A recombinant saccharomyces cerevisiae strain with high yield of cannabigerolic acid and a construction method and application thereof

Abstract

The present application relates to the technical field of metabolic engineering, and in particular to a recombinant Saccharomyces cerevisiae strain for high-yield of cannabigerolic acid and a construction method and application thereof. The present application first knocks out the beta-oxidation pathway gene POX1, solves the problem of degradation of hexanoic acid substrate; then strengthens the expression of OA pathway enzymes TKS and / or OAC, replaces the expression promoter of fatty acid synthase FAS1, and improves the conversion rate of hexanoic acid substrate; further expresses the endoplasmic reticulum membrane synthesis related gene INO2, and expresses the cannabigerolic acid synthase gene SOD1-tCsPT4n or CUP1-tCsPT4n containing a fusion protein, thereby improving the synthesis efficiency of cannabigerolic acid. Finally, by regulating the expression level of part of genes in the Saccharomyces cerevisiae cell through the above gene editing, the purpose of high-yield of cannabigerolic acid is achieved.

Description

Technical Field

[0001] This invention relates to the field of metabolic engineering technology, specifically to a recombinant Saccharomyces cerevisiae strain that is metabolically engineered to produce high levels of cannabinoid phenolic acids, its construction method, and its application. Background Technology

[0002] The plant-derived cannabinoid family, isolated from cannabis, comprises over 100 species and has garnered widespread attention from academia and the business community due to its unique medicinal properties. Its potential medical applications have been extensively studied, including antibacterial, anti-inflammatory, antitumor, anti-anxiety, and antidepressant effects. Furthermore, it shows significant clinical potential for treating various human diseases such as epilepsy, diabetes, and Parkinson's syndrome. To date, cannabinoid therapeutics for various indications have been approved and used, demonstrating the medical and commercial value of cannabinoids. Cannabinoid glycosides (CBGA), as an important intermediate, can be converted into various other cannabinoids by cannabinoid synthases, and after decarboxylation, cannabinoid glycosides can be obtained. However, the content of CBGA in cannabis plants is low, making it difficult to obtain. Therefore, the efficient synthesis of CBGA products using Saccharomyces cerevisiae strains has significant application value.

[0003] Cannabinoids are found in low abundance in plants, making it difficult to obtain pure samples from plants through plant extraction methods due to the presence of multiple cannabinoids. Furthermore, the complex chemical structures of cannabinoids make chemical synthesis of cannabinoids and their derivatives complex, expensive, and inefficient. In contrast, microbial cell synthesis of cannabinoids shows great potential. Luo et al. synthesized cannabinoid phenolic acids and other cannabinoid products by introducing a heterologous cannabinoid synthesis pathway into a chassis strain of *Saccharomyces cerevisiae*. Luo et al. designed a natural mevalonic acid pathway to provide high-throughput geranylide pyrophosphate (GPP) and introduced a heterologous oleic acid (OA) synthesis pathway. They also introduced the *CsPT4* gene, a geranylide pyrophosphate:oleic acid geranylide transferase derived from cannabis plants, to catalyze the synthesis of cannabinoid phenolic acid products from GPP and OA substrates. Using hexanoic acid as a substrate, 7.2 mg / L CBGA was produced.

[0004] Existing synthetic pathways for cannabinoid phenolic acids include: Figure 1 As shown, sugar (i.e., the carbon source) produces the intermediate acetyl-CoA via glycolysis and acetyl-CoA synthase. Acetyl-CoA then proceeds via the mevalonate pathway to geranyl pyrophosphate, one of the precursors to cannabinoid phenolic acids. Simultaneously, hexanoic acid provides hexanoyl-CoA via acylation. Hexanoyl-CoA, catalyzed by tetraketon synthase and oleuropenic acid cyclase, yields oleic acid, another precursor to cannabinoid phenolic acids. These two precursors, geranyl pyrophosphate and oleic acid, are then catalyzed by geranyl pyrophosphate:oleic acid geranyl transferase derived from hemp plants to generate cannabinoid phenolic acids.

[0005] In patent (CN110914416A), feeding hexanoic acid to yeast can produce 22.9-72.9 mg / L of CBGA. However, existing cannabinoid phenolic acid synthesis pathways still have technical problems such as degradation of the metabolic precursor hexanoic acid, low conversion rate of hexanoic acid substrate, and low yield of cannabinoid phenolic acid synthesis.

[0006] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0007] In view of the shortcomings of the prior art, this invention first knocks out the β-oxidation pathway gene POX1, solving the problem of hexanoic acid substrate degradation; then, it enhances the expression of OA pathway enzymes TKS and / or OAC, and replaces the expression promoter of fatty acid synthase FAS1, improving the conversion rate of hexanoic acid substrate; furthermore, it expresses the endoplasmic reticulum membrane synthesis-related gene INO2, and the cannabinoid phenolic acid synthase genes SOD1-tCsPT4n and CUP1-tCsPT4n containing fusion proteins, improving the synthesis efficiency of cannabinoid phenolic acids. Finally, by regulating the expression levels of some genes in Saccharomyces cerevisiae cells through the above gene editing, the goal of high-yield cannabinoid phenolic acid is achieved.

[0008] It should be noted that this invention references the research of Luo, X. et al. (Complete biosynthesis of cannabinoids and their unnatural analogues in yeast) in 2019, using their research result yCAN31 as the original Saccharomyces cerevisiae strain. Based on the original Saccharomyces cerevisiae strain yCAN31, TKS and OAC were overexpressed, and the β-oxidation pathway gene POX1 was knocked out to construct the recombinant Saccharomyces cerevisiae strain yS413; FAS1 expression was downregulated to construct the recombinant Saccharomyces cerevisiae strain yS489; INO2 expression was upregulated to construct the recombinant Saccharomyces cerevisiae strain yS576; tCsPT4n was overexpressed and the gene was linked to the promoter through the protein tag SOD1 to construct the recombinant Saccharomyces cerevisiae strain yS628 which produces high levels of cannabinoids; tCsPT4n was overexpressed and the gene was linked to the promoter through the protein tag CUP1 to construct the recombinant Saccharomyces cerevisiae strain yS629 which produces high levels of cannabinoids.

[0009] Overexpression refers to the upregulation of gene expression, that is, the gene is excessively transcribed and translated, and the final gene expression product exceeds the normal level.

[0010] Downregulation refers to the restriction of gene expression, that is, the transcription and translation of the gene are restricted, resulting in the final gene expression product being lower than the normal level.

[0011] The knockout refers to the instruction to lose the function of a specific gene.

[0012] The technical solution of the present invention is as follows:

[0013] This invention provides a recombinant Saccharomyces cerevisiae strain, using yeast strains expressing enzymes in the cannabinoid phenolic acid (CBGA) synthesis pathway and capable of synthesizing CBGA as the starting strain. Some endogenous genes are overexpressed, downregulated, or knocked out. These endogenous genes include the fatty acid synthase FAS1 gene and the β-oxidation pathway POX1 gene. Simultaneously, some exogenous genes are overexpressed, including the oleuropeinate cyclase OAC gene, the tetraketon synthase TKS gene, the cannabinoid phenolic acid synthase tCsPT4n gene, and the INO2 gene related to endoplasmic reticulum membrane synthesis.

[0014] The nucleotide sequence of the TKS gene is shown in SEQ ID NO:1, the nucleotide sequence of the OAC gene is shown in SEQ ID NO:2, and the nucleotide sequence of the INO2 gene is shown in SEQ ID NO:4.

[0015] Furthermore, the recombinant Saccharomyces cerevisiae strain is based on Saccharomyces cerevisiae strain yCAN31, and any one of the following modifications (a) to (e) is made to the genetic basis of Saccharomyces cerevisiae strain yCAN31:

[0016] (a): Overexpression of the tetraketon synthase TKS gene and / or olive methyl cyclase OAC gene;

[0017] (b): Based on (a), the POX1 gene of the β-oxidation pathway was knocked out;

[0018] (c): Based on (b), overexpress the tetraketon synthase TKS gene and / or olive methyl cyclase OAC gene, and downregulate the fatty acid synthase FAS1 gene.

[0019] (d): Based on (c), overexpress the INO2 gene related to endoplasmic reticulum membrane synthesis;

[0020] (e): Based on (d), overexpress the truncated cannabinoid phenolic acid synthase tCsPT4n gene containing the SOD1 or CUP1 protein tag.

[0021] This invention yields a recombinant Saccharomyces cerevisiae strain that is metabolically engineered to produce high levels of cannabinoid phenolic acids. A series of transformed strains are shown in Table 1, and their sequences are listed in the sequence listing.

[0022] In (a) and (c), when simultaneously overexpressing the tetraketon synthase TKS gene and the oleuropetolate cyclase OAC gene, different codon formats are selected for expression, such as: TKS0-SCOAC0 (as shown in SEQ ID NO:10), TKS4-SCOAC4 (as shown in SEQ ID NO:6), and TKS5-SCOAC5 (as shown in SEQ ID NO:7). Comparison shows that TKS0, TKS4, and TKS5 have the same amino acid sequence (as shown in SEQ ID NO:9); SCOAC0, SCOAC4, and SCOAC5 have the same amino acid sequence (as shown in SEQ ID NO:8).

[0023] The modified strain (a) yielded strain yS022;

[0024] The modification described in (b) yielded strain yS291;

[0025] The modification described in (c) yielded strains yS349, yS413, and yS489.

[0026] The (d) modification yielded strain yS576;

[0027] The modification described in (e) yielded strains yS628 and yS629.

[0028] Optionally, when both the TKS gene and the OAC gene are overexpressed, the nucleotide sequence of the linker fragment connecting the TKS gene and the OAC gene is as shown in SEQ ID NO:3.

[0029] Optionally, in (b), after knocking out the POX1 gene, the olive benzoic acid cyclase OAC gene is inserted into the Saccharomyces cerevisiae genome and expressed by the pHSP26 promoter.

[0030] Optionally, the INO2 gene overexpression is achieved by inserting the INO2 gene into the Saccharomyces cerevisiae genome and initiating expression via the pPGK1 promoter.

[0031] Optionally, the overexpression of the truncated tCsPT4n gene containing the SOD1 or CUP1 protein tag is achieved by fusing the truncated tCsPT4n gene with the SOD1 or CUP1 protein tag and then inserting it into the Saccharomyces cerevisiae genome and initiating expression via the pSeGal2 promoter.

[0032] The present invention also provides a method for constructing the recombinant brewer's yeast, comprising the following steps:

[0033] (1) PCR amplification of the expression cassette of the gene to be overexpressed and integration into the Saccharomyces cerevisiae genome; and / or PCR amplification of the homologous fragment for the knockout gene and replacement of the gene to be knocked out on the Saccharomyces cerevisiae genome with the homologous fragment.

[0034] Gene knockout and / or insertion on the genome of Saccharomyces cerevisiae was achieved using CRISPR-Cas9 technology;

[0035] (2) Screening to obtain recombinant Saccharomyces cerevisiae strains with positive clones.

[0036] The present invention also provides the application of the recombinant Saccharomyces cerevisiae strain in the production of cannabinoid phenolic acids.

[0037] Optionally, the method for producing cannabinoid phenolic acid includes the following steps:

[0038] (1) The recombinant Saccharomyces cerevisiae strain was activated and cultured to obtain a seed culture of the recombinant Saccharomyces cerevisiae strain;

[0039] (2) The seed culture of the recombinant Saccharomyces cerevisiae strain was transferred to a suitable culture medium and the recombinant Saccharomyces cerevisiae strain was cultured under suitable conditions to obtain the fermentation culture product;

[0040] (3) The fermentation culture product is extracted and purified to obtain cannabinoid phenolic acid.

[0041] The recombinant Saccharomyces cerevisiae strain of the present invention can also be used to produce cannabinoids. Specifically, the cannabinoid acid produced by the recombinant Saccharomyces cerevisiae strain of the present invention is decarboxylated in vitro using an enzyme catalyst or a chemical catalyst to obtain cannabinoids.

[0042] The recombinant Saccharomyces cerevisiae strain described in this invention can also be used to produce downstream products of cannabinoids such as cannabinoids. Specifically, the cannabinoids produced by the recombinant Saccharomyces cerevisiae strain of this invention can be converted in vitro using enzyme catalysts or chemical catalysts to obtain various other cannabinoids.

[0043] The beneficial effects of this invention: This invention, through a series of gene editing modifications including overexpression of TKS and / or OAC, knockout of the POX1 gene in *Saccharomyces cerevisiae* cells, regulation of FAS1 and INO2 expression levels in *Saccharomyces cerevisiae* strains, and overexpression of cannabinoid phenolic acid synthase, ultimately provides a metabolically engineered *Saccharomyces cerevisiae* strain that produces high levels of cannabinoid phenolic acids, along with its construction method and applications. This achieves the goal of high cannabinoid phenolic acid production, solving the technical problems of low conversion rate and low yield of cannabinoid phenolic acid synthesized using hexanoic acid as a substrate in existing methods. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the synthetic pathway of cannabinoid phenolic acid in Saccharomyces cerevisiae strains in the prior art.

[0045] Figure 2 This is a schematic diagram comparing the hexanoic acid conversion rates of Saccharomyces cerevisiae strain yS022 and Saccharomyces cerevisiae strain yS291 in Example 1.

[0046] Figure 3 This is a schematic diagram comparing the CBGA yields of Saccharomyces cerevisiae strain yS022 and Saccharomyces cerevisiae strain yS291 in Example 1.

[0047] Figure 4 This is a schematic diagram comparing the hexanoic acid conversion rates of Saccharomyces cerevisiae strains yS349, yS413, and yS489 in Example 2.

[0048] Figure 5 This is a schematic diagram comparing the CBGA yields of Saccharomyces cerevisiae strains yS349, yS413, and yS489 in Example 2.

[0049] Figure 6 This is a schematic diagram comparing the CBGA yields of Saccharomyces cerevisiae strains yS576, yS629, and yS628 in Example 3. Detailed Implementation

[0050] This invention provides a recombinant Saccharomyces cerevisiae strain that is metabolically engineered to produce high levels of cannabinoid phenolic acids, along with its construction method and applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0051] Example 1

[0052] To address the issue of hexanoic acid substrate degradation, strain yS291 was constructed: Based on the original Saccharomyces cerevisiae strain yCAN31, overexpression of tetraketon synthase TKS and oleuropetolate cyclase OAC yielded strain yS022. Then, by knocking out the β-oxidation pathway gene POX1 in yS022, strain yS291 was obtained. The specific construction steps included:

[0053] The integrated fragment was amplified by PCR using 2×Phanta Max Master Mix (Phanta DNA polymerase): Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, the upstream homologous arm 1622b-UP and the downstream homologous arm 1622b-DOWN fragment were obtained; using the genome of a strain possessing the pGAL1 promoter as a template, the promoter fragment pGAL1 was obtained; using a plasmid containing the TKS0-SCOAC0 gene as a template, the fragment TKS0-SCOAC0 was obtained; and using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, the terminator ScENO1t was obtained. The final expression cassette was 1622b-UP-pGAL1-TKS0-SCOAC0-ScENO1t-1622b-DOWN. The 1622b-UP-pGAL1-TKS0-SCOAC0-ScENO1t-1622b-DOWN expression cassette fragment was then transformed into the original host yCAN31 to obtain strain yS022. Strains with correctly sequenced genes were then used for further culture.

[0054] The POX1 gene knockout was achieved by linking the upstream and downstream components of the POX1 gene. The integrated fragment was amplified by PCR using 2×Phanta Max MasterMix (Phanta DNA polymerase). Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, the upstream homologous arm POX1-UP and the downstream homologous arm POX1-DOWN were obtained. This combination of fragments was then transformed into strain yS022 to obtain strain yS291. Strains with correctly sequenced genes were then used for further culture.

[0055] Strains yS022 and yS291 were cultured separately. Ultimately, compared to its host strain yS022, strain yS291, which had the β-oxidation pathway gene POX1 knocked out using gene editing technology, showed a 7.05% increase in hexanoic acid conversion rate under the same culture conditions (see...). Figure 2 As shown), the CBGA conversion rate increased by 44.6% (see figure). Figure 3 ).

[0056] Example 2

[0057] To improve the conversion rate of hexanoic acid substrate, strain yS489 was constructed: Based on strain yS022 constructed in Example 1, the POX1 gene was knocked out while the olive oleanoic acid cyclase gene OAC was overexpressed to obtain strain yS349; based on yS349, the tetraketon synthase gene TKS and the olive oleanoic acid cyclase gene OAC were overexpressed to obtain strain yS413; based on yS413, the FAS1 promoter was replaced with pRPS25A to obtain strain yS489. The specific construction steps included:

[0058] The integrated fragment was amplified by PCR using 2×Phanta Max Master Mix (Phanta DNA polymerase): Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, the upstream homologous arm DPOX1-UP, the downstream homologous arm DPOX1-DOWN, the promoter pHSP26, and the terminator tHSP26 fragments were obtained; using a plasmid containing the OAC gene as a template, the OAC fragment was obtained; finally, the DPOX1-UP-pHSP26-OAC-HSP26t-DPOX1-DOWN expression cassette was obtained. The DPOX1-UP-pHSP26-OAC-tHSP26-DPOX1-DOWN expression cassette fragment was then transformed into the host yS022 to obtain strain yS349.

[0059] The integrated fragment was amplified by PCR using 2×Phanta Max Master Mix (Phanta DNA polymerase): Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, the following fragments were obtained: upstream homologous arm NDT80-UP fragment, downstream homologous arm NDT80-DOWN fragment, promoter pTDH3 fragment, terminator tHSP26 fragment, promoter pTDH1 fragment, terminator tADH1 fragment, promoter pPGK1 fragment, terminator tCYC1 fragment, promoter pSED1 fragment, and terminator tTDH1 fragment; [The text abruptly ends here, likely due to an incomplete translation or missing information.] Using plasmids of the SCOAC4-tCYC1, pSED1-TKS5-SCOAC5-tTDH1, and 4xOAC genes as templates, fragments of pPGK1-TKS4-SCOAC4-tCYC1, pSED1-TKS5-SCOAC5-tTDH1, and 4xOAC were obtained; ultimately, the NDT80-UP-4xOAC-pPGK1-TKS4-SCOAC4-tCYC1 / pSED1-TKS5-SCOAC5-tTDH1-NDT80-DOWN expression cassette was obtained. This NDT80-UP-4xOAC-pPGK1-TKS4-SCOAC4-tCYC1 / pSED1-TKS5-SCOAC5-tTDH1-NDT80-DOWN expression cassette was transformed into the host yS349 to obtain strain yS413.

[0060] The integrated fragment was amplified by PCR using 2×Phanta Max Master Mix (Phanta DNA polymerase). Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, the upstream homologous arm pfas1-UP fragment, the downstream homologous arm FAS1 gene fragment, and the promoter pRPS25A fragment were amplified, ultimately yielding the pfas1-UP-pRPS25A-FAS1 expression cassette fragment. This pfas1-UP-pRPS25A-FAS1 expression cassette fragment was then transformed into the host yS413 to obtain strain yS489. Strains with correctly sequenced genes were used for subsequent culturing.

[0061] Strains yS349, yS413, and yS489 were cultured separately. Finally, through gene editing technology, the expression of the OA pathway enzymes OAC and TKS was enhanced, and the expression promoter of fatty acid synthase FAS1 was replaced. Compared with their host strain (yS349), under the same culture conditions, the hexanoic acid conversion rate of strains yS349 and yS489 increased by 3.80% (yS413) and 33.52% (yS489), respectively (see...). Figure 4 CBGA production increased by 142.3% (yS413) and 380.0% (yS489) respectively (see...). Figure 5 ).

[0062] Example 3

[0063] To improve the synthesis efficiency of cannabinoid phenolic acids, strains yS628 and yS629 were constructed: Based on strain yS489 constructed in Example 2, the endoplasmic reticulum membrane synthesis-related gene INO2 was overexpressed to obtain strain yS576; based on yS576, the cannabinoid phenolic acid synthase gene tCsPT4n was overexpressed by linking the protein tags SOD1 and CUP1 to the promoter pSeGal2, respectively, to obtain strains yS628 and yS629. The specific construction steps included:

[0064] The integrated fragment was amplified by PCR using 2×Phanta Max Master Mix (Phanta DNA polymerase): Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the 1014a-UP fragment (upstream homologous arm of the 1014a site), the 1014a-DOWN fragment (downstream homologous arm of the 1014a site), the pPGK1 promoter fragment, and the INO2-INO2t fragment were obtained by PCR amplification. Finally, the 1014a-UP-pPGK1-INO2-INO2t-1014a-DOWN expression cassette was obtained, and then the 1014a-UP-pPGK1-INO2-INO2t-1014a-DOWN expression cassette fragment was transformed into the host yS489 to obtain strain yS576.

[0065] Through 2×Phanta Max Master Mix (Phanta DNA polymerase was used to amplify the integrated fragments via PCR: using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the upstream homologous arm HO-UP fragment, the downstream homologous arm HO-DOWN fragment, and the terminator THD1t fragment were obtained by PCR amplification; using the genome containing the promoter pSeGal2 gene as a template, the pSeGal2 fragment was amplified; using the genome containing the SOD1 and CUP1 genes as a template, the SOD1 and CUP1 fragments were amplified; using the plasmid containing the tCsPT4n gene as a template, the tCsPT4n fragment was amplified; finally, the HO-UP-pSeGal2-SOD1-tCsPT4n-tTDH1-HO-DOWN expression cassette and the HO-UP-pSeGal2-CUP1-tCsPT4n-tTDH1-HO-DOWN expression cassette were obtained, and then these two expression cassette fragments were transformed into the host yS576 to obtain strains yS628 and yS629, respectively. Strains yS576, yS628, and yS629 with correct gene sequencing were used for subsequent culture.

[0066] Strains yS576, yS628, and yS629 were cultured separately. Finally, through gene editing technology, the endoplasmic reticulum membrane synthesis-related genes INO2, SOD1-tCsPT4n, and CUP1-tCsPT4n were overexpressed. Compared with their host strain yS576, under the same culture conditions, strain yS629 showed a 42.2% (yS629) and strain yS628 showed a 66.9% (yS628) increase in CBGA production, respectively (see...). Figure 6 ).

[0067] The yeast transformation methods in Examples 1-3 above all use the lithium acetate / PEG3350 transformation method, specifically including: inoculating the host strain into 1×YPD medium and culturing overnight; then inoculating into fresh 2×YPD medium to an initial OD value of 0.2, continuing to culture for 4 hours, taking 5OD of bacterial culture, centrifuging, discarding the supernatant, and washing twice with sterile ultrapure water to obtain yeast cells; preparing a DNA mixing system, each system containing 5OD of host cells, and mixing it with the DNA mixture to resuspend the cells; the DNA mixture is composed of the inserted fragment, tool plasmid, and ddH2O; adding the lithium acetate transformation mixture to the suspended cells, obtaining transformed cells after heat shock, plating onto the corresponding auxotrophic selection plates, and culturing in an incubator for the appropriate number of days; obtaining single colonies, which are the recombinant Saccharomyces cerevisiae strains; after sequencing to verify the transformation, taking the correctly sequenced strains for streak plating and cryopreservation in glycerol.

[0068] 2×YPD medium formulation: yeast extract 20.0 g / L, peptone 40.0 g / L, glucose 40.0 g / L.

[0069] Lithium acetate conversion mixture: 50% W / V PEG3350 260 μL, 1 mol / L LiOAc 36 μL, denatured salmon sperm DNA 10 μL (denatured salmon sperm DNA was placed in a 95℃ metal bath for 5 min before use), ddH2O 4 μL.

[0070] The cultivation methods in Examples 1-3 above include the following steps: Single colonies of the strain are cultured overnight for 16 hours in 2 mL of 1×YPD (2% glucose, 1% yeast extract, 2% peptone) 24-well plates at 30°C and 800 rpm. The overnight culture is then diluted 10-fold with 1×YPD, and the OD of the culture is measured using a UV spectrophotometer at 600 nm. The initial OD is then transferred to 2 mL of 1×YPG medium (2% galactose, 1% yeast extract, 2% peptone) for further cultivation. The feeding method is as follows: after transfer, hexanoic acid (0.2 mM) is added every 12 hours for a total of five times. Starting 24 hours after transfer, galactose (2% w / v) is added every 24 hours for a total of three times, with a final concentration of 2%.

[0071] The sample preparation methods in Examples 1-3 above include the following steps: After culturing for 84 hours, collect 200 μL of bacterial culture as the sample. After sample collection, based on the sample OD600, add 0.2 mL of 0.5 mm diameter glass beads and 0.4 mL of ethyl acetate:formic acid (0.05%). In a high-speed tissue homogenizer, process at 68 Hz for 180 s, with 30 s intervals, repeating four times. After short-term centrifugation, take 0.28 mL of the upper organic layer into a 1.5 mL centrifuge tube, repeating twice, and combine the collected upper organic phases. The organic layers from the three extractions are dried using a vacuum dryer at 45°C for 1 hour until no solvent residue remains. The sample was resuspended in 140 μL of AHF (acetonitrile:H2O:formic acid = 80:20:0.05%) containing 15 μM internal standard PHB (propyl p-hydroxybenzoate solution, standard substance), filtered through a 0.22 μm PVDF membrane into the inner tube of the liquid chromatography detection bottle, and used as the test sample. Three parallel samples were prepared for each sample.

[0072] The sample detection methods in Examples 1-3 above include the following detection conditions: see Table 2.

[0073] The strain construction information for Examples 1-3 above is shown in Table 1 below.

[0074] Table 1. Strain Construction Information

[0075]

[0076]

[0077] Table 2. HPLC detection conditions

[0078]

[0079]

[0080] It should be noted that in the nucleotide and amino acid sequence listing of this invention, SEQ ID NO:5 is the nucleotide sequence of 4xOAC, SEQ ID NO:11 is the nucleotide sequence of pRPS25A, SEQ ID NO:12 is the nucleotide sequence of pSeGal2, SEQ ID NO:13 is the nucleotide sequence of pPGK1, SEQ ID NO:14 is the nucleotide sequence of SOD1-tCsPT4n, SEQ ID NO:15 is the amino acid sequence of tCsPT4n, SEQ ID NO:16 is the nucleotide sequence of CUP1-tCsPT4n, SEQ ID NO:17 is the nucleotide sequence of pHSP26, SEQ ID NO:18 is the nucleotide sequence of tHSP26, SEQ ID NO:19 is the nucleotide sequence of PTDH3, SEQ ID NO:20 is the nucleotide sequence of pGAL1, SEQ ID NO:21 is the nucleotide sequence of tCYC1, SEQ ID NO:22 is the nucleotide sequence of pTDH1, SEQ ID NO:23 is the nucleotide sequence of tADH1, and SEQ ID NO:24 is the nucleotide sequence of pGAL1. SEQ ID NO:24 is the nucleotide sequence of tENO1, SEQ ID NO:25 is the nucleotide sequence of pSED1, and SEQ ID NO:26 is the nucleotide sequence of tTDH1.

[0081] In summary, this invention addresses three problems in the synthesis of cannabinoid phenolic acids through a series of gene editing modifications: improving the supply of propionyl-CoA and oleic acid; increasing the endoplasmic reticulum area; and enhancing the efficiency of CBGA synthesis by overexpressing geranyl pyrophosphate:olate geranyl transferase using a protein tag.

[0082] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A recombinant Saccharomyces cerevisiae strain metabolically engineered to produce high levels of cannabinoid phenolic acids, characterized in that, The recombinant Saccharomyces cerevisiae strain is based on Saccharomyces cerevisiae strain yCAN31, and any one of the following modifications (b) to (e) is made to the genes of Saccharomyces cerevisiae strain yCAN31: (a): Overexpression of the tetraketon synthase TKS gene and the olive methyl cyclase OAC gene; (b): Based on (a), knock out the POX1 gene of the β-oxidation pathway; (c): Based on (b), overexpress the tetraketon synthase TKS gene and the olive oleate cyclase OAC gene, and downregulate the fatty acid synthase FAS1 gene. (d): Based on (c), overexpress the INO2 gene, which is related to endoplasmic reticulum membrane synthesis; (e): Based on (d), overexpress the truncated cannabinoid phenolic acid synthase tCsPT4n gene containing the SOD1 or CUP1 protein tag; In (a) and (c), when both the tetraketon synthase (TKS) gene and the oleuropeyl benzoate cyclase (OAC) gene are overexpressed, The modification in (a) yielded strain yS022, which contains TKS0-SCOAC0, and the nucleotide sequence of TKS0-SCOAC0 is shown in SEQ ID NO:

10. The modification described in (b) yielded strain yS291; The modification in (c) yielded strains yS349, yS413, and yS489. Strain yS413 comprises TKS4-SCOAC4 and TKS5-SCOAC5. The nucleotide sequence of TKS4-SCOAC4 is shown in SEQ ID NO:6, and the nucleotide sequence of TKS5-SCOAC5 is shown in SEQ ID NO:

7. The (d) modification yielded strain yS576; The modification described in (e) yielded strain yS628 or strain yS629.

2. The recombinant Saccharomyces cerevisiae strain with high cannabinoid phenolic acid production, as described in claim 1, is characterized in that... When both the TKS gene and the OAC gene are overexpressed, the nucleotide sequence of the linker fragment connecting the TKS gene and the OAC gene is shown in SEQ ID NO:

3.

3. The recombinant Saccharomyces cerevisiae strain with high cannabinoid phenolic acid production, as described in claim 1, is characterized in that... In (b), after knocking out the POX1 gene, the olive benzoic acid cyclase OAC gene is inserted into the Saccharomyces cerevisiae genome and expressed by the pHSP26 promoter.

4. The recombinant Saccharomyces cerevisiae strain with high cannabinoid phenolic acid production, as described in claim 1, is characterized in that... The INO2 gene overexpression is achieved by inserting the INO2 gene into the genome of Saccharomyces cerevisiae and initiating expression via the pPGK1 promoter.

5. The recombinant Saccharomyces cerevisiae strain with high cannabinoid phenolic acid production, as described in claim 1, is characterized in that... The overexpression of the truncated tCsPT4n gene containing the SOD1 or CUP1 protein tag is achieved by fusing the truncated tCsPT4n gene with the SOD1 or CUP1 protein tag, inserting it into the Saccharomyces cerevisiae genome, and then initiating expression via the pSeGal2 promoter.

6. A method for constructing a recombinant Saccharomyces cerevisiae strain with high production of cannabinoid phenolic acids through metabolic engineering as described in any one of claims 1 to 5, characterized in that, Includes the following steps: (1) PCR amplification to obtain the expression cassette of the gene to be overexpressed, and integration of the expression cassette into the Saccharomyces cerevisiae genome; and / or PCR amplification to obtain a homologous fragment for knocking out the gene, and replacement of the gene to be knocked out on the Saccharomyces cerevisiae genome with the homologous fragment; Gene knockout and / or insertion on the genome of Saccharomyces cerevisiae was achieved using CRISPR-Cas9 technology; (2) Screening to obtain recombinant Saccharomyces cerevisiae strains with positive clones.

7. The use of any of the recombinant Saccharomyces cerevisiae strains according to claims 1 to 5 in the production of cannabinoid phenolic acids.

8. The application according to claim 7, characterized in that, The method for producing cannabinoid phenolic acid includes the following steps: (1) The recombinant Saccharomyces cerevisiae strain was activated and cultured to obtain a seed culture of the recombinant Saccharomyces cerevisiae strain; (2) The seed culture of the recombinant Saccharomyces cerevisiae strain was transferred to a suitable culture medium for producing cannabinoid phenolic acid and cultured to obtain cannabinoid phenolic acid.

9. The use of any of the recombinant Saccharomyces cerevisiae strains according to claims 1 to 5 in the production of cannabinoids, a downstream form of cannabinoids, from cannabinoid phenolic acids.

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