Biosynthesis method and application of cannabinoid compound

Abstract

The invention discloses a biosynthesis method and application of cannabinoid compounds. The specific method comprises the following steps: constructing a non-reductive polyketide synthase (NRPKS) AtnG from a fungus Arthrinum sp.NF2194, an isopentenyl transferase NphBV49W / Y288P from an actinomycetes Streptomyces sp.CL190, a cyclase Svz9 from an actinomycetes Streptoviensis NA431, a GPP synthase AgGPPS from a plant Abis grandis and a terpene synthesis enhancement path MEV into a heterologous expression vector, and sequentially introducing the heterologous expression vector into escherichia coli, so as to successfully realize the production of the cannabinoid compounds OA, CBGA and CBCA. The cannabinoid compound disclosed by the invention has important medical and industrial values.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosynthesis, and particularly relates to a biosynthesis method and application of a cannabinoid compound. BACKGROUND

[0002] Cannabis sativa L. is an annual erect herbaceous plant of Cannabaceae and Cannabis. Humans have used cannabis for a long history, and cannabis is planted as an important food, fiber and medicinal crop. Cannabinoids are a class of biologically active terpenes, which have important medical and industrial values. At present, more than 150 kinds of cannabinoids have been isolated from cannabis, some of which are active cannabinoids such as Δ9-tetrahydrocannabinol (Δ9-THC), a famous hallucinogenic addictive agent; cannabidiol (CBD), which has been approved by FDA for the treatment of pediatric epilepsy and colon cancer; cannabichromene (CBC), an effective analgesic, also has anti-inflammatory and antidepressant effects. In addition, studies have shown that CBC can improve the viability of neural stem / progenitor cells, thereby promoting brain neurogenesis, and these active cannabinoids have been widely used in the medical field, the food field and the cosmetic field.

[0003] The traditional preparation method of cannabinoids is extraction and purification of Cannabis sativa. This method is limited by factors such as unstable cannabinoid yield in plants, slow production cycle, complex composition of crude extract and difficulty in separating active ingredients, which cannot meet the market demand for active cannabinoids. For cannabinoids with low natural abundance (CBD and CBC), these problems further limit their application. For years, researchers have completed the analysis of the path of secondary metabolites of cannabis.

[0004] Plant type III polyketide synthase (Olivetol synthase, OLS) and cyclase (Olivetolic acid cyclase, OAC) complete the synthesis of the cannabinoid polyketide precursor OA. Then, isopentenyltransferase CsPT4 transfers geranyl diphosphate synthase (GPP) to the C-3 position of OA to generate CBGA. Finally, under the action of different cannabinoid cyclases, the corresponding cannabinoids THCA, CBDA and CBCA are generated. This path has been reconstituted in Saccharomyces cerevisiae, but the yield cannot meet the growing demand. SUMMARY

[0005] The first object of the present application is to provide a biosynthesis method and application of a cannabinoid compound.

[0006] To solve the problems in the prior art, the technical scheme adopted by the present application is as follows: in a first aspect, the present application provides a biosynthesis method of a cannabinoid compound; in a second aspect, the present application provides a cannabinoid compound; and in a third aspect, the present application provides a composition.

[0007] The first aspect of the present application provides a biosynthesis method of a cannabinoid compound, comprising the following steps:

[0008] (1) constructing a non-reducing polyketide synthase NRPKS from a fungus into a vector pET22b, constructing an isopentenyltransferase from an actinomycete into a vector pET28a, constructing a GPP synthase from a plant into a vector pET28a, constructing a cyclase from an actinomycete into a vector pET28a, and constructing a terpene synthesis enhancement pathway MEV into a vector pCDFDuet-1;

[0009] (2) extracting an intron-free cDNA sequence of a non-reducing polyketide synthase from a fungus, and using the same for construction into the vector pET22b;

[0010] (3) sequentially transforming the three heterologous expression vectors constructed above into an Escherichia coli host BAP1, activating through an LB culture medium, and then transferring to a TB culture medium for fermentation;

[0011] (4) after the fermentation is completed, centrifuging to separate the fermentation broth and the bacterial body, extracting, sampling after extraction for analysis, and preparing the cannabinoid compounds olivetolic acid OA, cannabigerolic acid CBGA, and cannabichromenic acid CBCA.

[0012] Further, in step (1), the non-reducing polyketide synthase from a fungus is AtnG from Arthrinium sp. NF2194; the isopentenyltransferase from an actinomycete is NphB_V49W / Y288P from Streptomyces sp. CL190; the GPP synthase from a plant is AgGPPS from Abies grandis; and the cyclase from an actinomycete is Svz9 from Streptomyces varsoviensis NA431.

[0013] Further, in step (1), the amino acid sequence of the non-reducing polyketide synthase AtnG is shown in SEQ ID No. 1; the amino acid sequence of the geranyl pyrophosphate synthase AgGPPS is shown in SEQ ID No. 2; and the amino acid sequence of the cyclase Svz9 is shown in SEQ ID No. 3. Since the optimal reaction temperature of the cyclase Svz9 is 37°C, a strategy of low-temperature induction at 16°C for 3 days and then warming to 37°C for one day was adopted for the fermentation of cannabinoids. The protein sequence of NphB_V49W / Y288P from Streptomyces sp. CL190 is a known sequence, reference: ACS Catal. 2022, 12, 4628-4639.

[0014] Further, in step (1), the terpene synthesis enhancement pathway MEV includes eight genes from E. coli and S. cerevisiae, namely atoB, HMGS, tHMGR, MVD1, idi, ERG8, ERG12 or ERG20_F69W / N127W. The eight genes are from the article included in the NLM database: Modulating Precursor and Terpene Synthase Supply for Whole-Cell Biocatalytic Production of Sesquiterpene (+)-Zizaene in Pathway-Engineered Escherichia coli. The nucleotide sequence of atoB is shown in SEQ ID No. 4, the nucleotide sequence of HMGS is shown in SEQ ID No. 5, the nucleotide sequence of tHMGR is shown in SEQ ID No. 6, the nucleotide sequence of MVD1 is shown in SEQ ID No. 7, the nucleotide sequence of idi is shown in SEQ ID No. 8, the nucleotide sequence of ERG8 is shown in SEQ ID No. 9, the nucleotide sequence of ERG12 is shown in SEQ ID No. 10, and the nucleotide sequence of ERG20_F69W / N127W is shown in SEQ ID No. 11.

[0015] The terpene synthesis enhancement pathway MEV refers to the method for enhancing the terpene synthesis pathway in the document Nat. Biotechnol. 2003, 21, 796-802. The method can increase the supply of endogenous isoprenyl units in E. coli, increase the yield of GPP, and further increase the yield of cannabinoids. The eight genes are constructed on the vector pCDFDuet. The reference documents for the eight genes are Nat. Biotechnol. 2003, 21, 796-802. ACS Synth. Biol. 2014, 3, 298-306. atoB, acetoacetyl-CoA synthase; HMGS, HMG-CoA synthase; tHMGR, truncated HMG-CoA reductase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; MVD1, mevalonate pyrophosphate decarboxylase; idi, IPP isomerase; ERG20_F69W / N127W, GPP synthase

[0016] Further, in step (1), taking the construction of the heterologous expression vector pET22b-AtnG as an example, the plasmid pET22b is linearized, double digestion is performed using restriction enzymes NdeI / HindIII, the linearized vector is recovered after incubation at 37°C for 1 hour; the target gene sequence is amplified by PCR, the fragment size is confirmed by nucleic acid gel electrophoresis, and the gene fragment is recovered; the linearized vector and the gene fragment are connected by the rapid cloning kit ClonExpress Ultra One Step Cloning Kit V2, and then transformed into E. coli DH5α and coated on an LB plate (Amp resistance) after incubation at 50°C for 5-10 min and overnight culture at 37°C. After overnight culture at 37°C, a single colony is selected for digestion verification.

[0017] Further, in step (2), the cDNA sequence is extracted using the FastPure Universal Plant Total RNA Isolation Kit fungal RNA extraction kit and the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) full-length cDNA single-strand synthesis kit.

[0018] Further, in step (3), the transformed E. coli host of the heterologous expression vector is activated by LB medium at 37℃, 220 rpm, and then transferred to TB medium for fermentation; in the TB medium (Trytone 12 g / L, Yeast extract 24 g / L and Glycerol 20 g / L), when the OD600 reaches 0.4-0.6 at 37℃, 220 rpm, the temperature is reduced to 16℃, 0.25 mM IPTG is added for induction, and fermentation is carried out for 3 days; in step (4), after fermentation, the fermentation broth and bacteria are separated by centrifugation at 8000 rpm, an equal volume of ethyl acetate is added to the fermentation broth, and after thorough shaking and extraction, samples are taken for HPLC or LC-MS analysis.

[0019] The second aspect of the application provides a cannabinoid compound synthesized by the method, and the cannabinoid compound is olivetolic acid OA, cannabigerolic acid CBGA or cannabichromenic acid CBCA.

[0020] The third aspect of the application provides a composition, which comprises a cannabinoid compound and at least one pharmaceutically acceptable carrier.

[0021] Beneficial effects: the application improves production efficiency, reduces cost, is environmentally friendly, and the purity of the microbially synthesized cannabinoid is higher, reducing impurities and harmful substances that may exist in the plant extraction process, improving product safety and quality controllability.

[0022] Compared with the prior art, the application has the following advantages:

[0023] (1) Innovative production platform: a new microbial production platform based on E. coli is developed for the de novo synthesis of cannabinoids, breaking through the limitations of traditional extraction of cannabinoids from cannabis plants, and solving the problems of unstable yield, long production cycle, complex composition and difficult separation in plant extraction method, which leads to the inability to meet market demand.

[0024] (2) Improve production efficiency: efficient synthesis of cannabinoid compounds OA, CBGA and CBCA is achieved, and experimental data show that the cannabinoid production strain can finally obtain 3.5 mg / L of CBCA, providing a feasible and efficient way for the industrial production of cannabinoids, which is expected to greatly improve the yield of cannabinoids.

[0025] (3) Reduce cost: producing cannabinoids by microbial fermentation can significantly reduce production costs, including raw material costs, production cycle costs and purification costs, making the production of cannabinoids more economical, and helping to promote the widespread application of cannabinoids in medicine, food, cosmetics and many other fields.

[0026] (4) Environmental protection: microbial fermentation production process is relatively more environmentally friendly, reducing a large number of waste and environmental pollution problems that may occur in the traditional plant extraction process, in line with the requirements of sustainable development. Stable supply: Escherichia coli grows rapidly and is easy to cultivate, enabling large-scale fermentation production, thereby ensuring the stable supply of cannabinoids to meet the growing demand of the market.

[0027] (5) Overcome regulatory restrictions: traditional cannabis cultivation is strictly regulated, while the present application can legally and stably produce cannabinoids through microbial synthesis, free from the regulatory restrictions of plant cultivation, opening up a new path for the legal production and application of cannabinoids. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0029] Figure 1 The present application is a new path for cannabinoid biosynthesis.

[0030] Figure 2 The present application is a total ion flow diagram or superimposed diagram of cannabinoid compounds.

[0031] Figure 3 The present application is a HPLC and mass spectrum data diagram of cannabinoid compound OA. Compound OA, HR-ESIMS (negative source mode) m / z 223.1199 [M-H]-(molecular formula is C 12 H 16 O4).

[0032] Figure 4 The present application is a HPLC and mass spectrum data diagram of cannabinoid CBGA. Compound CBGA, HR-ESIMS (negative source mode) m / z 359.2408 [M-H]-(molecular formula is C 22 H 32 O4).

[0033] Figure 5 The present application is a HPLC and mass spectrum data diagram of cannabinoid CBCA. Compound CBCA, HR-ESIMS (negative source mode) m / z 357.2157 [M-H]-(molecular formula is C 22 H 30 O4).

[0034] Figure 6 The present application is a synthesis path diagram of cannabinoid compound CBCA. DETAILED DESCRIPTION

[0035] The specific embodiments described herein are presented for purposes of illustration and description. They are not intended to be exhaustive of the scope and spirit of the application.

[0036] The present application can be better understood in accordance with the following examples. However, one skilled in the art will readily appreciate that the following examples are included merely for purposes of illustration and are not intended to limit the application, as described in the claims.

[0037] In the present application, the term "and / or", describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B, which can represent the following three cases: A exists alone, A and B exist together, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the front and rear associated objects.

[0038] In the present application, "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or the like means any combination of these items, including any combination of single item or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can represent a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, wherein a, b, and c can be single or multiple.

[0039] It should be understood that the size of the sequence number of the above-mentioned processes in various embodiments of the present application does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence. The execution order of each process should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0040] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0041] The weight of the related components mentioned in the specification of the embodiments of the present application can not only refer to the specific content of each component, but also represent the weight ratio between each component. Therefore, as long as the content of the related components in the specification of the embodiments of the present application is enlarged or reduced in proportion, it is within the scope disclosed in the specification of the embodiments of the present application. Specifically, the mass mentioned in the specification of the embodiments of the present application can be μg, mg, g, kg and other mass units commonly known in the chemical field.

[0042] The first aspect of the embodiment of the present application provides a biosynthesis method of cannabinoid compounds, comprising the following steps:

[0043] (1) Constructing a non-reducing polyketide synthase NRPKS from a fungus to a vector pET22b, constructing an isopentenyltransferase from an actinomycete to a vector pET28a, constructing a GPP synthase from a plant to a vector pET28a, constructing a cyclase from an actinomycete to a vector pET28a, and constructing a terpene synthesis enhancement pathway MEV to a vector pCDFDuet-1;

[0044] (2) Extracting an intron-free cDNA sequence of a non-reducing polyketide synthase from a fungus, and using it for construction to a vector pET22b;

[0045] (3) Transforming the three heterologous expression vectors constructed above into an Escherichia coli host BAP1 in turn, activating through a LB medium, and then transferring to a TB medium for fermentation;

[0046] (4) After fermentation, centrifuging the fermentation broth and the bacterial body, extracting, sampling for analysis, and preparing cannabinoid compounds olivetolic acid (OA), cannabigerolic acid CBGA, and cannabichromenic acid CBCA.

[0047] In some embodiments, in step (1), the non-reducing polyketide synthase from a fungus is AtnG from Arthrinium sp. NF2194; the isopentenyltransferase from an actinomycete is NphB_V49W / Y288P from Streptomyces sp. CL190; the GPP synthase from a plant is AgGPPS from Abies grandis; and the cyclase from an actinomycete is Svz9 from Streptomyces varsoviensis NA431.

[0048] In some embodiments, in step (1), the amino acid sequence of the non-reducing polyketide synthase AtnG is shown in SEQ ID No. 1; the amino acid sequence of the geranyl pyrophosphate synthase AgGPPS is shown in SEQ ID No. 2; and the amino acid sequence of the cyclase Svz9 is shown in SEQ ID No. 3. Since the optimum reaction temperature of the cyclase Svz9 is 37℃, a strategy of low-temperature induction at 16℃ for 3 days and then warming to 37℃ for one day is adopted for the fermentation of the cannabinoid.

[0049] In some embodiments, in step (1), the terpene synthesis enhanced pathway MEV includes eight genes from E. coli and S. cerevisiae, which are atoB, HMGS, tHMGR, MVD1, idi, ERG8, ERG12 or ERG20_F69W / N127W. The nucleotide sequence of atoB is shown in SEQ ID No. 4, the nucleotide sequence of HMGS is shown in SEQ ID No. 5, the nucleotide sequence of tHMGR is shown in SEQ ID No. 6, the nucleotide sequence of MVD1 is shown in SEQ ID No. 7, the nucleotide sequence of idi is shown in SEQ ID No. 8, the nucleotide sequence of ERG8 is shown in SEQ ID No. 9, the nucleotide sequence of ERG12 is shown in SEQ ID No. 10, and the nucleotide sequence of ERG20_F69W / N127W is shown in SEQ ID No. 11.

[0050] In some embodiments, in step (1), the construction of the heterologous expression vector pET22b-AtnG is taken as an example, the plasmid pET22b is linearized, double digestion is performed using restriction enzymes NdeI / HindIII, the linearized vector is recovered after incubation at 37°C for 1 hour; the target gene sequence is amplified by PCR, the fragment size is confirmed by nucleic acid gel electrophoresis, and the gene fragment is recovered; the linearized vector and the gene fragment are connected by a rapid cloning kit ClonExpress Ultra One Step Cloning Kit V2, and are transformed into E. coli DH5a after incubation at 50°C for 5-10 min and coating on an LB plate (Amp resistance) and incubation at 37°C overnight. After incubation at 37°C overnight, a single colony is selected for enzyme digestion verification.

[0051] In some embodiments, in step (2), the cDNA sequence is extracted using a FastPure Universal Plant Total RNA Isolation Kit fungal RNA extraction kit and a HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) full-length cDNA single-strand synthesis kit.

[0052] In some embodiments, in step (3), the transformed E. coli host of the heterologous expression vector is activated by LB medium at 37°C and 220 rpm, and then transferred to TB medium for fermentation; in the TB medium (Trytone 12 g / L, yeast extract 24 g / L and glycerol 20 g / L), when the OD600 reaches 0.4-0.6, the temperature is reduced to 16°C, 0.25 mM IPTG is added for induction, and fermentation is carried out for 3 days.

[0053] In some embodiments, in step (4), after fermentation, the fermentation broth and the bacterial cells are centrifuged at 8000 rpm, an equal volume of ethyl acetate is added to the fermentation broth, and after sufficient shaking and extraction, a sample is taken for HPLC or LC-MS analysis.

[0054] The second aspect of the embodiments of the present application provides a cannabinoid compound synthesized by the method, and the cannabinoid compound is olivetolic acid OA, cannabigerolic acid CBGA or cannabichromenic acid CBCA.

[0055] The third aspect of the embodiments of the present application provides a composition, and the composition comprises the cannabinoid compound of claim 1 and at least one pharmaceutically acceptable carrier.

[0056] The inventors found that the non-reducing polyketide synthase (NRPKS) AtnG from the fungus Arthrinium sp. NF2194 successfully produced the key precursor of cannabinoids OA when heterologously expressed in E. coli through extensive screening, and further screened the downstream genes in the cannabinoid biosynthesis pathway to construct a non-plant-derived cannabinoid biosynthesis pathway, and successfully achieved de novo synthesis of cannabinoids in E. coli.

[0057] Embodiment 1

[0058] As shown in Figure 1 , Figure 1 is a schematic diagram of the non-plant pathway for synthesizing cannabinoids in E. coli of the present application.

[0059] The biosynthesis method of the cannabinoid compound of the present application comprises the following steps:

[0060] (1) the non-reducing polyketide synthase NRPKS from fungi is constructed into vector pET22b, the prenyltransferase from actinomycetes is constructed into vector pET28a, the GPP synthase from plants is constructed into vector pET28a, the cyclase from actinomycetes is constructed into vector pET28a, and the terpenoid synthesis enhancement pathway MEV is constructed into vector pCDFDuet-1; the non-reducing polyketide synthase from fungi is AtnG from Arthrinium sp. NF2194, the prenyltransferase from actinomycetes is NphB_V49W / Y288P from Streptomyces sp. CL190, the GPP synthase from plants is AgGPPS from Abies grandis, and the cyclase from actinomycetes is Svz9 from Streptomyces varsoviensis NA431. The amino acid sequence of the non-reducing polyketide synthase AtnG is shown in SEQ ID No. 1, the amino acid sequence of the GPP synthase AgGPPS is shown in SEQ ID No. 2, and the amino acid sequence of the cyclase Svz9 is shown in SEQ ID No. 3.

[0061] Since the optimal reaction temperature of the cyclase Svz9 is 37℃, a strategy of low-temperature induction at 16℃ for 3 days and then warming to 37℃ for one day is adopted for the fermentation of cannabinoids. The terpenoid synthesis enhancement pathway MEV includes eight genes atoB, HMGS, tHMGR, MVD1, idi, ERG8, ERG12 or ERG20_F69W / N127W from Escherichia coli and Saccharomyces cerevisiae, the nucleotide sequence of atoB is shown in SEQ ID No. 4, the nucleotide sequence of HMGS is shown in SEQ ID No. 5, the nucleotide sequence of tHMGR is shown in SEQ ID No. 6, the nucleotide sequence of MVD1 is shown in SEQ ID No. 7, the nucleotide sequence of idi is shown in SEQ ID No. 8, the nucleotide sequence of ERG8 is shown in SEQ ID No. 9, the nucleotide sequence of ERG12 is shown in SEQ ID No. 10, and the nucleotide sequence of ERG20_F69W / N127W is shown in SEQ ID No. 11.

[0062] Taking the construction of the heterologous expression vector pET22b-AtnG as an example, the plasmid pET22b was linearized and double-digested with restriction endonucleases NdeI / HindIII. After incubation at 37°C for 1 hour, the linearized vector was recovered. The target gene sequence was amplified by PCR, and the fragment size was confirmed by nucleic acid gel electrophoresis, and the gene fragment was recovered. The linearized vector and gene fragment were ligated using the rapid cloning kit ClonExpress Ultra One Step Cloning KitV2, incubated at 50°C for 5-10 minutes, and then transformed into E. coli DH5α and spread on LB plates (Amp resistance). After overnight culture at 37°C, single clones were picked for enzyme digestion verification.

[0063] (2) The intronless cDNA sequence of the fungal non-reducing polyketide synthase was extracted and used to construct the vector pET22b; the cDNA sequence was extracted using the FastPure Universal Plant Total RNA Isolation Kit and the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper).

[0064] (3) The three heterologous expression vectors constructed above were sequentially transformed into the Escherichia coli host BAP1, activated by LB medium, and then transferred to TB medium for fermentation; the Escherichia coli host transformed with the heterologous expression vector was activated by LB medium at 37°C, 220 rpm, and then transferred to TB medium for fermentation; in TB medium (Trytone 12 g / L, Yeast extract 24 g / L and glycerol 20 g / L), at 37°C, 220 rpm, when the OD600 reached 0.4-0.6, the temperature was lowered to 16°C, 0.25 mM IPTG was added for induction, and fermentation was carried out for 3 days.

[0065] (4) After the fermentation is completed, the fermentation liquid and the bacterial cells are separated by centrifugation, and samples are taken for analysis after extraction to obtain the cannabinoid compounds oleic acid (OA), cannabigerolic acid CBGA, and cannabichromenic acid CBCA. Figure 6 Schematic diagram of the synthesis of the cannabinoid compound CBCA of the present invention.

[0066] The present invention discloses a cannabinoid compound synthesized by a method, wherein the cannabinoid compound is oleic acid OA, cannabigerolic acid CBGA or cannabichromenic acid CBCA.

[0067]

[0068] Example 2

[0069] A composition of the present application, the composition comprising a cannabinoid compound and at least one pharmaceutically acceptable carrier.

[0070] Example 3

[0071] In order to achieve the de novo synthesis of cannabinoids in E. coli, the non-ribosomal PKS (NRPKS) AtnG from the fungus Arthrinium sp. NF2194, the prenyltransferase NphB_V49W / Y288P from the actinomycete Streptomyces sp. CL190, the cyclase Svz9 from the actinomycete Streptomyces varsoviensis NA431, the GPP synthase AgGPPS from the plant Abies grandis and the terpene synthesis enhancement pathway MEV (including the 8 genes atoB, HMGS, tHMGR, MVD1, idi, ERG8, ERG12 and ERG20_F69W / N127W from E. coli and S. cerevisiae) were constructed into different heterologous expression vectors, the nucleotide sequence of atoB is shown in SEQ ID No. 4, the nucleotide sequence of HMGS is shown in SEQ ID No. 5, the nucleotide sequence of tHMGR is shown in SEQ ID No. 6, the nucleotide sequence of MVD1 is shown in SEQ ID No. 7, the nucleotide sequence of idi is shown in SEQ ID No. 8, the nucleotide sequence of ERG8 is shown in SEQ ID No. 9, the nucleotide sequence of ERG12 is shown in SEQ ID No. 10, and the nucleotide sequence of ERG20_F69W / N127W is shown in SEQ ID No. 11.

[0072] The application selects different screening marker vectors, constructs the above-mentioned genes, that is, constructs AtnG into vector pET22b, constructs NphB_V49W / Y288P, AgGPPS and Svz9 into vector pET28a, and constructs 8 genes involved in the terpene synthesis enhancement pathway MEV into vector pCDFDuet-1. Here, taking the heterologous expression vector pET22b-AtnG as an example: linearize the plasmid pET22b, double-digest it with restriction endonuclease NdeI / HindIII, recover the linearized vector after 37°C incubation for 1 hour; perform PCR amplification on the target gene sequence, confirm the fragment size by nucleic acid gel electrophoresis, and recover the gene fragment; connect the linearized vector and the gene fragment through the rapid cloning kit ClonExpress Ultra One Step Cloning Kit V2, transform it into E. coli DH5α after 50°C incubation for 5-10 min and coat it on an LB plate (Amp resistance), and after 37°C overnight culture, pick a single colony for digestion verification.

[0073] It should be noted here that the expression of AtnG of fungal origin in E. coli requires an intron-free cDNA sequence. Here, the application extracts the cDNA sequence of AtnG through the FastPure Universal Plant Total RNA Isolation Kit fungal RNA extraction kit and the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) full-length cDNA single-strand synthesis kit.

[0074] The application transforms the three constructed heterologous expression vectors into E. coli host BAP1 in turn, activates them through LB medium at 37°C and 220 rpm, and then transfers them to TB medium for fermentation. In the TB medium (Trytone 12 g / L, yeast extract 24 g / L and glycerol 20 g / L), when the OD600 reaches 0.4-0.6 at 37°C and 220 rpm, the temperature is lowered to 16°C, 0.25 mM IPTG is added for induction, and fermentation is carried out for 3 days. Since the optimal reaction temperature of cyclase Svz9 is 37°C, the strategy of low-temperature induction at 16°C for 3 days and then warming to 37°C for one day is adopted for cannabinoid fermentation. After fermentation, the fermentation broth and the bacterial body are separated by centrifugation at 8000 rpm, an equal volume of ethyl acetate is added to the fermentation broth, and after thorough shaking and extraction, samples are taken for HPLC or LC-MS analysis.

[0075] The results show that the cannabinoid production strain can finally obtain 3.5 mg / L of CBCA. The yield of CBCA of the present application is 3.5 mg / L, which is higher than the reported path of the original plant source, Saccharomyces cerevisiae as the chassis (31.7 pg / L, ACS Synth. Biol. 2025, 14, 2, 531-541.)

[0076] Example 4

[0077] The difference between Example 4 and Example 1 is that:

[0078] In step (4), after fermentation, the fermentation broth and bacteria are centrifuged at 8000 rpm, an equal volume of ethyl acetate is added to the fermentation broth, and an equal volume of ethyl acetate is selected as the extractant. After extraction three times, a concentrated sample is obtained. The extracted ethyl acetate is combined and concentrated using a rotary evaporator to obtain a crude paste. After weighing the crude paste, 2 times the weight of reverse phase filler is weighed for sample mixing. After the sample is completely dried, it is ground and eluted with a methanol-water system from 10% methanol to 100% for gradient elution. The eluted fractions and compound standards are analyzed together by HPLC to confirm. Figure 2 The HPLC analysis chart of the target compound of the present application.

[0079] Example 5

[0080] The difference between Example 5 and Example 1 is that:

[0081] In step (4), the fermentation broth is collected, and an equal volume of ethyl acetate is selected as the extractant. After extraction three times, a concentrated sample is obtained. The extracted ethyl acetate is combined and concentrated using a rotary evaporator to obtain a crude paste. After weighing the crude paste, 2 times the weight of reverse phase filler is weighed for sample mixing. After the sample is completely dried, it is ground and eluted with a methanol-water system from 10% methanol to 100% for gradient elution. The eluted fractions and OA standards are analyzed together by HPLC and mass spectrometry to confirm. Figure 3 The HPLC and mass spectrometry data chart of the cannabinoid compound OA of the present application. Compound OA, HR-ESIMS (negative source mode) m / z 223.1199 [M-H]-(molecular formula is C 12 H 16 O4).

[0082] Example 6

[0083] The difference between Example 6 and Example 1 is that:

[0084] In step (4), the fermentation broth is collected, and an equal volume of ethyl acetate is selected as the extractant. After extraction three times, the concentrated sample is obtained by concentration. The extracted ethyl acetate is combined and concentrated using a rotary evaporator to obtain a crude paste. After weighing the crude paste, 2 times the weight of reverse phase filler is weighed and mixed with the sample. After the sample is completely dried, it is ground and eluted using a methanol-water system with a gradient from 10% methanol to 100%. The eluted fractions are analyzed by HPLC and mass spectrometry together with the CBGA standard. Figure 4 The HPLC and mass spectrometry data of the cannabinoid CBGA of the present application are shown in the figure. Compound CBGA, HR-ESIMS (negative source mode) m / z 359.2408 [M-H]-(molecular formula C 22 H 32 O4). Figure 5 The HPLC and mass spectrometry data of the cannabinoid CBCA of the present application are shown in the figure. Compound CBCA, HR-ESIMS (negative source mode) m / z 357.2157 [M-H]-(molecular formula C 22 H 30 O4).

[0085] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and the scope of protection of the present application is defined by the appended claims, the specification and their equivalents.

Claims

1. A method for biosynthesis of cannabinoid compounds, characterized in that The steps include: (1) The fungal non-reducing polyketide synthase NRPKS was constructed into the vector pET22b, the actinomycete isopentenyl transferase was constructed into the vector pET28a, the plant GPP synthase was constructed into the vector pET28a, the actinomycete cyclase was constructed into the vector pET28a, and the terpene biosynthesis enhancement pathway MEV was constructed into the vector pCDFDuet-1; (2) extracting the intron-free cDNA sequence of the fungal non-reducing polyketide synthase and using it to construct the vector pET22b; (3) The three heterologous expression vectors constructed above were sequentially transformed into the Escherichia coli host BAP1, activated using LB medium, and then transferred to TB medium for fermentation; (4) After the fermentation is completed, the fermentation liquid and the bacterial cells are separated by centrifugation, and samples are taken for analysis after extraction to obtain the cannabinoid compounds oleic acid OA, cannabigerolic acid CBGA, and cannabichromenic acid CBCA.

2. The method for biosynthesis of cannabinoid compounds according to claim 1, characterized in that: In step (1), the fungal non-reducing polyketide synthase is the non-reducing polyketide synthase AtnG derived from Arthrinium sp. NF2194; the actinomycete-derived isopentenyl transferase is NphB_V49W / Y288P derived from Streptomyces sp. CL190; the plant-derived GPP synthase is the geranyl pyrophosphate synthase AgGPPS derived from Abies grandis; and the actinomycete-derived cyclase is the cyclase Svz9 derived from Streptomyces varsoviensis NA431.

3. The method for biosynthesis of cannabinoid compounds according to claim 2, characterized in that: In step (1), the amino acid sequence of the non-reducing polyketide synthase AtnG is shown in SEQ ID No. 1; the amino acid sequence of the geranyl pyrophosphate synthase AgGPPS is shown in SEQ ID No. 2; and the amino acid sequence of the cyclase Svz9 is shown in SEQ ID No.

3.

4. The method for biosynthesis of cannabinoid compounds according to claim 1, characterized in that: In step (1), the terpene synthesis enhancement pathway MEV includes 8 genes derived from Escherichia coli and Saccharomyces cerevisiae, namely atoB, HMGS, tHMGR, MVD1, idi, ERG8, ERG12 or ERG20_F69W / N127W, the nucleotide sequence of atoB is shown in SEQ ID No.4, the nucleotide sequence of HMGS is shown in SEQ ID No.5, the nucleotide sequence of tHMGR is shown in SEQ ID No.6, the nucleotide sequence of MVD1 is shown in SEQ ID No.7, the nucleotide sequence of idi is shown in SEQ ID No.8, the nucleotide sequence of ERG8 is shown in SEQ ID No.9, the nucleotide sequence of ERG12 is shown in SEQ ID No.10, and the nucleotide sequence of ERG20_F69W / N127W is shown in SEQ ID No.

11.

5. The method for biosynthesis of cannabinoid compounds according to claim 2, characterized in that: In step (1), taking the construction of the heterologous expression vector pET22b-AtnG as an example, the plasmid pET22b is linearized, double-digested with restriction endonucleases NdeI / HindIII, and the linearized vector is recovered after incubation at 37°C for 1 hour; the target gene sequence is amplified by PCR, and the gene fragment is recovered after the fragment size is confirmed by nucleic acid gel electrophoresis; the linearized vector and the gene fragment are connected using the rapid cloning kit ClonExpressUltra One Step Cloning KitV2, incubated at 50°C for 5-10 minutes, and then transformed into E.coli DH5α and coated on an LB plate. After overnight culture at 37°C, a single clone is picked for enzyme digestion verification.

6. The method for biosynthesis of cannabinoid compounds according to claim 1, characterized in that: In step (2), the cDNA sequence was extracted using the FastPure Universal Plant Total RNA Isolation Kit fungal RNA extraction kit and the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) full-length cDNA single-strand synthesis kit.

7. The method for biosynthesis of cannabinoid compounds according to claim 2, characterized in that: In step (3), the Escherichia coli host transformed with the heterologous expression vector is activated by LB medium at 37°C and 220 rpm, and then transferred to TB medium for fermentation; in the TB medium, at 37°C and 220 rpm, when the OD600 reaches 0.4-0.6, the temperature is lowered to 16°C, 0.25 mM IPTG is added for induction, and fermentation is carried out for 3 days; in step (4), after the fermentation is completed, the fermentation broth and the bacterial cells are separated by centrifugation at 8000 rpm, an equal volume of ethyl acetate is added to the fermentation broth, and after sufficient shaking and extraction, a sample is taken for HPLC or LC-MS analysis.

8. A cannabinoid compound synthesized by the method of claim 1, characterized in that: The cannabinoid compound is oleic acid OA, cannabigerolic acid CBGA or cannabichromenic acid CBCA.

9. A composition, characterized in that: The composition comprises the cannabinoid compound according to claim 1 and at least one pharmaceutically acceptable carrier.

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