CN117903960A - Recombinant saccharomyces cerevisiae strain for producing cannabidiol and construction method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of synthetic biology, and particularly discloses a recombinant saccharomyces cerevisiae strain for producing cannabidiol, and a construction method and application thereof. The strain comprises CsAAE-PEX 15, csTKS-CsOAC-PEX15, csPT-PEX 15 and CsCBDASm-PEX15 fusion genes; the olive alkyd OA biosynthesis is carried out near the peroxisome membrane by utilizing the peroxisome membrane anchoring motif Pex15, GPP biosynthesis is carried out in cytoplasm, the membrane area of the peroxisome is enlarged, the OA and the GPP filled in cells are fully contacted and reacted, CBGA is efficiently synthesized under the action of CsPT-PEX 15 fusion genes, meanwhile, csCBDASm is expressed on the peroxisome membrane, and the obtained product rapidly reacts with cannabigerol CBGA near the membrane to efficiently synthesize cannabidiol CBDA.

Description

A recombinant saccharomyces cerevisiae strain producing cannabidiol acid and its construction method and application

Technical Field

The present invention belongs to the field of synthetic biotechnology, and specifically relates to a recombinant brewer's yeast strain producing cannabidiol acid, and a construction method and application thereof.

Background technique

Among the more than 130 known cannabinoids, the non-hallucinogenic cannabinoid CBG (cannabigerolic acid) has attracted attention due to its unique medical properties and relative rarity. Current studies have shown that CBG can reduce the side effects caused by chemotherapy, relieve neuropathic pain, has anti-tumor properties, and has antibacterial effects similar to vancomycin, and has lower drug resistance. In plants, many cannabinoids exist at low levels. Since various cannabinoids are mixed together, it is difficult to extract pure monomeric cannabinoids from plants. The growth cycle of industrial hemp plants is long, and they cannot be planted on a large scale due to the limitations of the natural environment and growth conditions, as well as the influence of relevant laws and regulations. Moreover, due to the complex chemical structure of cannabinoids and the existence of isomers, the cost of chemical synthesis is expensive and the synthesis method is not environmentally friendly. Therefore, the use of biosynthesis methods can obtain high-purity monomeric cannabinoids.

In industrial hemp plants, the biosynthesis pathway of CBG includes geranyl pyrophosphate (GPP) of olivetolic acid (OA) and mevalonate pathway (MVA pathway) with malonyl-CoA and hexanoyl-CoA as precursors. Subsequently, olivetolic acid OA and geranyl pyrophosphate GPP are catalyzed by geranyl pyrophosphate-olivetylgeranyltransferase (isopentenyltransferase, CsPT4) in industrial hemp to form cannabigerol acid (CBGA). Subsequently, cannabigerol (CBG) is obtained by decarboxylation during the extraction process. Luo et al. first used galactose to synthesize various cannabinoids and derivatives in Saccharomyces cerevisiae (Nature volume 567, pages123–126 (2019)), but the yield of various cannabinoids was very low. Zhang et al. successfully used glucose to produce high-yield cannabigerol acid CBGA in Saccharomyces cerevisiae based on the work of Luo et al. (Metabolic Engineering, Volume 80, 2023, Pages 232-240). Related technologies have proposed the production of cannabinoid acid (CBGA) from microbial culture, but there are several problems:

(1) Low hexanoyl-CoA and malonyl-CoA pool levels in Saccharomyces cerevisiae limit the synthesis of oleic acid OA;

(2) In order to increase the intracellular hexanoyl-CoA pool level, when exogenous hexanoic acid or hexanoate is added, Saccharomyces cerevisiae cells will consume hexanoic acid or hexanoate for β-oxidation, competing with the olivetol biosynthesis pathway for substrates;

(3) Geranyl pyrophosphate-geranyl olivetate transferase (CsPT4) is unable to exert high catalytic activity in Saccharomyces cerevisiae cells.

Therefore, how to eliminate the competition between hexanoic acid or hexanoate and the oleic acid synthesis pathway without destroying the β-oxidation metabolic pathway in the peroxisome of Saccharomyces cerevisiae, thereby increasing the utilization of hexanoic acid or hexanoate to synthesize oleic acid, and further increasing the production of cannabidiolic acid (CBDA) in Saccharomyces cerevisiae cell factories, is a technical problem that scientific researchers urgently need to solve.

Summary of the invention

The purpose of the present invention is to provide a recombinant Saccharomyces cerevisiae strain producing cannabidiolic acid and its construction method and application in view of the above-mentioned deficiencies in the prior art, construct a double mutation (CsCBDASm, i.e., CsCBDAS ^F382R/H184R ), and fuse it with the PEX15 anchor motif to form a CsCBDASm-PEX15 fusion gene, so that CsCBDASm is expressed on the peroxisome membrane, reacts rapidly with the cannabidiolic acid CBGA near the membrane, and efficiently synthesizes cannabidiolic acid CBDA.

To achieve the above object, the present invention adopts the following technical solution:

The first object of the present invention is to provide a recombinant Saccharomyces cerevisiae strain producing cannabidiol acid, wherein the recombinant yeast contains CsAAE1-PEX15, CsTKS-CsOAC-PEX15, CsPT4-PEX15 and CsCBDASm-PEX15 fusion genes; the nucleotide sequence of the CsAAE1-PEX15 fusion gene is shown in SEQ ID NO.1, the nucleotide sequence of the CsTKS-CsOAC-PEX15 fusion gene is shown in SEQ ID NO.2, the nucleotide sequence of the CsPT4-PEX15 fusion gene is shown in SEQID NO.3, and the nucleotide sequence of the CsCBDASm-PEX15 fusion gene is shown in SEQ ID NO.4.

The second object of the present invention is to provide a method for constructing the above-mentioned recombinant Saccharomyces cerevisiae strain, comprising the following specific steps:

S1, using the engineered strain of Saccharomyces cerevisiae CEN.PK2-1D as the chassis, overexpressing all genes of the mevalonate pathway, and introducing the ERG20 ^F96W/N127W mutant to obtain strain N1;

S2, the carnitine-O-acetyltransferase CAT2 gene and the acetyl-CoA carboxylase ACC1 ^{S659A/S686A/S1157A} mutant gene were fused with the natural Pex15 anchor motif to form CAT2-PEX15 and ACC1 ^{S659A/S686A/S1157A} -PEX15 fusion genes, respectively, and the strain N1 was used as the chassis strain, and the CRISPR/Cas9 gene editing technology was used to integrate them into the genome of the strain N1 to obtain the strain DN02;

S3, the activating enzyme CsAAE1 gene, tetraketide synthase-olivine alcohol acid cyclase CsTKS-CsOAC gene and geranyl pyrophosphate-olivine alcohol acid geranyl transferase CsPT4 gene in industrial hemp were fused with the natural Pex15 anchor motif to form CsAAE1-PEX15, CsTKS-CsOAC-PEX15 and CsPT4-PEX15 fusion genes, and the strain DN02 was used as the chassis strain, and the CRISPR/Cas9 gene editing technology was used to integrate the CsAAE1-PEX15, CsTKS-CsOAC-PEX15 and CsPT4-PEX15 gene expression cassettes into the genome of the strain DN02 to obtain the strain DN05;

S4, integrating the overexpression GAL4 gene expression cassette into the genome of strain DN05 to obtain strain DN06;

S5, integrating the overexpression PEX11 gene expression cassette into the genome of strain DN06 to obtain the recombinant Saccharomyces cerevisiae strain, which is recorded as strain DN07;

S6. Using strain DN07 as the chassis, the CsCBDASm-PEX15 fusion gene was integrated into the genome of strain DN07 to obtain the recombinant Saccharomyces cerevisiae strain, which was recorded as DN12.

Furthermore, the plasmid vector of the CsAAE1-PEX15 gene expression cassette is the pCsAAE1-PEX15 plasmid. Using the pOE-AAE1 plasmid as a template, PCR amplification is performed to obtain a DNA fragment with a partial homologous sequence of the PEX15 peroxisome anchor motif; using the genomic DNA of Saccharomyces cerevisiae S288C as a template, PCR amplification is performed to obtain a PEX15 anchor motif DNA fragment; the above-mentioned fragments are integrated by homologous recombination to obtain the pAAE1-PEX15 plasmid.

Furthermore, the plasmid vector of the CsTKS-CsOAC-PEX15 gene expression cassette is the pCsTKS-CsOAC-PEX15 plasmid. Using the pOE-CsTKS-CsOAC plasmid as a template, PCR amplification is performed to obtain a DNA fragment with a partial homologous sequence of the PEX15 peroxisome anchor motif; using the genomic DNA of Saccharomyces cerevisiae S288C as a template, PCR amplification is performed to obtain a PEX15 anchor motif DNA fragment; the above-mentioned fragments are integrated by homologous recombination to obtain the pCsTKS-CsOAC-PEX15 plasmid.

Furthermore, the plasmid vector of the CsPT4-PEX15 gene expression cassette is a CsPT4-PEX15 plasmid. Using the pOE-PT4 plasmid as a template, PCR amplification is performed to obtain a DNA fragment with a partial homologous sequence of the PEX15 peroxisome anchor motif; using the genomic DNA of Saccharomyces cerevisiae S288C as a template, PCR amplification is performed to obtain a PEX15 anchor motif DNA fragment; the above-mentioned fragments are integrated by homologous recombination to obtain the pCsPT4-PEX15 plasmid.

Furthermore, the plasmid vector for overexpressing the GAL4 gene expression cassette is a pOE-GAL4 plasmid. The genome of Saccharomyces cerevisiae CEN.PK2-1D is used as a template, and PCR amplification is performed to obtain the homologous left arm and homologous right arm of the integration site GAL1/7/10; the genomic DNA of Saccharomyces cerevisiae S288C is used as a template, and PCR expansion is performed to obtain the GAL4 gene and its promoter fragment; the addgene#43802 plasmid is used as a template, and PCR amplification is performed to obtain the CYC1 terminator fragment; the plasmid pRS426-URA is used as a template, and PCR amplification is performed to obtain the plasmid backbone fragment; the above fragments are integrated by homologous recombination to obtain the plasmid pOE-GAL4.

Furthermore, the plasmid vector for overexpressing the PEX11 gene expression cassette is a pOE-PEX11 plasmid. The genome of Saccharomyces cerevisiae CEN.PK2-1D is used as a template, and PCR amplification is performed to obtain the homologous left arm and the homologous right arm of the integration site AAT1; the addgene#43802 plasmid is used as a template, and PCR amplification is performed to obtain the TEF1 promoter fragment; the genomic DNA of Saccharomyces cerevisiae S288C is used as a template, and PCR amplification is performed to obtain the PEX11 gene fragment; the addgene#43802 plasmid is used as a template, and PCR amplification is performed to obtain the CYC1 terminator fragment; the pRS426-URA plasmid is used as a template, and PCR amplification is performed to obtain the plasmid backbone fragment; the above fragments are integrated by homologous recombination to obtain the plasmid pOE-PEX11.

Furthermore, the plasmid vector of the CsCBDASm-PEX15 gene expression cassette is pCsCBDASm-PEX15. Using the pCsCBDASm plasmid as a template, PCR amplification is performed to obtain a plasmid skeleton carrying the CsCBDASm gene expression cassette and the left and right homologous arms at the integration site; using the genomic DNA of Saccharomyces cerevisiae S288C as a template, PCR amplification is performed to obtain a PEX15 gene fragment, and the above fragment is integrated by homologous recombination to obtain the plasmid pCsCBDASm-PEX15.

Furthermore, the pCsCBDASm plasmid uses the single mutation plasmid pCsCBDAS ^F382R as a template, and PCR amplifies to obtain a linearized plasmid fragment carrying CsCBDASm; the above fragments are integrated by homologous recombination to obtain the plasmid pCsCBDASm; the single mutation plasmid pCsCBDAS ^F382R uses the genomic DNA of Saccharomyces cerevisiae CEN.PK2-1D as a template, and PCR amplifies to obtain the homologous left arm and the homologous right arm of the integration site 11-6; uses the genomic DNA of Saccharomyces cerevisiae S288C as a template, and PCR amplifies to obtain the GAL1 promoter fragment; uses the pESC-URA-CsCBDAS ^F382R plasmid as a template, and PCR amplifies to obtain the CsCBDAS ^F382R gene fragment; uses the addgene#43802 plasmid as a template, and PCR amplifies to obtain the CYC1 terminator fragment; uses the pRS426-URA plasmid as a template, and PCR amplifies to obtain the plasmid backbone fragment; and the above fragments are integrated by homologous recombination.

The third object of the present invention is to provide a method for producing cannabidiol acid, which comprises fermenting and culturing the above-mentioned recombinant Saccharomyces cerevisiae strain to obtain cannabidiol acid from the culture.

Furthermore, the fermentation culture process is to carry out shake flask fermentation culture in YPD medium for 96 h to 144 h.

Furthermore, the bacterial cells obtained by fermentation culture are subjected to microwave heating for decarboxylation, the radiation temperature of the microwave treatment is 55-60° C., the power of the microwave treatment is 500-550 W, and the duration is 25-30 min.

Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

(1) The present invention provides a recombinant Saccharomyces cerevisiae strain producing cannabidiolic acid, which comprises CsAAE1-PEX15, CsTKS-CsOAC-PEX15, CsPT4-PEX15 fusion gene and CsCBDASm-PEX15 fusion gene. The peroxisomal membrane anchoring motif Pex15 is used to carry out the biosynthesis of oleic acid OA near the peroxisomal membrane, and the biosynthesis of geranyl pyrophosphate (GPP) is carried out in the cytoplasm, thereby expanding the membrane area of the peroxisome, allowing OA to fully contact and react with the GPP filled in the cell. Under the action of the CsPT4-PEX15 fusion gene, CBGA is efficiently synthesized. At the same time, CsCBDASm is expressed on the peroxisomal membrane, reacts rapidly with cannabidiolic acid CBGA near the membrane, and cannabidiolic acid CBDA is efficiently synthesized.

(2) The present invention provides a method for constructing a recombinant Saccharomyces cerevisiae strain producing cannabinoid acid, which comprises randomly mutating the CsCBDAS gene to construct a mutant library, and finding that mutants at two sites (CsCBDAS ^F382R and CsCBDAS ^H184R ) can better catalyze the conversion of CBGA to CBDA; and constructing a double mutant gene (CsCBDASm, i.e., CsCBDAS ^F382R/H184R ) and fusion with the PEX15 anchor motif, the catalytic efficiency is significantly improved compared with the wild type.

(3) Compared with the production of cannabidiol acid by expressing the CBGA biosynthetic pathway in the prior art, the production of cannabidiol acid by the recombinant Saccharomyces cerevisiae strain producing cannabidiol acid provided by the present invention is increased by nearly 12 times.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a comparison of the cannabidiol content generated by decarboxylation of the fermentation products of strains DN08, DN09, DN10, DN11, and DN12;

Figure 2 is a typical spectrum of HPLC detection of the fermentation sample of strain DN12;

FIG3 is a reaction pathway diagram of cannabidiol acid decarboxylation by microwave.

Detailed ways

In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are further described in detail below in conjunction with specific examples and drawings. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

The specific process of constructing the recombinant Saccharomyces cerevisiae strain provided by the present invention is as follows:

(1) The present invention uses the engineered strain of Saccharomyces cerevisiae CEN.PK2-1D as the chassis, overexpresses all genes of the mevalonate pathway, and introduces the ERG20 ^F96W/N127W mutant, so that the strain can efficiently synthesize geranyl pyrophosphate (GPP);

(2) The carnitine-O-acetyltransferase CAT2 gene and the acetyl-CoA carboxylase ACC1 ^{S659A/S686A/S1157A} mutant gene were fused with the natural Pex15 anchor motif to form the CAT2-PEX15 and ACC1 ^{S659A/S686A/S1157A} -PEX15 fusion genes, so that the acetyl-carnitine in the peroxisome is directly converted into acetyl-CoA when passing through the peroxisomal membrane, and then converted into malonyl-CoA under the action of the acetyl-CoA carboxylase mutant, thereby increasing the malonyl-CoA content in Saccharomyces cerevisiae;

(3) The acyl activating enzyme CsAAE1 gene in industrial hemp was fused with the natural Pex15 anchoring motif to form the CsAAE1-PEX15 fusion gene, so that hexanoic acid or hexanoate is converted into hexanoyl-CoA before entering the peroxisome, thereby increasing the hexanoyl-CoA content in Saccharomyces cerevisiae;

(4) The tetraketide synthase-olivine alcohol cyclase CsTKS-CsOAC gene in industrial hemp is fused with the natural Pex15 anchor motif to form the CsTKS-CsOAC-PEX15 fusion gene, so that CsTKS-CsOAC is expressed on the peroxisomal membrane. Hexanoyl-CoA and malonyl-CoA near the peroxisomal membrane directly participate in the reaction with the protein expressed by the CsTKS-CsOAC gene to synthesize olive alcohol acid OA;

(5) The geranyl pyrophosphate-olive geranyl transferase (isopentenyl transferase, CsPT4) in industrial hemp is fused with the natural Pex15 anchor motif to form a CsPT4-PEX15 fusion gene, so that CsPT4 is expressed on the peroxisomal membrane and rapidly reacts with the high level of intracellular GPP and oleic acid OA near the peroxisomal membrane to synthesize cannabinoid phenolic acid CBGA;

(6) Overexpression of the peroxisome biogenesis protein Pex11 to increase the peroxisome membrane area, thereby improving the catalytic efficiency of the entire cannabigerol acid (CBGA) biosynthesis pathway;

(7) The CsCBDAS gene was randomly mutated to construct a mutant library, and highly efficient catalytic mutants were screened. Mutants at two sites (CsCBDAS ^F382R and CsCBDAS ^H184R ) were screened out that could better catalyze the conversion of CBGA into CBDA. Subsequently, a double mutation (CsCBDASm, i.e., CsCBDAS ^F382R/H184R ) was constructed and fused with the PEX15 anchor motif to form the CsCBDASm-PEX15 fusion gene, which allowed CsCBDASm to be expressed on the peroxisome membrane and rapidly react with cannabigerolic acid CBGA near the membrane to efficiently synthesize cannabidiolic acid CBDA.

It should be noted that, compared with the wild-type CsCBDAS, the F at position 382 of the mutant CsCBDAS ^F382R is replaced by R; compared with the wild-type CsCBDAS, the H at position 184 of the mutant CsCBDAS ^H184R is replaced by R; and compared with the wild-type CsCBDAS, the F at position 382 of the double mutant CsCBDASm is replaced by R and the H at position 184 is replaced by R.

In some embodiments, the recombinant Saccharomyces cerevisiae strain producing cannabidiol acid provided by the present invention comprises CsAAE1-PEX15, CsTKS-CsOAC-PEX15, CsPT4-PEX15 and CsCBDASm-PEX15 fusion genes; it is obtained by directly adding the PEX15 sequence after removing the termination codons at the ends of the CsAAE1 gene, CsTKS-CsOAC gene, CsPT4 gene and CsCBDASm, and the PEX15 gene is a natural Pex15 anchor motif, and its nucleotide sequence is shown in SEQ ID NO.93.

Specifically, the nucleotide sequence of the CsAAE1-PEX15 fusion gene is shown in SEQ ID NO.1, the nucleotide sequence of the CsTKS-CsOAC-PEX15 fusion gene is shown in SEQ ID NO.2, the nucleotide sequence of the CsPT4-PEX15 gene is shown in SEQ ID NO.3, and the nucleotide sequence of the CsCBDASm-PEX15 fusion gene is shown in SEQ ID NO.4.

The detailed information of the plasmids used in the present invention is shown in Table 1 and Table 2:

Table 1.

Table 2.

The detailed information of the strains used in the present invention is shown in Table 3:

table 3.

The primers used in the process of constructing the strains in the present invention are shown in Table 4-5.

Table 4. Primers involved in strain construction

Table 5. Primers involved in strain construction (continued)

The method for constructing the gene expression cassette in the present invention is yeast assembly (lithium acetate transformation method), and the specific steps are as follows:

1. Pretreatment method:

The pretreatment process of each fragment of yeast assembly is as follows: 300 ng of each fragment is calculated, the volume is calculated, mixed evenly, the total volume is calculated, 3 M sodium acetate is added at a ratio of 10% volume/volume fraction, mixed evenly, the total volume after the addition of sodium acetate is calculated, and then 10 mg/mL glycogen solution is added at a ratio of 2% volume/volume fraction, mixed evenly, the total volume after the addition of glycogen solution is calculated, and then 2 times the total volume of anhydrous ethanol is added, mixed evenly, and the DNA is precipitated at -80 ℃ for 2h. Centrifuge at 13200rpm, 4 ℃ for 20 min, remove the supernatant, add 500 μL of -40 ℃ precooled 70% ethanol, gently invert and wash the precipitate, centrifuge at 13200 rpm, 4 ℃ for 6 min, remove the supernatant, add 8 μL of ddH20 to dissolve the precipitate, and store at -40 ℃.

2. Lithium acetate conversion method:

(1) Take an appropriate amount of Saccharomyces cerevisiae liquid or a single clone from a seed tube or streaked plate stored at -80°C and transfer to 5 mL of YPD liquid medium. Incubate overnight at 30°C and 220 rpm in a shaking incubator (18-24 h).

(2) The next day, transfer an appropriate amount of the Saccharomyces cerevisiae culture medium to 50 mL of freshly prepared YPD liquid medium (freshly prepared to ensure accurate volume) so that the initial OD600 of the culture medium after transfer is about 0.2. Incubate the culture medium in a shaking incubator at 30°C and 220 rpm for 4 to 6 h until the OD600 reaches 0.6 to 0.8.

(3) Centrifuge at 3500 rpm for 6-10 min at room temperature;

(4) Remove the supernatant in the clean bench and add 1×TE (20 mL) to resuspend the cells;

(5) Centrifuge at 3500 rpm for 6-10 min at room temperature;

(6) Remove the supernatant in the clean bench and add 1×LiAc/0.5×TE (2 mL);

(7) Place in a 25°C water bath for 10 min. During this period, boil the single-stranded fish sperm DNA (salmon sperm DNA) in boiling water for 5 min to fully denature it, then quickly remove it and place it on ice (to keep the fish sperm DNA in a single-stranded state).

(8) After 10 min in a 25°C water bath, pipette 100 μL of Saccharomyces cerevisiae competent cell suspension into a clean bench, add 20 μL of single-stranded fish sperm DNA (5 μg/μL) boiled in boiling water, mix well, and add the substance to be transformed:

Pretreated yeast assembly DNA fragments (8 μL);

or donor DNA fragment (linearized vector);

sgRNA plasmid, Cas9 plasmid (43802)

Mix well, add 1×LiAc/40%PEG-4000/1×TE (700 μL), mix well by gently pipetting with the pipette tip, and then culture in a 30 ℃ incubator for 30 min;

(9) Add 88 μL of DMSO (filter-sterilized) to the system, gently invert to mix evenly, and heat shock in a 42 °C water bath for 9 min;

(10) Centrifuge at 12,000 rpm for 2 to 3 minutes at room temperature;

(11) Remove the supernatant in a clean bench, add 1 mL of YPD liquid medium to wash the cells, centrifuge at 10,000 rpm for 1 min at room temperature, remove the supernatant in a clean bench, add 1 mL of YPD liquid medium to resuspend the cells, and place in a 30 °C incubator for pre-culture for 1 h;

(12) Centrifuge at 10,000 rpm for 1 min at room temperature, remove the supernatant in a clean bench, add 1 mL of 1×TE to wash the cells, centrifuge at 10,000 rpm for 1 min at room temperature, remove the supernatant in a clean bench, and add 1×TE (200 μL) to resuspend the cells. Spread the plates evenly on the corresponding resistance plates or amino acid-deficient plates. After the plates are dried, place them upside down in a 30°C incubator and culture for 2–3 days.

The specific operation method of the CRISPR/Cas9 gene editing method used in the present invention is:

The CRISPR/Cas9 vectors used in the present invention are Cas9 protein vector-addgene#43802 plasmid and sgRNA-addgene#43803 plasmid. In order to be suitable for different integration sites, the first 20bp sequence of the gRNA scaffold sequence is replaced by PCR amplification during the operation design process, and the corresponding sequence of the integration site is shown in Table 6 below. At the same time, in order to make the used sgRNA easier to discard, 5-FOA (pentafluoroorotic acid) is used for reverse screening, and strains containing sgRNA plasmids (there is a URA screening marker on the plasmid, and yeast strains with this marker cannot survive on a medium containing 5-FOA) cannot grow normally.

Table 6. Integration site sequence list

Preparation of solution and culture medium used in the present invention:

(1) 5’FOA solid medium

Accurately weigh 6.7 g of amino acid-free yeast nitrogen source YNB, 20 g of glucose, 1.3 g of corresponding amino acid powder, and 18 g of agar powder. Before adding agar powder, dilute the above components to 1L and adjust the pH to 6.5 with NaOH solution. Add agar powder and mix well, then sterilize with high-pressure steam at 115 ℃ for 30 min. Wait for the culture medium to cool slightly, add sterile 5'FOA solution in the dark, mix well, pour into a plate, and store in the dark at 4 ℃.

(2) DNA fragment pretreatment solution for yeast assembly

Preparation of 3 M sodium acetate: Accurately weigh 24.61 g of anhydrous sodium acetate, dissolve it in distilled water and make up to 100 mL, sterilize it by high-pressure steam at 115°C for 30 min, and store it at 4°C.

Preparation of 10 mg/mL glycogen: Accurately weigh 100 mg of glycogen, mix with distilled water and make up to 10 mL, sterilize with high-pressure steam at 115℃ for 30 min, and store at room temperature.

(3) Preparation of Saccharomyces cerevisiae competent cells and solutions required for yeast lithium acetate transformation

10×TE solution: Accurately weigh 12.114 g of Tris base and 3.7224 g of disodium ethylenediaminetetraacetate, dissolve them in distilled water and make up to 1 L, adjust the pH to 7.5 with dilute HCl, sterilize with high-pressure steam at 115℃ for 30 min, and store at room temperature.

10×LiAc solution: Accurately weigh 10.2 g of lithium acetate, fully dissolve it with distilled water, adjust the pH to 7.5 with glacial acetic acid, and then make up to 100 mL with distilled water. Filter and sterilize using a 0.22 μm sterile filter membrane and store at 4°C.

40% PEG4000 solution: Accurately weigh 80 g of PEG4000, dissolve it in distilled water and make up to 160 mL. Sterilize it with high pressure steam at 115°C for 30 min and store it at 4°C.

(4) Cell wall breaking buffer for genome extraction of Saccharomyces cerevisiae cells

Accurately weigh 20 g Triton-100, 10 g SDS, 5.844 g sodium chloride, 1.211 g Tris-Hcl, and 0.3695 g ethylenediaminetetraacetic acid, fully dissolve in distilled water and make up to 1 L, adjust the pH to 8.0 with hydrochloric acid, and store at room temperature.

(5) 4 M ammonium acetate solution for genome extraction from Saccharomyces cerevisiae cells

Accurately weigh 30.83 g of ammonium acetate, fully dissolve it in distilled water, make up to 100 mL, mix well, sterilize with high-pressure steam at 115°C for 30 min, and store at 4°C.

The primers used to construct the yeast chassis of the present invention include: pD1 assembly, pD2 assembly, pD5 assembly, pHXT1-pERG20 assembly, pOE-AAE1 assembly, pOE-CsTKS-CsOAC (pOE-TKSOAC) assembly, pOE-PT4 assembly, pCAT2-PEX15 assembly, pACC1*-PEX15 assembly, pCsAAE1-PEX15 assembly, pCsTKS-CsOAC-PEX15 assembly, pCsPT4-PEX15 assembly, pOE-GAL4 assembly, pOE-PEX11 assembly, pACC1* assembly, pCAT2 assembly, pCSCBDAS assembly, pCsCBDAS ^F382R assembly, CsCBDAS ^H184R assembly, CsCBDAS ^F382R/H184R assembly, and pCsCBDASm-PEX15 assembly. The detailed information of the primer sequences are shown in Tables 4-6.

It should be noted that the gene sequences and primer sequences used in the embodiments of the present invention were commissioned to be synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and other biological materials and reagents can be obtained commercially.

Acetyl-CoA carboxylase "ACC1 ^{S659A/S686A/S1157A} mutant" is abbreviated as "ACC1*"; CsCBDASm is CsCBDAS ^F382R/H184R .

The ERG20 ^F96W/N127W mutant gene used in the present invention is referenced to Codruta Ignea "Engineering Monoterpene Production in Yeast Using a Synthetic Dominant Negative Geranyl Diphosphate Synthase"; the ACC1 ^{S659A/S686A/S1157A} mutant gene is referenced to Xiaoxu Chen "Screening Phosphorylation Site Mutations in Yeast Acetyl-CoA Carboxylase Using Malonyl-CoASensor to Improve Malonyl-CoA-Derived Product", both of which were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd. The wild-type CsCBDAS has Gene ID: 115697762 in the NCBI database.

Example 1

This embodiment provides the construction of a recombinant Saccharomyces cerevisiae strain that produces cannabidiol acid.

Specific steps are as follows:

Step S1: Construction of high-yield GPP Saccharomyces cerevisiae chassis strain N1

Using the Saccharomyces cerevisiae CEN.PK2-1D strain as the chassis, all MVA pathway genes and ERG20F96W/N127W mutant genes were overexpressed to enable the strain to have high GPP production capacity. The construction process involved pD1, pD2, pD5, pHXT1-pERG20 plasmids, which were constructed according to the methods in Thomas Siemon "Semisynthesis of Plant-Derived Englerin A Enabledby Microbe Engineering of Guaia-6,10(14)-diene as Building Block" and Xiao Kong "Efficient Synthesis of Limonene in Saccharomyces cerevisiae UsingCombinatorial Metabolic Engineering Strategies". The gene characteristics of pD1, pD2, pD5, pHXT1-pERG20 plasmids are shown in Table 1.

The successfully constructed pD1, pD2, pD5, and pHXT1-pERG20 plasmids were linearized with NotⅠ restriction endonuclease to obtain a gene expression cassette with homology arms, which were sequentially integrated into Saccharomyces cerevisiae CEN.PK2-1D using CRISPR/Cas9 gene editing technology to obtain strain N0, which overexpressed all genes in the MVA pathway and introduced the ERG20 ^{F96W /N127W} mutant. Finally, by replacing the endogenous ERG20 gene promoter and inhibiting the expression of the endogenous ERG20 gene, the precursor GPP could be better accumulated, resulting in the high-GPP-yielding Saccharomyces cerevisiae strain N1.

Step S2: Using the high-GPP-producing Saccharomyces cerevisiae strain N1 as the chassis, using CRISPR/Cas9 gene editing technology, the pCAT2-PEX15 and pACC1*-PEX15 gene expression cassettes were integrated into the chromosomal DNA to obtain strain DN02.

References for construction of pCAT2-PEX15 plasmid and pACC1*-PEX15 plasmid

《Enhanced production of acetyl-CoA-based products via peroxisomal surface display in Saccharomyces cerevisiae》https://doi.org/10.1073/pnas.2214941119. The pCAT2-PEX15 and pACC1*-PEX15 plasmids constructed above were linearized with NotⅠ restriction enzyme to obtain a gene expression cassette with homology arms, which was then integrated into the Saccharomyces cerevisiae strain N1 using CRISPR/Cas9 gene editing technology to obtain strain DN02.

Step S3: Using strain DN02 as the chassis, using CRISPR/Cas9 gene editing technology, the CsAAE1-PEX15, CsTKS-CsOAC-PEX15 and CsPT4-PEX15 gene expression cassettes were integrated into the chromosomal DNA to obtain strain DN05.

Construction process of plasmid pCsAAE1-PEX15: Using pOE-AAE1 plasmid as template, primers 201-F and 201-R were used to PCR amplify a DNA fragment with a partial homologous sequence of the PEX15 peroxisome anchor motif (with left and right homologous arms of the integration site and the CsAAE1 gene expression cassette); using Saccharomyces cerevisiae S288C genomic DNA as template, primers 202-F and 202-R were used to PCR amplify a DNA fragment with the PEX15 anchor motif; using a one-step cloning kit (Hieff Clone® Plus One StepCloning Kit), the above fragments were integrated by homologous recombination to obtain plasmid pAAE1-PEX15.

pOE-AAE1 is the expression cassette of the acyl activating enzyme CsAAE1 gene. The plasmid construction process is as follows: the genome of Saccharomyces cerevisiae CEN.PK2-1D is used as a template, and primers 111-F and 111-R, 115-F and 115-R are used to PCR amplify the homologous left arm and homologous right arm of the integration site LPP1; the genome of Saccharomyces cerevisiae S288C The genome was used as a template, and primers 112-F and 112-R were used to PCR amplify the GAL1 promoter fragment; pESC-URA-CsAAE1 plasmid (synthesized by Sangon Biotech Co., Ltd.) was used as a template, and primers 113-F and 113-R were used to PCR amplify the CsAAE1 gene fragment; pESC-HIS plasmid was used as a template, and primers 114-F and 114-R were used to PCR amplify the CYC1 terminator fragment; pRS426-URA plasmid was used as a template, and primers 116-F and 116-R were used to PCR amplify the plasmid backbone fragment. After pretreatment, the above fragments were assembled in yeast lithium acetate transformants to form pOE-AAE1 plasmid.

Construction process of plasmid pCsTKS-CsOAC-PEX15: Using pOE-CsTKS-CsOAC plasmid as template, primers 211-F and 211-R were used to PCR amplify a DNA fragment with a partial homologous sequence of the PEX15 peroxisome anchor motif (with left and right homologous arms of the integration site and the CsTKS-CsOAC gene expression cassette); using Saccharomyces cerevisiae S288C genomic DNA as template, primers 212-F and 212-R were used to PCR amplify a DNA fragment with the PEX15 anchor motif; using a one-step cloning kit (Hieff Clone® Plus One Step Cloning Kit), the above fragments were integrated by homologous recombination to obtain plasmid pCsTKS-CsOAC-PEX15.

pOE-CsTKS-CsOAC is the expression cassette of the tetraketide synthase-olivoline cyclase CsTKS-CsOAC fusion gene. The plasmid construction process is as follows: the genome of Saccharomyces cerevisiae CEN.PK2-1D is used as a template, and primers 121-F and 121-R, 125-F and 125-R are used to PCR amplify the homologous left arm and homologous right arm of the integration site DPP1; the genome of Saccharomyces cerevisiae S288C The genome was used as a template, and primers 122-F and 122-R were used to PCR amplify the GAL1 promoter fragment; pESC-TRP-CsTKS-CsOAC plasmid (synthesized by Sangon Biotech Co., Ltd.) was used as a template, and primers 123-F and 123-R were used to PCR amplify the CsTKS-CsOAC gene fragment; pESC-HIS plasmid was used as a template, and primers 124-F and 124-R were used to PCR amplify the CYC1 terminator fragment; pRS426-URA plasmid was used as a template, and primers 126-F and 126-R were used to PCR amplify the plasmid backbone fragment. After pretreatment, the above fragments were assembled in yeast lithium acetate transformants to form pOE-CsTKS-CsOAC plasmid.

Construction process of plasmid pCsPT4-PEX15: Using pOE-PT4 plasmid as template, primers 221-F and 221-R were used to PCR amplify a DNA fragment with partial homologous sequence of PEX15 peroxisome anchor motif (with left and right homologous arms of integration site and CsPT4 gene expression cassette); using Saccharomyces cerevisiae S288C genomic DNA as template, primers 222-F and 222-R were used to PCR amplify a DNA fragment with PEX15 anchor motif; using a one-step cloning kit (Hieff Clone® Plus One StepCloning Kit), the above fragments were integrated by homologous recombination to obtain plasmid pCsPT4-PEX15.

pOE-PT4 is the gene expression cassette of geranyl pyrophosphate-olivogeranyltransferase CsPT4. The plasmid construction process is as follows: the genome of Saccharomyces cerevisiae CEN.PK2-1D is used as a template, and primers 131-F and 131-R, 135-F and 135-R are used to PCR amplify the homologous left arm and homologous right arm of the integration site 10-21b; the genome of Saccharomyces cerevisiae S288C The genome was used as a template, and primers 132-F and 132-R were used to PCR amplify the GAL1 promoter fragment; pESC-TRP-CsPT4 plasmid (synthesized by Sangon Biotech Co., Ltd.) was used as a template, and primers 133-F and 133-R were used to PCR amplify the CsPT4 gene fragment; pESC-HIS plasmid was used as a template, and primers 134-F and 134-R were used to PCR amplify the CYC1 terminator fragment; pRS426-URA plasmid was used as a template, and primers 136-F and 136-R were used to PCR amplify the plasmid backbone fragment. After pretreatment, the above fragments were assembled in yeast lithium acetate transformants to form pOE-PT4 plasmid.

Using CRISPR/Cas9 gene editing technology, the above-mentioned CsAAE1-PEX15, CsTKS-CsOAC-PEX15 and CsPT4-PEX15 gene expression cassettes were integrated into the genome of strain DN02 to obtain strain DN05.

Step S4, using strain DN05 as the chassis, overexpressing the GAL4 gene, knocking in the GAL1/7/10 site, and increasing the promoter strength of the galactose-inducible promoter pGAL to obtain strain DN06.

Construction process of plasmid pOE-GAL4 overexpressing GAL4 gene:

Using the genome of Saccharomyces cerevisiae CEN.PK2-1D as a template, primers 161-F and 161-R, 164-F and 164-R were used to PCR amplify the homologous left arm and homologous right arm of the integration site GAL1/7/10; using the genomic DNA of Saccharomyces cerevisiae S288C as a template, primers 162-F and 162-R were used to PCR amplify the GAL4 gene and its promoter fragment; using addgene#43802 plasmid as a template, primers 163-F and 163-R were used to PCR amplify the CYC1 terminator fragment; using plasmid pRS426-URA as a template, primers 165-F and 165-R were used to PCR amplify the plasmid backbone fragment; using a one-step cloning kit (Hieff Clone®Plus One Step Cloning Kit), the above fragments were integrated by homologous recombination to obtain plasmid pOE-GAL4.

Using CRISPR/Cas9 gene editing technology, the pOE-GAL4 gene expression cassette was integrated into the genome of strain DN05 to obtain strain DN06.

Step S5: Using strain DN06 as the chassis, overexpressing the PEX11 gene, and knocking in the gene at the AAT1 site to obtain strain DN07.

Construction process of plasmid pOE-PEX11 overexpressing PEX11 gene:

Using the genome of Saccharomyces cerevisiae CEN.PK2-1D as a template, primers 171-F and 171-R, 175-F and 175-R were used to PCR amplify the homologous left arm and homologous right arm of the integration site AAT1; using addgene#43802 plasmid as a template, primers 172-F and 172-R were used to PCR amplify the TEF1 promoter fragment; using Saccharomyces cerevisiae S288C genomic DNA as a template, primers 173-F and 173-R were used to PCR amplify the PEX11 gene fragment; using addgene#43802 plasmid as a template, primers 174-F and 174-R were used to PCR amplify the CYC1 terminator fragment; using pRS426-URA plasmid as a template, primers 176-F and 176-R were used to PCR amplify the plasmid backbone fragment; using a one-step cloning kit (Hieff Clone® Plus One Step The above fragments were integrated by homologous recombination using the GenBank Cloning Kit to obtain the plasmid pOE-PEX11.

Using CRISPR/Cas9 gene editing technology, the pOE-PEX11 gene expression cassette was integrated into the genome of strain DN06 to obtain strain DN07. By overexpressing the GAL4 gene and the PEX11 gene, the expression of the corresponding genes was enhanced and the catalytic area of the enzymatic reaction was increased, thereby increasing the yield of CBGA.

Step S6: Using strain DN07 as the chassis, the CsCBDASm-PEX15 fusion gene was integrated into the genome of strain DN07 to obtain strain DN12.

The construction process of pCsCBDASm-PEX15 plasmid vector is as follows:

Using CsCBDASm plasmid as template, primers 501-F and 501-R were used to PCR amplify the plasmid backbone with the CsCBDASm gene expression cassette and the left and right homologous arms at the integration site; using Saccharomyces cerevisiae S288C genomic DNA as template, primers 502-F and 502-R were used to PCR amplify the PEX15 gene fragment; using a one-step cloning kit (Hieff Clone® PlusOne Step Cloning Kit), the above fragments were integrated by homologous recombination to obtain the plasmid pCsCBDASm-PEX15.

Using the Saccharomyces cerevisiae strain DN07 as the chassis, the pCsCBDASm-PEX15 plasmid was linearized by NotⅠ, and then yeast lithium acetate transformation was performed using CRISPR/Cas9 gene editing technology. After verification, strain DN12 was obtained.

The construction process of pCsCBDASm plasmid vector is as follows:

Using the single mutant plasmid pCsCBDAS ^F382R as a template, primers 401-F and 401-R were used to PCR amplify a linearized plasmid fragment containing CsCBDASm (with homology series at the 5-end and 3-end); the above fragment was integrated by homologous recombination using a one-step cloning kit (HieffClone® Plus One Step Cloning Kit) to obtain the plasmid pCsCBDASm.

The construction process of the single mutant pCsCBDAS ^F382R plasmid vector is as follows:

The mutant library constructed above was used to construct the corresponding expression vector for integration into the chromosome of Saccharomyces cerevisiae. The specific process was as follows: using Saccharomyces cerevisiae CEN.PK2-1D genomic DNA as a template, primers 301-F and 301-R, 306-F and 306-R were used to PCR amplify the homologous left arm and homologous right arm of the integration site 11-6; using Saccharomyces cerevisiae S288C genomic DNA as a template, primers 302-F and 302-R were used to PCR amplify the GAL1 promoter fragment; using pESC-URA-CsCBDAS ^F382R plasmid as a template, primers 303-F and 303-R were used to PCR amplify the CsCBDAS ^F382R gene fragment; using addgene#43802 plasmid as template, primers 304-F and 304-R, PCR amplification obtained CYC1 terminator fragment; using pRS426-URA plasmid as template, primers 305-F and 305-R, PCR amplification obtained plasmid backbone fragment; using a one-step cloning kit (Hieff Clone® Plus OneStep Cloning Kit), the above fragments were integrated by homologous recombination to obtain plasmid pCsCBDAS ^F382R .

The applicant used the pESC-URA-CsCBDAS plasmid vector synthesized by Sangon Biotechnology and the TakaraDiversify™ PCR Random Mutagenesis Kit to construct a mutation library and found that the catalytic activity of the single mutants CsCBDAS ^F382R and CsCBDAS ^H184R was improved compared with the wild-type CsCBDAS. The catalytic activity of the double mutant CsCBDASm (i.e., CsCBDAS ^F38R/H184R ) obtained by fusing the above two mutants was further improved. It was unexpectedly found that the catalytic activity of the double mutant CsCBDASm was significantly improved by fusing it with the PEX15 anchor motif, forming a CsCBDASm-PEX15 fusion gene, which allowed CsCBDASm to be expressed on the peroxisome membrane, react rapidly with the cannabinoid phenolic acid CBGA near the membrane, and efficiently synthesize cannabigerolic acid CBDA.

Comparative Example 1

Using the Saccharomyces cerevisiae strain DN07 as the chassis, the plasmid vector pCsCBDAS was linearized with NotⅠ, and then transformed with yeast lithium acetate using CRISPR/Cas9 gene editing technology and integrated into the genome of strain DN07 to obtain strain DN08.

Wild-type CsCBDAS plasmid vector construction method:

Using Saccharomyces cerevisiae CEN.PK2-1D genomic DNA as a template, primers 301-F and 301-R, 306-F and 306-R were used to PCR amplify the homologous left arm and homologous right arm of integration site 11-6; using Saccharomyces cerevisiae S288C genomic DNA as a template, primers 302-F and 302-R were used to PCR amplify the GAL1 promoter fragment; using pESC-URA-CsCBDAS plasmid as a template, primers 303-F and 303-R were used to PCR amplify the CsCBDAS gene fragment; using addgene#43802 plasmid as a template, primers 304-F and 304-R were used to PCR amplify the CYC1 terminator fragment; using pRS426-URA plasmid as a template, primers 305-F and 305-R were used to PCR amplify the plasmid backbone fragment; using a one-step cloning kit (Hieff Clone® Plus One StepCloning Kit) Kit), and the above fragments were integrated by homologous recombination to obtain plasmid pCsCBDAS.

Comparative Example 2

Using the Saccharomyces cerevisiae strain DN07 as the chassis, the plasmid vector pCsCBDAS ^F382R was linearized with NotⅠ, and then transformed with yeast lithium acetate using CRISPR/Cas9 gene editing technology and integrated into the genome of strain DN07 to obtain strain DN09.

The construction method of plasmid vector pCsCBDAS ^F382R is the same as that in Example 1.

Comparative Example 3

Using the Saccharomyces cerevisiae strain DN07 as the chassis, the plasmid vector pCsCBDAS ^H184R was linearized with NotⅠ, and then transformed with yeast lithium acetate using CRISPR/Cas9 gene editing technology and integrated into the genome of strain DN07 to obtain strain DN10.

Single mutant CsCBDAS ^H184R vector construction method:

The mutant library constructed above was used to construct the corresponding expression vector for integration into the chromosome of Saccharomyces cerevisiae. The specific process was as follows: using Saccharomyces cerevisiae CEN.PK2-1D genomic DNA as a template, primers 301-F and 301-R, 306-F and 306-R were used to PCR amplify the homologous left arm and homologous right arm of the integration site 11-6; using Saccharomyces cerevisiae S288C genomic DNA as a template, primers 302-F and 302-R were used to PCR amplify the GAL1 promoter fragment; using pESC-URA-CsCBDAS ^H184R plasmid as a template, primers 303-F and 303-R were used to PCR amplify the CsCBDAS ^H184R gene fragment; using addgene#43802 plasmid as template, primers 304-F and 304-R, PCR amplification obtained CYC1 terminator fragment; using pRS426-URA plasmid as template, primers 305-F and 305-R, PCR amplification obtained plasmid backbone fragment; using a one-step cloning kit (Hieff Clone® Plus OneStep Cloning Kit), the above fragments were integrated by homologous recombination to obtain plasmid pCsCBDAS ^H184R .

Comparative Example 4

This comparative example provides the construction of a recombinant Saccharomyces cerevisiae strain DN11 that produces cannabidiol acid.

Using the Saccharomyces cerevisiae strain DN07 as the chassis, the plasmid vector pCsCBDASm (i.e., pCsCBDAS ^F382R/H184R ) was linearized with NotⅠ and then transformed with yeast lithium acetate using CRISPR/Cas9 gene editing technology and integrated into the genome of strain DN07 to obtain strain DN11.

The construction method of plasmid vector pCsCBDASm is the same as that in Example 1.

The strains constructed in Example 1 and Comparative Examples 1-4 were fermented and cultured to examine the contents of olivetolic acid and cannabigerol in the fermented products.

Strain fermentation method:

Cultivate overnight in YPD medium (2% glucose, 1% yeast extract, 2% tryptone), then transfer to 50 mL YPG liquid medium (2% galactose, 1% yeast extract, 2% tryptone) at 1 OD/mL, and culture in a shaking incubator at 220 rpm and 30 ℃. Add caproic acid at 48 h, 64 h, 80 h, and 96 h (to a final concentration of 0.4 µM caproic acid). From 60 h, add glucose every 12 h, 5 g/L each time, until 120 h. Cultivate for 144 h and end the fermentation. Take 5 mL of the cells and wash twice with sorbitol buffer.

Preparation method of cannabidiol: After the fermented bacteria are subjected to microwave heating decarboxylation treatment (microwave radiation temperature 60°C, power 500W, duration 30min), 500 µL 100mg/mL snail enzyme is added and incubated at 37°C for 2h. 500uL 0.25mm glass beads are added, and the ultrasonic cell disruptor is 800W for 15 min. 5 mL ethyl acetate is added for extraction twice, and the upper organic phase is combined. The vacuum dryer is evaporated until no solvent remains, resuspended with methanol, and filtered through a 0.22 µm filter membrane to obtain the sample to be tested.

HPLC is used to test the samples to be tested:

(1) Preparation of the sample to be tested: Take 5 mL of bacteria and wash twice with sorbitol buffer. After microwave heating decarboxylation treatment (microwave radiation temperature 60°C, power 500W, duration 30min), add 500 µL 100mg/mL snail enzyme and incubate at 37°C for 2h. Add 500uL 0.25mm glass beads and ultrasonic cell disruptor 800W for 15 min. Add 5 mL ethyl acetate and extract twice, and combine the upper organic phase. Evaporate in a vacuum dryer until no solvent remains, resuspend in methanol, and filter through a 0.22 µm filter membrane to obtain the sample to be tested. Microwave heating decarboxylation treatment can completely decarboxylate cannabidiol acid. The process of decarboxylation of cannabidiol acid to cannabidiol is shown in Figure 3. The content of cannabidiol can be characterized by detecting the content of cannabidiol. The calculation formula is as follows:

(2) HPLC detection: UV absorption wavelength was set to 220 nm;

The mobile phase for HPLC-UV was acetonitrile (solvent A) and 0.02% v/v aqueous ammonium acetate (solvent C), with gradient elution:

0 min 48% solvent A, 52% solvent C; 6 min 43% solvent A, 57% solvent C; 10 min 100% solvent C, 16.5 min 100% solvent C.

Reference Figure 2 is a typical HPLC spectrum of the fermentation sample of strain DN12.

The results are shown in Table 7:

Table 7.

Using strain DN07 as the chassis, the CsCBDAS mutants and wild types screened in the mutant library were integrated separately, and it was found that DN11 (CsCBDAS ^F382R/H184R double mutant) converted the most cannabidiol. Next, the CsCBDAS ^F382R/H184R double mutant was fused with the PEX15 anchor motif to construct strain DN12, and the production of cannabidiol was greatly improved, indicating that the production of cannabidiol acid produced by the metabolism of strain DN12 was significantly improved, indicating that the peroxisomal membrane anchor motif PEX15 in the present invention is suitable for the production of cannabinoids.

In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

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 principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A recombinant saccharomyces cerevisiae strain for producing cannabidiol, which is characterized in that CsAAE-PEX 15, csTKS-CsOAC-PEX15, csPT-PEX 15 and CsCBDASm-PEX15 fusion genes are contained in the recombinant saccharomyces cerevisiae; the nucleotide sequence of CsAAE-PEX 15 fusion gene is shown as SEQ ID NO.1, the nucleotide sequence of CsTKS-CsOAC-PEX15 fusion gene is shown as SEQ ID NO.2, the nucleotide sequence of CsPT-PEX 15 fusion gene is shown as SEQ ID NO.3, and the nucleotide sequence of CsCBDASm-PEX15 fusion gene is shown as SEQ ID NO. 4.

2. A method of constructing a recombinant saccharomyces cerevisiae strain according to claim 1 comprising the steps of:

S1, taking a Saccharomyces cerevisiae CEN.PK2-1D engineering strain as a chassis, over-expressing all genes of a mevalonate pathway, and simultaneously introducing an ERG20 ^F96W/N127W mutant to obtain a strain N1;

S2, respectively fusing a carnitine-O-acetyltransferase CAT2 gene and an acetyl coenzyme A carboxylase ACC1 ^{S659A/S686A/S1157A} mutant gene with a natural Pex15 anchoring motif to respectively form CAT2-PEX15 and ACC1 ^{S659A/S686A/S1157A} -PEX15 fusion genes, taking a strain N1 as a chassis strain, and integrating the fusion genes onto a strain N1 genome by utilizing a CRISPR/Cas9 gene editing technology to obtain a strain DN02;

S3, fusing an activating enzyme CsAAE gene, a tetraketone synthase-oleyl cyclase CsTKS-CsOAC gene and a geranyl pyrophosphate-oleyl geranyl transferase CsPT4 gene in industrial hemp with a natural Pex15 anchoring motif to form CsAAE-PEX 15, csTKS-CsOAC-PEX15 and CsPT-PEX 15 fusion genes, taking a strain DN02 as a chassis strain, and integrating CsAAE-PEX 15, csTKS-CsOAC-PEX15 and CsPT-PEX 15 gene expression cassettes on a strain DN02 genome by using a CRISPR/Cas9 gene editing technology to obtain a strain DN05;

s4, integrating the overexpression GAL4 gene expression cassette into a genome of the strain DN05 to obtain a strain DN06;

s5, integrating the over-expressed PEX11 gene expression cassette into a strain DN06 genome to obtain DN07;

S6, integrating CsCBDASm-PEX15 gene expression cassettes on a bacterial strain DN07 genome by taking bacterial strain DN07 as a chassis to obtain the recombinant saccharomyces cerevisiae bacterial strain which is recorded as DN12.

3. The construction method as claimed in claim 2, wherein the plasmid vector of CsAAE-PEX 15 gene expression cassette is pCsAAE-PEX 15 plasmid, the genome of saccharomyces cerevisiae cen.pk2-1D is used as a template, the gene fragment of promoter GAL1, acyl activating enzyme gene CsAAE and terminator CYC1 is copied to obtain pOE-AAE1 plasmid, and DNA fragment with homologous sequence of peroxisome anchoring motif part of PEX15 is obtained by PCR amplification using pOE-AAE1 plasmid as a template; PCR amplification is carried out by taking S288C genome DNA of saccharomyces cerevisiae as a template to obtain a PEX15 anchoring motif DNA fragment; homologous recombination and integration are carried out on the fragments to obtain pAAE-PEX 15 plasmid.

4. The construction method as claimed in claim 2, wherein the plasmid vector of CsTKS-CsOAC-PEX15 gene expression cassette is pCsTKS-CsOAC-PEX15 plasmid, the genome of saccharomyces cerevisiae cen.pk2-1D is used as a template, the fusion gene CsTKS-CsOAC of promoter GAL1, tetraon synthase-olive alkyd cyclase and the terminator CYC1 gene fragment are copied to obtain a pOE-CsTKS-CsOAC plasmid, the pOE-CsTKS-CsOAC plasmid is used as a template, and the DNA fragment with the peroxisome anchoring motif part homologous sequence of PEX15 is obtained by PCR amplification; PCR amplification is carried out by taking S288C genome DNA of saccharomyces cerevisiae as a template to obtain a PEX15 anchoring motif DNA fragment; homologous recombination and integration are carried out on the fragments to obtain pCsTKS-CsOAC-PEX15 plasmid.

5. The construction method as claimed in claim 2, wherein the plasmid vector of CsPT-PEX 15 gene expression cassette is CsPT-PEX 15 plasmid, the genome of saccharomyces cerevisiae cen.pk2-1D is used as a template, the promoter GAL1, geranyl pyrophosphate-oliv oleyl geranyl transferase gene CsPT and terminator CYC1 gene fragment are copied to obtain a pOE-PT4 plasmid, and the pOE-PT4 plasmid is used as a template, and the DNA fragment with the homologous sequence of the peroxisome anchor motif part of PEX15 is obtained by PCR amplification; PCR amplification is carried out by taking S288C genome DNA of saccharomyces cerevisiae as a template to obtain a PEX15 anchoring motif DNA fragment; homologous recombination and integration are carried out on the fragments to obtain pCsPT-PEX 15 plasmid.

6. The construction method according to claim 2, wherein the plasmid vector for overexpressing the GAL4 gene expression cassette is a ploe-GAL 4 plasmid, and the homologous left arm and the homologous right arm of the integration site GAL1/7/10 are obtained by PCR amplification using the genome of saccharomyces cerevisiae cen.pk2-1D as a template; taking Saccharomyces cerevisiae S288C genome DNA as a template, and performing PCR expansion to obtain GAL4 genes and promoter fragments thereof; taking addgene #43802 plasmid as a template, and carrying out PCR amplification to obtain a CYC1 terminator fragment; PCR amplification is carried out by taking plasmid pRS426-URA as a template to obtain a plasmid skeleton fragment; homologous recombination and integration are carried out on the fragments to obtain plasmid pOE-GAL4.

7. The construction method of claim 2, wherein the plasmid vector of the over-expressed PEX11 gene expression cassette is a pOE-PEX11 plasmid, and the homologous left arm and the homologous right arm of the integration site AAT1 are obtained by PCR amplification using the genome of saccharomyces cerevisiae cen.pk2-1D as a template; taking addgene #43802 plasmid as a template, and carrying out PCR amplification to obtain a TEF1 promoter fragment; PCR amplification is carried out by taking S288C genome DNA of saccharomyces cerevisiae as a template to obtain a PEX11 gene fragment; taking addgene #43802 plasmid as a template, and carrying out PCR amplification to obtain a CYC1 terminator fragment; PCR amplification is carried out by taking pRS426-URA plasmid as a template to obtain a plasmid skeleton fragment; the fragment is subjected to homologous recombination and integration to obtain plasmid pOE-PEX11.

8. The construction method of claim 2, wherein the plasmid vector of CsCBDASm-PEX15 gene expression cassette is pCsCBDASm-PEX15, and the plasmid backbone with CsCBDASm gene expression cassette and left and right homology arms at integration site is obtained by PCR amplification using pCsCBDASm plasmid as template; and (3) taking the saccharomyces cerevisiae S288C genome DNA as a template, carrying out PCR amplification to obtain a PEX15 gene fragment, and carrying out homologous recombination and integration on the fragment to obtain the plasmid pCsCBDASm-PEX15.

9. The construction method according to claim 8, wherein the pCsCBDASm plasmid is PCR amplified with single mutant plasmid pCsCBDAS ^F382R as template to obtain a linearized plasmid fragment with CsCBDASm; homologous recombination and integration are carried out on the fragments to obtain plasmid pCsCBDASm; the single mutation plasmid pCsCBDAS ^F382R uses Saccharomyces cerevisiae CEN.PK2-1D genome DNA as a template, and the homologous left arm and the homologous right arm of the integration site 11-6 are obtained through PCR amplification; taking Saccharomyces cerevisiae S288C genome DNA as a template, and carrying out PCR amplification to obtain a GAL1 promoter fragment; PCR amplification is carried out by taking pESC-URA-CsCBDAS ^F382R plasmid as a template to obtain CsCBDAS ^F382R gene fragment; taking addgene #43802 plasmid as a template, and carrying out PCR amplification to obtain a CYC1 terminator fragment; PCR amplification is carried out by taking pRS426-URA plasmid as a template to obtain a plasmid skeleton fragment; and (3) carrying out homologous recombination and integration on the fragments.

10. A method for producing cannabidiol, characterized in that the recombinant saccharomyces cerevisiae strain as claimed in claim 1 is fermented and cultured, and cannabidiol is obtained from the culture.