Improved enzymes and methods for the synthesis of cannabinoids

Abstract

Disclosed herein are novel CBGA and CBGVA synthases and methods for improving their overall activity for synthesizing CBGA and CBGVA from their respective precursors, olivetolic acid (OA) or divaleric acid (DVA), and GPP. Fusion proteins for enhancing the synthesis of CBGA and CBGVA are also disclosed. The methods described herein also increase the titer and purity of CBGA and CBGVA produced by cells by: 1) reducing the formation of the by-products FCBGA and FCBGVA, which are synthesized from the prenylation of OA and DVA, respectively, with FPP; 2) increasing the intracellular availability of OA and DVA; and 3) increasing the rate of CBGA and CBGVA formation while reducing the accumulation of intermediates.

Description

[Technical Field]

[0001] Inventors: Spiros Kambourakis, Russell Scott Komor, Nicky Christopher Caiazza, Nicholas Donald Keul, Caleb Marshall Walker Related Applications This application claims priority to and the benefit of co-pending U.S. Provisional Application No. 63 / 433,676, filed December 19, 2022. The disclosure of said provisional application is incorporated herein by reference in its entirety. [Background technology]

[0002] Background of the Invention Plants in the Cannabaceae family produce numerous different cannabinoids (>=120) in variable relative amounts over a 7-10 week flowering period. Many of these cannabinoids have been and are currently being studied as therapeutic agents in chordates (e.g., mammals), and as a result, most have been approved for medical and / or recreational use in the United States (Abrams DI Eur J Int Med 2018, 49, 7-11). Specifically, the most sought-after (phyto)cannabinoids are tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and cannabichromenic acid (CBCA). These phytocannabinoids and their related chemical analogs are all biosynthesized in varying amounts from the same precursor: cannabigerolic acid (CBGA). Therefore, to mass-produce any particular phytocannabinoid (e.g., THCA / CBDA / CBCA), both the rate and total amount of CBGA biosynthesized must be enhanced and increased (Blatt-Janmaat K, Qu, Y. Int J Mol Sci 2021, 22(5), 2454). One of the major obstacles to increasing CBGA titer is the activity of prenyltransferase (PT), the enzyme that combines geranyl pyrophosphate (GPP) and olivetolic acid (OA) to form CBGA. Various soluble and membrane-bound prenyltransferases that perform this reaction have been described, with one of the most successful being MPT4, a membrane-bound enzyme from cannabis. However, even this enzyme needs to be improved to increase the titer and productivity of CBGA and its derived cannabinoids. In addition to CBGA, cannabinoids derived from cannabigerovaric acid (CBGVA) are commercially available, with tetrahydrocannabirarinic acid (THCVA) being the most common. Therefore, increasing the titer and productivity of CBGVA is crucial for creating industrial organisms for the synthesis of these types of cannabinoids (e.g., THCVA, CBCVA, and CBDVA). The activity of prenyltransferase, which condenses GPP and DVA to form CBGVA, is one of the major limitations for increasing CBGVA titer. Asaprenyltransferase MPT4 has 3-5-fold lower activity in prenylating DVA, and as a result, a better prenyltransferase for producing CBGVA is needed. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Abrams DI Eur J Int Med 2018,49,7-11 [Non-patent document 2] Blatt-Janmaat K,Qu,Y.Int J Mol Sci 2021,22(5),2454 Summary of the Invention [Means for solving the problem]

[0004] Summary of the Invention The present inventors disclose both soluble and membrane-bound prenyltransferases with increased activity for the synthesis of CBGA and CBVA. Other host strain modifications that increase the availability of GPP and DVA from butyryl-CoA have been achieved in the present inventors' laboratories and are described in co-owned PCT application no. PCT / US2022 / 046926, the entire contents of which are incorporated herein by reference. Described herein are novel CBGA and cannabigerovarinic acid (CBGVA) synthases and methods for improving their overall activity for synthesizing CBGA and CBGVA from their respective precursors, olivetolic acid (OA) or divaleric acid (DVA) and geranyl pyrophosphate (GPP). In some embodiments, the methods described herein also increase the titer and purity of CBGA and CBGVA produced by cells by: i) reducing the formation of the by-products farnesylcannabigerolic acid (FCBGA) and farnesylcannabigerovarilic acid (FCBGVA) (e.g., Figure 1), which are synthesized from the prenylation of OA and DVA, respectively, with farnesyl pyrophosphate (FPP), and ii) increasing the rate of CBGA and CBGVA formation while reducing the accumulation of intermediates. In addition to providing mutant membrane-bound prenyltransferases (MPTs) and soluble aromatic prenyltransferases (APTs) that exhibit improved selectivity for GPP and enhanced production of CBGA and CBVA, fusion proteins containing prenyltransferases and GPP synthases are provided, resulting in an overall increase in CBGA production. Thus, the present invention relates in some embodiments to mutant prenyltransferases that are binary fusion proteins between GPP synthase and prenyltransferase (soluble or membrane-bound) to achieve increased titer and purity of the mutant prenyltransferases, CBGA and CBGVA.

[0005] Some embodiments of the present disclosure are directed to an aromatic prenyltransferase (APT) that has been engineered to transfer geranyl pyrophosphate (GPP) to olivetolic acid and / or divalanic acid with at least two-fold increased efficiency compared to mutant aromatic prenyltransferase APT73.77 (SEQ ID NO: 38), wherein the engineered APT contains an amino acid sequence having at least two amino acid modifications compared to SEQ ID NO: 38, wherein a first amino acid modification of the at least two amino acid modifications corresponds to a substitution, deletion, or insertion at amino acid position R162 of SEQ ID NO: 38.

[0006] In some embodiments, the second amino acid modification of the at least two amino acid modifications compared to the amino acid sequence APT73.77 is selected from a substitution, deletion, or insertion at an amino acid position corresponding to position H39, V41, Q127, E130, V131, I156, R162, N164, R205, A223, H225, Q227, G254, G260, L276, G280, R282, C283, L284, D285, or G286 of SEQ ID NO: 38.

[0007] In some embodiments, the at least two amino acid modifications comprise second, third, fourth, and fifth or more amino acid modifications selected from substitutions, deletions, or insertions at four or more amino acid positions corresponding to positions H39, V41, Q127, E130, V131, I156, R162, N164, R205, A223, H225, Q227, G254, G260, L276, G280, R282, C283, L284, D285 and / or G286 of SEQ ID NO: 38, wherein the modification is not the substitution R205S.

[0008] In some embodiments, the engineered APTs are APT73.179 (SEQ ID NO: 1), APT73.119 (SEQ ID NO: 2), APT73.159 (SEQ ID NO: 3), APT73.160 (SEQ ID NO: 4), APT73.161 (SEQ ID NO: 5), APT73.162 (SEQ ID NO: 6), APT73.163 (SEQ ID NO: 7), APT73.165 (SEQ ID NO: 8), APT73.166 (SEQ ID NO: 9), AP T73.168 (SEQ ID NO: 10), APT73.169 (SEQ ID NO: 11), APT73.170 (SEQ ID NO: 12), APT73.172 (SEQ ID NO: 13), APT73.182 (SEQ ID NO: 14), APT73.183 (SEQ ID NO: 15), APT73.184 (SEQ ID NO: 16), APT73.185 (SEQ ID NO: 17), APT73.186 (SEQ ID NO: 18), APT73.187 (SEQ ID NO: 19), APT73.188 (SEQ ID NO:20), APT73.189 (SEQ ID NO:21), APT73.190 (SEQ ID NO:22), APT73.191 (SEQ ID NO:23), APT73.192 (SEQ ID NO:24), APT73.193 (SEQ ID NO:25), APT73.194 (SEQ ID NO:26), APT73.195 (SEQ ID NO:27), APT73.196 (SEQ ID NO:28), APT73.197 (SEQ ID NO:29) APT73.199 (SEQ ID NO:31), APT73.200 (SEQ ID NO:32), APT73.201 (SEQ ID NO:33), APT73.202 (SEQ ID NO:34), APT73.203 (SEQ ID NO:35), APT73.204 (SEQ ID NO:36), APT73.248 (SEQ ID NO:96), or a functional fragment or variant thereof. In some embodiments, the engineered APT comprises an amino acid sequence at least 90% identical to the amino acid sequence of APT73.187 (SEQ ID NO:96) or APT73.248 (SEQ ID NO:96).

[0009] In some embodiments, the at least two amino acid modifications include three or more amino acid modifications including the substitution R162Q, one or more substitutions selected from the group consisting of Q127E, E130R, V131I and A280G, and one or more substitutions selected from the group consisting of H39V, V41C and V41L, all relative to the amino acid sequence of APT73.77 (SEQ ID NO: 38).

[0010] In some embodiments, the engineered APTs are APT73.179 (SEQ ID NO: 1), APT73.119 (SEQ ID NO: 2), APT73.159 (SEQ ID NO: 3), APT73.160 (SEQ ID NO: 4), APT73.161 (SEQ ID NO: 5), APT73.162 (SEQ ID NO: 6), APT73.163 (SEQ ID NO: 7), APT73.165 (SEQ ID NO: 8), APT73.166 (SEQ ID NO: 9), AP T73.168 (SEQ ID NO: 10), APT73.169 (SEQ ID NO: 11), APT73.170 (SEQ ID NO: 12), APT73.172 (SEQ ID NO: 13), APT73.182 (SEQ ID NO: 14), APT73.183 (SEQ ID NO: 15), APT73.184 (SEQ ID NO: 16), APT73.185 (SEQ ID NO: 17), APT73.186 (SEQ ID NO: 18), APT73.187 (SEQ ID NO: 19), APT73.188 (SEQ ID NO: 20), APT73.189 (SEQ ID NO: 21), APT73.190 (SEQ ID NO: 22), APT73.191 (SEQ ID NO: 23), APT73.192 (SEQ ID NO: 24), APT73.193 (SEQ ID NO: 25), APT73.194 (SEQ ID NO: 26), APT73.195 (SEQ ID NO: 27), APT73.196 (SEQ ID NO: 28), APT73.197 (SEQ ID NO: 29), 9), APT73.199 (SEQ ID NO:31), APT73.200 (SEQ ID NO:32), APT73.201 (SEQ ID NO:33), APT73.202 (SEQ ID NO:34), APT73.203 (SEQ ID NO:35), APT73.204 (SEQ ID NO:36), APT73.248 (SEQ ID NO:96), or a functional fragment or variant thereof.

[0011] In another aspect, the disclosure is directed to a recombinant membrane-bound prenyltransferase (rMPT), wherein the rMPT has been engineered to transfer geranyl pyrophosphate (GPP) to olivetolic acid and / or divalanic acid with greater efficiency than naturally occurring MPT, and wherein the rMPT has an amino acid sequence that includes at least one amino acid modification compared to the amino acid sequence of MPT4.1 (SEQ ID NO: 46), MPT48 (SEQ ID NO: 47), or MPT69 (SEQ ID NO: 48).

[0012] In some embodiments, the rMPT has at least one amino acid modification at a position corresponding to amino acid position 29, 72, 135 and / or 254 of SEQ ID NO: 46 (MPT4.1). In some embodiments, the at least one amino acid modification compared to SEQ ID NO: 46 (MPT4.1) is selected from at least one of M29I, M29V, I72C, I72V, I72A, A135M and / or V254L.

[0013] In some embodiments, the rMPT comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of MPT4.33 (SEQ ID NO: 39), MPT4.29 (SEQ ID NO: 40), MPT4.30 (SEQ ID NO: 41), MPT4.32 (SEQ ID NO: 42), MPT4.34 (SEQ ID NO: 43), MPT4.40 (SEQ ID NO: 44), or MPT4.41 (SEQ ID NO: 45).

[0014] In some embodiments, rMPT comprises an amino acid sequence having at least one amino acid modification compared to SEQ ID NO: 47 (MPT48) that produces a variant with one or both of: i) increased activity with respect to prenylation of OA and DVA to form CBGA and CBGVA, respectively; and ii) increased selectivity for GPP over FPP.

[0015] In some embodiments, at least one amino acid modification compared to MPT48 (SEQ ID NO: 47) is at least one of the first 50 amino acids M1 to G50 at the N-terminus of SEQ ID NO: 47 and / or H54, M88, R89, C93, A94, N96, D97, V98, V99, D100, Q101, D102, F103, D104, R109, R113, S121, A136, C147, Q148, V154, K165, Q172, L175, T1 78, L179, I181, L201, T207, V214, Y217, D218, V219, Y221, T253, L254, T262, V267, N269, P271, L281, A284, A287, S304, G305, W306, N314, L316, G317, G318, V327, M329 and / or L330.

[0016] In some embodiments, rMPT comprises an amino acid sequence having at least one amino acid modification compared to SEQ ID NO: 48 (MPT69) that produces a variant with one or both of: i) increased activity with respect to prenylation of OA and DVA to form CBGA and CBGVA, respectively; and ii) increased selectivity for GPP over FPP.

[0017] In some embodiments, at least one amino acid modification compared to MPT69 (SEQ ID NO: 48) is within the first 45 amino acids M1 to G45 at the N-terminus of SEQ ID NO: 48, and / or any of H49, M83, R84, C88, A89, N91, D92, N93, I94, D95, Q96, D97, F98, D99, R104, R108, S116, A131, C142, H143, V149, K160, Q167, L170, T173 , L174, A176, R196, T202, V209, Y212, D213, V214, Y216, T248, C249, V257, V262, N264, P266, L276, A279, A282, S299, G300, W301, N309, M311, S312, G313, A322, L324 and / or L325.

[0018] In some embodiments, the rMPT comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of MPT69.2 (SEQ ID NO: 98), MPT69.5 (SEQ ID NO: 99), MPT69.6 (SEQ ID NO: 100), MPT69.7 (SEQ ID NO: 101), MPT69.8 (SEQ ID NO: 102), MPT69.9 (SEQ ID NO: 103) or MPT69.10 (SEQ ID NO: 104).

[0019] In some embodiments, the present disclosure is directed to a cell comprising the APT and / or rMPT described herein, wherein the cell is capable of producing CBGA in the presence of GPP and OA. In some embodiments, the cell is capable of producing CBGA in the presence of GPP and DVA. In some embodiments, the cell is capable of producing cannabinoids in the presence of a carbon source and, optionally, hexanoic acid or butyric acid.

[0020] In some embodiments, the cells express an exogenous membrane transporter that improves OA or DVA uptake. In some embodiments, one or more genes in the cells that encode proteins that excrete OA or DVA are downregulated or inactivated. In some embodiments, the cells encode exogenous hexanoyl-CoA synthetase and / or butyryl-CoA synthetase.

[0021] In some embodiments, the cell is a yeast cell or a bacterial cell. In some embodiments, the yeast cell is a Yarrowia strain or a Saccharomyces strain.

[0022] In another aspect, the present disclosure is directed to a method for producing CBGA, CBGVA, or a derivative thereof, comprising culturing a cell described herein under suitable conditions for producing CBGA, CBGVA, or a derivative thereof. In some embodiments, the suitable conditions include adding at least one of butyric acid, valeric acid, isovaleric acid, hexanoic acid, hexanol, butanol, oleic acid, glycerol, or glucose to the culture medium in which the cells are cultured.

[0023] Some embodiments of the present disclosure are directed to a fusion protein comprising a polypeptide having geranyl diphosphate synthase (GPS) activity fused directly or indirectly to a polypeptide having prenyltransferase activity, wherein the polypeptide having prenyltransferase activity comprises a) a polypeptide sequence having at least 85% identity to the polypeptide sequence of MPT48 (SEQ ID NO: 47) or MPT69 (SEQ ID NO: 48), b) a polypeptide sequence comprising at least one amino acid modification compared to the amino acid sequence of MPT4.1 (SEQ ID NO: 46), or c) a polypeptide comprising i) an amino acid sequence having at least two amino acid modifications compared to SEQ ID NO: 38, wherein a first of the at least two amino acid modifications corresponds to a substitution, deletion, or insertion at amino acid position R162 of SEQ ID NO: 38, and wherein the N-terminus of the polypeptide having prenyltransferase activity is fused to the C-terminus of GPS, or the C-terminus of the polypeptide having prenyltransferase activity is fused to the N-terminus of GPS.

[0024] In some embodiments, the fusion protein is selected from the group consisting of GPS1.1-L18-APT73.119 (SEQ ID NO: 52), GPS1.1-L18-APT73.159 (SEQ ID NO: 53), GPS1.1-L18-APT73.160 (SEQ ID NO: 54), GPS1.1-L18-APT73.161 (SEQ ID NO: 55), GPS1.1-L18-APT73.162 (SEQ ID NO: 56), GPS1.1-L18-APT73.163 (SEQ ID NO: 57), GPS1.1-L18-APT73.165 (SEQ ID NO: 58), GPS1.1-L18-APT73.166 (SEQ ID NO: 59), No. 59), GPS1.1-L18-APT73.168 (SEQ ID NO: 60), GPS1.1-L18-APT73.169 (SEQ ID NO: 61), GPS1.1-L18-APT73.170 (SEQ ID NO: 62), GPS1.1-L18-APT73.172 (SEQ ID NO: 63), GPS1.1-L18-APT73.179 (SEQ ID NO: 64), GPS1.1-L18-APT73.182 (SEQ ID NO: 65), GPS1.1-L18-APT73.183 (SEQ ID NO: 66), GPS1.1-L18-APT73.184 (SEQ ID NO: 67), GPS1.1-L18-A PT73.185 (SEQ ID NO: 68), GPS1.1-L18-APT73.186 (SEQ ID NO: 69), GPS1.1-L18-APT73.187 (SEQ ID NO: 70), GPS1.1-L18-APT73.188 (SEQ ID NO: 71), GPS1.1-L18-APT73.189 (SEQ ID NO: 72), GPS1.1-L18-APT73.190 (SEQ ID NO: 73), GPS1.1-L18-APT73.191 (SEQ ID NO: 74), GPS1.1-L18-APT73.192 (SEQ ID NO: 75), GPS1.1-L18-APT73.193 (SEQ ID NO: 76), GPS1.1-L18-APT73.194 (SEQ ID NO: 77), GPS1.1-L18-APT73.195 (SEQ ID NO: 78), GPS1.1-L18-APT73.196 (SEQ ID NO: 79), GPS1.1-L18-APT73.197 (SEQ ID NO: 80), GPS1.1-L18-APT73.199 (SEQ ID NO: 82), GPS1.1-L18-APT73.200 (SEQ ID NO: 83), GPS1.1-L18-APT73.201 (SEQ ID NO: 84), GPS1.1-L18-APT73.202 (SEQ ID NO: 85), GPS1.1-L18-APT73.and a polypeptide sequence having at least 90% identity to GPS1.1-L18-APT73.203 (SEQ ID NO: 86), GPS1.1-L18-APT73.204 (SEQ ID NO: 87), or GPS1.1-L18-APT73.248 (SEQ ID NO: 97).

[0025] In some embodiments, the fusion protein comprises a polypeptide sequence having at least 90% identity to GPS1.1-L11-MPT4.1 (sequence number 88), GPS1.1-L11-MPT4.29 (sequence number 89), GPS1.1-L11-MPT4.30 (sequence number 90), GPS1.1-L11-MPT4.32 (sequence number 91), GPS1.1-L11-MPT4.33 (sequence number 92), GPS1.1-L11-MPT4.34 (sequence number 93), GPS1.1-L11-MPT4.40 (sequence number 94), or GPS1.1-L11-MPT4.41 (sequence number 95).

[0026] In some embodiments, the polypeptide having prenyltransferase activity has improved selectivity for GPP over FPP compared to a control membrane-bound prenyltransferase or a soluble aromatic prenyltransferase. In some embodiments, the polypeptide having geranyl diphosphate synthase activity comprises the polypeptide of SEQ ID NO: 49 (GPS1.1), or a functional fragment or functional variant thereof.

[0027] In some embodiments, the fusion protein further comprises a linker polypeptide between the polypeptide having geranyl diphosphate synthase activity and the polypeptide having prenyltransferase activity, in some embodiments, the linker comprises a polypeptide selected from the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 51.

[0028] In some embodiments, the fusion protein is selected from the group consisting of GPS1.1-L18-APT73.119 (SEQ ID NO: 52), GPS1.1-L18-APT73.159 (SEQ ID NO: 53), GPS1.1-L18-APT73.160 (SEQ ID NO: 54), GPS1.1-L18-APT73.161 (SEQ ID NO: 55), GPS1.1-L18-APT73.162 (SEQ ID NO: 56), GPS1.1-L18-APT73.163 (SEQ ID NO: 57), GPS1.1-L18-APT73.165 (SEQ ID NO: 58), GPS1.1-L18-APT73.166 (SEQ ID NO: 59), No. 59), GPS1.1-L18-APT73.168 (SEQ ID NO: 60), GPS1.1-L18-APT73.169 (SEQ ID NO: 61), GPS1.1-L18-APT73.170 (SEQ ID NO: 62), GPS1.1-L18-APT73.172 (SEQ ID NO: 63), GPS1.1-L18-APT73.179 (SEQ ID NO: 64), GPS1.1-L18-APT73.182 (SEQ ID NO: 65), GPS1.1-L18-APT73.183 (SEQ ID NO: 66), GPS1.1-L18-APT73.184 (SEQ ID NO: 67), GPS1.1-L18-A PT73.185 (SEQ ID NO: 68), GPS1.1-L18-APT73.186 (SEQ ID NO: 69), GPS1.1-L18-APT73.187 (SEQ ID NO: 70), GPS1.1-L18-APT73.188 (SEQ ID NO: 71), GPS1.1-L18-APT73.189 (SEQ ID NO: 72), GPS1.1-L18-APT73.190 (SEQ ID NO: 73), GPS1.1-L18-APT73.191 (SEQ ID NO: 74), GPS1.1-L18-APT73.192 (SEQ ID NO: 75), GPS1.1-L18-APT73.193 (SEQ ID NO: 76), GPS1.1-L18-APT73.194 (SEQ ID NO: 77), GPS1.1-L18-APT73.195 (SEQ ID NO: 78), GPS1.1-L18-APT73.196 (SEQ ID NO: 79), GPS1.1-L18-APT73.197 (SEQ ID NO: 80), GPS1.1-L18-APT73.199 (SEQ ID NO: 82), GPS1.1-L18-APT73.200 (SEQ ID NO: 83), GPS1.1-L18-APT73.201 (SEQ ID NO: 84), GPS1.1-L18-APT73.202 (SEQ ID NO: 85), GPS1.1-L18-APT73.The polypeptide sequence of GPS1.1-L18-APT73.203 (SEQ ID NO: 86), GPS1.1-L18-APT73.204 (SEQ ID NO: 87), or GPS1.1-L18-APT73.248 (SEQ ID NO: 97), or a functional fragment or variant thereof.

[0029] In some embodiments, the fusion protein comprises the polypeptide sequence of GPS1.1-L11-MPT4.1 (SEQ ID NO: 88), GPS1.1-L11-MPT4.29 (SEQ ID NO: 89), GPS1.1-L11-MPT4.30 (SEQ ID NO: 90), GPS1.1-L11-MPT4.32 (SEQ ID NO: 91), GPS1.1-L11-MPT4.33 (SEQ ID NO: 92), GPS1.1-L11-MPT4.34 (SEQ ID NO: 93), GPS1.1-L11-MPT4.40 (SEQ ID NO: 94), GPS1.1-L11-MPT4.41 (SEQ ID NO: 95), or a functional fragment or variant thereof.

[0030] In another aspect, the present disclosure is directed to a cell comprising the fusion protein described herein, wherein the cell is capable of producing CBGA in the presence of OA and GPP. In some embodiments, the cell is capable of producing CBGA in the presence of DVA and GPP. In some embodiments, the cell is capable of producing cannabinoids in the presence of a carbon source and, optionally, hexanoic acid or butyric acid. In some embodiments, the cell expresses an exogenous membrane transporter that improves OA or DVA uptake. In some embodiments, one or more genes in the cell that encode proteins that export OA or DVA are downregulated or inactivated.

[0031] In some embodiments, the cell encodes an exogenous hexanoyl-CoA synthetase and / or butyryl-CoA synthetase. In some embodiments, the cell is a yeast cell or a bacterial cell. In some embodiments, the yeast cell is a Yarrowia strain or a Saccharomyces strain.

[0032] In another aspect, the present disclosure is directed to a method for producing CBGA, CBGVA, or a derivative thereof, comprising culturing a cell described herein under suitable conditions for producing CBGA, CBGVA, or a derivative thereof. In some embodiments, the suitable conditions include adding at least one of butyric acid, valeric acid, isovaleric acid, hexanoic acid, hexanol, butanol, oleic acid, glycerol, or glucose to the culture medium in which the cells are cultured. [Brief explanation of the drawings]

[0033] BRIEF DESCRIPTION OF THE DRAWINGS The following figures further illustrate embodiments of the present invention.

[0034] [Figure 1] FIG. 1A illustrates the CBGA derivatives synthesized by the CBGA synthase described herein.

[0035] FIG. 1B illustrates the FCBGA derivatives synthesized by the CBGA synthase described herein. DETAILED DESCRIPTION OF THE INVENTION

[0036] Detailed Description of the Invention Some embodiments of the present disclosure are directed to an aromatic prenyltransferase (APT) that has been engineered to transfer geranyl pyrophosphate (GPP) to olivetolic acid and / or divalanic acid with at least two-fold increased efficiency compared to mutant aromatic prenyltransferase APT73.77 (SEQ ID NO: 38), wherein the engineered APT contains an amino acid sequence having at least two amino acid modifications compared to SEQ ID NO: 38, wherein a first amino acid modification of the at least two amino acid modifications corresponds to a substitution, deletion, or insertion at amino acid position R162 of SEQ ID NO: 38.

[0037] Another aspect of the present disclosure is directed to an aromatic prenyltransferase (APT) that has been engineered to transfer geranyl pyrophosphate (GPP) to olivetolic acid and / or divalanic acid with at least 20-fold greater efficiency than the naturally occurring aromatic prenyltransferase APT73 (SEQ ID NO: 37), wherein the engineered APT contains an amino acid sequence having at least two amino acid modifications compared to SEQ ID NO: 37, wherein a first amino acid modification of the at least two amino acid modifications corresponds to a substitution, deletion, or insertion at amino acid position R162 of SEQ ID NO: 37.

[0038] Another aspect of the present invention is directed to an aromatic prenyltransferase (APT) that has been engineered to transfer geranyl pyrophosphate (GPP) to olivetolic acid and / or divalanic acid with equal or greater efficiency than mutant aromatic prenyltransferase APT73.119 (SEQ ID NO:2), wherein the engineered APT comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:2, and at least four of the amino acids of the engineered APT corresponding to the amino acids at positions 41, 127, 130, 131, 162, and 280 of SEQ ID NO:2 are identical.

[0039] Amino acid modifications can be amino acid substitutions, amino acid deletions, and / or amino acid insertions. Amino acid substitutions can be conservative or non-conservative amino acid substitutions. Conservative substitutions (also called conservative mutations, conservative substitutions, or conservative variations) are amino acid substitutions in proteins that change a given amino acid into a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). As used herein, "conservative variations" refer to the replacement of an amino acid residue with another biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another hydrophobic residue, or the substitution of one polar residue for another polar residue, such as the substitution of lysine for arginine, aspartic acid for glutamic acid, or asparagine for glutamine. Other examples of conservative substitutions include alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine ​​to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine, and the like.

[0040] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 1 (APT73.179). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1 (APT73.179). In some embodiments, the APT comprises SEQ ID NO: 1 (APT73.179) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 1 (APT73.179). In some embodiments, a functional fragment of SEQ ID NO:1 (APT73.179) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0041] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:2 (APT73.119). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:2 (APT73.119). In some embodiments, the APT comprises SEQ ID NO:2 (APT73.119) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:2 (APT73.119). In some embodiments, a functional fragment of SEQ ID NO:2 (APT73.119) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0042] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:3 (APT73.159). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:3 (APT73.159). In some embodiments, the APT comprises SEQ ID NO:3 (APT73.159) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:3 (APT73.159). In some embodiments, a functional fragment of SEQ ID NO:3 (APT73.159) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0043] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:4 (APT73.160). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:4 (APT73.160). In some embodiments, the APT comprises SEQ ID NO:4 (APT73.160) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:4 (APT73.160). In some embodiments, a functional fragment of SEQ ID NO:4 (APT73.160) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0044] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:5 (APT73.161). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:5 (APT73.161). In some embodiments, the APT comprises SEQ ID NO:5 (APT73.161) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:5 (APT73.161). In some embodiments, a functional fragment of SEQ ID NO:5 (APT73.161) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0045] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:6 (APT73.162). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:6 (APT73.162). In some embodiments, the APT comprises SEQ ID NO:6 (APT73.162) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:6 (APT73.162). In some embodiments, a functional fragment of SEQ ID NO:6 (APT73.162) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0046] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:7 (APT73.163). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:7 (APT73.163). In some embodiments, the APT comprises SEQ ID NO:7 (APT73.163) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 96%, 97%, 98%, 98.5%, 95%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:7 (APT73.163). In some embodiments, a functional fragment of SEQ ID NO:7 (APT73.163) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0047] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:8 (APT73.165). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:8 (APT73.165). In some embodiments, the APT comprises SEQ ID NO:8 (APT73.165) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:8 (APT73.165). In some embodiments, a functional fragment of SEQ ID NO:8 (APT73.165) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0048] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:9 (APT73.166). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:9 (APT73.166). In some embodiments, the APT comprises SEQ ID NO:9 (APT73.166) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:9 (APT73.166). In some embodiments, a functional fragment of SEQ ID NO:9 (APT73.166) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0049] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 10 (APT73.168). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 10 (APT73.168). In some embodiments, the APT comprises SEQ ID NO: 10 (APT73.168) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 10 (APT73.168). In some embodiments, a functional fragment of SEQ ID NO: 10 (APT73.168) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0050] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 11 (APT73.169). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 11 (APT73.169). In some embodiments, the APT comprises SEQ ID NO: 11 (APT73.169) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 11 (APT73.169). In some embodiments, a functional fragment of SEQ ID NO: 11 (APT73.169) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0051] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 12 (APT73.170). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 12 (APT73.170). In some embodiments, the APT comprises SEQ ID NO: 12 (APT73.170) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 12 (APT73.170). In some embodiments, a functional fragment of SEQ ID NO: 12 (APT73.170) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0052] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 13 (APT73.172). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 13 (APT73.172). In some embodiments, the APT comprises SEQ ID NO: 13 (APT73.172) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 13 (APT73.172). In some embodiments, a functional fragment of SEQ ID NO: 13 (APT73.172) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0053] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 14 (APT73.182). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 14 (APT73.182). In some embodiments, the APT comprises SEQ ID NO: 14 (APT73.182) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 14 (APT73.182). In some embodiments, a functional fragment of SEQ ID NO: 14 (APT73.182) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0054] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 15 (APT73.183). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 15 (APT73.183). In some embodiments, the APT comprises SEQ ID NO: 15 (APT73.183) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 15 (APT73.183). In some embodiments, a functional fragment of SEQ ID NO: 15 (APT73.183) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0055] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 16 (APT73.184). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 16 (APT73.184). In some embodiments, the APT comprises SEQ ID NO: 16 (APT73.184) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 16 (APT73.184). In some embodiments, a functional fragment of SEQ ID NO: 16 (APT73.184) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0056] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 17 (APT73.185). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 17 (APT73.185). In some embodiments, the APT comprises SEQ ID NO: 17 (APT73.185) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 17 (APT73.185). In some embodiments, a functional fragment of SEQ ID NO: 17 (APT73.185) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0057] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 18 (APT73.186). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 18 (APT73.186). In some embodiments, the APT comprises SEQ ID NO: 18 (APT73.186) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 18 (APT73.186). In some embodiments, a functional fragment of SEQ ID NO: 18 (APT73.186) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0058] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 19 (APT73.187). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 19 (APT73.187). In some embodiments, the APT comprises SEQ ID NO: 19 (APT73.187) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 19 (APT73.187). In some embodiments, a functional fragment of SEQ ID NO: 19 (APT73.187) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0059] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:20 (APT73.188). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:20 (APT73.188). In some embodiments, the APT comprises SEQ ID NO:20 (APT73.188) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:20 (APT73.188). In some embodiments, a functional fragment of SEQ ID NO:20 (APT73.188) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0060] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:21 (APT73.189). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:21 (APT73.189). In some embodiments, the APT comprises SEQ ID NO:21 (APT73.189) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:21 (APT73.189). In some embodiments, a functional fragment of SEQ ID NO:21 (APT73.189) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0061] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:22 (APT73.190). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:22 (APT73.190). In some embodiments, the APT comprises SEQ ID NO:22 (APT73.190) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:22 (APT73.190). In some embodiments, a functional fragment of SEQ ID NO:22 (APT73.190) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0062] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:23 (APT73.191). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:23 (APT73.191). In some embodiments, the APT comprises SEQ ID NO:23 (APT73.191) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:23 (APT73.191). In some embodiments, a functional fragment of SEQ ID NO:23 (APT73.191) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0063] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:24 (APT73.192). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:24 (APT73.192). In some embodiments, the APT comprises SEQ ID NO:24 (APT73.192) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:24 (APT73.192). In some embodiments, a functional fragment of SEQ ID NO:24 (APT73.192) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0064] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:25 (APT73.193). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:25 (APT73.193). In some embodiments, the APT comprises SEQ ID NO:25 (APT73.193) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:25 (APT73.193). In some embodiments, a functional fragment of SEQ ID NO:25 (APT73.193) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0065] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:26 (APT73.194). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:26 (APT73.194). In some embodiments, the APT comprises SEQ ID NO:26 (APT73.194) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:26 (APT73.194). In some embodiments, a functional fragment of SEQ ID NO:26 (APT73.194) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0066] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:27 (APT73.195). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:27 (APT73.195). In some embodiments, the APT comprises SEQ ID NO:27 (APT73.195) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:27 (APT73.195). In some embodiments, a functional fragment of SEQ ID NO:27 (APT73.195) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0067] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:28 (APT73.196). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:28 (APT73.196). In some embodiments, the APT comprises SEQ ID NO:28 (APT73.196) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:28 (APT73.196). In some embodiments, a functional fragment of SEQ ID NO:28 (APT73.196) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0068] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO:29 (APT73.197). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO:29 (APT73.197). In some embodiments, the APT comprises SEQ ID NO:29 (APT73.197) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:29 (APT73.197). In some embodiments, a functional fragment of SEQ ID NO:29 (APT73.197) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0069] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 30 (APT73.198). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 30 (APT73.198). In some embodiments, the APT comprises SEQ ID NO: 30 (APT73.198) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 30 (APT73.198). In some embodiments, a functional fragment of SEQ ID NO: 30 (APT73.198) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0070] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 31 (APT73.199). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 31 (APT73.199). In some embodiments, the APT comprises SEQ ID NO: 31 (APT73.199) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 31 (APT73.199). In some embodiments, a functional fragment of SEQ ID NO: 31 (APT73.199) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0071] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 32 (APT73.200). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 32 (APT73.200). In some embodiments, the APT comprises SEQ ID NO: 32 (APT73.200) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 32 (APT73.200). In some embodiments, a functional fragment of SEQ ID NO: 32 (APT73.200) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0072] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 33 (APT73.201). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 33 (APT73.201). In some embodiments, the APT comprises SEQ ID NO: 33 (APT73.201) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 33 (APT73.201). In some embodiments, a functional fragment of SEQ ID NO: 33 (APT73.201) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0073] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 34 (APT73.202). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 34 (APT73.202). In some embodiments, the APT comprises SEQ ID NO: 34 (APT73.202) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 34 (APT73.202). In some embodiments, a functional fragment of SEQ ID NO: 34 (APT73.202) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0074] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 35 (APT73.203). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 35 (APT73.203). In some embodiments, the APT comprises SEQ ID NO: 35 (APT73.203) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 35 (APT73.203). In some embodiments, a functional fragment of SEQ ID NO: 35 (APT73.203) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0075] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 36 (APT73.204). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 36 (APT73.204). In some embodiments, the APT comprises SEQ ID NO: 36 (APT73.204) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 36 (APT73.204). In some embodiments, a functional fragment of SEQ ID NO: 36 (APT73.204) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0076] In some embodiments, the APT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.9%, or 100% identity to SEQ ID NO: 96 (APT73.248). In some embodiments, the APT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 96 (APT73.248). In some embodiments, the APT comprises SEQ ID NO: 96 (APT73.248) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 96 (APT73.248). In some embodiments, a functional fragment of SEQ ID NO:96 (APT73.248) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the carboxy terminus.

[0077] In some embodiments, the engineered APT comprises an amino acid sequence containing at least three amino acid modifications at three or more positions corresponding to any three or more amino acids of APT73.77 (SEQ ID NO: 38). In some embodiments, the engineered APT comprises an amino acid sequence containing at least four amino acid modifications at four or more positions corresponding to any four or more amino acids of APT73.77 (SEQ ID NO: 38). In some embodiments, the engineered APT comprises an amino acid sequence containing at least five amino acid modifications at five or more positions corresponding to any five or more amino acids of APT73.77 (SEQ ID NO: 38). In some embodiments, the engineered APT comprises an amino acid sequence containing at least six amino acid modifications at six or more positions corresponding to any six or more amino acids of APT73.77 (SEQ ID NO: 38). In some embodiments, the engineered APT comprises an amino acid sequence containing at least seven amino acid modifications at seven or more positions corresponding to any seven or more amino acids of APT73.77 (SEQ ID NO: 38). In some embodiments, the engineered APT comprises an amino acid sequence containing at least eight amino acid modifications at eight or more positions corresponding to any eight or more amino acids of APT73.77 (SEQ ID NO: 38). In some embodiments, the engineered APT comprises an amino acid sequence containing at least nine amino acid modifications at nine or more positions corresponding to any nine or more amino acids of APT73.77 (SEQ ID NO: 38). In some embodiments, the engineered APT comprises an amino acid sequence containing at least ten amino acid modifications at ten or more positions corresponding to any ten or more amino acids of APT73.77 (SEQ ID NO: 38).

[0078] In some embodiments, an engineered APT comprises an amino acid sequence that contains at least 2, 3, 4, 5, 6, or more amino acid modifications at 2, 3, 4, 5, 6, or more positions corresponding to any 2, 3, 4, 5, 6, or more amino acids of a naturally occurring APT. In some embodiments, an engineered APT comprises an amino acid sequence that contains 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid modifications compared to the amino acid sequence of a naturally occurring APT. In a preferred embodiment, an engineered APT comprises an amino acid sequence that contains 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid modifications compared to the amino acid sequence of a naturally occurring APT. In a most preferred embodiment, the engineered APT comprises an amino acid sequence containing 27, 28, 29, 30, 31, 32, or 33 amino acid modifications relative to the amino acid sequence of a naturally occurring APT. In some embodiments, the amino acid modifications are relative to naturally occurring APT73 (SEQ ID NO: 37). In some embodiments, the engineered APT comprises a truncation of 3 to 45 amino acids at the C-terminus relative to a naturally occurring APT.

[0079] In some embodiments, the engineered APT comprises at least three amino acid modifications, the modifications comprising the substitutions R162Q, one or more substitutions selected from the group consisting of Q127E, E130R, and V131I, and one or more substitutions selected from the group consisting of H39V, V41C, V41L, and A280G, all relative to the amino acid sequence of mutant APT73.77 (SEQ ID NO: 38) or naturally occurring APT73 (SEQ ID NO: 37). In some embodiments, the engineered APT comprises the substitutions R162R and three or more of A280G, Q127E, E130R, V131I, H39V, V41C, and / or V41L. In some embodiments, the engineered APT comprises four or more of the substitutions A280G, Q127E, E130R, V131I, R162Q, H39V, V41C, and / or V41L. In some embodiments, the engineered APT comprises five or more of the substitutions A280G, Q127E, E130R, V131I, H39V, V41C, R162Q, and / or V41L. In some embodiments, the engineered APT comprises all six of the substitutions A280G, Q127E, E130R, R162Q, V131I, H39V and one of the substitutions V41C or V41L. In some further embodiments, the amino acid sequence comprises the addition of R / H / Y287, R / Y288, R / G289 compared to the amino acid sequence of SEQ ID NO: 38.

[0080] In some embodiments, the engineered APT does not comprise a substitution selected from I156A, R205S, A223S, H225K, G260A, L276Y, R282G, C283A, L284S, and D285N. In some embodiments, the engineered APT does not comprise the substitution R205S. In some embodiments, the engineered APT does not comprise the substitution I156A. In some embodiments, the engineered APT does not comprise the substitution A223S. In some embodiments, the engineered APT does not comprise the substitution H225K. In some embodiments, the engineered APT does not comprise the substitution G260A. In some embodiments, the engineered APT does not comprise the substitution L276Y. In some embodiments, the engineered APT does not comprise the substitution R282G. In some embodiments, the engineered APT does not comprise the substitution C283A. In some embodiments, the engineered APT does not comprise the substitution L284S. In some embodiments, the engineered APT does not comprise the substitution D285N.

[0081] In some embodiments, the engineered APT does not comprise two or more substitutions selected from I156A, R205S, A223S, H225K, G260A, L276Y, R282G, C283A, L284S and D285N.

[0082] In some embodiments, the engineered APT comprises an amino acid sequence containing at least two amino acid modifications at two positions in APT73.119. In some embodiments, the engineered APT comprises an amino acid sequence containing at least three amino acid modifications at three positions in APT73.119. In some embodiments, the engineered APT comprises an amino acid sequence containing at least four amino acid modifications at four positions corresponding to any four of APT73.119. In some embodiments, the engineered APT comprises an amino acid sequence containing 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid modifications compared to the amino acid sequence of APT73.119. In preferred embodiments, the engineered APT comprises an amino acid sequence that contains 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid modifications relative to APT73.119. In most preferred embodiments, the engineered APT comprises an amino acid sequence that contains 27, 28, 29, 30, 31, 32 or 33 amino acid modifications relative to the amino acid sequence of APT73.119. In some embodiments, the engineered APT comprises amino acid modifications at one or more amino acid positions corresponding to one or more of C41, E127, R130, 1156, Q162, N164, R205, A223, H225, Q227, G254, G260, L276, G280, C283, R282, C283, L284, D285 or G286 of SEQ ID NO:2. In another embodiment, the amino acid sequence of the engineered APT comprises at least three additional amino acids at the C-terminus compared to the amino acid sequence of APT73.119, hi some embodiments, the amino acid sequence comprises the additions R / H / Y287, R / Y288, and R / G289 compared to the amino acid sequence of SEQ ID NO:2.

[0083] In some embodiments, the APT is selected from the group consisting of APT73.179 (SEQ ID NO: 1), APT73.119 (SEQ ID NO: 2), APT73.159 (SEQ ID NO: 3), APT73.160 (SEQ ID NO: 4), APT73.161 (SEQ ID NO: 5), APT73.162 (SEQ ID NO: 6), APT73.163 (SEQ ID NO: 7), APT73.165 (SEQ ID NO: 8), APT73.166 (SEQ ID NO: 9), APT73.168 (SEQ ID NO: 10), APT73.169 (SEQ ID NO: 11), APT73.170 (SEQ ID NO: 12), APT73.171 (SEQ ID NO: 13), APT73.172 (SEQ ID NO: 14), APT73.173 (SEQ ID NO: 15), APT73.174 (SEQ ID NO: 16), APT73.175 (SEQ ID NO: 17), APT73.176 (SEQ ID NO: 18), APT73.177 (SEQ ID NO: 19), APT73.178 (SEQ ID NO: 20), APT73.179 (SEQ ID NO: 21), APT73.180 (SEQ ID NO: 22), APT73.181 (SEQ ID NO: 23), APT73.182 (SEQ ID NO: 24), APT73.183 (SEQ ID NO: 25), APT73.184 (SEQ ID NO: 26), APT73.185 (SEQ ID NO: 27), APT73.186 (SEQ ID NO: 28), APT ), APT73.169 (SEQ ID NO: 11), APT73.170 (SEQ ID NO: 12), APT73.172 (SEQ ID NO: 13), APT73.182 (SEQ ID NO: 14), APT73.183 (SEQ ID NO: 15), APT73.184 (SEQ ID NO: 16), APT73.185 (SEQ ID NO: 17), APT73.186 (SEQ ID NO: 18), APT73.187 (SEQ ID NO: 19), APT73.188 (SEQ ID NO: 20), APT73.189 (SEQ ID NO: 21), APT73.190 (SEQ ID NO: 22), APT73.191 (SEQ ID NO: 23), APT73.192 (SEQ ID NO: 24), APT73.193 (SEQ ID NO: 25), APT73.194 (SEQ ID NO: 26), APT73.195 (SEQ ID NO: 27), APT73.196 (SEQ ID NO: 28), APT73.197 (SEQ ID NO: 29), APT73.198 (SEQ ID NO: 30), APT73.199 (SEQ ID NO: 31), APT73.199 (SEQ ID NO: 32), APT73.191 (SEQ ID NO: 33), APT73.192 (SEQ ID NO: 34), APT73.193 (SEQ ID NO: 35), APT73.194 (SEQ ID NO: 36), APT73.195 (SEQ ID NO: 37), APT73.196 (SEQ ID NO: 38), APT73. APT73.3.188 (SEQ ID NO: 20), APT73.189 (SEQ ID NO: 21), APT73.190 (SEQ ID NO: 22), APT73.191 (SEQ ID NO: 23), APT73.192 (SEQ ID NO: 24), APT73.193 (SEQ ID NO: 25), APT73.194 (SEQ ID NO: 26), APT73.195 (SEQ ID NO: 27), APT73.196 (SEQ ID NO: 28), APT73.197 (SEQ ID NO: 29), APT73.198 ( In some embodiments, the amino acid sequence comprises an amino acid sequence having at least one amino acid modification compared to APT73.199 (SEQ ID NO:31), APT73.200 (SEQ ID NO:32), APT73.201 (SEQ ID NO:33), APT73.202 (SEQ ID NO:34), APT73.203 (SEQ ID NO:35), APT73.204 (SEQ ID NO:36), or APT73.248 (SEQ ID NO:96), or a functional fragment or variant thereof. In some embodiments, the at least one amino acid modification comprises at least two, at least three, at least four, at least five, or at least six amino acid modifications.

[0084] In another aspect, the disclosure is directed to a recombinant membrane-bound prenyltransferase (rMPT), wherein the rMPT has been engineered to transfer geranyl pyrophosphate (GPP) to olivetolic acid and / or divalanic acid with greater efficiency than naturally occurring MPT, and wherein the rMPT has an amino acid sequence that includes at least one amino acid modification compared to the amino acid sequence of MPT4.1 (SEQ ID NO: 46), MPT48 (SEQ ID NO: 47), or MPT69 (SEQ ID NO: 48). In some embodiments, the at least one amino acid modification includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid modifications compared to the amino acid sequence of MPT4.1 (SEQ ID NO: 46), MPT48 (SEQ ID NO: 47) or MPT69 (SEQ ID NO: 48).

[0085] In some embodiments, at least one amino acid modification compared to MPT48 (SEQ ID NO: 47) is the first 50 amino acids M1 to G50 at the N-terminus of SEQ ID NO: 47 and / or H54, M88, R89, C93, A94, N96, D97, V98, V99, D100, Q101, D102, F103, D104, R109, R113, S121, A136, C147, Q148, V154, K165, Q172, L1 75, T178, L179, I181, L201, T207, V214, Y217, D218, V219, Y221, T253, L254, T262, V267, N269, P271, L281, A284, A287, S304, G305, W306, N314, L316, G317, G318, V327, M329 and / or L330. In some embodiments, the rMPT comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or more amino acid modifications. In some embodiments, the rMPT further comprises a truncation (e.g., 1-10 amino acids) at the C-terminus and / or N-terminus.

[0086] In some embodiments, at least one amino acid modification compared to MPT69 (SEQ ID NO: 48) is within the first 45 amino acids M1 to G45 at the N-terminus of SEQ ID NO: 48, and / or any of the following amino acids: H49, M83, R84, C88, A89, N91, D92, N93, I94, D95, Q96, D97, F98, D99, R104, R108, S116, A131, C142, H143, V149, K160, Q167, L170 , T173, L174, A176, R196, T202, V209, Y212, D213, V214, Y216, T248, C249, V257, V262, N264, P266, L276, A279, A282, S299, G300, W301, N309, M311, S312, G313, A322, L324 and / or L325. In some embodiments, the rMPT comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or more amino acid modifications. In some embodiments, the rMPT further comprises a truncation (e.g., 1-10 amino acids) at the C-terminus and / or N-terminus.

[0087] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 98 (MPT69.2). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 98 (MPT69.2). In some embodiments, the rMPT comprises SEQ ID NO: 98 (MPT69.2) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 98 (MPT69.2). In some embodiments, a functional fragment of SEQ ID NO:98 (MPT69.2) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO:98 (MPT69.2) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids deleted from the carboxy terminus.

[0088] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 99 (MPT69.5). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 99 (MPT69.5). In some embodiments, the rMPT comprises SEQ ID NO: 99 (MPT69.5) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 99 (MPT69.5). In some embodiments, a functional fragment of SEQ ID NO:99 (MPT69.5) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO:99 (MPT69.5) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids deleted from the carboxy terminus.

[0089] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 100 (MPT69.6). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 100 (MPT69.6). In some embodiments, the rMPT comprises SEQ ID NO: 100 (MPT69.6) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 100 (MPT69.6). In some embodiments, a functional fragment of SEQ ID NO: 100 (MPT69.6) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO: 100 (MPT69.6) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids deleted from the carboxy terminus.

[0090] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 101 (MPT69.7). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 101 (MPT69.7). In some embodiments, the rMPT comprises SEQ ID NO: 101 (MPT69.7) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 101 (MPT69.7). In some embodiments, the functional fragment of SEQ ID NO: 101 (MPT69.7) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO: 101 (MPT69.7) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids deleted from the carboxy terminus.

[0091] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 102 (MPT69.8). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 102 (MPT69.8). In some embodiments, the rMPT comprises SEQ ID NO: 102 (MPT69.8) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 102 (MPT69.8). In some embodiments, the functional fragment of SEQ ID NO: 102 (MPT69.8) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO: 102 (MPT69.8) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids deleted from the carboxy terminus.

[0092] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 103 (MPT69.9). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 103 (MPT69.9). In some embodiments, the rMPT comprises SEQ ID NO: 103 (MPT69.9) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 103 (MPT69.9). In some embodiments, the functional fragment of SEQ ID NO: 103 (MPT69.9) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO: 103 (MPT69.9) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids deleted from the carboxy terminus.

[0093] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 104 (MPT69.10). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 104 (MPT69.10). In some embodiments, the rMPT comprises SEQ ID NO: 104 (MPT69.10) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 104 (MPT69.10). In some embodiments, the functional fragment of SEQ ID NO: 104 (MPT69.10) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO: 104 (MPT69.10) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids deleted from the carboxy terminus.

[0094] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 40 (MPT4.29). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 40 (MPT4.29). In some embodiments, the rMPT comprises SEQ ID NO: 40 (MPT4.29) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 40 (MPT4.29). In some embodiments, a functional fragment of SEQ ID NO:40 (MPT4.29) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO:40 (MPT4.29) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids deleted from the carboxy terminus.

[0095] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 41 (MPT4.30). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 41 (MPT4.30). In some embodiments, the rMPT comprises SEQ ID NO: 41 (MPT4.30) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 41 (MPT4.30). In some embodiments, a functional fragment of SEQ ID NO:41 (MPT4.30) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO:41 (MPT4.30) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the carboxy terminus.

[0096] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 42 (MPT4.32). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 42 (MPT4.32). In some embodiments, the rMPT comprises SEQ ID NO: 42 (MPT4.32) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 42 (MPT4.32). In some embodiments, a functional fragment of SEQ ID NO:42 (MPT4.32) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO:42 (MPT4.32) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the carboxy terminus.

[0097] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 39 (MPT4.33). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 39 (MPT4.33). In some embodiments, the rMPT comprises SEQ ID NO: 39 (MPT4.33) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 39 (MPT4.33). In some embodiments, a functional fragment of SEQ ID NO:39 (MPT4.33) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO:39 (MPT4.33) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the carboxy terminus.

[0098] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 43 (MPT4.34). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 43 (MPT4.34). In some embodiments, the rMPT comprises SEQ ID NO: 43 (MPT4.34) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 43 (MPT4.34). In some embodiments, a functional fragment of SEQ ID NO:43 (MPT4.34) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO:43 (MPT4.34) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the carboxy terminus.

[0099] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 44 (MPT4.40). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 44 (MPT4.40). In some embodiments, the rMPT comprises SEQ ID NO: 44 (MPT4.40) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 44 (MPT4.40). In some embodiments, a functional fragment of SEQ ID NO:44 (MPT4.40) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO:44 (MPT4.40) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the carboxy terminus.

[0100] In some embodiments, the rMPT comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 45 (MPT4.41). In some embodiments, the rMPT comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 45 (MPT4.41). In some embodiments, the rMPT comprises SEQ ID NO: 45 (MPT4.41) or a functional fragment of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 45 (MPT4.41). In some embodiments, a functional fragment of SEQ ID NO:45 (MPT4.41) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the amino terminus. In some embodiments, a functional fragment of SEQ ID NO:45 (MPT4.41) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted from the carboxy terminus.

[0101] "Identity" refers to the degree to which two or more nucleic acid or polypeptide sequences are identical. In some embodiments, the percent identity between a sequence of interest and a second sequence over a window of evaluation, for example, the length of the sequence of interest, can be calculated by aligning these sequences, determining the number of residues (nucleotides or amino acids) within the window of evaluation that are identical, allowing for the introduction of gaps to maximize identity, dividing by the total number of residues in the sequence of interest or the second sequence that fall within that window (whichever is greater), and multiplying by 100. When calculating the number of identical residues required to achieve a specific percent identity, fractions should be rounded to the nearest integer. Percent identity can be calculated using various computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, and Gapped BLAST generate alignments and provide percent identity between sequences of interest. The algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:22264-2268, 1990), as modified in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993, has been incorporated into the NBLAST and XBLAST programs of Altschul et al. (Altschul et al., J. Mol. Biol. 215:403-410, 1990). To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (Altschul, et al. Nucleic Acids Res. 25:3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program can be used. PAM250 or BLOSUM62 matrices can be used. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI).For these programs, see the website with the URL ncbi.nlm.nih.gov. In certain embodiments, percent identity is calculated using BLAST2 with the default parameters provided by NCBI.

[0102] In some embodiments, the rMPT comprises a fusion domain, which in some embodiments improves the expression and / or overall activity of the enzyme.

[0103] In some embodiments, rMPT can convert olivetolic acid (OA) and geranyl diphosphate (GPP) into one or more products, including cannabigerolic acid (CBGA). In some embodiments, rMPT can produce CBGA in a cell-free system, yeast cells, bacterial cells, algae cells, or plant cells. In some embodiments, the one or more products comprise at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or substantially 100% CBGA. In some embodiments, at least about 50% of the one or more products is CBGA. In some embodiments, more than about 90% of the one or more products is CBGA. In some embodiments, rMPT has a rate of cannabigerolic acid (CBGA) formation from olivetolic acid (OA) and geranyl diphosphate (GPP) that is greater than the rate of CBGA formation from OA and GPP by MPT4.1, MPT48, and / or MPT69 under the same conditions. In some embodiments, the rate of CBGA formation from OA and GPP is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 5-fold, 10-fold, or more, compared to the rate of CBGA formation from OA and GPP by MPT4 under the same conditions.

[0104] In some embodiments, rMPT can convert olivetolic acid (OA) and farnesyl pyrophosphate (FPP) to one or more cannabinoids, cannabinoid derivatives, or cannabinoid analogs. In some embodiments, rMPT can produce cannabinoids, cannabinoid derivatives, or cannabinoid analogs in a cell-free system, in yeast cells, in bacterial cells, in algae cells, or in plant cells. In some embodiments, the activity of rMPT to convert OA and FPP to one or more cannabinoids, cannabinoid derivatives, or cannabinoid analogs is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or substantially 100% of the activity of rMPT to convert OA and GPP to one or more cannabinoids, cannabinoid derivatives, or cannabinoid analogs.

[0105] In some embodiments, rMPT produces CBGA and FCBGA from olivetolic acid (OA) and geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) at a CBGA / FCBGA ratio greater than the ratio of CBGA / FCBGA formation from OA and GPP and FPP by MPT4.1, MPT48, or MPT69 under the same conditions. As used herein and in some embodiments, "greater" is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 5-fold, 10-fold, or more, compared to a relevant control.

[0106] Recombinant rMPT with reduced or no FCBGA-forming activity may be advantageous in some situations. In some embodiments, rMPT has a rate of cannabigerovarinic acid (CBGVA) formation from divaleric acid (DVA) and geranyl diphosphate (GPP) that is greater than the rate of CBGVA formation from DVA and GPP by MPT4.1, MPT48 or MPT69 under the same conditions. In some embodiments, rMPT has a ratio of CBGVA formation to F-CBGVA formation from DVA and GPP and FPP that is greater than the ratio of CBGVA formation to F-CBGVA formation from DVA and GPP and FPP by MPT4 under the same conditions. As used herein and in some embodiments, "greater" means at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 5-fold, 10-fold, or more, compared to a relevant control. In some embodiments, rMPT does not form F-CBGVA.

[0107] In some embodiments, rMPT has a rate of CBGA formation from OA and GPP that is at least 1.2 times greater (e.g., 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 5 times, 10 times, or more) than the rate of CBGA formation from OA and GPP by MPT4.1, MPT48, or MPT69 under the same conditions.

[0108] The cannabinoids, cannabinoid derivatives, and cannabinoid analogs described herein are not limited. In some embodiments, the cannabinoid is a cannabichromene (CBC) type (e.g., cannabichromene acid), a cannabigerol (CBG) type (e.g., cannabigerolic acid), a cannabidiol (CBD) type (e.g., cannabidiolic acid), a Δ9-trans-tetrahydrocannabinol (Δ9-THC) type (e.g., Δ9-tetrahydrocannabinolic acid), a Δ8-trans-tetrahydrocannabinol (Δ8-THC) type, a cannabicyclol (CBL) type, a cannabielsoin (CBE) type, a cannabinol (CBN) type, cannabinodiol (CBND) type, cannabidiol (CBT) type, cannabigerolic acid (CBGA), cannabigerolic acid monomethyl ether (CBGAM), cannabigerol (CBG), cannabigerol monomethyl ether (CBGM), cannabigerovaric acid (CBGVA), cannabigerovarin (CBGV), cannabichromene acid (CBCA), cannabichromene (CBC), cannabichromevaric acid (CBCVA), cannabichromevarin (CBCV), cannabidiolic acid (CBDA), Cannabidiol (CBD), cannabidiol monomethyl ether (CBDM), cannabidiol-C4 (CBD-C4), cannabidivaric acid (CBDVA), cannabidivarin (CBDV), cannabidiolcol (CBD-C1), Δ9-tetrahydrocannabinolic acid A (THCA-A), Δ9-tetrahydrocannabinolic acid B (THCA-B), Δ9-tetrahydrocannabinol (THC), Δ9-tetrahydrocannabinolic acid-C4 (THCA-C4), Δ9-tetrahydrocannabinol-C4 ( THC-C4), Δ9-tetrahydrocannabivarinic acid (THCVA), Δ9-tetrahydrocannabivarin (THCV), Δ9-tetrahydrocannabiorcholic acid (THCA-C1), Δ9-tetrahydrocannabiorcholic acid (THC-C1), Δ7-cis-iso-tetrahydrocannabivarin, Δ8-tetrahydrocannabinolic acid (Δ8-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabicyclolic acid (CBLA), cannabicyclol (CBL), cannabicyclovalin (CBLV),Cannabielsonic acid A (CBEA-A), cannabielsonic acid B (CBEA-B), cannabielsoin (CBE), cannabielsoic acid, cannabicitranic acid, cannabinolic acid (CBNA), cannabinol (CBN), cannabinol methyl ether (CBNM), cannabinol-C4 (CBN-C4), cannabivarin (CBV), cannabinol-C2 (CNB-C2), cannabiolcol (CBN-C1), cannabinodiol (CBND), cannabinodivarin (CBVD), cannabiditriol (CBT), 10-ethyoxy-9-hydroxy-delta-6a-tetrahydrocannabinol, 8,9-dihydroxyl-delta-6a-tetrahydrocanna Cannabidiol, cannabidiol valine (CBTVE), dehydrocannabifuran (DCBF), cannabifuran (CBF), cannabichromanone (CBCN), cannabiditran (CBT), 10-oxo-delta-6a-tetrahydrocannabinol (OTHC), delta-9-cis-tetrahydrocannabinol (cis-THC), 3,4,5,6-tetrahydro-7-hydroxy-alpha-alpha-2-trimethyl-9-n-propyl-2,6-methano-2H-1-benzoxocine-5-methanol (OH-iso-HHCV), cannabilipsol (CBR), and trihydroxy-delta-9-tetrahydrocannabinol (tri-OH-THC).

[0109] In some embodiments, rMPT can convert divaleric acid (DVA) and GPP into one or more cannabinoids, cannabinoid derivatives, or cannabinoid analogs. The cannabinoids are not limited and can be any of those disclosed herein. In some embodiments, rMPT can produce cannabinoids, cannabinoid derivatives, or cannabinoid analogs in a cell-free system, in yeast cells, in bacterial cells, in algae cells, or in plant cells. In some embodiments, the activity of rMPT to convert DVA and FPP to one or more cannabinoids, cannabinoid derivatives or cannabinoid analogs is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or substantially 100% of the activity of rMPT to convert OA and GPP to one or more cannabinoids, cannabinoid derivatives or cannabinoid analogs.

[0110] fusion proteins

[0111] Some embodiments of the present disclosure are directed to a fusion protein comprising a polypeptide having geranyl diphosphate (GPP) synthase activity and a polypeptide having prenyltransferase activity. As used herein, "GPP synthase activity" refers to the ability to catalyze the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to geranyl diphosphate. As used herein, "prenyltransferase activity" refers to the ability to catalyze the transfer of a prenyl group from one compound (donor) to another compound (acceptor).

[0112] Some embodiments of the present disclosure are directed to a fusion protein comprising a polypeptide having geranyl diphosphate synthase activity and a polypeptide having prenyltransferase activity, wherein the polypeptide having prenyltransferase activity comprises: a) a polypeptide sequence having at least 85% identity to the polypeptide sequence of MPT48 (SEQ ID NO: 47) or MPT69 (SEQ ID NO: 48); b) a polypeptide sequence comprising at least one amino acid modification compared to the amino acid sequence of MPT4.1 (SEQ ID NO: 46); or c) a polypeptide comprising: i) an amino acid sequence having a C-terminal truncation of at least 8 amino acids compared to naturally occurring aromatic prenyltransferase (APT) and ii) an amino acid sequence comprising at least one amino acid modification compared to naturally occurring APT at a position corresponding to one of the first 135 amino acids of naturally occurring APT, or a functional fragment thereof.

[0113] In some embodiments, the polypeptide having prenyltransferase activity is APT73.179 (SEQ ID NO: 1), APT73.119 (SEQ ID NO: 2), APT73.159 (SEQ ID NO: 3), APT73.160 (SEQ ID NO: 4), APT73.161 (SEQ ID NO: 5), APT73.162 (SEQ ID NO: 6), APT73.163 (SEQ ID NO: 7), APT73.165 (SEQ ID NO: 8), APT73.166 (SEQ ID NO: 9), APT73.168 (SEQ ID NO: 10), APT73.169 (SEQ ID NO: 11), APT73.170 (SEQ ID NO: 12), APT73.172 (SEQ ID NO: 13), APT73.182 (SEQ ID NO: 14), APT73.183 (SEQ ID NO: 15), APT73.184 (SEQ ID NO: 16), APT73.185 (SEQ ID NO: 17), APT73.186 (SEQ ID NO: 18), APT73.187 (SEQ ID NO: 19), APT73.188 (SEQ ID NO: 20), APT73.189 (SEQ ID NO: 21), APT73.189 (SEQ ID NO: 22), APT73.189 (SEQ ID NO: 23), APT73.189 (SEQ ID NO: 24), APT73.189 (SEQ ID NO: 25), APT73.189 (SEQ ID NO: 26), APT73.189 (SEQ ID NO: 27), APT73.189 (SEQ ID NO: 28), APT73.189 (SEQ ID NO: 29), APT73.189 (SEQ ID NO: 30), APT73.189 (SEQ ID NO: 31), APT73.189 (SEQ ID NO: 32), APT73.189 (SEQ ID NO: 33), APT73.189 (SEQ ID NO: 34), APT73.189 (SEQ ID NO: 35), APT73.189 (SEQ ID NO: 36), APT73.189 (SEQ ID NO: 37), APT73.189 (SEQ ID NO: No. 21), APT73.190 (SEQ ID NO: 22), APT73.191 (SEQ ID NO: 23), APT73.192 (SEQ ID NO: 24), APT73.193 (SEQ ID NO: 25), APT73.194 (SEQ ID NO: 26), APT73.195 (SEQ ID NO: 27), APT73.196 (SEQ ID NO: 28), APT73.197 (SEQ ID NO: 29), APT73.198 (SEQ ID NO: 30), APT73.199 (SEQ ID NO: 31), APT73.200 (SEQ ID NO: 32), APT73.201 (SEQ ID NO: 33), APT73.202 (SEQ ID NO: 34), APT73.203 (SEQ ID NO: 35), APT73.204 (SEQ ID NO: 36), APT73.205 (SEQ ID NO: 37), APT73.206 (SEQ ID NO: 38), APT73.207 (SEQ ID NO: 39), APT73.208 (SEQ ID NO: 40), APT73.209 (SEQ ID NO: 41), APT73.210 (SEQ ID NO: 42), APT73.211 (SEQ ID NO: 43), APT73.212 (SEQ ID NO: 44), APT73.213 (SEQ ID NO: 45), APT73.214 (SEQ ID NO: 46), APT73.215 (SEQ ID NO: 47), APT73.216 (SEQ ID NO: 48), APT73.217 (SEQ ID NO: 49), A 2), APT73.201 (SEQ ID NO:33), APT73.202 (SEQ ID NO:34), APT73.203 (SEQ ID NO:35), APT73.204 (SEQ ID NO:36), or APT73.248 (SEQ ID NO:96), or a fragment or variant thereof.

[0114] In some embodiments, the polypeptide having prenyltransferase activity is APT73.179 (SEQ ID NO: 1), APT73.119 (SEQ ID NO: 2), APT73.159 (SEQ ID NO: 3), APT73.160 (SEQ ID NO: 4), APT73.161 (SEQ ID NO: 5), APT73.162 (SEQ ID NO: 6), APT73.163 (SEQ ID NO: 7), APT73.165 (SEQ ID NO: 8), APT73.166 (SEQ ID NO: 9), APT73.167 (SEQ ID NO: 10), APT73.168 (SEQ ID NO: 11), APT73.169 (SEQ ID NO: 12), APT73.170 (SEQ ID NO: 13), APT73.171 (SEQ ID NO: 14), APT73.172 (SEQ ID NO: 15), APT73.173 (SEQ ID NO: 16), APT73.174 (SEQ ID NO: 17), APT73.175 (SEQ ID NO: 18), APT73.176 (SEQ ID NO: 19), APT73.177 (SEQ ID NO: 20), APT73.178 (SEQ ID NO: 21), APT73.179 (SEQ ID NO: 22), APT73.179 (SEQ ID NO: 23), APT73.179 (SEQ ID NO: 24), APT73.179 (SEQ ID NO: 25), APT73.179 (SEQ ID NO: 26), APT73.171 (SEQ ID NO: 27), APT73.172 (SEQ ID NO: 28), A APT73.168 (SEQ ID NO: 10), APT73.169 (SEQ ID NO: 11), APT73.170 (SEQ ID NO: 12), APT73.172 (SEQ ID NO: 13), APT73.182 (SEQ ID NO: 14), APT73.183 (SEQ ID NO: 15), APT73.184 (SEQ ID NO: 16), APT73.185 (SEQ ID NO: 17), APT73.186 (SEQ ID NO: 18), APT73.187 (SEQ ID NO: 19), APT73.188 (SEQ ID NO: 20), APT 73.189 (SEQ ID NO: 21), APT73.190 (SEQ ID NO: 22), APT73.191 (SEQ ID NO: 23), APT73.192 (SEQ ID NO: 24), APT73.193 (SEQ ID NO: 25), APT73.194 (SEQ ID NO: 26), APT73.195 (SEQ ID NO: 27), APT73.196 (SEQ ID NO: 28), APT73.197 (SEQ ID NO: 29), APT73.198 (SEQ ID NO: 30), APT73.199 (SEQ ID NO: 31), A APT73.200 (SEQ ID NO: 32), APT73.201 (SEQ ID NO: 33), APT73.202 (SEQ ID NO: 34), APT73.203 (SEQ ID NO: 35), APT73.204 (SEQ ID NO: 36), or APT73.248 (SEQ ID NO: 96), or a fragment or variant thereof.

[0115] In some embodiments, the polypeptide having prenyltransferase activity comprises a polypeptide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 99.9% identity to MPT4.1 (SEQ ID NO:46), MPT4.29 (SEQ ID NO:40), MPT4.30 (SEQ ID NO:41), MPT4.32 (SEQ ID NO:42), MPT4.33 (SEQ ID NO:40), MPT4.34 (SEQ ID NO:43), MPT4.40 (SEQ ID NO:44) or MPT4.41 (SEQ ID NO:45).

[0116] In some embodiments, the polypeptide having prenyltransferase activity comprises a polypeptide sequence having at least 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 99.9% identity to MPT4.1 (SEQ ID NO:46), MPT4.29 (SEQ ID NO:40), MPT4.30 (SEQ ID NO:41), MPT4.32 (SEQ ID NO:42), MPT4.33 (SEQ ID NO:39), MPT4.34 (SEQ ID NO:43), MPT4.40 (SEQ ID NO:44) or MPT4.41 (SEQ ID NO:45).

[0117] In some embodiments, the polypeptide having prenyltransferase activity has improved selectivity for GPP over FPP compared to a control membrane-bound prenyltransferase or a soluble aromatic prenyltransferase. In some embodiments, the polypeptide having prenyltransferase activity has an amino acid sequence comprising a portion of the amino acid sequence of SEQ ID NO: 46 (MPT4.1) and SEQ ID NO: 47 (MPT48) or SEQ ID NO: 48 (MPT69). In some embodiments, the polypeptide having geranyl diphosphate synthase activity comprises the polypeptide of SEQ ID NO: 49 (GPS1.1), or a functional fragment or functional variant thereof.

[0118] In some embodiments, the fusion protein is selected from the group consisting of GPS1.1-L18-APT73.119 (SEQ ID NO: 52), GPS1.1-L18-APT73.159 (SEQ ID NO: 53), GPS1.1-L18-APT73.160 (SEQ ID NO: 54), GPS1.1-L18-APT73.161 (SEQ ID NO: 55), GPS1.1-L18-APT73.162 (SEQ ID NO: 56), GPS1.1-L18-APT73.163 (SEQ ID NO: 57), GPS1.1-L18-APT73.165 (SEQ ID NO: 58), GPS1.1-L18-APT73.166 (SEQ ID NO: 59), No. 59), GPS1.1-L18-APT73.168 (SEQ ID NO: 60), GPS1.1-L18-APT73.169 (SEQ ID NO: 61), GPS1.1-L18-APT73.170 (SEQ ID NO: 62), GPS1.1-L18-APT73.172 (SEQ ID NO: 63), GPS1.1-L18-APT73.179 (SEQ ID NO: 64), GPS1.1-L18-APT73.182 (SEQ ID NO: 65), GPS1.1-L18-APT73.183 (SEQ ID NO: 66), GPS1.1-L18-APT73.184 (SEQ ID NO: 67), GPS1.1-L18-A PT73.185 (SEQ ID NO: 68), GPS1.1-L18-APT73.186 (SEQ ID NO: 69), GPS1.1-L18-APT73.187 (SEQ ID NO: 70), GPS1.1-L18-APT73.188 (SEQ ID NO: 71), GPS1.1-L18-APT73.189 (SEQ ID NO: 72), GPS1.1-L18-APT73.190 (SEQ ID NO: 73), GPS1.1-L18-APT73.191 (SEQ ID NO: 74), GPS1.1-L18-APT73.192 (SEQ ID NO: 75), GPS1.1-L18-APT73.193 (SEQ ID NO: 76), GPS1.1-L18-APT73.194 (SEQ ID NO: 77), GPS1.1-L18-APT73.195 (SEQ ID NO: 78), GPS1.1-L18-APT73.196 (SEQ ID NO: 79), GPS1.1-L18-APT73.197 (SEQ ID NO: 80), GPS1.1-L18-APT73.198 (SEQ ID NO: 81), GPS1.1-L18-APT73.199 (SEQ ID NO: 82), GPS1.1-L18-APT73.200 (SEQ ID NO: 83), GPS1.1-L18-APT73.201 (SEQ ID NO: 84), GPS1.1-L18-APT73.and a polypeptide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 99.9% identity to the polypeptide sequence of GPS1.1-L18-APT73.202 (SEQ ID NO: 85), GPS1.1-L18-APT73.203 (SEQ ID NO: 86), GPS1.1-L18-APT73.204 (SEQ ID NO: 87), GPS1.1-L18-APT73.248 (SEQ ID NO: 97), or a functional fragment or variant thereof.

[0119] In some embodiments, the fusion protein is selected from the group consisting of GPS1.1-L11-MPT4.1 (SEQ ID NO: 88), GPS1.1-L11-MPT4.29 (SEQ ID NO: 89), GPS1.1-L11-MPT4.30 (SEQ ID NO: 90), GPS1.1-L11-MPT4.32 (SEQ ID NO: 91), GPS1.1-L11-MPT4.33 (SEQ ID NO: 92), GPS1.1-L11-MPT4. .34 (SEQ ID NO: 93), GPS1.1-L11-MPT4.40 (SEQ ID NO: 94), GPS1.1-L11-MPT4.41 (SEQ ID NO: 95), or a functional fragment or variant thereof.

[0120] In some embodiments, the polypeptide having prenyltransferase activity has improved selectivity for GPP over FPP compared to the same unfused prenyltransferase. In some embodiments, the polypeptide having prenyltransferase activity has at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 5-fold, 10-fold, or more selectivity for GPP over FPP compared to the same unfused prenyltransferase. In some embodiments, the polypeptide produces CBGA and FCBGA at a higher CBGA / FCBGA ratio compared to the same unfused prenyltransferase.

[0121] In some embodiments, the fusion protein further comprises a linker polypeptide between the polypeptide having geranyl diphosphate synthase activity (GPS) and the polypeptide having prenyltransferase activity. The linker is not limited and can be any suitable linker. For example, the linker can be a short polypeptide (e.g., 15 to 52 amino acids). Often, the linker is composed of small amino acid residues such as serine, glycine, and / or alanine. The heterologous domain can include a transmembrane domain, a secretory signal domain, etc. In some embodiments, the linker is a polypeptide. In some embodiments, the polypeptide is 5 to 52 amino acids in length. In some embodiments, the linker comprises a polypeptide selected from SEQ ID NO: 50 or SEQ ID NO: 51.

[0122] In some embodiments, geranyl diphosphate is fused to the N-terminus of the prenyltransferase, hi other embodiments, geranyl diphosphate is fused to the C-terminus of the prenyltransferase.

[0123] Recombinant Cells and Cell Culture

[0124] Some aspects of the present disclosure are directed to cells that express the rMPT described herein. Some aspects of the present disclosure are directed to cells that have an exogenous nucleic acid sequence encoding the rMPT described herein.

[0125] Some embodiments of the present disclosure are directed to cells comprising engineered APT and / or rMPT, wherein the cells are capable of producing CBGA in the presence of GPP and OA. Some embodiments of the present disclosure are directed to cells comprising engineered APT and / or rMPT, wherein the cells are capable of producing CBGA in the presence of GPP and DVA. Some embodiments of the present disclosure are directed to cells comprising APT and / or rMPT, wherein the cells are capable of producing cannabinoids in the presence of a carbon source and, optionally, hexanoic acid or butyric acid.

[0126] In some embodiments, the cell is a yeast cell or a bacterial cell. In some embodiments, the yeast cell is a Yarrowia strain or a Saccharomyces strain.

[0127] Some aspects of the present disclosure are directed to methods for producing CBGA, CBGVA, or derivatives thereof, comprising culturing cells containing APT or rMPT under conditions suitable for producing CBGA, CBGVA, or derivatives thereof. In some embodiments, the suitable conditions include adding at least one of butyric acid, valeric acid, isovaleric acid, hexanoic acid, hexanol, butanol, oleic acid, glycerol, or glucose to the culture medium in which the cells are cultured.

[0128] Some aspects of the present disclosure are directed to cells that express the fusion proteins described herein. Some aspects of the present disclosure are directed to cells that have an exogenous nucleic acid sequence encoding the fusion proteins described herein.

[0129] Some embodiments of the present disclosure are directed to cells comprising a fusion protein, wherein the cells are capable of producing CBGA in the presence of OA and GPP. Some embodiments of the present disclosure are directed to cells comprising a fusion protein, wherein the cells are capable of producing CBGA in the presence of DVA and GPP. Some embodiments of the present disclosure are directed to cells comprising a fusion protein, wherein the cells are capable of producing cannabinoids in the presence of a carbon source and, optionally, hexanoic acid or butyric acid.

[0130] In some embodiments, the cells are capable of forming acyl-CoA from a carboxylic acid, hi some embodiments, the cells encode exogenous hexanoyl-CoA synthetase and / or butyryl-CoA synthetase.

[0131] In some embodiments, the cell is a yeast cell or a bacterial cell. In some embodiments, the yeast cell is a Yarrowia strain or a Saccharomyces strain.

[0132] Some aspects of the present disclosure are directed to methods for producing CBGA, CBGVA, or derivatives thereof, comprising culturing cells containing a fusion protein under conditions suitable for producing CBGA, CBGVA, or derivatives thereof. In some embodiments, the suitable conditions include adding at least one of butyric acid, valeric acid, isovaleric acid, hexanoic acid, hexanol, butanol, oleic acid, glycerol, or glucose to a culture medium in which the cells are cultured.

[0133] The cell is not limited and can be any suitable cell for expression. In some embodiments, the cell can be a microorganism or a plant. In some embodiments, the microorganism is a bacterium (e.g., E. coli), algae, or yeast. In some embodiments, the yeast is an oleaginous yeast (e.g., a Yarrowia lipolytica strain). In some embodiments, the bacterium is Escherichia coli.

[0134] Suitable cells are Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia species, Saccharomyces cerevisiae, Saccharomyces species, Hansenula polymorpha (now known as Pichia angusta), Kluyveromyces species, Kluyveromyces lactis, Kluyveromyces marxianus, Schizosaccharomyces pompe, Dekkera bruxellensis, Arxula adeninivorans, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium species, Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas reinhardtii, Yarrowia lipolytica, and the like. In some embodiments, the cell is a protease-deficient strain of Saccharomyces cerevisiae. In some embodiments, the cell is a eukaryotic cell other than a plant cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a plant cell, and the plant cell does not normally produce cannabinoids, cannabinoid derivatives or analogs, cannabinoid precursors, or cannabinoid precursor derivatives or analogs. In some embodiments, the cell is Saccharomyces cerevisiae. In some embodiments, the cells disclosed herein are cultured in vitro.

[0135] In some embodiments, the cell is a prokaryotic cell. Suitable prokaryotic cells may include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus species, Salmonella species, Shigella species, and the like. See, for example, Carrier et al. (1992) J. Immunol. 148:1176-1181; U.S. Patent No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302. Examples of Salmonella strains that can be used include, but are not limited to, Salmonella typhi and S. typhimurium. Suitable Shigella strains may include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Typically, laboratory strains are non-pathogenic strains. Non-limiting examples of other suitable bacteria may include, but are not limited to, Bacillus subtilis, Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and the like.

[0136] One or more expression vectors can be constructed to contain an exogenous nucleotide sequence encoding the rMPT or APT described herein operably linked to an expression control sequence functional in a cell. Applicable expression vectors include, for example, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which contain vectors and selection sequences or markers operable for stable integration into a host chromosome. In addition, the expression vector can contain one or more selectable marker genes and appropriate expression control sequences. For example, selectable marker genes that provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not present in the culture medium can also be included. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or into separate expression vectors. For single-vector expression, the encoding nucleic acids can be operably linked to a common expression control sequence or to different expression control sequences, such as one inducible promoter and one constitutive promoter. Transformation of an exogenous nucleic acid sequence can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of a gene product, or other suitable analytical methods for testing expression of the introduced nucleic acid sequence or its corresponding gene product. It will be understood by those skilled in the art that the exogenous nucleic acid will be expressed in an amount sufficient to produce the desired product, and it will further be understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and disclosed herein.

[0137] The term "exogenous" is intended to mean that the referenced molecule or referenced activity is introduced into a cell. The molecule can be introduced by introduction of an encoding nucleic acid into the host genetic material, for example, by integration into a host chromosome, or as non-chromosomal genetic material such as a plasmid. Thus, when used with respect to expression of an encoding nucleic acid, the term refers to the introduction of an expressible form of the encoding nucleic acid into a cell. When used with respect to a biosynthetic activity, the term refers to an activity introduced into a host. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity after introduction into the cell. Thus, the term "endogenous" refers to a referenced molecule or activity that is present in a cell. Similarly, when used with respect to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid contained within a microorganism. The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species, whereas "homologous" refers to a molecule or activity derived from the host microorganism. Thus, exogenous expression of an encoding nucleic acid can utilize either or both heterologous or homologous encoding nucleic acid.

[0138] In some embodiments, cells expressing rMPT or APT can produce CBGA in the presence of GPP and OA (e.g., metabolically produced by the cells or via the presence of OA). In some embodiments, cells expressing rMPT or APT can produce CBGA from a carbon source. In some embodiments, cells expressing rMPT or APT can produce CBGA from a carbon source in the presence of hexanoic acid.

[0139] In some embodiments, cells expressing rMPT or APT can produce CBGVA in the presence of GPP and DVA (e.g., metabolically produced by the cells or via the presence of one or more DVAs in the medium). In some embodiments, cells expressing rMPT or APT can produce CBGVA from a carbon source. In some embodiments, cells expressing rMPT or APT can produce CBGVA from a carbon source in the presence of butyrate.

[0140] In some embodiments, cells expressing rMPT or APT are capable of making cannabinoids or analogs thereof in the presence of a carbon source and, optionally, hexanoic acid or butyric acid.

[0141] In some embodiments, cells expressing the fusion protein can produce CBGA in the presence of GPP and OA (e.g., metabolically produced by the cells or via the presence of OA in the medium). In some embodiments, cells expressing the fusion protein can produce CBGA from a carbon source. In some embodiments, cells expressing the fusion protein can produce CBGA from a carbon source in the presence of hexanoic acid.

[0142] In some embodiments, cells expressing the fusion protein are capable of producing CBGVA in the presence of GPP and DVA (e.g., metabolically produced by the cells or via the presence of DVA in the medium). In some embodiments, cells expressing the fusion protein are capable of producing CBGVA from a carbon source. In some embodiments, cells expressing the fusion protein are capable of producing CBGVA from a carbon source in the presence of butyrate.

[0143] In some embodiments, cells expressing the fusion protein can produce cannabinoids or analogs thereof in the presence of a carbon source and, optionally, hexanoic acid or butyric acid. Exemplary carbon sources include sugar carbons such as sucrose, glucose, mannitol, galactose, fructose, mannose, isomaltose, xylose, pannose, maltose, arabinose, cellobiose, and their 3-, 4-, or 5-oligomers. Other carbon sources include alcoholic carbon sources such as ethanol and glycerol. Other carbon sources may contain combinations of the above carbon sources, such as glucose / mannitol or glucose / ethanol. Other carbon sources include acids and esters, such as acetate or formate, or fatty acids or fatty acid esters having 4 to 22 carbon atoms. Other carbon sources can include renewable feedstocks and biomass. Exemplary renewable feedstocks include cellulosic biomass, hemicellulosic biomass, and lignin feedstocks. Mixed carbon sources, such as fatty acids and sugars, as described herein, can also be used.

[0144] Depending on the cell, an appropriate culture medium can be used. For example, descriptions of various culture media can be found in the "Manual of Methods for General Bacteriology" (Washington DC, USA, 1981) of the American Society for Bacteriology. As used herein, "culture medium" in relation to a growth source refers to the starting medium, whether in solid or liquid form. On the other hand, as used herein, "cultured medium" refers to a medium (e.g., a liquid medium) containing fermentatively grown microorganisms and may contain other cellular biomass. A culture medium generally includes one or more carbon sources, nitrogen sources, inorganic salts, vitamins, and / or trace elements.

[0145] Culture conditions can include, for example, liquid culture procedures and fermentation and other large-scale culture procedures.Under aerobic culture conditions, useful yields of products can be obtained.Exemplary growth conditions for achieving one or more cannabinoid products include aerobic culture or fermentation conditions.In certain embodiments, microorganisms can be maintained, cultured or fermented under aerobic conditions.

[0146] Substantially aerobic conditions include, for example, cultures, batch fermentations, or continuous fermentations in which the dissolved oxygen concentration in the medium remains between 5% and 100% of saturation. The percentage of dissolved oxygen can be maintained, for example, by sparging with air, pure oxygen, or a mixture of air and oxygen.

[0147] Culture conditions can be scaled up and continuously grown to produce cannabinoid products. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation; or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of cannabinoid products. Generally, and similar to non-continuous culture procedures, continuous and / or near-continuous production of cannabinoid products involves culturing a cannabinoid-producing organism on nutrients and medium sufficient to maintain and / or nearly maintain exponential growth. Continuous culture under such conditions can include, for example, 1, 2, 3, 4, 5, 6, or 7 days or more. In addition, continuous culture can include 1, 2, 3, 4, or 5 weeks or more, and up to several months. Alternatively, if suitable for a particular application, the desired microorganism can be cultured for several hours. It should be understood that continuous and / or near-continuous culture conditions can also include all time intervals between these exemplary periods. It is further understood that the time for culturing the microorganism is for a period of time sufficient to produce a sufficient amount of product for the desired purpose.

[0148] Fermentation procedures are well known in the art. Briefly, for example, fermentation for the biosynthetic production of cannabinoid products can be used in fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

[0149] In some embodiments, the methods include providing a cell described herein comprising an exogenous nucleotide sequence encoding rMPT or APT described herein, and culturing the cell to produce a cannabinoid, cannabinoid derivative, or cannabinoid analogue thereof.

[0150] In some embodiments, a method comprises providing a cell described herein comprising an exogenous nucleotide sequence encoding a fusion protein described herein, and culturing the cell to produce a cannabinoid, cannabinoid derivative, or cannabinoid analog thereof. In some embodiments, a method comprises providing a cell described herein comprising an exogenous nucleotide sequence encoding a fusion protein described herein, and culturing the cell to produce a cannabinoid, cannabinoid derivative, or cannabinoid analog thereof.

[0151] In some embodiments, a method comprises providing a cell described herein comprising an exogenous nucleotide sequence encoding an OA or DVA uptake transporter protein described herein, and culturing the cell to produce a cannabinoid, cannabinoid derivative, or cannabinoid analog thereof. In some embodiments, the method comprises providing a cell described herein comprising an exogenous nucleotide sequence encoding an OA or DVA uptake transporter protein described herein, and culturing the cell to produce a cannabinoid, cannabinoid derivative, or cannabinoid analog thereof.

[0152] In some embodiments, a method comprises providing a cell described herein comprising an inactivated or deleted nucleotide sequence encoding an OA or DVA efflux transporter protein described herein, and culturing the cell to produce a cannabinoid, cannabinoid derivative, or cannabinoid analog thereof. In some embodiments, a method comprises providing a cell described herein comprising an inactivated or deleted nucleotide sequence encoding an OA or DVA efflux transporter protein described herein, and culturing the cell to produce a cannabinoid, cannabinoid derivative, or cannabinoid analog thereof.

[0153] The cannabinoids, cannabinoid derivatives and cannabinoid analogs produced by the methods disclosed herein are not limited and can be any of the disclosed cannabinoids. In some embodiments, the cannabinoids, cannabinoid derivatives and cannabinoid analogs are selected from cannabigerolic acid, tetrahydrocannabinolic acid, tetrahydrocannabinol, cannabidiolic acid, cannabidiol, cannabigerol, cannabichromene acid, cannabichromene, or acids or derivatives or analogs thereof.

[0154] In some embodiments, the method further comprises purifying or isolating cannabinoids, derivatives or analogs thereof from the culture.Isolation method is not limited and can be any suitable method known in the art.Purification method includes, for example, extraction procedures (for example, using supercritical carbon dioxide, ethanol or a mixture of these two), as well as continuous liquid-liquid extraction, pervaporation, evaporation, filtration, membrane filtration (including reverse osmosis, nanofiltration, ultrafiltration and microfiltration), membrane filtration with diafiltration, membrane separation, reverse osmosis, electrodialysis, distillation, extractive distillation, reactive distillation, azeotropic distillation, crystallization and recrystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, carbon adsorption, hydrogenation, and ultrafiltration or centrifugal partition chromatography (CPC).

[0155] In some embodiments, cells are grown in stirred tank fermenters with feed addition (sugars with or without organic acids), where dissolved oxygen, temperature, and pH are adjusted according to optimal growth and production processes. In some embodiments, aqueous-immiscible organic solvents are added to dissolve the added organic acids or to extract the cannabinoid product as it is synthesized. In some embodiments, these solvents may include, but are not limited to, isopropyl myristate (IPM), diisobutyl adipate, bis(2-ethylhexyl) adipate, decane, dodecane, hexadecane, or anther organic solvents with a logP > 5. The latter number (logP) is defined as the logarithm of the partitioning of a compound between water and octanol and is a standard parameter of a compound's hydrophobicity (the higher the logP, the less soluble it is in water). Depending on the fermentation process, the product may be isolated and purified using different methods.

[0156] If no organic co-solvent is used, the targeted cannabinoids will precipitate with the cell biomass after centrifugation or be isolated in a solid form after water removal using spray drying or other methods that remove water (i.e., freeze-drying, ultrafiltration, etc.). In one embodiment, an aqueous-miscible organic solvent (ethanol, acetonitrile, etc.) is added to the cannabinoid-containing cell pellet to dissolve the product. In some embodiments, simple filtration, ultrafiltration, or centrifugation can remove the cells, and the aqueous / organic medium is evaporated to dryness or to a small volume from which the cannabinoid product precipitates or crystallizes. Alternatively, to extract cannabinoids, the cannabinoid-containing cell pellet can be extracted with an aqueous-immiscible organic solvent (ethyl acetate, heptane, decane, etc.) or supercritical carbon dioxide (with 0-10% ethanol). Evaporation of the organic solvent and possible recrystallization produces pure cannabinoids. If the cannabinoid product was not extracted by the previous method and is trapped inside the cells, cell lysis may be required prior to the above extraction method. In some embodiments, the cells are disrupted using mechanical methods or by suspension in an appropriate lysis buffer that allows the cannabinoids to be extracted with an organic, aqueous-immiscible solvent (ethyl acetate, hexane, decane, methylene chloride, etc.). In other embodiments, the cells may be suspended in an organic solvent (ethanol, methanol, methylene chloride, etc.) that extracts the cannabinoids from the cells.

[0157] In some embodiments, an organic solvent is required during growth, which is separated at the end of fermentation. Back-extraction with an alkaline aqueous solvent or a different organic solvent with a low boiling point and high polarity (ethanol, acetonitrile, etc.) removes the cannabinoids. Isolation can then involve a simple pH shift if water is used, or evaporation if an organic solvent is used. In either case, a final recrystallization step may be required to improve the purity of the product. [Example]

[0158] Example Example 1: Activity of MPT4 and APT73 mutants Plasmids pYE01.GPS1.1.L18.APT73.119, pYE01.GPS1.1.L11.MPT4.1, and their respective mutants were transformed into strain SB-1334. Multiple colonies per transformation were pre-cultured in YNB containing glucose (6%), casamino acids (1%), MES (100 mM pH 6.5), and hygromycin (1 mg / mL) at 30 °C with shaking at 1000 rpm for 24 h. Each pre-culture was used to inoculate two separate cultures in assay medium consisting of YNB containing glucose (6%), casamino acids (1%), MES (100 mM pH 6.5), and hygromycin (1 mg / mL). One of these two separate cultures was supplemented with 5 mM OA after 24 h, and the other was supplemented with 5 mM DVA after 24 h. Cultures (0.5 mL) were incubated in 96-well blocks at 30°C with shaking at 1000 rpm and quenched with an equal volume of EtOH after 48 h (OA) or 72 h (DVA) of total growth. Samples were analyzed by HPLC / MS, and the major product, CBG(V)A, is shown in the table below. Mean values ​​and standard deviations were calculated from replicate experiments. FCBG(V)A was not detected. [Table 1-1] [Table 1-2]

[0159] Example 2: Activity of MPT48 and MPT69 Plasmids pYE01.MPT48 and pYE01.MPT69 and their respective mutants were transformed into strains SB-1459-5.2 and SB-1714-5.3. Multiple colonies per transformation were pre-cultured in YNB containing glucose (6%), casamino acids (1%), MES (100 mM pH 6.5), and hygromycin (1 mg / mL) for 24 hours at 30°C with shaking at 1000 rpm. Each pre-culture was used to inoculate two separate cultures of assay medium composed of YNB containing glucose (6%), casamino acids (1%), MES (100 mM pH 6.5), and hygromycin (1 mg / mL). One of these two separate cultures contained 2 mM OA and one contained 5 mM DVA. Cultures (0.5 mL) were incubated in 96-well blocks at 30°C with shaking at 1000 rpm and quenched with an equal volume of EtOH after 38 h (OA) or 48 h (DVA) of total growth. Samples were analyzed by HPLC / MS, and the major products, CBG(V)A and FCBG(V)A, are shown in the table below. Mean values ​​and standard deviations were calculated from replicate experiments. [Table 2] [Table 3]

[0160] The enzymes were screened in two different Yarrowia strains: one containing a native Erg20 in its genome (SB-1714-5.3), while the other strain (SB-1459-5.2) was engineered to contain the Erg20.A28 mutation (overexpression of the ERG20.F88W.N119W allele, expression of the ERG20.A28 allele, and disruption of endogenous Erg20), as described in co-owned PCT application PCT / US2022 / 046926. As expected, the A28 strain, which produces increased amounts of GPP over FPP, improves the CBG(V)A / FCBG(V)A ratio, especially when OA is the prenylation substrate.

[0161] Mutagenesis of these enzymes as described herein improves the ratio of CBG(V)A to FCBG(V)A and also increases overall activity.

[0162] Example 3: Activity of APT73 mutants Plasmid pYE01.GPS1.1.L18.APT73.165 and mutants were transformed into strain SB-1334. Multiple colonies per transformation were pre-cultured in YNB containing glucose (6%), casamino acids (1%), MES (100 mM pH 6.5), and hygromycin (1 mg / mL) at 30 °C with shaking at 1000 rpm for 24 h. Each pre-culture was used to inoculate two separate cultures in assay medium consisting of YNB containing glucose (6%), casamino acids (1%), MES (100 mM pH 6.5), and hygromycin (1 mg / mL). One of these two separate cultures was supplemented with 5 mM OA after 24 h, and the other was supplemented with 5 mM DVA after 24 h. Cultures (0.5 mL) were incubated in 96-well blocks at 30°C with shaking at 1000 rpm and quenched with an equal volume of EtOH after 40 h of total growth. Samples were analyzed by HPLC / MS, and the major product, CBG(V)A, is shown in the table below. Mean values ​​and standard deviations were calculated from replicate experiments. FCBG(V)A was not detected. [Table 4-1] [Table 4-2]

[0163] Example 4: Activity of APT73.187 and MPT69 mutants Plasmids pYE01.GPS1.1.L18.APT73.187 and pYE01.GPS1.1.L18.APT73.248 were transformed into strain SB-3589. Multiple colonies per transformation were pre-cultured in YNB containing glucose (6%), casamino acids (1%), MES (100 mM pH 6.5), and hygromycin (1 mg / mL) for 48 hours at 30 °C with shaking at 1000 rpm. Each pre-culture was used to inoculate two separate cultures in assay medium consisting of YNB containing glucose (6%), casamino acids (1%), MES (100 mM pH 6.5), and hygromycin (1 mg / mL). One of these two separate cultures was supplemented with 2 mM OA after 24 hours, and the other was supplemented with 2 mM DVA after 24 hours. Cultures (0.5 mL) were incubated in 96-well blocks at 30°C with shaking at 1000 rpm and quenched with an equal volume of EtOH after 48 h of total growth. Samples were analyzed by HPLC / MS, and the major product, CBG(V)A + THC(V)A (THCAS expressed from the genome converts some CBG(V)A to THC(V)A), is shown in the table below. Mean values ​​and standard deviations were calculated from replicate experiments. No FCBGVA was detected, and only trace amounts (less than 2% of the total products) of FCBGA were detected. [Table 5]

[0164] Plasmid pYE01.MPT69 and mutants were transformed into strain SB-3589. Multiple colonies per transformation (except for pYE01.MPT69.6, pYE01.MPT69.9, and pYE01.MPT69.10 transformations) were precultured in YNB containing glucose (6%), casamino acids (1%), MES (100 mM pH 6.5), and hygromycin (1 mg / mL) for 48 hours at 30 °C with shaking at 1000 rpm. Each preculture was used to inoculate assay medium consisting of YNB containing glucose (6%), casamino acids (1%), MES (100 mM pH 6.5), and hygromycin (1 mg / mL) supplemented with 2 mM DVA after 24 hours. Cultures (0.5 mL) were incubated in 96-well blocks at 30°C with shaking at 1000 rpm and quenched with an equal volume of EtOH after 48 h of total growth. Samples were analyzed by HPLC / MS, and the major products, CBGVA + THCVA (THCAS expressed from the genome converts some CBGVA to THCVA) and FCBGVA, are shown in the table below. Means and standard deviations were calculated from replicate experiments. [Table 6] [Table 7]

[0165] Experimental Method Cloning methods, vectors and strains

[0166] Yarrowia expression plasmids

[0167] The genes for each enzyme were optimized for expression in Yarrowia, synthesized (Codex DNA), and cloned into the pYE01 vector. Plasmids were transformed into chemically competent E. coli NEB 10 beta cells (NEB), plated onto LB agar plates containing 50 μg / mL kanamycin, and grown overnight at 37°C. Colony PCR was used to confirm gene fragment insertion, and positive colonies were inoculated into liquid LB medium containing 50 μg / mL kanamycin. Cultures were grown overnight at 37°C and then used to isolate plasmid DNA (Qiagen).

[0168] Analysis method

[0169] All samples after quenching with an equal volume of EtOH were centrifuged and analyzed by HPLC-MS.

[0170] All samples were quenched with an equal volume of EtOH containing 0.2 mg / mL of internal standard (3,5-diisopropyl-2-hydroxybenzoic acid CAS#2215-21-6), centrifuged, and the clarified solutions were analyzed by HPLC-MS.

[0171] Method A

[0172] Column: 2.1 x 50 mm COSMOCORE PBr (Nacalai USA, Inc.)

[0173] Mobile phase: A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile

[0174] Flow rate: 0.45mL / min

[0175] Temperature:%50°C

[0176] Gradient: 20% B at 0 min, 70% B at 2.3 min, 89% B at 4.2 min, 20% B at 4.3 min, 20% B at 6 min

[0177] [Table 8]

[0178] All compounds were confirmed by retention time, UV profile, and MS comparison based on authentic standards. For FCBGA and FCBGA, authentic standards were not available, so these compounds were identified based on UV profile and MS analysis (molecular ion and fragmentation pattern). Process Development for Producing Cannabinoids Through Fermentation The CBGA synthases described above can be used in cell-free reactions (in vitro) to produce CBGA and analogs by providing appropriate substrates, or they can be introduced into recombinant organisms (yeast, bacteria, fungi, algae, or plants) to improve the flux of either CBGA or its analogs. These recombinant organisms contain the optimized genes described herein, and the mevalonate pathway or MEP pathway is modified to increase the flux toward GPP or FPP to synthesize olivetolic acid and CBGA (or their analogs). To improve the flux and increase the intracellular concentration of GPP, mutant farnesyl pyrophosphate synthases can be used as described in yeast (Jian GZ et al., Metabolic Engineering, 2017, 41, 57), or GPP-specific synthases can be introduced (Schmidt A, Gershenzon J. Phytochemistry, 2008, 69, 49). Other enzymes in the mevalonate pathway (e.g., HMG-CoA reductase) may need to be engineered (truncated or mutated) or overexpressed. Formation of GPP / FPP and OA can occur when the organism is grown using a simple carbon source such as glucose, sucrose, glycerol, or another simple or complex sugar mixture. Exogenous organic acids with carbon chains varying from 4 to more than 12 (linear or branched) can also be added during growth. Along with the addition, introduction of an appropriate acid-CoA synthase may be required to produce the corresponding organic acid-CoA, which can then be used by the PKS and PKC to produce the OA analog. The organism can also express an appropriate synthase to cyclize CBGA or any of its analogs to other cannabinoids. A description of these enzymes is provided in more detail in co-owned PCT application PCT / US22 / 46926, incorporated herein by reference in its entirety.

[0179] Enumeration of Amino Acid Sequences Mutant and native (APT73) soluble aromatic prenyltransferase enzymes APT73.179 (SEQ ID NO: 1)

[0180] [ka]

[0181] APT73.119 (SEQ ID NO: 2)

[0182] [ka] [ka]

[0183] APT73.159 (SEQ ID NO: 3)

[0184] [ka]

[0185] APT73.160 (SEQ ID NO: 4)

[0186] [ka]

[0187] APT73.161 (SEQ ID NO: 5)

[0188] [ka]

[0189] APT73.162 (SEQ ID NO: 6)

[0190] [ka]

[0191] APT73.163 (SEQ ID NO: 7)

[0192] [ka]

[0193] APT73.165 (SEQ ID NO: 8)

[0194] [ka]

[0195] APT73.166 (SEQ ID NO: 9)

[0196] [ka]

[0197] APT73.168 (SEQ ID NO: 10)

[0198] [ka]

[0199] APT73.169 (SEQ ID NO: 11)

[0200] [ka] [ka]

[0201] APT73.170 (SEQ ID NO: 12)

[0202] [ka]

[0203] APT73.172 (SEQ ID NO: 13)

[0204] [ka]

[0205] APT73.182 (SEQ ID NO: 14)

[0206] [ka]

[0207] APT73.183 (SEQ ID NO: 15)

[0208] [ka]

[0209] APT73.184 (SEQ ID NO: 16)

[0210] [ka]

[0211] APT73.185 (SEQ ID NO: 17)

[0212] [ka]

[0213] APT73.186 (SEQ ID NO: 18)

[0214] [ka]

[0215] APT73.187 (SEQ ID NO: 19)

[0216] [ka]

[0217] APT73.188 (SEQ ID NO: 20)

[0218] [ka] [ka]

[0219] APT73.189 (SEQ ID NO: 21)

[0220] [ka]

[0221] APT73.190 (SEQ ID NO: 22)

[0222] [ka]

[0223] APT73.191 (SEQ ID NO: 23)

[0224] [ka]

[0225] APT73.192 (SEQ ID NO: 24)

[0226] [ka]

[0227] APT73.193 (SEQ ID NO: 25)

[0228] [ka]

[0229] APT73.194 (SEQ ID NO: 26)

[0230] [ka]

[0231] APT73.195 (SEQ ID NO: 27)

[0232] [ka]

[0233] APT73.196 (SEQ ID NO: 28)

[0234] [ka]

[0235] APT73.197 (SEQ ID NO: 29)

[0236] [ka] [ka]

[0237] APT73.198 (SEQ ID NO: 30)

[0238] [ka]

[0239] APT73.199 (SEQ ID NO: 31)

[0240] [ka]

[0241] APT73.200 (SEQ ID NO: 32)

[0242] [ka]

[0243] APT73.201 (SEQ ID NO: 33)

[0244] [ka]

[0245] APT73.202 (SEQ ID NO: 34)

[0246] [ka]

[0247] APT73.203 (SEQ ID NO: 35)

[0248] [ka]

[0249] APT73.204 (SEQ ID NO: 36)

[0250] [ka]

[0251] APT73 (Natural aromatic Prentyl transferase) (SEQ ID NO: 37)

[0252] [ka]

[0253] APT73.77 (mutant aromatic prenyltransferase) (SEQ ID NO: 38)

[0254] [ka] [ka]

[0255] APT73.248 (SEQ ID NO: 96)

[0256] [ka]

[0257] Membrane-bound prenyltransferase enzymes MPT4.33 (SEQ ID NO: 39)

[0258] [ka]

[0259] MPT4.29 (SEQ ID NO: 40)

[0260] [ka]

[0261] MPT4.30 (SEQ ID NO: 41)

[0262] [ka] [ka]

[0263] MPT4.32 (SEQ ID NO: 42)

[0264] [ka]

[0265] MPT4.34 (SEQ ID NO: 43)

[0266] [ka]

[0267] MPT4.40 (SEQ ID NO: 44)

[0268] [ka]

[0269] MPT4.41 (SEQ ID NO: 45)

[0270] [ka]

[0271] MPT4.1 (SEQ ID NO: 46)

[0272] [ka]

[0273] MPT48 (SEQ ID NO: 47)

[0274] [ka]

[0275] MPT69 (SEQ ID NO: 48)

[0276] [ka]

[0277] MPT69.2 (SEQ ID NO: 98)

[0278] [ka]

[0279] MPT69.5 (SEQ ID NO: 99)

[0280] [ka]

[0281] MPT69.6 (SEQ ID NO: 100)

[0282] [ka]

[0283] MPT69.7 (SEQ ID NO: 101)

[0284] [ka]

[0285] MPT69.8 (SEQ ID NO: 102)

[0286] [ka]

[0287] MPT69.9 (SEQ ID NO: 103)

[0288] [ka]

[0289] MPT69.10 (SEQ ID NO: 104)

[0290] [ka]

[0291] Geranyl diphosphate synthase protein GPS1.1 (SEQ ID NO: 49)

[0292] [ka]

[0293] Linker peptide L11 linker (SEQ ID NO: 50)

[0294] [ka]

[0295] L18 linker (SEQ ID NO: 51)

[0296] [ka]

[0297] Geranyl pyrophosphate synthase / soluble aromatic prenyltransferase fusion protein GPS1.1-L18-APT73.119 (SEQ ID NO: 52)

[0298] [ka]

[0299] GPS1.1-L18-APT73.159 (SEQ ID NO: 53)

[0300] [ka] [ka]

[0301] GPS1.1-L18-APT73.160 (SEQ ID NO: 54)

[0302] [ka]

[0303] GPS1.1-L18-APT73.161 (SEQ ID NO: 55)

[0304] [ka] [ka]

[0305] GPS1.1-L18-APT73.162 (SEQ ID NO: 56)

[0306] [ka]

[0307] GPS1.1-L18-APT73.163 (SEQ ID NO: 57)

[0308] [ka]

[0309] GPS1.1-L18-APT73.165 (SEQ ID NO: 58)

[0310] [ka]

[0311] GPS1.1-L18-APT73.166 (SEQ ID NO: 59)

[0312] [ka]

[0313] GPS1.1-L18-APT73.168 (SEQ ID NO: 60)

[0314] [ka]

[0315] GPS1.1-L18-APT73.169 (SEQ ID NO: 61)

[0316] [ka]

[0317] GPS1.1-L18-APT73.170 (SEQ ID NO: 62)

[0318] [ka]

[0319] GPS1.1-L18-APT73.172 (SEQ ID NO: 63)

[0320] [ka]

[0321] GPS1.1-L18-APT73.179 (SEQ ID NO: 64)

[0322] [ka]

[0323] GPS1.1-L18-APT73.182 (SEQ ID NO: 65)

[0324] [ka]

[0325] GPS1.1-L18-APT73.183 (SEQ ID NO: 66)

[0326] [ka]

[0327] GPS1.1-L18-APT73.184 (SEQ ID NO: 67)

[0328] [ka]

[0329] GPS1.1-L18-APT73.185 (SEQ ID NO: 68)

[0330] [ka]

[0331] GPS1.1-L18-APT73.186 (SEQ ID NO: 69)

[0332] [ka]

[0333] GPS1.1-L18-APT73.187 (SEQ ID NO: 70)

[0334] [ka]

[0335] GPS1.1-L18-APT73.188 (SEQ ID NO: 71)

[0336] [ka]

[0337] GPS1.1-L18-APT73.189 (SEQ ID NO: 72)

[0338] [ka]

[0339] GPS1.1-L18-APT73.190 (SEQ ID NO: 73)

[0340] [ka]

[0341] GPS1.1-L18-APT73.191 (SEQ ID NO: 74)

[0342] [ka]

[0343] GPS1.1-L18-APT73.192 (SEQ ID NO: 75)

[0344] [ka]

[0345] GPS1.1-L18-APT73.193 (SEQ ID NO: 76)

[0346] [ka]

[0347] GPS1.1-L18-APT73.194 (SEQ ID NO: 77)

[0348] [ka]

[0349] GPS1.1-L18-APT73.195 (SEQ ID NO: 78)

[0350] [ka]

[0351] GPS1.1-L18-APT73.196 (SEQ ID NO: 79)

[0352] [ka]

[0353] GPS1.1-L18-APT73.197 (SEQ ID NO: 80)

[0354] [ka]

[0355] GPS1.1-L18-APT73.198 (SEQ ID NO: 81)

[0356] [ka]

[0357] GPS1.1-L18-APT73.199 (SEQ ID NO: 82)

[0358] [ka]

[0359] GPS1.1-L18-APT73.200 (SEQ ID NO: 83)

[0360] [ka]

[0361] GPS1.1-L18-APT73.201 (SEQ ID NO: 84)

[0362] [ka]

[0363] GPS1.1-L18-APT73.202 (SEQ ID NO: 85)

[0364] [ka]

[0365] GPS1.1-L18-APT73.203 (SEQ ID NO: 86)

[0366] [ka]

[0367] GPS1.1-L18-APT73.204 (SEQ ID NO: 87)

[0368] [ka]

[0369] GPS1.1-L18-APT73.248 (SEQ ID NO: 97)

[0370] [ka]

[0371] Geranyl pyrophosphate synthase / membrane-bound prenyltransferase fusion protein GPS1.1-L11-MPT4.1 (SEQ ID NO: 88)

[0372] [ka] [ka]

[0373] GPS1.1-L11-MPT4.29 (SEQ ID NO: 89)

[0374] [ka]

[0375] GPS1.1-L11-MPT4.30 (SEQ ID NO: 90)

[0376] [ka]

[0377] GPS1.1-L11-MPT4.32 (SEQ ID NO: 91)

[0378] [ka]

[0379] GPS1.1-L11-MPT4.33 (SEQ ID NO: 92)

[0380] [ka]

[0381] GPS1.1-L11-MPT4.34 (SEQ ID NO: 93)

[0382] [ka]

[0383] GPS1.1-L11-MPT4.40 (SEQ ID NO: 94)

[0384] [ka]

[0385] GPS1.1-L11-MPT4.41 (SEQ ID NO: 95)

[0386] [ka]

[0387] Farnesyl diphosphate synthase GPS1.A28 (sequence number 105)

[0388] [ka]

Claims

1. An aromatic prenyltransferase (APT) engineered to transfer geranyl pyrophosphate (GPP) to olivetolic acid and / or divalanic acid with at least two-fold increased efficiency compared to mutant aromatic prenyltransferase APT73.77 (SEQ ID NO: 38), wherein the engineered APT contains an amino acid sequence having at least two amino acid modifications compared to SEQ ID NO: 38, the first of the at least two amino acid modifications corresponding to a substitution, deletion, or insertion at amino acid position R162 of SEQ ID NO:

38.

2. The engineered APT of claim 1, wherein the second of the at least two amino acid modifications compared to the amino acid sequence APT73.77 is selected from substitutions, deletions or insertions at amino acid positions corresponding to positions H39, V41, Q127, E130, V131, I156, N164, R205, A223, H225, Q227, G254, G260, L276, G280, R282, C283, L284, D285 or G286 of SEQ ID NO:

38.

3. 2. The engineered APT of claim 1, wherein the at least two amino acid modifications include second, third, fourth and fifth or more amino acid modifications selected from substitutions, deletions or insertions at four or more amino acid positions corresponding to positions H39, V41, Q127, E130, V131, I156, N164, R205, A223, H225, Q227, G254, G260, L276, G280, R282, C283, L284, D285 and / or G286 of SEQ ID NO: 38, and wherein the modifications do not include the substitution R205S.

4. The engineered APTs are APT73.179 (SEQ ID NO: 1), APT73.119 (SEQ ID NO: 2), APT73.159 (SEQ ID NO: 3), APT73.160 (SEQ ID NO: 4), APT73.161 (SEQ ID NO: 5), APT73.162 (SEQ ID NO: 6), APT73.163 (SEQ ID NO: 7), APT73.165 (SEQ ID NO: 8), APT73.166 (SEQ ID NO: 9), APT73.168 (SEQ ID NO: 10). , APT73.169 (SEQ ID NO: 11), APT73.170 (SEQ ID NO: 12), APT73.172 (SEQ ID NO: 13), APT73.182 (SEQ ID NO: 14), APT73.183 (SEQ ID NO: 15), APT73.184 (SEQ ID NO: 16), APT73.185 (SEQ ID NO: 17), APT73.186 (SEQ ID NO: 18), APT73.187 (SEQ ID NO: 19), APT73.188 (SEQ ID NO: 20), AP T73.73.189 (SEQ ID NO: 21), APT73.190 (SEQ ID NO: 22), APT73.191 (SEQ ID NO: 23), APT73.192 (SEQ ID NO: 24), APT73.193 (SEQ ID NO: 25), APT73.194 (SEQ ID NO: 26), APT73.195 (SEQ ID NO: 27), APT73.196 (SEQ ID NO: 28), APT73.197 (SEQ ID NO: 29), APT73.199 (SEQ ID NO: 31), A The engineered APT of any one of claims 1 to 3, comprising an amino acid sequence having at least 90% identity to the amino acid sequence of APT73.200 (SEQ ID NO: 32), APT73.201 (SEQ ID NO: 33), APT73.202 (SEQ ID NO: 34), APT73.203 (SEQ ID NO: 35), APT73.204 (SEQ ID NO: 36), or APT73.248 (SEQ ID NO: 96), or a functional fragment or variant thereof.

5. The engineered APT of claim 4, wherein the engineered APT comprises an amino acid sequence having at least 90% identity to the amino acid sequence of APT73.187 (SEQ ID NO: 96) or APT73.248 (SEQ ID NO: 96).

6. The engineered APT of claim 2, wherein the at least two amino acid modifications include three or more amino acid modifications including the substitution R162Q, one or more substitutions selected from the group consisting of Q127E, E130R, V131I and A280G, and one or more substitutions selected from the group consisting of H39V, V41C and V41L, all relative to the amino acid sequence of APT73.77 (SEQ ID NO: 38).

7. The engineered APTs are APT73.179 (SEQ ID NO: 1), APT73.119 (SEQ ID NO: 2), APT73.159 (SEQ ID NO: 3), APT73.160 (SEQ ID NO: 4), APT73.161 (SEQ ID NO: 5), APT73.162 (SEQ ID NO: 6), APT73.163 (SEQ ID NO: 7), APT73.165 (SEQ ID NO: 8), APT73.166 (SEQ ID NO: 9), APT73.168 (SEQ ID NO: 10), APT73.169 (SEQ ID NO: 11), APT73.170 (SEQ ID NO: 12), APT73.171 (SEQ ID NO: 13), APT73.172 (SEQ ID NO: 14), APT73.173 (SEQ ID NO: 15), APT73.174 (SEQ ID NO: 16), APT73.175 (SEQ ID NO: 17), APT73.176 (SEQ ID NO: 18), APT73.177 (SEQ ID NO: 19), APT73.178 (SEQ ID NO: 20), APT73.179 (SEQ ID NO: 21), APT73.180 (SEQ ID NO: 22), APT73.181 (SEQ ID NO: 23), APT73.182 (SEQ ID NO: 24), APT73.183 (SEQ ID NO: 25), APT73.184 (SEQ ID NO: 26), APT73.185 (SEQ ID NO: 27), APT73.186 (SEQ ID NO: 28), APT73.187 ( 10), APT73.169 (SEQ ID NO: 11), APT73.170 (SEQ ID NO: 12), APT73.172 (SEQ ID NO: 13), APT73.182 (SEQ ID NO: 14), APT73.183 (SEQ ID NO: 15), APT73.184 (SEQ ID NO: 16), APT73.185 (SEQ ID NO: 17), APT73.186 (SEQ ID NO: 18), APT73.187 (SEQ ID NO: 19), APT73.188 (SEQ ID NO:

4. The engineered APT of claim 3, comprising an amino acid sequence having at least one amino acid modification compared to APT73.189 (SEQ ID NO:21), APT73.190 (SEQ ID NO:22), APT73.191 (SEQ ID NO:23), APT73.192 (SEQ ID NO:24), APT73.193 (SEQ ID NO:25), APT73.194 (SEQ ID NO:26), APT73.195 (SEQ ID NO:27), APT73.196 (SEQ ID NO:28), APT73.197 (SEQ ID NO:29), APT73.199 (SEQ ID NO:31), APT73.200 (SEQ ID NO:32), APT73.201 (SEQ ID NO:33), APT73.202 (SEQ ID NO:34), APT73.203 (SEQ ID NO:35), APT73.204 (SEQ ID NO:36), APT73.248 (SEQ ID NO:96), or a functional fragment or variant thereof.

8. A recombinant membrane-bound prenyltransferase (rMPT), which has been engineered to transfer geranyl pyrophosphate (GPP) to olivetolic acid and / or divalanic acid with higher efficiency than naturally occurring MPT, and which has an amino acid sequence that includes at least one amino acid modification compared to the amino acid sequence of MPT4.1 (SEQ ID NO: 46), MPT48 (SEQ ID NO: 47) or MPT69 (SEQ ID NO: 48).

9. 9. The rMPT of claim 8, wherein the MPT has at least one amino acid modification at a position corresponding to amino acid position 29, 72, 135 and / or 254 of SEQ ID NO: 46 (MPT4.1).

10. 10. The rMPT of claim 9, wherein the at least one amino acid modification compared to sequence number 46 (MPT4.1) is selected from at least one of M29I, M29V, I72C, I72V, I72A, A135M and / or V254L.

11. The rMPT of claim 9, wherein the rMPT comprises an amino acid sequence having at least 90% identity to the amino acid sequences of MPT4.33 (sequence number 39), MPT4.29 (sequence number 40), MPT4.30 (sequence number 41), MPT4.32 (sequence number 42), MPT4.34 (sequence number 43), MPT4.40 (sequence number 44), and MPT4.41 (sequence number 45).

12. The rMPT of claim 8, wherein the rMPT comprises an amino acid sequence having at least one amino acid modification compared to SEQ ID NO: 47 (MPT48), which produces a variant having one or both of: i) increased activity with respect to prenylation of OA and DVA to form CBGA and CBGVA, respectively; and ii) increased selectivity for GPP over FPP.

13. The at least one amino acid modification compared to MPT48 (SEQ ID NO: 47) is selected from the group consisting of the first 50 amino acids M1 to G50 at the N-terminus of SEQ ID NO: 47 and / or H54, M88, R89, C93, A94, N96, D97, V98, V99, D100, Q101, D102, F103, D104, R109, R113, S121, A136, C147, Q148, V154, K165, Q172, L175, T178, L179, I181 , L201, T207, V214, Y217, D218, V219, Y221, T253, L254, T262, V267, N269, P271, L281, A284, A287, S304, G305, W306, N314, L316, G317, G318, V327, M329 and / or L330.

14. The rMPT of claim 8, wherein the rMPT comprises an amino acid sequence having at least one amino acid modification compared to SEQ ID NO: 48 (MPT69), which produces a variant having one or both of: i) increased activity with respect to prenylation of OA and DVA to form CBGA and CBGVA, respectively; and ii) increased selectivity for GPP over FPP.

15. The at least one amino acid modification compared to MPT69 (SEQ ID NO:48) is selected from the group consisting of the first 45 amino acids M1 to G45 at the N-terminus of SEQ ID NO:48, and / or H49, M83, R84, C88, A89, N91, D92, N93, I94, D95, Q96, D97, F98, D99, R104, R108, S116, A131, C142, H143, V149, K160, Q167, L170, T173, L174, A176, R 15. The rMPT of claim 14, comprising one or more deletions, insertions or substitutions at one or more amino acid positions corresponding to 196, T202, V209, Y212, D213, V214, Y216, T248, C249, V257, V262, N264, P266, L276, A279, A282, S299, G300, W301, N309, M311, S312, G313, A322, L324 and / or L325.

16. The rMPT of claim 14, wherein the rMPT comprises an amino acid sequence having at least 90% identity to the amino acid sequence of MPT69.2 (sequence number 98), MPT69.5 (sequence number 99), MPT69.6 (sequence number 100), MPT69.7 (sequence number 101), MPT69.8 (sequence number 102), MPT69.9 (sequence number 103) or MPT69.10 (sequence number 104).

17. A cell comprising the APT and / or rMPT according to claims 1 to 16, said cell being capable of producing CBGA in the presence of GPP and OA.

18. A cell comprising the APT and / or rMPT according to claims 1 to 16, said cell being capable of producing CBGVA in the presence of GPP and DVA.

19. A cell comprising the APT and / or rMPT of claims 1 to 16, said cell being capable of producing cannabinoids in the presence of a carbon source and, optionally, hexanoic acid or butyric acid.

20. The cell of claims 17 to 19, wherein the cell expresses an exogenous membrane transporter that improves OA or DVA uptake.

21. 21. The cell of claims 17 to 20, wherein one or more genes in the cell that encode proteins that expel OA or DVA are downregulated or inactivated.

22. 22. The cell of claims 17-21, wherein the cell encodes an exogenous hexanoyl-CoA synthetase and / or butyryl-CoA synthetase.

23. The cell according to claims 17 to 22, wherein the cell is a yeast cell or a bacterial cell.

24. 24. The cell of claim 23, wherein the yeast cell is a Yarrowia or Saccharomyces strain.

25. A method for producing CBGA, CBGVA or a derivative thereof, comprising culturing the cells according to claims 17 to 24 under appropriate conditions to produce CBGA, CBGVA or a derivative thereof.

26. 26. The method of claim 25, wherein the suitable conditions comprise adding at least one of butyric acid, valeric acid, isovaleric acid, hexanoic acid, hexanol, butanol, oleic acid, glycerol, or glucose to a culture medium in which the cells are cultured.

27. A fusion protein comprising a polypeptide having geranyl diphosphate synthase (GPS) activity fused directly or indirectly to a polypeptide having prenyltransferase activity, wherein the polypeptide having prenyltransferase activity comprises: a) a polypeptide sequence having at least 85% identity to the polypeptide sequence of MPT48 (SEQ ID NO: 47) or MPT69 (SEQ ID NO: 48); b) a polypeptide sequence comprising at least one amino acid modification compared to the amino acid sequence of MPT4.1 (SEQ ID NO: 46); or c) a polypeptide comprising i) an amino acid sequence having at least two amino acid modifications compared to SEQ ID NO: 38, wherein a first of the at least two amino acid modifications corresponds to a substitution, deletion or insertion at amino acid position R162 of SEQ ID NO: 38; and wherein the N-terminus of the polypeptide having prenyltransferase activity is fused to the C-terminus of the GPS, or the C-terminus of the polypeptide having prenyltransferase activity is fused to the N-terminus of the GPS.

28. The fusion protein is selected from the group consisting of GPS1.1-L18-APT73.119 (SEQ ID NO: 52), GPS1.1-L18-APT73.159 (SEQ ID NO: 53), GPS1.1-L18-APT73.160 (SEQ ID NO: 54), GPS1.1-L18-APT73.161 (SEQ ID NO: 55), GPS1.1-L18-APT73.162 (SEQ ID NO: 56), GPS1.1-L18-APT73.163 (SEQ ID NO: 57), GPS1.1-L18-APT73.165 (SEQ ID NO: 58), GPS1.1-L18-APT73.166 (SEQ ID NO: 59), GPS1.1 -L18-APT73.168 (SEQ ID NO: 60), GPS1.1-L18-APT73.169 (SEQ ID NO: 61), GPS1.1-L18-APT73.170 (SEQ ID NO: 62), GPS1.1-L18-APT73.172 (SEQ ID NO: 63), GPS1.1-L18-APT73.179 (SEQ ID NO: 64), GPS1.1-L18-APT73.182 (SEQ ID NO: 65), GPS1.1-L18-APT73.183 (SEQ ID NO: 66), GPS1.1-L18-APT73.184 (SEQ ID NO: 67), GPS1.1-L18-APT73.185 (SEQ ID NO: 68), No. 68), GPS1.1-L18-APT73.186 (SEQ ID NO: 69), GPS1.1-L18-APT73.187 (SEQ ID NO: 70), GPS1.1-L18-APT73.188 (SEQ ID NO: 71), GPS1.1-L18-APT73.189 (SEQ ID NO: 72), GPS1.1-L18-APT73.190 (SEQ ID NO: 73), GPS1.1-L18-APT73.191 (SEQ ID NO: 74), GPS1.1-L18-APT73.192 (SEQ ID NO: 75), GPS1.1-L18-APT73.193 (SEQ ID NO: 76), GPS1.1-L18- APT73.194 (SEQ ID NO: 77), GPS1.1-L18-APT73.195 (SEQ ID NO: 78), GPS1.1-L18-APT73.196 (SEQ ID NO: 79), GPS1.1-L18-APT73.197 (SEQ ID NO: 80), GPS1.1-L18-APT73.199 (SEQ ID NO: 82), GPS1.1-L18-APT73.200 (SEQ ID NO: 83), GPS1.1-L18-APT73.201 (SEQ ID NO: 84), GPS1.1-L18-APT73.202 (SEQ ID NO: 85), GPS1.1-L18-APT73.203 (SEQ ID NO: 86),The fusion protein of claim 27, comprising a polypeptide having at least 90% identity to GPS1.1-L18-APT73.204 (SEQ ID NO: 87) or GPS1.1-L18-APT73.248 (SEQ ID NO: 97).

29. The fusion protein of claim 27, wherein the fusion protein comprises a polypeptide having at least 90% identity to GPS1.1-L11-MPT4.1 (SEQ ID NO: 88), GPS1.1-L11-MPT4.29 (SEQ ID NO: 89), GPS1.1-L11-MPT4.30 (SEQ ID NO: 90), GPS1.1-L11-MPT4.32 (SEQ ID NO: 91), GPS1.1-L11-MPT4.33 (SEQ ID NO: 92), GPS1.1-L11-MPT4.34 (SEQ ID NO: 93), GPS1.1-L11-MPT4.40 (SEQ ID NO: 94), or GPS1.1-L11-MPT4.41 (SEQ ID NO: 95).

30. 28. The fusion protein of claim 27, wherein the polypeptide having prenyltransferase activity has improved selectivity for GPP over FPP compared to a control membrane-bound prenyltransferase or a soluble aromatic prenyltransferase.

31. The fusion protein of claims 27 to 30, wherein the polypeptide having geranyl diphosphate synthase activity comprises the polypeptide of SEQ ID NO: 49 (GPS1.1), or a functional fragment or functional variant thereof.

32. 28. The fusion protein of claim 27, wherein the fusion protein further comprises a linker polypeptide between the polypeptide having geranyl diphosphate synthase activity and the polypeptide having prenyltransferase activity.

33. 33. The fusion protein of claim 32, wherein the linker comprises a polypeptide selected from the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO:

51.

34. The fusion protein is selected from the group consisting of GPS1.1-L18-APT73.119 (SEQ ID NO: 52), GPS1.1-L18-APT73.159 (SEQ ID NO: 53), GPS1.1-L18-APT73.160 (SEQ ID NO: 54), GPS1.1-L18-APT73.161 (SEQ ID NO: 55), GPS1.1-L18-APT73.162 (SEQ ID NO: 56), GPS1.1-L18-APT73.163 (SEQ ID NO: 57), GPS1.1-L18-APT73.165 (SEQ ID NO: 58), GPS1.1-L18-APT73.166 (SEQ ID NO: 59), GPS1.1 -L18-APT73.168 (SEQ ID NO: 60), GPS1.1-L18-APT73.169 (SEQ ID NO: 61), GPS1.1-L18-APT73.170 (SEQ ID NO: 62), GPS1.1-L18-APT73.172 (SEQ ID NO: 63), GPS1.1-L18-APT73.179 (SEQ ID NO: 64), GPS1.1-L18-APT73.182 (SEQ ID NO: 65), GPS1.1-L18-APT73.183 (SEQ ID NO: 66), GPS1.1-L18-APT73.184 (SEQ ID NO: 67), GPS1.1-L18-APT73.185 (SEQ ID NO: 68), No. 68), GPS1.1-L18-APT73.186 (SEQ ID NO: 69), GPS1.1-L18-APT73.187 (SEQ ID NO: 70), GPS1.1-L18-APT73.188 (SEQ ID NO: 71), GPS1.1-L18-APT73.189 (SEQ ID NO: 72), GPS1.1-L18-APT73.190 (SEQ ID NO: 73), GPS1.1-L18-APT73.191 (SEQ ID NO: 74), GPS1.1-L18-APT73.192 (SEQ ID NO: 75), GPS1.1-L18-APT73.193 (SEQ ID NO: 76), GPS1.1-L18- APT73.194 (SEQ ID NO: 77), GPS1.1-L18-APT73.195 (SEQ ID NO: 78), GPS1.1-L18-APT73.196 (SEQ ID NO: 79), GPS1.1-L18-APT73.197 (SEQ ID NO: 80), GPS1.1-L18-APT73.199 (SEQ ID NO: 82), GPS1.1-L18-APT73.200 (SEQ ID NO: 83), GPS1.1-L18-APT73.201 (SEQ ID NO: 84), GPS1.1-L18-APT73.202 (SEQ ID NO: 85), GPS1.1-L18-APT73.203 (SEQ ID NO: 86),The fusion protein of claim 28, comprising the polypeptide sequence of GPS1.1-L18-APT73.204 (SEQ ID NO: 87), GPS1.1-L18-APT73.248 (SEQ ID NO: 97), or a functional fragment or variant thereof.

35. The fusion protein of claim 28, wherein the fusion protein comprises the polypeptide sequence of GPS1.1-L18-APT73.187 (SEQ ID NO: 70) or GPS1.1-L18-APT73.248 (SEQ ID NO: 97).

36. 30. The fusion protein of claim 29, wherein the fusion protein comprises the polypeptide sequence of GPS1.1-L11-MPT4.1 (SEQ ID NO:88), GPS1.1-L11-MPT4.29 (SEQ ID NO:89), GPS1.1-L11-MPT4.30 (SEQ ID NO:90), GPS1.1-L11-MPT4.32 (SEQ ID NO:91), GPS1.1-L11-MPT4.33 (SEQ ID NO:92), GPS1.1-L11-MPT4.34 (SEQ ID NO:93), GPS1.1-L11-MPT4.40 (SEQ ID NO:94), GPS1.1-L11-MPT4.41 (SEQ ID NO:95), or a functional fragment or variant thereof.

37. A cell comprising the fusion protein of claims 27 to 36, wherein the cell is capable of producing CBGA in the presence of OA and GPP.

38. A cell comprising the fusion protein of claims 27 to 36, wherein the cell is capable of producing CBGVA in the presence of DVA and GPP.

39. 39. A cell comprising the fusion protein of claims 37-38, wherein the cell is capable of producing cannabinoids in the presence of a carbon source and, optionally, hexanoic acid or butyric acid.

40. The cell of claims 37 to 39, wherein the cell expresses an exogenous membrane transporter that improves OA or DVA uptake.

41. 41. The cell of claims 37-40, wherein one or more genes in the cell that encode proteins that expel OA or DVA are downregulated or inactivated.

42. 42. The cell of claims 37-41, wherein the cell encodes an exogenous hexanoyl-CoA synthetase and / or butyryl-CoA synthetase.

43. The cell according to claims 37 to 42, wherein the cell is a yeast cell or a bacterial cell.

44. 44. The cell of claim 43, wherein the yeast cell is a Yarrowia or Saccharomyces strain.

45. A method for producing CBGA, CBGVA or a derivative thereof, comprising culturing the cells according to claims 37 to 44 under appropriate conditions to produce CBGA, CBGVA or a derivative thereof.

46. 46. ​​The method of claim 45, wherein the suitable conditions comprise adding at least one of butyric acid, valeric acid, isovaleric acid, hexanoic acid, hexanol, butanol, oleic acid, glycerol, or glucose to a culture medium in which the cells are cultured.

Images