METHOD FOR PRODUCING HETEROGENOUS CANNABICHROMENE FROM SACCHAROMYCES CEREVISIAE

Abstract

A recombinant host cell capable of biosynthesizing cannabichromenic acid and a construction method thereof, and a method for biosynthesizing cannabichromenic acid through the recombinant host cell. Saccharomyces cerevisiae is taken as a host. First, cannabigerolic acid synthase and cannabichromenic acid synthase are over-expressed in the host; then, a metabolic pathway of a precursor compound, olivetolic acid, synthesizing cannabichromenic acid from saccharides is constructed in the host, a metabolic pathway for hexanoic acid to olivetolic acid is further constructed in the host, an endogenous mevalonate pathway of the host and a metabolic pathway of acetyl-CoA are optimized, cannabichromenic acid synthase is rationally designed, highly active cannabichromenic acid synthase is screened out, and finally, a cannabichromene pathway is located to peroxisomes and lipid droplets by using the cell compartmentalization principle to obtain recombinant Saccharomyces cerevisiae capable of biosynthesizing cannabichromenic acid.

Claims

1-18. (canceled)

19. A construction method of a recombinant Saccharomyces cerevisiae, wherein cannabigerolic acid synthase polypeptide and cannabichromenic acid synthase polypeptide are heterologously expressed by the recombinant Saccharomyces cerevisiae strain; p-ketothiolase polypeptide, 3-hydroxybutyryl coenzyme A dehydrogenase polypeptide, crotonase polypeptide, trans-2-enoyl-CoA reductase polypeptide, type III polyketide synthase polypeptide and olivetolic acid cyclase polypeptide are heterologously expressed by the recombinant Saccharomyces cerevisiae; acyl activating enzyme polypeptide and acetyl-CoA carboxylase polypeptide are heterologously expressed by the recombinant Saccharomyces cerevisiae; wherein the construction method comprising the following steps: 1) respectively constructing expression cassettes of cannabigerolic acid synthase polypeptide and cannabichromenic acid synthase polypeptide, and inserting the above expression cassettes into a Saccharomyces cerevisiae genome through homologous recombination; 2) respectively constructing expression cassettes of p-ketothiolase polypeptide, 3-hydroxybutyryl coenzyme A dehydrogenase polypeptide, crotonase polypeptide, trans-2-enoyl-CoA reductase polypeptide, type III polyketide synthase polypeptide and olivetolic acid cyclase polypeptide, and inserting the above expression cassettes into the Saccharomyces cerevisiae genome obtained in step (1) through homologous recombination; 3) respectively constructing expression cassettes of acyl activating enzyme polypeptide and acetyl-CoA carboxylase polypeptide, and inserting the above expression cassettes into the Saccharomyces cerevisiae genome obtained in step (2) through homologous recombination; 4) respectively constructing expression cassettes of truncated HMG-CoA reductase polypeptide, acetoacetyl-CoA thiolase polypeptide, HMG-CoA synthase polypeptide, mutants of farnesyl pyrophosphate synthetase polypeptide, mevalonate kinase polypeptide ERG12, phosphomevalonate kinase polypeptide ERG8, mevalonate pyrophosphate decarboxylase polypeptide and isopentenyl-pyrophosphate delta isomerase polypeptide, and inserting the above expression cassettes into the Saccharomyces cerevisiae genome obtained in step (3) through homologous recombination; 5) respectively constructing expression cassettes of acetaldehyde dehydrogenase polypeptide, acetyl-CoA synthetase polypeptide and alcohol dehydrogenase polypeptide, and inserting the above expression cassettes into the Saccharomyces cerevisiae genome obtained in step (4) through homologous recombination.

20. The construction method of the recombinant Saccharomyces cerevisiae according to claim 19, comprising the following steps: knocking out alcohol dehydrogenase polypeptide (ADH1) and YPL062W; and over-expressing pyruvate decarboxylase polypeptide PDC from Zea mays, endogenous acyl-CoA synthetase polypeptide FAA2, and acetyl-CoA synthetase polypeptide SeACS from Salmonella enterica.

21. A construction method of a recombinant Saccharomyces cerevisiae, wherein cannabigerolic acid synthase polypeptide and cannabichromenic acid synthase polypeptide are heterologously expressed by the recombinant Saccharomyces cerevisiae strain; p-ketothiolase polypeptide, 3-hydroxybutyryl coenzyme A dehydrogenase polypeptide, crotonase polypeptide, trans-2-enoyl-CoA reductase polypeptide, type III polyketide synthase polypeptide and olivetolic acid cyclase polypeptide are heterologously expressed by the recombinant Saccharomyces cerevisiae; acyl activating enzyme polypeptide and acetyl-CoA carboxylase polypeptide are heterologously expressed by the recombinant Saccharomyces cerevisiae; wherein the construction method comprises the following steps: (1) screening out promiscuous kinase polypeptide to be used from Salmonella enterica subsp and Saccharomyces cerevisiae; and screening out isopentenyl phosphate kinase polypeptide which can be successfully expressed in the Saccharomyces cerevisiae from multiple species of Thermoplasma acidophilum, Methanococcus vannielii, Methanolobus tindarius, Methanosalsum zhilinae, Methanococcus maripaludis, Methanococcoides burtonii and Arabidopsis thaliana; (2) locating the promiscuous kinase polypeptide screened out, isopentenyl phosphokinase, acetoacetyl-CoA thiolase polypeptide, HMG-CoA synthase polypeptide, mevalonate kinase polypeptide ERG12, phosphomevalonate kinase polypeptide ERG8, mevalonate pyrophosphate decarboxylase polypeptide, isopentenyl-pyrophosphate delta isomerase polypeptide IDI, mutants of farnesyl pyrophosphate synthetase polypeptide ERG20mut, cannabigerolic acid synthase polypeptide CsPT4 and cannabichromenic acid synthase polypeptide CBCAS to peroxisomes; (3) in order to increase the number and volume of the peroxisomes, knocking out peroxisomal membrane protein PEX31 and PEX32; and over-expressing peroxisomal membrane protein PEX3, PEX19, PEX11 and PEX34 so as to increase the production of cannabichromenic acid.

22. The construction method of the recombinant Saccharomyces cerevisiae according to claim 21, wherein: in order to ensure the proper folding of CBCAS, a variety of molecular chaperones, foldases and transcription activators are screened simultaneously to improve the expression activity of CBCAS, including proteins involved in folding and endoplasmic reticulum quality control, cofactor FAD1p, UPR albumen IRE1*p, UPR activator Hac1s and endoplasmic reticulum size regulator INO1, wherein the proteins involved in folding and endoplasmic reticulum quality control comprise CNE1p, KAR2p, PDI1p and ERO1p.

23. The construction method of the recombinant Saccharomyces cerevisiae according to claim 21, wherein: a plurality of mutants, respectively H292L, H292R, Y417H, Y417R, N89Q-N499Q, R463C-D488C, T379S, K377R, N196Q, F171Y, S170T, R349K, F365Y, V138A, R532K, L524I, Y472F, N528Q, F353Y and a combination of double mutants, triple mutants and multiple mutants, are produced by site-directed mutagenesis of cannabichromenic acid synthase, and the mutant with the best activity is screened out to produce cannabichromenic acid.

24. The construction method of the recombinant Saccharomyces cerevisiae according to claim 21, wherein: in order to increase intracellular ATP and dissolved oxygen supply, the adenylate kinase polypeptide ADK1, phosphite dehydrogenase polypeptide ptxD from Pseudomonas stutzeri and Vibrio hyaluronicus hemoglobin polypeptide VHB are over-expressed to increase the synthesis rate of ATP, so as to promote cell growth and cannabinoid production.

25. The construction method of the recombinant Saccharomyces cerevisiae according to claim 21, wherein: cannabichromenic acid synthase polypeptide is located to the lipid droplets in order to obtain higher reaction rate and higher productivity by increasing the local concentration of substrate and enzyme, meanwhile, in order to improve the solubility of cannabinoid, the key enzyme polypeptides of DAG acyltransferase polypeptide DGA1, G3P dehydrogenase polypeptide GPD1 and phosphatidic acid phosphatase polypeptide PAH1 in the triacylglycerol pathway TAG are over-expressed, and the key protein polypeptide SEI1 for lipid droplet synthesis is knocked out to increase lipid level and lipid droplet aggregation; and cannabichromenic acid synthase polypeptide is located to the lipid droplets in order to obtain higher reaction rate and higher productivity and expand the storage capacity of engineered yeast to synthesize cannabinoids by increasing the local concentration of substrate and enzyme.

26. The construction method of the recombinant Saccharomyces cerevisiae according to claim 21, wherein: the key enzyme polypeptides in the metabolic pathway of cannabichromene are simultaneously integrated to the rDNA locus of the Saccharomyces cerevisiae to achieve multi-copy expression of a plurality of key enzyme polypeptides.

27. The construction method of the recombinant Saccharomyces cerevisiae according to claim 21, wherein: Gal80 is knocked out to remove the inhibitory effect thereof on Gal4, and meanwhile, the promoter of Gal4 is replaced to remove the inhibitory effect of glucose; and galactose is no longer needed as an inducer, and the yield of cannabichromenic acid CBCA is optimized through mixed fermentation of glucose and ethanol.

28. A method for producing cannabichromenic acid by fermenting recombinant Saccharomyces cerevisiae, wherein cannabigerolic acid synthase polypeptide and cannabichromenic acid synthase polypeptide are heterologously expressed by the recombinant Saccharomyces cerevisiae strain; β-ketothiolase polypeptide, 3-hydroxybutyryl coenzyme A dehydrogenase polypeptide, crotonase polypeptide, trans-2-enoyl-CoA reductase polypeptide, type III polyketide synthase polypeptide and olivetolic acid cyclase polypeptide are heterologously expressed by the recombinant Saccharomyces cerevisiae; acyl activating enzyme polypeptide and acetyl-CoA carboxylase polypeptide are heterologously expressed by the recombinant Saccharomyces cerevisiae; wherein the method comprising the following steps: 1) culturing the recombinant Saccharomyces cerevisiae of claim 19 in an appropriate culture medium for a period of time; 2) recycling cannabichromenic acid generated by fermentation; 3) decarboxylating cannabichromenic acid by heating or storage to form cannabichromene.

29. The method according to claim 28, wherein the culture medium contains one or a mixture of more of glucose, galactose, glycerine, ethanol, starch, hexanoic acid and olivetolic acid.

30. The method according to claim 28, wherein the culture conditions are as follows: the revolving speed is 50-300 rpm, the temperature is 28-32° C., and the culture time is 24-120 h.

31. The method according to claim 30, wherein the process of recycling cannabichromenic acid generated by fermentation comprises the step of extracting cannabichromenic acid from fermentation liquor or cell crushing liquid with an organic solvent.

32. The method according to claim 31, wherein the organic solvent is one or a mixture of more of ethyl acetate, hexane, heptane, petroleum ether and chloroform.

33. The method according to claim 32, wherein the cell crushing liquid is obtained by crushing host cells through high-pressure homogenization crushing, ultrasonic crushing, ball-milling brushing, repeated freezing and thawing crushing or enzymatic solubilization crushing.

34. The method according to claim 33, wherein the cell crushing liquid is obtained by crushing host cells through high-pressure homogenization crushing, ultrasonic crushing, ball-milling brushing, repeated freezing and thawing crushing or enzymatic solubilization crushing.

35. The method according to claim 29, wherein the culture conditions are as follows: the revolving speed is 50-300 rpm, the temperature is 28-32° C., and the culture time is 24-120 h.

Description

DESCRIPTION OF DRAWINGS

[0070] FIG. 1 shows a biosynthetic pathway of cannabichromenic acid by recombinant Saccharomyces cerevisiae, wherein CsAAE1: acyl activating enzyme polypeptide, ACC1: acetyl-CoA carboxylase polypeptide, RebktB: β-ketothiolase polypeptide, CnpaaH1: 3-hydroxybutyryl coenzyme A dehydrogenase polypeptide, Cacrt: crotonase polypeptide, Tdter: trans-2-enoyl-CoA reductase polypeptide, CsTKS: polyketide synthase polypeptide, CsOAC: olivetolic acid cyclase polypeptide, CsPT4: olivetolic acid geranyl transferase, and CBCAS: cannabichromenic acid synthase polypeptide.

[0071] FIG. 2 is a schematic diagram of a GPP biosynthetic pathway.

[0072] FIG. 3 is a liquid chromatogram of cannabichromenic acid produced by fermenting recombinant Saccharomyces cerevisiae, wherein the upper figure is an LC-MS liquid chromatogram of cannabichromenic acid standard, the abscissa is retention time, and the ordinate is abundance; and the lower figure is an LC-MS liquid chromatogram of a fermentation sample of a recombinant genetically engineered strain of cannabichromenic acid.

[0073] FIG. 4 is a mass spectrum of cannabichromenic acid produced by fermenting recombinant Saccharomyces cerevisiae, wherein the abscissa is M/Z, and the ordinate is abundance. The upper figure is an LC-MS mass spectrogram of cannabichromenic acid standard; and the lower figure is an LC-MS mass spectrogram of a fermentation sample of a recombinant genetically engineered strain of cannabichromenic acid.

DETAILED DESCRIPTION

[0074] The present invention is described below in detail in combination with embodiments, but the embodiments of the present invention are limited thereto. Obviously, the embodiments in the following description are merely part of the embodiments of the present invention, and for those skilled in the art, other similar embodiments obtained without contributing creative labor fall into the protection scope of the present invention.

Embodiment 1

[0075] Construction of Recombinant Yeast Strain Capable of Expressing Cannabigerolic Acid Synthase Polypeptide and Cannabichromenic Acid Synthase Polypeptide

[0076] The host bacterium of the present invention is Saccharomyces cerevisiae INVSc1, diplontic INVSc1 has high robustness, and the complicated gene regulatory network thereof is beneficial to expression and catalysis of enzymes in adverse environmental conditions. The present invention constructs two gene expression cassettes: a codon-optimized CsPT4 gene expression cassette and a codon-optimized CBCAS gene expression cassette, based on the findings that cannabigerolic acid (CBGA) is produced by geranyl pyrophosphate (GPP) and olivetolic acid (OA) under the action of cannabigerolic acid synthase polypeptide (CsPT4) and cannabichromenic acid (CBCA) is formed by cannabigerolic acid (CBGA) under the catalysis of cannabichromenic acid synthase polypeptide (CBCAS), a GAL10 promoter and a CYC1 terminator are used for the CsPT4 gene expression cassette, a GAL10 promoter and a CYC1 terminator are used for the CBCAS gene expression cassette, and the CsPT4 gene expression cassette is integrated to 416d, CAN1y and YOLCd1b genome loci of Saccharomyces cerevisiae through the genome editing technology; and the CBCAS gene expression cassette is respectively integrated to 308a, HIS3b and 511b genome loci of Saccharomyces cerevisiae to express three copies of CsPT4 and CBCAS, so as to obtain the recombinant Saccharomyces cerevisiae strain capable of expressing cannabigerolic acid synthase polypeptide and cannabichromenic acid synthase polypeptide.

Embodiment 2

[0077] Construction of Recombinant Yeast Strain Capable of Producing Cannabichromenic Acid from Saccharides

[0078] In order to enable the biosynthesis of olivetolic acid in the recombinant Saccharomyces cerevisiae in embodiment 1, a biometabolic synthesis pathway by which olivetolic acid is produced from saccharides through hexanoyl-CoA is constructed in the above recombinant Saccharomyces cerevisiae. Six codon-optimized gene expression cassettes: a RebktB gene expression cassette, a CnpaaH1 gene expression cassette, a Cacrt gene expression cassette, a Tdter gene expression cassette, a CsTKS gene expression cassette and a CsOAC gene expression cassette, are constructed. A TEF1 promoter and a TEF1 terminator are used for the RebktB gene expression cassette, a GAL10 promoter and a CYC1 terminator are used for the CnpaaH1 gene expression cassette, a GAL1 promoter and an ADH1 terminator are used for the Cacrt gene expression cassette, a PGK1 promoter and an HXT7 terminator are used for the Tdter gene expression cassette, a GAL10 promoter and a CYC1 terminator are used for the CsTKS gene expression cassette, and a GAL1 promoter and an ADH1 terminator are used for the CsOAC gene expression cassette. Through the gene editing technology, the RebktB gene expression cassette is integrated to the SAP155b genome locus of Saccharomyces cerevisiae, the CnpaaH1 gene expression cassette and the Cacrt gene expression cassette are integrated to the SAP155c genome locus of Saccharomyces cerevisiae, and the Tdter gene expression cassette is integrated to the YPRCδ15c genome locus of Saccharomyces cerevisiae, respectively expressing one copy of the above polypeptides. The CsOAC gene expression cassette and the CsTKS gene expression cassette are formed into one expression cassette to be respectively integrated to 1622b, X4, XI3 and XII5 genome loci of Saccharomyces cerevisiae to express four copies of the above polypeptides, finally obtaining the recombinant Saccharomyces cerevisiae strain capable of producing cannabichromenic acid from saccharides.

Embodiment 3

[0079] Construction of Biometabolic Synthesis Pathway for Hexanoic Acid to Olivetolic Acid in Recombinant Saccharomyces cerevisiae

[0080] In order to increase the flux of hexanoyl-CoA of the recombinant Saccharomyces cerevisiae in embodiment 2, a biometabolic synthesis pathway for hexanoic acid to olivetolic acid in the recombinant Saccharomyces cerevisiae is constructed, two gene expression cassettes: a codon-optimized CsAAE1 gene expression cassette and a codon-optimized ACC1 gene expression cassette, are constructed, a GPD promoter and a CYC1 terminator are used for the CsAAE1 and ACC1 gene expression cassettes, and the ACC1 gene expression cassette is integrated to the X3 genome locus of Saccharomyces cerevisiae through the gene editing technology to over-express one copy of the above polypeptides; and the CsAAE1 gene expression cassette is integrated to 208a, 911b and 106a genome loci of Saccharomyces cerevisiae to express three copies of the above polypeptides, and hexanoic acid is converted to hexanoyl-CoA by feeding hexanoic acid under the catalysis of CsAAE1. Meanwhile, acetyl-CoA is catalyzed by ACC1 to produce malonyl-CoA, and olivetolic acid is produced by hexanoyl-CoA and malonyl-CoA under the action of type III polyketide synthase polypeptide (CsTKS) and olivetolic acid cyclase polypeptide (CsOAC) so as to increase the yield of cannabichromenic acid biosynthesized by recombinant Saccharomyces cerevisiae.

Embodiment 4

[0081] Optimization of Endogenous Mevalonate Pathway of Recombinant Saccharomyces cerevisiae

[0082] In order to increase the flux of GPP of the recombinant Saccharomyces cerevisiae in embodiment 3, the endogenous mevalonate pathway of the recombinant Saccharomyces cerevisiae is further optimized, and eight gene expression cassettes: a truncated tHMG1 gene expression cassette, a codon-optimized mvaE gene expression cassette, an mvaS gene expression cassette, an ERG20mut (F96W, N127W) gene expression cassette, an ERG12 gene expression cassette, an ERG8 gene expression cassette, an ERG19 gene expression cassette and an IDI gene expression cassette, are constructed. A GAL1 promoter and an ADH1 terminator are used for the tHMG1 gene expression cassette, a GAL1 promoter and an ADH1 terminator are used for the mvaE gene expression cassette, a GAL10 promoter and a CYC1 terminator are used for the mvaS gene expression cassette, a GAL10 promoter and a CYC1 terminator are used for the ERG20mut gene expression cassette, a GAL10 promoter and a CYC1 terminator are used for the ERG12 gene expression cassette, a GAL1 promoter and an ADH1 terminator are used for the ERG8 gene expression cassette, a GAL10 promoter and a CYC1 terminator are used for the ERG19 gene expression cassette, and a GAL1 promoter and an ADH1 terminator are used for the IDI gene expression cassette. Through the gene editing technology, the tHMG1 gene expression cassette and the ERG20mut gene expression cassette are integrated to the 1021b genome locus of Saccharomyces cerevisiae, the mvaE gene expression cassette and the mvaS gene expression cassette are integrated to the 1414a genome locus of Saccharomyces cerevisiae, the ERG12 gene expression cassette and the IDI gene expression cassette are integrated to the 1114a genome locus of Saccharomyces cerevisiae, and the ERG8 gene expression cassette and the ERG19 gene expression cassette are integrated to the 1014a genome locus of Saccharomyces cerevisiae to over-express one copy of the above polypeptides, so as to ensure the supply of geranyl pyrophosphate in the mevalonate downstream pathway in the recombinant Saccharomyces cerevisiae.

Embodiment 5

[0083] Optimization of Metabolic Pathway of Acetyl-CoA of Recombinant Saccharomyces cerevisiae

[0084] In order to increase the metabolic flux of acetyl-CoA in the cytoplasm of the recombinant Saccharomyces cerevisiae in embodiment 4, acetaldehyde dehydrogenase polypeptide (ALD6) from Saccharomyces cerevisiae, acetyl-CoA synthetase polypeptide (ACS2) and alcohol dehydrogenase polypeptide (ADH2) are obtained from the NCBI database, and three gene expression cassettes: an ALD6 gene expression cassette, an ACS2 gene expression cassette and an ADH2 gene expression cassette, are constructed. A GAL10 promoter and a CYC1 terminator are used for the ALD6 gene expression cassette, a GAL1 promoter and an ADH1 terminator are used for the ACS2 gene expression cassette, and a GPD promoter and a CYC1 terminator are used for the ADH2 gene expression cassette. Through the gene editing technology, the ALD6 gene expression cassette and the ACS2 gene expression cassette are integrated to the 1309a genome locus of Saccharomyces cerevisiae, and the ADH2 gene expression cassette is integrated to the X2 genome locus of Saccharomyces cerevisiae to over-express one copy of the above polypeptides, so as to increase the flux of acetyl-CoA in the cytoplasm of the recombinant Saccharomyces cerevisiae and provide precursor compounds for biosynthesis of mevalonate pathway, olivetolic acid and cannabichromenic acid.

Embodiment 6

[0085] Construction of Biosynthetic Pathway of Cannabichromene in Peroxisome

[0086] First, a gene expression cassette which can be located to peroxisome is constructed; and for codon-optimized IPK gene, choline kinase (ScCK), acetoacetyl-CoA thiolase polypeptide (EfmvaE), HMG-CoA synthase polypeptide (EfmvaS), mevalonate kinase polypeptide (ERG12), phosphomevalonate kinase polypeptide (ERG8), mevalonate pyrophosphate decarboxylase polypeptide (ERG19), isopentenyl-pyrophosphate delta isomerase polypeptide (IDI), mutants of farnesyl pyrophosphate synthetase polypeptide (ERG20mut), codon-optimized cannabigerolic acid synthase polypeptide (CsPT4) and cannabichromenic acid synthase polypeptide (CBCAS), a PTS1 signal peptide sequence KL-X5-QL of peroxisome is added to N terminals, and a PTS2 signal peptide sequence SKL of peroxisome is added to C terminals. A TEF1 promoter and a TEF1 terminator are used for the IPK and choline kinase (ScCK) gene expression cassettes. A GAL10 promoter and a CYC1 terminator are both used for the EfmvaE, EfmvaS, ERG8, ERG19, ERG12, IDI, ERG20mut, CsPT4 and CBCAS gene expression cassettes. Through the gene editing technology, the EfmvaE-EfmvaS-SKL gene expression cassette is integrated to the YIRCA6 genome locus of Saccharomyces cerevisiae; the ERG19-ERG8-SKL gene expression cassette is integrated to the YMRWΔ15 genome locus of Saccharomyces cerevisiae; the ERG12-SKL gene expression cassette is integrated to the YNRCΔ9 genome locus of Saccharomyces cerevisiae; the ScCK-SKL gene expression cassette is integrated to the YGLCτ3 genome locus of Saccharomyces cerevisiae; the atIPK-SKL gene expression cassette is integrated to the YORWΔ17 genome locus of Saccharomyces cerevisiae; the IDI-SKL gene expression cassette is integrated to the YPRCτ3 genome locus of Saccharomyces cerevisiae; and the ERG20 mut (F96W, N127W)-SKL gene expression cassette is integrated to the SPB1/PBN1 genome locus of Saccharomyces cerevisiae.

Embodiment 7

[0087] In order to further the supply of acetyl-CoA in cytoplasm, the pyruvate decarboxylase polypeptide (PDC) from Zea mays and the endogenous acyl-CoA synthetase polypeptide (FAA2) are over-expressed. The codon-optimized pyruvate decarboxylase polypeptide zmPDC, the endogenous acyl-CoA synthetase polypeptide (FAA2) and the acetyl-CoA synthetase polypeptide (SeACS) from Salmonella enterica are respectively constructed to a TEF1 promoter and a TEF1 terminator. Through the gene editing technology, the zmPDC, FAA2 and acetyl-CoA synthetase polypeptide (SeACS) expression cassettes are respectively integrated to X2, XII4 and YORWΔ22 genome loci of Saccharomyces cerevisiae, and meanwhile, ADH1 is knocked out.

Embodiment 8

[0088] In order to increase intracellular ATP and dissolved oxygen supply, adenylate kinase polypeptide (ADK1), phosphite dehydrogenase polypeptide ptxD from Pseudomonas stutzeri and Vibrio hyaluronicus hemoglobin polypeptide (VHB) are over-expressed. The adenylate kinase polypeptide (ADK1), the codon-optimized phosphite dehydrogenase polypeptide ptxD and the Vibrio hyaluronicus hemoglobin polypeptide (VHB) are respectively constructed to a TEF1 promoter and a TEF1 terminator. Through the gene editing technology, the ADK1, ptxD and VHB gene expression cassettes are respectively integrated to YARC88, YCRWSI1 and YBRW816 genome loci of Saccharomyces cerevisiae.

Embodiment 9

[0089] In order to ensure the proper folding of CBCAS, proteins (CNE1p, KAR2p, PDI1p, ERO1p and IRE1p) involved in folding and endoplasmic reticulum quality control, cofactor (FAD1p), UPR activator Hac1s and endoplasmic reticulum size regulator INO1 are amplified from the Saccharomyces cerevisiae genome. Expression cassettes are constructed respectively by using PGK1 promoters and HXT7 terminators. Through the gene editing technology, the CNE1 gene expression cassette is integrated to the I12 genome locus of Saccharomyces cerevisiae; the KAR2 gene expression cassette is integrated to the I4 and I32 genome loci of Saccharomyces cerevisiae; the PDI1 gene expression cassette is integrated to the I10 genome locus of Saccharomyces cerevisiae; the ERO1 gene expression cassette is integrated to the X3 genome locus of Saccharomyces cerevisiae; the IRE1 gene expression cassette is integrated to the I8 genome locus of Saccharomyces cerevisiae; the Hac1s gene expression cassette is integrated to the I28 genome locus of Saccharomyces cerevisiae; and the INO1 gene expression cassette is integrated to the I3 genome locus of Saccharomyces cerevisiae.

Embodiment 10

[0090] In order to increase the intracellular solubility of cannabinoid, DAG acyltransferase polypeptide DGA1, G3P dehydrogenase polypeptide GPD1 and phosphatidic acid phosphatase polypeptide (PAH1) in the triacylglycerol synthesis pathway (TAG) are over-expressed. GPD1, DGA1 and PAH1 are respectively constructed to a TEF1 promoter and a TEF1 terminator. Through the gene editing technology, the GPD1, DGA1 and PAH1 gene expression cassettes are respectively integrated to the YORWΔ22, YCRWδ12 and YERCδ8 genome loci of Saccharomyces cerevisiae.

Embodiment 11

[0091] A plurality of mutants H292L, H292R, Y417H, Y417R, N89Q-N499Q, R463C-D488C, T379S, K377R, N196Q, F171Y, S170T, R349K, F365Y, V138A, R532K, L524I, Y472F, N528Q and F353Y produced by site-directed mutagenesis of cannabichromenic acid synthase and a combination of double mutants, triple mutants and multiple mutants thereof are respectively constructed to GAL10 promoters and CYC1 terminators and respectively integrated to HIS3b and 511b genome loci through the gene editing technology.

Embodiment 12

[0092] Production of Cannabichromenic Acid by Fermenting Recombinant Saccharomyces cerevisiae Strain

[0093] Recombinant Saccharomyces cerevisiae strains with high yield of cannabichromenic acid obtained in embodiment 2 are selected into test tubes containing 3-5 mL of YPD (10 g/L yeast extract, 20 g/L peptone and 20 g/L dextroglucose). Culture is carried out at 200 rpm at 30° C. for 24 h until glucose in the culture medium is depleted. Saturated strains are centrifuged at 4500 rpm and subcultured to a new culture medium containing 100 mL of YPG (10 g/L yeast extract, 20 g/L peptone and 20 g/L galactose), and culture is carried out at 200 rpm at 30° C. for 24-48 h. Fermentation liquor or high-pressure homogenization crushed bacteria liquid is centrifuged at 4500 rpm for 5 min, the supernatant is taken and extracted with an organic solvent, ethyl acetate, of ⅕ volume of supernatant (1 volume of ethyl acetate added to 5 volumes of fermentation liquor), and an organic layer is taken by centrifugation for rotary evaporation to obtain crude cannabichromenic acid which is heated at 105° C. for 15 min and then heated at 145° C. for 55 min, thus obtaining cannabichromene.

Embodiment 13

[0094] Production of Cannabichromenic Acid by Fermenting Recombinant Yeast Strain

[0095] Recombinant Saccharomyces cerevisiae strains with high yield of cannabichromenic acid obtained in embodiment 5 are selected into test tubes containing 3-5 mL of YPD (10 g/L yeast extract, 20 g/L peptone and 20 g/L dextroglucose). Cells are cultured at 200 rpm at 30° C. for 24 h until glucose in the culture medium is depleted. Saturated strains are centrifuged at 4500 rpm and subcultured to a new culture medium containing 100 mL of YPG (10 g/L yeast extract, 20 g/L peptone, 20 g/L galactose, 1 mM olivetolic acid or 2 mM hexanoic acid), and culture is carried out at 200 rpm at 30° C. for 24-48 h. Fermentation liquor or high-pressure homogenization crushed bacteria liquid is centrifuged at 4500 rpm for 5 min, the supernatant is taken and extracted with an organic solvent, ethyl acetate, of ⅕ volume of supernatant (1 volume of ethyl acetate added to 5 volumes of fermentation liquor), and an organic layer is taken by centrifugation for rotary evaporation to obtain crude cannabichromenic acid which is heated at 105° C. for 15 min and then heated at 145° C. for 55 min, thus obtaining cannabichromene.

Embodiment 14

[0096] Identification Method of Cannabichromenic Acid

[0097] Fermentation liquor or high-pressure homogenization crushed bacteria liquid is centrifuged at 4500 rpm for 5 min, the supernatant is taken and extracted with an organic solvent, ethyl acetate, of ⅕ volume of supernatant (1 volume of ethyl acetate added to 5 volumes of fermentation liquor), an organic layer is taken by centrifugation for rotary evaporation, the evaporated substance is resuspended in a mixed solution of acetonitrile/0.05% aqueous formic acid solution (v/v of 80%/20%) and then filtered by an organic filter membrane to obtain high-concentration organic phase containing cannabigerolic acid, and detection and analysis are performed through a liquid chromatography/time-of-flight mass spectrometer. Results are shown in FIG. 3 and FIG. 4.

[0098] The related instruments, equipment and experimental parameters for detection and analysis of cannabichromenic acid are as follows: the instrument is Agilent 6224 TOF LC/MS, the column temperature is 25° C., the chromatographic column is Agilent C18 chromatographic column, the flow velocity is 0.2 mL/min, the mobile phase is aqueous solution (A) containing 0.05% formic acid and acetonitrile solution (B), and gradient elution conditions are: 0-40 min, 30%-98% acetonitrile; 40-50 min, 98% acetonitrile; and 50-51 min, 98%-30% acetonitrile, and the sampling volume is 20 μL.

[0099] Finally, it should be noted that the above embodiments are only used for describing the technical solution of the present invention rather than limiting the present invention. Although the present invention is described in detail by referring to the above embodiments, those ordinary skilled in the art should understand that: the technical solution recorded in each of the above embodiments can be still amended, or part or all of technical features therein can be replaced equivalently; and the amendments or replacements do not enable the essence of the corresponding technical solution to depart from the scope of the technical solution of various embodiments of the present invention.