CN116987603A - Recombinant saccharomyces cerevisiae strain for high yield of cannabigerolic acid as well as construction method and application thereof - Google Patents

Recombinant saccharomyces cerevisiae strain for high yield of cannabigerolic acid as well as construction method and application thereof Download PDF

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CN116987603A
CN116987603A CN202211356510.3A CN202211356510A CN116987603A CN 116987603 A CN116987603 A CN 116987603A CN 202211356510 A CN202211356510 A CN 202211356510A CN 116987603 A CN116987603 A CN 116987603A
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saccharomyces cerevisiae
strain
acid
cannabigerol
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罗小舟
张云丰
郜佩桢
郭九龙
颜威
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Senris Biotechnology Shenzhen Co ltd
Shenzhen Institute of Advanced Technology of CAS
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Senris Biotechnology Shenzhen Co ltd
Shenzhen Institute of Advanced Technology of CAS
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Abstract

The invention relates to the technical field of metabolic engineering, in particular to a recombinant saccharomyces cerevisiae strain for high-yield cannabigerol acid, a construction method and application thereof. The invention firstly knocks out the beta-oxidation pathway gene POX1, thereby solving the problem of caproic acid substrate degradation; then strengthening the expression of OA pathway enzyme TKS and/or OAC, replacing the expression promoter of fatty acid synthetase FAS1, and improving the conversion rate of caproic acid substrate; and further expresses gene INO2 related to endoplasmic reticulum membrane synthesis and expresses cannabigerol acid synthase gene SOD1-tCsPT4n or CUP1-tCsPT4n containing fusion protein, thereby improving the synthesis efficiency of cannabigerol acid. Finally, the expression level of partial genes in the saccharomyces cerevisiae cells is regulated and controlled through the gene editing, so that the purpose of high yield of cannabigerol acid is achieved.

Description

Recombinant saccharomyces cerevisiae strain for high yield of cannabigerolic acid as well as construction method and application thereof
Technical Field
The invention relates to the technical field of metabolic engineering, in particular to a recombinant saccharomyces cerevisiae strain with high yield of cannabigerol acid by metabolic engineering and a construction method and application thereof.
Background
The family of phytocannabinoids isolated from cannabis contains over 100, and is of great interest to the academia and commercial industries for its unique pharmaceutical properties. The potential medical uses thereof have been widely studied, such as antibacterial, anti-inflammatory, antitumor, anxiolytic, antidepressant, etc. Meanwhile, the traditional Chinese medicine composition has great clinical potential for various human diseases such as epilepsy, diabetes, parkinsonism and the like. To date, cannabinoid therapeutics for a variety of indications have been approved and used, and the medical and commercial value of cannabinoids is readily apparent. Wherein, the cannabigerol acid (CBGA) is used as an important intermediate, can be converted into other various cannabinoids by cannabinoid synthase, and can be subjected to decarboxylation treatment to obtain the cannabigerol product. However, the cannabigerolic acid content in cannabis plants is low and is difficult to obtain. Therefore, the efficient synthesis of the cannabigerol acid product by using the saccharomyces cerevisiae strain cells has important application value.
Cannabinoids are very low in abundance in plants, and in plant extraction methods, it is difficult to obtain pure samples from plants due to the presence of a variety of cannabinoids. Meanwhile, the chemical structure of the cannabinoid is complex, and the method for chemically synthesizing the cannabinoid and the derivative thereof is complex, expensive and low in efficiency. In contrast, microbial cells have great potential for the synthesis of cannabinoids. Luo et al synthesized cannabigerolic acid and other cannabigenic acid products by introducing a heterologous cannabinoid synthesis pathway into the chassis strain of Saccharomyces cerevisiae. The study of Luo et al designed the natural mevalonate pathway to provide high throughput geranyl pyrophosphate (GPP) and introduced a heterologous Olive Acid (OA) synthesis pathway. Meanwhile, geranyl pyrophosphate from cannabis plant is introduced, namely olive acid ester geranyl transferase CsPT4 gene is introduced, and the cannabigolic acid product is synthesized by catalyzing GPP and OA substrates through the CsPT4 gene. With caproic acid as a substrate, 7.2mg/L CBGA was produced.
The existing synthetic pathway of cannabigerolic acid is shown in figure 1, and sugar (i.e. carbon source) generates intermediate acetyl-coa via glycolytic pathway and acetyl-coa synthetase. Acetyl-coa gives geranyl pyrophosphate, a precursor of cannabigerol acid, via the mevalonate pathway. At the same time, caproic acid provides caproyl-coa by acylation, which is catalyzed by tetraketone synthase and olive toluate cyclase to obtain cannabigerol acid as another precursor, olive acid. Two precursors: geranyl pyrophosphate and olive acid are catalyzed by geranyl pyrophosphate from cannabis plant, olive acid ester geranyl transferase, and cannabigerol acid is produced.
In the patent (CN 110914416A), feeding caproic acid to yeast can produce 22.9-72.9mg/L of CBGA. However, the existing cannabigerol acid synthesis pathway still has the technical problems of degradation of metabolic precursor caproic acid, low conversion rate of caproic acid substrate and low synthesis yield of cannabigerol acid.
Accordingly, the prior art is still in need of improvement and development.
Disclosure of Invention
In view of the defects in the prior art, the beta-oxidation pathway gene POX1 is knocked out, so that the problem of caproic acid substrate degradation is solved; then strengthening the expression of OA pathway enzyme TKS and/or OAC, replacing the expression promoter of fatty acid synthetase FAS1, and improving the conversion rate of caproic acid substrate; and further expresses gene INO2 related to endoplasmic reticulum membrane synthesis and expresses cannabigerol acid synthase genes SOD1-tCsPT4n and CUP1-tCsPT4n containing fusion proteins, thereby improving the synthesis efficiency of cannabigerol acid. Finally, the expression level of partial genes in the saccharomyces cerevisiae cells is regulated and controlled through the gene editing, so that the purpose of high yield of cannabigerol acid is achieved.
It should be noted that, the invention refers to the research content (Complete biosynthesis of cannabinoids and their unnatural analogues in yeast) of Luo, X.et al in 2019, takes the research result yCAN31 as the original Saccharomyces cerevisiae strain, over-expresses TKS and OAC on the basis of the original Saccharomyces cerevisiae strain yCAN31, knocks out the beta-oxidation pathway gene POX1, and constructs a recombinant Saccharomyces cerevisiae strain yS413; down-regulating FAS1 expression, and constructing a recombinant saccharomyces cerevisiae strain yS489; upregulating INO2 expression, constructing recombinant Saccharomyces cerevisiae strain yS576; over-expressing tCsPT4n, connecting a gene and a promoter through a protein tag SOD1, and constructing a recombinant saccharomyces cerevisiae strain yS628 with high yield of cannabigerolic acid; the tCsPT4n is over-expressed, and the gene and the promoter are connected through a protein tag CUP1, so that a recombinant saccharomyces cerevisiae strain yS629 with high yield of cannabigerolic acid is constructed.
Wherein, the over-expression refers to up-regulating expression of the gene, namely, the gene is transcribed and translated excessively, and the final gene expression product exceeds the normal level.
By downregulating expression is meant downregulating expression of a gene, i.e., limited transcription, translation of the gene, and a lower than normal level of the final gene expression product.
The knockout is a loss of function of the instruction specific gene.
The technical scheme of the invention is as follows:
the invention provides a recombinant saccharomyces cerevisiae strain, which takes yeast which expresses enzymes of a cannabigerol acid (CBGA) synthesis pathway and can synthesize CBGA as an original strain, wherein part of endogenous genes are over-expressed or down-regulated or knocked out, and the part of endogenous genes comprise: fatty acid synthase FAS1 gene, beta-oxidation pathway POX1 gene. Meanwhile, a part of exogenous genes are over-expressed, wherein the part of exogenous genes comprise olive toluate cyclase OAC gene, tetraketone synthase TKS gene, cannabigerolic acid synthase tCsPT4n gene and INO2 gene related to endoplasmic reticulum membrane synthesis.
Wherein the nucleotide sequence of the TKS gene is shown as SEQ ID NO. 1, the nucleotide sequence of the OAC gene is shown as SEQ ID NO. 2, and the nucleotide sequence of the INO2 gene is shown as SEQ ID NO. 4.
Further, the recombinant saccharomyces cerevisiae strain is based on a saccharomyces cerevisiae strain yCAN31, and any one of the following (a) to (e) modifications is made on the basis of the gene of the saccharomyces cerevisiae strain yCAN 31:
(a) The method comprises the following steps Overexpression of the tetraketone synthase TKS gene and/or the olive toluate cyclase OAC gene;
(b) The method comprises the following steps Knocking out the beta-oxidation pathway POX1 gene on the basis of (a);
(c) The method comprises the following steps Over-expressing the tetraon synthase TKS gene and/or olive toluate cyclase OAC gene on the basis of (b), down-regulating the fatty acid synthase FAS1 gene;
(d) The method comprises the following steps Over-expressing the INO2 gene associated with endoplasmic reticulum membrane synthesis on the basis of (c);
(e) The method comprises the following steps Based on (d), overexpressing a truncated cannabigerol acid synthase tCsPT4n gene comprising SOD1 or CUP1 protein tag.
The invention obtains the recombinant saccharomyces cerevisiae strain with metabolic engineering and high yield of cannabigerolic acid. A series of transformed strains are shown in Table 1, and the sequences are shown in a sequence table.
In the (a) and (c), when the tetraketone synthase TKS gene and the olive toluate cyclase OAC gene are simultaneously overexpressed, different codons are respectively selected for expression, and the codons are as follows: TKS0-SCOAC0 (shown as SEQ ID NO: 10), TKS4-SCOAC4 (shown as SEQ ID NO: 6) and TKS5-SCOAC5 (shown as SEQ ID NO: 7); the comparison shows that the amino acid sequences of TKS0, TKS4 and TKS5 are the same (shown as SEQ ID NO: 9); the amino acid sequences of SCOAC0 and SCOAC4 are identical with those of SCOAC5 (shown as SEQ ID NO: 8);
the (a) is modified to obtain a strain yS022;
the modification of (b) gives strain yS291;
the (c) is modified to obtain a strain yS349, a strain yS413 and a strain yS489;
said (d) engineering to give strain yS576;
the engineering of (e) results in strain yS628 and strain yS629.
Alternatively, when the TKS gene and the OAC gene are overexpressed simultaneously, the nucleotide sequence of the connecting fragment connecting the TKS gene and the OAC gene is shown as SEQ ID NO. 3.
Alternatively, in said (b), said knockout of the POX1 gene is followed by insertion of the olive toluate cyclase OAC gene into the Saccharomyces cerevisiae genome to initiate expression by the pHSP26 promoter.
Alternatively, the INO2 gene overexpression is by inserting the INO2 gene into the Saccharomyces cerevisiae genome to initiate expression via the pPGK1 promoter.
Alternatively, the overexpression of the truncated tCsPT4n gene comprising a SOD1 or CUP1 protein tag is performed by inserting the truncated tCsPT4n gene into the saccharomyces cerevisiae genome to initiate expression by the pSeGal2 promoter after fusion with the SOD1 or CUP1 protein tag.
The invention also provides a method for constructing the recombinant saccharomyces cerevisiae, which comprises the following steps:
(1) PCR amplification to obtain an expression cassette of a gene to be over-expressed, and integrating the expression cassette onto a saccharomyces cerevisiae genome; and/or, PCR amplification to obtain a homologous fragment for knocking out the gene, and replacing the gene to be knocked out on the saccharomyces cerevisiae genome with the homologous fragment;
gene knockout and/or insertion on the saccharomyces cerevisiae genome is achieved using CRISPR-Cas9 technology;
(2) Screening to obtain positive cloned recombinant Saccharomyces cerevisiae strain.
The invention also provides application of the recombinant saccharomyces cerevisiae strain in production of cannabigerol acid.
Optionally, the method for producing cannabigerolic acid comprises the following steps:
(1) Activating and culturing the recombinant saccharomyces cerevisiae strain to obtain a recombinant saccharomyces cerevisiae strain seed solution;
(2) Transferring the recombinant saccharomyces cerevisiae strain seed solution into a proper culture medium, and culturing the recombinant saccharomyces cerevisiae strain under proper conditions to obtain a fermentation culture product;
(3) And extracting and purifying the fermentation culture product to obtain the cannabigerol acid.
The recombinant saccharomyces cerevisiae strain can also be used for producing cannabigerol, and concretely, the cannabigerol acid produced by the recombinant saccharomyces cerevisiae strain is subjected to decarboxylation in vitro by using an enzyme catalyst or a chemical catalyst to obtain the cannabigerol.
The recombinant saccharomyces cerevisiae strain can also be used for producing a downstream product of the cannabigerol acid, such as cannabinoid, and specifically, the cannabigerol acid produced by the recombinant saccharomyces cerevisiae strain is converted into other various cannabinoids in vitro by using an enzyme catalyst or a chemical catalyst.
The invention has the beneficial effects that: the invention finally provides a saccharomyces cerevisiae strain with high yield of cannabigerol acid through metabolic engineering modification and a construction method and application thereof by over-expressing TKS and/or OAC, knocking out POX1 genes in cells of the saccharomyces cerevisiae, regulating and controlling FAS1 expression level and INO2 expression level in cells of the saccharomyces cerevisiae strain, over-expressing cannabigerol acid synthetase and the like. The method achieves the aim of high yield of the cannabigerol acid and solves the technical problems of low conversion rate and low yield of the cannabigerol acid synthesized by taking the caproic acid as a substrate.
Drawings
FIG. 1 is a schematic diagram of the synthetic pathway of cannabigerol acid in Saccharomyces cerevisiae strains in the prior art.
FIG. 2 is a comparative schematic diagram of caproic acid conversion of Saccharomyces cerevisiae strain yS022 and Saccharomyces cerevisiae strain yS291 of example 1.
FIG. 3 is a schematic diagram showing comparison of CBGA production of Saccharomyces cerevisiae strain yS022 and Saccharomyces cerevisiae strain yS291 in example 1.
FIG. 4 is a comparative schematic diagram of caproic acid conversion rates of Saccharomyces cerevisiae strain yS349, saccharomyces cerevisiae strain yS413, and Saccharomyces cerevisiae strain yS489 of example 2.
FIG. 5 is a schematic diagram showing comparison of CBGA production of Saccharomyces cerevisiae strain yS349, saccharomyces cerevisiae strain yS413, and Saccharomyces cerevisiae strain yS489 in example 2.
FIG. 6 is a schematic diagram showing a comparison of CBGA production of Saccharomyces cerevisiae strain yS576, saccharomyces cerevisiae strain yS629, and Saccharomyces cerevisiae strain yS628 of example 3.
Detailed Description
The invention provides a recombinant saccharomyces cerevisiae strain with metabolic engineering modified high-yield cannabigerol acid, a construction method and application thereof, and the invention is further described in detail below in order to make the purposes, technical schemes and effects of the invention clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
To solve the problem of caproic acid substrate degradation, strain yS291 was constructed: the tetraketone synthase TKS and olive toluate cyclase OAC are over-expressed on the basis of the original saccharomyces cerevisiae strain yCAN31 to obtain a strain yS022, and the beta-oxidation pathway gene POX1 is knocked out on the basis of yS022 to obtain a yS291 strain. The specific construction steps comprise:
PCR amplification of the integrated fragment was performed by 2X Phanta Max Master Mix (Phanta DNA polymerase): taking the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template to obtain a homology arm 1622b-UP fragment at the upstream of the integration site and a homology arm 1622b-DOWN fragment at the downstream of the integration site; taking a genome with pGAL1 promoter strain as a template to obtain a promoter fragment pGAL1; taking a plasmid containing the TKS0-SCOAC0 gene as a template to obtain a segment TKS0-SCOAC0; the terminator ScENO1t was obtained using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template. Finally, a 1622b-UP-pGAL1-TKS0-SCOAC0-ScENO1t-1622b-DOWN expression cassette is obtained. Then the 1622b-UP-pGAL1-TKS0-SCOAC0-ScENO1t-1622b-DOWN expression cassette fragment was transformed into the original host yCAN31 to obtain the strain yS022. And taking the strain with correct gene sequencing for subsequent culture.
The knockout of the gene POX1 is achieved by ligating upstream and downstream of the POX1 gene. The integrated fragment was amplified by PCR with 2X Phanta Max Master Mix (Phanta DNA polymerase). The genome of Saccharomyces cerevisiae CEN.PK2-1C is used as a template to obtain an upstream homology arm POX1-UP fragment and a downstream homology arm POX1-DOWN fragment. The combination of fragments was then transformed into strain yS022 to obtain strain yS291. And taking the strain with correct gene sequencing for subsequent culture.
Strain yS022 and strain yS291 were each cultured. Finally, compared with the host strain yS022, the strain yS291 of the beta-oxidation pathway gene POX1 knocked out by the gene editing technology has the advantages that the caproic acid conversion rate is improved by 7.05 percent (shown in figure 2) and the CBGA conversion rate is improved by 44.6 percent (shown in figure 3) under the same culture condition.
Example 2
To increase the conversion of caproic acid substrate, strain yS489 was constructed: on the basis of constructing the strain yS022 in example 1, knocking out the POX1 gene and simultaneously overexpressing the olive toluate cyclase gene OAC to obtain the strain yS349; over-expressing a tetraon synthase gene TKS and an olive toluate cyclase gene OAC on the basis of yS349 to obtain a strain yS413; substitution of the FAS1 promoter for prs 25A on the basis of yS413 gave the yS489 strain. The specific construction steps comprise:
PCR amplification of the integrated fragment was performed by 2X Phanta Max Master Mix (Phanta DNA polymerase): taking a genome of Saccharomyces cerevisiae CEN.PK2-1C as a template to obtain an upstream homology arm DPOX1-UP fragment of an integration site, a downstream homology arm DPOX1-DOWN fragment of the integration site, a promoter pHSP26 fragment and a terminator tHSP26 fragment; taking a plasmid containing an OAC gene as a template to obtain a fragment OAC; finally obtaining the DPOX1-UP-pHSP26-OAC-HSP26t-DPOX1-DOWN expression cassette. The DPOX1-UP-pHSP26-OAC-tHSP26-DPOX1-DOWN expression cassette fragment was then transformed into host yS022 to obtain strain yS349.
PCR amplification of the integrated fragment was performed by 2X Phanta Max Master Mix (Phanta DNA polymerase): taking a genome of Saccharomyces cerevisiae CEN.PK2-1C as a template to obtain an upstream homology arm NDT80-UP fragment of an integration site, a downstream homology arm NDT80-DOWN fragment of the integration site, a promoter pTDH3 fragment, a terminator tHSP26 fragment, a promoter pTDH1 fragment, a terminator tADH1 fragment, a promoter pPGK1 fragment, a terminator tCYC1 fragment, a promoter pSED1 fragment and a terminator tTDH1 fragment; taking plasmids containing pPGK1-TKS4-SCOAC4-tCYC1, pSED1-TKS5-SCOAC5-tTDH1 and 4xOAC genes as templates to obtain pPGK1-TKS4-SCOAC4-tCYC1, pSED1-TKS5-SCOAC5-tTDH1 and 4xOAC fragments; finally, the NDT80-UP-4xOAC-pPGK1-TKS4-SCOAC4-tCYC1/pSED1-TKS5-SCOAC5-tTDH1-NDT80-DOWN expression cassette is obtained. The NDT80-UP-4xOAC-pPGK1-TKS4-SCOAC4-tCYC1/pSED1-TKS5-SCOAC5-tTDH1-NDT80-DOWN expression cassette fragment was transformed into a host yS349 to obtain a strain yS413.
The integrated fragment was amplified by PCR with 2X Phanta Max Master Mix (Phanta DNA polymerase). Amplifying and obtaining a pfas1-UP fragment of an upstream homology arm of a pfas1 promoter, a FAS1 gene fragment of a downstream homology arm and a pRPS25A fragment of the promoter by taking a genome of Saccharomyces cerevisiae CEN.PK2-1C as a template; finally obtaining the pfas1-UP-pRPS25A-FAS1 expression cassette fragment. The pfas1-UP-pRPS25A-FAS1 expression cassette fragment was then transferred into a host yS413 to obtain a strain yS489. And taking the strain with correct gene sequencing for subsequent culture.
Strain yS349, strain yS413 and strain yS489 were each cultured. Finally, the expression of OA pathway enzymes OAC and TKS and the expression promoter of fatty acid synthase FAS1 are enhanced by a gene editing technology, and compared with a host strain (yS 349), the caproic acid conversion rate of the strain is respectively improved by 3.80 percent (yS 413) and 33.52 percent (yS 489) (see FIG. 4), and the CBGA yield is respectively improved by 142.3 percent (yS 413) and 380.0 percent (yS 489) (see FIG. 5) under the same culture conditions.
Example 3
In order to improve the synthesis efficiency of cannabigerolic acid, strains yS628 and yS629 are constructed: based on the construction of the strain yS489 in example 2, the endoplasmic reticulum membrane synthesis related gene INO2 was overexpressed to obtain a yS576 strain; based on yS576, the strain yS628 and yS629 were obtained by connecting the protein tag SOD1 and CUP1 with the promoter pSeGal2, and overexpressing the cannabigerol acid synthase gene tCsPT4 n. The specific construction steps comprise:
PCR amplification of the integrated fragment was performed by 2X Phanta Max Master Mix (Phanta DNA polymerase): the genome of Saccharomyces cerevisiae CEN.PK2-1C is used as a template, and a 1014a upstream homology arm 1014a-UP fragment of 1014a locus, a 1014a-DOWN fragment of 1014a downstream homology arm, a promoter pPGK1 fragment and an INO2-INO2t fragment are obtained through PCR amplification; finally, 1014a-UP-pPGK1-INO2-INO2t-1014a-DOWN expression cassette is obtained, and then 1014a-UP-pPGK1-INO2-INO2t-1014a-DOWN expression cassette fragment is transformed into a host yS489 to obtain a strain yS576.
PCR amplification of the integrated fragment was performed by 2X Phanta Max Master Mix (Phanta DNA polymerase): taking a genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, and obtaining a HO-UP fragment of a homologous arm at the upstream of a HO locus, a HO-DOWN fragment of a homologous arm at the downstream of the HO locus and a THD1t fragment of a terminator through PCR amplification; amplifying to obtain pSeGal2 fragments by taking a genome containing a promoter pSeGal2 gene as a template; taking a genome containing SOD1 and CUP1 genes as a template, and amplifying to obtain SOD1 and CUP1 fragments; using plasmid containing tCsPT4n gene as template, amplifying to obtain tCsPT4n fragment; finally obtaining HO-UP-pSeGal2-SOD1-tCspT4n-tTDH1-HO-DOWN expression cassette and HO-UP-pSeGal2-CUP1-tCspT4n-tTDH1-HO-DOWN expression cassette, and then respectively transforming the two expression cassette fragments into a host yS576 to obtain strains yS628 and yS629. The strains yS576, yS628 and yS629 with correct gene sequencing are taken for subsequent culture.
Strain yS576, strain yS628 and strain yS629 were each cultured. Finally, by the gene editing technology, the genes INO2, SOD1-tCspT4n and CUP1-tCspT4n related to the synthesis of the endoplasmic reticulum membrane are over-expressed, and compared with the host strain yS576, the strain has the CBGA yield improved by 42.2% (yS 629) and 66.9% (yS 628) respectively under the same culture conditions (see FIG. 6).
The yeast transformation method in the above examples 1-3 comprises the steps of lithium acetate/PEG 3350 transformation method, specifically comprising: the host strain was inoculated into 1 XYPD medium and cultured overnight. Then inoculating into a new 2 XYPD culture medium to make the initial OD value be 0.2, continuously culturing for 4 hours, taking 5OD bacterial liquid, centrifuging, discarding the supernatant, and washing twice with sterilized ultrapure water to obtain yeast cells; preparing DNA mixture systems, each comprising host cells of 5OD and mixing with DNA mixture consisting of the insert, tool plasmid and ddH 2 And mixing O. Adding lithium acetate transformation mixture into suspended cells, obtaining transformed cells after heat shock, coating the transformed cells on a corresponding auxotroph screening plate, and culturing in an incubator for a proper number of days. Obtaining single colony, namely recombinant Saccharomyces cerevisiae strain, sequencing, verifying transformation, and streaking the strain with correct sequencingPreserving and freezing glycerol.
2 XYPD medium formulation: yeast extract 20.0g/L, peptone 40.0g/L, and glucose 40.0g/L.
Lithium acetate conversion mixture: 50% W/V PEG3350 260. Mu.L, 1mol/L LiOAc 36. Mu.L, denatured salmon sperm DNA 10. Mu.L (denatured salmon sperm DNA was denatured in a metal bath at 95℃for 5min before use), ddH 2 O 4μL。
The cultivation method in the above examples 1 to 3, comprising the steps of: after single colonies of the strain were cultured overnight in 2mL of 1 XYPD (glucose 2%, yeast extract 1%, peptone 2%) 24-well plate at 30℃in a shaker at 800rpm for 16 hours, the overnight bacterial liquid was diluted 10 times with 1 XYPD and then the bacterial liquid OD was detected by an ultraviolet spectrophotometer at a wavelength of 600nm. The starting od=0.2 was then transferred to 2ml of 1×ypg medium (galactose 2%, yeast extract 1%, peptone 2%) for cultivation. The material supplementing mode is as follows: after transfer, hexanoic acid (0.2 mM) was added every 12h for a total of five times. Beginning 24h after transfer, galactose (2% w/v) was added every 24h for a total of three times with a final concentration of 2%.
The sample preparation method in the above examples 1 to 3, comprising the steps of: after 84 hours of culture, 200ul of bacterial liquid is collected as a sample. After sample collection, according to sample OD600, 0.2mL of 0.5mm diameter glass beads and 0.4mL of ethyl acetate were then added: formic acid (0.05%) was treated in a high speed tissue mill at 68Hz for 180s at 30s intervals, repeated four times, after short centrifugation, the upper organic layer was taken up in 0.28mL to 1.5mL centrifuge tubes, repeated twice, and the collected upper organic phases were pooled. The three extracted organic layers were evaporated using a vacuum dryer at 45 degrees celsius for 1 hour to no solvent residue. By resuspension of AHF (acetonitrile: H) 2 O: formic acid=80:20:0.05%, 140. Mu.L of PHB (propyl p-hydroxybenzoate standard substance, 15. Mu.M) containing internal standard, was resuspended, and 0.22 μm PVDF filter was filtered into the cannula in the liquid phase detection vial as detection sample. Three samples in parallel each.
The sample detection method in the above examples 1 to 3, the detection conditions include: the detection conditions are shown in Table 2.
The strain construction information for examples 1-3 above is shown in Table 1 below.
TABLE 1 Strain construction information
TABLE 2 HPLC detection conditions
In the nucleotide and amino acid sequence listing of the present invention, SEQ ID NO. 5 is the nucleotide sequence of 4xOAC, SEQ ID NO. 11 is the nucleotide sequence of pRPS25A, SEQ ID NO. 12 is the nucleotide sequence of pSeGal2, SEQ ID NO. 13 is the nucleotide sequence of pPGK1, SEQ ID NO. 14 is the nucleotide sequence of SOD1-tCsPT4n, SEQ ID NO. 15 is the amino acid sequence of tCsPT4n, SEQ ID NO. 16 is the nucleotide sequence of CUP1-tCsPT4n, SEQ ID NO. 17 is the nucleotide sequence of pHSP26, SEQ ID NO. 18 is the nucleotide sequence of tHSP26, SEQ ID NO. 19 is the nucleotide sequence of PTDH3, SEQ ID NO. 20 is the nucleotide sequence of pGAL1, SEQ ID NO. 21 is the nucleotide sequence of tCYC1, SEQ ID NO. 22 is the nucleotide sequence of pTDH1, SEQ ID NO. 23 is the nucleotide sequence of tCsPT4n, SEQ ID NO. 17 is the nucleotide sequence of pHSP26, SEQ ID NO. 18 is the nucleotide sequence of pSeDH 1.
In conclusion, the invention improves the problems existing in three ways of synthesizing cannabigerol acid through a series of gene editing and transformation, namely improves the supply of propionyl coenzyme A and olive acid; the endoplasmic reticulum area is increased, and the protein label is used for over-expressing geranyl pyrophosphate, olive oleate geranyl transferase, so that the synthesis efficiency of CBGA is improved.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.

Claims (10)

1. A recombinant saccharomyces cerevisiae strain with metabolic engineering modified high yield of cannabigerolic acid, which is characterized in that the recombinant saccharomyces cerevisiae strain is based on a saccharomyces cerevisiae strain yCAN31, and any one of the following (a) - (e) modification is performed on the basis of the gene of the saccharomyces cerevisiae strain yCAN 31:
(a) The method comprises the following steps Overexpression of the tetraketone synthase TKS gene and/or the olive toluate cyclase OAC gene;
(b) The method comprises the following steps Knocking out the beta-oxidation pathway POX1 gene on the basis of (a);
(c) The method comprises the following steps Over-expressing the tetraon synthase TKS gene and/or olive toluate cyclase OAC gene on the basis of (b), down-regulating the fatty acid synthase FAS1 gene;
(d) The method comprises the following steps Over-expressing the INO2 gene associated with endoplasmic reticulum membrane synthesis on the basis of (c);
(e) The method comprises the following steps Based on (d), overexpressing a truncated cannabigerol acid synthase tCsPT4n gene comprising SOD1 or CUP1 protein tag.
2. The metabolically engineered high yielding strain of Saccharomyces cerevisiae of claim 1, wherein upon simultaneous overexpression of the tetraketo synthase TKS gene and the olive toluate cyclase OAC gene in (a) and (c),
the strain yS022 is obtained by transformation in the step (a), wherein the strain yS022 comprises TKS0-SCOAC0, and the nucleotide sequence of the TKS0-SCOAC0 is shown as SEQ ID NO. 10;
the modification of (b) gives strain yS291;
the strain yS349, the strain yS413 and the strain yS489 are obtained through transformation in the step (c), wherein the strain yS413 comprises TKS4-SCOAC4 and TKS5-SCOAC5, the nucleotide sequence of the TKS4-SCOAC4 is shown as SEQ ID NO. 6, and the nucleotide sequence of the TKS5-SCOAC5 is shown as SEQ ID NO. 7;
said (d) engineering to give strain yS576;
the engineering of (e) gives strain yS628 or strain yS629.
3. The recombinant saccharomyces cerevisiae strain with metabolically engineered high yield of cannabigerolic acid according to claim 1 or 2, wherein when TKS gene and OAC gene are over expressed simultaneously, the nucleotide sequence of the connecting fragment connecting the TKS gene and OAC gene is shown as SEQ ID No. 3.
4. The recombinant saccharomyces cerevisiae strain with metabolically engineered high yield of cannabigerol according to claim 1 or 2, wherein in (b), the insertion of olive toluate cyclase OAC gene into saccharomyces cerevisiae genome after the knockout of POX1 gene is started to express by pHSP26 promoter.
5. The recombinant saccharomyces cerevisiae strain of metabolically engineered high yield of cannabigerolic acid according to claim 1 or 2, wherein the INO2 gene over-expression is by inserting the INO2 gene onto the saccharomyces cerevisiae genome to initiate expression by pPGK1 promoter.
6. The recombinant saccharomyces cerevisiae strain of metabolically engineered high yield of cannabigerolic acid according to claim 1 or 2, wherein the overexpression of truncated tCsPT4n gene comprising SOD1 or CUP1 protein tag is initiated by pseagl 2 promoter expression after fusion of truncated tCsPT4n gene with SOD1 or CUP1 protein tag.
7. A method of constructing a metabolically engineered recombinant saccharomyces cerevisiae strain of high yield cannabigerolic acid according to any of claims 1-6, comprising the steps of:
(1) PCR amplification to obtain an expression cassette of a gene to be over-expressed, and integrating the expression cassette into a saccharomyces cerevisiae genome; and/or, PCR amplification to obtain a homologous fragment for knocking out the gene, and replacing the gene to be knocked out on the saccharomyces cerevisiae genome with the homologous fragment;
gene knockout and/or insertion on the saccharomyces cerevisiae genome is achieved using CRISPR-Cas9 technology;
(2) Screening to obtain positive cloned recombinant Saccharomyces cerevisiae strain.
8. Use of the recombinant saccharomyces cerevisiae strain according to any of claims 1-6 for the production of cannabigerol acid.
9. The use according to claim 8, wherein the method for producing cannabigerol acid comprises the steps of:
(1) Activating and culturing the recombinant saccharomyces cerevisiae strain to obtain recombinant saccharomyces cerevisiae strain seed liquid;
(2) And transferring the recombinant saccharomyces cerevisiae strain seed solution into a culture medium suitable for producing cannabigerol acid, and culturing to obtain the cannabigerol acid.
10. Use of the recombinant saccharomyces cerevisiae strain according to any of claims 1-6 for the production of cannabinoids downstream of cannabigerol acids.
CN202211356510.3A 2022-11-01 2022-11-01 Recombinant saccharomyces cerevisiae strain for high yield of cannabigerolic acid as well as construction method and application thereof Pending CN116987603A (en)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN117363504A (en) * 2023-12-04 2024-01-09 潍坊医学院 Saccharomyces cerevisiae engineering bacteria for simultaneously producing brown cyanidin and eupatorium, construction method and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117363504A (en) * 2023-12-04 2024-01-09 潍坊医学院 Saccharomyces cerevisiae engineering bacteria for simultaneously producing brown cyanidin and eupatorium, construction method and application thereof
CN117363504B (en) * 2023-12-04 2024-02-23 潍坊医学院 Saccharomyces cerevisiae engineering bacteria for simultaneously producing brown cyanidin and eupatorium, construction method and application thereof

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