Enzymes for cannabinoid synthesis and methods for their preparation and use

Abstract

Provided are various enzymes for cannabinoid synthesis and methods for their preparation and use. Also provided are recombinant polypeptides having prenyltransferase activity and / or geranyl diphosphate:olivetolate geranyltransferase (GOT) activity, and engineered recombinant microorganisms for the production of CBGA, and methods for producing cannabigerolic acid (CBGA) or an analogue thereof, the methods comprising the step of reacting a heterologously expressed prenyltransferase with geranyl diphosphate (GPP) and an acid such as olivetolic acid (OA).

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application Serial No. 62 / 909,227, filed on October 1, 2019, U.S. Provisional Patent Application Serial No. 62 / 941,689, filed on November 27, 2019, and U.S. Provisional Patent Application Serial No. 62 / 942,198, filed on December 1, 2019, which are hereby incorporated by reference in their entirety.

[0003] Reference to a Sequence Listing

[0004] This application contains a sequence listing in computer - readable form, which is hereby incorporated by reference in its entirety. Technical Field

[0005] This application relates to molecular biology and, more particularly, to enzymes for cannabinoid synthesis. Background Art

[0006] Cannabinoid compounds act on other targets of cannabinoid receptors and significantly affect the release of neurotransmitters in the brain. The use of cannabinoid compounds, such as cannabidiol for medical purposes, has expanded globally, and its legislation has become increasingly accepted. Thus, cannabinoid compounds are becoming a new class of drugs. However, there are still many challenges in the production of cannabinoid compounds, which are traditionally extracted and purified from plants. In addition, it is difficult to produce multiple cannabinoid analogs using currently known methods. Therefore, new ways to synthesize and produce cannabinoid compounds are needed. Summary of the Invention

[0007] According to some aspects, this application relates to enzymes that can be used to produce cannabinoids or cannabinoid intermediates in a microbial host or a yeast host, as well as methods for preparing and using such enzymes.

[0008] In certain embodiments, the claimed methods for producing cannabinoids are improvements over currently known methods, such as plant extraction. Extracting cannabinoids from plants involves growing and harvesting plants that naturally contain cannabinoids and then using various extraction methods known in the art to extract the compounds. Since cannabinoids are naturally mixed in plants, it is often difficult to reproduce the same extraction and purification characteristics for each extraction sample. The unique genetic origin and growth conditions of each plant further complicate this. The resulting different cannabinoid profiles lead to each sample having different drug characteristics - a problem from a safety or regulatory perspective. Finally, due to the nature of the mixture of compounds with similar structures and sizes, the ability to purify a high - purity single product is very challenging.

[0009] Accordingly, in certain embodiments, the present application provides enzymes that can be used to produce cannabinoids in a microbial or yeast host. Example embodiments include novel prenyltransferases and methods of preparing and using such enzymes in the production of cannabigerolic acid (CBGA) or its analogs from a prenyl group donor and an acid.

[0010] In certain embodiments, the present application also provides methods of using such enzymes to produce compounds in the cannabinoid pathway, such as cannabigerolic acid (CBGA) or its analogs produced from a prenyl group donor and an acid, by heterologous expression of plant and microbial genes in a microbial host or a yeast host.

[0011] In certain embodiments, the present application provides a method of producing cannabigerolic acid (CBGA) or its analogs, the method comprising the step of reacting a prenyltransferase with a prenyl group donor and an acid.

[0012] In one embodiment, the prenyltransferase has cannabigerolic acid synthase (CBGAS) activity.

[0013] In one embodiment, the prenyltransferase is obtained from a whole, purified cell extract or a combination thereof.

[0014] In one embodiment, the prenyl group donor is selected from the group consisting of prenyl moieties derived from allylic isoprenyl diphosphate (including but not limited to dimethylallyl diphosphate (DMAPP; C5), geranyl diphosphate (GPP; C10), and farnesyl diphosphate (FPP; C15)).

[0015] In one embodiment, the prenyl group donor is GPP.

[0016] In one embodiment, the acid is selected from the group consisting of orsellinic acid (OSA), divarinolic acid (DVA), apigenin, daidzein, genistein, naringenin, olivetol, OA, and resveratrol.

[0017] In one embodiment, the acid is olivetolic acid.

[0018] In one embodiment, the prenyltransferase has an activity with increased synthetic ability and reduced byproduct formation compared to the activity of native CBGAS.

[0019] In one embodiment, the prenyltransferase has a reaction rate greater than 12 μg / mL CBGA.

[0020] In one embodiment, the prenyltransferase has a sequence identity of less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% with the sequence of the native nphB protein.

[0021] In one embodiment, the prenyltransferase has at least about 80%, 85%, 90% or 95% sequence identity with all or a fragment of SEQ ID NOs: 1-56.

[0022] In one embodiment, CBGA is a specific isomeric form, wherein the isomeric form is a specific structural isomer or stereoisomer.

[0023] In one embodiment, the prenyl group donor, acid (or fatty acid), and CBGA analog are as shown in the following table:

[0024] Isoprenyl group donor Fatty acid CBGA analog GPP Oleic acid CBGA GPP Propylpyrogallol carboxylic acid CBGAVA

[0025] In certain embodiments, provided are methods for producing cannabinoids, the methods comprising the steps of: (1) reacting a prenyltransferase with a prenyl group donor and an acid to produce cannabigerolic acid (CBGA) or an analog thereof; and (2) reacting CBGA or an analog thereof with a cannabinoid synthase to form an acidic form of a cannabinoid.

[0026] In one embodiment, the cannabinoid synthase is one of two oxidoreductases, tetrahydrocannabinolic acid synthase (THCAS) or cannabidiolic acid synthase (CBDAS), and the cannabinoid is THC or CBD.

[0027] In certain embodiments, provided are recombinant microorganisms engineered to produce CBGA, wherein the microorganisms overexpress a prenyltransferase having at least about 80%, 85%, 90% or 95% sequence identity with all or a fragment of SEQ ID NOs: 1-56.

[0028] In certain embodiments, provided is an isolated polypeptide having cannabigerolic acid synthase (CBGAS) activity, the polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90% or 95% sequence identity with all or a fragment of SEQ ID NOs: 1-56.

[0029] In one embodiment, the polypeptide is expressed in a microbial host or a plant host, wherein the microbial host includes but is not limited to Escherichia coli, Yarrowia lipolytica, and Saccharomyces cerevisiae, and the plant host includes but is not limited to Cannabis (species and genus).

[0030] In one embodiment, the method is an in vitro method.

[0031] In one embodiment, the method is an in vivo cell-based assay.

[0032] According to some aspects, the present application relates to enzymes that can be used to produce cannabinoids or cannabinoid intermediates in a microbial host, and methods for preparing and using such enzymes. In some embodiments, the microbial host is yeast, such as Saccharomyces cerevisiae and Yarrowia lipolytica.

[0033] In certain embodiments, the claimed method for producing cannabinoids is an improvement over currently known methods, such as plant extraction. Extracting cannabinoids from plants involves growing and harvesting plants that naturally contain cannabinoids, and then using various extraction methods known in the art to extract the compounds. Since cannabinoids are naturally mixed in plants, it is often difficult to reproduce the same extraction and purification characteristics for each extraction sample. The unique genetic origin and growth conditions of each plant further complicate this. The resulting different cannabinoid profiles lead to each sample having a different drug profile - a problem from a safety or regulatory perspective. Finally, due to the nature of the mixture of compounds with similar structures and sizes, the ability to purify a high-purity single product is very challenging.

[0034] Thus, in certain embodiments, the present application provides enzymes that can be used to produce cannabinoids in a microbial host. In some embodiments, the microbial host is yeast, such as Saccharomyces cerevisiae and Yarrowia lipolytica. Example embodiments include novel enzymes having cannabigerolic acid synthase (CBGAS) activity and methods for preparing and using such enzymes in the production of cannabigerolic acid (CBGA) or its analogs. In some example embodiments, the enzyme is an isoprenyltransferase. In some example embodiments, the enzyme is geranylpyrophosphate:olivatolate geranyltransferase (GOT) or its analog.

[0035] In certain embodiments, the present application also provides methods of using such enzymes to produce compounds in the cannabinoid pathway, such as cannabigerolic acid (CBGA) or its analogs produced from an isoprenyl group donor and an acid, by heterologous expression of a gene encoding such enzyme in a microbial host.

[0036] In certain embodiments, a method for producing cannabigerolic acid (CBGA) or its analogs is provided, the method comprising the step of using an enzyme having cannabigerolic acid synthase (CBGAS) activity.

[0037] In one embodiment, the enzyme is an isoprenyltransferase that reacts with an isoprenyl group donor and an acid.

[0038] In one embodiment, the prenyltransferase has geranyl pyrophosphate:oleate geranyltransferase (GOT) activity.

[0039] In one embodiment, the prenyltransferase is obtained from intact, purified cell extracts or combinations thereof.

[0040] In one embodiment, the prenyl group donor is selected from the group consisting of: isoprenyl moieties derived from allylic isoprenyl diphosphates (including but not limited to dimethylallyl diphosphate (DMAPP; C5), geranyl diphosphate (GPP; C10), and farnesyl diphosphate (FPP; C15)).

[0041] In one embodiment, the prenyl group donor is GPP.

[0042] In one embodiment, the acid is selected from the group consisting of: orsellinic acid (OSA), propyl-orcinol carboxylic acid (DVA), apigenin, daidzein, genistein, naringenin, oleanol, OA, and resveratrol.

[0043] In one embodiment, the acid is oleic acid.

[0044] In one embodiment, the prenyl group donor is GPP, the fatty acid is selected from the group consisting of: oleic acid, propyl-orcinol carboxylic acid, butyric acid, valeric acid, hexanoic acid, and heptanoic acid, and the CBGA analogs are selected from the group consisting of CBGA and CBGAVA.

[0045] In certain embodiments, a method for producing cannabigerolic acid (CBGA) or an analog thereof is provided, the method comprising the steps of: reacting a heterologously expressed prenyltransferase having geranyl pyrophosphate:oleate geranyltransferase (GOT) activity with GPP and OA.

[0046] In one embodiment, the prenyltransferase has an activity with increased synthetic ability and reduced byproduct formation compared to the activity of native CBGAS.

[0047] In one embodiment, the prenyltransferase is heterologously expressed in yeast.

[0048] In one embodiment, the prenyltransferase has an improved reaction rate for forming CBGA compared to the NphB wild type.

[0049] In one embodiment, the prenyltransferase has at least about 80%, 85%, 90%, or 95% sequence identity with all or a fragment of SEQ ID NOs: 57 - 103.

[0050] In one embodiment, CBGA is a specific isomeric form, wherein the isomeric form is a specific structural isomer or stereoisomer.

[0051] In certain embodiments, methods for producing cannabinoids are provided, the methods comprising the steps of: (1) reacting a heterologously expressed prenyltransferase with a prenyl group donor and an acid to produce cannabigerolic acid (CBGA) or an analog thereof; and (2) reacting CBGA or an analog thereof with a cannabinoid synthase to form an acidic form of a cannabinoid.

[0052] In one embodiment, the prenyltransferase has at least about 80%, 85%, 90% or 95% sequence identity with all or a fragment of SEQ ID NOs: 57 - 103.

[0053] In one embodiment, the cannabinoid synthase is one of two oxidoreductases, tetrahydrocannabinolic acid synthase (THCAS) or cannabidiolic acid synthase (CBDAS), and the cannabinoid is THC or CBD.

[0054] In one embodiment, the method is an in vitro method.

[0055] In one embodiment, the method is a cell-based in vivo assay.

[0056] In certain embodiments, recombinant microorganisms engineered to produce CBGA are provided, wherein the microorganisms overexpress a prenyltransferase having at least about 80%, 85%, 90% or 95% sequence identity with all or a fragment of SEQ ID NOs: 57 - 103.

[0057] In certain embodiments, recombinant microorganisms are provided that comprise at least one heterologous nucleotide sequence or a codon-degenerate nucleotide sequence thereof, the heterologous nucleotide sequence having at least about 80%, 85%, 90% or 95% sequence identity with SEQ ID NOs: 104 - 197.

[0058] In certain embodiments, isolated polypeptides having cannabigerolic acid synthase (CBGAS) activity are provided, the polypeptides comprising an amino acid sequence having at least about 80%, 85%, 90% or 95% sequence identity with all or a fragment of SEQ ID NOs: 57 - 103.

[0059] In one embodiment, the polypeptide is expressed in a microbial host, wherein the microbial host includes but is not limited to yeast.

[0060] In one embodiment, the polypeptide is expressed in Yarrowia lipolytica or Saccharomyces cerevisiae.

[0061] In some aspects, the present application relates to enzymes that can be used to produce cannabinoids or cannabinoid intermediates in a microbial host or a yeast host, as well as methods for preparing and using such enzymes.

[0062] In certain embodiments, the claimed methods for producing cannabinoids are improvements over currently known methods such as plant extraction. Extracting cannabinoids from plants involves growing and harvesting plants that naturally contain cannabinoids and then using various extraction methods known in the art to extract the compounds. Since cannabinoids are naturally mixed in plants, it is often difficult to reproduce the same extraction and purification characteristics for each extraction sample. The unique genetic origin and growth conditions of each plant further complicate this. The resulting different cannabinoid profiles lead to each sample having a different pharmaceutical profile - a problem from a safety or regulatory perspective. Finally, due to the nature of the mixture of compounds with similar structures and sizes, the ability to purify a high-purity single product is very challenging.

[0063] Accordingly, in certain embodiments, the present application provides enzymes that can be used to produce cannabinoids in a microbial host. Example embodiments include novel prenyltransferases and methods for preparing and using such enzymes in the production of cannabigerolic acid (CBGA) or its analogs from a prenyl group donor and an acid.

[0064] In certain embodiments, the present application also provides methods of using such enzymes to produce compounds in the cannabinoid pathway, such as cannabigerolic acid (CBGA) or its analogs produced from a prenyl group donor and an acid, by heterologous expression of plant and microbial genes in a microbial host. Various embodiments provide engineered enzymes with improved characteristics for producing compounds in the cannabinoid pathway. Such improved characteristics include, but are not limited to, better kinetics (e.g., kM and kCAT), higher tolerance to solvents, the ability to function at higher temperatures, and improved ability to use different substrates and group donors.

[0065] In certain embodiments, a method for producing cannabigerolic acid (CBGA) or its analogs is provided, the method comprising the step of reacting a prenyltransferase with a prenyl group donor and an acid.

[0066] In one embodiment, the prenyltransferase has cannabigerolic acid synthase (CBGAS) activity.

[0067] In one embodiment, the prenyltransferase is obtained from a whole, purified cell extract or a combination thereof.

[0068] In one embodiment, the isoprenyl group donor is selected from the group consisting of: isoprenyl moieties derived from allylic isoprenyl diphosphate (including but not limited to dimethylallyl diphosphate (DMAPP; C5), geranyl diphosphate (GPP; C10), and farnesyl diphosphate (FPP; C15)).

[0069] In one embodiment, the isoprenyl group donor is GPP.

[0070] In one embodiment, the acid is selected from the group consisting of orsellinic acid (OSA), divanillic acid (DVA), apigenin, daidzein, genistein, naringenin, oleanol, OA, and resveratrol.

[0071] In one embodiment, the acid is oleanic acid.

[0072] In one embodiment, the isoprenyltransferase has an activity with significantly enhanced synthesis ability and / or reduced by-product formation.

[0073] In one embodiment, the isoprenyltransferase has a sequence identity of less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% with the sequence of the native nphB protein.

[0074] In one embodiment, the isoprenyltransferase has at least about 80%, 85%, 90%, or 95% sequence identity with all or a fragment of SEQ ID NOs: 198 - 253.

[0075] In one embodiment, CBGA is a specific isomeric form, wherein the isomeric form is a specific structural isomer or stereoisomer.

[0076] In one embodiment, the isoprenyl group donor is GPP, the fatty acid is selected from the group consisting of oleanic acid, divanillic acid, butyric acid, valeric acid, caproic acid, and heptanoic acid, and the CBGA analog is selected from the group consisting of CBGA and CBGAVA.

[0077] In certain embodiments, provided is a method for producing cannabinoids, the method comprising the steps of: (1) reacting an isoprenyltransferase with an isoprenyl group donor and an acid to produce cannabigerolic acid (CBGA) or an analog thereof; and (2) reacting CBGA or an analog thereof with a cannabinoid synthase to form an acidic form of a cannabinoid.

[0078] In one embodiment, the cannabinoid synthase is one of two oxidoreductases, tetrahydrocannabinolic acid synthase (THCAS) or cannabidiolic acid synthase (CBDAS), and the cannabinoid is THC or CBD.

[0079] In certain embodiments, recombinant microorganisms engineered to produce CBGA are provided, wherein the microorganisms overexpress an isoprenyl transferase having at least about 80%, 85%, 90%, or 95% sequence identity to all or a fragment of SEQ ID NOs: 198 - 335.

[0080] In certain embodiments, an isolated polypeptide having cannabigerolic acid synthase (CBGAS) activity is provided, the polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, or 95% sequence identity to all or a fragment of SEQ ID NOs: 198 - 335.

[0081] In one embodiment, the polypeptide is expressed in a microbial host or a plant host, wherein the microbial host includes but is not limited to Escherichia coli, Yarrowia lipolytica, and Saccharomyces cerevisiae, and the plant host includes but is not limited to Cannabis (species and genus).

[0082] In one embodiment, the method is an in vitro method.

[0083] In one embodiment, the method is an in vivo cell - based assay.

[0084] In certain embodiments, a recombinant polypeptide is provided that comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 246 or SEQ ID NO: 232, wherein the amino acid sequence comprises at least one amino acid substitution of a conserved amino acid between SEQ ID NO: 246 and SEQ ID NO: 232.

[0085] In one embodiment, the conserved amino acid is replaced with an amino acid having similar chemical properties to the conserved amino acid.

[0086] In certain embodiments, a recombinant polypeptide is provided that comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 246, wherein the amino acid sequence comprises at least one amino acid substitution at ID No: 246.

[0087] In one embodiment, at least one amino acid substitution is selected from the group consisting of: Y46W, Y46A, Y46L, Y46C, Y46D, Y46E, Y46F, Y46G, Y46H, Y46I, Y46K, Y46P, Y46M, Y46N, Y46Q, Y46R, Y46S, Y46T, Y46V, Q51G, Q51A, Q51C, Q51D, Q51E, Q51F, Q51G, Q51H, Q51I, Q51K, Q51L, Q51M, Q51N, Q51P, Q51R, Q51S, Q51T, Q51V, Q51W, Q51Y, L55V, L55F, L55A, L55Q, L55W, L66V, L66F, L66A, L66Q, L66W, G67L, G67M, G67E, R100E, R100Q, D115E, K186N, K186Q, K186A, K186D, Y188W, Y188A, Y188L, Y188C, Y188, Y188E, Y188F, Y188G, Y188H, Y188I, Y188K, Y188P, Y188M, Y188N, Y188Q, Y188R, Y188S, Y188T, Y188V, D256E, R264E, R264Q, V265L, V265S, V265E, V265A, V265F, V265N, Y268W, Y268A, Y268L, Y268C, Y268D, Y268E, Y268F, Y268G, Y268H, Y268I, Y268K, Y268P, Y268M, Y268N, Y268Q, Y268R, Y268S, Y268T, Y268V, L269V, L269F, L269A, L269Q, L269W, L285V, L285F, L285A, L285Q, L285W, G286L, G286M, G286E, G287L, G287M, G287E, R288E, R288Q, G313L, G313M, G313E, Y352W, Y352A, Y352L, Y352C, Y352D, Y352E, Y352F, Y352G, Y352H, Y352I, Y352K, Y352P, Y352M, Y352N, Y352Q, Y352R, Y352S, Y352T, Y352V, Y383W, Y383A, Y383L, Y383C, Y383D, Y383E, Y383F, Y383G, Y383H, Y383I, Y383K, Y383P, Y383M, Y383N, Y383Q, Y383R, Y383S, Y383T, Y383V, S407W, S407A, S407L, S407C, S407D, S407E,S407F, S407G, S407H, S407I, S407K, S407P, S407M, S407N, S407Q, S407R, S407S, S407T, S407V, Y420W, Y420A, Y420L, Y420C, Y420D, Y420E, Y420F, Y420G, Y420H, Y420I, Y420K, Y420P, Y420M, Y420N, Y420Q, Y420R, Y420S, Y420T, and Y420V.

[0088] In certain embodiments, a recombinant polypeptide is provided, the recombinant polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 232, wherein the amino acid sequence comprises at least one amino acid substitution.

[0089] In one embodiment, at least one amino acid substitution is selected from the group consisting of: Y24W, Y24A, Y24L, Y24C, Y24D, Y24E, Y24F, Y24G, Y24H, Y24I, Y24K, Y24P, Y24M, Y24N, Y24Q, Y24R, Y24S, Y24T, Y24V, Q29G, Q29A, Q29C, Q29D, Q29E, Q29F, Q29G, Q29H, Q29I, Q29K, Q29L, Q29M, Q29N, Q29P, Q29R, Q29S, Q29T, Q29V, Q29W, Q29Y, L33V, L33F, L33A, L33Q, L33W, L44V, L44F, L44A, L44Q, L44W, G45L, G45M, G45E, R81E, R81Q, D96E, K165N, K165Q, K165A, K165D, Y167W, Y167A, Y167L, Y167C, Y167D, Y167E, Y167F, Y167G, Y167H, Y167I, Y167K, Y167P, Y167M, Y167N, Y167Q, Y167R, Y167S, Y167T, Y167V, D255E, R236E, R236Q, V237L, V237S, V237E, V237A, V237F, V237N, Y239W, Y239A, Y239L, Y239C, Y239D, Y239E, Y239F, Y239G, Y239H, Y239I, Y239K, Y239P, Y239M, Y239N, Y239Q, Y239R, Y239S, Y239T, Y239V, L241V, L241F, L241A, L241Q, L241W, L254V, L254F, L254A, L254Q, L254W, G258L, G258M, G258E, G259L, G259M, G259E, R260E, R260Q, G286L, G286M, G286E, Y330W, Y330A, Y330L, Y330C, Y330D, Y330E, Y330F, Y330G, Y330H, Y330I, Y330K, Y330P, Y330M, Y330N, Y330Q, Y330R, Y330S, Y330T, Y330V, Y364W, Y364A, Y364L, Y364C, Y364D, Y364E, Y364F, Y364G, Y364H, Y364I, Y364K, Y364P, Y364M, Y364N, Y364Q, Y364R, Y364S, Y364T, Y364V, S384W, S384A, S384L, S384C, S384D, S384E,S384F, S384G, S384H, S384I, S384K, S384P, S384M, S384N, S384Q, S384R, S384S, S384T, S384V, Y398W, Y398A, Y398L, Y398C, Y398D, Y398E, Y398F, Y398G, Y398H, Y398I, Y398K, Y398P, Y398M, Y398N, Y398Q, Y398R, Y398S, Y398T, and Y398V.

[0090] In certain embodiments, a recombinant polypeptide is provided, the recombinant polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 198, wherein the amino acid sequence comprises at least one amino acid substitution.

[0091] In one embodiment, the at least one amino acid substitution is selected from the group consisting of: A17T_Q159W_A230S; A51T_M104E_Q159S; A51Q_S175W_L217F; L217F_V292N_Q235A; A51T_D164E_Q293W; V47A_Q159S_I292A; and A51Q_S175Y_Y286H.

[0092] In certain embodiments, a method for producing cannabigerolic acid (CBGA) or an analogue thereof is provided, the method comprising the step of reacting a heterologously expressed prenyltransferase with geranyl diphosphate (GPP) and an acid.

[0093] In one embodiment, the prenyltransferase has prenyltransferase activity and / or geranyl pyrophosphate:olivetolate geranyltransferase (GOT) activity.

[0094] In one embodiment, the prenyltransferase has an activity with increased synthetic ability and reduced byproduct formation compared to the activity of native CBGAS; or has an improved reaction rate for forming CBGA compared to NphB wild type.

[0095] In one embodiment, the prenyltransferase has a reaction rate greater than 12 μg / mL CBGA.

[0096] In one embodiment, the acid is selected from the group consisting of orsellinic acid (OSA), divanillic acid (DVA), apigenin, daidzein, genistein, naringenin, oleanol, oleanic acid (OA), and resveratrol.

[0097] In certain embodiments, the prenyltransferase has at least about 80%, 85%, 90%, or 95% sequence identity with all or a fragment of an amino acid sequence selected from the group consisting of SEQ ID No: 01-103, 198-335, or the prenyltransferase is expressed by a nucleotide sequence having at least about 80%, 85%, 90%, or 95% sequence identity with all or a fragment of a nucleic acid sequence selected from the group consisting of SEQ ID No: 104-197, 336-583.

[0098] In one embodiment, wherein the amino acid sequence is SEQ ID No: 01, 02, 85, or 179.

[0099] In certain embodiments, provided is a method for producing cannabinoids, the method comprising the steps of: (1) reacting a heterologously expressed prenyltransferase with a prenyl group donor and an acid to produce cannabigerolic acid (CBGA) or an analogue thereof; and (2) reacting the CBGA or an analogue thereof with a cannabinoid synthase to form an acidic form of a cannabinoid.

[0100] In one embodiment, wherein the prenyltransferase has an activity with increased synthetic ability and reduced byproduct formation compared to the activity of native CBGAS.

[0101] In one embodiment, wherein the prenyltransferase has a reaction rate greater than 12 μg / mL CBGA.

[0102] In one embodiment, wherein the acid is selected from the group consisting of orsellinic acid (OSA), divanillic acid (DVA), apigenin, daidzein, genistein, naringenin, oleanol, oleanolic acid (OA), resveratrol, butyric acid, valeric acid, hexanoic acid, and heptanoic acid.

[0103] In certain embodiments, wherein the enzyme has at least about 80%, 85%, 90%, or 95% sequence identity with all or a fragment of a sequence selected from the group consisting of SEQ ID NO: 01-103, 198-335; or the enzyme is expressed by a nucleotide sequence having at least about 80%, 85%, 90%, or 95% sequence identity with all or a fragment of SEQ ID No: 104-197, 336-583.

[0104] In certain embodiments, provided is a recombinant microorganism engineered to produce CBGA, wherein the microorganism overexpresses an enzyme having at least about 80%, 85%, 90%, or 95% sequence identity with all or a fragment of a sequence selected from the group consisting of SEQ ID NO: 01-103 and 198-335.

[0105] In certain embodiments, recombinant microorganisms engineered to produce CBGA are provided, wherein the microorganisms comprise at least one heterologous nucleotide sequence or a codon-degenerate nucleotide sequence thereof, and the heterologous nucleotide sequence has at least about 80%, 85%, 90%, or 95% sequence identity to all or a fragment of a nucleic acid sequence selected from the group consisting of SEQ ID No: 104-197, 336-583.

[0106] In certain embodiments, an isolated polypeptide having cannabigerolic acid synthase (CBGAS) activity is provided; wherein the polypeptide comprises an amino acid sequence having at least about 80%, 85%, 90%, or 95% sequence identity to all or a fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 01-103, 198-335; or the polypeptide is expressed from a nucleotide sequence having at least about 80%, 85%, 90%, or 95% sequence identity to all or a fragment of a nucleotide sequence selected from the group consisting of SEQ ID No: 104-197 and 336-583.

[0107] In one embodiment, the polypeptide is expressed in a microbial host selected from the group consisting of Escherichia coli, Yarrowia lipolytica, and Saccharomyces cerevisiae.

[0108] The isolated polypeptide according to claim 15, wherein the polypeptide is expressed in a Cannabis sp.

[0109] In certain embodiments, the use of an enzyme for the production of CBGA is provided, wherein the enzyme has at least about 80%, 85%, 90%, or 95% sequence identity to all or a fragment of a sequence selected from the group consisting of SEQ IDNO: 01-103 and 198-335, or the enzyme is expressed from a nucleotide sequence having at least about 80%, 85%, 90%, or 95% sequence identity to all or a fragment of a group consisting of SEQ ID No: 104-197 and 336-583.

[0110] In certain embodiments, a recombinant polypeptide is provided, the recombinant polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 246, wherein the amino acid sequence comprises at least one amino acid substitution to a conserved amino acid.

[0111] In one embodiment, wherein the at least one amino acid substitution is selected from the group consisting of: Y46W, Y46A, Y46L, Y46C, Y46D, Y46E, Y46F, Y46G, Y46H, Y46I, Y46K, Y46P, Y46M, Y46N, Y46Q, Y46R, Y46S, Y46T, Y46V, Q51G, Q51A, Q51C, Q51D, Q51E, Q51F, Q51G, Q51H, Q51I, Q51K, Q51L, Q51M, Q51N, Q51P, Q51R, Q51S, Q51T, Q51V, Q51W, Q51Y, L55V, L55F, L55A, L55Q, L55W, L66V, L66F, L66A, L66Q, L66W, G67L, G67M, G67E, R100E, R100Q, D115E, K186N, K186Q, K186A, K186D, Y188W, Y188A, Y188L, Y188C, Y188, Y188E, Y188F, Y188G, Y188H, Y188I, Y188K, Y188P, Y188M, Y188N, Y188Q, Y188R, Y188S, Y188T, Y188V, D256E, R264E, R264Q, V265L, V265S, V265E, V265A, V265F, V265N, Y268W, Y268A, Y268L, Y268C, Y268D, Y268E, Y268F, Y268G, Y268H, Y268I, Y268K, Y268P, Y268M, Y268N, Y268Q, Y268R, Y268S, Y268T, Y268V, L269V, L269F, L269A, L269Q, L269W, L285V, L285F, L285A, L285Q, L285W, G286L, G286M, G286E, G287L, G287M, G287E, R288E, R288Q, G313L, G313M, G313E, Y352W, Y352A, Y352L, Y352C, Y352D, Y352E, Y352F, Y352G, Y352H, Y352I, Y352K, Y352P, Y352M, Y352N, Y352Q, Y352R, Y352S, Y352T, Y352V, Y383W, Y383A, Y383L, Y383C, Y383D, Y383E, Y383F, Y383G, Y383H, Y383I, Y383K, Y383P, Y383M, Y383N, Y383Q, Y383R, Y383S, Y383T, Y383V, S407W, S407A, S407L, S407C, S407DS407E, S407F, S407G, S407H, S407I, S407K, S407P, S407M, S407N, S407Q, S407R, S407S, S407T, S407V, Y420W, Y420A, Y420L, Y420C, Y420D, Y420E, Y420F, Y420G, Y420H, Y420I, Y420K, Y420P, Y420M, Y420N, Y420Q, Y420R, Y420S, Y420T, and Y420V.

[0112] In certain embodiments, a recombinant polypeptide is provided, the recombinant polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 232, wherein the amino acid sequence comprises at least one amino acid substitution to a conserved amino acid.

[0113] In one embodiment, where the at least one amino acid substitution is selected from the group consisting of: Y24W, Y24A, Y24L, Y24C, Y24D, Y24E, Y24F, Y24G, Y24H, Y24I, Y24K, Y24P, Y24M, Y24N, Y24Q, Y24R, Y24S, Y24T, Y24V, Q29G, Q29A, Q29C, Q29D, Q29E, Q29F, Q29G, Q29H, Q29I, Q29K, Q29L, Q29M, Q29N, Q29P, Q29R, Q29S, Q29T, Q29V, Q29W, Q29Y, L33V, L33F, L33A, L33Q, L33W, L44V, L44F, L44A, L44Q, L44W, G45L, G45M, G45E, R81E, R81Q, D96E, K165N, K165Q, K165A, K165D, Y167W, Y167A, Y167L, Y167C, Y167D, Y167E, Y167F, Y167G, Y167H, Y167I, Y167K, Y167P, Y167M, Y167N, Y167Q, Y167R, Y167S, Y167T, Y167V, D255E, R236E, R236Q, V237L, V237S, V237E, V237A, V237F, V237N, Y239W, Y239A, Y239L, Y239C, Y239D, Y239E, Y239F, Y239G, Y239H, Y239I, Y239K, Y239P, Y239M, Y239N, Y239Q, Y239R, Y239S, Y239T, Y239V, L241V, L241F, L241A, L241Q, L241W, L254V, L254F, L254A, L254Q, L254W, G258L, G258M, G258E, G259L, G259M, G259E, R260E, R260Q, G286L, G286M, G286E, Y330W, Y330A, Y330L, Y330C, Y330D, Y330E, Y330F, Y330G, Y330H, Y330I, Y330K, Y330P, Y330M, Y330N, Y330Q, Y330R, Y330S, Y330T, Y330V, Y364W, Y364A, Y364L, Y364C, Y364D, Y364E, Y364F, Y364G, Y364H, Y364I, Y364K, Y364P, Y364M, Y364N, Y364Q, Y364R, Y364S, Y364T, Y364V, S384W, S384A, S384L, S384C, S384D,S384E, S384F, S384G, S384H, S384I, S384K, S384P, S384M, S384N, S384Q, S384R, S384S, S384T, S384V, Y398W, Y398A, Y398L, Y398C, Y398D, Y398E, Y398F, Y398G, Y398H, Y398I, Y398K, Y398P, Y398M, Y398N, Y398Q, Y398R, Y398S, Y398T, and Y398V.

[0114] In certain embodiments, a recombinant polypeptide is provided, the recombinant polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 198, wherein the amino acid sequence comprises at least one amino acid substitution.

[0115] In one embodiment, the at least one amino acid substitution is selected from the group consisting of: A17T_Q159W_A230S; A51T_M104E_Q159S; A51Q_S175W_L217F; L217F_V292N_Q235A; A51T_D164E_Q293W; V47A_Q159S_I292A; and A51Q_S175Y_Y286H. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 1 Showing the results of titrating CBGA at four different concentrations according to an exemplary embodiment.

[0117] Figure 2 According to an exemplary embodiment, the CBGAS activities of four control samples (nphB gene) and two enzymes of the present invention were compared using LC-MS in terms of their ability to form CBGA.

[0118] Figure 3 According to an exemplary embodiment, the biological pathway of cannabinoid production is detailed.

[0119] Figure 4 According to another exemplary embodiment, the biological pathway of cannabinoid production is detailed.

[0120] Figure 5 According to an exemplary embodiment, the GOT activities and the CBGA / CBGA-isomer ratios of a control sample (nphB gene) and two enzymes of the present invention (eCAN20005 and eCAN20006) were compared using LC-MS in terms of their ability to form CBGA.

[0121] Figure 6AAccording to the exemplary embodiments, the GOT activities of various enzymes of the present invention (yCAN30003 to yCAN30049) expressed in E. coli were compared using LC-MS in terms of their ability to form CBGA. Figure 6B According to the exemplary embodiments, the GOT activities of various enzymes of the present invention (yCAN30003 to yCAN30049) expressed in yeast were compared using LC-MS in terms of their ability to form CBGA. Detailed Description

[0122] Definitions

[0123] As used herein and in the claims, "comprising" means including the following elements but not excluding other elements.

[0124] As used herein and in the claims, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. For example, as used above, "a" gene means one or more genes, which may be the same or different.

[0125] "Prenyl transferase enzymes or prenyl transferase" refers to aromatic prenyl transferases (PTases) that catalyze the transfer of C5 (dimethylallyl), C10 (geranyl), or C15 (farnesyl) prenyl groups, for example, derived from corresponding prenyl diphosphate metabolites, to a variety of electron-rich aromatic acceptors.

[0126] "Prenyl group" is a functional group present in bioactive natural products of various microbial and plant origins, including amino acids, stilbenes, alkaloids, polyketides, and phenylpropanoids (such as flavonoids), and its production results in natural product hybrids with altered or enhanced bioactivity.

[0127] "Prenylation" refers to the transfer of a prenyl group to an electron-rich aromatic acceptor. Prenylation appears to provide higher levels of bioactivity in many cases compared to non-prenylated precursors, such as by increasing the affinity for biological membranes and the interaction with cellular targets.

[0128] "GPP pathway" refers to the pathway for the production of geranyl diphosphate (GPP) through the MVA or MEP pathway. Microorganisms naturally produce GPP through the MVA or MEP pathway in yeast and bacteria, respectively.

[0129] The "OA pathway" or "olivetolic acid pathway" refers to the pathway for synthesizing OA, in which hexanoic acid (a simple fatty acid naturally produced in yeast) is converted to hexanoyl-CoA by hexanoyl-CoA synthase. The synthesis of OA is a two-step fusion of hexanoyl-CoA and 3 malonyl-CoA, and the enzymes responsible for these reactions are olivetol synthase (OLS) and olivetolic acid cyclase (OAC). The source of these coding sequences is from Cannabis (C. sativa). Feeding hexanoic acid by adding it to the growth medium has been shown to increase OA production.

[0130] "Cannabigerolic acid" or "CBGA" refers to the molecule produced from olivetolic acid and the mevalonate pathway intermediate geranyl pyrophosphate (GPP) by geranyl pyrophosphate:olivetolate geranyltransferase (GOT). CBGA is also a precursor of Δ-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and many other cannabinoids.

[0131] The "CBGA pathway" refers to the cannabigerolic acid synthase (CBGAS) activity shown by the geranyl pyrophosphate:olivetolate geranyltransferase activity (GOT) of Cannabis and the prenyltransferase activity of the nphB enzyme, where GPP and olivetolic acid fuse to form cannabigerolic acid (CBGA).

[0132] "nphB" or "nphB enzyme" refers to an aromatic prenyltransferase that catalyzes the attachment of a 10-carbon geranyl group to an aromatic substrate (such as CBGA). This enzyme class drives the first biochemical step of the cannabinoid pathway to form CBGA.

[0133] "Geranyl pyrophosphate:olivetolate geranyltransferase" or "GOT" refers to a prenyltransferase that is part of the cannabinoid biosynthetic pathway of the plant Cannabis and catalyzes the reaction between geranyl diphosphate and 2,4-dihydroxy-6-pentylbenzoate to form cannabigerol ester and diphosphate.

[0134] A "codon-degenerate nucleotide sequence" refers to a nucleotide sequence that encodes the same set of amino acids as another nucleotide sequence with different codons in a polypeptide sequence. For example, a codon-degenerate nucleotide sequence of a sequence containing GAA (encoding glutamate) will be the same as a sequence in which GAA is replaced by GAG (which also encodes glutamate).

[0135] Figure 3Describes the biosynthetic pathway of cannabinoids in cannabis. The precursors GPP and OA are converted to the central intermediate CBGA of the cannabinoid pathway. CBGA is converted to the acidic forms of THC and CBD by two oxidoreductases, tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS). Heterologously expressed enzymes are shown in green. Intermediates of primary metabolism are shown in grey (Zirpel et al., 2017).

[0136] Figure 4 Describes the biosynthetic pathway of cannabinoids in cannabis. The precursors GPP and OA are converted to the central intermediate CBGA of the cannabinoid pathway. CBGA is converted to the acidic forms of THC and CBD by two oxidoreductases, tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS).

[0137] This document provides enzymes that catalyze the same reaction at a surprisingly higher reaction rate than NphB.

[0138] In certain embodiments, this document provides enzymes that catalyze the same reaction at a surprisingly higher reaction rate, engineered organisms that express such enzymes, methods of using such engineered organisms to prepare such enzymes, and methods of using such enzymes to prepare cannabinoids or intermediates for the preparation of cannabinoids.

[0139] In some embodiments, the enzyme has an improved reaction rate for the formation of CBGA compared to NphB wild type (wt). In some embodiments, the enzyme is selected from those listed in Table 1 herein.

[0140] Examples

[0141] Cloning, expression, and purification of prenyltransferases

[0142] In certain embodiments, gene candidates are expressed in E. coli, protein extracts are prepared, and purified enzymes for testing are delivered by affinity enzyme purification using HIS tags to form CBGA. In still other exemplary embodiments, gene candidates are expressed in yeast. The expressed protein extracts are purified and used for testing. Exemplary embodiments demonstrating specific methods for cloning, expressing, and purifying the enzymes disclosed herein are described in the examples herein.

[0143] Methods for preparing cannabinoids

[0144] Cannabinoids can be prepared using heterologous expression in microorganisms or yeast. Microorganisms can be genetically engineered to express cannabinoids or cannabinoid precursor molecules. Methods for heterologous expression of cannabinoid compounds are known in the art and are incorporated herein by reference, for example, as described in Carvalho et al., 2017.

[0145] Figure 3 A figure showing a biological pathway detailing cannabinoid production, said biological pathway including precursors such as CBGA formed from olivetolic acid (OA) and geranyl diphosphate (GPP) by the prenyltransferase NphB. The prenylation of OA with NphB is non-specific and generates 2-O-geranylolivetolate (as a by-product) (Valliere et al., 2019). Prenylation can be carried out by methods known to those skilled in the art and includes but is not limited to Valliere et al., 2019 and Luo et al., 2019, which are hereby incorporated by reference.

[0146] Figure 4 A figure showing a biological pathway detailing cannabinoid production, said biological pathway including precursors such as CBGA formed from olivetolic acid (OA) and geranyl diphosphate (GPP) by CBGA-like enzymes (especially represented by the prenyltransferase NphB family). The prenylation of OA with NphB is non-specific and generates 2-O-geranylolivetolate (as a by-product) (Valliere et al., 2019). Prenylation can be carried out by methods known to those skilled in the art and includes but is not limited to Valliere et al., 2019 and Luo et al., 2019, which are hereby incorporated by reference.

[0147] Example 1: Cloning, Expression, and Purification of Enzyme

[0148] Candidate genes were purchased as gene blocks from General Biosystems (Anhui, China) Corporation Limited and cloned between the restriction enzyme sites NdeI (CATATG) and XhoI (C TCGAG) of pET28a(+) expressing an N-terminal His-tagged enzyme. All plasmids were transformed into BL21(DE3) and named eCAN20005 to eCAN20060 (see Table 1).

[0149] A 0.5 mL saturated culture in LB medium with 50 μg / mL kanamycin was inoculated into 50 mL TB medium with 50 μg / mL kanamycin. The culture was grown at 37 °C to an OD600 of 0.5 - 0.8, induced with 0.5 mM IPTG, and then expressed at 220 rpm at 25 °C for 18 hours.

[0150] Cells were harvested by centrifugation at 2500×g, washed with lysis buffer (50 mM Tris-HCl, 500 mM NaCl, [pH 8.0], 10% (v / v) glycerol), and resuspended in 10 ml of lysis buffer to an OD550 of approximately 100. At 4 °C, the cells were lysed using an ultrasonic cell disruptor for 10 min. The lysate was clarified by centrifugation at 12,000×g for 30 min at 4 °C. The supernatant containing the soluble protein fraction was recovered, filtered through a 0.45 μm filter, and bound to 0.5 mL of Ni-affinity resin in a rotator (or roller) at 25 °C for 1 h. The resin was transferred to a gravity flow column. The resin was washed with 10 column volumes of wash buffer (50 mM Tris-HCl, 500 mM NaCl, (pH 8.0), 10% (v / v) glycerol and 20 mM imidazole), and then eluted with 1 mL of elution buffer (50 mM Tris-HCl, 500 mM NaCl, (pH 8.0), 10% (v / v) glycerol and 250 mM imidazole). The elution buffer was replaced with reaction buffer (50 mM HEPES (pH 7.5), 5 mM Mg) and the protein sample was concentrated using an Amicon filter column (10K cut-off). The protein sample was initially concentrated to 200 μL.

[0151] Example 2: In Vitro Enzyme Assay

[0152] The reaction conditions for the enzyme assay consisted of: 50 mM HEPES (pH = 7.5) with 5 mM MgCl2, 2 mM GPP, 2 mM oleic acid, and 1 mg / ml of the purified candidate enzyme, in a final volume of 200 μL. After incubation at room temperature for 18 h, the reaction mixture was extracted twice with 200 μL of ethyl acetate / formic acid (0.05% (v / v)). The organic extracts from each reaction were combined and the solvent was removed using a block heater. Prior to LC-MS analysis, the samples were dissolved in 100 μL of resuspension solution (acetonitrile / H2O / formic acid (80% / 20% / 0.05% (v / v / v))) and filtered through a 0.22 μm PVDF membrane.

[0153] Figure 1 The enzyme assay was validated by showing the results of titrating CBGA at four different concentrations. CBGA titration curve concentrations: (A) 10 ng / mL, (B) 100 ng / mL, (C) 1 μg / mL, (D) 10 μg / mL. Table 2 also shows the LC-MS chromatogram of CBGA at m / z 359.5 with a retention time of 5.43 min.

[0154] Example 3: LC-MS Analysis of Cannabigerolic Acid (CBGA)

[0155] Equipped with an RP-C18 column (BEH130, 1.7 μm, 2.1 mm x 50 mm, Waters) UPLC-MS (Waters H-class with SQ detector 2). Mobile phase A is water containing 0.1% formic acid, and mobile phase B is acetonitrile containing 0.1% formic acid. Gradient elution starts with 50% B maintained for 1 min, then increases to 90% B in 10 min, decreases to 50% B in 1 min, and is maintained at 50% B for 1 min. The flow rate is 0.4 ml / min, and the running time is 13 min. The sample injection volume is 2 μl, and the sample tray and column oven are set to 10 °C and 30 °C, respectively. Cannabidiolic acid is detected by electrospray ionization in the negative ion mode, and the MS scan mode is SIR 359.5 m / z, with the capillary voltage and cone voltage set to 3,800 V and 30 V, respectively. The desolvation gas and temperature are set to 1,000 l / h and 500 °C, respectively.

[0156] Example 4

[0157] Results

[0158] Prepare the protein samples in Table 1 (below) according to the method described in Example 1, and test the CBGAS enzyme activity of the protein samples according to the determination in Example 2 and the LC-MS method in Example 3.

[0159] Table 1 provides a list of protein sequences of prenyltransferases, which may have an improved reaction rate for forming CBGA compared to NphB wt. Note that the following sequences include the following polyhistidine tags at the N-terminus: MGSSHHHHHHSSGLVPRGSH.

[0160] Table 1

[0161]

[0162]

[0163]

[0164]

[0165] Table 2

[0166] Sample Group Gene RCAN-0001 Group A nphB wt RCAN-0002 Group B nphB M23 RCAN-0003 Group C nphB M30 RCAN-0004 Group D nphB-G286S RCAN-0006 Group E SActPT06

[0167] Table 2 is related to Figure 2Four control sample groups A, B, C, and D (nphB gene, wild type, and mutants) and the novel enzyme (group E: SActPT06) were compared using LC-MS for their CBGAS activities in terms of their ability to form CBGA. The LC-MS chromatograms showed the activity of native nphB and confirmed enhanced activities of the M23, M30, and G286S mutants. It showed that SActPT06 also forms CBGA. The LC-MS chromatogram results of the samples at m / z 359.5, with a retention time of 5.43 minutes for CBGA, showed several isomers with R.T. of 3.47, 5.94, 6.30, and 8.70 min in our samples. R.T. 5.4 corresponded to CBGA.

[0168] Table 3 provides the CBGAS activities, shown in the form of the calculated yields of CBGA formed by the novel enzymes of the present invention. Surprisingly, the results showed that several prokaryotic enzymes expressed in E. coli have CBGAS activity (or GOT activity), and some enzymes (eCAN2005 and eCAN2006) even have higher CGGAS / GOT activities than the wild-type NphB enzyme (eCAN20001) and some mutant NphB enzymes (eCAN20002 and eCAN20004).

[0169] Figure 5 The GOT activities of control samples (nphB, wild type) and two novel enzymes (eCAN2005 and eCAN2006) were shown in terms of their ability to form CBGA using LC-MS. The LC-MS chromatograms showed the activity of native nphB and confirmed the activities of samples eCAN20005 and eCAN2006. It showed that SaPT05 and SActPT06 also form CBGA. The LC-MS chromatogram results showed that at m / z 359.5, the retention time of CBGA was 5.43 minutes, and several isomers with R.T. of 3.47 and 5.90 min were observed in the control and samples. The results also showed that the CBGA / CBGA-isomer ratios of samples eCAN20005 and eCAN2006 were higher than that of NpHB, indicating that both enzymes showed higher specificity than the control and thus produced more of the desired isomeric form. In summary, samples eCAN20005 (i.e., Streptomyces antibioticus SaPT05) (SEQ ID NO:01) and eCAN20006 (i.e., Streptomyces species SActPT06) (SEQ ID NO:02) are surprisingly useful in the production of CBGA or analogs.

[0170] Table 3: CBGA Yields

[0171]

[0172]

[0173] Example 4: Protein Identity of nphB Compared to Other Enzymes and Mutants

[0174] Table 4 shows the protein identity of the enzyme of the present invention compared to nphB using CLUSTAL W of UniProt and LALAIGN of the Expasy program. Table 4 indicates that the protein sequence of the enzyme of the present invention is very different from nphB, showing an overall percentage identity far below 50%. Thus, although the enzyme disclosed herein has a different structure from nphB, it has surprisingly high CBGAS activity. In addition, some of the enzymes of the present invention are surprisingly more effective in catalyzing the formation of CBGA.

[0175] Table 4: Sequence alignment showing the level of protein identity

[0176]

[0177]

[0178] Example 5: Cloning and Yeast Transformation

[0179] Cloning

[0180] Candidate genes were purchased from General Biosystems (Anhui, China) Co., Ltd. as gene blocks and cloned between the restriction enzyme sites EcoRI (GAATTC) and SpeI (ACTAGT) of pESC-TRP (containing the TRP1 yeast-selective marker). All plasmids were transformed into at least one yeast strain and named yCAN20003 to yCAN20053 (see Table 5 below).

[0181] Table 5 provides a list of the protein sequences of the isoprenyltransferases disclosed herein.

[0182] Protein samples in Table 5 were prepared according to the methods described in Examples 5 and 6, and the CBGAS enzyme activity of the protein samples was tested according to the assays of Examples 7(A), 7(B) and the LC-MS method of Example 8.

[0183] Table 6 provides a list of the gene sequences of the isoprenyltransferases disclosed herein. Note that SEQ ID No. 151 to SEQ ID No. 197 are gene sequences optimized for expression in yeast, and the sequences correspond to their native counterparts SEQ ID No. 104 to SEQ ID No. 150, respectively.

[0184] Table 5 List of Protein Sequences of the Isoprenyltransferases Disclosed in This Disclosure.

[0185]

[0186]

[0187]

[0188]

[0189]

[0190] Table 6 List of Gene Sequences of the Isoprenyltransferases Disclosed in This Disclosure.

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198] Yeast transformation

[0199] Reagents for yeast transformation

[0200] YPD medium:

[0201] 1% (w / v) Bacto yeast extract, 2% (w / v) Bacto peptone, 2% (w / v) glucose. For YPD agar plates, an additional 18 g / L Bacto agar is required.

[0202] Selection medium Synthetic complete tryptophan - deficient medium (SC / -Trp):

[0203] SC / -Trp broth was purchased from Coolaber Co., Ltd (Beijing, China). 8 g of the medium was mixed with 900 mL of distilled water and then adjusted to pH 5.8 with 1.0 N sodium hydroxide (NaOH) and autoclaved. For agar plates, an additional 18 g / L Bacto agar is required.

[0204] Lithium acetate (1.0 M), dissolution:

[0205] Dissolve 102 g of lithium acetate dihydrate in 100 ml of water (in a flask) and autoclave for 15 min. Alternatively, a filter unit (Nalgene) and a vacuum pump can be used to filter-sterilize the solution. Store the solution at room temperature (about 20 °C).

[0206] PEG MW 3350 (50% w / v):

[0207] Add 50 g of PEG 3350 to approximately 30 ml of distilled or deionized water (in a 150 ml beaker). Stir the mixture until it dissolves. If necessary, gently heat the solution with a hot plate to aid dissolution. In a 100 ml graduated cylinder, bring the volume of the solution up to 100 ml and mix the solution well. Transfer the solution to a glass storage bottle and autoclave for 15 min. Alternatively, a filter unit (Nalgene) and a vacuum pump can be used to filter-sterilize the solution. The solution can be sealed and stored at room temperature for several months.

[0208] Single-stranded carrier DNA (2.0 mg / ml)

[0209] Using a magnetic stirrer plate, completely dissolve 200 mg of salmon sperm DNA in 100 ml of sterile TE buffer (10 mM Tris-HCl, 1 mM Na 2 EDTA, pH 8.0) at 4 °C for several hours. Aliquot 1.0 ml of the solution into 1.5 ml microcentrifuge tubes, and the remaining solution can be aliquoted into 15 ml screw-cap plastic centrifuge tubes and stored at -20 °C. Denature the carrier DNA in a boiling water bath for 5 min and cool it immediately in an ice-water bath before use.

[0210] Yeast transformation procedure

[0211] Inoculate a single colony of yeast strain grown on a fresh YPD plate into 5 mL of YPD medium with a sterile inoculation loop and incubate overnight, about 12 - 16 hours, at 30 °C with a rotary shaker at 200 rpm.

[0212] Determine the cell density of the yeast culture. Dilute approximately 2.5x10 8 cells with 50 ml of YPD to obtain a cell suspension with a final density of approximately 5x10 6 cells / ml.

[0213] Incubate the cell suspension in the flask in an orbital shaker at 200 rpm at 30 °C for about 4 hours until the cell density reaches at least 2x10 7 cells / ml.

[0214] Denature 1.0 ml of carrier DNA in a boiling water bath for 5 min and immediately cool it in an ice water bath. Alternatively, pre-denatured carrier DNA stored at -20 °C can be used. Thaw it and keep it on ice until use.

[0215] Harvest yeast cells by centrifuging at 3,000 g for 5 min and resuspend the cell pellet in 25 ml of sterile water. At 20 °C, centrifuge the suspension at 3,000 g for 5 min to pellet the cells. Repeat this wash with an additional 25 ml of sterile water by resuspending the cells and pelleting them again by centrifugation. Resuspend the cells in 1.0 ml of sterile water.

[0216] Transfer the cell suspension to a 1.5 ml microcentrifuge tube and centrifuge at 13,000 g for 30 s. Discard the supernatant.

[0217] Resuspend the cells in 1.0 ml of sterile water and aliquot 100 μl of the suspension containing approximately 10 8 cells into 1.5 ml microcentrifuge tubes using a pipette. Use each aliquot for each transformation. Centrifuge the microcentrifuge tubes containing the suspension at 13,000 g for 30 s in a microcentrifuge to remove the supernatant. Name the microcentrifuge tubes containing the pellet "transformation tubes".

[0218] As shown in Table 7, mix the following components for each transformation reaction.

[0219] Table 7. Components of the transformation mixture for yeast transformation.

[0220] Transformation mixture components Volume (ul) PEG MW 3350 (50% (w / v)) 240 Lithium acetate, 1.0 M 36 Single-stranded carrier DNA (2.0 mg / ml) 50 Plasmid DNA in sterile water (30 ng / μl) 34 Total volume 360

[0221] Add 360 μl of the transformation mixture to each transformation tube and resuspend the cells by vigorous vortexing. A negative control without plasmid DNA can be included.

[0222] Place the transformation tubes in a water bath at 42 °C and incubate for 40 min.

[0223] Centrifuge the transformation tubes at 13,000 g for 30 s in a microcentrifuge and remove the supernatant using a micropipette. Pipette 1.0 ml of sterile water into each transformation tube. Stir the pellet with a sterile micropipette tip to disrupt the cell pellet and then vortex to resuspend the cell pellet evenly.

[0224] Plate 200 μl of the cell suspension onto an appropriate medium, such as SC / -TRP selection medium.

[0225] Incubate the agar plates at 30 °C for 3 - 4 days and count the number of colonies (transformants).

[0226] Example 6: Preparation of Yeast Cell Cultures and Expression of Enzyme

[0227] Reagents

[0228] YPD medium:

[0229] 1% (w / v) yeast extract for bacteria, 2% (w / v) peptone for bacteria, 2% (w / v) glucose. For YPD agar plates, an additional 18 g / L agar for bacteria is required.

[0230] Selective medium Synthetic complete tryptophan-deficient medium (SC / -Trp):

[0231] SC / -Trp broth was purchased from Coolaber Co., Ltd. (Beijing, China). 8 g of the medium was mixed with 900 mL of distilled water and then adjusted to pH 5.8 with 1.0 N sodium hydroxide (NaOH) and autoclaved. For agar plates, an additional 18 g / L agar for bacteria is required.

[0232] Lysis buffer:

[0233] 50 mM Tris-HCl (pH 7.4), containing 0.1 M KCl, 1 mM DTT, 10 mM PMSF and 1x protease inhibitor cocktail (PIC), without EDTA

[0234] Isolate the target single colonies (i.e., transformants) from the SC / -Trp medium containing 2% (w / v) glucose plates and inoculate them into 10 ml of SC / -Trp medium containing 2% (w / v) glucose overnight. Then harvest the cell culture by centrifugation at 3,000 g for 10 min and wash the cells twice with SC / -Trp medium and 2% galactose. Add the cell pellet to a 250-ml baffled flask (containing 50 ml of SC / -Trp medium and 2% galactose) with an initial optical density (OD600nm) of 0.1. Then incubate the culture in an orbital shaker at 28 °C at 200 rpm overnight for about 12 - 14 hours until the OD reaches about 3 - 4.

[0235] Then at 4 °C, harvest the cells by centrifugation at 8,000 rpm for 10 min. Discard the supernatant and resuspend the cell pellet in ice-cold lysis buffer. Wash the cell pellet once more and measure the biomass.

[0236] Cell lysis

[0237] Perform the following steps for preparing microsomes at 4 °C.

[0238] Resuspend the cell pellet from the previous step in ice-cold lysis buffer at a ratio of 1:3 (w:v), and transfer the cell suspension to a new tube. Add an equal amount of glass beads to the cell suspension, vortex at the highest speed for 1 min, and cool it on ice for 1 min. Repeat this step 10 - 12 times. Then examine the cells under a microscope to obtain the disruption efficiency.

[0239] Transfer the lysed or disrupted cell suspension to a new test tube, avoiding transferring any glass beads. Wash the glass beads with lysis buffer until most of the lysed or disrupted cells are removed from the glass beads. Collect all samples and proceed to the next centrifugation step.

[0240] Microsomal enzyme preparation

[0241] Reagents:

[0242] Reaction buffer:

[0243] 10 mM Tris-HCl, 10 mM MgCl2, pH 8.0, 10% glycerol

[0244] Perform the following steps for preparing microsomes at 4°C.

[0245] Centrifuge the sample at 17,000 g for 10 min to remove cell debris and unbroken cells. Then, pour the supernatant into an ultracentrifuge tube and centrifuge at 160,000 g for 1 h at 4°C. Discard the supernatant, and mix the pellet containing microsomes with reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 8.5).

[0246] Add 1x phosphate-buffered saline (PBS) containing 5% glycerol at a ratio of 1:10 (w:v). First, add 1 - 2 ml of the buffer, and resuspend the pellet with a pipette until the pellet is broken into small fragments. Transfer the microsome suspension to a pre-cooled Douncer homogenizer and add the remaining buffer. Gently homogenize the mixture with a Douncer homogenizer for 10 - 12 strokes. Measure the absorbance at 280 nm.

[0247] Example 7: In Vitro Enzyme Assay

[0248] Reagents:

[0249] Substrate solution buffer:

[0250] 50 mM Tris-HCl, 10 mM MgCl 2 , (pH 8.5), containing 2 mM oleic acid and 2 mM GPP.

[0251] Mix 100 ul of the microsomal preparation with 100 ul of substrate solution buffer (50 mM Tris-HCl, 10 mM MgCl 2 , (pH 8.5), containing 2 mM oleic acid and 2 mM GPP) to make a total volume of 200 ul. Incubate the sample at room temperature. After incubating for 18 hours at room temperature, extract the reaction mixture twice with 200 μL of ethyl acetate / formic acid (0.05% (v / v)). Combine the organic extracts of each reaction and remove the solvent using a block heater. Before LC-MS analysis, dissolve the sample in 100 μL of resuspension solution (acetonitrile / H2O / formic acid (80% / 20% / 0.05% (v / v / v))) and filter through a 0.22 μm PVDF membrane.

[0252] Example 8: LC-MS Analysis of Cannabigerolic Acid (CBGA)

[0253] Equip with an RP-C18 column (BEH130, 1.7 μm, 2.1 mm x 50 mm, Waters) for UPLC-MS (Waters H-Class with SQ detector 2). Mobile phase A is water containing 0.1% formic acid and mobile phase B is acetonitrile containing 0.1% formic acid. Gradient elution starts with 50% B held for 1 min, then increases to 90% B in 10 min, decreases to 50% B in 1 min, and is held at 50% B for 1 min. The flow rate is 0.4 ml / min and the run time is 13 min. The sample injection volume is 2 μl, and the sample tray and column oven are set to 10 °C and 30 °C, respectively. Cannabigerolic acid is detected by electrospray ionization in the negative ion mode, and the MS scan mode is SIR 359.5 m / z, with capillary voltage and cone voltage set to 3,800 V and 30 V, respectively. The desolvation gas and temperature are set to 1000 l / h and 500 °C, respectively.

[0254] Results

[0255] Figure 6AShow the GOT activities of various samples (yCAN30003 to yCAN30049) expressed in E. coli using the methods described in Examples 1-3 (where the enzymes are derived from various plants). The results showed that some samples (eCAN30003, eCAN30005, eCAN30006, eCAN30007, eCAN30008, eCAN30009, eCAN30010, eCAN30011, eCAN30013, eCAN30017, eCAN30018, eCAN30028, eCAN30029, eCAN30030, eCAN30033, eCAN30034, eCAN30035, eCAN30037, eCAN30041, eCAN30045, eCAN30046, eCAN30047, eCAN30048) indeed had different levels of positive GOT activity. Among the samples tested, sample yCAN30035 (i.e., cucumber CPHPT2) showed the highest GOT activity (about 2.8 mg / L). Figure 6B Show the GOT activities of various samples (yCAN30003 to yCAN30049) expressed in yeast using the methods described in Examples 5-8 (where the enzymes are derived from various plants). Surprisingly, when testing the in vivo GOT activities of these samples, only sample yCAN30035 (i.e., cucumber CPHPT2) showed positive GOT activity (about 40 mg / L). The other samples showed no GOT activity. In summary, sample yCAN30035 (i.e., cucumber CPHPT2) (SEQ ID NO: 85) is surprisingly useful in the production of CBGA or analogs. The artificial sequence SEQ ID NO: 179 based on cucumber CPHPT2 (SEQ ID NO: 85) with an optimized gene sequence design for yeast expression is particularly suitable for the production of CBGA or analogs by heterologous expression in yeast.

[0256] Example 9: Cloning, Expression, and Purification of Enzyme

[0257] The candidate genes were purchased as gene blocks from General Biosystems (Anhui, China) Co., Ltd. and cloned between the restriction enzyme sites NdeI (CATATG) and XhoI (C TCGAG) of pET28a(+) expressing the N-terminal His-tagged enzyme. All plasmids were transformed into BL21(DE3) and named eCAN20005 to eCAN20060 (see Table 9).

[0258] Table 8 provides a list of the protein sequences of the isoprenyltransferases of the present disclosure. Note that the following protein sequences may include sequences with or without the following polyhistidine tag at the N-terminus: MGSSHHHHHHSSGLVPRGSH. Also note that SEQ ID Nos. 310 to SEQ ID.335 are protein sequences with the corresponding native protein sequences truncated by 2 amino acids.

[0259] Prepare a protein sample according to the method described in Example 9, and test the CBGAS enzyme activity of the protein sample according to the assay of Example 10 and the LC-MS method of Example 11.

[0260] Table 9 provides a list of the gene sequences of the isoprenyltransferases of the present disclosure. Note that the following gene sequences may include sequences with or without a polyhistidine tag at the 5' end. Note that SEQ ID Nos. 472 to SEQ ID No. 583 are gene sequences optimized for expression in E. coli. Also note that SEQ ID Nos. 446 to SEQ ID No. 471 are protein sequences with the respective original gene sequences (SEQ ID Nos. 310 - 335) corresponding to the truncation in Table 8 truncated by 6 bases.

[0261] Table 8 lists the protein sequences of the isoprenyltransferases of the present disclosure.

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272] Table 9 lists the gene sequences of the isoprenyltransferases of the present disclosure.

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291] Inoculate 0.5 mL of a saturated culture in LB medium with 50 μg / mL kanamycin into 50 mL of TB medium with 50 μg / mL kanamycin. Grow the culture at 37 °C until the OD600 reaches 0.5 - 0.8, induce with 0.5 mM IPTG, and then express at 25 °C at 220 rpm for 18 hours.

[0292] Cells were harvested by centrifugation at 2500×g, washed with lysis buffer (50 mM Tris-HCl, 500 mM NaCl, [pH 8.0], 10% (v / v) glycerol), and resuspended in 10 ml of lysis buffer to an OD550 of approximately 100. At 4 °C, the cells were lysed for 10 min using an ultrasonic cell disruptor. The lysate was clarified by centrifugation at 12,000×g for 30 min at 4 °C. The supernatant containing the soluble protein fraction was recovered, filtered through a 0.45 μm filter, and bound to 0.5 mL of Ni affinity resin in a rotator (or roller) at 25 °C for 1 h. The resin was transferred to a gravity flow column. The resin was washed with 10 column volumes of wash buffer (50 mM Tris-HCl, 500 mM NaCl, (pH 8.0), 10% (v / v) glycerol and 20 mM imidazole), and then eluted with 1 mL of elution buffer (50 mM Tris-HCl, 500 mM NaCl, (pH 8.0), 10% (v / v) glycerol and 250 mM imidazole). The elution buffer was replaced with reaction buffer (50 mM HEPES (pH 7.5), 5 mM Mg) and the protein sample was concentrated using an Amicon filter column (10K cut-off). The protein sample was initially concentrated to 200 μL.

[0293] Example 10: In Vitro Enzyme Assay

[0294] The reaction conditions for the enzyme assay consisted of: 50 mM HEPES (pH = 7.5) with 5 mM MgCl2, 2 mM GPP, 2 mM oleic acid, and 1 mg / ml of the purified candidate enzyme, in a final volume of 200 μL. After incubation at room temperature for 18 h, the reaction mixture was extracted twice with 200 μL of ethyl acetate / formic acid (0.05% (v / v)). The organic extracts from each reaction were combined and the solvent was removed using a block heater. Prior to LC-MS analysis, the sample was dissolved in 100 μL of resuspension solution (acetonitrile / H2O / formic acid (80% / 20% / 0.05% (v / v / v))) and filtered through a 0.22 μm PVDF membrane.

[0295] Example 11: LC-MS Analysis of Cannabigerolic Acid (CBGA)

[0296] Equipped with an RP-C18 column (BEH130, 1.7 μm, 2.1 mm x 50 mm, UPLC-MS of Waters (Waters H-Class with SQ detector 2). Mobile phase A is water containing 0.1% formic acid, and mobile phase B is acetonitrile containing 0.1% formic acid. Gradient elution starts with 50% B maintained for 1 min, then increases to 90% B within 10 min, decreases to 50% B within 1 min, and is maintained at 50% B for 1 min. The flow rate is 0.4 ml / min, and the running time is 13 min. The sample injection volume is 2 μl, and the sample tray and column oven are set to 10 °C and 30 °C, respectively. Cannabigerolic acid is detected by electrospray ionization (ESI) in the negative ion mode, and the MS scan mode is SIR 359.5 m / z. The capillary voltage and cone voltage are 3,800 V and 30 V, respectively. The desolvation gas and temperature are set to 1000 l / h and 500 °C, respectively.

[0297] Example 12: Sequence Alignment

[0298] The protein identity of nphB with other enzymes was compared using CLUSTAL W of UniProt and LALIGN of the Expasy program, respectively.

[0299] Results

[0300] Example 13: Verification Results of CBGA by LC-MS

[0301] Figure 1 Using the method described in Example 3, the enzyme assay was verified by showing the results of titrating CBGA at four different concentrations. CBGA titration curve concentrations: (A) 10 ng / mL, (B) 100 ng / mL, (C) 1 μg / mL, (D) 10 μg / mL. Figure 2 The LC-MS chromatogram of CBGA at m / z 359.5 with a retention time of 5.43 minutes is also shown.

[0302] Table 10 and Figure 2 The CBGA activities of four control sample groups A, B, C, and D (nphB gene, wild type, and mutants) in terms of their ability to form CBGA were compared using LC-MS together. The LC-MS chromatogram shows the activity of native nphB and confirms the enhanced activities of the M23, M30, and G286S mutants. The results of the LC-MS chromatogram of the samples at m / z 359.5 with a CBGA retention time (R.T.) of approximately 5.43 minutes, and the R.T. of several isomers at approximately 3.47, 5.94, 6.30, and 8.70 min were also observed. R.T. 5.4 corresponds to CBGA.

[0303] Table 10 lists the groups and the corresponding genes

[0304] Sample Group Gene eCAN-0001 Group A nphB wt eCAN-0002 Group B nphB M23 eCAN-0003 Group C nphB M30 eCAN-0004 Group D nphB-G286S

[0305] Example 14: CBGA Yield

[0306] Table 3 provides the CBGAS activity, shown as the calculated yield of CBGA formed by the novel enzymes of the present invention. Table 3 lists the prenyltransferases with CBGA activity. Table 11 also shows that eCAN20003 (nphB-m30), eCAN20006 (SActPT06), eCAN20005 (SaPT05), eCAN20002 (nphB-m23), and eCAN20004 (nphB-G286S) have significantly higher CBGA yields compared to eCAN20001 (NphB-wt).

[0307] Table 11: CBGA Yields

[0308]

[0309]

[0310]

[0311] Example 15: Protein Identity of nphB Compared to Other Enzymes

[0312] Table 12 shows the protein identity of the enzymes of the present invention compared to nphB using CLUSTAL W of UniProt and LALIGN of the Expasy program. Table 12 indicates that the protein sequences of the enzymes of the present invention are very different from nphB, showing an overall percentage identity far below 50%. Thus, although the enzymes disclosed herein have a different structure from nphB, they have significant CBGAS activity. In addition, some of the enzymes of the present invention are surprisingly more effective in catalyzing the formation of CBGA.

[0313] Table 12: Sequence Alignments Showing Protein Identity Levels

[0314]

[0315]

[0316] Example 16: Generation of Mutants for Producing Compounds in the Cannabinoid Pathway

[0317] A pairwise sequence alignment was performed between the protein sequence of eCAN20053 (which has a plant origin) and the protein sequence of eCAN200039 (which has a fungal origin) using UNIPROT Clustal Omega to find conserved amino acids or conserved domains.

[0318] Table 13 provides mutant enzymes in which the conserved amino acids described above are replaced by other amino acids having similar chemical properties (such as polar, non-polar, charged, uncharged, etc.).

[0319] Amino acid mutation sites of mutants of eCAN200053 and eCAN200039 in Table 13.

[0320]

[0321]

[0322] Mutations on the protein sequence of eCAN20005 are listed in Table 14.

[0323] Mutations on the protein sequence of mutants of eCAN20005 in Table 14

[0324]

[0325]

[0326] Although the description refers to specific embodiments, it will be apparent to those skilled in the art that the invention can be practiced with variations of these specific details. Therefore, the invention should not be construed as limited to the claims set forth below or the embodiments described herein.

Claims

1. A method for producing cannabigerolic acid (CBGA) or an analogue thereof, the method comprising the steps of: reacting a heterologously expressed prenyltransferase with geranyl diphosphate (GPP) and an acid; wherein the amino acid sequence of the prenyltransferase is selected from SEQ ID No: 02, SEQ ID No. 199, SEQ ID No. 311, and SEQ ID No. 324, or the prenyltransferase is expressed by a nucleotide sequence selected from SEQ ID No. 337, SEQ ID No. 447, SEQ ID No. 460, SEQ ID No. 473, and SEQ ID No.

529.

2. The method according to claim 1, wherein the prenyltransferase has prenyltransferase activity and / or geranyl pyrophosphate:olivetolate geranyltransferase (GOT) activity.

3. The method according to claim 1, wherein the prenyltransferase has an activity with increased synthetic ability and reduced byproduct formation compared to the activity of native CBGAS; or has an improved reaction rate for forming CBGA compared to NphB wild type.

4. The method according to claim 1, wherein the prenyltransferase has a reaction rate for CBGA greater than 12 μg / mL.

5. The method according to claim 1, wherein the acid is selected from the group consisting of orsellinic acid (OSA), divanillic acid (DVA), apigenin, daidzein, genistein, naringenin, oleanol, oleic acid (OA), and resveratrol.

6. A method for producing cannabinoids, the method comprising the steps of: a) reacting a heterologously expressed prenyltransferase with a prenyl group donor and an acid to produce cannabigerolic acid (CBGA) or an analogue thereof; and b) reacting the CBGA or an analogue thereof with a cannabinoid synthase to form an acidic form of the cannabinoid; wherein the amino acid sequence of the prenyltransferase is selected from SEQ ID No: 02, SEQ ID No. 199, SEQ ID No. 311, and SEQ ID No. 324, or the prenyltransferase is expressed by a nucleotide sequence selected from SEQ ID No. 337, SEQ ID No. 447, SEQ ID No. 460, SEQ ID No. 473, and SEQ ID No.

529.

7. The method according to claim 6, wherein the prenyltransferase has an activity with increased synthetic ability and reduced byproduct formation compared to the activity of native CBGAS.

8. The method according to claim 6, wherein the prenyltransferase has a reaction rate for CBGA greater than 12 μg / mL.

9. The method according to claim 6, wherein the acid is selected from the group consisting of orsellinic acid (OSA), divanillic acid (DVA), apigenin, daidzein, genistein, naringenin, oleanol, oleanolic acid (OA), resveratrol, butyric acid, valeric acid, caproic acid, and heptanoic acid.

10. A recombinant microorganism engineered to produce CBGA, wherein the recombinant microorganism overexpresses an enzyme of a sequence selected from the group consisting of SEQ ID No: 02, SEQ ID No. 199, SEQ ID No. 311, and SEQ ID No.

324.

11. A recombinant microorganism engineered to produce CBGA, wherein the recombinant microorganism comprises at least one heterologous nucleotide sequence or its codon-degenerate nucleotide sequence, and the heterologous nucleotide sequence is selected from the group of nucleic acid sequences consisting of SEQ ID No. 337, SEQ ID No. 447, SEQ ID No. 460, SEQ ID No. 473, and SEQ ID No.

529.

12. An isolated polypeptide having cannabigerolic acid synthase (CBGAS) activity, wherein the polypeptide is of an amino acid sequence selected from the group consisting of SEQ ID No: 02, SEQ ID No. 199, SEQ ID No. 311, and SEQ ID No. 324; or the polypeptide is expressed from a nucleotide sequence selected from the group of nucleotide sequences consisting of SEQ ID No. 337, SEQ ID No. 447, SEQ ID No. 460, SEQ ID No. 473, and SEQ ID No.

529.

13. The isolated polypeptide according to claim 12, wherein the polypeptide is expressed in a microbial host selected from the group consisting of Escherichia coli, Yarrowia lipolytica, and Saccharomyces cerevisiae.

14. The isolated polypeptide according to claim 12, wherein the polypeptide is expressed in a Cannabis species.

15. Use of an enzyme for producing CBGA, wherein the enzyme is of a sequence selected from the group consisting of SEQ ID No: 02, SEQ ID No. 199, SEQ ID No. 311, and SEQ ID No. 324, or the enzyme is expressed from a nucleotide sequence selected from the group consisting of SEQ ID No. 337, SEQ ID No. 447, SEQ ID No. 460, SEQ ID No. 473, and SEQ ID No. 529.

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