CN113584089A - Application of prenyl transferase in catalytic synthesis of cannabigerol or cannabigerol acid - Google Patents

Application of prenyl transferase in catalytic synthesis of cannabigerol or cannabigerol acid Download PDF

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CN113584089A
CN113584089A CN202110749746.2A CN202110749746A CN113584089A CN 113584089 A CN113584089 A CN 113584089A CN 202110749746 A CN202110749746 A CN 202110749746A CN 113584089 A CN113584089 A CN 113584089A
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CN113584089B (en
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陈贤情
杨月
夏文豪
李珍珠
王筱
逯晓云
刘诗梦
江会锋
王文
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Abstract

The invention provides the use of an isopentenyl transferase enzyme in the catalytic synthesis of a phytocannabinoid or an analogue thereof, wherein the isopentenyl transferase enzyme has an amino acid sequence as set forth in any one of SEQ ID nos. 1-8. The present invention also provides a method for preparing phytocannabinoids or analogues thereof, using prenyltransferase to catalyse a substrate; wherein said prenyltransferase has the amino acid sequence shown in any of SEQ ID Nos. 1-8, and said 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 prenyl transferase in catalytic synthesis of cannabigerol or cannabigerol acid
Technical Field
The invention relates to the field of chemical synthesis, in particular to application of prenyltransferase in-vitro high-efficiency catalytic synthesis of phytocannabinoids or analogues thereof (such as cannabigerol or cannabigerolic acid).
Background
Cbg (cannabibergenol), cannabigerol, is one of cannabinoids, has a very high medical value, has great potential in reducing intraocular pressure in 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, assisting sleep and mood regulation, improving immunity and the like, but is mainly detected in industrial cannabis at present, has a very low content and does not have extraction value.
The main source of CBG still relies on traditional plant extraction at present, and its step is loaded down with trivial details, uses a large amount of organic reagent simultaneously, and the reaction process has strong acid, strong alkali and high temperature high pressure environment, and the environment is not friendly, and is inefficient simultaneously. While chemical synthesis of CBG once becomes the eosin of the market, the chemical synthesis process of CBG also involves the use of a plurality of strong acids and catalysts, so that the problems of more byproducts, difficult separation, low production efficiency and the like are solved. At present, in addition to the traditional chemical extraction, a recently emerging production method is a production approach of utilizing synthetic biology, searching and synthesizing key genes by genome assembly annotation and gene mining of industrial cannabis sativa, introducing target genes into saccharomyces cerevisiae and reconstructing CBGA in the saccharomyces cerevisiae. And further heating to produce CBG. Compared with chemical synthesis, the biosynthesis method has the advantages of mild reaction conditions, single product, easiness in separation and the like.
Prenyltransferases (NPHB) catalyze the transfer of isopentenyl structures to another compound, leaving two diphosphate groups, NPHB perhaps being capable of catalyzing olivetol and GPP to CBG, or 2, 4-dihydroxy-6-pentylbenzoic acid and GPP to CBGA.
The currently reported NPHB catalyzing and synthesizing CBGA is AltPT which is derived from CL190 sequence of Streptomyces coelicolor, but the catalytic synthesis efficiency of CBGA is extremely low.
The amino acid sequence of AltPT is:
MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLEAESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQRGLLKAFDSLED(SEQ ID NO:9)。
the method for synthesizing CBG and CBGA by NPHB in vitro catalysis is simple and efficient, so that more NPHB capable of synthesizing CBG and CBGA in vitro catalysis is urgently needed to be developed. The NPHB capable of efficiently catalyzing and synthesizing the CBGA and/or the CBG is obtained through screening, has the amino acid sequence shown in any one of SEQ ID NO 1-8, on one hand, the NPHB can directly catalyze and synthesize the CBG, and compared with the traditional approach, the steps are simpler and faster; on the other hand, the catalytic efficiency is higher in the process of catalytically synthesizing CBGA.
Disclosure of Invention
The object of the present invention is to provide a method for the in vitro catalytic synthesis of phytocannabinoids or analogues thereof (e.g. CBG and CBGA) in a simple and efficient manner. Specifically, the inventors analyzed the amino acid skeleton and conserved region of the sequence SEQ ID NO. 9 reported in the prior art, analyzed the CBGA and CBG structures by combining the activation energy of substrate docking and protein homeostasis, and selected multiple optimal sequences from 1000 sequences for verification, in hope of finding genes capable of better catalyzing CBG and/or CBGA. Finally, NPHB capable of efficiently catalyzing and synthesizing CBGA and/or CBG is obtained, and has an amino acid sequence shown in any one of SEQ ID NO. 1-8.
NPHB obtained by screening the invention is connected to plasmid, and is massively expressed and purified in bacteria, and then CBG is synthesized by catalysis in vitro by using olive alcohol and GPP as substrates, or CBGA is synthesized by catalysis by using 2, 4-dihydroxy-6-pentylbenzoic acid and GPP as substrates. The invention has the advantages of less steps in the process of catalytically producing the CBG and the CBGA, convenient operation, shortened production path, improved yield and reduced production cost.
Specifically, the present invention provides the following scheme:
1. use of an prenyltransferase enzyme having an amino acid sequence as set forth in any of SEQ ID nos. 1-8 in the catalytic synthesis of a phytocannabinoid or an analog thereof.
2. The use according to clause 1, wherein the phytocannabinoid or analog thereof is cannabigerol CBG or cannabigerolic acid CBGA.
3. The use according to item 2, wherein when the phytocannabinoid or analog thereof is CBG, the prenyltransferase used has an amino acid sequence as set forth in any one of SEQ ID No. 1-5; when the phytocannabinoid or analog thereof is CBGA, the prenyltransferase used has an amino acid sequence as set forth in any of SEQ ID Nos. 6-8;
optionally, the prenyltransferase can be artificially synthesized or obtained via prokaryotic (e.g., e.coli) expression;
optionally, the prenyltransferase can catalyze the synthesis of phytocannabinoids or analogues thereof via in vitro catalysis, intracellular catalysis or enzyme immobilization reactions.
4. The use according to item 2, wherein when the phytocannabinoid or analog thereof is CBG, the substrate used is olivetol 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 analogue thereof comprising catalytically reacting a substrate with an isopentenyl transferase, wherein said isopentenyl transferase has an amino acid sequence as set forth in any one of SEQ ID nos. 1-8;
preferably, the phytocannabinoid or analog thereof is CBG or CBGA.
6. The production method according to item 5, wherein when the phytocannabinoid or an analog thereof is CBG, the prenyltransferase used has an amino acid sequence represented by any one of SEQ ID Nos. 1 to 5; when the phytocannabinoid or analog thereof is CBGA, the prenyltransferase used has an amino acid sequence as set forth in any of SEQ ID Nos. 6-8.
7. The method of claim 5, wherein the prenyltransferase further comprises a signal peptide; optionally, the signal peptide includes, but is not limited to, signal peptides for purification, localization, and solubilizing functions.
8. The process according to item 5, wherein the amount of isopentenyl transferase used 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 method according to item 5, wherein the pH in the catalytic reaction is from 6 to 10, preferably from 7 to 9.
10. The 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 preparation process according to item 5, wherein when the phytocannabinoid or an analogue thereof is CBG, the substrate used is olive alcohol and GPP, wherein the final concentration of olive alcohol is 1 to 10mM, preferably 5 to 7 mM; the final concentration of GPP is 0.5-8mM, preferably 1-5 mM.
12. The preparation process according to item 5, wherein when the phytocannabinoid or an 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 7 mM; the final concentration of GPP is 0.5-8mM, preferably 1-5 mM.
The invention has the technical effects that:
compared with NPHB (SEQ ID NO: 9) used in the prior art, the NPHB obtained by screening can be used for efficiently synthesizing CBG and CBGA, and the catalytic efficiency of the sequence obtained by screening is improved by 8-28 times aiming at the CBG and/or 18-28 times aiming at the CBGA. The improvement of the catalytic activity can greatly reduce the production cost of CBG and CBGA, provide a data base for promoting the industrial production of CBG and CBGA, accelerate the medicinal research and development of CBG and CBGA, and meet the health requirements of people.
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FIG. 1 shows a plasmid map of the NPHB gene constructed in example 1 of the present invention.
FIG. 2 shows the SDS-PAGE gel of protein purification of SEQ ID NO. 1. Wherein, lane 1, the broken pellet of the bacterial liquid; 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 the SDS-PAGE gel of protein purification of SEQ ID NO 6. Wherein, lane 1, the broken pellet of the bacterial liquid; 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 a standard of CBG and HPLC high performance liquid chromatogram of the product of catalytic synthesis using SEQ ID NO: 1. Wherein, FIG. 4A is a chromatogram of CBG standard, and FIG. 4B is a chromatogram of catalytic synthesis product CBG using SEQ ID NO. 1.
FIG. 5 shows the efficiency of catalytic CBG production by different sequences compared to the wild sequence.
FIG. 6 shows HPLC high performance liquid chromatograms of CBGA standards and products synthesized using the catalysis of SEQ ID NO 6. Wherein A is the chromatogram of CBGA standard, and B is the chromatogram of CBGA synthesized by using the catalysis of SEQ ID NO. 6.
FIG. 7 shows the comparison of the efficiency of catalytic CBGA production by different sequences with the wild sequence.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further illustrated in the following examples, but not limited to the scope of the present invention. The details of the partial molecular cloning method vary depending on the reagents, enzymes or kits provided by the supplier, and should be conducted according to the product instructions, and will not be described in detail in the examples.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The Valliere (2019) literature discloses that NPHB with the sequence of SEQ ID NO:9 may be capable of catalyzing the synthesis of CBGA. Because CBGA and CBG are similar in structure, the inventor conjectures that whether certain NPHB can have the function of catalyzing CBG, so that a great amount of NPHB is screened and verified, and the NPHB with the sequences of SEQ ID NO. 1-SEQ ID NO. 5 can be used for efficiently catalyzing and synthesizing CBG, and the SEQ ID NO. 6-SEQ ID NO. 8 can be used for efficiently catalyzing and synthesizing CBGA.
Example 1 purification of NPHB enzyme
1. Construction of expression plasmids
A coding sequence of a target gene (for example, SEQ ID NO:1 derived from Streptomyces sp. S4.7) is codon-optimized according to codon preference of Escherichia coli (E.coli), and then ligated to a pET-28a plasmid (purchased from Sorbombo Biotech, Inc., Beijing) according to FIG. 1, transformed into E.coli competence (purchased from Nanjing Nozaki Biotech, Ltd.), cultured for 8-12h, streaked and plated, and a monoclonal strain of pET-28a-NphB is prepared.
TABLE 1 comparison of amino acid similarity of sequences involved in the present invention (where SEQ ID NO:9 is the wild type control sequence)
Figure BDA0003144080500000061
2. Expression and purification of protein:
preparing a protein purification buffer solution: 50mM Tris, 150mM NaCl, pH 8
a. A monoclonal strain of pET-28a-NphB or a strain stored at-80 ℃ thereof was selected and inoculated into a small tube containing 5mL of LB liquid medium (Kan +, 100. mu.g/mL) and cultured overnight at 220rpm at 37 ℃ to prepare a seed solution.
b. The seed solution was transferred to 50mL of LB liquid medium (Kan +, 100. mu.g/mL), incubated at 37 ℃ and 220rpm, and shaken and then activated again.
c. The reactivated bacterial suspension was transferred to 800mL of 2YT broth (Kan +, 100. mu.g/mL) at 1% inoculum size, and shake-cultured at 37 ℃ and 220rpm until OD 600 was about 0.6-0.8.
d. The temperature of the shaking table is reduced to 16-18 ℃, after the temperature of the cultured bacterial liquid is reduced, isopropyl thio-beta-D-galactoside (IPTG) is added to the final concentration of 0.5mM, and the induction expression is carried out for 14-16 h.
e. After the expression is completed, the above culture solution is collected into a bottle, and the centrifuge is pre-cooled to 4 ℃, 5500rpm, and centrifuged for 10 min.
f. The supernatant was removed, 30mL of protein purification buffer was added, and the cells were resuspended in a vortex shaker.
g. The resuspended cells were again centrifuged at 5500rpm for 10 min. The supernatant was decanted, 30mL of protein purification buffer was added, the cells were resuspended (no solid particles were present) using a vortex shaker, and poured into a 50mL centrifuge tube and stored in a freezer at-80 ℃.
3. Protein purification
a. Breaking the bacteria: and (3) breaking the collected bacterial liquid for 3-5min by adopting a high-pressure low-temperature breaker under the conditions of the pressure of 800-.
b. Centrifuging: centrifuging the crushed bacterial liquid in a precooled 4 ℃ centrifuge at 8000rpm for 60min, taking the centrifuged precipitate and supernatant, preparing a sample, and collecting the supernatant;
c. and (3) purification: and (3) carrying out nickel affinity chromatography purification on the supernatant, and specifically comprising the following steps:
(1) column balancing: first using dd H2The Ni affinity column (ex GE Healthcare) was washed 2 column volumes with O and then equilibrated 1 column volume with protein buffer.
(2) Loading: the supernatant was sampled 50ul, then slowly passed through a Ni affinity column, run through (repeated once) and a few drops were taken through the sample.
(3) Eluting the target protein: using 30mL of protein buffer solutions containing 20mM, 50mM, 100mM, 200mM and 300mM imidazole to elute the bound impure proteins, taking several samples to run through, sampling, detecting by 12% SDS-PAGE, and the results are shown in fig. 2 (taking SEQ ID NO:1 as an example, the SDS-PAGE of the proteins with other sequences is not shown), wherein, lane 1, the pellet broken by the bacterial solution has a large amount of protein expression in 33KD, which indicates that most of the proteins expressed by escherichia coli in the pellet broken by the bacterial solution is probably caused by the limited adsorption capacity of the nickel column; lane 2, the supernatant after disruption also showed a heavy band around 33KD, indicating that the supernatant of the disrupted bacterial liquid also contains a large amount of the target protein; lane 3, protein marker, which we refer to as a position intermediate 26KD and 35KD in size; lane 4, 10mM imidazole eluate, in which a small amount of the protein of interest, partially impure protein, was eluted, indicating that a low concentration of imidazole can elute a portion of the impure protein and a small amount of the protein of interest that was not adsorbed to the nickel column; lane 5, 20mM imidazole eluate, eluting large amounts of contaminating proteins and a portion of the protein of interest; lane 6, 50mM imidazole eluate, eluting a small amount of the protein of interest; lanes 7(100mM imidazole eluate) and 8(200mM imidazole eluate), both eluted large amounts of target protein that bound well to the nickel column and showed thick bands around 33 kD; lane 9, 300mM imidazole eluate, eluted a small amount of the protein of interest, indicating that most of the protein of interest had been eluted by the 100mM and 200mM imidazole eluate.
From the SDS-PAGE gel of the protein purification of SEQ ID NO.1 shown in FIG. 2, it was found that the protein of SEQ ID NO.1 was successfully expressed by analysis, and from the SDS-PAGE gel of the protein purification of SEQ ID NO.6 shown in FIG. 3, the protein of SEQ ID NO.6 was successfully expressed by analysis.
d. Concentrating and replacing liquid: the protein eluate containing the target protein was concentrated by centrifugation (4 ℃ C., 3400r/min) using a 50mL Amicon ultrafiltration tube (10kDa, Millipore Co.) to 1 mL. 10mL of protein buffer was added and concentrated to 1mL, and the process was repeated 1 time to ensure removal of imidazole from the protein, resulting in the purified protein NPHB.
4. Protein concentration determination
Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
The initial measurement of protein concentration is first carried out with the protein having an absorbance at 280nm, and then the protein concentration is diluted to 0.5-1mg/mL according to the initial measurement value. Reagent A and reagent B in the BCA Protein Assay Kit are prepared according to the proportion of 50:1 to be used as reaction liquid. And (3) placing 200uL of reaction liquid in an enzyme label plate, adding 25uL of diluted protein into the reaction liquid, uniformly blowing and sucking by using a gun, placing at 37 ℃ and reacting for 30 min. And (3) placing the ELISA plate into an ELISA reader to measure the 562nm light absorption value, and performing data processing according to a protein standard curve to obtain the protein concentration.
The procedures for determining 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 expression of natural products and applications to biological products [ J ]. Nature communications,2019,10(1): 1-9.
This example illustrates the preparation of purified NPHB containing the amino acid sequence shown in SEQ ID NO.1, using NPHB containing the amino acid sequence shown in SEQ ID NO.1 as an example, and the same procedures were used for the preparation and purification of NPHB containing other sequences as claimed in the claims of the invention.
Example 2 enzymatic Synthesis of CBG
NPHB enzymes obtained by screening the obtained NPHB enzymes having an amino acid sequence of any of SEQ ID NOS: 1 to 5 according to the method described in example 1 and catalytically synthesizing CBG in vitro using olivetol and GPP (i.e., geranyl pyrophosphate) as substrates according to the following reaction formula and reaction conditions.
Figure BDA0003144080500000091
In vitro enzyme catalysis reaction conditions:
buffer for reaction: 50mM Tris-HCl, pH 8.0
MgCl2The final concentration was 5mM
GPP final concentration of 2.5mM
The final concentration of the olive alcohol is as follows: 5mM
Amount of NPHB protease: 10ng, 20ng, 50ng, 70ng or 100ng (i.e., the amount of NPHB protease added is 0.2ng/uL, 0.4ng/uL, 1ng/uL, 1.4ng/uL, 2ng/uL)
Reaction temperature: 24 deg.C, 30 deg.C, 40 deg.C, 50 deg.C or 60 deg.C
Reaction time: 12h
The total reaction system is 50uL
After the reaction was extracted 2 times with ethyl acetate, the solution was evaporated to dryness by a vacuum rotary evaporator and finally dissolved in 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 subjected to HPLC high performance liquid chromatography for detecting CBG therein, wherein the conditions of the high performance liquid chromatography were as follows:
mobile phase: A. water (containing 0.1% formic acid) B. acetonitrile (containing 0.1% formic acid)
C18 liquid phase column (from Wo Tech science and technology (Shanghai) Co., Ltd.)
Temperature: 35 deg.C
Flow rate: 1ml/min
Sample introduction amount: 10uL
HPLC 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, the results of the HPLC analysis of the CBG synthesis using the NPHB enzyme of SEQ ID NO.1 as an example show that CBG was successfully synthesized by the method of examples 1 to 2 using the NPHB enzyme having the amino acid sequence of SEQ ID NO.1 as shown in FIG. 4.
The same conclusion can be drawn by detecting the catalytic reaction products of NPHB enzymes with the sequences of SEQ ID NO. 2-5 respectively according to the same method, namely the NPHB enzymes with the sequences of SEQ ID NO. 2-5 can 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 to SEQ ID NO:5 can catalyze the synthesis of CBG.
Example 4 determination of catalytic efficiency of NPHB
Using the purified protein and the measured protein concentration in example 1, the enzyme-catalyzed reaction was performed according to the procedure of example 2, and CBG detection was performed according to the procedure of example 3. The catalytic efficiency of the enzyme is characterized by calculating the amount of catalytic CBG per unit mass of the enzyme per unit time based on the nmol amount of CBG produced by the enzyme-catalyzed reaction 5 minutes before the catalytic reaction, divided by min and divided by mg of protein.
The catalytic activity of the NPHB enzymes of SEQ ID NO.1-5 claimed in the present invention in the catalytic synthesis of CBG was measured with reference to wild-type SEQ ID NO. 9 reported in the literature as a control. FIG. 5 shows the fold increase in catalytic activity of catalytic synthesis of CBG compared to SEQ ID NO 9 for NPHB 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 Nos. 1-5 claimed in the present invention can be used for the high-efficiency catalytic synthesis of CBG, compared to the prenyltransferase of SEQ ID No. 9 of the prior art.
Example 5 enzymatic Synthesis of CBGA
NPHB enzymes having an amino acid sequence of any of SEQ ID NOS 6 to 8 were obtained by screening according to the method described in example 1, and CBGA was synthesized in vitro using 2, 4-dihydroxy-6-pentylbenzoic acid and GPP as substrates under the reaction conditions described in the following reaction scheme and example 2.
The in vitro enzyme catalysis reaction conditions are as follows:
buffer for reaction: 50mM Tris-HCl, pH 8.0
MgCl2The final concentration was 5mM
GPP final concentration of 2.5mM
The final concentration of the 2, 4-dihydroxy-6-pentylbenzoic acid is as follows: 5mM
Amount of NPHB protease: 10ng, 20ng, 50ng, 70ng or 100ng (i.e., the amount of NPHB protease added is 0.2ng/uL, 0.4ng/uL, 1ng/uL, 1.4ng/uL, 2ng/uL)
Reaction temperature: 24 deg.C, 30 deg.C, 40 deg.C, 50 deg.C or 60 deg.C
Reaction time: 12h
The total reaction system is 50uL
Figure BDA0003144080500000111
Then, CBGA was assayed according to the method described in example 3, and NPHB comprising the amino acid sequence of any one of SEQ ID NOS: 6 to 8 was assayed for the catalytic activity of synthesized CBGA by the method described in example 4.
As shown in FIG. 6, it can be seen that NPHB containing the sequence of SEQ ID NO.6 is capable of catalyzing the synthesis of CBGA, and the detection of the catalytic reaction products of NPHB enzymes respectively containing the sequences of SEQ ID NO. 7-8 according to the same method leads to the same conclusion that NPHB enzymes containing the sequences of SEQ ID NO. 7-8 can also successfully synthesize CBGA.
The catalytic activity of the NPHB enzymes comprising the sequences of SEQ ID NOS: 6-8, respectively, as claimed in the present invention in the catalytic synthesis of CBGA was measured with the wild-type SEQ ID NO:9 reported in the literature as a control. FIG. 7 shows the fold increase in catalytic activity of catalytic synthesis of CBGA of NPHB compared to SEQ ID NO 9 in the examples of the invention. As can be seen from FIG. 7, the NPHB enzymes comprising the sequences of SEQ ID NOS: 6-8 as claimed in the present invention can be used for the high-efficiency catalytic synthesis of CBGA, as compared with the prenyltransferase of the prior art of SEQ ID NO: 9.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Figure IDA0003144080560000011
Figure IDA0003144080560000021
Figure IDA0003144080560000031
Figure IDA0003144080560000041
Figure IDA0003144080560000051
Figure IDA0003144080560000061
Figure IDA0003144080560000071
Figure IDA0003144080560000081

Claims (12)

1. Use of an prenyltransferase enzyme having an amino acid sequence as set forth in any of SEQ ID nos. 1-8 in the catalytic synthesis of a phytocannabinoid or an analog thereof.
2. The use as claimed in claim 1, wherein the phytocannabinoid or analog thereof is cannabigerol CBG or cannabigerolic acid CBGA.
3. Use according to claim 2, wherein when the phytocannabinoid or analog thereof is CBG, the prenyltransferase used has an amino acid sequence as set forth in any one of SEQ ID nos. 1-5; when the phytocannabinoid or analog thereof is CBGA, the prenyltransferase used has an amino acid sequence as set forth in any of SEQ ID Nos. 6-8;
optionally, the prenyltransferase can be artificially synthesized or obtained via prokaryotic (e.g., e.coli) expression;
optionally, the prenyltransferase can catalyze the synthesis of phytocannabinoids or analogues thereof via in vitro catalysis, intracellular catalysis or enzyme immobilization reactions.
4. Use according to claim 2, when the phytocannabinoid or analog thereof is CBG, the substrate used is olivetol 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 analogue thereof comprising catalytically reacting a substrate with an isopentenyl transferase, wherein said isopentenyl transferase has an amino acid sequence as set forth in any one of SEQ ID nos. 1-8;
preferably, the phytocannabinoid or analog thereof is CBG or CBGA.
6. The method according to claim 5, wherein when the phytocannabinoid or an analog thereof is CBG, the prenyltransferase used has an amino acid sequence as set forth in any one of SEQ ID NO. 1-5; when the phytocannabinoid or analog thereof is CBGA, the prenyltransferase used has an amino acid sequence as set forth in any of SEQ ID Nos. 6-8.
7. The method of claim 5, wherein said prenyltransferase further comprises a signal peptide; optionally, the signal peptide includes, but is not limited to, signal peptides for purification, localization, and solubilizing functions.
8. The process according to claim 5, wherein the amount of prenyltransferase in the catalytic reaction is 0.2-8ng/uL (preferably 0.5-8ng/uL, more preferably 1-3 ng/uL).
9. The process according to claim 5, wherein the pH in the catalytic reaction is from 6 to 10, preferably from 7 to 9.
10. The preparation method according to claim 5, wherein, in the catalytic reaction, the reaction temperature is 18-60 ℃ (preferably 18-40 ℃, more preferably 20-30 ℃) and the reaction time is 4-24h (preferably 8-16 h).
11. The preparation method according to claim 5, wherein when the phytocannabinoid or analog thereof is CBG, the substrate used is olive alcohol and GPP, wherein the final concentration of olive alcohol is 1-10mM, preferably 5-7 mM; the final concentration of GPP is 0.5-8mM, preferably 1-5 mM.
12. The method according to claim 5, wherein 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 1-10mM, preferably 5-7 mM; the final concentration of GPP is 0.5-8mM, preferably 1-5 mM.
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