation, and extracting the culture medium with ethyl acetate to obtain cycloastragenol.

10. The use according to claim 9, characterized in that: The OD value of the seed solution is 0.06-0.1.

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