A cannabinoid-producing microbial cell and its construction method and application

Abstract

The present application relates to the field of synthetic biology technology, and specifically discloses a cannabinol-producing microbial cell, its construction method, and application. A cannabinol-producing microbial cell includes a host cell, and the host cell includes a synthetic pathway for geranyl pyrophosphate, a synthetic pathway for olivate, and an isopentenyl transferase mutant. The construction method is as follows: based on the geranyl pyrophosphate-producing host cell yCAN10, the olivate synthesis pathway is constructed to form yCG02; a copy gene of the olivate synthase OLS is integrated into yCG02 to obtain yCG03; an acetyl-CoA carboxylase mutant ACC1 (S659A, S1157A) is integrated into yCG03 to obtain yCG04; and an isopentenyl transferase mutant is integrated into yCG04 to obtain yCG05, i.e., a cannabinol-producing microbial cell. The application is to produce cannabinol from a carbon source, which alleviates the problem of a large gap in cannabinol resources in the current market.

Description

Technical Field

[0001] The present application relates to the field of synthetic biology technology, and more specifically, to a cannabinoid-producing microbial cell and a construction method and application thereof. Background Art

[0002] Cannabigerol (CBG) is a non-psychoactive cannabinoid found in trace amounts in most cannabis plants, with common strains containing less than 1%. It has attracted considerable attention for its potential anti-tumor, anti-inflammatory, antioxidant, neuroprotective, immunomodulatory, and cancer therapeutic properties after binding to G protein-coupled receptors CB1 and CB2 in the human body. CBG also has antibacterial effects against methicillin-resistant Staphylococcus aureus.

[0003] Due to the complex chemical structure and existence of isomers of cannabinoids, chemical synthesis is difficult. Currently, the most common method for preparing cannabigerol is still extraction from cannabis plants, primarily through decarboxylation of the naturally occurring synthetic product, cannabigerolic acid. In the basal cells of the glandular trichomes of cannabis plants, the polyketide pathway in the vacuole utilizes fatty acids to synthesize dihydroxyvaleric acid, while the mevalonate pathway in the plastid utilizes acetyl-CoA to synthesize geranyl pyrophosphate. Furthermore, geranyltransferase condenses dihydroxyvaleric acid and geranyl pyrophosphate to form cannabigerolic acid. Finally, cannabigerolic acid, acting as a precursor to cannabinoid derivatives, is catalyzed by cannabinoid synthase in the secretory sac to produce cannabinoid products. However, cannabigerolic acid is present in very low concentrations in cannabis plants, and there are also issues such as long cultivation cycles, low production efficiency, high production costs, low purity of the final product, and waste of biomass resources.

[0004] Therefore, the related art proposes a method for producing cannabigerol acid from microbial culture or cell-free system culture, and then obtaining cannabigerol through heating and decarboxylation.

[0005] However, the above-mentioned related technologies have the following disadvantages: the cost of producing cannabigerol acid from microbial cells and cell-free systems is very high. At the same time, the use of heating to decarboxylate cannabigerol acid to produce cannabigerol has the accompanying problems of low yield and many by-products. Therefore, there is a large gap in cannabigerol resources in the current market. Using synthetic biology technology, a new microbial cell is constructed that can synthesize cannabigerol without decarboxylation of cannabigerol acid, which has important research and application significance. Summary of the Invention

[0006] In order to alleviate the problem of a large shortage of cannabinoid resources in the current market, the present application provides a cannabinoid-producing microbial cell and a construction method and application thereof.

[0007] In a first aspect, the present application provides a cannabinoid-producing microbial cell, comprising a host cell, wherein the host cell comprises a geranyl pyrophosphate synthesis pathway, an olive alcohol synthesis pathway, and an isopentenyl transferase mutant.

[0008] By adopting the above technical scheme, the synthesis pathways of geranyl pyrophosphate and olivetol are introduced into the host cell. Using geranyl pyrophosphate and olivetol as substrates, an isopentenyl transferase mutant is introduced to condense the two to produce cannabigerol, thereby forming a new microbial cell that can synthesize cannabigerol without the decarboxylation of cannabigerolic acid, thereby alleviating the problem of the large gap in cannabigerol resources in the current market.

[0009] Optionally, the host cell is one of Saccharomyces cerevisiae, Yarrowia lipolitica, Pichia pastoris, Aspergillus niger and Escherichia coli.

[0010] By adopting the above technical solution, when using the above host cells, the prepared microbial cells are easier to scale up for production, thereby obtaining high-purity and high-yield cannabinoids.

[0011] Alternatively, the synthesis pathway of olivetol is obtained by expressing acyl kinase (CsAAE1) and olivetol synthase (OLS) in host cells;

[0012] The DNA sequence of the CsAAE1 is SEQ ID NO. 1, which contains the amino acid sequence of SEQ ID NO. 2;

[0013] The DNA sequence of the OLS is SEQ ID NO.3, which contains the amino acid sequence of SEQ ID NO.4.

[0014] By adopting the above technical scheme, the synthesis pathway of olivetol can be obtained by expressing acyl kinase (CsAAE1) and olivetol synthase (OLS) in host cells.

[0015] Optionally, the isopentenyl transferase mutant is one of NphB1, NphB2 and CsPT4n;

[0016] NphB1, the gene sequence is SEQ ID NO. 7, which contains the amino acid sequence of SEQ ID NO. 8;

[0017] NphB2, the gene sequence is SEQ ID NO. 9, which contains the amino acid sequence of SEQ ID NO. 10;

[0018] CsPT4n, the gene sequence is SEQ ID NO.11, which contains the amino acid sequence of SEQ ID NO.12.

[0019] By adopting the above technical solution, when the above isopentenyl transferase mutant is used, geranyl pyrophosphate and olivetol can be better condensed to produce cannabinoids.

[0020] Optionally, the host cell further expresses one or both of a copy gene of olivetol synthase OLS and an acetyl-CoA carboxylase mutant ACC1 (S659A, S1157A);

[0021] The gene sequence of the ACC1 (S659A, S1157A) is SEQ ID NO. 5, which contains the amino acid sequence of SEQ ID NO. 6.

[0022] By adopting the above technical solution, the effective utilization of metabolite precursors is a key step in the synthesis of olivetol. Therefore, optimizing the synthesis pathway in the above manner can promote the metabolism of olivetol and increase the yield of olivetol.

[0023] In a second aspect, the present application provides a method for constructing a microbial cell that produces cannabinoids, using the following technical solution:

[0024] A method for constructing a cannabinoid-producing microbial cell comprises the following steps:

[0025] S1. Based on the host cell yCAN10 that produces geranyl pyrophosphate, the biosynthesis pathway of olivetol was constructed to form the microbial cell yCG02.

[0026] S2. Integrate an expression cassette containing a copy of the gene encoding olivetol synthase (OLS) into the microbial cell yCG02 to obtain the microbial cell yCG03.

[0027] S3. Integrate the expression cassette of the acetyl-CoA carboxylase mutant ACC1 (S659A, S1157A) into the microbial cell yCG03 to obtain the microbial cell yCG04;

[0028] S4. Integrate the expression cassette of the isopentenyl transferase mutant into the microbial cell yCG04 to obtain the microbial cell yCG05, i.e., the cannabinoid-producing microbial cell.

[0029] By adopting the above technical solution, a new synthetic pathway for cannabigerol was designed and cannabigerol-producing microbial cells were constructed. Specifically, a cannabigerol synthesis route was established in microbial cells, using geranyl pyrophosphate and olivetol as substrates and initiating the reaction with a prenyltransferase mutant. This eliminates the need for post-decarboxylation treatment of cannabigerol acid to produce cannabigerol, thus simplifying the production route, reducing production costs, and increasing the yield of cannabigerol, alleviating the significant shortage of cannabigerol in the market. Integrating another copy of the OLS gene and the acetyl-CoA carboxylase mutant ACC1 (S659A, S1157A) into the host cell chromosome further increased olivetol yield, thereby increasing the yield of cannabigerol.

[0030] In a third aspect, the present application provides an application of a microbial cell producing cannabinoids, using the following technical solution:

[0031] The invention discloses an application of a cannabinoid-producing microbial cell, wherein the cannabinoid-producing microbial cell produces cannabinoid from a carbon source.

[0032] By adopting the above technical solution, cannabinoid-producing microbial cells can obtain cannabinoid products directly from carbon sources, which has abundant carbon sources, reduces production costs, increases the yield of cannabinoids, and alleviates the problem of a large gap in cannabinoids in the market.

[0033] In summary, this application has the following beneficial effects:

[0034] 1. This application constructs a new pathway for producing cannabinol in microbial cells, reducing the accompanying problems of low yield and high by-products in the decarboxylation of cannabinoid acid to produce cannabinol.

[0035] 2. By designing a new synthetic pathway for cannabinol, a synthetic route for cannabinol is constructed in biological cells, and the cannabinol product is obtained directly from the carbon source without the need for decarboxylation treatment, which simplifies the steps and improves the yield.

[0036] 3. The microbial cells of the present application can be scaled up to produce high-purity cannabinoids in large quantities without being restricted by the environment and land resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the synthesis pathway of cannabinoids in Saccharomyces cerevisiae.

[0038] Figure 2 This is a graph showing the detection content of olivetol, a precursor of cannabinoid synthesis in this application. The left value for each strain is 72h, and the right value is 96h.

[0039] Figure 3 This is an analysis diagram of the cannabinoid product of this application. DETAILED DESCRIPTION

[0040] The present application is further described in detail below with reference to the accompanying drawings and examples. It should be noted that if no specific conditions are specified in the following examples, the experiments were carried out according to conventional conditions or those recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0041] Example Example

[0042] A cannabinoid-producing microbial cell comprises a host cell, wherein the host cell comprises a geranyl pyrophosphate synthesis pathway, an olivetol synthesis pathway, and an isopentenyl transferase mutant. The isopentenyl transferase mutant is selected from NphB1, whose gene sequence is SEQ ID NO. 7 and contains the amino acid sequence of SEQ ID NO. 8.

[0043] The host cell may be one of Saccharomyces cerevisiae, Yarrowia lipolitica, Pichia pastoris, Aspergillus niger and Escherichia coli. In this embodiment, the host cell is Saccharomyces cerevisiae.

[0044] Olivoletol is synthesized by expressing an acyl kinase (CsAAE1) and an olivetol synthase (OLS) in Saccharomyces cerevisiae. CsAAE1 encodes an acyl kinase gene from C. sativa (DNA sequence: SEQ ID NO. 1, containing the amino acid sequence of SEQ ID NO. 2), while OLS encodes an olivetol synthase gene (DNA sequence: SEQ ID NO. 3, containing the amino acid sequence of SEQ ID NO. 4).

[0045] Since the olivetol synthesis pathway requires the consumption of three malonyl-CoA molecules and one hexanoyl-CoA molecule to synthesize one molecule of olivetol product, and acetyl-CoA carboxylase ACC1 catalyzes the synthesis of malonyl-CoA from acetyl-CoA, to increase the accumulation of olivetol products, the host cell also expresses a copy of the olivetol synthase OLS gene and an acetyl-CoA carboxylase mutant ACC1 (S659A, S1157A). The gene sequence of ACC1 (S659A, S1157A) is SEQ ID NO. 5, which contains the amino acid sequence of SEQ ID NO. 6.

[0046] A method for constructing a cannabinoid-producing microbial cell comprises the following steps:

[0047] S1. Based on the geranyl pyrophosphate-producing Saccharomyces cerevisiae host yCAN10 (Luo et al., 2019), a biosynthesis pathway for olivetol was constructed to form the microbial cell yCG02:

[0048] The genetic information of the Saccharomyces cerevisiae host yCAN10 is as follows:

[0049] Microbial cell host cell genetic information

[0050] yCAN10 CEN. PK2-1C CEN. PK2-1C

[0051] (308a::GAL1p-ERG20(F96W-N127W)-TDH1t; erg9::KanMX / CTR3p-ERG9;leu2-3,112::His3MX6-GAL1p-ERG19 / GAL10p-ERG8;ura3- 52::GAL1p-mvaS(A110G) / GAL10p-MvaE;his3_1::hphMX4 / GAL1p-ERG12 / GAL10p-IDI1)

[0052] PCR amplification of the integrated gene fragments was performed using DNA polymerase: using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the homology arms upstream of the integration site, 911b-UP, and downstream of the integration site, 911b-DOWN, were obtained. The promoter fragment pHHF1 was obtained using the genome of a strain harboring the pHHF1 promoter as a template. The fragment CsAAE1 was obtained using a plasmid containing the CsAAE1 gene as a template. The terminator tTDH1 was obtained using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, and the promoter fragment pGal1 was obtained using the genome of a strain harboring the pGal1 promoter as a template. The fragment OLS was obtained using a plasmid containing the OLS gene as a template. The terminator tADH1 was obtained using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template. The resulting expression cassette consisted of 911b-Up-pHHF1-CsAAE1-tTDH1 / pGal1-OLS-tADH1-911b-Dp. The 911b-Up-pHHF1-CsAAE1-tTDH1 / pGal1-OLS-tADH1-911b-Dp expression cassette fragment was then transformed into the original host yCAN10 to obtain strain yCG02.

[0053] Therefore, the 911b-Up-pHHF1-CsAAE1-tTDH1 / pGal1-OLS-tADH1-911b-Dp expression cassette was transformed into the Saccharomyces cerevisiae host yCAN10, and a synthetic pathway for accumulating geranyl pyrophosphate and olivetol was constructed on yCAN10 to form the microbial cell yCG02.

[0054] S2. Integrate an expression cassette containing a copy of the olivetol synthase (OLS) gene into the microbial cell yCG02 to obtain the microbial cell yCG03:

[0055] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the YPRCd15C-UP fragment, containing the homology arm upstream of the integration site, and the YPRCd15C-DOWN fragment, containing the homology arm downstream of the integration site, were generated. The promoter fragment pGal1 was generated using the genome of a strain harboring the pGal1 promoter as a template. The OLS fragment was generated using a plasmid containing the OLS gene as a template. The terminator tENO1 was generated using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template. The resulting YPRCd15C-Up-pGal1-OLS-tENO1-YPRCd15C-Dp expression cassette was then transformed into the host cell yCG02 to generate the microbial cell yCG03. Strains with correctly sequenced genes were selected for subsequent culture.

[0056] S3. Integrate the expression cassette of the acetyl-CoA carboxylase mutant ACC1 (S659A, S1157A) into the microbial cell yCG03 to obtain the microbial cell yCG04:

[0057] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the 1414a-UP fragment, which is the homology arm upstream of the integration site, and the 1414a-DOWN fragment, which is the homology arm downstream of the integration site, were generated. The promoter fragment pHSP26 was generated using the genome of a strain harboring the pHSP26 promoter as a template. The ACC1(S659A, S1157A) fragment was generated using a plasmid containing the ACC1(S659A, S1157A) gene as a template. The terminator tACC1 was also generated using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template. Ultimately, the 1414a-Up-pHSP26-ACC1(S659A, S1157A)-tACC1-1414a-Dp expression cassette was generated. The 1414a-Up-pHSP26-ACC1(S659A, S1157A)-tACC1-1414a-Dp expression cassette fragment was then transformed into the host yCG03 to obtain microbial cells yCG04. Strains with correct gene sequencing were subsequently cultured.

[0058] S4. Integrate the expression cassette of the isopentenyl transferase mutant NphB1 into the microbial cell yCG04 to obtain the microbial cell yCG05, i.e., the cannabinoid-producing microbial cell:

[0059] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the fragments 1014a-UP, containing the homology arm upstream of the integration site, and 1014a-DOWN, containing the homology arm downstream of the integration site, were generated. A plasmid harboring the pSeGal2 promoter was used as a template to generate the promoter fragment pSeGal2. A plasmid containing the NphB1 gene was used as a template to generate the NphB1 fragment. The terminator tTEF1 was also generated using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template. The resulting expression cassette, 1014a-Up-pSeGal-NphB1-tTEF1-1014a-Dp, was then transformed into the host cell yCG04 to generate the cannabinoid-producing microbial cell yCG05. Strains with correctly sequenced genes were cultured.

[0060] Example 2

[0061] The difference between this embodiment and embodiment 1 is that the isopentenyl transferase mutant is NphB2, the gene sequence is SEQ ID NO. 9, and it contains the amino acid sequence of SEQ ID NO. 10.

[0062] Example 3

[0063] The difference between this embodiment and embodiment 1 is that the isopentenyl transferase mutant is CsPT4n, the gene sequence is SEQ ID NO.11, which contains the amino acid sequence of SEQ ID NO.12.

[0064] Application Examples

[0065] The invention discloses an application of a cannabinoid-producing microbial cell, wherein the cannabinoid-producing microbial cell produces cannabinoid from a carbon source.

[0066] The cannabinoid-producing microbial cells prepared in the above examples were cultured overnight in YPD medium (2% glucose, 1% yeast extract, 2% peptone) and then transferred to 2 ml of YPG liquid medium (2% galactose, 1% yeast extract, 2% peptone) at a concentration of 10 D / ml. Hexanoic acid (0.2 mM) was added to the YPG medium every 12 hours for a total of 1 mM. Starting 24 hours after the transfer, 2% galactose was added every 24 hours for 84 hours. Cannabinoid production occurred during the culture process.

[0067] Performance testing

[0068] Detection method

[0069] 1. Quantitative detection of olivetol

[0070] Single colonies of yCG02, yCG03, and yCG04 constructed in Example 1 were cultured in 24-well plates in 2 ml of 1xYPD (2% glucose, 1% yeast extract, 2% peptone) at 30°C and 800 rpm in a shaker for 16 hours. The culture was diluted 10-fold with 1xYPD and the OD600 value was measured using a UV spectrophotometer. A cell with an initial OD600 of 0.2 was then transferred to 3 ml of 1xYPD (2% galactose, 1% yeast extract, 2% peptone) and cultured.

[0071] Hexanoic acid feeding was performed as follows: After transferring 3 ml of 1xYPD culture medium, hexanoic acid (0.2 mM) was added every 12 hours for five times. Starting 24 hours after transfer, galactose (2% w / v) was added every 12 hours for three times, to a final concentration of 2%. After 84 hours of culture, 200 μL of the culture medium was collected as a sample.

[0072] Sample extraction: After sample collection, add 0.2 mL of 0.5 mm glass beads and 0.4 mL of ethyl acetate (containing 0.05% formic acid). Crush and extract the sample using a high-speed tissue grinder. Take 0.28 mL of the upper organic layer and transfer it to a 1.5 mL centrifuge tube. Repeat twice and combine the collected upper organic phases. Use a vacuum dryer to evaporate the three extracted organic phases at 45°C for 1 hour until no solvent remains. Resuspend the evaporated sample with acetonitrile / H2O (80 / 20) and then filter it with a 0.22 μm PVDF filter membrane as the test sample. Olive alcohol was quantified using a high-performance liquid chromatography system (HPLC). The test results are detailed in the following table. Figure 2 .

[0073] Depend on Figure 2 It was found that olivetol was produced by adding caproic acid to the culture medium of the above strains. In particular, yCG02 produced 222.95 µM olivetol from the addition of 1 mM caproic acid.

[0074] The expression cassette YPRCd15C-Up-pGal1-OLS-tENO1-YPRCd15C-Dp was further integrated into yCG03, so the olivetol production was increased to 343 μM, which was 1.5-fold higher than that obtained with yCG02.

[0075] Olivoletol synthesis requires malonyl-CoA. yCG04 further expressed the acetyl-CoA carboxylase mutant ACC1, which increased the olivate production to 495 µM (89.2 mg / L), a 2.22-fold increase compared to the olivate production obtained with yCG02.

[0076] 2. Analysis of Cannabinol Products

[0077] The cannabinoid-producing microbial cells prepared in different embodiments were collected and cultured using the method of the application example to obtain bacterial liquid, and the cannabinoid product was extracted and analyzed as follows:

[0078] 200 μl of each bacterial suspension was collected, 0.2 mL of 0.5 mm glass beads and 0.4 mL of ethyl acetate (containing 0.05% formic acid) were added, and the cells were disrupted using a tissue grinder to extract the CBG product. 0.28 mL of the upper organic layer was transferred to a 1.5 mL centrifuge tube. The collected upper organic layer was evaporated to dryness and resuspended in acetonitrile / H₂O (80 / 20) to obtain a cannabinol-producing microbial cell sample. The cannabinol-producing microbial cell sample and a 10 mg / L CBG standard were analyzed and identified by LC-MS / MS on an Agilent 6470B time-of-flight (TOF) instrument. The mass spectrometer detection mode was negative ion full scan mode. The mass / charge ratio of cannabinol in negative ion mode was 315. Using the CBG standard as a control, the characteristic ion peak with a mass / charge ratio of 315 was extracted to identify the presence of CBG in the sample. CBG product was detected in all examples.

[0079] The product analysis of cannabinoids prepared by the application example in Example 1 is detailed in Figure 3 ,like Figure 3 As shown, the cannabinol-producing microbial cell sample and the CBG standard have the same mass spectrum peak at a retention time of 7.3 minutes and a mass / charge ratio of 315. The results indicate that the cannabinol-producing microbial cells use hexanoic acid as a substrate to synthesize cannabinol CBG at a content of 1.113 mg / L.

[0080] The strain prepared in Example 2 had a cannabinoid CBG content of 1.015 mg / L; the strain prepared in Example 3 had a cannabinoid CBG content of 1.034 mg / L.

[0081] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A cannabinoid-producing microbial cell, characterized in that The invention comprises a host cell, wherein the host cell comprises a geranyl pyrophosphate synthesis pathway, an olivetol synthesis pathway and an isopentenyl transferase mutant; The synthesis pathway of olivetol is obtained by expressing acyl kinase (CsAAE1) and olivetol synthase (OLS) in host cells; The host cell also expresses a copy gene of olivetol synthase OLS and acetyl-CoA carboxylase mutant ACC1 (S659A, S1157A); The host cell is Saccharomyces cerevisiae (S. cerevisiae); The isopentenyl transferase mutant is one of NphB1, NphB2 and CsPT4n; NphB1, the nucleotide sequence is SEQ ID NO. 7, and the amino acid sequence is SEQ ID NO. 8; NphB2, the nucleotide sequence is SEQ ID NO.9, and the amino acid sequence is SEQ ID NO.10; CsPT4n, the nucleotide sequence is SEQ ID NO. 11, and the amino acid sequence is SEQ ID NO. 12; The nucleotide sequence of CsAAE1 is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2; The nucleotide sequence of the OLS is SEQ ID NO.3, and the amino acid sequence is SEQ ID NO.4; The nucleotide sequence of the ACC1 (S659A, S1157A) is SEQ ID NO. 5, and the amino acid sequence is SEQ ID NO.

6.

2. An application of a microbial cell producing cannabinoids, characterized in that: The cannabinol-producing microbial cell of claim 1 produces cannabinol from a carbon source.

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