Prenyltransferase variants and methods for production of prenylated aromatic compounds

Abstract

Described herein are prenyltransferases including non-natural variants thereof having at least one amino acid substitution as compared to its corresponding natural or unmodified prenyltransferases and that are capable of at least two-fold greater rate of formation of cannabinoids such as cannabigerolic acid, cannabigerovarinic acid, cannabigerorcinic acid, and cannabigerol, as compared to a wild type control. Prenyltransferase variants also demonstrated regioselectivity to desired cannabinoid isomers such as CDBA (3-GOLA), 3-GDVA, 3-GOSA, and CBG (2-GOL). The prenyltransferase variants can be used to form prenylated aromatic compounds, and can be expressed in an engineered microbe having a pathway to such compounds, which include 3-GOLA, 3-GDVA, 3-GOSA, and CBG. 3-GOLA can be used for the preparation of cannabigerol (CBG), which can be used in therapeutic compositions.

What is claimed is: 
     
         1 . A non-natural prenyltransferase comprising at least one amino acid variation as compared to a wild type prenyltransferase, and enzymatically capable of:
 (al) at least two-fold greater rate of formation of 3-geranyl-olivetolate (3-GOLA) from geranyl pyrophosphate and olivetolic acid, as compared to the wild type prenyltransferase; (a2) providing regioselectivity to 3-GOLA; or both (al) and (a2)   (b1) at least two-fold greater rate of formation of cannabigerovarinic acid (CBGVA) from geranyl pyrophosphate and divarinolic acid (DVA), as compared to the wild type prenyltransferase; (b2) 50% or greater regioselectivity to 3-geranyl-divarinolic acid (3-GDVA), or both (b1) and (b2);   (c1) at least two-fold greater rate of formation of cannabigerorcinic acid (CBGOA) from geranyl pyrophosphate and orsellinic acid (OSA), as compared to the wild type prenyltransferase; (c2) 50% or greater regioselectivity to 3-geranyl-orsellinate (3-GOSA); or both (c1) and (c2); or   (d1) enzymatically capable of regioselectively forming a 2-prenylated 5-alkylbenzene-1,3-diol from geranyl pyrophosphate and 5-alkylbenzene-1,3-diol.   
     
     
         2 . The non-natural prenyltransferase of  claim 1  enzymatically capable of
 (a) at least five-fold greater rate of formation of 3-GOLA from geranyl pyrophosphate and olivetolic acid, as compared to the wild type prenyltransferase 
 (b) 90% or greater regioselectivity to 3-GOLA, or both (a) and (b). 
 
     
     
         3 . The non-natural prenyltransferase of  claim 1  enzymatically capable of
 (i) at least fifty-fold greater rate of formation of: (a) cannabigerovarinic acid (CBGVA) from geranyl pyrophosphate and divarinolic acid (DVA), or (b) cannabigerorcinic acid (CBGOA) from geranyl pyrophosphate and orsellinic acid (OSA), as compared to the wild type prenyltransferase, 
 (ii) 90% or greater regioselectivity to 3-geranyl-divarinolic acid (3-GDVA) or 3-geranyl-orsellinate (3-GOSA), or 
 both (i) and (ii). 
 
     
     
         4 . The non-natural prenyltransferase of  claim 1  comprising at least two amino acid variations as compared to a wild type prenyltransferase. 
     
     
         5 . The non-natural prenyltransferase  claim 1  having 50% or greater identity to SEQ ID NO:1 or to any one of SEQ ID NO:2-15. 
     
     
         6 . The non-natural prenyltransferase of  claim 5  having 90% or greater identity to SEQ ID NO:1 or to any one of SEQ ID NO:2-15. 
     
     
         7 . The non-natural prenyltransferase of  claim 5  comprising one or more amino acid variations at position(s) selected from the group consisting of: 49, 121, 124, 159, 175, 212, 232, 267, 268, 285, 286, 293, and 300, relative to SEQ ID NO:1, wherein optionally the one or more amino acid variations at position(s) is selected from the group consisting of: S49T, F121L, T124R, Q159H, Q159R, Q159S, Q159T, Q159Y, Q159A, Q159F, Q159G, Q1591, Q159K, Q159L, Q159M, Q159A, S175H, S175K, S175R, S212H, 1232H, T267W, L268Y, A285Y, Y286A, Y286F, Y286L, Y286M, Y286P, Y2861, Y286T, Y286V, Q293F, Q293W, Q293H, Q293C, Q293A, Q293S, Q293V, Q293D, Q293Y, Q293E, Q2931, Q293M, Q293T, and F300K. 
     
     
         8 . The non-natural prenyltransferase of  claim 7  comprising at least two amino acid variations at positions selected from:
 (i) Q159A and (ii) Q293F, Q293M, Q293F, Q293F; 
 (i) Q159F and (ii) Q293F, Q293W, or Q293H; 
 (i) Q159G and (ii) Q293F; 
 (i) Q159H and (ii) Q293W, Q293H, Q293C, Q293A, Q293S, Q293V, Q293D, Q293Y, or Q293E; 
 (i) Q1591 and (ii) Q293F; 
 (i) Q159K and (ii) Q293V or Q293V; 
 (i) Q159L and (ii) Q293W or Q293F; 
 (i) Q159M and (ii) Q293F or Q293W; 
 (i) Q159R and (ii) Q293V, Q293M, or Q293T; 
 (i) Q159S and (ii) Y2861; and 
 (i) S175H and (ii) Q293V. 
 
     
     
         9 . The non-natural prenyltransferase of  claim 8  comprising at least three amino acid variations at positions selected from
 (i) Q159H, (ii) Y286A, and (iii) Q293F, Q293M, or Q293V; 
 (i) Q159H, (ii) Y286I, and (iii) Q293M or Q293V; 
 (i) Q159H, (ii) Y286V, and (iii) Q293F, Q293M, Q293V, or Q293W; 
 (i) Q159L, (ii) S175H, and (iii) Q293F; 
 (i) S175H, (ii), Y286V, and (iii) Q293M; 
 (i) S175H, (ii), Y286I, and (iii) Q293M or Q293V; 
 (i) Q159S, (ii) S175H, and (iii) Y286I; 
 (i) Q159S, (ii) S175R, and (iii) Y286V; 
 (i) Q159S, (ii) S175S, and (iii) Y2861; and 
 (i) Q159S, (ii) S212H, (iii) Y286A or Y286V. 
 
     
     
         10 . The non-natural prenyltransferase of  claim 8  comprising at least four amino acid variations at positions selected from:
 (i) Q159H, (ii) S175H, (iii) Y286A, and (iv) Q293V; 
 (i) Q159H, (ii) S175H, (iii) Y286V, and (iv) Q293M or Q293V; 
 (i) Q159H, (ii) S175R, (iii) Y286I, and (iv) Q293M; 
 (i) Q159L, (ii) S175K, (iii) Y286A, and (iv) Q293V; 
 (i) Q159M, (ii) S175H, (iii) Y286V, and (iv) Q293F; 
 (i) Q159R, (ii) S175H, (iii) Y286I, and (iv) Q293Q; 
 (i) Q159S, (ii) S175H, (iii) Y286V, and (iv) Q293F; 
 (i) Q159S, (ii) S175K, (iii) Y286V, and (iv) Q293V; and 
 (i) Q159S, (ii) S212H, (iii) Y286V, and (iv) Q293M, or comprising at least five amino acid variations at positions selected from: 
 (i) Q159H, (ii) S175R, (iii) S212H, (iv) Y286A, and (v) Q293V; and 
 (i) Q159R, (ii) S175R, (iii) S212H, (iv) Y2861, and (v) Q293M. 
 
     
     
         11 . The non-natural prenyltransferase of  claim 5  comprising one or more amino acid variations at position(s) selected from the group consisting of: 51, 123, 126, 161, 177, 214, 234, 269, 270, 287, 288, 295, and 302, relative to SEQ ID NO:2, wherein optionally the one or more amino acid variations at position(s) selection from the group consisting of: S49T, F121L, T124R, Q159H, Q159R, Q159S, Q159T, Q159Y, Q159A, Q159F, Q159G, Q1591, Q159K, Q159L, Q159M, Q159A, S175H, S175K, S175R, S212H, 1232H, T267W, L268Y, A285Y, Y286A, Y286F, Y286L, Y286M, Y286P, Y2861, Y286T, Y286V, Q293F, Q293W, Q293H, Q293C, Q293A, Q293S, Q293V, Q293D, Q293Y, Q293E, Q2931, Q293M, Q293T, and F300K. 
     
     
         12 . The non-natural prenyltransferase of  claim 11  comprising one or more amino acid variations at position(s) selection from the group consisting of:
 (a) Q161H; 
 (b) S214H; 
 (c) Y288V; 
 (d) Q161R and Q295V; 
 (e) Q161H and Q295W; 
 (f) Q161H and Q295V 
 (g) Y2881 and Q295V; 
 (h) Q161S, S214H, and Y288V; 
 (i) Q161H, Y2881, and Q295W; and 
 (j) Q161H, Y288V, and Q295M. 
 
     
     
         13 . A non-natural prenyltransferase having 50% or greater identity to any one of SEQ ID NOs:1-15 and having one or more of amino acid motifs (a)-(j) as follows:
 (a) FX 3 M (SEQ ID NO:16) or VFX 3 MA (SEQ ID NO:17) wherein; X 3  is T;   (b) X 4 FPX 5  (SEQ ID NO:18), wherein X 4  is F or L, and wherein X 5  is T or R, with the proviso that in a single motif, X 4  is not F, and X 5  is not T;   (c) VX 6 M (SEQ ID NO:19) or KVX 6 MT (SEQ ID NO:20) wherein X 6  is selected from the group consisting of H, R, S, T, Y, A, F, G, I, K, L, M, and A;   (d) FX 7 E (SEQ ID NO:21) or YFX 7 EL (SEQ ID NO:22) wherein X 7  is selected from the group consisting of H, K, and R;   (e) FX 8 V (SEQ ID NO:23) or SFX 8 VY (SEQ ID NO:24) wherein X 8  is H;   (f) VX 9 S (SEQ ID NO:25) or AVX 9 SN (SEQ ID NO:26) wherein X 9  is H;   (g) RX 10 × 11 V (SEQ ID NO:27), wherein, X 10  is T or W, and wherein X 11  is L or Y;   with the proviso that in a single motif, X 10  is not T and X 11  is not L;   (h) GX 12 × 13 Y (SEQ ID NO:28), wherein, X 12  is A or Y, and wherein X 13  is Y, A, F, L, M, P, T, or V; with the proviso that in a single motif, X 12  is not A and X 13  is not Y;   (i) X 14 × 15 R (SEQ ID NO:29) or DX 14 × 15 R (SEQ ID NO:30), wherein, X 14  is V or I, and wherein X 15  is F, W, H, C, A, S, V, D, Y, E, I, M, or T; and   (j) AX 16 D (SEQ ID NO:31) or KAX 16 D (SEQ ID NO:32) wherein X 16  is K.   
     
     
         14 . A nucleic acid encoding the non-natural prenyltransferase of  claim 1 , wherein the nucleic acid is optionally in the form of an expression construct. 
     
     
         15 . An engineered cell comprising the non-natural prenyltransferase of  claim 1 . 
     
     
         16 . The engineered cell of  claim 15  comprising a non-natural prenyltransferase comprising one or more amino acid variations at position(s) selected from the group consisting of: 49, 121, 124, 159, 175, 212, 232, 267, 268, 285, 286, 293, and 300, relative to SEQ ID NO:1, wherein optionally the one or more amino acid variations at position(s) is selected from the group consisting of: S49T, F121L, T124R, Q159H, Q159R, Q159S, Q159T, Q159Y, Q159A, Q159F, Q159G, Q1591, Q159K, Q159L, Q159M, Q159A, S175H, S175K, S175R, S212H, 1232H, T267W, L268Y, A285Y, Y286A, Y286F, Y286L, Y286M, Y286P, Y2861, Y286T, Y286V, Q293F, Q293W, Q293H, Q293C, Q293A, Q293S, Q293V, Q293D, Q293Y, Q293E, Q293I, Q293M, Q293T, and F300K. 
     
     
         17 . The engineered cell of  claim 15  comprising
 (i) an olivetolic acid pathway, optionally comprising a polyketide synthase/olivetol synthase (condensation of hexanoyl coenzyme A (CoA) and malonyl CoA); 
 (ii) a DVA or OSA pathway; 
 (iii) an olivetol pathway, optionally comprising polyketide synthase; or 
 (iv) a geranyl pyrophosphate pathway (GPP), optionally comprising geranyl pyrophosphate synthase, and optionally comprising a mevalonate (MVA) pathway, a MEP pathway, or 
 any two or more of (i)-(iv). 
 
     
     
         18 . The engineered cell of  claim 15  selected from the group consisting of  Escherichia, Corynebacterium, Bacillus, Ralstonia , and  Staphylococcus.    
     
     
         19 . A cell extract, cell culture medium, therapeutic composition, or medicinal composition comprising cannabinoid derived from the engineered cell of  claim 15 . 
     
     
         20 . A non-natural prenyltransferase having 50% or greater identity to SEQ ID NO:1 or to any one of SEQ ID NO:2-15 and comprising one or more amino acid variations selected from the group consisting of: S49T, F121L, T124R, Q159H, Q159R, Q159S, Q159T, Q159Y, Q159A, Q159F, Q159G, Q1591, Q159K, Q159L, Q159M, Q159A, S175H, S175K, S175R, S212H, 1232H, T267W, L268Y, A285Y, Y286A, Y286F, Y286L, Y286M, Y286P, Y2861, Y286T, Y286V, Q293F, Q293W, Q293H, Q293C, Q293A, Q293S, Q293V, Q293D, Q293Y, Q293E, Q2931, Q293M, Q293T, and F300K relative to SEQ ID NO:1.