WO2022127481A1 - 利用酿酒酵母生产异源大麻环萜酚的方法 - Google Patents

利用酿酒酵母生产异源大麻环萜酚的方法 Download PDF

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WO2022127481A1
WO2022127481A1 PCT/CN2021/131047 CN2021131047W WO2022127481A1 WO 2022127481 A1 WO2022127481 A1 WO 2022127481A1 CN 2021131047 W CN2021131047 W CN 2021131047W WO 2022127481 A1 WO2022127481 A1 WO 2022127481A1
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gene
saccharomyces cerevisiae
acid
synthase
coa
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French (fr)
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薛闯
齐明明
张文强
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大连理工大学
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Definitions

  • the invention belongs to the fields of biotechnology and medicine, and in particular relates to a host cell capable of biosynthesizing cannabidiol (CBC), a construction method thereof, and a method for biosynthesizing cannabidiol.
  • CBC biosynthesizing cannabidiol
  • Cannabis (Cannabis sativa) has been used for thousands of years due to its richness in various pharmacologically active cannabinoids.
  • CBDs cannabigerols
  • CBDs cannabidiols
  • ⁇ 9 -tetrahydrocannabinols ⁇ 9-tetrahydrocannabinols, ⁇ 9 - THCs
  • ⁇ 8 -tetrahydrocannabinols ⁇ 8 -tetrahydrocannabinols
  • cannabidiols cannabicyclols (CBLs), cannabielsoins (CBEs)
  • cannabinols cannabinols, CBNs
  • dehydrocannabinoids cannabinols
  • cannabidiol CBC
  • CBG cannabinol
  • cannabinol CBG
  • Decarboxylation forms neutral cannabinoids (eg, cannabinol to form cannabinol, which forms cannabinol acid).
  • cannabinol cannabinol
  • CBG cannabinol
  • cannabinol have antibacterial activity against Staphylococcus aureus (Appendino, G.; Gibbons, S.; Giana, A.; Pagani, A.; Grassi, G. Stavri, M.; Smith, E.; Rahman, MM, Antibacterial cannabinoids from Cannabis sativa: a structure-activity study. Journal of Natural Products 2008, 71(8), 1427-30.).
  • CBG is also significantly active on several ligand-gated cation channels of the TRP superfamily, and can act as an agonist of TRPV1 (TRP-type vanillin 1) and TRPA1 (TRP-type ankyrin 1), as well as TRPM8 (TRP-type ankyrin 1)
  • TRPV1 TRP-type vanillin 1
  • TRPA1 TRP-type ankyrin 1
  • TRPM8 TRP-type ankyrin 1
  • a potent inhibitor of melatonin 8) (Pollastro, F.; Taglialatela-Scafati, O.; Allarà, M.; E.; Di Marzo, V.; De Petrocellis, L.; Appendino, G., Bioactive prenylogous cannabinoid from fiber hemp (Cannabis sativa). Journal of Natural Products 2011, 74(9), 2019-22.).
  • CBC inhibits endocannabinoid inactivation and activates TRPA1, resulting in protection against intestinal inflammation in experimental model systems.
  • CBC has multiple pharmacological and biological effects, including analgesic, antinociceptive, anti-inflammatory, and anti-inflammatory. inflammatory activity.
  • cannabidiol CBC
  • CBG cannabinol
  • the agricultural cultivation of cannabis faces several challenges, such as the sensitivity of the plant to climate and disease, the lack of GAP standardization, the low content of cannabinol and cannabinol in cannabis, the large area of arable land and the long period of time, and the combination of rich
  • the coexistence of other types of cannabinoids, obtaining pure samples from the plant is time-consuming and labor-intensive, which seriously affects the study of its therapeutic potential.
  • Microbial fermentation has the advantages of high production efficiency and short cycle, and provides a method for producing a large number of high value-added products from cheap carbon sources.
  • Microorganisms are also a research hotspot in recent years. At present, there is no relevant research on obtaining host cells capable of biosynthesizing cannabinoids and their construction methods through genetic engineering methods and using them for the biosynthesis of cannabinoids report.
  • IUP isoprenol utilization pathway
  • IP or DMAP isoprenol or prenol are phosphorylated to form isopentenyl monophosphate (IP) or dimethylallyl monophosphate (DMAP), respectively; then, IP or DMAP is phosphorylated again Form IPP or DMAPP.
  • IPK isopentenyl phosphokinase
  • the second step of the pathway is catalyzed by isopentenyl phosphokinase (IPK), part of the archaeal mevalonate pathway.
  • IPK isopentenyl phosphokinase
  • the present invention screened several kinases, including IPK homologues, for prenol kinase activity, and some IPK variants can convert prenol to DMAP through promiscuous activity.
  • Eukaryotic cells control the complexity of their metabolism by utilizing organelles to sequester biochemical pathways.
  • peroxisomes are suitable targets for organelle engineering because they are not essential for cell viability under most culture conditions, and their number and size can be modified in various ways for better meet the needs of engineering.
  • Yeast peroxisomes are the site of ⁇ -oxidation of fatty acids, forming a pool of acetyl-CoA that can provide the isoprenoid precursors IPP and DMAPP through heterologous pathways, such as the mevalonate (MVA) pathway.
  • MVA mevalonate
  • peroxisomes have monolayer membranes that allow passage of a large number of small-molecule compounds passively or via channel proteins. Peroxisomes are also detoxifying organelles that process and sequester more toxic molecules from the rest of the cell. Furthermore, because cannabinoids are hydrophobic compounds their solubility in the cytoplasm is limited. However, they have high solubility in hydrophobic liquids such as lipids, oils or fats. It is speculated that CBGA, the precursor for the synthesis of cannabidiolic acid, is likely to be sequestered in lipid droplets. Localization of cannabinoid synthase into lipid droplets is therefore expected to yield faster reaction rates and higher productivity by increasing local concentrations of substrate and enzyme.
  • the present invention now provides the following technical solutions in order to solve the problems of complex preparation methods, high cost and low yield of cannabidiol and cannabidiol.
  • the present invention constructs a recombinant Saccharomyces cerevisiae strain capable of biosynthesizing cannabinoid phenolic acid, and the biosynthetic pathway of cannabinoid phenolic acid includes multiple genes.
  • a metabolic pathway from hexanoic acid to hexanoyl-CoA was constructed to convert hexanoic acid to hexanoic acid using acyl activating enzyme (CsAAE1) by replenishing hexanoic acid Acyl-CoA, while acetyl-CoA carboxylase (ACC1) catalyzes acetyl-CoA to produce malonyl-CoA, hexanoyl-CoA and trimolecular malonyl-CoA via polyketide synthase (CsTKS) and olive acid ring urease (CsOAC) to produce olive acid (OA); in addition, ⁇ -ketothiolase (RebktB), 3-hydroxybutyryl-CoA dehydrogenase (CnpaaH1), crotonase (Cacrt
  • CsTKS polyketide synthase
  • CsOAC olivetate cyclase
  • OA olive alkyd
  • GPP geranyl pyrophosphate
  • OA olive acid
  • CBGA cannabinoid acid
  • CsPT4 cannabinoid synthase
  • the invention provides a recombinant Saccharomyces cerevisiae strain for synthesizing cannabinoid phenolic acid (CBCA), the recombinant Saccharomyces cerevisiae strain heterologously expressing cannabinoid synthase gene (CsPT4) and cannabinoid phenolic acid synthase gene (CBCAS) .
  • CBCA cannabinoid phenolic acid
  • CsPT4 cannabinoid synthase gene
  • CBCAS cannabinoid phenolic acid synthase gene
  • the recombinant Saccharomyces cerevisiae strain overexpresses ⁇ -ketothiolase gene (RebktB), 3-hydroxybutyryl-CoA dehydrogenase gene (CnpaaH1), crotonase gene (Cacrt) and trans-2-
  • the recombinant S. cerevisiae strain overexpresses the acyl activase gene (CsAAE1) and the acetyl-CoA carboxylase gene (ACC1).
  • CsAAE1 acyl activase gene
  • ACC1 acetyl-CoA carboxylase gene
  • the recombinant Saccharomyces cerevisiae strain overexpresses HMG-CoA reductase gene (tHMG1), acetyl-CoA acetyltransferase gene (mvaE), hydroxymethylglutarate-CoA synthase gene (mvaS), incense Leaf-based diphosphate synthase gene (ERG20mut), mevalonate kinase gene (ERG12), mevalonate kinase gene (ERG8), mevalonate pyrophosphate decarboxylase gene (ERG19), and isopentenyl pyrophosphate Isomerase gene (IDI).
  • HMG-CoA reductase gene tHMG1
  • mvaE acetyl-CoA acetyltransferase gene
  • mvaS hydroxymethylglutarate-CoA synthase gene
  • incense Leaf-based diphosphate synthase gene ERG20mut
  • the recombinant Saccharomyces cerevisiae strain overexpresses acetaldehyde dehydrogenase gene (ALD6), acetyl-CoA synthase gene (ACS2) and alcohol dehydrogenase gene (ADH2).
  • ALD6 acetaldehyde dehydrogenase gene
  • ACS2 acetyl-CoA synthase gene
  • ADH2 alcohol dehydrogenase gene
  • cannabinoid acid synthase gene cannabinoid phenolic acid synthase gene, ⁇ -ketothiolase gene, 3-hydroxybutyryl-CoA dehydrogenase gene, crotonase gene, trans-2-ene Acyl-CoA reductase gene, cannabis polyketide synthase gene, olive acid cyclase gene, acyl activase gene, acetyl-CoA carboxylase gene, HMG-CoA reductase gene, acetyl-CoA acetyltransferase gene , hydroxymethylglutarate coenzyme A synthase gene, geranyl diphosphate synthase gene, mevalonate kinase gene (ERG12), mevalonate kinase gene (ERG8), mevalonate pyrophosphate decarboxylase
  • the cannabinoid terpene acid synthase gene is derived from cannabis
  • the cannabinoid phenolic acid synthase gene is derived from cannabis
  • the ⁇ -ketothiolase gene is derived from Ralstonia, and 3-hydroxybutyryl-CoA dehydrogenation
  • the enzyme gene is derived from the hookworm copper greedy fungus
  • the crotonase gene is derived from Clostridium acetobutylicum
  • the trans-2-enoyl-CoA reductase gene is derived from Treponema dentate
  • the cannabis polyketide synthase gene is derived from cannabis
  • the olive The acid cyclase gene is derived from cannabis
  • the acyl activase gene is derived from cannabis
  • the acetyl-CoA carboxylase gene is derived from Saccharomyces cerevisiae
  • the truncated HMG-CoA reductase gene is derived from
  • nucleotide sequence of the cannabinoid acid synthase gene is shown in SEQ ID NO: 1, and the nucleotide sequence of the cannabinoid phenolic acid synthase gene is shown in SEQ ID NO: 2; ⁇ -ketothiol
  • the nucleotide sequence of the enzyme gene is shown in SEQ ID NO: 3
  • the nucleotide sequence of the 3-hydroxybutyryl-CoA dehydrogenase gene is shown in SEQ ID NO: 4
  • nucleotide sequence of the crotonase gene As shown in SEQ ID NO: 5, the nucleotide sequence of the trans-2-enoyl-CoA reductase gene is shown in SEQ ID NO: 6, and the nucleotide sequence of the cannabinoid synthase gene is shown in SEQ ID NO: : 7, the nucleotide sequence of the olive acid cyclase gene is shown in SEQ ID NO: 8; the nucleotide sequence of
  • nucleotide sequence of the hydroxymethylglutarate-CoA synthase gene is shown in SEQ ID NO: 13
  • nucleotide sequence of the geranyl diphosphate synthase gene is shown in SEQ ID NO: 14
  • nucleotide sequence of the mevalonate kinase gene (ERG12) is shown in SEQ ID NO: 15
  • nucleotide sequence of the mevalonate kinase gene (ERG8) is shown in SEQ ID NO: 16
  • the nucleotide sequence of the mevalonate pyrophosphate decarboxylase gene is shown in SEQ ID NO: 17
  • nucleotide sequence of the isopentenyl pyrophosphate isomerase gene is shown in SEQ ID NO: 18
  • the nucleotide sequence of the hydrogenase gene is shown in SEQ ID NO: 19, the nucleotide sequence of the acetyl-CoA synthase gene is shown in SEQ ID NO: 20, and
  • cannabinoid acid synthase gene cannabinoid phenolic acid synthase gene, ⁇ -ketothiolase gene, 3-hydroxybutyryl-CoA dehydrogenase gene, crotonase gene, trans-2-ene Acyl-CoA reductase gene, cannabis polyketide synthase gene, olive acid cyclase gene, acyl activase gene, acetyl-CoA carboxylase gene, HMG-CoA reductase gene, acetyl-CoA acetyltransferase gene , hydroxymethylglutarate coenzyme A synthase gene, geranyl diphosphate synthase gene, mevalonate kinase gene (ERG12), mevalonate kinase gene (ERG8), mevalonate pyrophosphate decarboxylase
  • cannabinoid acid synthase gene cannabinoid phenolic acid synthase gene, ⁇ -ketothiolase gene, 3-hydroxybutyryl-CoA dehydrogenase gene, crotonase gene, trans-2-ene Acyl-CoA reductase gene, cannabis polyketide synthase gene, olive acid cyclase gene, acyl activase gene, acetyl-CoA carboxylase gene, HMG-CoA reductase gene, acetyl-CoA acetyltransferase gene , hydroxymethylglutarate coenzyme A synthase gene, geranyl diphosphate synthase gene, mevalonate kinase gene (ERG12), mevalonate kinase gene (ERG8), mevalonate pyrophosphate decarboxylase Gene, isopentenyl pyrophosphate isomerase gene, acetaldehyde
  • cannabinoid acid synthase gene cannabinoid phenolic acid synthase gene, ⁇ -ketothiolase gene, 3-hydroxybutyryl-CoA dehydrogenase gene, crotonase gene, trans-2-ene Acyl-CoA reductase gene, cannabis polyketide synthase gene, olive acid cyclase gene, acyl activase gene, acetyl-CoA carboxylase gene, HMG-CoA reductase gene, acetyl-CoA acetyltransferase gene , hydroxymethylglutarate coenzyme A synthase gene, geranyl diphosphate synthase gene, mevalonate kinase gene (ERG12), mevalonate kinase gene (ERG8), mevalonate pyrophosphate decarboxylase
  • cannabinoid acid synthase gene cannabinoid phenolic acid synthase gene, ⁇ -ketothiolase gene, 3-hydroxybutyryl-CoA dehydrogenase gene, crotonase gene, trans-2-ene Acyl-CoA reductase gene, cannabis polyketide synthase gene, olive acid cyclase gene, acyl activase gene, acetyl-CoA carboxylase gene, HMG-CoA reductase gene, acetyl-CoA acetyltransferase gene , hydroxymethylglutarate coenzyme A synthase gene, geranyl diphosphate synthase gene, mevalonate kinase gene (ERG12), mevalonate kinase gene (ERG8), mevalonate pyrophosphate decarboxylase
  • the cannabinoid synthase gene insertion site is located at the 416d site, the CAN1y site or the YOLCd1b site in the yeast genome; the cannabinoid phenolic acid synthase gene insertion site is located in the yeast genome 308a site, HIS3b site or 511b site site.
  • the insertion site of ⁇ -ketothiolase gene is located at SAP155b site of yeast genome; the insertion site of 3-hydroxybutyryl-CoA dehydrogenase gene and crotonase gene is located at site SAP155c of yeast genome; trans-2-enoyl
  • the insertion site of coenzyme A reductase gene is located at YPRC ⁇ 15c site of yeast genome; the insertion site of cannabinoid synthase gene and olive acid cyclase gene is located at site 1622b, X4 site, XI site 3 or XII5 site of yeast genome ;
  • Acyl activase gene insertion site is located at site 911b of yeast genome; acetyl-CoA carboxylase gene insertion site is located at site X3 of yeast genome; truncated HMG-CoA reductase gene and mutated geranyl diphosphate
  • Another aspect of the present invention provides a construction method of the above-mentioned recombinant Saccharomyces cerevisiae strain, which mainly comprises the following steps:
  • HMG-CoA reductase gene HMG-CoA reductase gene, acetyl-CoA acetyltransferase gene, hydroxymethylglutarate-CoA synthase gene, geranyl diphosphate synthase gene, mevalonate kinase gene (ERG12 ), mevalonate kinase gene (ERG8), mevalonate pyrophosphate decarboxylase gene and isopentenyl pyrophosphate isomerase gene expression cassette, the above-mentioned expression cassette is inserted into step (3) by homologous recombination to obtain in the genome of Saccharomyces cerevisiae;
  • homologous recombination uses zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR/Cas systems.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • CRISPR/Cas systems CRISPR/Cas systems.
  • homologous recombination uses the CRISPR/Cas system.
  • promoters of the above genes are constitutive promoters or inducible promoters, respectively.
  • the promoter is GAL1, GAL10, GPD, TEF1, PGK1 or ADH.
  • YPL062W was the core promoter of ALD6, and the expression level of ALD6 was negatively correlated with terpenoid production.
  • ADH1 alcohol dehydrogenase gene
  • YPL062W was knocked out; Zea mays-derived pyruvate decarboxylase gene (PDC), endogenous acyl-CoA synthase gene (FAA2) and Acetyl-CoA synthase gene (SeACS) from Salmonella enterica.
  • PDC Zea mays-derived pyruvate decarboxylase gene
  • FAA2 endogenous acyl-CoA synthase gene
  • SeACS Acetyl-CoA synthase gene
  • the present invention provides another construction method of the above-mentioned recombinant Saccharomyces cerevisiae strain, which mainly includes the following steps:
  • the promiscuous kinases used were screened from Salmonella enterica subsp (PhoN) and Saccharomyces cerevisiae (ScCK).
  • CBCAS a variety of molecular chaperones, foldases, and transcriptional activators were screened to improve the expression activity of CBCAS. Including proteins involved in folding and endoplasmic reticulum quality control (CNE1p, KAR2p, PDI1p, ERO1p), cofactors (FAD1p), UPR protein IRE1*p, UPR activator Hac1s and endoplasmic reticulum size regulator INO1.
  • site-directed mutagenesis of cannabinol synthase produces multiple mutants, namely H292L, H292R, Y417H, Y417R, N89Q-N499Q, R463C-D488C, T379S, K377R, N196Q, F171Y, S170T, R349K, F365Y, V138A , R532K, L524I, Y472F, N528Q, F353Y and their combination of double mutants, triple mutants and multiple mutants, and screened out the mutants with the best activity for the production of cannabidiolic acid.
  • ADK1 adenylate kinase gene
  • phosphite dehydrogenase gene ptxD from Pseudomonas stutzeri and hemoglobin gene (VHB) of Vibrio hygienicum increased the rate of ATP synthesis. , thereby promoting cell growth and cannabinoid production.
  • cannabinoid synthase into lipid droplets is expected to result in faster reaction rates and higher productivity by increasing local concentrations of substrates and enzymes.
  • the key enzyme genes in the triacylglycerol pathway TAG were overexpressed. Synthesizes the key protein gene SEI1, increases lipid levels and lipid droplet aggregation. Simultaneous localization of cannabinoid synthase into lipid droplets is expected to expand the storage capacity of engineered yeast to synthesize cannabinoids by increasing the local concentrations of substrates and enzymes, resulting in faster reaction rates and higher productivity.
  • the key enzyme genes in the cannabinol metabolic pathway were simultaneously integrated into the rDNA site of Saccharomyces cerevisiae to achieve multi-copy expression of multiple key enzyme genes.
  • Gal80 was knocked out to relieve its inhibitory effect on Gal4, and the promoter of Gal4 was replaced to relieve the glucose repression effect. Therefore, galactose was no longer needed as an inducer, and the mixed fermentation of glucose and ethanol was used to optimize cannabis. Yield of cycloterpene phenol (CBCA).
  • CBCA cycloterpene phenol
  • promiscuous kinase genes isopentenyl phosphokinase genes, pyruvate decarboxylase genes, acyl-CoA synthase genes, related genes involved in folding and endoplasmic reticulum quality control, adenylate kinase genes, phosphite decarboxylase genes Hydrogenase gene, hemoglobin gene, diacylglycerol acyltransferase gene, glyceraldehyde triphosphate dehydrogenase gene, phosphatidic acid phosphatase gene, endoplasmic reticulum quality control proteins (CNE1p, KAR2p, PDI1p, ERO1p), cofactors (FAD1p), the UPR protein IRE1*p, the UPR activator Hac1s, and the endoplasmic reticulum size regulator INO1 were either homologous or heterologous.
  • the promiscuous kinase gene is derived from Saccharomyces cerevisiae; the isopentenyl phosphokinase gene is derived from Pyroplasma acidophilus, Methanococcus methyle and Arabidopsis thaliana; the pyruvate decarboxylase gene is derived from corn; acyl-CoA synthase The gene was derived from Saccharomyces cerevisiae; the acetyl-CoA synthase gene was derived from Salmonella; the related genes involved in folding and endoplasmic reticulum quality control and the adenylate kinase gene were derived from Saccharomyces cerevisiae; the phosphite dehydrogenase gene was derived from Pseudomonas stutzeri The hemoglobin gene is derived from Vibrio vibrio, diacylglycerol acyltransferase gene, glyceraldehyde tri
  • nucleotide sequence of the hybrid kinase gene is shown in SEQ ID NO: 22
  • nucleotide sequence of the isopentenyl phosphokinase gene (thaIPK) is shown in SEQ ID NO: 23
  • isopentenyl The nucleotide sequence of the base phosphokinase gene (mbIPK) is shown in SEQ ID NO: 24, the nucleotide sequence of the isopentenyl phosphokinase gene (atIPK) is shown in SEQ ID NO: 25, and the pyruvate decarboxylase gene
  • the nucleotide sequence of (ZmPDC) is shown in SEQ ID NO: 26
  • nucleotide sequence of the acyl-CoA synthase gene is shown in SEQ ID NO: 27
  • nucleotide sequence of the acetyl-CoA synthase gene The sequence is shown in SEQ ID NO: 28, the nucleotide sequence of the gene
  • the nucleotide sequence of the gene (PDI1) is shown in SEQ ID NO: 32
  • the nucleotide sequence of the unfolded protein response (UPR) gene (IRE1*) is shown in SEQ ID NO: 33
  • the unfolded protein response (UPR) ) gene (HAC1s) nucleotide sequence is shown in SEQ ID NO: 34
  • the nucleotide sequence of endoplasmic reticulum size-related gene (INO1) is shown in SEQ ID NO: 35
  • the nucleotide sequence of adenylate kinase gene is shown in SEQ ID NO: 35.
  • the acid sequence is shown in SEQ ID NO: 36
  • the nucleotide sequence of the phosphite dehydrogenase gene ptxD is shown in SEQ ID NO: 37
  • the nucleotide sequence of the hemoglobin gene is shown in SEQ ID NO: 38.
  • the nucleotide sequence of the acylglycerol acyltransferase gene is shown in SEQ ID NO: 39
  • the nucleotide sequence of the phosphatidic acid phosphatase gene is shown in SEQ ID NO: 40
  • the nucleus of the glyceraldehyde triphosphate dehydrogenase gene The nucleotide sequence is shown in SEQ ID NO:41.
  • promiscuous kinase genes isopentenyl phosphokinase genes, pyruvate decarboxylase genes, acyl-CoA synthase genes, related genes involved in folding and endoplasmic reticulum quality control, adenylate kinase genes, phosphite decarboxylase genes Hydrogenase gene, hemoglobin gene, diacylglycerol acyltransferase gene, glyceraldehyde triphosphate dehydrogenase gene, phosphatidic acid phosphatase gene, endoplasmic reticulum quality control proteins (CNE1p, KAR2p, PDI1p, ERO1p), cofactors
  • the nucleotide sequences of (FAD1p), UPR protein IRE1*p, UPR activator Hac1s and endoplasmic reticulum size regulator INO1 have at least 70% and 80% of the nucleotides shown in SEQ ID NOs:
  • promiscuous kinase genes isopentenyl phosphokinase genes, pyruvate decarboxylase genes, acyl-CoA synthase genes, related genes involved in folding and endoplasmic reticulum quality control, adenylate kinase genes, phosphite decarboxylase genes Hydrogenase gene, hemoglobin gene, diacylglycerol acyltransferase gene, glyceraldehyde triphosphate dehydrogenase gene, phosphatidic acid phosphatase gene, endoplasmic reticulum quality control proteins (CNE1p, KAR2p, PDI1p, ERO1p), cofactors
  • the nucleotide sequences of (FAD1p), UPR protein IRE1*p, UPR activator Hac1s and endoplasmic reticulum size regulator INO1 are SEQ ID NOs: 22-41 through one or more nucleotides, respectively The
  • promiscuous kinase genes isopentenyl phosphokinase genes, pyruvate decarboxylase genes, acyl-CoA synthase genes, related genes involved in folding and endoplasmic reticulum quality control, adenylate kinase genes, phosphite decarboxylase genes Hydrogenase gene, hemoglobin gene, diacylglycerol acyltransferase gene, glyceraldehyde triphosphate dehydrogenase gene, phosphatidic acid phosphatase gene, endoplasmic reticulum quality control proteins (CNE1p, KAR2p, PDI1p, ERO1p), cofactors
  • the nucleotide sequences of (FAD1p), the UPR protein IRE1*p, the UPR activator Hac1s and the endoplasmic reticulum size regulator INO1, respectively, are able to interact with those shown in SEQ ID NOs: 22-
  • promiscuous kinase genes isopentenyl phosphokinase genes, pyruvate decarboxylase genes, acyl-CoA synthase genes, related genes involved in folding and endoplasmic reticulum quality control, adenylate kinase genes, phosphite decarboxylase genes Hydrogenase gene, hemoglobin gene, diacylglycerol acyltransferase gene, glyceraldehyde triphosphate dehydrogenase gene, phosphatidic acid phosphatase gene, endoplasmic reticulum quality control proteins (CNE1p, KAR2p, PDI1p, ERO1p), cofactors
  • the nucleotide sequences of (FAD1p), UPR protein IRE1*p, UPR activator Hac1s and endoplasmic reticulum size regulator INO1 are partially or fully codon-optimized nucleotide sequences.
  • the EfmvaE-EfmvaS-SKL gene insertion site is located at the YIRC ⁇ 6 site of the Saccharomyces cerevisiae genome; the ERG19-ERG8-SKL gene insertion site is located at the Saccharomyces cerevisiae genome YMRW ⁇ 15 site; the ERG12-SKL gene insertion site is located in the Saccharomyces cerevisiae genome YNRC ⁇ 9 site
  • the ScCK-SKL gene insertion site is located at the YGLC ⁇ 3 site of the Saccharomyces cerevisiae genome; the atIPK-SKL gene insertion site is located at the YORW ⁇ 17 site of the Saccharomyces cerevisiae genome; the IDI-SKL gene insertion site is located at the Saccharomyces cerevisiae genome YPRC ⁇ 3 site; ERG20-SKL The gene insertion site is located at the SPB1/PBN1 site of the Saccharomyces cerevisia
  • the above-mentioned key enzyme genes are constructed into a high-copy plasmid vector for episomal expression.
  • the present invention provides a method for utilizing the above-mentioned recombinant Saccharomyces cerevisiae to ferment and produce cannabinol, the method mainly comprises the following steps:
  • the medium is YPD medium.
  • the culture medium contains one or more mixtures of glucose, galactose, glycerol, ethanol, starch, caproic acid or olivetolic acid.
  • the culturing conditions are the rotational speed of 50-300 rpm, the temperature of 28-32° C., and the culturing time of 24-120 h.
  • the process of recovering the cannabinoid phenolic acid produced by the fermentation includes the step of extracting the cannabinoid phenolic acid in the fermentation broth with an organic solvent.
  • the organic solvent is one or more mixtures of ethyl acetate, hexane, heptane, petroleum ether, or chloroform.
  • the process of recovering the cannabinoid phenolic acid produced by fermentation includes the process of recombinant Saccharomyces cerevisiae obtained by crushing and fermentation.
  • the crushing method is high pressure homogenization crushing method, ultrasonic crushing method, ball milling crushing method, repeated freeze-thaw crushing method or enzymatic crushing method.
  • volume ratio of the organic solvent to the fermentation broth in the extraction process is 1:1 to 1:20.
  • the present invention discloses a recombinant Saccharomyces cerevisiae strain capable of biosynthesizing cannabinol, and provides a new way to produce a large amount of high value-added cannabinol from a cheap carbon source.
  • the present invention has an accurate and efficient method for constructing a recombinant Saccharomyces cerevisiae strain for biosynthesizing cannabinoid phenolic acid, and the obtained recombinant Saccharomyces cerevisiae strain has stable genetic performance.
  • the method for producing cannabinol by fermentation with recombinant Saccharomyces cerevisiae disclosed in the present invention has high production efficiency, short cycle and low cost, which is beneficial to the large-scale production of cannabinol and the expansion of applications in the field of medicine.
  • Figure 1 is a map of the biosynthetic pathway of cannabinoid phenolic acid in recombinant Saccharomyces cerevisiae, wherein CsAAE1: acyl activating enzyme, ACC1: acetyl-CoA carboxylase, RebktB: ⁇ -ketothiolase, CnpaaH1: 3-hydroxybutyryl coenzyme A dehydrogenase, Cacrt: crotonase, Tdter: trans-2-enoyl-CoA reductase, CsTKS: polyketide synthase, CsOAC: olive acid cyclase, CsPT4: olive acid geranyl transferase , CBCAS: Cannabidiol phenolic acid synthase.
  • CsAAE1 acyl activating enzyme
  • ACC1 acetyl-CoA carboxylase
  • RebktB ⁇ -ketothiolase
  • FIG. 2 is a schematic diagram of the GPP biosynthesis pathway.
  • Fig. 3 is the liquid chromatogram of the cannabinoid phenolic acid produced by recombinant Saccharomyces cerevisiae fermentation, wherein, the upper figure: the liquid chromatogram of the cannabinoid phenolic acid standard product LC-MS, the abscissa is the retention time, and the ordinate is Abundance; bottom panel: Liquid chromatogram of LC-MS of fermentation samples of cannabinoid phenolic acid recombinant genetically engineered strains.
  • Figure 4 is a mass spectrogram of cannabinoid phenolic acid produced by recombinant Saccharomyces cerevisiae fermentation, the abscissa is M/Z, and the ordinate is abundance.
  • Top image LC-MS mass spectrum of cannabidiolic acid standard
  • Bottom image LC-MS mass spectrum of fermented samples of cannabinoid phenolic acid recombinant genetically engineered strains.
  • the host bacteria of the present invention is Saccharomyces cerevisiae INVSc1, and the diploid INVSc1 has high robustness, and its complex gene regulation network is beneficial to the expression and catalysis of enzymes under unfavorable environmental conditions.
  • the present invention is based on the discovery that geranyl pyrophosphate (GPP) and olivetolic acid (OA) generate cannabidiolic acid (CBGA) under the action of cannabinoid synthase (CsPT4).
  • CBCAS Terpene phenol synthase catalyzes the formation of cannabinoid phenolic acid (CBCA), and constructs two gene expression cassettes: the codon-optimized CsPT4 gene expression cassette and the codon-optimized CBCAS gene expression cassette.
  • the CsPT4 gene expression cassette uses GAL10 promoter and CYC1 terminator
  • CBCAS gene expression cassette uses GAL10 promoter and CYC1 terminator
  • the CsPT4 gene expression cassette is integrated into the 416d, CAN1y and YOLCd1b genomic loci of Saccharomyces cerevisiae by gene editing technology; It was integrated into the 308a, HIS3b and 511b genomic loci of Saccharomyces cerevisiae and expressed 3 copies of the CsPT4 gene and CBCAS gene to obtain a recombinant Saccharomyces cerevisiae strain capable of expressing cannabinoid synthase and cannabinoid synthase.
  • RebktB gene expression cassette uses TEF1 promoter and TEF1 terminator
  • CnpaaH1 gene expression cassette uses GAL10 promoter and CYC1 terminator
  • Cacrt gene expression cassette uses GAL1 promoter and ADH1 terminator
  • Tdter gene expression cassette uses PGK1 promoter and HXT7 termination
  • the CsTKS gene expression cassette uses the GAL10 promoter and CYC1 terminator
  • the CsOAC gene expression cassette uses the GAL1 promoter and ADH1 terminator.
  • the RebktB gene expression cassette was integrated into the Saccharomyces cerevisiae SAP155b genomic locus
  • the CnpaaH1 gene expression cassette and the Cacrt gene expression cassette were integrated into the Saccharomyces cerevisiae SAP155c genomic locus
  • the Tdter gene expression cassette was integrated into the Saccharomyces cerevisiae YPRC ⁇ 15c genomic locus Points, respectively express 1 copy of the above genes.
  • the CsOAC gene expression cassette and the CsTKS gene expression cassette formed an expression cassette group and were respectively integrated into the 1622b, X4, XI3, and XII5 genomic sites of Saccharomyces cerevisiae to express 4 copies of the above genes, and finally obtain the ability to use sugar to produce cannabidiol. Acidic recombinant strains of Saccharomyces cerevisiae.
  • the ACC1 gene expression cassette is integrated into the Saccharomyces cerevisiae X3 genome locus to overexpress 1 copy of the above genes; CsAAE1
  • the gene expression cassette was integrated into the genome sites of Saccharomyces cerevisiae 208a, 911b and 106a to express 3 copies of the above genes, and through the supply of caproic acid, caproic acid was converted to caproyl-CoA under the catalysis of CsAAE1.
  • acetyl-CoA is catalyzed by ACC1 to generate malonyl-CoA, hexanoyl-CoA and malonyl-CoA, under the action of cannabis polyketide synthase (CsTKS) and olive acid cyclase (CsOAC), to generate olivetol acid, thereby increasing the yield of cannabidiolic acid biosynthesized by recombinant Saccharomyces cerevisiae.
  • CsTKS cannabis polyketide synthase
  • CsOAC olive acid cyclase
  • the endogenous mevalonate pathway of the recombinant Saccharomyces cerevisiae was further optimized, and 8 gene expression cassettes were constructed: the truncated tHMG1 gene expression cassette, and the codon-optimized mvaE gene expression cassette.
  • tHMG1 gene expression cassette uses GAL1 promoter and ADH1 terminator
  • mvaE gene expression cassette uses GAL1 promoter and ADH1 terminator
  • mvaS gene expression cassette uses GAL10 promoter and CYC1 terminator
  • ERG20mut gene expression cassette uses GAL10 promoter and CYC1 terminator
  • ERG12 gene expression cassette uses GAL10 promoter and CYC1 terminator
  • ERG8 gene expression cassette uses GAL1 promoter and ADH1 terminator
  • ERG19 gene expression cassette uses GAL10 promoter and CYC1 terminator
  • IDI gene expression cassette uses GAL1 promoter and ADH1 terminator.
  • the tHMG1 gene expression cassette and ERG20mut gene expression cassette were integrated into the 1021b genome site of Saccharomyces cerevisiae
  • the mvaE gene expression cassette and mvaS gene expression cassette were integrated into the 1414a genome site of Saccharomyces cerevisiae
  • the ERG12 gene expression cassette was integrated into the 1414a genome site of Saccharomyces cerevisiae.
  • the IDI gene expression cassette was integrated into the 1114a genome site of Saccharomyces cerevisiae, the ERG8 gene expression cassette and the ERG19 gene expression cassette were integrated into the 1014a genome site of Saccharomyces cerevisiae, and one copy of the above genes was overexpressed, thereby ensuring that the recombinant S. Supply of geranyl pyrophosphate in the downstream pathway of valerate.
  • ALD6 acetaldehyde dehydrogenase
  • ACS2 acetyl-CoA synthase
  • ADH2 alcohol dehydrogenase
  • the ALD6 gene expression cassette and ACS2 gene expression cassette were integrated into the 1309a genome locus of Saccharomyces cerevisiae, and the ADH2 gene expression cassette was integrated into the X2 genome locus of Saccharomyces cerevisiae, overexpressing 1 copy of the above genes to improve recombination Acetyl-CoA flux in the cytoplasm of Saccharomyces cerevisiae, providing precursor compounds for the mevalonate pathway, oleicolic acid, and cannabinolic acid biosynthesis.
  • the IPK, choline kinase (ScCK) gene expression cassette uses the TEF1 promoter and TEF1 terminator.
  • EfmvaE, EfmvaS, ERG8, ERG19, ERG12, IDI, ERG20mut, CsPT4, CBCAS gene expression cassettes all use GAL10 promoter and CYC1 terminator.
  • the EfmvaE-EfmvaS-SKL gene expression cassette was integrated into the YIRC ⁇ 6 genomic locus of Saccharomyces cerevisiae by gene editing technology; the ERG19-ERG8-SKL gene expression cassette was integrated into the YMRW ⁇ 15 genomic locus of Saccharomyces cerevisiae; the ERG12-SKL gene expression cassette was integrated to the YNRC ⁇ 9 genomic locus of Saccharomyces cerevisiae; integrate the ScCK-SKL gene expression cassette into the YGLC ⁇ 3 genomic locus of Saccharomyces cerevisiae; integrate the atIPK-SKL gene expression cassette into the YORW ⁇ 17 genomic locus of Saccharomyces cerevisiae; integrate the IDI-SKL gene expression cassette Integrated into the YPRC ⁇ 3 genomic locus of Saccharomyces cerevisiae; integrated the ERG20-SKL gene expression cassette into the SPB1/PBN1 genomic locus of Saccharomyces cerevisi
  • Zea mays-derived pyruvate decarboxylase gene (PDC) and endogenous acyl-CoA synthase gene (FAA2) were overexpressed.
  • the codon-optimized pyruvate decarboxylase gene zmPDC, the endogenous acyl-CoA synthase gene (FAA2) and the Salmonella enterica-derived acetyl-CoA synthase gene (SeACS) were constructed into the TEF1 promoter and TEF1 terminator, respectively .
  • the zmPDC, FAA2 gene and acetyl-CoA synthase gene (SeACS) expression cassettes were integrated into the X2, XII4 and YORW ⁇ 22 genomic loci of Saccharomyces cerevisiae by gene editing technology, and ADH1 was knocked out at the same time.
  • the adenylate kinase gene (ADK1), the phosphite dehydrogenase gene ptxD from Pseudomonas stutzeri, and the Vibrio vitreous hemoglobin gene (VHB) were overexpressed.
  • the adenylate kinase gene (ADK1), the codon-optimized phosphite dehydrogenase gene ptxD, and the Vibrio vibrio hemoglobin gene (VHB) were constructed into the TEF1 promoter and TEF1 terminator, respectively.
  • the ADK1, ptxD and VHB gene expression cassettes were integrated into the YARC ⁇ 8, YCRW ⁇ 11 and YBRW ⁇ 16 genomic loci of Saccharomyces cerevisiae, respectively, by gene editing technology.
  • CBCAS proteins involved in folding and endoplasmic reticulum quality control (CNE1p, KAR2p, PDI1p, ERO1p, IRE1p), cofactors (FAD1p), UPR activator Hac1s, and endoplasmic reticulum were amplified from the Saccharomyces cerevisiae genome, respectively. Size regulator INO1. Expression cassettes were constructed using the PGK1 promoter and HXT7 terminator, respectively.
  • the CNE1 gene expression cassette was integrated into the I12 genomic locus of Saccharomyces cerevisiae by gene editing technology; the KAR2 gene expression cassette was integrated into the I4 and I32 genomic loci of Saccharomyces cerevisiae; the PDI1 gene expression cassette was integrated into the I10 genomic locus of Saccharomyces cerevisiae ; Integrate the ERO1 gene expression cassette into the X3 genomic locus of Saccharomyces cerevisiae; Integrate the IRE1 gene expression cassette into the I8 genomic locus of Saccharomyces cerevisiae; Integrate the Hac1s gene expression cassette into the I28 genomic locus of Saccharomyces cerevisiae; Express the INO1 gene The cassette integrates into the I3 genomic locus of Saccharomyces cerevisiae.
  • the diacylglycerol acyltransferase gene DGA1 the glyceraldehyde triphosphate dehydrogenase gene GPD1 and the phosphatidic acid phosphatase gene (PAH1) in the triacylglycerol synthesis pathway TAG were overexpressed.
  • GPD1, DGA1 and PAH1 were constructed into the TEF1 promoter and TEF1 terminator, respectively.
  • the GPD1, DGA1 and PAH1 gene expression cassettes were integrated into the YORW ⁇ 22, YCRW ⁇ 12 and YERC ⁇ 8 genomic loci of Saccharomyces cerevisiae, respectively, by gene editing technology.
  • the high-yielding cannabinol acid recombinant Saccharomyces cerevisiae strain obtained in Example 2 was picked into a test tube containing 3-5mL YPD (10g/L yeast extract, 20g/L peptone, 20g/L dextrose). Incubate at 200rpm and 30°C for 24h until the glucose in the medium is exhausted. The strains with saturated growth were subcultured by centrifugation at 4500 rpm into a new medium containing 100 mL of YPG (10 g/L yeast extract, 20 g/L peptone, 20 g/L galactose) at 200 rpm and 30 °C for 24-48 h.
  • YPG g/L yeast extract, 20 g/L peptone, 20 g/L galactose
  • the high-yielding cannabinol acid recombinant Saccharomyces cerevisiae strain obtained in Example 5 was picked into a test tube containing 3-5mL YPD (10g/L yeast extract, 20g/L peptone, 20g/L dextrose). Cells were incubated at 200 rpm and 30°C for 24 h until glucose in the medium was depleted.
  • YPD yeast extract, 20g/L peptone, 20g/L dextrose
  • strains with saturated growth were subcultured by centrifugation at 4500 rpm into a new medium containing 100 mL of YPG (10 g/L yeast extract, 20 g/L peptone, 20 g/L galactose, 1 mM oleicolic acid or 2 mM caproic acid), 200 rpm, Incubate at 30°C for 24-48h.
  • YPG 10 g/L yeast extract, 20 g/L peptone, 20 g/L galactose, 1 mM oleicolic acid or 2 mM caproic acid
  • the relevant equipment and experimental parameters for the detection and analysis of cannabinoid phenolic acid are as follows: the instrument is an Agilent 6224 TOF LC/MS, the column temperature is 25°C, the chromatographic column is an Agilent C 18 chromatographic column, the flow rate is 0.2mL/min, and the mobile phase contains 0.05 % Formic acid in water (A) and acetonitrile solution (B), gradient elution conditions: 0-40min, 30%-98% acetonitrile; 40-50min, 98% acetonitrile; 50-51min, 98%-30% acetonitrile, The injection volume was 20 ⁇ L.

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Abstract

提供一种能够生物合成大麻环萜酚酸的重组酿酒酵母以及通过该细胞生物合成大麻环萜酚酸的方法。首先在宿主中过表达大麻萜酸合酶和大麻环萜酚酸合酶，然后在宿主中构建利用糖类合成大麻环萜酚酸的前体化合物橄榄醇酸的代谢途径，进一步在宿主中构建己酸到橄榄醇酸代谢途径，优化宿主内源甲羟戊酸途径和乙酰辅酶A的代谢途径，对大麻环萜酚合酶进行理性设计，筛选出高活性的大麻环萜酚合酶，最后利用细胞区室化原理，将大麻环萜酚途径定位到过氧化物酶体和脂滴，获得能够生物合成大麻环萜酚酸的重组酿酒酵母。

Description

利用酿酒酵母生产异源大麻环萜酚的方法 技术领域
本发明属于生物技术与医药领域，具体涉及能够生物合成大麻环萜酚(CBC)的宿主细胞及其构建方法和大麻环萜酚的生物合成方法。
背景技术
大麻(Cannabis sativa)由于其富含多种药理活性的大麻素已有数千年的应用历史。目前，已经从大麻植物中分离鉴定出超过113种大麻素并且被分为不同的类型，例如大麻萜酚型(cannabigerols，CBGs)，大麻环萜酚型(cannabichromenes，CBCs)，大麻二酚型(cannabidiols，CBDs)，Δ 9-四氢大麻酚型(Δ 9-tetrahydrocannabinols，Δ 9-THCs)，Δ 8-四氢大麻酚型(Δ 8-tetrahydrocannabinols，Δ 8-THCs)，大麻环酚型(cannabicyclols，CBLs)，大麻脂(cannabielsoins，CBEs)，大麻醇(cannabinols，CBNs)，脱氢大麻二酚型(cannabinodiol，CBNDs)，大麻三酚型(cannabitriols，CBTs)和其他大麻素(Elsohly,M.A.；Slade,D.,Chemical constituents of marijuana:the complex mixture of natural cannabinoids.Life Sciences 2005,78(5),539-48.)。其中，大麻环萜酚(CBC)以及其酸形式大麻环萜酚和大麻萜酚(CBG)以及其酸形式大麻萜酚酸是大麻素的主要成分，在加热或者长期储存会导致这些酸性大麻素脱羧形成中性大麻素(如大麻环萜酚酸形成大麻环萜酚，大麻萜酚形成大麻萜酚酸)。研究表明，大麻环萜酚(CBC)和大麻萜酚(CBG)对金黄色葡萄球菌具有抗菌活性(Appendino,G.；Gibbons,S.；Giana,A.；Pagani,A.；Grassi,G.；Stavri,M.；Smith,E.；Rahman,M.M.,Antibacterial cannabinoids from Cannabis sativa:a structure-activity study.Journal of Natural Products 2008,71(8),1427-30.)。CBG对TRP超家族的几个配体门控的阳离子通道也具有显著活性，并且可以充当TRPV1(TRP型香草素1)和TRPA1(TRP型锚蛋白1)的激动剂，能够作为TRPM8(TRP型褪黑素8)的有效抑制剂(Pollastro,F.；Taglialatela-Scafati,O.；Allarà,M.；
Figure PCTCN2021131047-appb-000001
E.；Di Marzo,V.；De Petrocellis,L.；Appendino,G.,Bioactive prenylogous cannabinoid from fiber hemp(Cannabis sativa).Journal of Natural Products 2011,74(9),2019-22.)。CBC能够抑制内源性大麻素失活并激活TRPA1，从而在实验模型系统中产生针对肠道炎症的保护作用，此外，CBC具有多种药理学和生物学效应，包括镇痛、抗伤害、抗炎活性。
目前，大麻环萜酚(CBC)和大麻萜酚(CBG)的获取途径主要通过从植物中提取或化学合成。然而化学合成工艺复杂、成本高并且产率很低。而大麻的农业种植面临若干挑战，例如植物对气候和疾病的敏感性、没有GAP标准化、大麻中的大麻环萜酚和大麻萜酚含量较低、占用耕地面积大且周期较长，而且与丰富的其他型大麻素共存，从植物中获得纯的样品费时费力，严重影响到其治疗潜力的研究。
微生物发酵具有生产效率高，周期短等优势，提供了一种从廉价的碳源生产大量高附加值产物的方法，同时，基于基因工程技术手段，通过对微生物的代谢通路改造获得具有特定功能的微生物也是近些年的研究热点，目前，通过基因工程手段获得能够生物合成大麻环萜酚酸的宿主细胞及其构建方法并将其用于大麻环萜酚酸的生物合成的相关研究还未见报道。
另一方面，为了进一步增加GPP的通量，一种从次级底物生物合成类异戊二烯的合成途径，称为异戊烯醇利用途径(IUP)，它从异戊二烯醇或泼尼醇产生类异戊二烯前体IPP和DMAPP。IUP是一种代谢途径，其通量可与一些最快的类异戊二烯途径竞争，并允许将类异戊二烯生物合成与中心碳代谢分离，这将极大地简化未来生产高价值类异戊二烯的工程努力。由于结构相似，异戊烯醇异构体被选为IPP和DMAPP的前体。首先，异戊二烯醇或异戊烯醇分别被磷酸化形成异戊烯基单磷酸酯(IP)或二甲基烯丙基单磷酸酯(DMAP)；然后，IP或DMAP再次被磷酸化形成IPP或DMAPP。该途径的第二步由异戊烯基磷酸激酶(IPK)催化，IPK是古细菌甲羟戊酸途径的一部分。尽管自然界中不会发生第一次磷酸化，但一些磷酸激酶可以表现出混杂的激酶活性。因此，本发明针对异戊烯醇激酶活性筛选了几种激酶，包括IPK同源物，一些IPK变体可以通过混杂活性将异戊烯醇转化为DMAP。
然而，竞争途径和代谢串扰经常阻碍目标化合物在细胞质中的有效合成。真核细胞通过利用细胞器来隔离生化途径控制其代谢的复杂性。在酿酒酵母中，过氧化物酶体是细胞器工程的合适目标，因为在大多数培养条件下，它们对细胞活力不是必需的，并且它们的数量和大小可以通过各种方式进行修改以更好地满足工程化的需要。酵母过氧化物酶体是脂肪酸发生β-氧化的场所，形成一个乙酰辅酶A库，可以通过异源途径，例如甲羟戊酸(MVA)途径，提供类异戊二烯前体IPP和DMAPP。此外，过氧化物酶体具有单层膜，允许大量小分子化合物被动或通过通道蛋白穿过。同时过氧化物酶体也是解毒细胞器，可以处理和隔离细胞其余部分的更多有毒分子。此外，由于大麻素是疏水性化合物使其在细胞质中溶解度有限。但是，它们在疏水性液体如脂质、油或脂肪中具有高溶解度。推测合成大麻环萜酚酸的前提物质CBGA很可能被隔离在脂滴中。因此将大麻环萜酚酸合酶定位到脂滴中以期通过增加底物和酶的局部浓度能够产生更快的反应速率和更高的生产力。
发明内容
本发明为解决大麻环萜酚和大麻环萜酚酸的制备方法复杂、成本高和产率低等问题，现提供以下技术方案。
本发明构建了能够生物合成大麻环萜酚酸重组酿酒酵母菌株，大麻环萜酚酸的生物合成途径中包含多个基因，一方面，优化酿酒酵母利用单糖通过其内源的甲羟戊酸途径合成大麻萜酚酸前体香叶基焦磷酸(GPP)；另一方面，构建了己酸到己酰基-CoA代谢途径，通过补给己酸利用酰基活化酶(CsAAE1)将己酸转化为己酰基-CoA，同时乙酰辅酶A羧化酶(ACC1)催化乙酰辅酶A产生丙二酰辅酶A，己酰基-CoA和三分子的丙二酰-CoA通过聚酮合酶(CsTKS)和橄榄酸环化酶(CsOAC)生成橄榄酸(OA)；此外，还构建了β-酮硫醇酶(RebktB)、3-羟基丁酰辅酶A脱氢酶(CnpaaH1)、巴豆酸酶(Cacrt)以及反式-2-烯酰辅酶A还原酶(Tdter)代谢通路，使得酿酒酵母利用单糖通过乙酰辅酶A生成己酰辅酶A，然后通过聚酮合酶(CsTKS)和橄榄酸环化酶(CsOAC)生成橄榄酸(OA)，提高橄榄醇酸的产量。最后，通过大麻萜酸合酶(CsPT4)将香叶基焦磷酸(GPP)和橄榄酸(OA)生成大麻萜酚酸(CBGA)，通过大麻环萜酚合酶催化生成大麻环萜酚酸(CBCA)(如图1所示)。
本发明提供了一种合成大麻环萜酚酸(CBCA)重组酿酒酵母菌株，所述重组酿酒酵 母菌株异源表达大麻萜酸合酶基因(CsPT4)和大麻环萜酚酸合酶基因(CBCAS)。
进一步地，所述重组酿酒酵母菌株过表达β-酮硫醇酶基因(RebktB)，3-羟基丁酰辅酶A脱氢酶基因(CnpaaH1)，巴豆酸酶基因(Cacrt)以及反式-2-烯酰辅酶A还原酶基因(Tdter)，大麻聚酮合酶基因(CsTKS)和橄榄酸环化酶基因(CsOAC)。
进一步地，所述重组酿酒酵母菌株过表达酰基活化酶基因(CsAAE1)和乙酰辅酶A羧化酶基因(ACC1)。
进一步地，所述重组酿酒酵母菌株过表达HMG-辅酶A还原酶基因(tHMG1)、乙酰辅酶A乙酰基转移酶基因(mvaE)、羟甲基戊二酸辅酶A合成酶基因(mvaS)、香叶基二磷酸合成酶基因(ERG20mut)、甲羟戊酸激酶基因(ERG12)、甲羟戊酸激酶基因(ERG8)、甲羟戊酸焦磷酸脱羧酶基因(ERG19)和异戊烯基焦磷酸异构酶基因(IDI)。
进一步地，所述重组酿酒酵母菌株过表达乙醛脱氢酶基因(ALD6)、乙酰辅酶A合成酶基因(ACS2)和乙醇脱氢酶基因(ADH2)。
进一步地，大麻萜酸合酶基因、大麻环萜酚酸合酶基因、β-酮硫醇酶基因、3-羟基丁酰辅酶A脱氢酶基因、巴豆酸酶基因、反式-2-烯酰辅酶A还原酶基因、大麻聚酮合酶基因、橄榄酸环化酶基因、酰基活化酶基因、乙酰辅酶A羧化酶基因、HMG-辅酶A还原酶基因、乙酰辅酶A乙酰基转移酶基因、羟甲基戊二酸辅酶A合成酶基因、香叶基二磷酸合成酶基因、甲羟戊酸激酶基因(ERG12)、甲羟戊酸激酶基因(ERG8)、甲羟戊酸焦磷酸脱羧酶基因、异戊烯基焦磷酸异构酶基因、乙醛脱氢酶基因、乙酰辅酶A合成酶基因和乙醇脱氢酶基因来源于同源的或者异源的。
进一步地，大麻萜酸合酶基因来源于大麻，大麻环萜酚酸合酶基因来源于大麻；β-酮硫醇酶基因来源于罗尔斯通氏菌，3-羟基丁酰辅酶A脱氢酶基因来源于钩虫贪铜菌，巴豆酸酶基因来源于丙酮丁醇梭菌，反式-2-烯酰辅酶A还原酶基因来源于齿密螺旋体；大麻聚酮合酶基因来源于大麻，橄榄酸环化酶基因来源于大麻；酰基活化酶基因来源于大麻，乙酰辅酶A羧化酶基因来源于酿酒酵母，截短的HMG-辅酶A还原酶基因来源于酿酒酵母，乙酰辅酶A乙酰基转移酶基因来源于粪肠球菌，羟甲基戊二酸辅酶A合成酶基因来源于粪肠球菌，香叶基二磷酸合成酶基因来源于酿酒酵母，甲羟戊酸激酶基因(ERG12)、甲羟戊酸激酶基因(ERG8)来源于酿酒酵母，甲羟戊酸焦磷酸脱羧酶基因来源于酿酒酵母，异戊烯基焦磷酸异构酶基因来源于酿酒酵母；乙醛脱氢酶基因、乙酰辅酶A合成酶基因和乙醇脱氢酶基因来源于酿酒酵母。
进一步地，大麻萜酸合酶基因的核苷酸序列如SEQ ID NO：1所示，大麻环萜酚酸合酶基因的核苷酸序列如SEQ ID NO：2所示；β-酮硫醇酶基因的核苷酸序列如SEQ ID NO：3所示，3-羟基丁酰辅酶A脱氢酶基因的核苷酸序列如SEQ ID NO：4所示，巴豆酸酶基因的核苷酸序列如SEQ ID NO：5所示，反式-2-烯酰辅酶A还原酶基因的核苷酸序列如SEQ ID NO：6所示，大麻聚酮合酶基因的核苷酸序列如SEQ ID NO：7所示，橄榄酸环化酶基因的核苷酸序列如SEQ ID NO：8所示；酰基活化酶基因的核苷酸序列如SEQ ID NO：9所示，乙酰辅酶A羧化酶基因的核苷酸序列如SEQ ID NO：10所示；HMG-辅酶A还原酶基因的核苷酸序列如SEQ ID NO：11所示，乙酰辅酶A乙酰基转移酶基 因的核苷酸序列如SEQ ID NO：12所示，羟甲基戊二酸辅酶A合成酶基因的核苷酸序列如SEQ ID NO：13所示，香叶基二磷酸合成酶基因的核苷酸序列如SEQ ID NO：14所示，甲羟戊酸激酶基因(ERG12)的核苷酸序列如SEQ ID NO：15所示，甲羟戊酸激酶基因(ERG8)的核苷酸序列如SEQ ID NO：16所示，甲羟戊酸焦磷酸脱羧酶基因的核苷酸序列如SEQ ID NO：17所示，异戊烯基焦磷酸异构酶基因的核苷酸序列如SEQ ID NO：18所示；乙醛脱氢酶基因的核苷酸序列如SEQ ID NO：19所示，乙酰辅酶A合成酶基因的核苷酸序列如SEQ ID NO：20所示，乙醇脱氢酶基因的核苷酸序列如SEQ ID NO：21所示。
进一步地，大麻萜酸合酶基因、大麻环萜酚酸合酶基因、β-酮硫醇酶基因、3-羟基丁酰辅酶A脱氢酶基因、巴豆酸酶基因、反式-2-烯酰辅酶A还原酶基因、大麻聚酮合酶基因、橄榄酸环化酶基因、酰基活化酶基因、乙酰辅酶A羧化酶基因、HMG-辅酶A还原酶基因、乙酰辅酶A乙酰基转移酶基因、羟甲基戊二酸辅酶A合成酶基因、香叶基二磷酸合成酶基因、甲羟戊酸激酶基因(ERG12)、甲羟戊酸激酶基因(ERG8)、甲羟戊酸焦磷酸脱羧酶基因、异戊烯基焦磷酸异构酶基因、乙醛脱氢酶基因、乙酰辅酶A合成酶基因和乙醇脱氢酶基因分别具有与SEQ ID NO：1-21所示的核苷酸具有至少70％、80％、90％、95％同源性且具有相应酶的活性功能。
进一步地，大麻萜酸合酶基因、大麻环萜酚酸合酶基因、β-酮硫醇酶基因、3-羟基丁酰辅酶A脱氢酶基因、巴豆酸酶基因、反式-2-烯酰辅酶A还原酶基因、大麻聚酮合酶基因、橄榄酸环化酶基因、酰基活化酶基因、乙酰辅酶A羧化酶基因、HMG-辅酶A还原酶基因、乙酰辅酶A乙酰基转移酶基因、羟甲基戊二酸辅酶A合成酶基因、香叶基二磷酸合成酶基因、甲羟戊酸激酶基因(ERG12)、甲羟戊酸激酶基因(ERG8)、甲羟戊酸焦磷酸脱羧酶基因、异戊烯基焦磷酸异构酶基因、乙醛脱氢酶基因、乙酰辅酶A合成酶基因和乙醇脱氢酶基因分别为SEQ ID NO：1-21所示的核苷酸经过一个或多个核苷酸序列的取代、替换或缺失得到的核苷酸序列，且具有相应酶的活性功能。
进一步地，大麻萜酸合酶基因、大麻环萜酚酸合酶基因、β-酮硫醇酶基因、3-羟基丁酰辅酶A脱氢酶基因、巴豆酸酶基因、反式-2-烯酰辅酶A还原酶基因、大麻聚酮合酶基因、橄榄酸环化酶基因、酰基活化酶基因、乙酰辅酶A羧化酶基因、HMG-辅酶A还原酶基因、乙酰辅酶A乙酰基转移酶基因、羟甲基戊二酸辅酶A合成酶基因、香叶基二磷酸合成酶基因、甲羟戊酸激酶基因(ERG12)、甲羟戊酸激酶基因(ERG8)、甲羟戊酸焦磷酸脱羧酶基因、异戊烯基焦磷酸异构酶基因、乙醛脱氢酶基因、乙酰辅酶A合成酶基因和乙醇脱氢酶基因分别为能够在中度或高度或极高度条件下与SEQ ID NO：1-21所示的核苷酸序列杂交互补的核苷酸序列，且具有相应酶的活性功能。
进一步地，大麻萜酸合酶基因、大麻环萜酚酸合酶基因、β-酮硫醇酶基因、3-羟基丁酰辅酶A脱氢酶基因、巴豆酸酶基因、反式-2-烯酰辅酶A还原酶基因、大麻聚酮合酶基因、橄榄酸环化酶基因、酰基活化酶基因、乙酰辅酶A羧化酶基因、HMG-辅酶A还原酶基因、乙酰辅酶A乙酰基转移酶基因、羟甲基戊二酸辅酶A合成酶基因、香叶基二磷酸合成酶基因、甲羟戊酸激酶基因(ERG12)、甲羟戊酸激酶基因(ERG8)、甲羟戊酸焦磷 酸脱羧酶基因、异戊烯基焦磷酸异构酶基因、乙醛脱氢酶基因、乙酰辅酶A合成酶基因和乙醇脱氢酶基因的核苷酸序列为部分或者全部经过密码子优化后的核苷酸序列。
进一步地，大麻萜酸合酶基因插入位点位于酵母基因组416d位点、CAN1y位点或YOLCd1b位点；大麻环萜酚酸合酶基因插入位点位于酵母基因组308a位点、HIS3b位点或511b位点。β-酮硫醇酶基因插入位点位于酵母基因组SAP155b位点；3-羟基丁酰辅酶A脱氢酶基因和巴豆酸酶基因插入位点位于酵母基因组SAP155c位点；反式-2-烯酰辅酶A还原酶基因插入位点位于酵母基因组YPRCδ15c位点；大麻聚酮合酶基因和橄榄酸环化酶基因插入位点位于酵母基因组1622b位点、X4位点、XI位点3或XII5位点；酰基活化酶基因插入位点位于酵母基因组911b位点；乙酰辅酶A羧化酶基因插入位点位于酵母基因组X3位点；截短的HMG-辅酶A还原酶基因和突变的香叶基二磷酸合成酶ERG20mut(F96W，N127W)插入位点位于酵母基因组1021b位点；乙酰辅酶A乙酰基转移酶基因和羟甲基戊二酸辅酶A合成酶基因插入位点位于酵母基因组1414a位点；甲羟戊酸激酶基因(ERG12)和异戊烯基焦磷酸异构酶基因插入位点位于酵母基因组1114a位点；甲羟戊酸激酶基因(ERG8)和甲羟戊酸焦磷酸脱羧酶基因插入位点位于酵母基因组1014a位点；乙醛脱氢酶基因和乙酰辅酶A合成酶基因插入位点位于酵母基因组1309a位点；乙醇脱氢酶基因插入位点位于酵母基因组X2位点。
进一步地，上述基因的拷贝数分别为1～10个。
本发明另一方面提供了一种上述重组酿酒酵母菌株的构建方法，主要包括以下步骤：
1)分别构建大麻萜酸合酶基因和大麻环萜酚酸合酶基因表达盒，通过同源重组技术将上述表达盒插入到酿酒酵母的基因组中；
2)分别构建β-酮硫醇酶基因、3-羟基丁酰辅酶A脱氢酶基因、巴豆酸酶基因、反式-2-烯酰辅酶A还原酶基因、大麻聚酮合酶基因和橄榄酸环化酶基因表达盒，通过同源重组将上述表达盒插入到步骤(1)得到的酿酒酵母的基因组中；
3)分别构建酰基活化酶基因、乙酰辅酶A羧化酶基因表达盒，通过同源重组将上述表达盒插入到步骤(2)得到的酿酒酵母的基因组中；
4)分别构建HMG-辅酶A还原酶基因、乙酰辅酶A乙酰基转移酶基因、羟甲基戊二酸辅酶A合成酶基因、香叶基二磷酸合成酶基因、甲羟戊酸激酶基因(ERG12)、甲羟戊酸激酶基因(ERG8)、甲羟戊酸焦磷酸脱羧酶基因和异戊烯基焦磷酸异构酶基因表达盒，通过同源重组将上述表达盒插入到步骤(3)得到的酿酒酵母的基因组中；
5)分别构建乙醛脱氢酶基因、乙酰辅酶A合成酶基因和乙醇脱氢酶基因表达盒，通过同源重组将上述表达盒插入到步骤(4)得到的酿酒酵母的基因组中。
进一步地，同源重组使用锌指核酸酶(ZFN)、转录激活样效应因子核酸酶(TALEN)和CRISPR/Cas系统。
进一步地，所述的同源重组使用CRISPR/Cas系统。
进一步地，上述基因的启动子分别为组成型启动子或诱导型启动子。
进一步地，所述启动子为GAL1、GAL10、GPD、TEF1、PGK1或ADH。
进一步地，生物信息学分析和序列启动子缺失分析表明，YPL062W是ALD6的核心 启动子，ALD6的表达水平与萜类产量呈负相关。为了进一步增加乙酰辅酶A通量，敲除乙醇脱氢酶基因(ADH1)和YPL062W；过表达Zea mays来源的丙酮酸脱羧酶基因(PDC)、内源的酰基辅酶A合成酶基因(FAA2)及Salmonella enterica来源的乙酰辅酶A合成酶基因(SeACS)。
为了增加大麻环萜酚酸重组酿酒酵母菌株中GPP及下游各产物通量，本发明提供了上述重组酿酒酵母菌株的另一种构建方法，主要包括以下步骤：
(1)一方面，从Salmonella enterica subsp(PhoN)和Saccharomyces cerevisiae(ScCK)筛选出使用的混杂激酶。另一方面，从多个物种来源Thermoplasma acidophilum(thaIPK),Methanococcus vannielii(mvIPK),Methanolobus tindarius(mt IPK),Methanosalsum zhilinae(mzIPK),Methanococcus maripaludis(mmIPK)and Methanococcoides burtonii(mbIPK)及Arabidopsis thaliana(atIPK)中筛选出能够在酿酒酵母中成功表达的异戊基磷酸激酶。
(2)将筛选出的混杂激酶基因与异戊烯基磷酸激酶、乙酰辅酶A乙酰基转移酶基因、羟甲基戊二酸辅酶A合成酶基因、甲羟戊酸激酶基因(ERG12)、甲羟戊酸激酶基因(ERG8)、甲羟戊酸焦磷酸脱羧酶基因、异戊烯基焦磷酸异构酶基因(IDI)、香叶基二磷酸合成酶基因(ERG20mut)、大麻萜酸合酶基因(CsPT4)和大麻环萜酚合酶基因(CBCAS)定位到过氧化物酶体。
(3)为了增加过氧化物酶体的数量及体积，敲除过氧化物酶体膜蛋白PEX31，PEX32；过表达过氧化物酶体膜蛋白PEX3，PEX19，PEX11和PEX34，从而增加大麻环萜酚的生产。
进一步地，为了确保CBCAS的正确折叠，同时筛选了多种分子伴侣，折叠酶，转录激活因子以提高CBCAS的表达活性。包括参与折叠和内质网质量控制的蛋白质(CNE1p、KAR2p、PDI1p、ERO1p)、辅因子(FAD1p)、UPR蛋白IRE1*p、UPR激活剂Hac1s及内质网大小调节因子INO1。
进一步地，定点突变大麻环萜酚合酶产生多个突变体，分别为H292L,H292R,Y417H,Y417R,N89Q-N499Q,R463C-D488C，T379S,K377R,N196Q,F171Y,S170T，R349K,F365Y,V138A,R532K,L524I,Y472F,N528Q,F353Y及其双突变体、三突变体和多突变体组合，筛选出活性最好的突变体用于生产大麻环萜酚酸。
进一步地，为了增加胞内ATP和溶氧供应，过表达腺苷酸激酶基因(ADK1)、Pseudomonas stutzeri来源的亚磷酸盐脱氢酶基因ptxD及透明颤菌血红蛋白基因(VHB)提高ATP的合成速率，从而促进细胞生长和大麻素的生产。
进一步地，将大麻环萜酚酸合酶定位到脂滴中以期通过增加底物和酶的局部浓度能够产生更快的反应速率和更高的生产力。同时，为了提高大麻素的溶解度，过表达三酰甘油途径TAG中关键酶基因二酰基甘油酰基转移酶基因DGA1、G3P脱氢酶基因GPD1和磷脂酸磷酸酯酶基因(PAH1)，敲除脂滴合成关键蛋白基因SEI1，增加脂质水平和脂滴聚集。同时将大麻环萜酚酸合酶定位到脂滴中以期通过增加底物和酶的局部浓度能够产生更快的反应速率和更高的生产力，扩大工程酵母合成大麻素的储存能力。
进一步地，将大麻环萜酚代谢途径中的关键酶基因同时整合到酿酒酵母的rDNA位 点，实现多个关键酶基因的多拷贝表达。
进一步地，为了节省发酵成本，敲除Gal80以解除其对Gal4的抑制作用，同时替换Gal4的启动子解除葡萄糖抑制作用，因此，不再需要半乳糖作为诱导剂，通过葡萄糖和乙醇混合发酵优化大麻环萜酚(CBCA)的产量。
进一步地，混杂激酶基因、异戊烯基磷酸激酶基因、丙酮酸脱羧酶基因、酰基辅酶A合成酶基因、参与折叠和内质网质量控制的相关基因、腺苷酸激酶基因、亚磷酸盐脱氢酶基因、血红蛋白基因、二酰基甘油酰基转移酶基因、三磷酸甘油醛脱氢酶基因,磷脂酸磷酸酯酶基因,内质网质量控制的蛋白质(CNE1p、KAR2p、PDI1p、ERO1p)、辅因子(FAD1p)、UPR蛋白IRE1*p、UPR激活剂Hac1s及内质网大小调节因子INO1来源于同源的或者异源的。
进一步地，混杂激酶基因来源于酿酒酵母；异戊烯基磷酸激酶基因来源于嗜酸热原体，甲基拟甲烷球菌和拟南芥；丙酮酸脱羧酶基因来源于玉米；酰基辅酶A合成酶基因来源于酿酒酵母；乙酰辅酶A合成酶基因来源于沙门氏菌；参与折叠和内质网质量控制的相关基因和腺苷酸激酶基因来源于酿酒酵母；亚磷酸盐脱氢酶基因来源于斯氏假单胞菌；血红蛋白基因来源于透明颤菌、二酰基甘油酰基转移酶基因、三磷酸甘油醛脱氢酶基因,磷脂酸磷酸酯酶基因,内质网质量控制的蛋白质(CNE1p、KAR2p、PDI1p、ERO1p)、辅因子(FAD1p)、UPR蛋白IRE1*p、UPR激活剂Hac1s及内质网大小调节因子INO1来源于酿酒酵母。
进一步地，混杂激酶基因(ScCK)的核苷酸序列如SEQ ID NO：22所示，异戊烯基磷酸激酶基因(thaIPK)的核苷酸序列如SEQ ID NO：23所示，异戊烯基磷酸激酶基因(mbIPK)的核苷酸序列如SEQ ID NO：24所示，异戊烯基磷酸激酶基因(atIPK)的核苷酸序列如SEQ ID NO：25所示，丙酮酸脱羧酶基因(ZmPDC)的核苷酸序列如SEQ ID NO：26所示，酰基辅酶A合成酶基因的核苷酸序列如SEQ ID NO：27所示，乙酰辅酶A合成酶基因(SeACS)的核苷酸序列如SEQ ID NO：28所示，参与折叠和内质网质量控制的基因(ERO1)的核苷酸序列如SEQ ID NO：29所示,参与折叠和内质网质量控制的基因(CEN1)的核苷酸序列如SEQ ID NO：30所示,参与折叠和内质网质量控制的基因(KAR2)的核苷酸序列如SEQ ID NO：31所示,参与折叠和内质网质量控制的基因(PDI1)的核苷酸序列如SEQ ID NO：32所示,非折叠蛋白响应(UPR)基因(IRE1*)的核苷酸序列如SEQ ID NO：33所示，非折叠蛋白响应(UPR)基因(HAC1s)的核苷酸序列如SEQ ID NO：34所示，内质网大小相关基因(INO1)的核苷酸序列如SEQ ID NO：35所示，腺苷酸激酶基因的核苷酸序列如SEQ ID NO：36所示，亚磷酸盐脱氢酶基因ptxD的核苷酸序列如SEQ ID NO：37所示，血红蛋白基因的核苷酸序列如SEQ ID NO：38所示，二酰基甘油酰基转移酶基因的核苷酸序列如SEQ ID NO：39所示，磷脂酸磷酸酯酶基因的核苷酸序列如SEQ ID NO：40所示，三磷酸甘油醛脱氢酶基因的核苷酸序列如SEQ ID NO：41所示。
进一步地，混杂激酶基因、异戊烯基磷酸激酶基因、丙酮酸脱羧酶基因、酰基辅酶A合成酶基因、参与折叠和内质网质量控制的相关基因、腺苷酸激酶基因、亚磷酸盐脱氢酶 基因、血红蛋白基因、二酰基甘油酰基转移酶基因、三磷酸甘油醛脱氢酶基因，磷脂酸磷酸酯酶基因，内质网质量控制的蛋白质(CNE1p、KAR2p、PDI1p、ERO1p)、辅因子(FAD1p)、UPR蛋白IRE1*p、UPR激活剂Hac1s及内质网大小调节因子INO1的核苷酸序列分别具有与SEQ ID NO：22-41所示的核苷酸具有至少70％、80％、90％、95％同源性且具有相应酶的活性功能。
进一步地，混杂激酶基因、异戊烯基磷酸激酶基因、丙酮酸脱羧酶基因、酰基辅酶A合成酶基因、参与折叠和内质网质量控制的相关基因、腺苷酸激酶基因、亚磷酸盐脱氢酶基因、血红蛋白基因、二酰基甘油酰基转移酶基因、三磷酸甘油醛脱氢酶基因，磷脂酸磷酸酯酶基因，内质网质量控制的蛋白质(CNE1p、KAR2p、PDI1p、ERO1p)、辅因子(FAD1p)、UPR蛋白IRE1*p、UPR激活剂Hac1s及内质网大小调节因子INO1的核苷酸序列分别为SEQ ID NO：22-41所示的核苷酸经过一个或多个核苷酸序列的取代、替换或缺失得到的核苷酸序列，且具有相应酶的活性功能。
进一步地，混杂激酶基因、异戊烯基磷酸激酶基因、丙酮酸脱羧酶基因、酰基辅酶A合成酶基因、参与折叠和内质网质量控制的相关基因、腺苷酸激酶基因、亚磷酸盐脱氢酶基因、血红蛋白基因、二酰基甘油酰基转移酶基因、三磷酸甘油醛脱氢酶基因，磷脂酸磷酸酯酶基因，内质网质量控制的蛋白质(CNE1p、KAR2p、PDI1p、ERO1p)、辅因子(FAD1p)、UPR蛋白IRE1*p、UPR激活剂Hac1s及内质网大小调节因子INO1的核苷酸序列分别为能够在中度或高度或极高度条件下与SEQ ID NO：22-41所示的核苷酸序列杂交互补的核苷酸序列，且具有相应酶的活性功能。
进一步地，混杂激酶基因、异戊烯基磷酸激酶基因、丙酮酸脱羧酶基因、酰基辅酶A合成酶基因、参与折叠和内质网质量控制的相关基因、腺苷酸激酶基因、亚磷酸盐脱氢酶基因、血红蛋白基因、二酰基甘油酰基转移酶基因、三磷酸甘油醛脱氢酶基因，磷脂酸磷酸酯酶基因，内质网质量控制的蛋白质(CNE1p、KAR2p、PDI1p、ERO1p)、辅因子(FAD1p)、UPR蛋白IRE1*p、UPR激活剂Hac1s及内质网大小调节因子INO1的核苷酸序列为部分或者全部经过密码子优化后的核苷酸序列。
进一步地，EfmvaE-EfmvaS-SKL基因插入位点位于酿酒酵母基因组YIRCΔ6位点；ERG19-ERG8-SKL基因插入位点位于酿酒酵母基因组YMRWΔ15位点；ERG12-SKL基因插入位点位于酿酒酵母基因组YNRCΔ9位点；ScCK-SKL基因插入位点位于酿酒酵母基因组YGLCτ3位点；atIPK-SKL基因插入位点位于酿酒酵母基因组YORWΔ17位点；IDI-SKL基因插入位点位于酿酒酵母基因组YPRCτ3位点；ERG20-SKL基因插入位点位于酿酒酵母基因组SPB1/PBN1位点；zmPDC基因插入位点位于酿酒酵母基因组YCRWδ11位点；FAA2基因插入位点位于酿酒酵母基因组XII4位点；乙酰辅酶A合成酶基因(SeACS)插入位点位于酿酒酵母基因组YORWΔ22位点；ADK1基因插入位点位于酿酒酵母基因组YARCδ8位点；ptxD基因插入位点位于酿酒酵母基因组YCRWδ11位点；VHB基因插入位点位于酿酒酵母基因组YBRWδ16位点；CNE1基因插入位点位于酿酒酵母基因组I12位点；KAR2基因插入位点位于酿酒酵母基因组I4和I32位点；PDI1基因插入位点位于酿酒酵母基因组I10位点；ERO1基因插入位点位于酿酒酵母基因组X3位点；IRE1基因插入位点位于酿酒酵母 基因组I8位点；将Hac1s基因插入位点位于酿酒酵母基因组I28位点；将INO1基因插入位点位于酿酒酵母基因组I3位点；将DGA1插入位点位于酿酒酵母基因组YCRWδ12位点；PAH1基因插入位点位于酿酒酵母基因组YERCδ8位点；GPD1基因插入位点位于酿酒酵母基因组YORWΔ22位点。
进一步地，上述关键酶基因构建到高拷贝质粒载体游离表达。
本发明提供了一种利用上述重组酿酒酵母发酵生产大麻环萜酚酸的方法，所述方法主要包括以下步骤：
1)在合适的培养基中培养上述重组酿酒酵母细胞一段时间；
2)回收发酵产生的大麻环萜酚酸；
3)通过加热或者长期储存使得大麻环萜酚酸脱羧形成大麻环萜酚。
进一步地，所述的培养基为YPD培养基。
进一步地，所述的培养基含有葡萄糖、半乳糖、甘油、乙醇、淀粉、己酸或橄榄醇酸中的一种或多种混合物。
进一步地，培养条件为转速50～300rpm，温度28～32℃，培养时间为24-120h。
进一步地，回收发酵产生的大麻环萜酚酸过程包括使用有机溶剂萃取发酵液中的大麻环萜酚酸步骤。
进一步地，所述的有机溶剂为乙酸乙酯、己烷、庚烷、石油醚、或氯仿中的一种或多种混合物。
进一步地，回收发酵产生的大麻环萜酚酸过程包括破碎发酵得到的重组酿酒酵母过程。
进一步地，所述的破碎方法为高压均质破碎法、超声波破碎法、球磨破碎法、反复冻融破碎法或酶溶破碎法。
进一步地，萃取过程中所述的有机溶剂与发酵液的体积比1:1～1:20。
本发明相对于现有技术具有的有益效果如下：
1.本发明公开了一种能够生物合成大麻环萜酚酸的重组酿酒酵母菌株，提供了一种从廉价的碳源生产大量高附加值大麻环萜酚的新途径。
2.本发明构建生物合成大麻环萜酚酸的重组酿酒酵母菌株方法准确高效，所得到的重组酿酒酵母菌株遗传性能稳定。
3.本发明公开的重组酿酒酵母发酵生产大麻环萜酚酸的方法生产效率高，周期短，成本低，有利于大麻环萜酚的大规模生产和医药领域应用的拓展。
附图说明
图1为重组酿酒酵母大麻环萜酚酸生物合成途径图谱，其中，CsAAE1：酰基活化酶，ACC1：乙酰辅酶A羧化酶，RebktB：β-酮硫醇酶，CnpaaH1：3-羟基丁酰辅酶A脱氢酶，Cacrt：巴豆酸酶，Tdter：反式-2-烯酰辅酶A还原酶，CsTKS：聚酮合酶，CsOAC：橄榄酸环化酶，CsPT4：橄榄醇酸香叶基转移酶，CBCAS：大麻环萜酚酸合酶。
图2为GPP生物合成通路示意图。
图3为重组酿酒酵母发酵生产的大麻环萜酚酸的液相色谱图，其中，上图：大麻环萜酚 酸标准品LC-MS的液相色谱图，横坐标为保留时间，纵坐标为丰度；下图：大麻环萜酚酸重组基因工程菌株发酵样品LC-MS的液相色谱图。
图4为重组酿酒酵母发酵生产的大麻环萜酚酸的质谱谱图，横坐标为M/Z，纵坐标为丰度。上图：大麻环萜酚酸标准品在的LC-MS质谱图；下图：大麻环萜酚酸重组基因工程菌株发酵样品的LC-MS质谱图。
具体实施方式
下面结合实施例对本发明进行详细的说明，但本发明的实施方式不限于此，显而易见地，下面描述中的实施例仅是本发明的部分实施例，对于本领域技术人员来讲，在不付出创造性劳动性的前提下，获得其他的类似的实施例均落入本发明的保护范围。
实施例1
构建能够表达大麻萜酸合酶和大麻环萜酚酸合酶的重组酵母菌株
本发明的宿主菌为酿酒酵母(Saccharomyces cerevisiae)INVSc1，二倍体的INVSc1具有较高的鲁棒性，其复杂的基因调控网络有利于不利环境条件下酶的表达与催化。本发明基于发现香叶基焦磷酸(GPP)和橄榄醇酸(OA)在大麻萜酸合酶(CsPT4)的作用下生成大麻萜酚酸(CBGA)，大麻萜酚酸(CBGA)通过大麻环萜酚合酶(CBCAS)催化形成大麻环萜酚酸(CBCA)，构建2个基因表达盒：经过密码子优化的CsPT4基因表达盒和经过密码子优化的CBCAS基因表达盒，CsPT4基因表达盒使用GAL10启动子和CYC1终止子，CBCAS基因表达盒使用GAL10启动子和CYC1终止子，通过基因编辑技术将CsPT4基因表达盒整合到酿酒酵母的416d，CAN1y和YOLCd1b基因组位点；将CBCAS基因表达盒分别整合到酿酒酵母的308a，HIS3b和511b基因组位点，表达3个拷贝的CsPT4基因和CBCAS基因，获得能够表达大麻萜酸合酶和大麻环萜酚酸合酶的重组酿酒酵母菌株。
实施例2
构建能够利用糖类产大麻环萜酚酸的重组酵母菌株
为使得实施例1的重组酿酒酵母中能够生物合成橄榄醇酸，在上述重组酿酒酵母中构建利用糖类通过己酰辅酶A生成橄榄醇酸生物代谢合成途径。构建6个经过密码子优化基因表达盒：RebktB基因表达盒，CnpaaH1基因表达盒，Cacrt基因表达盒，Tdter基因表达盒，CsTKS基因表达盒和CsOAC基因表达盒。RebktB基因表达盒使用TEF1启动子和TEF1终止子，CnpaaH1基因表达盒使用GAL10启动子和CYC1终止子，Cacrt基因表达盒使用GAL1启动子和ADH1终止子，Tdter基因表达盒使用PGK1启动子和HXT7终止子，CsTKS基因表达盒使用GAL10启动子和CYC1终止子，CsOAC基因表达盒使用GAL1启动子和ADH1终止子。通过基因编辑技术，将RebktB基因表达盒整合到酿酒酵母SAP155b基因组位点，将CnpaaH1基因表达盒和Cacrt基因表达盒整合到酿酒酵母SAP155c基因组位点，将Tdter基因表达盒整合到酿酒酵母YPRCδ15c基因组位点，分别表达1个拷贝上述基因。将CsOAC基因表达盒和CsTKS基因表达盒组成一个表达盒组分别整合到酿酒酵母的1622b，X4，XI3，XII5基因组位点，表达4个拷贝上述基因，最终获得能够利用糖类产大麻环萜酚酸的重组酿酒酵母菌株。
实施例3
构建重组酿酒酵母中己酸到橄榄醇酸生物代谢合成途径
为了增加实施例2的重组酿酒酵母的己酰辅酶A的通量，构建重组酿酒酵母中己酸到橄榄醇酸生物代谢合成途径，构建2个基因表达盒：经过密码子优化CsAAE1基因表达盒和ACC1基因表达盒，CsAAE1和ACC1基因表达盒均使用GPD启动子和CYC1终止子，通过基因编辑技术，将ACC1基因表达盒整合到酿酒酵母X3基因组位点，过表达1个拷贝上述基因；将CsAAE1基因表达盒整合到酿酒酵母208a，911b和106a基因组位点表达3个拷贝上述基因，通过补给己酸，在CsAAE1的催化下将己酸转化为己酰辅酶A。同时，乙酰辅酶A通过ACC1催化产生的丙二酰辅酶A，己酰辅酶A与丙二酰辅酶A在大麻聚酮合酶(CsTKS)和橄榄酸环化酶(CsOAC)的作用下生成橄榄醇酸，从而提高重组酿酒酵母生物合成大麻环萜酚酸的产量。
实施例4
优化重组酿酒酵母内源甲羟戊酸途径
为了增加实施例3的重组酿酒酵母GPP的通量，进一步优化重组酿酒酵母内源甲羟戊酸途径，构建8个基因表达盒：截短的tHMG1基因表达盒，经过密码子优化的mvaE基因表达盒，mvaS基因表达盒，ERG20mut(F96W，N127W)基因表达盒，ERG12基因表达盒，ERG8基因表达盒，ERG19基因表达盒和IDI基因表达盒。tHMG1基因表达盒使用GAL1启动子和ADH1终止子，mvaE基因表达盒使用GAL1启动子和ADH1终止子，mvaS基因表达盒使用GAL10启动子和CYC1终止子，ERG20mut基因表达盒使用GAL10启动子和CYC1终止子，ERG12基因表达盒使用GAL10启动子和CYC1终止子，ERG8基因表达盒使用GAL1启动子和ADH1终止子，ERG19基因表达盒使用GAL10启动子和CYC1终止子，IDI基因表达盒使用GAL1启动子和ADH1终止子。通过基因编辑技术，将tHMG1基因表达盒和ERG20mut基因表达盒整合到酿酒酵母的1021b基因组位点，将mvaE基因表达盒和mvaS基因表达盒整合到酿酒酵母的1414a基因组位点，将ERG12基因表达盒和IDI基因表达盒整合到酿酒酵母的1114a基因组位点，将ERG8基因表达盒和ERG19基因表达盒整合到酿酒酵母的1014a基因组位点，过表达1个拷贝上述基因，从而保证重组酿酒酵母中甲羟戊酸下游途径中香叶基焦磷酸的供给。
实施例5
优化重组酿酒酵母乙酰辅酶A的代谢途径
为了增加实施例4的重组酿酒酵母细胞质中乙酰辅酶A的代谢通量，从NCBI数据库获得Saccharomyces cerevisiae来源的乙醛脱氢酶(ALD6)、乙酰辅酶A合成酶(ACS2)和乙醇脱氢酶(ADH2)，构建3个基因表达盒：ALD6基因表达盒、ACS2基因表达盒和ADH2基因表达盒。ALD6基因表达盒使用GAL10启动子和CYC1终止子，ACS2基因表达盒使用GAL1启动子和ADH1终止子，ADH2基因表达盒使用GPD启动子和CYC1终止子。通过基因编辑技术，将ALD6基因表达盒和ACS2基因表达盒整合到酿酒酵母的1309a基因组位点，将ADH2基因表达盒整合到酿酒酵母的X2基因组位点，过表达1个拷贝上述基因，提高重组酿酒酵母细胞质中乙酰辅酶A通量，为甲羟戊酸途径、橄榄醇酸以及大麻环萜酚酸的生 物合成提供前体化合物。
实施例6
构建过氧化物酶体中大麻环萜酚生物合成途径
首先构建能够定位到过氧化物酶体的基因表达盒，分别在经过密码子优化的IPK基因、胆碱激酶(ScCK)、乙酰辅酶A乙酰基转移酶基因(EfmvaE)、羟甲基戊二酸辅酶A合成酶基因(EfmvaS)、甲羟戊酸激酶基因(ERG12)、甲羟戊酸激酶基因(ERG8)、甲羟戊酸焦磷酸脱羧酶基因(ERG19)、异戊烯基焦磷酸异构酶基因(IDI)、香叶基二磷酸合成酶基因(ERG20mut)、经过密码子优化的大麻萜酸合酶基因(CsPT4)和大麻环萜酚合酶基因(CBCAS)的N端加上过氧化物酶体PTS1信号肽序列KL-X5-QL，C端加上过氧化物酶体PTS2信号肽序列SKL。IPK、胆碱激酶(ScCK)基因表达盒使用TEF1启动子和TEF1终止子。EfmvaE,EfmvaS,ERG8,ERG19,ERG12,IDI、ERG20mut、CsPT4、CBCAS基因表达盒均使用GAL10启动子和CYC1终止子。通过基因编辑技术将EfmvaE-EfmvaS-SKL基因表达盒整合到酿酒酵母的YIRCΔ6基因组位点；将ERG19-ERG8-SKL基因表达盒整合到酿酒酵母的YMRWΔ15基因组位点；将ERG12-SKL基因表达盒整合到酿酒酵母的YNRCΔ9基因组位点；将ScCK-SKL基因表达盒整合到酿酒酵母的YGLCτ3基因组位点；将atIPK-SKL基因表达盒整合到酿酒酵母的YORWΔ17基因组位点；将IDI-SKL基因表达盒整合到酿酒酵母的YPRCτ3基因组位点；将ERG20-SKL基因表达盒整合到酿酒酵母的SPB1/PBN1基因组位点。
实施例7
为了进一步增加细胞质中乙酰辅酶A的供应，过表达Zea mays来源的丙酮酸脱羧酶基因(PDC)及内源的酰基辅酶A合成酶基因(FAA2)。将经过密码子优化的丙酮酸脱羧酶基因zmPDC、内源的酰基辅酶A合成酶基因(FAA2)和Salmonella enterica来源的乙酰辅酶A合成酶基因(SeACS)分别构建到TEF1启动子和TEF1终止子中。通过基因编辑技术将zmPDC、FAA2基因和乙酰辅酶A合成酶基因(SeACS)表达盒分别整合到酿酒酵母的X2，XII4和YORWΔ22基因组位点，同时对ADH1进行敲除。
实施例8
为了增加胞内ATP和溶氧供应，过表达腺苷酸激酶基因(ADK1)、Pseudomonas stutzeri来源的亚磷酸盐脱氢酶基因ptxD及透明颤菌血红蛋白基因(VHB)。将腺苷酸激酶基因(ADK1)、经过密码子优化的亚磷酸盐脱氢酶基因ptxD以及透明颤菌血红蛋白基因(VHB)分别构建到TEF1启动子和TEF1终止子中。通过基因编辑技术将ADK1、ptxD和VHB基因表达盒分别整合到酿酒酵母的YARCδ8、YCRWδ11和YBRWδ16基因组位点。
实施例9
为了确保CBCAS的正确折叠，分别从酿酒酵母基因组扩增参与折叠和内质网质量控制的蛋白质(CNE1p、KAR2p、PDI1p、ERO1p、IRE1p)、辅因子(FAD1p)、UPR激活剂Hac1s以及内质网大小调节因子INO1。分别利用PGK1启动子和HXT7终止子构建表达盒。通过基因编辑技术将CNE1基因表达盒整合到酿酒酵母的I12基因组位点；将KAR2基因表达盒整合到酿酒酵母的I4和I32基因组位点；将PDI1基因表达盒整合到酿酒酵母的I10基因组位点；将ERO1基因表达盒整合到酿酒酵母的X3基因组位点；将IRE1基因表达盒整合到酿酒 酵母的I8基因组位点；将Hac1s基因表达盒整合到酿酒酵母的I28基因组位点；将INO1基因表达盒整合到酿酒酵母的I3基因组位点。
实施例10
为了提高大麻素在细胞内的溶解度，过表达三酰甘油合成途径TAG中的二酰基甘油酰基转移酶基因DGA1，三磷酸甘油醛脱氢酶基因GPD1和磷脂酸磷酸酯酶基因(PAH1)。将GPD1，DGA1和PAH1分别构建到TEF1启动子和TEF1终止子中。通过基因编辑技术将GPD1，DGA1和PAH1基因表达盒分别整合到酿酒酵母的YORWΔ22,YCRWδ12和YERCδ8基因组位点。
实施例11
将定点突变大麻环萜酚合酶产生多个突变体H292L,H292R,Y417H,Y417R,N89Q-N499Q,R463C-D488C,T379S,K377R,N196Q,F171Y,S170T，R349K,F365Y,V138A,R532K,L524I,Y472F,N528Q,F353Y及其双突变体、三突变体和多突变体组合分别构建到GAL10启动子和CYC1终止子中，通过基因编辑技术分别整合到HIS3b和511b基因组位点。
实施例12
重组酿酒酵母菌株发酵产大麻环萜酚酸
将实施例2获得的高产大麻环萜酚酸重组酿酒酵母菌株挑到含有3-5mL YPD(10g/L酵母提取物、20g/L蛋白胨、20g/L右旋葡萄糖)的试管中。于200rpm，30℃培养24h，直到培养基中葡萄糖耗尽。将生长饱和的菌株4500rpm离心传代培养到含有100mL YPG(10g/L酵母提取物、20g/L蛋白胨、20g/L半乳糖)的新的培养基中，200rpm，30℃培养24-48h。将发酵液或高压匀浆破碎的菌体液于4500rpm离心5min后取上清液，使用上清液1/5体积的有机溶剂乙酸乙酯萃取(5体积的发酵液加1体积的乙酸乙酯)，离心取有机层进行旋蒸，得到大麻环萜酚酸粗品，于105℃加热15分钟，然后加热至145℃持续55分钟后获得大麻环萜酚。
实施例13
重组酵母菌株发酵产大麻环萜酚酸
将实施例5获得的高产大麻环萜酚酸重组酿酒酵母菌株挑到含有3-5mL YPD(10g/L酵母提取物、20g/L蛋白胨、20g/L右旋葡萄糖)的试管中。将细胞在于200rpm，30℃培养24h，直到培养基中葡萄糖耗尽。将生长饱和的菌株4500rpm离心传代培养到含有100mL YPG(10g/L酵母提取物、20g/L蛋白胨、20g/L半乳糖、1mM橄榄醇酸或2mM己酸)的新的培养基中，200rpm，30℃培养24-48h。将发酵液或高压匀浆破碎的菌体液于4500rpm离心5min后取上清液，使用上清液1/5体积的有机溶剂乙酸乙酯萃取(5体积的发酵液加1体积的乙酸乙酯)，离心取有机层进行旋蒸，得到大麻环萜酚酸粗品，于105℃加热15分钟，然后加热至145℃持续55分钟后获得大麻环萜酚。
实施例14
大麻环萜酚的鉴定方法
将发酵液或高压匀浆破碎的菌体液于4500rpm离心5min后取上清液，使用上清液 1/5体积的有机溶剂乙酸乙酯萃取(5体积的发酵液加1体积的乙酸乙酯)，离心取有机层进行旋蒸，将蒸发后的物质重悬于乙腈/0.05％甲酸水溶液(80％/20％v/v)的混合溶液中，用有机滤膜过滤后，获得含有大麻萜酚酸的高浓度有机相，通过液相色谱/飞行时间质谱联用仪进行检测分析，结果如图3和图4所示。
大麻环萜酚酸检测分析相关仪器设备和实验参数如下：仪器为安捷伦6224 TOF LC/MS，柱温25℃，色谱柱为安捷伦C 18色谱柱，流速为0.2mL/min，流动相为含有0.05％甲酸的水溶液(A)和乙腈溶液(B)，梯度洗脱条件为：0-40min，30％-98％乙腈；40-50min，98％乙腈；50-51min，98％-30％乙腈，进样体积为20μL。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (18)

  1. 一种能够合成大麻环萜酚酸的重组酿酒酵母，其特征在于，所述重组酿酒酵母菌株异源表达大麻萜酸合酶基因和大麻环萜酚酸合酶基因。
  2. 根据权利要求1所述的重组酿酒酵母，其特征在于，所述重组酿酒酵母异源表达β-酮硫醇酶基因、3-羟基丁酰辅酶A脱氢酶基因、巴豆酸酶基因、反式-2-烯酰辅酶A还原酶基因、大麻聚酮合酶基因和橄榄酸环化酶基因。
  3. 根据权利要求2所述的重组酿酒酵母，其特征在于，所述重组酿酒酵母异源表达酰基活化酶基因和乙酰辅酶A羧化酶基因。
  4. 权利要求1-3所述的重组酿酒酵母的构建方法，其特征在于，主要包括以下步骤：
    1)分别构建大麻萜酸合酶基因和大麻环萜酚酸合酶基因表达盒，通过同源重组将上述表达盒插入到酿酒酵母的基因组中；
    2)分别构建β-酮硫醇酶基因、3-羟基丁酰辅酶A脱氢酶基因、巴豆酸酶基因、反式-2-烯酰辅酶A还原酶基因、大麻聚酮合酶基因和橄榄酸环化酶基因表达盒，通过同源重组将上述表达盒插入到步骤(1)得到的酿酒酵母的基因组中；
    3)分别构建酰基活化酶基因和乙酰辅酶A羧化酶基因表达盒，通过同源重组将上述表达盒插入到步骤(2)得到的酿酒酵母的基因组中；
    4)分别构建HMG-辅酶A还原酶基因、乙酰辅酶A乙酰基转移酶基因、羟甲基戊二酸辅酶A合成酶基因、香叶基二磷酸合成酶基因、甲羟戊酸激酶基因ERG12、甲羟戊酸激酶基因ERG8、甲羟戊酸焦磷酸脱羧酶基因和异戊烯基焦磷酸异构酶基因表达盒，通过同源重组将上述表达盒插入到步骤(3)得到的酿酒酵母的基因组中；
    5)分别构建乙醛脱氢酶基因、乙酰辅酶A合成酶基因和乙醇脱氢酶基因表达盒，通过同源重组将上述表达盒插入到步骤(4)得到的酿酒酵母的基因组中。
  5. 根据权利要求4所述的重组酿酒酵母的构建方法，其特征在于，包括以下步骤：
    敲除乙醇脱氢酶基因ADH1和YPL062W；过表达Zea mays来源的丙酮酸脱羧酶基因PDC、内源的酰基辅酶A合成酶基因FAA2及Salmonella enterica来源的乙酰辅酶A合成酶基因SeACS。
  6. 权利要求1-3所述的重组酿酒酵母的构建方法，其特征在于，主要包括以下步骤：
    (1)从Salmonella enterica subsp和Saccharomyces cerevisiae筛选出使用的混杂激酶；从多个物种来源Thermoplasma acidophilum,Methanococcus vannielii,Methanolobus tindarius,Methanosalsum zhilinae,Methanococcus maripaludisand Methanococcoides burtonii及Arabidopsis thaliana中筛选出能够在酿酒酵母中成功表达的异戊基磷酸激酶；
    (2)将筛选出的混杂激酶基因与异戊烯基磷酸激酶、乙酰辅酶A乙酰基转移酶基因、羟甲基戊二酸辅酶A合成酶基因、甲羟戊酸激酶基因ERG12、甲羟戊酸激酶基因ERG8、甲羟戊酸焦磷酸脱羧酶基因、异戊烯基焦磷酸异构酶基因IDI，香叶基二磷酸合成酶基因ERG20mut，大麻萜酸合酶基因CsPT4和大麻环萜酚合酶基因CBCAS定位到过氧化物酶体；
    (3)为了增加过氧化物酶体的数量及体积，敲除过氧化物酶体膜蛋白PEX31和PEX32；过表达过氧化物酶体膜蛋白PEX3、PEX19、PEX11和PEX34，从而增加大麻环萜酚的生产。
  7. 根据权利要求6所述的重组酿酒酵母的构建方法，其特征在于：
    确保CBCAS的正确折叠，同时筛选了多种分子伴侣，折叠酶，转录激活因子以提高CBCAS的表达活性，包括参与折叠和内质网质量控制的蛋白质、辅因子FAD1p、UPR蛋白IRE1*p、UPR激活剂Hac1s及内质网大小调节因子INO1，其中与折叠和内质网质量控制的蛋白质包括CNE1p、KAR2p、PDI1p和ERO1p。
  8. 根据权利要求6所述的重组酿酒酵母的构建方法，其特征在于：
    定点突变大麻环萜酚合酶产生多个突变体，分别为H292L,H292R,Y417H,Y417R,N89Q-N499Q,R463C-D488C，T379S,K377R,N196Q,F171Y,S170T，R349K,F365Y,V138A,R532K,L524I,Y472F,N528Q,F353Y及其双突变体、三突变体和多突变体组合，筛选出活性最好的突变体用于生产大麻环萜酚酸。
  9. 根据权利要求6所述的重组酿酒酵母的构建方法，其特征在于：
    为了增加胞内ATP和溶氧供应，过表达腺苷酸激酶基因ADK1、Pseudomonas stutzeri来源的亚磷酸盐脱氢酶基因ptxD及透明颤菌血红蛋白基因VHB提高ATP的合成速率，从而促进细胞生长和大麻素的生产。
  10. 根据权利要求6所述的重组酿酒酵母的构建方法，其特征在于：
    将大麻环萜酚酸合酶定位到脂滴中以期通过增加底物和酶的局部浓度能够产生更快的反应速率和更高的生产力；同时，为了提高大麻素的溶解度，过表达三酰甘油途径TAG中关键酶基因二酰基甘油酰基转移酶基因DGA1、G3P脱氢酶基因GPD1和磷脂酸磷酸酯酶基因PAH1，同时敲除脂滴合成关键蛋白基因SEI1，增加脂质水平和脂滴聚集；将大麻环萜酚酸合酶定位到脂滴中以期通过增加底物和酶的局部浓度能够产生更快的反应速率和更高的生产力，扩大工程酵母合成大麻素的储存能力。
  11. 根据权利要求6所述的重组酿酒酵母的构建方法，其特征在于：
    将大麻环萜酚代谢途径中的关键酶基因同时整合到酿酒酵母的rDNA位点，实现多个关键酶基因的多拷贝表达。
  12. 根据权利要求6所述的重组酿酒酵母的构建方法，其特征在于：
    敲除Gal80以解除其对Gal4的抑制作用，同时替换Gal4的启动子解除葡萄糖抑制作用，不再需要半乳糖作为诱导剂，通过葡萄糖和乙醇混合发酵优化大麻环萜酚CBCA的产量。
  13. 一种利用重组酿酒酵母发酵生产大麻环萜酚的方法，其特征在于，主要包括以下步骤：
    1)在合适的培养基中培养权利要求1-3任一项所述的重组酿酒酵母一段时间；
    2)回收发酵产生的大麻环萜酚酸；
    3)通过加热或者储存使大麻环萜酚酸形成大麻环萜酚。
  14. 根据权利要求13所述的方法，其特征在于，所述的培养基含有葡萄糖、半乳糖、甘油、乙醇、淀粉、己酸或橄榄醇酸中的一种或多种混合物。
  15. 根据权利要求13-14任一项所述的方法，其特征在于，所述培养的条件为转速50～300rpm，温度28～32℃，培养时间为24-120h。
  16. 根据权利要求15所述的方法，其特征在于，所述回收发酵产生的大麻环萜酚酸过程包括使用有机溶剂萃取发酵液或细胞破碎液中的大麻环萜酚酸步骤。
  17. 根据权利要求16所述的方法，其特征在于，所述的有机溶剂为乙酸乙酯、己烷、庚烷、石油醚、或氯仿中的一种或多种混合物。
  18. 根据权利要求16或17所述的方法，其特征在于，所述的细胞破碎液通过高压均质破碎法、超声波破碎法、球磨破碎法、反复冻融破碎法或酶溶破碎法破碎宿主细胞后得到。
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