CN113584089B

CN113584089B - Application of isopentenyl transferase in catalytic synthesis of cannabigerol or cannabigerol acid

Info

Publication number: CN113584089B
Application number: CN202110749746.2A
Authority: CN
Inventors: 陈贤情; 杨月; 夏文豪; 李珍珠; 王筱; 逯晓云; 刘诗梦; 江会锋; 王文
Original assignee: Jiaxing Synbiolab Biotechnology Co ltd
Current assignee: Jiaxing Synbiolab Biotechnology Co ltd
Priority date: 2021-07-01
Filing date: 2021-07-01
Publication date: 2023-11-24
Anticipated expiration: 2041-07-01
Also published as: CN113584089A

Abstract

The present invention provides the use of an prenyl transferase in the catalytic synthesis of a phytocannabinoid or an analog thereof, wherein the prenyl transferase has the amino acid sequence shown in any one of SEQ ID NO. 1-8. The present invention also provides a process for the preparation of phytocannabinoids or analogues thereof using a substrate catalysed by an prenyl transferase; wherein the prenyl transferase has an amino acid sequence shown in any one of SEQ ID NO.1-8, and the phytocannabinoid or analog thereof is CBG or CBGA. The NPHB obtained by screening can be used for efficiently synthesizing CBG and CBGA, and has the advantages of few synthesis steps, simple and convenient approach, high synthesis efficiency and high single-step conversion rate.

Description

Application of isopentenyl transferase in catalytic synthesis of cannabigerol or cannabigerol acid

Technical Field

The invention relates to the field of chemical synthesis, in particular to an application of prenyl transferase in-vitro high-efficiency catalytic synthesis of phytocannabinoids or analogues thereof (such as cannabigerol or cannabigerol acid).

Background

CBG (Cannabigerol) the cannabigerol is a kind of cannabinoid, has extremely high medical value, has great potential in reducing intraocular pressure of glaucoma, treating inflammatory bowel disease, inhibiting colon cancer, treating huntington's disease, multiple sclerosis, resisting bacteria, diminishing inflammation, treating psoriasis and the like, and has the effects of resisting depression, regulating appetite, helping sleep and emotion regulation, improving immunity and the like, but is mainly detected in industrial cannabis at present, has extremely low content and has no extraction value.

The main source of CBG is the traditional plant extraction, the steps are complicated, a large amount of organic reagents are used, strong acid, strong alkali and high-temperature and high-pressure environment exist in the reaction process, the environment is not friendly, and the efficiency is low. However, the chemical synthesis of CBG involves the use of various strong acids and catalysts, so that the CBG is difficult to separate and has low production efficiency. At present, in addition to the traditional chemical extraction, a recently emerging production method is to utilize synthetic biology, which searches for a synthetic key gene through genome assembly annotation and gene mining of industrial hemp, introduces a target gene into saccharomyces cerevisiae, and reconstructs a production path of CBGA in the saccharomyces cerevisiae. Further heat treatment is carried out to produce CBG. Compared with chemical synthesis, the biosynthesis method has the advantages of mild reaction conditions, single product, easy separation and the like.

Prenyl transferases (NPHB) catalyze the transfer of the prenyl structure to another compound, leaving behind two biphosphoric acid groups, NPHB may be capable of catalyzing oleyl alcohol and GPP to CBG, or 2, 4-dihydroxy-6-pentylbenzoic acid and GPP to CBGA.

The NPHB reported to date for the catalytic synthesis of CBGA has AltPT derived from CL190 sequence of streptomyces coelicolor, but its catalytic synthesis of CBGA is extremely inefficient.

The amino acid sequence of AltPT is:

MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLEAESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQRGLLKAFDSLED(SEQ ID NO:9)。

the method for synthesizing CBG and CBGA by utilizing NPHB in vitro catalysis is simple and efficient, so that more NPHB capable of synthesizing CBG and CBGA in vitro catalysis needs to be developed. According to the invention, NPHB capable of efficiently catalyzing and synthesizing CBGA and/or CBG is obtained through screening, and has an amino acid sequence shown in any one of SEQ ID NO 1-8, on one hand, the NPHB can directly catalyze and synthesize CBG, and compared with the traditional approach, the NPHB has more concise and rapid steps; on the other hand, the catalytic efficiency is higher in the process of synthesizing CBGA in a catalytic mode.

Disclosure of Invention

The object of the present invention is to provide a method for the catalytic synthesis of phytocannabinoids or analogues thereof (e.g. CBG and CBGA) in vitro which is simple and efficient. Specifically, the present inventors analyzed the amino acid backbone and conserved regions thereof according to the sequence SEQ ID NO 9 reported in the prior art, and combined with the activation energy and protein homeostasis of substrate docking, analyzed CBGA and CBG structures, selected a plurality of optimal sequences from 1000 sequences for verification, and expected to find genes that better catalyze CBG and/or CBGA. Finally, NPHB capable of efficiently catalyzing and synthesizing CBGA and/or CBG is obtained, and the NPHB has an amino acid sequence shown in any one of SEQ ID NO 1-8.

NPHB obtained by screening of the invention is connected to a plasmid, expressed in a large amount in bacteria and purified, and then in vitro, the CBG is synthesized by catalyzing with olive alcohol and GPP as substrates, or the CBGA is synthesized by catalyzing with 2, 4-dihydroxyl-6-amyl benzoic acid and GPP as substrates. The method has the advantages of few steps in the process of producing CBG and CBGA by catalysis, convenient operation, shortened production path, improved yield and reduced production cost.

Specifically, the invention provides the following scheme:

1. use of an prenyl transferase in the catalytic synthesis of a phytocannabinoid or an analog thereof, wherein the prenyl transferase has an amino acid sequence as shown in any one of SEQ ID nos. 1 to 8.

2. The use of item 1, wherein the phytocannabinoid or analog thereof is cannabigerol CBG or cannabigerol CBGA.

3. According to the use of item 2, when the phytocannabinoid or analog thereof is CBG, the prenyltransferase used has the amino acid sequence shown in any one of SEQ ID NO. 1-5; when the phytocannabinoid or analog thereof is CBGA, the prenyltransferase used has the amino acid sequence shown in any one of SEQ ID NO. 6-8;

optionally, the prenyltransferase may be synthetic or obtained via expression by a prokaryote (e.g., E.coli);

optionally, the prenyl transferase may catalyze the synthesis of phytocannabinoids or analogs thereof via in vitro catalysis, intracellular catalysis, or enzyme immobilization reactions.

4. According to the use of item 2, when the phytocannabinoid or analog thereof is CBG, the substrate used is oleuropein and GPP; when the phytocannabinoid or analog thereof is CBGA, the substrates used are 2, 4-dihydroxy-6-pentylbenzoic acid and GPP.

5. A process for the preparation of a phytocannabinoid or an analog thereof, characterized in that a substrate is subjected to a catalytic reaction using an isopentenyl transferase having an amino acid sequence as set forth in any one of SEQ ID nos. 1 to 8;

preferably, the phytocannabinoid or analog thereof is CBG or CBGA.

6. The production method according to item 5, wherein when the phytocannabinoid or the analog thereof is CBG, the prenyltransferase used has the amino acid sequence shown in any one of SEQ ID NO. 1-5; when the phytocannabinoid or analog thereof is CBGA, the prenyltransferase used has the amino acid sequence shown in any one of SEQ ID NO. 6-8.

7. The production method according to item 5, wherein the prenyltransferase further comprises a signal peptide; optionally, the signal peptide includes, but is not limited to, a signal peptide for purification, localization, and solubilization functions.

8. The production method according to item 5, wherein the amount of prenyltransferase in the catalytic reaction is 0.2 to 8ng/uL (preferably 0.5 to 8ng/uL, more preferably 1 to 3 ng/uL).

9. The production process according to item 5, wherein the pH in the catalytic reaction is 6 to 10, preferably 7 to 9.

10. The production process according to item 5, wherein, in the catalytic reaction, the reaction temperature is 18 to 60 ℃ (preferably 18 to 40 ℃, more preferably 20 to 30 ℃) and the reaction time is 4 to 24 hours (preferably 8 to 16 hours).

11. The production method according to item 5, wherein when the phytocannabinoid or analog thereof is CBG, the substrate used is oleuropein and GPP, wherein the final concentration of oleuropein is 1-10mM, preferably 5-7mM; the final concentration of GPP is 0.5-8mM, preferably 1-5mM.

12. According to the production method of item 5, when the phytocannabinoid or analog thereof is CBGA, the substrates used are 2, 4-dihydroxy-6-pentylbenzoic acid and GPP, wherein the final concentration of 2, 4-dihydroxy-6-pentylbenzoic acid is 1 to 10mM, preferably 5 to 7mM; the final concentration of GPP is 0.5-8mM, preferably 1-5mM.

The invention has the technical effects that:

the NPHB obtained by screening can be used for efficiently synthesizing CBG and CBGA, wherein compared with NPHB used in the prior art, namely SEQ ID NO:9, the catalytic efficiency of the sequence obtained by screening is improved by 8-28 times for CBG, and/or the catalytic efficiency is improved by 18-28 times for CBGA. The improvement of the catalytic activity can greatly reduce the production cost for producing the CBG and the CBGA, provide a data base for promoting the industrial production of the CBG and the CBGA, accelerate the pharmaceutical development of the CBG and the CBGA and meet the health requirements of people.

Drawings

FIG. 1 shows a plasmid map of the NPHB gene constructed in example 1 of the invention.

FIG. 2 shows SDS-PAGE gel of protein purification of SEQ ID NO. 1. Wherein, lane 1, bacterial liquid broken sediment; 2, crushing the supernatant; 3, protein marker;4, 10mM imidazole eluent; 5, 20mM imidazole eluent; 6, 50mM imidazole eluent; 7,100 mM imidazole eluent; 8,200 mM imidazole eluent; 9,300 mM imidazole eluent.

FIG. 3 shows SDS-PAGE gel of protein purification of SEQ ID NO. 6. Wherein, lane 1, bacterial liquid broken sediment; 2, crushing the supernatant; 3, 10mM imidazole eluent; 4, 20mM imidazole eluent; 5, 50mM imidazole eluent; 6,100 mM imidazole eluent; 7, protein marker;8,200 mM imidazole eluent; 9,300 mM imidazole eluent.

FIG. 4 shows the HPLC high performance liquid chromatograms of the standard for CBG and the catalytic synthesis product using SEQ ID NO: 1. Wherein FIG. 4A is a chromatogram of a CBG standard and FIG. 4B is a chromatogram of a CBG product synthesized catalytically using SEQ ID NO: 1.

FIG. 5 shows the results of comparison of the efficiency of catalytic production of CBG with wild-type sequences.

FIG. 6 shows the HPLC high performance liquid chromatograms of CBGA standards and products synthesized catalytically using SEQ ID NO: 6. Wherein A is the chromatogram of the CBGA standard substance, and B is the chromatogram of the CBGA product synthesized by using the catalysis of SEQ ID NO: 6.

FIG. 7 shows the results of comparison of the efficiency of catalytic production of CBGA by different sequences with wild-type sequences.

Detailed Description

The invention is further illustrated in the following examples for the purpose of making the objects, technical solutions and advantages of the invention more apparent, but without limiting the scope of the invention. Some molecular cloning method details vary from reagent, enzyme or kit provider to kit provider, and should be performed according to product instructions, and will not be described in detail in the examples.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The Valliere (2019) literature discloses that NPHB having the sequence SEQ ID NO:9 may be capable of catalyzing the synthesis of CBGA. Because CBGA and CBG are similar in structure, the inventor largely guesses whether some NPHB can have the function of catalyzing CBG or not, and screening and verification are carried out on a large number of NPHB, so that the NPHB with the sequences of SEQ ID NO. 1-SEQ ID NO.5 can catalyze and synthesize CBG efficiently, and the NPHB with the sequences of SEQ ID NO. 6-SEQ ID NO. 8 can catalyze and synthesize CBGA efficiently.

Example 1 purification of NPHB enzyme

1. Construction of expression plasmids

The coding sequence of a gene of interest (e.g., SEQ ID NO:1 from Streptomyces sp.S4.7 species) was codon optimized according to the codon preference of E.coli, then ligated to the pET-28a plasmid (available from Beijing Soy Biotechnology Co., ltd.) according to FIG. 1, transformed into E.coli competence (available from Nannuno-Zan Biotechnology Co., ltd.) and plated by streaking after 8-12 hours of cultivation to prepare a monoclonal strain of pET-28 a-NphB.

TABLE 1 amino acid similarity comparison of sequences involved in the present invention (wherein SEQ ID NO:9 is a wild-type control sequence)

2. Expression and purification of proteins:

preparing a protein purification buffer solution: 50mM Tris,150mM NaCl,PH =8

a. The monoclonal strain of pET-28a-NphB or the strain stored at-80℃was selected and inoculated into a small tube containing 5mL of LB liquid medium (Kan+, 100. Mu.g/mL), and cultured overnight at 37℃and 220rpm as a seed solution.

b. The seed solution was transferred to 50mL of LB liquid medium (Kan+, 100. Mu.g/mL), and the mixture was again subjected to shaking culture at 37℃and 220 rpm.

c. The re-activated bacterial liquid is transferred into 800mL 2YT liquid culture medium (Kan+, 100 mug/mL) according to the inoculation amount of 1 percent, and the temperature is 37 ℃ and 220rpm, and the shaking culture is carried out until the OD 600 is about 0.6-0.8.

d. Reducing the temperature of the shaking table to 16-18 ℃, adding isopropyl thio-beta-D-galactoside (IPTG) to a final concentration of 0.5mM after the temperature of the bacterial liquid to be cultured is reduced, and inducing the expression for 14-16h.

e. After the expression was completed, the above-mentioned culture broth was collected in a bottle, and the centrifuge was pre-cooled to 4℃at 5500rpm and centrifuged for 10min.

f. The supernatant was removed, 30mL of protein purification buffer was added, and the cells were resuspended with a vortex.

g. The resuspended cells were centrifuged again at 5500rpm for 10min. The supernatant was decanted, 30mL of protein purification buffer was added, the cells were resuspended (solid particles were not available) with a vortex, and poured into a 50mL centrifuge tube and stored at-80℃in a refrigerator.

3. Protein purification

a. And (3) breaking bacteria: and (3) carrying out bacteria breaking on the collected bacterial liquid by adopting a high-pressure low-temperature breaker under the conditions of the pressure of 800-1000bar and the temperature of 4 ℃ for 3-5min to fully lyse cells, thereby releasing and dissolving the expressed target protein in a protein buffer solution.

b. And (3) centrifuging: centrifuging the crushed bacterial liquid in a centrifugal machine with precooled temperature of 4 ℃ at 8000rpm for 60min, taking the sediment and supernatant after centrifugation, preparing a sample, and collecting the supernatant;

c. purifying: the supernatant is purified by nickel affinity chromatography, and the specific steps are as follows:

(1) Column balance: first using dd H ₂ 2 column volumes of the Ni affinity chromatography column (purchased from GE Healthcare) were O-washed, and 1 column volume of the Ni affinity chromatography column was equilibrated with the protein buffer.

(2) Loading: after 50ul of the supernatant was taken, it was slowly passed through a Ni affinity column, run through (which may be repeated once) and the first few run through samples were taken.

(3) Eluting the target protein: bound hybrid proteins were eluted with 30mL of protein buffer containing 20mM, 50mM, 100mM, 200mM and 300mM imidazole, respectively, the first few samples were run through, and were prepared, and the results were shown in FIG. 2 (protein SDS-PAGE of other sequences is not shown, as exemplified by SEQ ID NO: 1), wherein a large amount of protein was expressed in 33KD in the bacterial liquid-disrupted precipitate in lane 1, indicating that most of the proteins expressed by E.coli were probably caused by the limited adsorption capacity of the nickel column in the bacterial liquid-disrupted precipitate; lane 2, broken supernatant, also showed a dense band around 33KD, indicating that the broken bacterial fluid supernatant also contained a large amount of the protein of interest; lane 3, protein marker, we refer to a position intermediate 26KD and 35KD in protein marker size; lane 4, 10mM imidazole eluate, where a small amount of protein of interest, a portion of the hybrid protein, indicates that low concentrations of imidazole can elute a portion of the hybrid protein and a small amount of the protein of interest that is not adsorbed to the nickel column; lane 5, 20mM imidazole eluent, eluting out a large amount of hetero-proteins and part of the target proteins; lane 6, 50mM imidazole eluent, eluting off a small amount of protein of interest; both lane 7 (100 mM imidazole eluent) and lane 8 (200 mM imidazole eluent) eluted a large amount of the target protein that bound well to the nickel column, and a thick band appeared around 33 KD; lane 9, 300mM imidazole eluent, a small amount of the protein of interest was eluted, indicating that most of the protein of interest had been eluted by 100mM and 200mM imidazole eluent.

From the SDS-PAGE gel diagram of the protein purification of SEQ ID NO.1 shown in FIG. 2, the protein of SEQ ID NO.1 was successfully expressed, and the same analysis of FIG. 3 shows the SDS-PAGE gel diagram of the protein purification of SEQ ID NO.6, the protein of SEQ ID NO.6 was successfully expressed.

d. Concentrating and changing liquid: the collected protein eluate containing the target protein was concentrated by centrifugation (4 ℃ C., 3400 r/min) using a 50mL Amicon ultrafiltration tube (10 kDa, millipore Co.) to 1mL. Then, 10mL of protein buffer was added, and the mixture was concentrated to 1mL, and the procedure was repeated 1 time to ensure removal of imidazole from the protein, thereby obtaining purified protein NPHB.

4. Protein concentration determination

Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

The initial measurement of protein concentration is first performed by using the absorbance of protein at 280nm, and then the protein concentration is diluted to 0.5-1mg/mL according to the initial measurement value. The reaction solution was prepared by mixing the reagent A and the reagent B in a ratio of 50:1 in BCA Protein Assay Kit. 200uL of reaction solution is placed in an ELISA plate, 25uL of diluted protein is added into the reaction solution, and the mixture is blown and sucked by a gun, and then the mixture is placed at 37 ℃ for reaction for 30min. And (3) placing the ELISA plate into an ELISA instrument to measure the light absorption value at 562nm, and performing data processing according to a protein standard curve to obtain the protein concentration.

The procedure for the determination of the protein concentration can be found in particular in Valliere M A, korman T P, woodall N B, et al A cell-free platform for the prenylation of natural products and application to cannabinoid production [ J ]. Nature communications,2019,10 (1): 1-9.

This example exemplifies the preparation of a purified NPHB comprising the amino acid sequence shown in SEQ ID NO.1 by using the same procedure as the preparation and purification of NPHB comprising the amino acid sequence shown in SEQ ID NO. 1.

EXAMPLE 2 enzymatic Synthesis of CBG

The NPHB enzymes obtained containing any of the amino acid sequences of SEQ ID NO:1-5 were screened as described in example 1 and catalytic synthesis of CBG was performed in vitro using olivine and GPP (i.e., geranyl pyrophosphate) as substrates according to the following reaction scheme and reaction conditions.

In vitro enzyme catalytic reaction conditions:

buffer solution for reaction: 50mM Tris-HCl, pH=8.0

MgCl ₂ Final concentration of 5mM

GPP final concentration of 2.5mM

The final concentration of olivetol is: 5mM

Amount of NPHB protease: 10ng, 20ng, 50ng, 70ng or 100ng (i.e., NPHB protease is added in an amount of 0.2ng/uL, 0.4ng/uL, 1ng/uL, 1.4ng/uL, 2 ng/uL)

Reaction temperature: 24 ℃, 30 ℃, 40 ℃, 50 ℃ or 60 DEG C

Reaction time: 12h

The total reaction system was 50uL

After 2 times extraction with ethyl acetate, the solution was evaporated to dryness by vacuum rotary evaporator and finally dissolved with 50uL of methanol.

EXAMPLE 3 detection of CBG by HPLC high Performance liquid chromatography

The reaction solution obtained after the catalytic reaction in example 2 was used for detecting CBG therein by HPLC high performance liquid chromatography under the following conditions:

mobile phase: A. water (0.1% formic acid) B. Acetonitrile (0.1% formic acid)

C18 liquid column (from Watersh technologies (Shanghai))

Temperature: 35 DEG C

Flow rate: 1ml/min

Sample injection amount: 10uL

HPLC high performance liquid chromatography procedure:

time (min)	Mobile phase a (%)	Mobile phase B (%)
			6	30	70
12	23	77
			22	23	77
22.2	30	70
			26	30	70

As shown in FIG. 4, it can be seen from FIG. 4 that CBG was successfully synthesized by the method of example 1-2 using NPHB enzyme having the amino acid sequence of SEQ ID NO.1, as exemplified by the NPHB enzyme of SEQ ID NO.1, whose result of catalytic synthesis of CBG was examined by HPLC high performance liquid chromatography.

The same conclusion was reached by examining the catalytic reaction products of NPHB enzymes having the sequences of SEQ ID NO. 2-5, respectively, according to the same method, i.e.NPHB enzymes having the sequences of SEQ ID NO. 2-5 also successfully catalyze the synthesis of CBG from olive alcohol and GPP. As can be seen from the above, NPHB having the sequences of SEQ ID NO. 1-SEQ ID NO.5 is capable of catalyzing the synthesis of CBG.

Example 4 determination of catalytic efficiency of NPHB

Using the purified protein and the measured protein concentration in example 1, an enzyme-catalyzed reaction was performed in accordance with the procedure of example 2, and CBG detection was performed in accordance with the procedure of example 3. The catalytic efficiency of the enzyme was characterized by calculating the amount of catalytic CBG per unit mass per unit time of the enzyme based on the nmol amount of CBG produced by the enzyme catalytic reaction 5 minutes before the catalytic reaction divided by each minute and divided by each mg of protein.

The catalytic activity of the NPHB enzymes of SEQ ID NOS 1-5 claimed in the present invention in the catalytic synthesis of CBG was measured with reference to the wild type SEQ ID NO 9 reported in the literature. FIG. 5 shows the improvement in catalytic activity of the catalytic synthesis CBG of NPHB compared with SEQ ID NO. 9 in the examples of the invention. As can be seen from FIG. 5, the NPHB enzymes comprising any of the amino acid sequences of SEQ ID NO:1-5 as claimed in the invention can be used for the efficient catalytic synthesis of CBG in comparison with the prior art prenyltransferase SEQ ID NO: 9.

EXAMPLE 5 enzymatic Synthesis of CBGA

NPHB enzymes containing any of the amino acid sequences of SEQ ID NO:6-8 were obtained by screening as described in example 1 and catalytically synthesizing CBGA in vitro using 2, 4-dihydroxy-6-pentylbenzoic acid and GPP as substrates according to the following reaction scheme and reaction conditions described in example 2.

The conditions for the in vitro enzyme-catalyzed reaction are as follows:

buffer solution for reaction: 50mM Tris-HCl, pH=8.0

MgCl ₂ Final concentration of 5mM

GPP final concentration of 2.5mM

The final concentration of 2, 4-dihydroxy-6-pentylbenzoic acid is: 5mM

Reaction temperature: 24 ℃, 30 ℃, 40 ℃, 50 ℃ or 60 DEG C

Reaction time: 12h

The total reaction system was 50uL

CBGA was then assayed as described in example 3 and the catalytic activity of NPHB comprising any of the amino acid sequences of SEQ ID NO:6-8 for the synthesis of CBGA was determined as described in example 4.

As a result, it was revealed from FIG. 6 that NPHB comprising the sequence of SEQ ID NO.6 is capable of catalyzing the synthesis of CBGA, and the same conclusion was reached by examining the catalytic reaction products of NPHB enzymes each comprising the sequence of SEQ ID NO. 7-8 according to the same method, that is, the NPHB enzymes comprising the sequence of SEQ ID NO. 7-8 can also successfully synthesize CBGA.

The catalytic activity of the NPHB enzymes of the invention, comprising the sequences of SEQ ID NOS.6-8, respectively, was measured in the catalytic synthesis of CBGA, as reported in the literature, with reference to wild type SEQ ID NO. 9. FIG. 7 shows the fold increase in catalytic activity of the catalytic synthesis CBGA of NPHB compared to SEQ ID NO. 9 in the examples of this invention. As can be seen from FIG. 7, the NPHB enzymes comprising the sequence of SEQ ID NO:6-8 claimed in the invention can be used for efficient catalytic synthesis of CBGA as compared to the prior art prenyltransferase SEQ ID NO: 9.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims

1. Use of an prenyl transferase in the catalytic synthesis of cannabigerol CBG, wherein the prenyl transferase has the amino acid sequence shown in SEQ ID No.5, and the substrate used is oleyl alcohol and GPP.

2. The use according to claim 1, wherein the prenyltransferase is synthetic or obtained via prokaryotic expression.

3. The use according to claim 2, wherein the prokaryote is e.

4. The use according to claim 1, wherein the prenyltransferase synthesizes cannabigerol CBG via in vitro or intracellular catalysis.

5. The use according to claim 1, wherein the prenyltransferase catalyzes the synthesis of cannabigerol CBG via an enzyme immobilization reaction.

6. A process for the preparation of phytocannabinoids or analogues thereof, characterized in that a substrate is subjected to a catalytic reaction using an prenyl transferase, wherein the prenyl transferase has the amino acid sequence shown in SEQ ID No.5 and the substrate used is oleyl alcohol and GPP;

wherein the phytocannabinoid or analog thereof is CBG.

7. The method of claim 6, wherein the prenyltransferase is further linked to a signal peptide.

8. The method of claim 7, wherein the signal peptide includes, but is not limited to, a signal peptide for purification, localization, and solubilization-aiding functions.

9. The process according to claim 6, wherein the amount of prenyltransferase in the catalytic reaction is 0.2 to 8ng/uL.

10. The preparation method according to claim 9, wherein the amount of prenyltransferase in the catalytic reaction is 0.5-8ng/uL.

11. The production method according to claim 9, wherein the amount of the prenyltransferase in the catalytic reaction is 1 to 3ng/uL.

12. The process according to claim 6, wherein the pH in the catalytic reaction is from 6 to 10.

13. The preparation method according to claim 12, wherein the pH in the catalytic reaction is 7 to 9.

14. The preparation method according to claim 6, wherein in the catalytic reaction, the reaction temperature is 18-60℃and the reaction time is 4-24 hours.

15. The production process according to claim 14, wherein, in the catalytic reaction, the reaction temperature is 18 to 40 ℃.

16. The production method according to claim 14, wherein, in the catalytic reaction, the reaction temperature is 20 to 30 ℃.

17. The preparation method according to claim 14, wherein in the catalytic reaction, the reaction time is 8 to 16 hours.

18. The method of claim 6, wherein the final concentration of olivetol is 1-10mM; the final concentration of GPP is 0.5-8mM.

19. The method of claim 18, wherein the final concentration of olivetol is 5-7mM; the final concentration of GPP is 1-5mM.