Prenyltransferase variants and methods for production of prenylated aromatic compounds

A non-natural prenyltransferase based on a template of a wild type prenyltransferase that has 75% or greater identity to SEQ ID NO:1 or to any one of SEQ ID NO:2-3, the non-natural prenyltransferase comprising

• (A) at least one amino acid variation as compared to the wild type prenyltransferase 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
• the variation at position 49 is optionally S49T,
• the variation at position 121 is F121L,
• the variation at position 124 is T124R,
• the variation at position 159 is selected from the group consisting of Q159H, Q159R, Q159S, Q159Y, Q159F, Q159G, Q159I, Q159K, Q159L, Q159M,
• the variation at position 175 is selected from the group consisting of S175H, S175K, S175R,
• the variation at position 212 is S212H,
• the variation at position 232 is optionally I232H,
• the variation at position 267 is T267W,
• the variation at position 268 is optionally L268Y,
• the variation at position 285 is A285Y,
• the variation at position 286 is selected from the group consisting of Y286F, Y286L, Y286M, Y286P, Y286I, Y286T, Y286V,
• the variation at position 293 is selected from the group consisting of Q293F, Q293W, Q293H, Q293C, Q293S, Q293V, Q293D, Q293Y, Q293E, Q293I, Q293M, Q293T, and
• the variation at position 300 is optionally F300K, or
• (B) at least two amino acid variations as compared to the wild type prenyltransferase, the at least two variations at positions 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 non-natural prenyltransferase is enzymatically capable of
• (a1) 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 (a1) 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.