WO2021063396A1

WO2021063396A1 - Enzymes for cannabinoids synthesis and methods of making and using thereof

Info

Publication number: WO2021063396A1
Application number: PCT/CN2020/119399
Authority: WO
Inventors: Guillaume Cottarel; Hao Cui; Jicong Cao
Original assignee: Hangzhou Enhe Biotechnology Co., Ltd.
Priority date: 2019-10-01
Filing date: 2020-09-30
Publication date: 2021-04-08
Also published as: CN114729386A

Abstract

Provided is various enzymes for cannabinoids synthesis and methods of making and using thereof. Also provided is the recombinant polypeptides having prenyl transferase activity and/or geranylpyrophosphate: olivatolate geranylttransfersae (GOT) activity and the recombinant microorganisms engineered for the production of CBGA, and the methods of producing cannabigerolic acid (CBGA) or analogs thereof comprising the step of reacting a heterologously expressed prenyltransferase enzyme with geranyl diphosphate (GPP) and an acid, such as olivetolic acid (OA).

Description

ENZYMES FOR CANNABINOIDS SYNTHESIS AND METHODS OF MAKING AND USING THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefits and priorities of U.S. Provisional Patent Application Serial No. 62/909,227, filed on 1 Oct 2019, U.S. Provisional Patent Application Serial No. 62/941,689 filed on 27 Nov 2019, and U.S. Provisional Patent Application Serial No. 62/942,198 filed on 1 Dec 2019, which are incorporated by reference herein.

REFERENCE TO SEQUENCE LISTING

This application contains a sequence listing in computer readable form, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present application relates to molecular biology, and more specifically to enzymes for cannabinoids synthesis.

BACKGROUND OF THE INVENTION

Cannabinoid compounds act on the cannabinoid receptors other targets and notably impact the release of neurotransmitters in the brain. The use of cannabinoid compounds, such as cannabidiol for medical purposes has expanded globally and with its legislation is becoming more accepted. As a result, the cannabinoid class of compounds is emerging as a novel class of pharmaceuticals. However, there are still many challenges in producing cannabinoid compounds, which are traditionally extracted and purified from plants. Furthermore, it is difficult to use currently known methods to produce a wide variety of cannabinoid analogs. Accordingly, there is a need for new ways of synthesizing and producing cannabinoid compounds.

SUMMARY OF THE INVENTION

According to some aspects, the present application relates to enzymes useful for the production of cannabinoids or cannabinoid intermediates in microbial or yeast hosts and methods of making and using such enzymes.

In certain embodiments, the claimed methods of producing cannabinoids are an improvement over currently known methods, such as plant extraction. Extraction of cannabinoids from plants involves growing and harvesting plants that naturally contain cannabinoids and then extracting the compounds using a variety of extraction methods known in the art. Due to the natural mix of cannabinoids in plants, it is often difficult to reproduce identical extraction and purification profiles for each extracted sample. This is further complicated by each plant’s unique genetic origin and growth conditions. The resulting diverse cannabinoid profiles leads to samples each having a different pharmaceutical profile -a problem from a safety or regulatory perspective. Finally, the ability to purify a highly pure single product is very challenging due to the nature of the mix of compounds with similar structure and size.

Accordingly, in certain embodiments, the present application provides enzymes useful for the production of cannabinoids in microbial or yeast hosts. Example embodiments include novel prenyl transferase enzymes and methods of making and using such enzymes in producing cannabigerolic acid (CBGA) or analogs thereof from a prenyl group donor and an acid.

In certain embodiments, the present application also provides methods of using such enzymes through the use of heterologous expression of plant and microbial genes in microbial or yeast hosts to produce compounds in the cannabinoid pathway, such as cannabigerolic acid (CBGA) or analogs thereof from a prenyl group donor and an acid.

In certain embodiments, the present application provides a method of producing cannabigerolic acid (CBGA) or analogs thereof comprising the step of reacting a prenyl transferase enzyme with a prenyl group donor and an acid.

In one embodiment, the prenyl transferase enzyme has cannabigerolic acid synthase (CBGAS) activity.

In one embodiment, the prenyl transferase enzyme is obtained from whole, purified cell extracts, or a combination thereof.

In one embodiment, the prenyl group donor is selected from the group consisting of a prenyl moiety derived from allylic isoprenyl diphosphates, including, but not limited to, dimethylallyl diphosphate (DMAPP; C5) , geranyl diphosphate (GPP; C10) and farnesyl diphos-phate (FPP; C15) .

In one embodiment, the prenyl group donor is GPP.

In one embodiment, the acid is selected from the group consisting of orsellinic (OSA) , divarinolic (DVA) , and olivetolic (OLA) , apigenin, diadzein, genestein, naringenin, olivetol, OA, and resveratrol.

In one embodiment, the acid is olivetolic acid.

In one embodiment, the prenyltransferase enzyme has activity with increased synthesis capacity and decreased by-products formation compared with the activity of native CBGAS.

In one embodiment, the prenyltransferase enzyme has a reaction rate of greater than 12 μg /mL of CBGA.

In one embodiment, the prenyltransferase enzyme has less than about 50, 45, 40, 35, 30, 25, 20, 15, 10%sequence identity to the sequence of native nphB protein.

In one embodiment, the prenyl transferase enzyme has at least about 80, 85, 90, or 95%sequence identity to all or a fragment of SEQ ID NO: 1-56.

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

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

Prenyl Group Donor	FattyAcid	CBGA Analog
GPP	olivetolic acid	CBGA
GPP	divarinolic acid	CBGAVA

In certain embodiments, provided is a method of producing a cannabinoid comprising the steps of: (1) reacting a prenyl transferase enzyme with a prenyl group donor and an acid to produce cannabigerolic acid (CBGA) or analogs thereof; and (2) reacting CBGA or analogs thereof with a cannabinoid synthase to form acidic forms of a cannabinoid.

In one embodiment, the cannabinoid synthaseis two oxidoreductase tetrahydrocannabinoic synthase (THCAS) or cannabidiolic acid synthase (CBDAS) and the cannabinoid is THC or CBD.

In certain embodiments, provided is a recombinant microorganism engineered for the production of CBGA, wherein said microorganism overexpresses a prenyl transferase enzyme having at least about 80, 85, 90, or 95%sequence identity to all or a fragment of SEQ ID NO: 1-56.

In certain embodiments, provided is an isolated polypeptide having cannabigerolic acid synthase (CBGAS) activity comprising an amino acid sequence having at least about 80, 85, 90, or 95%sequence identity to all or a fragment of SEQ ID NO: 1-56.

In one embodiment, the polypeptide is expressed in a microcrobial or a plant host, wherein the microbial host includes but not limited to E. coli, Y. lipolytica and S. cerevisiae and the plant host includes but not limited to cannabis (species &genus) .

In one embodiment, the method is an in vitro method.

In one embodiment, the method is an in vivo cell based assay.

According to some aspects, the present application relates to enzymes useful for the production of cannabinoids or cannabinoid intermediates in microbial hosts and methods of making and using such enzymes. In some embodiments, the microbial hosts are yeasts, such as Saccharomyces cerevisiae and Yarrowia lipolytica.

Accordingly, in certain embodiments, the present application provides enzymes useful for the production of cannabinoids in microbial hosts. In some embodiments, the microbial hosts are yeasts, such as Saccharomyces cerevisiae and Yarrowia lipolytica. Example embodiments include novel enzymes with cannabigerolic acid synthase (CBGAS) activity and methods of making and using such enzymes in producing cannabigerolic acid (CBGA) or analogs thereof. In some example embodiments, the enzymes are prenyl transferases. In some example embodiments, the enzymes are geranylpyrophosphate: olivatolate geranyltransferase (GOT) or analogs thereof.

In certain embodiments, the present application also provides methods of using such enzymes through the use of heterologous expression of genes encoding such enzymes in microbial hosts to produce compounds in the cannabinoid pathway, such as cannabigerolic acid (CBGA) or analogs thereof from a prenyl group donor and an acid.

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

In one embodiment, the enzyme is a prenyl transferase enzyme which reacts with a prenyl group donor and an acid.

In one embodiment, the prenyl transferase enzyme has geranylpyrophosphate: olivatolate geranyltransferase (GOT) activity.

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

In one embodiment, the prenyl group donor is GPP.

In one embodiment, the acid is olivetolic acid.

In one embodiment, the prenyl group donor is GPP, the fatty acid is selected from a group consisting of olivetolic acid, divarinolic acid, butanoic acid, pentanoic acid, hexanoic acid and heptanoic acid, and the CBGA analog is selected from a group consisting of CBGA and CBGAVA.

In certain embodiments, provided is a method of producing cannabigerolic acid (CBGA) or analogs thereof comprising the step of reacting a heterologously expressed prenyltransferase enzyme having geranylpyrophosphate: olivatolate geranyltransferase (GOT) activity with GPP and OA.

In one embodiment, the prenyltransferase enzyme is heterologously expressed in yeast.

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

In one embodiment, the prenyl transferase enzyme has at least about 80, 85, 90, or 95%sequence identity to all or a fragment of SEQ ID NO: 57-103.

In certain embodiments, provided is a method of producing a cannabinoid comprising the steps of: (1) reacting a heterologously expressed prenyl transferase enzyme with a prenyl group donor and an acid to produce cannabigerolic acid (CBGA) or analogs thereof; and (2) reacting CBGA or analogs thereof with a cannabinoid synthase to form acidic forms of a cannabinoid.

In one embodiment, the cannabinoid synthase is two oxidoreductase tetrahydrocannabinoic synthase (THCAS) or cannabidiolic acid synthase (CBDAS) and the cannabinoid is THC or CBD.

In one embodiment, the method is an in vitro method.

In one embodiment, the method is an in vivo cell based assay.

In certain embodiments, provided is a recombinant microorganism engineered for the production of CBGA, wherein said microorganism overexpresses a prenyl transferase enzyme having at least about 80, 85, 90, or 95%sequence identity to all or a fragment of SEQ ID NO: 57-103.

In certain embodiments, provided is a recombinant microorganism comprising at least one heterologous nucleotide sequence having at least about 80, 85, 90, or 95%sequence identity to SEQ ID NO: 104-197 or a codon degenerate nucleotide sequence thereof.

In certain embodiments, provided is an isolated polypeptide having cannabigerolic acid synthase (CBGAS) activity comprising an amino acid sequence having at least about 80, 85, 90, or 95%sequence identity to all or a fragment of SEQ ID NO: 57-103.

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

In one embodiment, the polypeptide is expressed in Y. lipolytica or S. cerevisiae.

Accordingly, in certain embodiments, the present application provides enzymes useful for the production of cannabinoids in microbial hosts. Example embodiments include novel prenyl transferase enzymes and methods of making and using such enzymes in producing cannabigerolic acid (CBGA) or analogs thereof from a prenyl group donor and an acid.

In certain embodiments, the present application also provides methods of using such enzymes through the use of heterologous expression of plant and microbial genes in microbial hosts to produce compounds in the cannabinoid pathway, such as cannabigerolic acid (CBGA) or analogs thereof from a prenyl group donor and an acid. Various embodiments provide engineered enzymes having improved features for producing compounds in the cannabinoid pathway. Such improved features include, but are not limited to, better kinetics (e.g. kM and kCAT) , higher resistance to solvent, ability to function at higher temperatures, and improved ability to use different substrates and groups donors.

In certain embodiments, provided is a method of producing cannabigerolic acid (CBGA) or analogs thereof comprising the step of reacting a prenyl transferase enzyme with a prenyl group donor and an acid.

In one embodiment, the prenyl group donor is GPP.

In one embodiment, the acid is olivetolic acid.

In one embodiment, the prenyltransferase enzyme has activity with significant synthesis capacity and/or decreased by-products formation.

In one embodiment, the prenyl transferase enzyme has at least about 80, 85, 90, or 95%sequence identity to all or a fragment of SEQ ID NO: 198-253.

In certain embodiments, provided is a recombinant microorganism engineered for the production of CBGA, wherein said microorganism overexpresses a prenyl transferase enzyme having at least about 80, 85, 90, or 95%sequence identity to all or a fragment of SEQ ID NO: 198-335.

In certain embodiments, provided is an isolated polypeptide having cannabigerolic acid synthase (CBGAS) activity comprising an amino acid sequence having at least about 80, 85, 90, or 95%sequence identity to all or a fragment of SEQ ID NO: 198-335.

In one embodiment, the method is an in vitro method.

In one embodiment, the method is an in vivo cell based assay.

In certain embodiments, provided is a recombinant polypeptide comprising an amino acid sequence with 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 to a conserved amino acid between SEQ ID NO: 246 and SEQ ID NO: 232.

In one embodiment, the conserved amino acid is replaced with an amino acid having similar chemical property with the conserved amino acid.

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

In one embodiment, 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, 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.

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

In one embodiment, 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.

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

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.

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

In one embodiment, the prenyltransferase enzyme has prenyl transferase activity and/or geranylpyrophosphate: olivatolate geranylttransfersae (GOT) activity.

In one embodiment, the prenyltransferase enzyme has activity with increased synthesis capacity and decreased by-products formation compared with the activity of native CBGAS; or has an improved reaction rate for forming CBGA compared to NphB wild type.

In one embodiment, the acid is selected from the group consisting of orsellinic (OSA) , divarinolic (DVA) , and olivetolic (OLA) , apigenin, diadzein, genestein, naringenin, olivetol, olivetolic acid (OA) , and resveratrol.

In certain embodiments, the prenyltransferase enzyme has 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 prenyltransferase 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 nucleic acid sequence selected from the group consisting of SEQ ID No: 104-197, 336-583.

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

In certain embodiments, provided is a method of producing a cannabinoid, comprising the steps of: (1) reacting a heterolously expressed prenyl transferase enzyme with a prenyl group donor and an acid to produce cannabigerolic acid (CBGA) or analogs thereof; and (2) reacting the CBGA or analogs thereof with a cannabinoid synthase to form acidic forms of a cannabinoid.

In one embodiment, wherein the prenyltransferase enzyme has activity with increased synthesis capacity and decreased by-products formation compared with the activity of native CBGAS.

In one embodiment, wherein the prenyltransferase enzyme has a reaction rate of greater than 12 μg/mL of CBGA.

In one embodiment, wherein the acid is selected from the group consisting of orsellinic (OSA) , divarinolic (DVA) , and olivetolic (OLA) , apigenin, diadzein, genestein, naringenin, olivetol, olivetolic acid (OA) , resveratrol, butanoic acid, pentanoic acid, hexaonic acid and heptanoic acid.

In certain embodiments, 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 ID NO: 01-103, 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 SEQ ID No: 104-197, 336-583.

In certain embodiments, provided is a recombinant microorganism engineered for the production of CBGA, wherein said microorganism overexpresses an enzyme having 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 ID NO: 01-103 and 198-335.

In certain embodiments, provided is a recombinant microorganism engineered for the production of CBGA, wherein said microorganism comprises at least one heterologous nucleotide sequence having 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 or a codon degenerate nucleotide sequence thereof.

In certain embodiments, provided is an isolated polypeptide having cannabigerolic acid synthase (CBGAS) activity; 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.

In one embodiment, wherein the polypeptide is expressed in a microcrobial host selected from the group consisting of E. coli, Y. lipolytica and S. cerevisiae.

The isolated polypeptide of claim 15, wherein the polypeptide is expressed in Cannabis sp.

In certain embodiments, provided is a use of an enzyme for the production of CBGA, 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 ID NO: 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 selected from the group consisting of SEQ ID No: 104-197 and 336-583.

In certain embodiments, provided is a recombinant polypeptide, comprising an amino acid sequence with 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.

In one embodiment, wherein said 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.

In certain embodiments, provided is a recombinant polypeptide, comprising an amino acid sequence with 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.

In one embodiment, wherein said 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.

In one embodiment, wherein 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 FIGURES

FIG. 1 shows the results of CBGA titrated at four difference concentrations, according to an example embodiment.

FIG. 2 compares CBGAS activity of four control samples (nphB gene) and two enzymes of the present invention in their ability to form CBGA using LC-MS, according to an example embodiment.

FIG. 3 details the biological pathway of cannabinoid production, according to an example embodiment.

FIG. 4 details the biological pathway of cannabinoid production, according to another example embodiment.

FIG. 5 compares GOT activity and the CBGA/CBGA-isomer ratio of control sample (nphB gene) and two enzymes (eCAN20005 and eCAN20006) of the present invention in their ability to form CBGA using LC-MS, according to an example embodiment.

FIG. 6A compares GOT activities of various enzymes (yCAN30003 to yCAN30049) of the present invention in their ability to form CBGA using LC-MS expressed in E. coli, according to an example embodiment. FIG. 6B compares GOT activities of various enzymes (yCAN30003 to yCAN30049) of the present invention in their ability to form CBGA using LC-MS expressed in yeast, according to an example embodiment.

DETAILED DESCRIPTION

Definitions

As used herein and in the claims, “comprising” means including the following elements but not excluding others.

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

“Prenyl transferase enzymes” or “prenyl transferase” refers to aromatic prenyltransferases (PTases) that catalyze the transfer of a, for example, C5 (dimethylallyl) , C10 (geranyl) or C15 (farnesyl) prenyl group derived from the corresponding isoprenyl diphosphate metabolites onto a variety of electron-rich aromatic acceptors.

“Prenyl groups” refers to a functional group that appear in a wide variety of bioactive natural products of microbial and plant origin, including amino acids, stilbenes, alkaloids, polyketides and phenylpropanoids such as flavonoids, creating natural product hybrids with altered or enhanced bioactivities.

“Prenylation” refers to the transfer of a prenyl group onto an electron-rich aromatic acceptor. Prenylation appears in many cases to provide a higher level of bioactivity compared to the non-prenylated precursor, such as by increasing affinity for biological membranes and interactions with cellular targets.

“GPP Pathway” refers to a pathway for producing geranyldiphospahe (GPP) via MVA or MEP pathways. Microbes naturally produce GPP via the MVA or MEP pathways in yeast and bacteria, respectively.

“OA pathway” or “Olivetolic Acid Pathway” refers to the pathway of synthesizing OA whereing hexanoic acid (a simple fatty acid naturally produced in yeast) is converted to hexanoyl-CoA by hexanoyl-CoA synthase. The synthesis of OA is the two-step fusion of hexanoyl-CoA and 3 malonyl-CoA, the enzyme responsible for these reactions are olivetol synthase (OLS) and olivetolic acid cyclase (OAC) . The source of these coding sequences are from C. sativa. Hexanoic acid feeding by the addition in the growth medium has shown increased of OA production.

“Cannabigerolic acid” or “CBGA” refers to a molecule that is produced from olivetolic acid and the mevalonate-pathway intermediate geranyl pyrophosphate (GPP) by a geranylpyrophosphate: olivetolate geranyltransferase (GOT) . CBGA is also the precursor to Δ -tetrahydrocannabinolic acid (THCA) , cannabidiolic acid (CBDA) and numerous other cannabinoids.

“CBGA Pathway” refers to the formation of cannabigerolic acid (CBGA) from the fusion of GPP and olivetolic acid via the activity of the cannabigerolic acid synthase (CBGAS) displayed by the Cannabis sativa geranylpyrophosphate: olivatolate geranylttransfersae activity (GOT) and the prenyltransferase activity of the nphB enzyme.

“nphB” or “nphB enzyme” refers to an aromatic prenyltransferase that catalyzes the attachment of a 10-carbon geranyl group to aromatic substrates, such as CBGA. This enzyme class drives the first biochemical step of the cannabinoid pathway to form CBGA.

“geranylpyrophosphate: olivatolate geranyltransferase” or "GOT” refers to a prenyl transferase that is part of the cannabinoids biosynthetic pathway of the plant Cannabis sativa which catalyzes the reaction between geranyl diphosphate and 2, 4-dihydroxy-6-pentylbenzoate to form cannabigerolate and diphosphate.

“codon degenerate nucleotide sequence” refers to a nucleotide sequence that encodes for the same set of amino acids in a polypeptide sequence as another nucleotide sequence having different codons. For example, a codon degenerate nucleotide sequence for a sequence comprising GAA, which encodes for glutamic acid, would be the same sequence with GAA replaced by GAG, which also codes for glutamic acid.

Figure 3 describes the biosynthetic pathway of cannabinoids in Cannabis sativa. The precursor GPP and OA are converted to the central intermediate of the cannabinoid pathway CBGA. CBGA is converted by two oxidoreductase tetrahydrocannabinoic synthase (THCAS) and cannabidiolic acid synthase (CBDAS) to the acidic forms of THC and CBD. Heterologously expressed enzymes are shown in green. Intermediates of the primary metabolism are displayed in grey (Zirpel et al., 2017) .

Figure 4 describes the biosynthetic pathway of cannabinoids in Cannabis sativa. The precursor GPP and OA are converted to the central intermediate of the cannabinoid pathway CBGA. CBGA is converted by two oxidoreductase tetrahydrocannabinoic synthase (THCAS) and cannabidiolic acid synthase (CBDAS) to the acidic forms of THC and CBD.

provided herein are enzymes that catalyze the same reaction at a surprisingly higher reaction rate than NphB.

In certain embodiments, provided herein are 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 make such enzymes, and methods of using such enzymes to make cannabinoids or intermediates for making cannabinoid.

In some embodiments, the enzymes have improved reactions rates for forming CBGA compared to NphB wild type (wt) . In some embodiments, the enzymes are selected from those listed in Table 1 herein.

EXAMPLES

Cloning, Expressing, and Purifying Prenyl Transferase Enzymes

In certain embodiments, the gene candidates were expressed in E. coli, protein extract made, enzyme purification via affinity using the HIS tag delivered purified enzymes used for testing to form CBGA. In yet other example embodiments, the gene candidates are expressed in yeast. The expressed protein extract is purified and used for testing. Example embodiments demonstrating specific methods of cloning, expressing, and purifying enzymes of the present disclosure are described in the Examples herein.

Methods of Making Cannabinoids

Cannabinoids can be made using heterologous expression in microbes or yeast. A microorganism can be genetically engineered to express cannabinoids or cannabinoids precursor molecules. Methods of expressing cannabinoid compounds heterologously is known in the art and are as described, for example, in Carvalho et al., 2017, hereby incorporated by reference.

Figure 3 shows a chart which details the biological pathway of cannabinoid production, including precursors such as CBGA, which is formed from olivetolic acid (OA) and geranyl diphosphate (GPP) via the prenyltransferase enzyme NphB. Prenylation of OA with NphB is non-specific and generates 2-O-geranyl olivetolate as a side product (Valliere et al., 2019) . Prenylation can be done by methods known to one of skill in the art, and include, but are not limited to, Valliere et al, 2019 and Luo et al., 2019, hereby incorporated by reference.

Figure 4 shows a chart which details the biological pathway of cannabinoid production, including precursors such as CBGA, which is formed from olivetolic acid (OA) and geranyl diphosphate (GPP) via the CBGAs class of enzymes notably represented by prenyltransferase enzyme NphB family. Prenylation of OA with NphB is non-specific and generates 2-O-geranyl olivetolate as a side product (Valliere et al., 2019) . Prenylation can be done by methods known to one of skill in the art, and include, but are not limited to, Valliere et al, 2019 and Luo et al., 2019, hereby incorporated by reference.

EXAMPLE 1: Cloning, Expression and Purification of Enzymes

The candidate gene was purchased as a gene block from General Biosystems (Anhui, China) Corporation Limited, and cloned between the restricted endonuclease site NdeI (CATATG) and XhoI (C TCGAG) of pET28a (+, which expressed N-terminal His-tagged enzyme. All plasmids were transformed into BL21 (DE3) , and named: eCAN20005 to eCAN20060 (See Table 1) .

0.5 mL of the saturated culture in LB media with 50 μg/mL kanamycin was seeded 50 mL TB media with 50 μg/mL kanamycin. The cultures were grown to an OD600 of 0.5–0.8 at 37 ℃ and induced with 0.5 mM IPTG, and then expressed at 25 ℃ in 220rpm for 18 hrs.

The cells were harvested by centrifugation at 2500×g, and washed with lysis buffer (50 mM Tris-HCl, 500 mM NaCl, [pH 8.0] , 10% (v/v) glycerol) , and resuspended in 10ml lysis buffer to an OD550 about of 100. Cells were lysed using Ultrasonic Cell Disruptor for 10min at 4℃. The lysate was clarified by centrifugation at 12,000×g for 30 minutes at 4℃. The supernatant containing soluble protein fraction is recovered and filtered through a 0.45 μm filter, and bound with 0.5 mL of Ni affinity resin in Rotator (or Roller) at 25℃ for 1 hr. 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) followed by elution 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 substitution of elution buffer and concentration of the protein sample with reaction buffer (50mM HEPES (pH 7.5) , 5mM Mg) via Amicon filter column (10K cutoff) . The protein sample was initially concentrated to 200 μL.

EXAMPLE 2: In vitro Enzyme Assay

Reaction conditions for enzyme assays consisted of 50 mM HEPES with 5 mM MgCl2 (pH=7.5) , 2 mM GPP, 2 mM olivetolic acid, and 1 mg/ml purified candidate enzyme in a final volume of 200 uL. After incubation at room temperature for 18 hrs, the reaction mixture was extracted 2 times with 200 μL of ethyl acetate/formic acid (0.05% (v/v) ) . The organic extract of each reaction was pooled, and the solvent was removed using block heater. The samples were dissolved in 100 μL resuspension solution (acetonitrile/H2O/formic acid (80%/20%/0.05% (v/v/v) ) ) and filtered with 0.22μm PVDF membrane before LC-MS analysis.

Figure 1 validates the enzyme assay by showing the results of CBGA titrated at four difference concentrations. CBGA Titration curve Concentration: (A) 10ng/mL (B) 100ng/mL (C) 1ug/mL (D) 10 ug/mL. Table 2 also shows that LC-MS chromatogram of CBGA at m/z 359.5, having a retention time of 5.43 minutes.

EXAMPLE 3: LC-MS Analysis of Cannabigerolic acid (CBGA)

UPLC-MS (Waters H-class with SQ Detector 2) equipped with RP-C18 column (BEH130,

1.7 μm, 2.1mm x 50mm, Waters) . Mobile phase A is water with 0.1%formic acid and mobile phase B is acetonitrile with 0.1%formic acid. Gradient elution starts from 50%B hold 1 min, then increase to 90%B in 10 min, decrease to 50%B in 1min, and hold at 50%B for 1min. The flow rate is 0.4 ml/min, run time 13 min. Sample injection volume is 2 μl, and the sample tray and column oven were set to 10 ℃ and 30 ℃, respectively. Cannabigerolic acid was detected by electrospray ionization in the negative ion mode and MS scan mode was SIR 359.5 m/z, capillary and cone voltage were 3,800 V and 30 V, respectively. Desolvation gas and temperature were set to 1000 l/h and 500 ℃, respectively.

EXAMPLE 4

RESULTS

The protein samples from Table 1 (below) were prepared according to the method described in Example 1, tested for CBGAS enzyme activity according to the assay of Example 2 and LC-MS method of Example 3.

Table 1 provides a list of protein sequences for prenyltransferase enzymes that may have improved reactions rates for forming CBGA compared to NphB wt. Note that the sequences below have included the following polyhistidine-tag at the N-terminus: MGSSHHHHHHSSGLVPRGSH.

Table 1

Table 2

Sample	Panel	gene
RCAN-0001	Panel A	nphB wt
RCAN-0002	Panel B	nphB M23
RCAN-0003	Panel C	nphB M30
RCAN-0004	Panel D	nphB-G286S
RCAN-0006	Panel E	SActPT06

Table 2 together with Figure 2 compare CBGAS activity of four control samples Panels A, B, C, and D (nphB genes, wild type and mutants) and a novel enzyme (Panel E: SActPT06) in their ability to form CBGA using LC-MS. The LC-MS chromatograms showed activity for the native nphB and confirms the enhanced activity for the M23, M30 and G286S mutants. It showed that SActPT06 also forms CBGA. LC-MS chromatograms result of samples at m/z 359.5, CBGA retention time is 5.43 minute, several isomers, R.T. 3.47, 5.94, 6.30 and 8.70 min were observed in our samples. R.T. 5.4 corresponds to CBGA.

Table 3 provides CBGAS activity, shown in the form of calculated yield of CBGA formed of new enzymes of the present invention. Surprisingly, results showed that several prokaryotic enzymes expressed in E. coli have CBGAS activities (or GOT activities) and some of the 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) .

Figure 5 shows the GOT activity of the control sample (nphB, wild type) and the two novel enzymes (eCAN2005 and eCAN2006) in their ability to form CBGA using LC-MS. The LC-MS chromatograms showed activity for the native nphB and confirms the activity for the samples eCAN20005 and eCAN2006. It showed that SaPT05 and SActPT06 also forms CBGA. LC-MS chromatograms results showed that at m/z 359.5, CBGA retention time is 5.43 minute, several isomers, R.T. 3.47 and 5.90 min were observed in the control and the samples. Results also showed that the CBGA/CBGA-isomer ratio of the samples eCAN20005 and eCAN2006 is higher than that of NpHB, indicating that both enzymes shows higher specificity than the control and thus produces more of the desired isomer form. In summary, sample eCAN20005 (i.e., SaPT05 of Streptomyces antibioticus) (SEQ ID NO: 01) and eCAN20006 (i.e., SActPT06 of Streptomyces sp. ) (SEQ ID NO: 02) are surprisingly useful in producing CBGA or analogs.

Table 3: CBGA yield

EXAMPLE 4: Protein Identify of nphB compared with other enzymes and mutants

Table 4 shows protein identity of the enzymes of the present invention compared with nphB using the CLUSTAL W from UniProt and LALAIGN from Expasy programs. Table 4 demonstrates that the protein sequences of the enzymes of the present invention are very different from nphB, showing overall percent identity well under 50%. Accordingly, the enzymes disclosed herein have surprising high CBGAS activity despite the structural difference from nphB. Furthermore, some of the present enzymes are surprisingly more efficient in catalyzing the formation of CBGA.

Table 4: Sequence alignment showing Level of Protein Identity

EXAMPLE 5: Cloning and Yeast Transformation

Cloning

The candidate genes are purchased as a gene block from General Biosystems (Anhui, China) Corporation Limited, and cloned between the restricted endonuclease site EcoRI (GAATTC) and SpeI (ACTAGT) of pESC-TRP (which contains TRP1 yeast-selectable markers) . All plasmids are transformed into at least one yeast strain, and named: yCAN20003 to yCAN20053 (See Table 5 below) .

Table 5 provides a list of protein sequences for prenyl transferase enzymes of the present disclosure.

The protein samples from Table 5 are prepared according to the methods described in Examples 5 and 6, tested for CBGAS enzyme activity according to the assay of Examples 7 (A) , 7 (B) and LC-MS method of Example 8.

Table 6 provides a list of gene sequences for prenyl transferase enzymes of the present disclosure. Note that SEQ ID No. 151 to SEQ ID No. 197 are gene sequences optimized for expression in yeast corresponding to their native counterparts SEQ ID No. 104 to SEQ ID No. 150 respectively.

Table 5 List of protein sequences for prenyl transferase enzymes of the present disclosure.

Table 6 List of gene sequences for prenyl transferase enzymes of the present disclosure.

Yeast Transformation

Reagents for Yeast Transformation

YPD medium:

(1% (w/v) Bacto yeast extract, 2% (w/v) Bacto peptone, 2% (w/v) glucose. For YPD agar plates, require additional 18 g/L of Bacto Agar.

Selection medium Synthetic complete drop-out Tryptophan medium (SC/-Trp) :

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

Lithium acetate (1.0 M) , Dissolved:

102 g of lithium acetate dihydrate is dissolved in 100 ml of water in a bottle, autoclaved for 15min. Alternatively, the solution may be filter-sterilized using a filter unit (Nalgene) and a vacuum pump. The solution is stored at room temperature (around 20 ℃) .

PEG MW 3350 (50%w/v) :

50 g of PEG 3350 is added in about 30 ml of distilled or deionized water in a 150 ml beaker. The mixture is stirred until it dissolves. The solution is gently heated with a hot plate to aid dissolving if necessary. The volume of the solution is made up to 100ml in a 100ml measuring cylinder and the solution is mixed thoroughly. The solution is transferred to a glass storage bottle and autoclaved for 15min. Alternatively, the solution may be filter sterilized using using a filter unit (Nalgene) and a vacuum pump. The solution may be sealed and stored at room temperature for a few months.

Single-stranded carrier DNA (2.0 mg/ml)

200 mg of salmon sperm DNA is fully dissolved in 100ml of sterile TE buffer (10mMTris–HCl, 1mM Na ₂EDTA, pH 8.0) using a magnetic stir plate at 4℃ for a few hours. 1.0ml of the solution is aliquoted into 1.5 ml microcentrifuge tubes and the remainder may be aliquoted in 15 ml screw-capped plastic centrifuge tubes and stored at -20 ℃. The carrier DNA is denatured in a boiling water bath for 5min and chilled immediately in an iced water bath before use.

Yeast Transformation Procedure

A single colony of a yeast strain grown from a fresh YPD plate is inoculated into 5 mL of YPD medium with a sterile inoculation loop and incubated overnight with a rotary shaker at 200rpm at 30℃ for around 12-16 hours.

The cell density of the yeast culture is determined. Around 2.5 x 10 ⁸ cells are diluted with 50 ml of YPD to obtain a cell suspension with a final density of around 5 x 10 ⁶ cells/ml.

The cell suspension in a flask is incubated in a shaking incubator at 200rpm at 30℃ around 4 hours until the cell density reached at least 2 x 10 ⁷ cells/ml.

1.0ml carrier DNA is denatured in a boiling water bath for 5 min. and chilled immediately in an iced water bath. Alternatively, pre-denatured carrier DNA stored at -20℃ may be used, thawed and kept on ice until use.

The yeast cells are harvested by centrifugation at 3,000g for 5 min. and the cell pellet is resuspended in 25ml of sterile water. The suspension is centrifuged at 3,000g for 5 min. at 20℃ to pellet the cells. This washing is repeated with another 25ml of sterile water by resuspending the cells and pelleting them again by centrifugation. The cells are resuspended in 1.0 ml of sterile water.

The cell suspension is transferred to 1.5ml microcentrifuge tube and centrifuged for 30 sec. at 13,000g and the supernatant is discarded.

The cells are resuspended in 1.0 ml of sterile water and 100μl suspension containing around 10 ⁸ cells is aliquoted into 1.5 ml microcentrifuge tubes with pipette. Each aliquot is used for each transformation. The microcentrifuge tubes containing the suspension are centrifuged in a microcentrifuge at 13,000g for 30 sec to remove the supernatant. The microcentrifuge tubes containing the pellet are named “transformation tubes” .

The following components are mixed for each transformation reaction as shown in Table 7.

Table 7. Transformation mix components for yeast transformation.

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

360 ml of transformation mix is added to each transformation tube and the cells are resuspended by vortexing vigorously. A negative control without plasmid DNA may be included.

The transformation tubes are placed in a water bath at 42 ℃ and incubated for 40 min.

The transformation tubes are centrifuged at 13,000g for 30 sec. in a microcentrifuge and the supernatants are removed with a micropipettor. 1.0 ml of sterile water is pipetted into each of the transformation tube. The pellets are stirred with a sterile micropipette tip to break the cell pellets and then vortexed to resuspend the cell pellets uniformly.

200ml of the cell suspensions are plated onto an appropriate medium such as SC/-TRP selection medium.

The agar plates are incubated at 30℃ for 3-4 days and the number of colonies (transformants) are counted.

EXAMPLE 6: Preparing Yeast Cell Cultures and Expressing the Enzymes

Reagents

YPD medium:

Selection medium Synthetic complete drop-out Tryptophan medium (SC/-Trp) :

Lysis buffer:

50 mM Tris-HCl (pH 7.4) containing 0.1 M KCl, 1 mM DTT, 10 mM PMSF and 1 x Protease Inhibitor Cocktail (PIC) , EDTA-free

A single colony in interest (i.e. a transformant) is isolated from a SC/-Trp medium containing 2% (w/v) glucose plate and inoculated in 10 ml of SC/-Trp media containing 2% (w/v) glucose overnight. The cell culture is then harvested by centrifugation at 3,000g for 10 min. and the cells are washed with SC/-Trp medium and 2%galactose twice. The cell pellet is added to a 250-ml baffled flask containing 50 ml SC/-Trp medium and 2%galactose with an initial optical density (OD 600nm) of 0.1. The culture is then incubated overnight in an orbital shaker at 28℃ 200rpm for around 12-14 hours until the OD reached around 3-4.

The cells are then harvested by centrifugation at 8,000 rpm for 10 min at 4 ℃. The supernatant is discarded and the cell pellet is resuspended with ice-cold lysis buffer. The cell pellet is washed once more time and the biomass is measured.

Cell Lysis

The following steps for preparation of microsomes are carried out at 4℃.

The cell pellet from the previous step is resuspended with ice-cold lysis buffer with the ratio 1: 3 (w: v) and the cell suspension is transferred into a new tube. Equal amount of glass beads are added to the cell suspension and vortexed at the highest speed for 1 min. and cool it down on ice for 1 min. This step is repeated for 10-12 times. The cells are then examined under microscope for accessing the disruption efficiency.

Lysed or broken cell suspension is transferred into a new tube, avoiding transferring any glass beads. The glass beads are washed with the lysis buffer until most of the lysed or broken cells are removed from the glass beads. All of samples are collected and proceed to the next centrifugation step.

Microsomal Enzymes Preparation

Reagents:

Reaction buffer:

10 mM Tris-HCl, 10 mM MgCl2, pH 8.0, 10%glycerol

The following steps for preparation of microsomes are carried out at 4℃.

Samples are centrifuged at 17,000 g for 10 min to remove the cell debris and unbroken cells. The supernatants are then poured into ultracentrifuge tubes and centrifuged for 1 h at 160,000 g at 4 ℃. The supernatants are discarded and the pellets containing microsomes are mixed with reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 8.5) .

1x phosphate buffered saline (PBS) containing 5%glycerol is added with a ratio of 1: 10 (w: v) . 1-2ml of the buffer is added first and the pellet is re-suspended with a pipette until the pellet breaks into small fragments. The microsome suspension is transferred into a pre-chilled Douncer or Dounce homogenizer and the rest of the buffer is added. The mixture is gently homogenized with 10-12 stroke of the Dounce homogenizer. The absorbance at 280nm is measured.

EXAMPLE 7: In vitro Enzyme Assay

Reagents:

Substrate solution buffer:

50 mM Tris-HCl, 10 mM MgCl ₂, (pH 8.5) containing 2mM olivetolic acid and 2mM GPP.

100ul microsomal preparations are mixed with 100ul substrate solution buffer (50 mM Tris-HCl, 10 mM MgCl ₂, (pH 8.5) containing 2mM olivetolic acid and 2mM GPP) to make up a total volume of 200ul. The samples are incubated at at room temperature. After incubation at room temperature for 18 hrs, the reaction mixture is extracted 2 times with 200 μL of ethyl acetate/formic acid (0.05% (v/v) ) . The organic extract of each reaction is pooled, and the solvent is removed using block heater. The samples are dissolved in 100 μL resuspension solution (acetonitrile/H2O/formic acid (80%/20%/0.05% (v/v/v) ) ) and filtered with 0.22μm PVDF membrane before LC-MS analysis.

EXAMPLE 8: LC-MS Analysis of Cannabigerolic acid (CBGA)

1.7 μm, 2.1mm x 50mm, Waters) . Mobile phase A is water with 0.1%formic acid and mobile phase B is acetonitrile with 0.1%formic acid. Gradient elution starts from 50%B hold 1 min, then increase to 90%B in 10 min, decrease to 50%B in 1min, and hold at 50%B for 1min. The flow rate is 0.4 ml/min, run time 13 min. Sample injection volume is 2 μl, and the sample tray and column oven are set to 10 ℃ and 30 ℃, respectively. Cannabigerolic acid is detected by electrospray ionization in the negative ion mode and MS scan mode is SIR 359.5 m/z, capillary and cone voltage are 3,800 V and 30 V, respectively. Desolvation gas and temperature are set to 1000 l/h and 500 ℃, respectively.

RESULTS

Figures 6A showed the GOT activities of various samples (yCAN30003 to yCAN30049) with enzymes originated from various plants, expressed in E. coli using methods described in EXAMPLES 1 -3. Results showed that some of the samples do have positive GOT activities in various levels (eCAN30003, eCAN30005, eCAN30006, eCAN30007, eCAN30008, eCAN30009, eCAN30010, eCAN30011, eCAN30013, eCAN30017, eCAN30018, eCAN30028, eCAN30029, eCAN30030, eCAN30033, eCAN30034, eCAN30035, eCAN30037, eCAN30041, eCAN30045, eCAN30046, eCAN30047, eCAN30048) . Sample yCAN30035 (i.e., CPHPT2 of Cucumis sativus) showed the highest GOT activity (about 2.8mg/L) among the samples tested. Figure 6B showed the GOT activities of various samples (yCAN30003 to yCAN30049) with enzymes originated from various plants, expressed in yeast using methods described in EXAMPLES 5 -8. Surprisingly, when these samples were tested for the GOT activity in vivo, only sample yCAN30035 (i.e., CPHPT2 of Cucumis sativus) showed positive GOT activity (about 40mg/L) . Other samples showed no GOT activity. In summary, sample yCAN30035 (i.e., CPHPT2 of Cucumis sativus) (SEQ ID NO: 85) is surprisingly useful in producing CBGA or analogs. Artificial sequence SEQ ID NO: 179 based on CPHPT2 of Cucumis sativus (SEQ ID NO: 85) with optimized gene sequence design for yeast expression is particularly useful in producing CBGA or analogs by heterologous expression in yeast.

EXAMPLE 9: Cloning, Expression and Purification of Enzymes

The candidate gene is purchased as a gene block from General Biosystems (Anhui, China) Corporation Limited, and cloned between the restricted endonuclease site NdeI (CATATG) and XhoI (C TCGAG) of pET28a (+, which expressed N-terminal His-tagged enzyme. All plasmids are transformed into BL21 (DE3) , and named: eCAN20005 to eCAN20060 (See Table 9) .

Table 8 provides a list of protein sequences for prenyltransferase enzymes of the present disclosure. Note that the protein sequences below may include sequences with or without the following polyhistidine-tag at the N-terminus: MGSSHHHHHHSSGLVPRGSH. Also note that SEQ ID No. 310 to SEQ ID. 335 are protein sequences with 2 amino acids truncated from the corresponding native protein sequences.

The protein samples are prepared according to the method described in Example 9, tested for CBGAS enzyme activity according to the assay of Example 10 and LC-MS method of Example 11.

Table 9 provides a list of gene sequences for prenyl transferase enzymes of the present disclosure. Note that the gene sequences below may include sequences with or without the gene sequence for polyhistidine-tag at the 5’ end. Note that SEQ ID No. 472 to SEQ ID No. 583 are gene sequences optimized for expression in E. coli. Also note that SEQ ID No. 446 to SEQ ID No. 471 are protein sequences with 6 bases truncated from the respective original gene sequences corresponding to the 2 truncated amino acids (SEQ ID No. 310-335) in Table 8.

Table 8 listed protein sequences for prenyltransferase enzymes of the present disclosure.

Table 9 listed gene sequences for prenyltransferase enzymes of the present disclosure.

0.5 mL of the saturated culture in LB media with 50 μg/mL kanamycin is seeded 50 mL TB media with 50 μg/mL kanamycin. The cultures are grown to an OD600 of 0.5–0.8 at 37 ℃ and induced with 0.5 mM IPTG, and then expressed at 25 ℃ in 220rpm for 18 hrs.

The cells are harvested by centrifugation at 2500×g, and washed with lysis buffer (50 mM Tris-HCl, 500 mM NaCl, [pH 8.0] , 10% (v/v) glycerol) , and resuspended in 10ml lysis buffer to an OD550 about of 100. Cells are lysed using Ultrasonic Cell Disruptor for 10min at 4℃. The lysate is clarified by centrifugation at 12,000×g for 30 minutes at 4℃. The supernatant containing soluble protein fraction is recovered and filtered through a 0.45 μm filter, and bound with 0.5 mL of Ni affinity resin in Rotator (or Roller) at 25℃ for 1 hr. The resin is transferred to a gravity flow column. The resin is 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) followed by elution 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 substitution of elution buffer and concentration of the protein sample with reaction buffer (50mM HEPES (pH 7.5) , 5mM Mg) via Amicon filter column (10K cutoff) . The protein sample is initially concentrated to 200 μL.

EXAMPLE 10: In vitro Enzyme Assay

Reaction conditions for enzyme assays consisted of 50 mM HEPES with 5 mM MgCl2 (pH=7.5) , 2 mM GPP, 2 mM olivetolic acid, and 1 mg/ml purified candidate enzyme in a final volume of 200 uL. After incubation at room temperature for 18 hrs, the reaction mixture is extracted 2 times with 200 μL of ethyl acetate/formic acid (0.05% (v/v) ) . The organic extract of each reaction is pooled, and the solvent is removed using block heater. The samples are dissolved in 100 μL resuspension solution (acetonitrile/H2O/formic acid (80%/20%/0.05% (v/v/v) ) ) and filtered with 0.22μm PVDF membrane before LC-MS analysis.

EXAMPLE 11: LC-MS Analysis of Cannabigerolic acid (CBGA)

1.7 μm, 2.1mm x 50mm, Waters) . Mobile phase A is water with 0.1%formic acid and mobile phase B is acetonitrile with 0.1%formic acid. Gradient elution starts from 50%B hold 1 min, then increase to 90%B in 10 min, decrease to 50%B in 1 min, and hold at 50%B for 1min. The flow rate is 0.4 ml/min, run time 13 min. Sample injection volume is 2 μl, and the sample tray and column oven are set to 10 ℃ and 30 ℃, respectively. Cannabigerolic acid is detected by electrospray ionization (ESI) in the negative ion mode and MS scan mode was SIR 359.5 m/z, capillary and cone voltage were 3,800 V and 30 V, respectively. Desolvation gas and temperature are set to 1000 l/h and 500 ℃, respectively.

EXAMPLE 12: Sequence Alignment

Protein Identity of nphB is compared with other enzymes using the CLUSTAL W from UniProt and LALAGN from Expasy programs, respectively.

Results

EXAMPLE 13: Validation results of CBGA by LC-MS

Figure 1 validates the enzyme assay by showing the results of CBGA titrated at four difference concentrations using method as described in EXAMPLE 3. CBGA Titration curve Concentration: (A) 10ng/mL (B) 100ng/mL (C) 1ug/mL (D) 10 ug/mL. Figure 2 also shows that LC-MS chromatogram of CBGA at m/z 359.5, having a retention time of 5.43 minutes.

Table 10 together with Figure 2 compare CBGA activity of four control samples Panels A, B, C, and D (nphB genes, wild type and mutants) in their ability to form CBGA using LC-MS. The LC-MS chromatograms showed activity for the native nphB and confirms the enhanced activity for the M23, M30 and G286S mutants. LC-MS chromatograms result of samples at m/z 359.5, CBGA retention time (R.T. ) is around 5.43 minute, several isomers, R.T. around 3.47, 5.94, 6.30 and 8.70 min were also observed. R.T. 5.4 corresponds to CBGA.

Table 10 listed the Panel and the corresponding genes

Sample	Panel	gene
eCAN-0001	Panel A	nphB wt
eCAN-0002	Panel B	nphB M23

eCAN-0003	Panel C	nphB M30
eCAN-0004	Panel D	nphB-G286S

EXAMPLE 14: CBGA Yield

Table 3 provides CBGAS activity, shown in the form of calculated yield of CBGA formed of new enzymes of the present invention. Table 3 lists the phrenyltransferase having CBGA activities. Table 11 also shows eCAN20003 (nphB-m30) , eCAN20006 (SActPT06) , eCAN20005 (SaPT05) , eCAN20002 (nphB-m23) and eCAN20004 (nphB-G286S) have significantly higher CBGA yield as compared to eCAN20001 (NphB-wt) .

Table 11: CBGA Yield

EXAMPLE 15: Protein Identity of nphB compared with other enzymes

Table 12 shows protein identity of the enzymes of the present invention compared with nphB using the CLUSTAL W from UniProt and LALIGN from Expasy programs. Table 12 demonstrates that the protein sequences of the enzymes of the present invention are very different from nphB, showing overall percent identity well under 50%. Accordingly, the enzymes disclosed herein havesignificant CBGAS activity despite the structural difference from nphB. Furthermore, some of the present enzymes are surprisingly more efficient in catalyzing the formation of CBGA.

Table 12: Sequence alignment showing Level of Protein Identity

EXAMPLE 16: Generation of Mutants to Produce Compounds in the Cannabinoid Pathway

PAIRWISE sequence alignment between protein sequences of eCAN20053 (which has a plant origin) and that of eCAN200039 (which has a fungal origin) was performed to find the conserved amino acids or conserved domains using UNIPROT Clustal Omega.

Table 13 provides mutant enzymes wherein the conserved amino acids described above are replaced with other amino acids having similar chemical properties (such as polar, non-polar, charged, non-charged etc. ) .

Table 13 Amino Acid Mutated Sites for mutants of eCAN200053 and eCAN200039.

Mutations on protein sequences of eCAN20005 are listed in Table 14.

Table 14 Mutations on protein sequences of mutants of eCAN20005

Clone no.	Mutants based on eCAN20005
1	A17T_Q159W_A230S
2	A51T_M104E_Q159S
3	A51Q_S175W_L217F
4	L217F_V292N_Q235A
5	A51T_D164E_Q293W
6	V47A_Q159S_I292A
7	A51Q_S175Y_Y286H

Although the description referred to particular embodiments, it will be clear to one skilled in the art that the present invention may be practiced with variations of these specific details. Hence this invention should not be construed as limited to the claims set forth below or embodiments set forth herein.

Claims

A method of producing cannabigerolic acid (CBGA) or analogs thereof comprising the step of reacting a heterologously expressed prenyltransferase enzyme with geranyl diphosphate (GPP) and an acid.
The method of claim 1, wherein the prenyltransferase enzyme has prenyl transferase activity and/or geranylpyrophosphate: olivatolate geranylttransfersae (GOT) activity.
The method of claim 1, wherein the prenyltransferase enzyme has activity with increased synthesis capacity and decreased by-products formation compared with the activity of native CBGAS; or has an improved reaction rate for forming CBGA compared to NphB wild type.
The method of claim 1, wherein the prenyltransferase enzyme has a reaction rate of greater than 12 μg /mL of CBGA.
The method of claim 1, wherein the acid is selected from the group consisting of orsellinic (OSA) , divarinolic (DVA) , and olivetolic (OLA) , apigenin, diadzein, genestein, naringenin, olivetol, olivetolic acid (OA) , and resveratrol.
The method of any one of claims 1-5, wherein the prenyltransferase enzyme has 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 prenyltransferase 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 nucleic acid sequence selected from the group consisting of SEQ ID No: 104-197, 336-583.
The method of claim 6, wherein the amino acid sequence is SEQ ID No: 01, 02, 85 or 179.
A method of producing a cannabinoid, comprising the steps of

a) reacting a heterolously expressed prenyl transferase enzyme with a prenyl group donor and an acid to produce cannabigerolic acid (CBGA) or analogs thereof; and

b) reacting the CBGA or analogs thereof with a cannabinoid synthase to form acidic forms of a cannabinoid.
The method of claim 8, wherein the prenyltransferase enzyme has activity with increased synthesis capacity and decreased by-products formation compared with the activity of native CBGAS.
The method of claim 8, wherein the prenyltransferase enzyme has a reaction rate of greater than 12 μg/mL of CBGA.
The method of claim 8, wherein the acid is selected from the group consisting of orsellinic (OSA) , divarinolic (DVA) , and olivetolic (OLA) , apigenin, diadzein, genestein, naringenin, olivetol, olivetolic acid (OA) , resveratrol, butanoic acid, pentanoic acid, hexaonic acid and heptanoic acid.
The method of any one of claims 8-11, 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 ID NO: 01-103, 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 SEQ ID No: 104-197, 336-583.
A recombinant microorganism engineered for the production of CBGA, wherein said microorganism overexpresses an enzyme having 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 ID NO: 01-103 and 198-335.
A recombinant microorganism engineered for the production of CBGA, wherein said microorganism comprises at least one heterologous nucleotide sequence having 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 or a codon degenerate nucleotide sequence thereof.
An isolated polypeptide having cannabigerolic acid synthase (CBGAS) activity; 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.
The isolated polypeptide of claim 15, wherein the polypeptide is expressed in a microcrobial host selected from the group consisting of E. coli, Y. lipolytica and S. cerevisiae.
The isolated polypeptide of claim 15, wherein the polypeptide is expressed in Cannabis sp.
A use of an enzyme for the production of CBGA, 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 ID NO: 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 selected from the group consisting of SEQ ID No: 104-197 and 336-583.
A recombinant polypeptide, comprising an amino acid sequence with 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.
The recombinant polypeptide of claim 19, wherein said 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.
A recombinant polypeptide, comprising an amino acid sequence with 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.
The recombinant polypeptide of claim 21, wherein said 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.
A recombinant polypeptide, comprising an amino acid sequence with at least 95%identity to SEQ ID NO: 198, wherein the amino acid sequence comprises at least one amino acid substitution.
The recombinant polypeptide of claim 23, wherein 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.