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Method for producing heterologous cannabichromenol using Saccharomyces cerevisiae

A technology of cannabinoid phenolic acid and recombinant Saccharomyces cerevisiae, which is applied in the fields of biotechnology and medicine, can solve the problems of high cost, complicated preparation methods of cannabinol and cannabinoid phenolic acid, low yield and the like, and achieves low cost , The method is accurate and efficient, and the genetic performance is stable.

Active Publication Date: 2021-10-26
DALIAN UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of complex preparation method, high cost and low yield of cannabichromene and cannabichromene phenolic acid, the present invention provides the following technical solutions

Method used

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  • Method for producing heterologous cannabichromenol using Saccharomyces cerevisiae
  • Method for producing heterologous cannabichromenol using Saccharomyces cerevisiae
  • Method for producing heterologous cannabichromenol using Saccharomyces cerevisiae

Examples

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Effect test

Embodiment 1

[0054] Construction of recombinant yeast strains capable of expressing cannabigeroid acid synthase and cannabichromene phenolic acid synthase

[0055] The host bacterium 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 olivolic acid (OA) generate cannabigerolic acid (CBGA) under the action of cannabinoid acid synthase (CsPT4), and cannabigerolic acid (CBGA) is passed through cannabinoid Terpene phenol synthase (CBCAS) catalyzes the formation of cannabichromene phenolic acid (CBCA), and constructs two gene expression cassettes: the codon-optimized CsPT4 gene expression cassette and the codon-optimized CBCAS gene expression cassette, and the CsPT4 gene expression cassette uses GAL10 promoter and CYC1 t...

Embodiment 2

[0057] Construction of Recombinant Yeast Strain Capable of Utilizing Sugars to Produce Cannabichromenic Acids

[0058] In order to enable the biosynthesis of olivolic acid in the recombinant Saccharomyces cerevisiae of Example 1, a biosynthetic pathway for olivetolic acid generation by using sugars through hexanoyl-CoA was constructed in the above-mentioned recombinant Saccharomyces cerevisiae. Six codon-optimized gene expression cassettes were constructed: RebktB gene expression cassette, CnpaaH1 gene expression cassette, Cacrt gene expression cassette, Tdter gene expression cassette, CsTKS gene expression cassette and CsOAC gene expression cassette. 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 terminator CsTKS gene expression cassette uses GAL10 pr...

Embodiment 3

[0060] Construction of biosynthetic pathway from hexanoic acid to olivolic acid in recombinant Saccharomyces cerevisiae

[0061] In order to increase the flux of hexanoyl-CoA in the recombinant Saccharomyces cerevisiae of Example 2, construct the biosynthetic pathway from hexanoic acid to olivolic acid in the recombinant Saccharomyces cerevisiae, and construct two gene expression cassettes: the codon-optimized CsAAE1 gene expression cassette and ACC1 gene expression cassette, both CsAAE1 and ACC1 gene expression cassettes use the GPD promoter and CYC1 terminator, and through gene editing technology, the ACC1 gene expression cassette is integrated into the Saccharomyces cerevisiae X3 genomic locus, and one copy of the above gene is overexpressed; CsAAE1 The gene expression cassettes were integrated into Saccharomyces cerevisiae 208a, 911b and 106a genome sites to express 3 copies of the above-mentioned genes, and by supplying hexanoic acid, hexanoic acid was converted into hexan...

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Abstract

The invention discloses a recombinant host cell capable of biosynthesizing cannabichromene phenolic acid, a construction method thereof, and a method for biosynthesizing cannabichromene phenolic acid through a recombinant host cell, belonging to the fields of biotechnology and medicine. The present invention takes Saccharomyces cerevisiae as a host, firstly overexpresses cannabigerene acid synthase and cannabichromenol acid synthase in the host, and then constructs the precursor compound olivate, which is a precursor compound of cannabichromenol acid, to be synthesized from sugars in the host Metabolic pathways, further constructing the metabolic pathways from hexanoic acid to olivolic acid in the host, and finally optimizing the metabolic pathways of the host's endogenous mevalonate pathway and acetyl-CoA, and obtaining recombinant Saccharomyces cerevisiae capable of biosynthesizing cannabichromene phenolic acid, providing A new way to produce cannabichromene with high added value from cheap carbon sources has been developed, which has high production efficiency, short cycle time and low cost, which is conducive to the large-scale production of cannabichromene.

Description

technical field [0001] The invention belongs to the field of biotechnology and medicine, and in particular relates to a host cell capable of biosynthesizing cannabichromene (CBC), a construction method thereof, and a biosynthesis method of cannabichromene. Background technique [0002] Cannabis (Cannabis sativa) has been used for thousands of years due to its rich variety of pharmacologically active cannabinoids. At present, more than 113 cannabinoids have been isolated and identified from cannabis plants and are divided into different types, such as cannabigerols (CBGs), cannabichromenes (CBCs), cannabidiol ( cannabidiols, CBDs), Δ 9 -THC-type (Δ 9 -tetrahydrocannabinols, Δ 9 -THCs), Δ 8 -THC-type (Δ 8 -tetrahydrocannabinols, Δ 8 -THCs), cannabicyclols (CBLs), cannabielsoins, CBEs, cannabinols, CBNs, cannabinodiol, CBNDs, cannabitriols, CBTs ) and other cannabinoids (Elsohly, M.A.; Slade, D., Chemical constituents of marijuana: the complex mixture of natural cannabin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/52C12N15/53C12N15/54C12N15/60C12N15/61C12P17/06C12R1/865
CPCC12N9/0004C12N9/1085C12N9/0006C12N9/88C12N9/001C12N9/93C12N9/1029C12N9/1025C12N9/1205C12N9/90C12N9/0008C12P17/06C12Y121/03008C12Y205/01039C12Y101/01157C12Y402/01017C12Y103/0104C12Y404/01026C12Y604/01002C12Y203/01009C12Y101/01034C12Y205/01029C12Y203/0301C12Y503/03002C12Y207/01036C12Y401/01033C12Y102/0101C12Y203/01086C12Y101/01001C12Y121/99C12N15/52C12Y103/01044C12P5/007
Inventor 薛闯齐明明
Owner DALIAN UNIV OF TECH
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