A recombinant Saccharomyces cerevisiae producing cannabigerolic acid, its construction method and application

Abstract

This application relates to a recombinant Saccharomyces cerevisiae for producing cannabigerolic acid, which is prepared from a basic Saccharomyces cerevisiae, wherein the following endogenous genes of the basic Saccharomyces cerevisiae are overexpressed, downregulated or knocked out: pep4 , gal80 , gal4 and fas1 ; exogenous genes encoding the following enzymes are overexpressed: tetraketo synthase ( Cs. TKS) gene, olivetolic acid cyclase ( Cs. OAC) gene, geranyl diphosphate:olivetolate geranyltransferase ( Cs. PT4n) gene and variant acetyl-CoA synthase ( Se. ACS) gene. This application uses Saccharomyces cerevisiae capable of synthesizing CBGA as the original strain, selectively overexpresses Cs. TKS, Cs. OAC, Cs. PT4, Se. ACS, selectively knocks out gal80 and overexpresses gal4 , selectively knocks out pep4 , selectively overexpresses OAC with or without linker fusion of Cs. , localizes Cs. PT4n enzyme to the plasma membrane with or without signal peptide, balances the metabolic flux, optimizes the heterologous synthesis pathway, and significantly improves the yield of CBGA.

Description

Technical Field

[0001] The present application relates to the technical fields of synthetic biology and genetic engineering, and particularly relates to a recombinant Saccharomyces cerevisiae for producing cannabigerolic acid and a construction method and application thereof. Background Art

[0002] Due to its medicinal properties, cannabis has been cultivated globally for thousands of years. To date, more than 100 plant-derived cannabinoids have been isolated from cannabis. Cannabinoids have potential medical uses (such as antibacterial, anti-inflammatory, anti-tumor, anti-anxiety, and antidepressant effects) and have great clinical potential in the treatment of various human diseases (such as epilepsy, diabetes, and Parkinson's syndrome). Currently, cannabinoid therapeutics for various indications have been approved and used.

[0003] Among them, cannabigerolic acid (CBGA) is an important precursor in the synthetic pathways of several other cannabinoids. Since CBGA can be converted into several other cannabinoids, for example, it can be converted into three other cannabinoids: tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and cannabichromenic acid (CBCA) through corresponding enzymes. After decarboxylation of these three cannabinoids, cannabigerol (CBG), tetrahydrocannabinol (THC), and cannabidiol (CBD) are obtained respectively.

[0004] However, cannabinoids are present in plants in low abundance and coexist with other relatively more abundant cannabinoids, making it difficult to obtain pure samples from plants. At the same time, due to the complex chemical structure of cannabinoids, the methods for chemically synthesizing cannabinoids and their derivatives are complex, expensive, and have low efficiency. Therefore, it is necessary to seek economically viable alternative sources for large-scale commercial production. Producing cannabinoids and their analogs by introducing heterologous biosynthetic pathways into chassis strains is considered a promising method for a stable cannabinoid supply chain.

[0005] Currently, there are research reports that Saccharomyces cerevisiae has completely synthesized several cannabinoids of interest using galactose, such as cannabigerolic acid (CBGA), Δ 9 -tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), Δ 9 -tetrahydrocannabidiolic acid (THCVA), and cannabidiolic acid (CBDVA). This study designed the natural mevalonate pathway to provide high-throughput geranyl pyrophosphate (GPP), and introduced heterologous, multi-biological-derived hexanoyl-CoA to supply oleic acid (OA) through the biosynthetic pathway. At the same time, the heterologous geranyl pyrophosphate:oleic acid geranyltransferase Cs.PT4 gene was introduced, and the important precursor compound: CBGA was synthesized using GPP and oleic acid OA as substrates.

[0006] However, there are multi-pathway competitions in the heterologous biosynthesis of CBGA, which can lead to metabolic imbalance and then result in a low yield of cannabinoids. Summary of the Invention

[0007] In order to improve the yield of cannabigerolic acid (CBGA) and reduce the impact of multi-pathway competition in the heterologous biosynthesis of CBGA on the yield of CBGA, the present application provides a recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid, a construction method thereof, and an application thereof.

[0008] In a first aspect, a recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid provided by the present application adopts the following technical solution: A recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid, wherein the recombinant Saccharomyces cerevisiae is prepared from a base Saccharomyces cerevisiae, and the following endogenous genes of the base Saccharomyces cerevisiae are overexpressed, down-regulated or knocked out: pep4, gal80, gal4, and fas1; exogenous genes encoding the following enzymes are overexpressed: tetraketo synthase (Cs.TKS) gene, olivetolic acid cyclase (Cs.OAC) gene, truncated geranyl diphosphate: oleate geranyltransferase (Cs.PT4n) gene, and variant acetyl-CoA synthase (Se.ACS) gene.

[0009] By adopting the above technical solution, the present application uses Saccharomyces cerevisiae capable of synthesizing CBGA as the original strain, selectively overexpresses Cs.TKS, Cs.OAC, Cs.PT4n, Se.ACS L641P , selectively knocks out gal80 and overexpresses gal4, selectively knocks out pep4, selectively overexpresses Cs.OAC fused with or without a linker, localizes the Cs.PT4 enzyme to the plasma membrane with or without a signal peptide, balances the metabolic flux, optimizes the heterologous synthesis pathway, and greatly improves the yield of CBGA.

[0010] In some embodiments, the overexpression, down-regulation or knockout of some endogenous genes is any one of (a) to (i):

[0011] (a) Overexpress the Cs.TKS gene;

[0012] (b) Overexpress the Cs.OAC gene;

[0013] (c) Overexpress Cs.PT4;

[0014] (d) Down-regulate fas1;

[0015] (e) Overexpress gal4;

[0016] (f) Simultaneously have (a), (b), (c), (d) and (e);

[0017] (g) On the basis of (f), knock out gal80;

[0018] (h) Based on (g), knockout pep4;

[0019] (i) Based on (g), overexpress the variant Se.ACS gene;

[0020] (j) Based on (g), fuse the Cs.PT4n gene with the SNC1 signal peptide.

[0021] In some embodiments, the basic Saccharomyces cerevisiae expresses the enzymes of the synthetic cannabigerolic acid (CBGA) pathway and is capable of synthesizing CBGA, wherein the enzymes include tetraketide synthase (Cs.TKS), Cs.OAC, truncated geranyl diphosphate:olivetolate geranyltransferase (Cs.PT4n), and Se.ACS L641P 。

[0022] In some embodiments, the nucleotide sequence of the truncated Cs.PT4 gene Cs.PT4n is SEQ ID NO:1; the truncation method includes: lacking the N-terminal amino acids 1-76 of the full-length amino acid sequence Cs.PT4 shown in SEQ ID NO:0.

[0023] In some embodiments, when overexpressing the Cs.OAC gene, it is ligated using linker (SEQ ID NO:2), and the coding gene is inserted into the basic Saccharomyces cerevisiae genome and expressed through the pHSP26 promoter.

[0024] In some embodiments, when overexpressing Cs.OAC, it is not ligated through linker (SEQ ID NO:2), and the coding gene is directly inserted into the basic Saccharomyces cerevisiae genome and expressed through a strong promoter.

[0025] In some embodiments, when overexpressing the Cs.OAC gene, it can be ligated with Cs.TKS through linker (SEQ ID NO:35), and then the coding gene is inserted into the basic Saccharomyces cerevisiae genome and expressed through an inducible promoter.

[0026] In some embodiments, the overexpression of Cs.OAC includes inserting the coding gene into the Saccharomyces cerevisiae genome and initiating expression through a strong promoter.

[0027] In some embodiments, the strong promoter is the endogenous pPGK1 promoter (the promoter sequence is 600 bp upstream of the pgk1 gene) or the pTDH3 promoter (the promoter sequence is 600 bp upstream of the tdh3 gene).

[0028] In some embodiments, the inducible promoter is the endogenous pGAL1 promoter (the promoter sequence is 535 bp upstream of the gal1 gene).

[0029] In some embodiments, expressing the gene encoding Cs.PT4n includes inserting the heterologous Cs.PT4n gene into the genome of Saccharomyces cerevisiae and initiating expression through the constitutive pTDH3 promoter.

[0030] In some embodiments, overexpression refers to upregulating the expression of a gene, that is, the gene is overly transcribed and translated, and the final gene expression product exceeds the normal level.

[0031] In some embodiments, downregulation refers to downregulating the expression of a gene, that is, the gene is restricted in transcription and translation, and the final gene expression product is lower than the normal level.

[0032] In some embodiments, knockout refers to rendering a specific gene functionless.

[0033] In some embodiments, the fas1 gene encodes a subunit of cytoplasmic fatty acid synthase and is required for the biosynthesis of long-chain fatty acids. Downregulation of fas1 refers to using pHXT1, which is not expressed in semi-lactose, to downregulate the expression of fas1.

[0034] In some embodiments, the pep4 gene encodes vacuolar protease. Knocking out pep4 refers to rendering pep4 on the genome of Saccharomyces cerevisiae non-expressed by deleting the coding region of pep4.

[0035] In some embodiments, the gal80 gene encodes a transcriptional regulatory factor involved in the repression of Gal genes. Knocking out gal80 refers to rendering gal80 on the genome of Saccharomyces cerevisiae non-expressed by deleting the coding region of gal80.

[0036] In some embodiments, the gal4 gene encodes a transcriptional regulatory factor involved in the activation of Gal genes, the gal4 gene is expressed by pGAL4(OC), and the nucleotide sequence of pGAL4(OC)-gal4 is SEQ ID NO:37.

[0037] In some embodiments, Se.ACS is Se.ACS from Salmonella enterica L641P , and the sequence is SEQ ID NO:3.

[0038] In some embodiments, the SNC1 signal peptide localizes the Cs.PT4n enzyme to the plasma membrane, and the sequence of the SNC1 signal peptide is SEQ ID NO:4.

[0039] Second aspect, the present application provides a method for constructing a recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid, adopting the following technical solution:

[0040] A method for constructing a recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid, comprising the following steps:

[0041] (1) PCR amplify the expression cassette of the gene to be overexpressed and integrate it into the genome of Saccharomyces cerevisiae; or,

[0042] PCR amplify the homologous fragment of the gene to be knocked out and replace the gene to be knocked out on the genome of Saccharomyces cerevisiae with the homologous fragment; achieve gene knockout and insertion on the genome of Saccharomyces cerevisiae;

[0043] (2) Screen and obtain positive clones.

[0044] Third aspect, the present application provides an application of a recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid in the production of cannabigerolic acid, comprising the following steps:

[0045] (1) Activate and culture the recombinant Saccharomyces cerevisiae, and culture to obtain a recombinant Saccharomyces cerevisiae seed solution,

[0046] (2) Transfer the recombinant Saccharomyces cerevisiae seed solution to a culture medium and culture the recombinant Saccharomyces cerevisiae to produce cannabigerolic acid.

[0047] Fourth aspect, the present application provides an application of a recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid in the production of downstream cannabinoids of cannabigerolic acid.

[0048] In some embodiments, the downstream cannabinoid of cannabigerolic acid is cannabigerol.

[0049] Specifically, it comprises the following steps: Separate and purify the cannabigerolic acid produced by the recombinant Saccharomyces cerevisiae, and then carry out decarboxylation in vitro using an enzyme catalyst or a chemical catalyst to obtain cannabigerol.

[0050] In some embodiments, the downstream cannabinoid of cannabigerolic acid is cannabidiol.

[0051] Specifically, it comprises the following steps: Express a decarboxylase in the recombinant Saccharomyces cerevisiae to decarboxylate cannabigerolic acid to obtain cannabidiol; or, separate and purify the cannabigerolic acid produced by the recombinant Saccharomyces cerevisiae, and then carry out decarboxylation in vitro using an enzyme catalyst or a chemical catalyst to obtain cannabidiol.

[0052] In summary, the present application includes at least one of the following beneficial technical effects:

[0053] The present application uses Saccharomyces cerevisiae capable of synthesizing CBGA as the original strain, and selectively overexpresses Cs.TKS, Cs.OAC, Cs.PT4n and Se.ACSL641P Selectively knockout gal80 and overexpress gal4, selectively knockout pep4, selectively overexpress Cs.OAC fused or not fused with a linker, and localize Cs.PT4n enzyme to the plasma membrane with or without a signal peptide, which balanced the metabolic flux, optimized the heterologous synthesis pathway, and significantly increased the yield of CBGA. Description of the Drawings

[0054] Figure 1 It is the synthesis pathway of cannabigerolic acid in Saccharomyces cerevisiae in the background art;

[0055] Figure 2 Based on the CBGA-producing strain yCAN31, the pathway enzymes Cs.TKS, Cs.OAC, Cs.PT4n, and Se.ACS L641P were modified to optimize the heterologous gene synthesis pathway in the chassis strain and further increase the yield of CBGA. Among them, the knockout of gal80 and the overexpression of gal4 as well as the knockout of the vacuolar protease pep4 are not marked in the figure.

[0056] Figure 3 It is the comparison of the cannabigerolic acid (CBGA) yields of each constructed strain. Detailed Description of the Invention

[0057] To make the above objects, features, and advantages of the present invention more understandable, the following provides a detailed description of the specific embodiments of the present invention. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the spirit of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0058] The terms in this application are described as follows:

[0059] 1. pep4: Encodes the vacuolar protease, namely protease A, which can activate the activities of some other proteases;

[0060] 2. gal80: A transcriptional regulatory factor involved in inhibiting the transcription of Gal genes, and gal4 is inhibited by gal80;

[0061] 3. gal4: Encodes a transcriptional activator protein that can recognize a 17-bp long sequence of the upstream activation sequence (UASg) of the gene promoter: 5'-CGGRNNRCYNYNYNCNCCG-3';

[0062] 4. fas1: Encodes a subunit of the cytoplasmic fatty acid synthase, which is required for the biosynthesis of long-chain fatty acids;

[0063] 5. Overexpression: It refers to the up-regulation of gene expression, that is, the gene is over-transcribed and translated, and the final gene expression product exceeds the normal level;

[0064] 6. Down-regulation: It refers to the down-regulation of gene expression, that is, the gene is restricted in transcription and translation, and the final gene expression product is lower than the normal level;

[0065] 7. Knockout: It is to use a DNA fragment containing a certain known sequence to carry out homologous recombination with the gene with the same or similar sequence in the receptor cell genome, so that the specific gene function in the receptor cell genome loses its effect.

[0066] In the examples of this application, the PCR amplification method, the fusion method of different fragments, the gene knockout and overexpression methods can adopt common technical means in the art, such as fusion PCR, homologous recombination, CRISPR-Cas9 technology. The enzymes and reagent kits used are all conventional commercially available products.

[0067] The experimental methods in the examples of this application are as follows:

[0068] 1. Transformation, using lithium acetate / PEG3350.

[0069] (1) First, activate the host strain in 1×YPD medium and culture it overnight at 30°C and 200 rpm;

[0070] (2) Inoculate into a new 2×YPD medium to make the initial OD value 0.2, and continue to culture at 30°C for 4.5 h;

[0071] (3) Take 5 OD of the bacterial liquid, centrifuge at 3000 rcf at room temperature for 5 min, discard the supernatant, and wash twice with sterilized ultrapure water to obtain yeast cells;

[0072] (4) Prepare a DNA mixing system. Each system contains 5 OD of host cells and is mixed with 50 μL of DNA mixture to resuspend the cells; among them, 50 μL of DNA mixture is composed of 2 μg of the inserted fragment, 250 ng of the tool plasmid, and sufficient ddH2O;

[0073] (5) Add the lithium acetate transformation mixture to the suspended cells, obtain transformed cells after heat shock at 42°C for 40 min, coat them on the screening plate, and culture them in an incubator at 30°C for an appropriate number of days to obtain single colonies, which are recombinant Saccharomyces cerevisiae;

[0074] (6) After sequencing verification of the transformation, take the strains with correct sequencing for streak preservation and glycerol cryopreservation.

[0075] 2. Colony PCR and sequencing verification

[0076] After monoclonal colonies grow on the plate to be screened, colony PCR and sequencing verification are carried out. The specific steps are as follows: Use a pipette tip to pick up a small amount of cells and place them into 20 μL of 20 mmol / L NaOH solution respectively, vortex to mix evenly, and incubate at 95 °C in a metal bath for 20 min for lysis. After vortexing and mixing, take 1 μL as the template for colony PCR reaction. The reaction primers vary according to different verification sequences. Compare the sizes of the cloned bands and the negative cloned bands, and select the bacterial liquid of the positive clone in colony PCR for sequencing verification.

[0077] 3. Cultivation of recombinant Saccharomyces cerevisiae strains Using 2 mL of 1×YPD as the medium, pick a single colony of recombinant yeast and culture it overnight for 16 h in a 24-well plate. The shaker parameters are 30 °C and 800 rpm. The next day, after diluting the overnight bacterial liquid by an appropriate multiple, measure the OD of the bacterial liquid using an ultraviolet spectrophotometer, with the wavelength set at 600 nm. Make the initial OD 0.2, and transfer the bacterial liquid to fresh 2 mL of 1×YPG medium for culture.

[0078] The feeding method is as follows: After transfer, add 0.4 mM caproic acid every 12 h and add 200 μL of 20% galactose every 24 h.

[0079] After culturing for 87 h, collect 200 μL of the bacterial liquid as a sample to detect the CBGA yield.

[0080] 4. Detection method for the amount of CBGA produced by recombinant Saccharomyces cerevisiae

[0081] After sample collection, according to the sample OD 600 , add 0.2 mL of glass beads with a diameter of 0.5 mm and 0.4 mL of a mixed solution of ethyl acetate and formic acid. The volume ratio of ethyl acetate to formic acid is 99.95:0.05. In a high-speed tissue grinder, process at 65 Hz for 180 s, with an interval of 30 s, and repeat four times; after centrifugation, take 0.28 mL of the upper organic layer into a 1.5 mL centrifuge tube, repeat twice, and combine the collected upper organic phases. Evaporate the organic layers extracted three times, mode V-AL, dry at 45 °C for 1 h until there is no solvent residue. Resuspend with 140 μL of resuspension solution AHF (acetonitrile: H2O + formic acid = 80:20 + 0.05% formic acid) containing an internal standard PHB (propyl p-hydroxybenzoate solution reference material, 15 μM), and filter through a 0.22 μm PVDF filter membrane into the inner insert of a liquid phase detection bottle as the detection sample. There are three parallels for each sample. After the samples are prepared, detect them by HPLC. The detection conditions are shown in Table 1.

[0082] Table 1 HPLC detection conditions

[0083]

[0084] 5.2 × YPD medium formula: yeast extract 20.0 g / L, peptone 40.0 g / L, glucose 40.0 g / L.

[0085] 6. Lithium acetate transformation mixture: 50% W / V PEG3350 260 μL, 1 mol / L LiOAc 36 μL, denatured salmon sperm DNA 10 μL (denature the denatured salmon sperm DNA in a metal bath at 95 °C for 5 min before use), ddH2O 4 μL.

[0086] 7. Screening plate formula lacking uracil: yeast nitrogen source stock solution 1.7 g / L, ammonium sulfate 5 g / L, agar 20 g / L, glucose 20 g / L, and various amino acids as shown in Table 2. Among them, glucose is sterilized separately.

[0087] Table 2 Contents of various amino acids in the screening plate

[0088] Amino acid Content (mg / L) Amino acid Content (mg / L) Adenine hemisulfate 18 L-Phenylalanine 76 L-Alanine 76 L-Proline 76 L-Argnine 76 L-Serine 76 L-Aspartic acid 76 L-Threonine 76 L-Asparagine 76 L-Tryptophane 76 L-Cysteine 76 L-Tyrosine 76 L-Glutamic acid 76 L-Valine 76 L-Glycine 76 L-Methionine 76 L-Isoleucine 76 L-Lysine 76 L-Glutamine 76 L-Leucine 360 L-Histidine 76

[0089] The gene information of the strain yCAN31 used in each example of this application is shown in Table 3.

[0090] Table 3 Gene information of yCAN31

[0091]

[0092] Example 1 Construction of Saccharomyces cerevisiae yG278 expressing the cannabinolic acid synthesis pathway

[0093] Based on the original strain yCAN31, overexpress the tetraketide synthase gene Cs.TKS, the olivetolic acid cyclase gene Cs.OAC, the truncated geranyl diphosphate:olivetolate geranyltransferase gene Cs.PT4n, downregulate the fatty acid synthase gene fas1, overexpress gal4, and knockout the gene gal80 to obtain the strain yG278.

[0094] Among them, overexpression of Cs.TKS and / or Cs.OAC, overexpression of gal4, and overexpression of Cs.PT4n include: PCR amplification of the integration fragment by 2×Phanta Max Master Mix (Phanta DNA polymerase). Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the upstream fragment 1622b-Up of the integration site 1622b, the downstream fragment 1622b-Down of the integration site 1622b, the upstream fragment 511b-Up of the integration site 511b, the downstream fragment 511b-Down of the integration site 511b, the upstream fragment 1414a-Up of the integration site 1414a, the downstream fragment 1414a-Down of the integration site 1414a, the upstream fragment 106a-Up of the integration site 106a, the downstream fragment 106a-Down of the integration site 106a, the promoter pTDH3, the pHSP26 fragment, the terminator ScENO1t, and the TDH1t fragment are obtained; using the genome containing the pGAL1 gene as a template, the pGAL1 fragment is amplified; using the genome containing the pGAL4(OC)-Gal4-TGal4 gene as a template, the pGAL4(OC)-Gal4-TGal4 fragment is amplified; using the plasmid containing the Cs.OAC3, Cs.OAC4, and Cs.OAC5 genes as a template, the Cs.OAC3, Cs.OAC4, and Cs.OAC5 fragments are amplified; using the genome containing the linker (SEQ ID NO:2) or linker (SEQ ID NO:35) gene as a template, the linker (SEQ ID NO:2) or linker (SEQ ID NO:35) fragment is obtained. Finally, the 1622b-Up-pGAL1-Cs.TKS-linker-Cs.OAC-ScENO1t-1622b-Dp expression cassette is obtained, where Cs.OAC is inserted into the basic Saccharomyces cerevisiae genome after being connected to Cs.TKS through linker (SEQ ID NO:35); and the 1414a-Up-pTDH3-Cs.OAC-ENO1t-1414a-Down expression cassette, where Cs.OAC is directly inserted into the Saccharomyces cerevisiae genome without using a linker; and the 511b-Up-pGal4(OC)-Gal4-TGal4 / pHSP26-Cs.OAC3-linker-Cs.OAC5 / pGal1-Cs.OAC4-511b-Down expression cassette, where Cs.OAC3 is inserted into the Saccharomyces cerevisiae genome after being connected to Cs.OAC5 through linker (SEQ ID NO:2). The 106a-Up-pTDH3-Cs.PT4-TDH1t-106a-Down expression cassette will be integrated into the corresponding site of the genome.Among them, the Cs.OAC3 sequence is as shown in SEQ ID NO:32, the Cs.OAC4 sequence is as shown in SEQ ID NO:33, and the Cs.OAC5 sequence is as shown in SEQ ID NO:34.

[0095] Among them, the truncation mode of Cs.PT4 is: lacking the N-terminal amino acids 1-76 of the full-length amino acid sequence Cs.PT4 shown in SEQ ID NO:36, and the truncated Cs.PT4n nucleotide sequence is as shown in SEQ ID NO:1.

[0096] Among them, knocking out the gene gal80 is achieved by connecting the upstream and downstream of the gal80 gene. The integration fragment is amplified by 2×Phanta Max Master Mix (Phanta DNA polymerase). Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the upstream homologous arm gal80-Up fragment and the downstream homologous arm gal80-Down fragment are amplified. The expression cassette gal80-Up-gal80-Down is obtained and integrated into the corresponding site.

[0097] Among them, downregulating fas1 is achieved by replacing the fas1 promoter. The integration fragment is amplified by 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 of the promoter pfas1 is amplified using primer 1 and primer 2 in Table 4, and the downstream homologous arm fas1 gene fragment is amplified using primer 5 and primer 6 in Table 4; using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the promoter pHXT1 fragment is amplified using primer 3 and primer 4 in Table 4, and finally the pfas1-Up-pHXT1-fas1 expression cassette fragment is obtained. Then the pfas1-Up-pHXT1-fas1 expression cassette fragment is transformed into the host Saccharomyces cerevisiae yCAN31 (the CBGA-producing strain recorded in the background art). Primer 1 and primer 7 in Table 4 are used for PCR reaction of the constructed strain to obtain the reaction solution of colony PCR positive clones for gene sequencing.

[0098] Cultivate yG278 and detect the content of CBGA. As Figure 3 shown, the CBGA of the original strain yCAN31 is low, only 20 μM. While the content of CBGA in the recombinant strain yG278 constructed in this example is 448 μM.

[0099] Table 4 Primer sequences for constructing high-CBGA-producing strains

[0100]

[0101] Example 2: The vacuolar protease Pep4 gene was knocked out in the genome of the high-yield cannabigerolic acid strain yG278 to obtain the recombinant Saccharomyces cerevisiae yG350

[0102] The knockout of the pep4 gene was achieved by ligating the upstream and downstream of the pep4 gene. The integration fragment was amplified by 2×PhantaMax MasterMix (Phanta DNA polymerase). Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the upstream homologous arm Pep4-Up fragment was amplified using primer 1 and primer 2 in Table 5, and the downstream homologous arm Pep4-Down fragment was amplified using primer 3 and primer 4 in Table 5. Then the combination of the fragments was transformed into the high-yield cannabigerolic acid strain yG278 to obtain strain yG350. Primer 1 and primer 4 in Table 5 were used for PCR reaction on strain ySC207 to obtain the reaction solution of colony PCR positive clones for gene sequencing.

[0103] Cultivate yG350 and detect the content of CBGA. As Figure 3 shown, the content of CBGA in yG350 is 650 μM.

[0104] Table 5 Primer sequences for knocking out Pep4

[0105]

[0106] Example 3: A variant encoding the acetyl-CoA synthase Se.ACS gene was overexpressed in the genome of the high-yield cannabigerolic acid strain yG278 to obtain the recombinant Saccharomyces cerevisiae yG361

[0107] The integration fragment was amplified by 2×Phanta Max Master Mix (Phanta DNA polymerase). Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the upstream homologous arm HO-Up fragment of the integration site was amplified using primer 1 and primer 2 in Table 6, and the downstream homologous arm HO-Down fragment of the integration site was amplified using primer 7 and primer 8 in Table 6; using the genome of the strain with the pSeGAL2 promoter as a template, the promoter pSeGAL2 was amplified by primer 3 and primer 4 in Table 6, and using the genome of the strain with the Se.ACS L641P gene (SEQ ID NO:3) and the terminator tADH1 already integrated as a template, the Se.ACSL641P-tADH1 fragment was amplified by primer 5 and primer 6 in Table 6, and finally HO-Up-pSeGAL2-Se.ACS L641P-tADH1-HO-Down expression cassette fragment. Then HO-Up-pSeGAL2-Se.ACS L641P The -tADH1-HO-Down expression cassette fragment was transformed into the high-yield cannabigerolic acid strain yG278 to obtain strain yG361. Primers 1 and 8 in Table 6 were used for PCR reaction on strain yG361 to obtain the reaction solution of colony PCR positive clones for gene sequencing.

[0108] Cultivate yG361 and detect the content of CBGA. As Figure 3 shown, the content of CBGA in yG361 was 585 μM.

[0109] Table 6 Overexpression of Se.ACS L641P Primer sequences

[0110]

[0111] Example 4 Overexpression of the gene encoding geranyl pyrophosphate:oleate geranyltransferase (Cs.PT4) and its targeting peptide in the genome of the high-yield cannabigerolic acid strain yG278 to obtain recombinant Saccharomyces cerevisiae yG391

[0112] The integration fragment was amplified by 2×Phanta Max Master Mix (Phanta DNA polymerase). Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the upstream homologous arm HO-Up fragment of the integration site was amplified using primers 1 and 2 in Table 7, and the downstream homologous arm HO-Down fragment of the integration site was amplified using primers 7 and 8 in Table 7; using the genome with the pSeGAL2 promoter and the truncated Cs.PT4 gene as a template, the promoter pSeGAL2 and the truncated Cs.PT4 gene (Cs.PT4n) were amplified by primers 3 and 4 in Table 7, and using the strain genome with the signal peptide SNC1 gene (SEQ ID NO:4) and the terminator tADH1 already integrated as a template, the SNC1-tADH1 fragment was amplified by primers 5 and 6 in Table 7, and finally the HO-Up-pSeGAL2-Cs.PT4-SNC1-tADH1-HO-Down expression cassette fragment was obtained. Then the HO-Up-pSeGAL2-Cs.PT4n-SNC1-tADH1-HO-Down expression cassette fragment was transformed into the high-yield cannabigerolic acid strain yG278 to obtain strain yG391. Primers 1 and 8 in Table 7 were used for PCR reaction on strain yG391 to obtain the reaction solution of colony PCR positive clones for gene sequencing.

[0113] Cultivate yG391 and detect the content of CBGA. As Figure 3As shown, the content of CBGA in yG391 is 775 μM.

[0114] Table 7 Construction of primers for overexpressing truncated Cs.PT4n and signal peptide

[0115] Table 8 Information on strain construction

[0116]

[0117] In summary, combined with Figure 3 It can be seen that in the present invention, by regulating the genes encoding the enzymes in the pathway for synthesizing cannabigerolic acid in yeast cells, selectively knocking out gal80 and overexpressing gal4, selectively knocking out pep4, selectively overexpressing Cs.OAC with or without linker fusion, and targeting truncated Cs.PT4n enzyme to the plasma membrane with or without signal peptide, the metabolic flux was balanced and the heterologous synthesis pathway was optimized. The engineered strain after transformation can reasonably utilize the intermediate products OA and GPP in the whole synthesis pathway to generate the target product CBGA, achieving the construction of a high-yield yeast strain for the key cannabinoid precursor cannabigerolic acid CBGA, which is of great significance for the biosynthesis and commercialization of CBGA and its downstream cannabinoids.

[0118] Example 5 Application of the recombinant Saccharomyces cerevisiae constructed in Example 1 in the production of cannabigerolic acid

[0119] The application of the recombinant Saccharomyces cerevisiae in the production of cannabigerol includes the following steps:

[0120] (1) Activate and culture the recombinant Saccharomyces cerevisiae constructed in Example 1 to obtain a recombinant Saccharomyces cerevisiae seed culture;

[0121] (2) Transfer the recombinant Saccharomyces cerevisiae seed culture obtained in step (1) to a suitable medium for producing cannabigerolic acid, and culture the recombinant Saccharomyces cerevisiae under suitable conditions to produce cannabigerolic acid.

[0122] Examples 6 - 8

[0123] Examples 6 - 8 are respectively the applications of the recombinant Saccharomyces cerevisiae constructed in Examples 2 - 4 in the production of cannabigerolic acid.

[0124] The differences between Examples 6 - 8 and Example 5 are that in step (1), Examples 6 - 8 respectively use the recombinant Saccharomyces cerevisiae constructed in Examples 2 - 4.

[0125] Example 9 Application of the recombinant Saccharomyces cerevisiae constructed in Example 1 in the production of cannabigerol

[0126] The cannabigerolic acid obtained in Example 5 was decarboxylated by heat treatment to obtain cannabigerol.

[0127] Examples 10 - 12

[0128] Examples 10 - 12 are respectively the applications of the recombinant Saccharomyces cerevisiae constructed in Examples 2 - 4 in the production of cannabigerol.

[0129] The differences between Examples 10 - 12 and Example 9 are that the cannabigerolic acids obtained in Examples 6 - 8 were respectively decarboxylated by heat treatment to obtain cannabigerol.

[0130] The above are all preferred embodiments of this application. The protection scope of this application is not limited hereby. Therefore, all equivalent changes made according to the structure, shape, and principle of this application shall be covered within the protection scope of this application.

Claims

1. A recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid, characterized in that, The recombinant Saccharomyces cerevisiae is prepared from the basic Saccharomyces cerevisiae strain yCAN31, in which the endogenous gene fas1 of the basic Saccharomyces cerevisiae is down-regulated, the endogenous gene gal4 is overexpressed, and the endogenous gene gal80 is knocked out; the following exogenous genes of the basic Saccharomyces cerevisiae are overexpressed: the tetrone synthase Cs.TKS gene, the olivetol benzoate cyclase Cs.OAC gene, the truncated geranyl diphosphate:oleate geranyltransferase Cs.PT4n gene, and the nucleotide sequence of the truncated Cs.PT4 gene Cs.PT4n is SEQ ID NO:

1.

2. The recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to claim 1, characterized in that, The endogenous gene pep4 of the basic Saccharomyces cerevisiae is knocked out.

3. The recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to claim 1, characterized in that, The exogenous gene variant acetyl-CoA synthase Se.ACS of the basic Saccharomyces cerevisiae L641P is overexpressed, and the nucleotide sequence of the variant acetyl-CoA synthase Se.ACS L641P is SEQ ID NO:

3.

4. The recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to claim 1, characterized in that, Overexpression of the truncated Cs.PT4 gene Cs.PT4n also includes inserting the truncated Cs.PT4 gene Cs.PT4n fused with the SNC1 signal peptide into the Saccharomyces cerevisiae genome and expressing it through the pSeGAL2 promoter.

5. The recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to claim 1, characterized in that, Overexpression of the Cs.OAC gene includes ligating Cs.OAC3 with Cs.OAC5 using a linker and then inserting it into the Saccharomyces cerevisiae genome and expressing it through the pHSP26 promoter. The sequence of Cs.OAC3 is shown in SEQ ID NO:32, and the sequence of Cs.OAC5 is shown in SEQ ID NO:

34.

6. The recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to claim 1, characterized in that, Overexpression of the Cs.OAC gene includes directly inserting the coding gene into the Saccharomyces cerevisiae genome without using a linker and initiating expression through the constitutive pTDH3 promoter.

7. The recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to claim 1, characterized in that, Overexpression of Cs.OAC includes ligating the coding gene with Cs.TKS using a linker and then inserting it into the Saccharomyces cerevisiae genome and expressing it through the pGal1 promoter.

8. The recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to claim 5, characterized in that, The sequence of the linker is SEQ ID NO:

2.

9. The recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to claim 7, characterized in that, The sequence of the linker is SEQ ID NO:

35.

10. The recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to claim 1, characterized in that, Overexpression of the Cs.TKS includes inserting the coding gene into the Saccharomyces cerevisiae genome and initiating expression through the pGal1 promoter.

11. The recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to claim 1, characterized in that, Down-regulation of the endogenous gene fas1 includes using pHXT1 that is not expressed in galactose to down-regulate the expression of fas1.

12. The recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to claim 4, characterized in that, The SNC1 signal peptide localizes the Cs.PT4n enzyme to the plasma membrane, and the sequence of the SNC1 signal peptide is SEQ ID NO:

4.

13. A method for constructing a recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to any one of claims 1 to 12, characterized in that, Including the following steps: PCR amplification to obtain the expression cassette of the gene to be overexpressed and integrating it into the Saccharomyces cerevisiae genome; or, PCR amplification to obtain the homologous fragment of the gene to be knocked out and replacing the gene to be knocked out on the Saccharomyces cerevisiae genome with the homologous fragment; realizing gene knockout and insertion on the Saccharomyces cerevisiae genome; Screening to obtain positive clones.

14. Use of the recombinant Saccharomyces cerevisiae capable of producing cannabigerolic acid according to any one of claims 1 to 12 in the production of cannabigerolic acid.

15. The use according to claim 14, characterized in that, Including the following steps: (1) Activate and culture the recombinant Saccharomyces cerevisiae to obtain a recombinant Saccharomyces cerevisiae seed solution; (2) Transfer the recombinant Saccharomyces cerevisiae seed solution to a suitable medium for producing cannabigerolic acid and culture the recombinant Saccharomyces cerevisiae to produce cannabigerolic acid.

16. Use of the recombinant Saccharomyces cerevisiae producing cannabigerolic acid according to any one of claims 1 to 12 in the production of downstream cannabinoids of cannabigerolic acid.

Images