CN115927029A

CN115927029A - Recombinant saccharomyces cerevisiae for producing cannabigerol acid and construction method and application thereof

Info

Publication number: CN115927029A
Application number: CN202211420285.5A
Authority: CN
Inventors: 罗小舟; 张云丰; 郜佩桢; 郭九龙; 颜威
Original assignee: Senris Biotechnology Shenzhen Co ltd; Shenzhen Institute of Advanced Technology of CAS
Current assignee: Senris Biotechnology Shenzhen Co ltd
Priority date: 2022-11-11
Filing date: 2022-11-11
Publication date: 2023-04-07

Abstract

This applicationRelates to a recombinant saccharomyces cerevisiae producing cannabigerolic acid, which is prepared from basic saccharomyces cerevisiae, wherein the following endogenous genes of the basic saccharomyces cerevisiae are overexpressed, downregulated or knocked out:pep4、gal80、gal4andfas1(ii) a Exogenous genes encoding the following enzymes were overexpressed: tetrone synthase (a)Cs.TKS) gene, olive toluate cyclase (TKS)Cs.OAC) gene, geranyl pyrophosphate, olive oleate geranyl transferase: (Cs.PT4 n) Gene and variant acetyl-coenzyme synthase (PT 4 n)Se.ACS) gene. The application takes Saccharomyces cerevisiae capable of synthesizing CBGA as an original strain to selectively over-expressCs.TKS、Cs.OAC、Cs.PT4、Se.ACS, selective knock-outgal80And over-expressgal4Selectively knock outpep4Selectively over-expressed with or without linker fusionCs.OAC, with or without localization by signal peptideCs.The PT4n enzyme reaches a plasma membrane, so that metabolic flow is balanced, a heterologous synthesis path is optimized, and the yield of CBGA is greatly improved.

Description

Recombinant saccharomyces cerevisiae for producing cannabigerol acid and construction method and application thereof

Technical Field

The application relates to the technical field of synthetic biology and the field of genetic engineering, in particular to recombinant saccharomyces cerevisiae for producing cannabigerol acid and a construction method and application thereof.

Background

Cannabis has been cultivated worldwide for thousands of years due to its medicinal properties, and over 100 phytocannabinoids have been isolated from cannabis to date. Cannabinoids have potential medical uses (antibacterial, anti-inflammatory, anti-tumour, anxiolytic and antidepressant) and have great clinical potential in the treatment of a variety of human diseases (epilepsy, diabetes and parkinsonism, etc.). Currently, cannabinoid therapeutics for a variety of indications have been approved and used.

Among them, cannabigerolic acid (CBGA) is an important precursor in several other cannabinoid synthesis pathways. Since CBGA can be converted into other various cannabinoids, for example, into three additional cannabinoids by corresponding enzymes: tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA), which are decarboxylated to yield Cannabigerol (CBG), tetrahydrocannabinol (THC) and Cannabidiol (CBD), respectively.

However, cannabinoids are very low abundant in plants and co-exist with other cannabinoids, which are relatively more abundant, making it difficult to obtain a pure sample from a plant. Meanwhile, since cannabinoids have complicated chemical structures, methods for chemically synthesizing cannabinoids and derivatives thereof are complicated, expensive, and inefficient. Therefore, there is a need to find economically viable alternative sources for large-scale commercial production. The production of cannabinoids and analogues thereof by introducing heterologous biosynthetic pathways in the chassis strain is considered to be a promising approach to a stable cannabinoid supply chain.

Currently, there are studies reporting that saccharomyces cerevisiae utilizes galactose to completely synthesize several cannabinoids of interest, such as cannabigerolic acid (CBGA), Δ ⁹ -tetrahydrocannabinic acid (THCA), cannabidiolic acid (CBDA), delta ⁹ Tetrahydrocannabidioic acid (THCVA) and cannabidioic acid (CBDVA). This study designed the native mevalonate pathway to provide high throughput geranyl pyrophosphate (GPP) and introduced a heterologous, multi-bio-derived hexanoyl-coa, via the biosynthetic pathway to supply Olivinic Acid (OA). Meanwhile, heterologous geranyl pyrophosphate was introduced: the olive oleate geranyltransferase Cs. PT4 gene synthesizes important precursor compounds by using GPP and olive acid OA as substrates: and (3) CBGA.

However, there is a multi-pathway competition in CBGA heterologous biosynthesis, which leads to a metabolic imbalance and thus to a lower yield of cannabinoids.

Disclosure of Invention

In order to improve the yield of cannabigerolic acid (CBGA) and reduce the influence of multi-pathway competition in CBGA heterologous biosynthesis on the yield of CBGA, the application provides a recombinant saccharomyces cerevisiae for producing cannabigerolic acid and a construction method and application thereof.

In a first aspect, the application provides a recombinant saccharomyces cerevisiae for producing cannabigerolic acid, which adopts the following technical scheme: a recombinant Saccharomyces cerevisiae producing cannabigerolic acid, said recombinant Saccharomyces cerevisiae made from a basal Saccharomyces cerevisiae, wherein the following endogenous genes of the basal Saccharomyces cerevisiae have been overexpressed, downregulated, or knocked out: pep4, gal80, gal4, and fas1; exogenous genes encoding the following enzymes were overexpressed: a tetraone synthase (cs.tks) gene, an olive toluate cyclase (cs.oac) gene, a truncated geranyl pyrophosphate, an olive oleate geranyl transferase (cs.pt4n) gene, and a variant acetyl-coenzyme synthase (se.acs) gene.

By adopting the technical scheme, the saccharomyces cerevisiae capable of synthesizing the CBGA is used as an original strain, and the Cs.TKS, cs.OAC, cs.PT4n and Se.ACS are selectively overexpressed ^L641P Selectively knocking out gal80 and over-expressing gal4, selectively knocking out pep4, selectively over-expressing Cs.OAC fused by or without linker, and positioning Cs.PT4 enzyme to plasma membrane by or without signal peptide, thereby balancing metabolic flow, optimizing heterologous synthesis pathway and greatly improving CBGA yield.

In some embodiments, the overexpression, downregulation, or knock-out of a portion of the endogenous gene is any one of (a) - (i):

(a) Overexpression of the cs.tks gene;

(b) (ii) overexpressing a cs.oac gene;

(c) Over-expressing cs.pt4;

(d) Down-regulating fas1;

(e) Overexpression of gal4;

(f) Having (a), (b), (c), (d) and (e) at the same time;

(g) Knocking out gal80 based on (f);

(h) Knocking out pep4 on the basis of (g);

(i) Over-expressing a variant se.acs gene on the basis of (g);

(j) And (g) fusing the Cs.PT4n gene with the SNC1 signal peptide.

In some embodiments, the s.cerevisiae expresses enzymes of the synthetic cannabigerolic acid (CBGA) pathway and is capable of synthesizing CBGA, wherein the enzymes comprise a tetraone synthase (Cs.TKS), cs.OAC, a truncated geranyl pyrophosphate, an olive oleate geranyl transfer, and the likeEnzyme (cs. Pt4n) and se. Acs ^L641P 。

In some embodiments, the nucleotide sequence expressing the truncated cs.pt4 gene cs.pt4n is SEQ ID NO 1; the truncation mode comprises the following steps: lacks SEQ ID NO:0, N-terminal amino acids 1-76 of the full-length amino acid sequence cs pt4.

In some embodiments, in overexpressing the Cs.OAC gene, the coding gene is inserted into the basal Saccharomyces cerevisiae genome using linker (SEQ ID NO: 2) for ligation and expression via the pHSP26 promoter.

In some embodiments, the coding gene is inserted directly into the basal s.cerevisiae genome without linker (SEQ ID NO: 2) ligation and expressed from a strong promoter when the Cs.OAC is overexpressed.

In some embodiments, where the Cs.OAC gene is overexpressed, the coding gene is inserted into the basal s.cerevisiae genome after ligation to the Cs.TKS by linker (SEQ ID NO: 35) and expressed by an inducible promoter.

Oac comprises inserting the coding gene into the s.cerevisiae genome, expression being initiated by a strong promoter.

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

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

In some embodiments, the expressing the cs.pt4n encoding gene comprises inserting a heterologous cs.pt4n gene into the saccharomyces cerevisiae genome, the expression being promoted by the constitutive pTDH3 promoter.

In some embodiments, the overexpression refers to up-regulation of gene expression, i.e., the gene is over-transcribed and translated, and the final gene expression product exceeds the normal level.

In some embodiments, the down-regulation refers to down-regulation of expression of a gene, i.e., the gene is restricted from transcription and translation, and the final gene expression product is below normal levels.

In some embodiments, the knockout is an instruction for loss of function of a particular gene.

In some embodiments, the fas1 gene encodes a subunit of cytoplasmic fatty acid synthase that is required for long chain fatty acid biosynthesis. The fas1 down-regulation refers to the down-regulation of the expression of the fas1 by using pHXT1 which is not expressed in lactose.

In some embodiments, the pep4 gene encodes vacuolar protease. The pep4 knockout means that pep4 on the saccharomyces cerevisiae genome is not expressed and is realized by deleting the coding region of pep 4.

In some embodiments, the Gal80 gene encodes a transcriptional regulator involved in Gal gene suppression. The gal80 knockout means that gal80 on the s.cerevisiae genome is not expressed and is achieved by deleting the coding region of gal 80.

In some embodiments, the Gal4 gene encodes a transcriptional regulator involved in activation of the Gal gene, the Gal4 gene is expressed from pGAL4 (OC), and the nucleotide sequence of pGAL4 (OC) -Gal4 is SEQ ID NO:37.

In some embodiments, the se.acs is se.acs from salmonella enterica ^L641P The sequence is SEQ ID NO. 3.

In some embodiments, the SNC1 signal peptide targets the cs.pt4n enzyme to the plasma membrane, and the sequence of the SNC1 signal peptide is SEQ ID No. 4.

In a second aspect, the application provides a construction method of recombinant saccharomyces cerevisiae for producing cannabigerol acid, which adopts the following technical scheme:

a construction method of recombinant saccharomyces cerevisiae for producing cannabigerolic acid comprises the following steps:

(1) Carrying out PCR amplification to obtain an expression cassette of a gene needing overexpression, and integrating the expression cassette to a saccharomyces cerevisiae genome; or the like, or, alternatively,

carrying out PCR amplification to obtain a homologous fragment of the gene to be knocked out, and replacing the gene to be knocked out on the saccharomyces cerevisiae genome with the homologous fragment; realizing gene knockout and insertion on a saccharomyces cerevisiae genome;

(2) Screening to obtain positive clones.

In a third aspect, the present application provides a use of recombinant saccharomyces cerevisiae producing cannabigerolic acid in the production of cannabigerolic acid, comprising the steps of:

(1) Activating and culturing the recombinant saccharomyces cerevisiae, culturing to obtain a recombinant saccharomyces cerevisiae seed solution,

(2) And transferring the recombinant saccharomyces cerevisiae seed liquid into a culture medium and culturing the recombinant saccharomyces cerevisiae to produce the cannabigerol acid.

In a fourth aspect, the present application provides the use of a recombinant saccharomyces cerevisiae producing cannabigerolic acid in the downstream cannabinoid production of cannabigerolic acid.

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

Specifically, the method comprises the following steps: separating and purifying cannabigerolic acid produced by recombinant saccharomyces cerevisiae, and then performing decarboxylation in vitro by using an enzyme catalyst or a chemical catalyst to obtain cannabigerolic acid.

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

Specifically, the method comprises the following steps: expressing decarboxylase in the recombinant saccharomyces cerevisiae, and decarboxylating cannabigerol acid to obtain cannabidiol; or separating and purifying cannabigerolic acid produced by recombinant saccharomyces cerevisiae, and then performing decarboxylation in vitro by using an enzyme catalyst or a chemical catalyst to obtain cannabidiol.

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

the application takes saccharomyces cerevisiae capable of synthesizing CBGA as an original strain, and selectively over-expresses Cs.TKS, cs.OAC, cs.PT4n and Se.ACS ^L641P Selectively knocking out gal80 and overexpressing gal4, selectively knocking out pep4, selectively overexpressing Cs.OAC fused by or without linker, and positioning Cs.PT4n enzyme to plasma membrane by or without signal peptide, thereby balancing metabolic flow, optimizing heterologous synthesis pathway and greatly improving CBGA yield.

Drawings

FIG. 1 is a synthesis pathway of cannabigerolic acid in Saccharomyces cerevisiae in the background art;

FIG. 2 shows the results of the comparison of pathway enzymes Cs.TKS, cs.OAC, cs.PT4n and Se.ACS on the basis of CBGA-producing strain yCAN31 ^L641P The modification is carried out to optimize the heterologous gene synthesis pathway in the chassis strain and further improve the yield of CBGA. Wherein the deletion of gal80 simultaneously overexpresses gal4 and the deletion of vacuolar protease pep4 is not shown.

FIG. 3 is a comparison of CBGA yields of cannabigerolic acid from each of the constructed strains.

Detailed Description

The present invention will be described in detail with reference to the following embodiments in order to make the aforementioned objects, features and advantages of the invention more comprehensible. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

The terms in this application are described as follows:

1.pep4: the coding vacuolar protease, protease A, can activate the activity of other proteases;

2.Gal80: a transcriptional regulator involved in the inhibition of the Gal gene, gal4 being inhibited by Gal80;

3.Gal4: a 17bp long stretch encoding a transcriptional activator protein capable of recognizing the upstream activating sequence (UASg) of the gene promoter: 5 '-CGGRNNRCYNYNYCCNCCG-3';

4.Fas1: a subunit encoding a cytoplasmic fatty acid synthase, required for long chain fatty acid biosynthesis;

5. overexpression: refers to up-regulated expression of a gene, i.e., excessive transcription and translation of the gene, with the final gene expression product exceeding normal levels;

6. Down-Regulation: refers to the down-regulation of expression of a gene, i.e., the restricted transcription and translation of the gene, with the final gene expression product being below normal levels;

7. knocking out: it uses DNA segment containing certain known sequence to make homologous recombination with gene whose sequence is identical or similar in receptor cell genome so as to make the specific gene function in receptor cell genome lose effect.

The PCR amplification method, the fusion method of different fragments, the gene knockout and overexpression method used in the embodiments of the present application can adopt the technical means common in the art, such as fusion PCR, homologous recombination, CRISPR-Cas9 technology. The enzyme and the kit are all conventional commercial products.

The experimental methods in the examples of the present application are as follows:

1. transformation was performed using lithium acetate/PEG 3350.

(1) Activating a host strain in a 1 XYPD culture medium, and culturing overnight at 30 ℃ and 200 rpm;

(2) Inoculating into new 2 × YPD culture medium to make initial OD value 0.2, and further culturing at 30 deg.C for 4.5 hr;

(3) Centrifuging 5OD bacterial liquid at the normal temperature of 3000rcf for 5min, discarding the supernatant, and washing twice with sterilized ultrapure water to obtain yeast cells;

(4) Preparing DNA mixing systems, each system containing 5OD host cells, and mixing with 50 μ L DNA mixture, so that the cells are resuspended; wherein 50 μ L of DNA mixture is mixed from 2 μ g of the insert, 250ng of the tool plasmid, and sufficient ddH 2O;

(5) Adding lithium acetate into the suspended cells to convert the mixture, thermally shocking at 42 ℃ for 40min to obtain transformed cells, coating the transformed cells on a screening plate, and culturing in an incubator at 30 ℃ for a proper number of days to obtain single colonies, namely recombinant saccharomyces cerevisiae;

(6) After sequencing verification and transformation, taking the strain with correct sequencing for streak storage and glycerol cryopreservation.

2. Colony PCR and sequencing verification

After the monoclonal bacteria grow on the screening plate, carrying out colony PCR and sequencing verification, and specifically comprising the following steps: a small amount of cells were picked up with a pipette tip and placed in 20. Mu.L of 20mmol/LNaOH solution, vortexed, mixed well, and incubated in a metal bath at 95 ℃ for 20min for lysis. After vortex mixing, 1 mu L of the mixture is taken as a template to carry out colony PCR reaction, reaction primers are different according to different verification sequences, the sizes of a clone strip and a negative clone strip are compared, and bacterial liquid of colony PCR positive clone is selected for sequencing verification.

3. The recombinant saccharomyces cerevisiae strain is cultured by using 2mL 1 XYPD as a culture medium, taking a recombinant yeast single colony to be cultured in a 24-well plate overnight for 16h, and the parameters of a shaking table are 30 ℃ and 800rpm. The next day, the overnight bacterial solution was diluted by a suitable amount and then the OD of the bacterial solution was measured with an ultraviolet spectrophotometer at a wavelength of 600nm. The initial OD was set to 0.2, and the cells were transferred to fresh 2mL of 1 XYPG medium and cultured.

The material supplementing mode is as follows: after the transfer, 0.4mM hexanoic acid was added every 12h, and 200. Mu.L of 20% galactose was added every 24 h.

After culturing for 87h, 200. Mu.L of bacterial liquid is collected as a sample to detect the CBGA yield.

4. Detection method for CBGA (CBGA) yield of recombinant saccharomyces cerevisiae

After sample collection, according to sample OD ₆₀₀ Adding 0.2mL of glass beads with the diameter of 0.5mm and 0.4mL of a mixed solution of ethyl acetate and formic acid, wherein the volume ratio of the ethyl acetate to the formic acid is 99.95:0.05, in a high speed tissue grinder, treat 180s at 65Hz, interval 30s, repeat four times; after centrifugation, the upper organic layer was taken from 0.28mL to 1.5mL centrifuge tube, repeated twice, and the collected upper organic layers were combined. Evaporating the organic layer obtained in the third extraction, evaporating at 45 deg.C for 1H to remove solvent, and adding heavy suspension AHF (acetonitrile: H) ₂ O + formic acid =80 +0.05% formic acid), 140 μ L of heavy suspension containing internal standard PHB (propylparaben solution standard, 15 μ M), filtered into a tube inserted in a liquid phase detection vial with 0.22 μ M PVDF filter, as a detection sample. Three of each sample were run in parallel. After the sample was prepared, the detection was performed by HPLC, and the detection conditions are shown in Table 1.

TABLE 1 HPLC detection conditions

5.2 XYPD Medium formulation: 20.0g/L of yeast extract, 40.0g/L of peptone and 40.0g/L of glucose.

6. Lithium acetate conversion mixture: 50% w/VPEG 3350260. Mu.L, 1mol/L LiOAc 36. Mu.L, 10. Mu.L of denatured salmon sperm DNA (denatured salmon sperm DNA was placed in a metal bath before use)Denaturation at 95 ℃ for 5 min), ddH ₂ O 4μL。

7. Screening plate formula for lacking uracil: yeast nitrogen source mother liquor 1.7g/L, ammonium sulfate 5g/L, agar 20g/L, glucose 20g/L and various amino acids as shown in Table 2. Wherein the glucose is separately sterilized.

TABLE 2 screening of the content of various amino acids in the plates

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

The genetic information of the strain yCAN31 used in each example in the present application is shown in table 3.

TABLE 3 Gene information for yCAN31

Example 1 construction of Saccharomyces cerevisiae yG278 expressing the cannabigerolic acid synthetic pathway

On the basis of an original strain yCAN31, overexpressing a tetrone synthase gene Cs.TKS, an olive toluate cyclase gene Cs.OAC and truncated geranyl pyrophosphate, namely an olive oleate geranyl transferase gene Cs.PT4n, down-regulating a fatty acid synthase gene fas1, overexpressing gal4, and knocking out a gene gal80 to obtain a strain yG278.

Wherein overexpressing cs.tks and/or cs.oac, overexpressing gal4, overexpressing cs.pt4n comprises: the integrated fragment was PCR amplified by 2X Phanta Max Master Mix (Phanta DNA polymerase). Using a genome of Saccharomyces cerevisiae CEN. PK2-1C as a template to obtain an integration site 1622b upstream fragment 1622b-Up, an integration site 1622b downstream fragment 1622b-Down, an integration site 511b upstream fragment 511b-Up, an integration site 511b downstream fragment 511b-Down, an integration site 1414a upstream fragment 1414a-Up, an integration site 1414a downstream fragment 1414a-Down, an integration site 106a upstream fragment 106a-Up, an integration site 106a downstream fragment 106a-Down, a promoter pTDH3, a pHSP26 fragment, a terminator ScENO1t and a TDH1t fragment; taking a genome containing pGAL1 gene as a template, and amplifying to obtain a pGAL1 fragment; taking a genome containing pGAL4 (OC) -Gal4-TGal4 gene as a template, and amplifying to obtain pGAL4 (OC) -Gal4-TGal4 fragments; using plasmids containing Cs.OAC3, cs.OAC4 and Cs.OAC5 genes as templates, and amplifying to obtain Cs.OAC3, cs.OAC4 and Cs.OAC5 fragments; the linker (SEQ ID NO: 2) or linker (SEQ ID NO: 35) fragment was obtained using the genome containing the linker (SEQ ID NO: 2) or linker (SEQ ID NO: 35) gene as a template. Finally obtaining 1622b-Up-pGAL1-Cs TKS-linker-Cs.OAC-ScENO1t-1622b-Dp expression cassette, wherein the Cs.OAC is inserted into the basic saccharomyces cerevisiae genome after being connected with the Cs.TKS through the linker (SEQ ID NO: 35); and 1414a-Up-pTDH3-Cs. OAC-ENO1t-1414a-Down expression cassette, wherein the Cs. OAC does not use linker, and the gene is directly inserted into the genome of the saccharomyces cerevisiae; and 511b-Up-pGal4 (OC) -Gal4-TGal4/pHSP26-Cs.OAC3-linker-Cs.OAC5/pGal1-Cs.OAC4-511 b-Down expression cassette, where Cs.OAC3 is inserted into the s.cerevisiae genome after being linked to Cs.OAC5 by a linker (SEQ ID NO: 2). 106a-Up-pTDH3-Cs. PT4-TDH1t-106a-Down expression cassette will be integrated into the corresponding site of the genome. Wherein, the sequence of Cs.OAC3 is shown in SEQ ID NO. 32, the sequence of Cs.OAC4 is shown in SEQ ID NO. 33, and the sequence of Cs.OAC5 is shown in SEQ ID NO. 34.

The truncation pattern of cs.pt4 is: lacks SEQ ID NO:36, and the N-terminal amino acids 1-76 of the full-length amino acid sequence Cs.PT4, and the truncated Cs.PT4n nucleotide sequence is shown as SEQ ID NO. 1.

Wherein, the gene gal80 is knocked out by connecting the upstream and downstream of the gal80 gene. The integrated fragment was PCR amplified by 2X Phanta Max Master Mix (Phanta DNA polymerase). And (3) amplifying to obtain an upstream homology arm gal80-Up fragment and a downstream homology arm gal80-Down fragment by taking a genome of saccharomyces cerevisiae CEN. PK2-1C as a template. The expression cassette gal80-Up-gal80-Down was obtained and integrated into the corresponding site.

Wherein down-regulation of fas1 is achieved by replacing the fas1 promoter. The integrated fragment was PCR-amplified by 2X Phanta Max Master Mix (Phanta DNA polymerase). Using a genome of saccharomyces cerevisiae CEN.PK2-1C as a template, amplifying by using a primer 1 and a primer 2 in a table 4 to obtain a promoter pfas1 upstream homology arm pfas1-Up fragment, and amplifying by using a primer 5 and a primer 6 in the table 4 to obtain a fas1 downstream homology arm fas1 gene fragment; a genome of Saccharomyces cerevisiae CEN. PK2-1C is used as a template, a promoter pHXT1 fragment is obtained by amplification by using a primer 3 and a primer 4 in a table 4, and a pfas1-Up-pHXT1-fas1 expression cassette fragment is finally obtained. The pfas1-Up-pHXT1-fas1 expression cassette fragment was then transformed into the host Saccharomyces cerevisiae yCAN31 (CBGA-producing strain described in the background). Primer 1 and primer 7 in table 4 were used to perform PCR reaction on the constructed strains to obtain reaction solution for colony PCR positive clone for gene sequencing.

And (5) culturing yG278, and detecting the content of CBGA. As shown in FIG. 3, the original strain yCAN31 had a low CBGA of only 20. Mu.M. The recombinant strain yG278 constructed in this example had a CBGA content of 448. Mu.M.

TABLE 4 primer sequences for construction of CBGA high producing strains

Example 2 knock-out of the vacuolar protease Pep4 Gene in the genome of a high-yield cannabigerolic acid strain yG278 to obtain a recombinant Saccharomyces cerevisiae yG350

The gene pep4 is knocked out by connecting the upstream and downstream of the pep4 gene. The integrated fragment was PCR amplified by 2X PhantaMax Master Mix (Phanta DNA polymerase). The upstream homology arm Pep4-Up fragment is obtained by using the genome of Saccharomyces cerevisiae CEN. PK2-1C as a template and by using the primer 1 and the primer 2 in the table 5 for amplification, and the downstream homology arm Pep4-Down fragment is obtained by using the primer 3 and the primer 4 in the table 5 for amplification. The combination of fragments was then transformed into strain yG278 with high cannabigerolic acid yield, yielding strain yG350. Primer 1 and primer 4 in table 5 were used to perform PCR reaction on the strain ySC207 to obtain a reaction solution for colony PCR positive clone for gene sequencing.

And (5) culturing yG350 and detecting the content of CBGA. As shown in FIG. 3, the content of CBGA in yG350 was 650. Mu.M.

TABLE 5 primer sequences for Pep4 knock-outs

Example 3 overexpression of a variant encoding the acetyl-CoA synthase Se.ACS Gene in the genome of the cannabigerolic acid producing Strain yG278, recombinant Saccharomyces cerevisiae yG361 was obtained

The integrated fragment was PCR-amplified by 2X Phanta Max Master Mix (Phanta DNA polymerase). Using genome of Saccharomyces cerevisiae CEN. PK2-1C as template, using primer 1 and primer 2 in Table 6 to amplify and obtain HO-Up fragment of homologous arm at upstream of integration site, using primer 7 and primer 8 in Table 6 to amplify and obtain HO-Up fragment at downstream of integration siteA source arm HO-Down segment; the promoter pSeGAL2 was obtained by amplification using the genome of the strain harboring the pSeGAL2 promoter as a template with primers 3 and 4 in Table 6 to harbor Se ^L641P The gene (SEQ ID NO: 3) and the genome of the strain into which the terminator tADH1 had been integrated were used as templates, and the primers 5 and 6 in Table 6 were used to amplify to obtain the Se.ACSL641P-tADH1 fragment, and HO-Up-pSeGAL2-Se.ACS was finally obtained ^L641P -tADH1-HO-Down expression cassette fragment. ACS was then added to HO-Up-pSeGAL2-Se ^L641P The fragment of the tADH1-HO-Down expression cassette is transformed into a strain yG278 with high cannabigerolic acid yield, and a strain yG361 is obtained. Primer 1 and primer 8 in table 6 were used to perform PCR reaction on strain yG361 to obtain reaction solution for colony PCR positive clone for gene sequencing.

And (5) culturing yG361 and detecting the content of CBGA. As shown in FIG. 3, the content of CBGA in yG361 was 585. Mu.M.

Table 6 overexpression of se ^L641P Primer sequences

Example 4 overexpression of the Gene encoding geranyl pyrophosphate, olive oleate geranyl transferase (Cs. PT4) and the localization peptide in the genome of the strain yG278 of highly cannabigerolic acid producing Strain to obtain the recombinant Saccharomyces cerevisiae yG391

The integrated fragment was PCR amplified by 2X Phanta Max Master Mix (Phanta DNA polymerase). Using genome of Saccharomyces cerevisiae CEN. PK2-1C as template, using primer 1 and primer 2 in Table 7 to amplify and obtain HO-Up fragment of homologous arm upstream of integration site, using primer 7 and primer 8 in Table 7 to amplify and obtain HO-Down fragment of homologous arm downstream of integration site; the genome with pSeGAL2 promoter and Cs.PT4 truncated gene is used as a template, the promoter pSeGAL2 and Cs.PT4 truncated gene (Cs.PT4n) is obtained by amplification through a primer 3 and a primer 4 in a table 7, the genome with a signal peptide SNC1 gene (SEQ ID NO: 4) and a strain integrated with a terminator tADH1 is used as a template, and an SNC1-tADH1 fragment is obtained by amplification through a primer 5 and a primer 6 in the table 7, and finally the HO-Up-pSeGAL2-Cs.PT4-SNC1-tADH1-HO-Down expression cassette fragment is obtained. The HO-Up-pSeGAL2-Cs. PT4n-SNC1-tADH1-HO-Down expression cassette fragment was then transformed into the highly cannabigerolic acid-producing strain yG278, obtaining strain yG391. Primer 1 and primer 8 in Table 7 were used for PCR reaction of strain yG391 to obtain a reaction solution for PCR positive clone of colony for gene sequencing.

And (5) culturing yG391, and detecting the content of CBGA. As shown in FIG. 3, the content of CBGA in yG391 is 775. Mu.M.

Table 7 constructs over-expression truncated Cs.PT4n and signal peptide primer sequences

TABLE 8 strain construction information

In conclusion, as can be seen from the combination of fig. 3, the gene of the cannabigerolic acid synthesis pathway enzyme is expressed and controlled in yeast cells, gal80 is selectively knocked out and gal4 is overexpressed, pep4 is selectively knocked out, cs.OAC fused by or without linker is selectively overexpressed, and the truncated Cs.PT4n enzyme is positioned to a plasma membrane by or without a signal peptide, so that metabolic flow is balanced, and a heterologous synthesis pathway is optimized. The transformed engineering strain can reasonably generate a target product CBGA by using intermediate products OA and GPP in the whole synthesis path, realizes the construction of the high-yield yeast strain of the CBGA, which is a key cannabinoid precursor, and has very important significance for the biosynthesis and commercialization of the CBGA and downstream cannabinoids.

Example 5 application of recombinant Saccharomyces cerevisiae constructed in example 1 to production of cannabigerolic acid

The application of the recombinant saccharomyces cerevisiae in the production of cannabigerol comprises the following steps:

(1) Activating and culturing the recombinant saccharomyces cerevisiae constructed in the embodiment 1, and culturing to obtain a recombinant saccharomyces cerevisiae seed solution;

(2) And (2) transferring the recombinant saccharomyces cerevisiae seed liquid obtained in the step (1) into a culture medium suitable for producing the cannabigerolic acid, and culturing the recombinant saccharomyces cerevisiae under a suitable condition to produce the cannabigerolic acid.

Examples 6 to 8

Examples 6 to 8 are applications of the recombinant saccharomyces cerevisiae constructed in examples 2 to 4 to the production of cannabigerolic acid, respectively.

Examples 6 to 8 differ from example 5 in that, in step (1), examples 6 to 8 employed recombinant s.cerevisiae constructed in examples 2 to 4, respectively.

Example 9 application of the recombinant Saccharomyces cerevisiae constructed in example 1 to the production of cannabigerol

The cannabigerolic acid produced in example 5 was decarboxylated by heat treatment to yield cannabigerol.

Examples 10 to 12

Examples 10 to 12 are applications of the recombinant saccharomyces cerevisiae constructed in examples 2 to 4 to the production of cannabigerol, respectively.

Examples 10 to 12 differ from example 9 in that cannabigerol acids produced in examples 6 to 8, respectively, were decarboxylated by heat treatment to yield cannabigerol.

The above embodiments are preferred embodiments of the present application, and the protection scope of the present application is not limited by the above embodiments, so: all equivalent changes made according to the structure, shape and principle of the present application shall be covered by the protection scope of the present application.

Claims

1. A recombinant Saccharomyces cerevisiae producing cannabigerolic acid, wherein the recombinant Saccharomyces cerevisiae is made from a basic Saccharomyces cerevisiae, wherein the following endogenous genes of the basic Saccharomyces cerevisiae have been overexpressed, downregulated, or knocked out:pep4、gal80、gal4andfas1(ii) a The following exogenous genes of the basic s.cerevisiae were overexpressed: tetrone synthase (a)Cs.TKS gene, olive toluate cyclase (TKS)Cs.OAC) gene, truncated geranyl pyrophosphate, olive oleate geranyl transferase: (Cs.PT 4) Gene and variant acetyl-CoA synthase: (Se.ACS) gene.

2. The recombinant saccharomyces cerevisiae for producing cannabigerolic acid as claimed in claim 1 or 2, wherein the overexpression, down-regulation or knock-out of the endogenous gene comprises any one of (a) - (i):

over-expressionCs.A TKS gene;

over-expressionCs.An OAC gene;

overexpression of truncatedCs.PT4 Gene: (Cs.PT4n）；

Down-regulation offas1；

Overexpressiongal4；

Having both (a), (b), (c), (d) and (e);

on the basis of (f), knocking outgal80；

On the basis of (g), knocking outpep4；

Over-expressing the variant on the basis of (g)Se.An ACS gene;

on the basis of (g), willCs.The PT4n gene is fused with SNC1 signal peptide.

3. The recombinant Saccharomyces cerevisiae producing cannabigerolic acid as claimed in claim 1, wherein the basal Saccharomyces cerevisiae expresses enzymes of the synthetic cannabigerolic acid (CBGA) pathway and is capable of synthesizing CBGA, wherein the enzymes comprise the tetraketone synthase (C: (C))Cs.TKS), olive toluate cyclase (C)Cs.OAC), truncated geranyl pyrophosphate: olive oleate geranyl transferase: (Cs.PT4 n) and variant acetyl-coenzyme synthase: (Se.ACS ^L641P ）。

4. The recombinant Saccharomyces cerevisiae producing cannabigerolic acid as claimed in claim 3, wherein the expression of truncatedCs.PT4 geneCs.The nucleotide sequence of PT4n is SEQ ID NO 1.

5. The recombinant saccharomyces cerevisiae for producing cannabigerolic acid as claimed in claim 1, wherein the recombinant saccharomyces cerevisiae is overexpressedCs.In the case of the OAC gene, the linker can be used for connection, and the coding gene can be inserted into the basic Saccharomyces cerevisiae genome,expressed by the pHSP26 promoter.

6. The recombinant saccharomyces cerevisiae capable of producing cannabigerolic acid as claimed in claim 1, wherein the overexpressionCs.OAC involves direct insertion of the coding gene into the genome of the basal s.cerevisiae without the use of linker ligation, and expression is driven by the constitutive pTDH3 promoter.

7. The recombinant Saccharomyces cerevisiae producing cannabigerolic acid as claimed in claim 3, wherein the overexpressionCs.OAC involves passing the coding gene through linkerCs.The TKS is inserted into a basic saccharomyces cerevisiae genome after being connected, and is expressed through a pGal1 promoter.

8. The recombinant saccharomyces cerevisiae for producing cannabigerolic acid as claimed in claim 5, wherein the sequence of the linker is SEQ ID NO. 2.

9. The recombinant saccharomyces cerevisiae for producing cannabigerolic acid as claimed in claim 7, wherein the sequence of the linker is SEQ ID NO 35.

10. The recombinant saccharomyces cerevisiae capable of producing cannabigerolic acid as claimed in claim 2, wherein the recombinant saccharomyces cerevisiae is characterized by being prepared from cannabigerolic acidCs.Overexpression of TKS involves insertion of the coding gene into the s.cerevisiae genome, and expression is promoted by pGal1 promoter.

11. The recombinant Saccharomyces cerevisiae producing cannabigerolic acid of claim 1, wherein the down-regulation isfas1Comprising downregulation with pHXT1 not expressed in galactosefas1Expression of (2).

12. The recombinant Saccharomyces cerevisiae producing cannabigerolic acid as claimed in claim 1, wherein the recombinant Saccharomyces cerevisiae is capable of producing cannabigerolic acidSe.ACS is derived from Salmonella entericaSe.ACS ^L641P The sequence is SEQ ID NO. 3.

13. The recombinant Saccharomyces cerevisiae producing cannabigerolic acid as claimed in claim 3, wherein the SNC1 signal peptide is localizedCs.PT4 enzyme reaches plasma membrane, and the sequence of SNC1 signal peptide is SEQ ID NO. 4.

14. A construction method of the recombinant saccharomyces cerevisiae for producing cannabigerol acid according to any one of claims 1 to 13, characterized by comprising the following steps:

carrying out PCR amplification to obtain an expression cassette of a gene needing overexpression, and integrating the expression cassette to a saccharomyces cerevisiae genome; or the like, or, alternatively,

carrying out PCR amplification to obtain a homologous fragment of the gene to be knocked out, and replacing the gene to be knocked out on the saccharomyces cerevisiae genome with the homologous fragment;

realizing gene knockout and insertion on a saccharomyces cerevisiae genome;

screening to obtain positive clones.

15. Use of the recombinant saccharomyces cerevisiae producing cannabigerolic acid according to any one of claims 1 to 13 in cannabigerolic acid production.

16. Use according to claim 15, characterized in that it comprises the following steps:

(1) Activating and culturing the recombinant saccharomyces cerevisiae, and culturing to obtain a recombinant saccharomyces cerevisiae seed solution;

(2) And transferring the recombinant saccharomyces cerevisiae seed liquid into a culture medium containing cannabigerolic acid, and culturing the recombinant saccharomyces cerevisiae to produce the cannabigerolic acid.

17. Use of a recombinant saccharomyces cerevisiae producing cannabigerolic acid as claimed in any of claims 1 to 13 in the production of downstream cannabinoids of cannabigerolic acid.