Compositions and methods for using genetically modified enzymes

Abstract

The disclosure relates to the biosynthesis of cannabinoids and related prenylated phenolic compounds using recombinant enzymes. In particular, the disclosure provides recombinant mutant ORF2 enzymes engineered to produce a greater amount of a desired product, or to have a greater ability to catalyze a reaction using a desired substrate, as compared to WT ORF2. The disclosure also provides methods of preparing such ORF2 mutant enzymes; as well as methods of use thereof in improving the biosynthesis of cannabinoids and related prenylated phenolic compounds.

1 .- 30 . (canceled) 
     
     
         31 . A recombinant polypeptide, comprising an amino acid sequence with at least 95% identity to SEQ ID NO: 1, wherein said amino acid sequence comprises an amino acid substitution to SEQ ID NO: 1 at a position selected from the group consisting of Q161Y, Q161F, Q161K, Q161T, S214R, S214D, S214E, S214N, S214K, S214Q, S214T, Y288F, Y288L, Y288M, Y288P, Y288T, Y288W, Y288C, Y288G, Q295F, Q295H, Q295I, Q295L, Q295Y, and Q295D, wherein said recombinant polypeptide converts DVA and geranyl diphosphate (GPP) to cannabigerovarinic acid (CBGVA) and 5-GDVA, and wherein said recombinant polypeptide has a CBGVA production potential that is higher than wild-type ORF2 enzyme under the same condition. 
     
     
         32 . The recombinant polypeptide of  claim 31 , wherein said amino acid substitution is selected from the group consisting of Q161Y, Q161F, Q161K, and Q161T. 
     
     
         33 . The recombinant polypeptide of  claim 31 , wherein said amino acid substitution is selected from the group consisting of Q295F, Q295H, Q295I, Q295L, Q295Y and Q295D. 
     
     
         34 . The recombinant polypeptide of  claim 31 , wherein said amino acid substitution is selected from the group consisting of S214R, S214D, S214E, S214N, S214K, S214Q, and S214T. 
     
     
         35 . The recombinant polypeptide of  claim 31 , wherein said amino acid substitution is selected from the group consisting of Y288F, Y288L, Y288M, Y288P, Y288T, Y288W, Y288C, and Y288G. 
     
     
         36 . The recombinant polypeptide of  claim 31 , wherein said amino acid sequence comprises two or more amino acid substitutions selected from the group consisting of: A53T and S214R; S177W and Q295A; S214R and Q295F; Q16S and S214R; S177W and S214R; Q161S and Q295L; Q161S and Q295F; V49A and S214R; A53T and Q295F; Q161S and S177W; Q161S, V294A and Q295A; A53T, Q161S and Q295W; A53T and S177W; A53T, V294A and Q295W; A53T and Q295W; Q161S, V294A and Q295W; V49A and Q295L; A53T, Q161S, V294A and Q295W; A53T, Q161S, V294N and Q295A; A53T, Q161S and Q295A; A53T, Q161S, V294A and Q295A; A53T, Q161S, V294N and Q295W; A53T and Q161S; A53T, Q161S and V294A; A53T, Q161S and V294N; and A53T, V294A and Q295A. 
     
     
         37 . The recombinant polypeptide of  claim 31 , wherein said amino acid sequence is selected from the group consisting of SEQ ID NOs: 99, 102, 103, 105, 107, 108, 110, 114, 116, 122, 126, 132, 133, 134, 137, 140, 142, 144, 145, 147, 148, 156, 157, 160, 161, 166, 169, 171, 172, 174, 272-274, 276-277, and 279-300. 
     
     
         38 . A recombinant polypeptide, comprising an amino acid sequence with at least 95% identity to SEQ ID NO: 1, wherein said amino acid sequence comprises an amino acid substitution to SEQ ID NO: 1 at a position selected from the group consisting of Q161F, Q161Y, Q161L, Q161M, Q161I, Q161C, Q161T, Q161D, Q161G, Q161W, S214Q, S214E, S214N, S214F, S214I, S214L, S214M, Y288F, Y288L, Y288M, Y288T, Y288C, Y288W, Q295H, Q295G, Q295F, and Q295C, wherein said recombinant polypeptide converts olivetol (O) and geranyl diphosphate (GPP) to cannabigerol (CBG) and 5-GO, and wherein said recombinant polypeptide has a CBG production potential that is higher than wild-type ORF2 enzyme under the same condition. 
     
     
         39 . The recombinant polypeptide of  claim 38 , wherein said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295H, Q295G, Q295F, and Q295C. 
     
     
         40 . The recombinant polypeptide of  claim 38 , wherein said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161F, Q161Y, Q161L, Q161M, Q161I, Q161C, Q161T, Q161D, Q161G, and Q161W. 
     
     
         41 . The recombinant polypeptide of  claim 38 , wherein said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214Q, S214E, S214N, S214F, S214I, S214L, and S214M. 
     
     
         42 . The recombinant polypeptide of  claim 38 , wherein said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Y288F, Y288L, Y288M, Y288T, Y288C, and Y288W. 
     
     
         43 . The recombinant polypeptide of  claim 38 , wherein said amino acid sequence comprises two or more amino acid substitutions selected from the group consisting of: A53T, Q161S, V294A and Q295W; A53T, Q161S and V294A; Q161S and Q295L; A53T and Q295F; A53T and Q295A; A53T, Q161S and V294N; Q161S and Q295F; A53T, Q161S and Q295W; A53T and Q161S; A53T and S177W; Q161S, V294A and Q295W; A53T and V294A; A53T, V294A and Q295W; S177W and Q295A; Q161S and S177W; and A53T and S214R. 
     
     
         44 . The recombinant polypeptide of  claim 38 , wherein said amino acid sequence is selected from the group consisting of SEQ ID NOs: 99, 102, 105, 107, 108, 114, 116, 119, 120, 122, 123, 125, 127, 128, 133, 134, 136, 137 139, 142, 143, 144, 148, 156, 161, 162, 165, 167, 168, 169, 171, 224, 272, 273, 275, 280, 282, 283, 284, 285, 287, 289, 290, 293, 294, 296, and 297. 
     
     
         45 . A method of producing a prenylation product, comprising: contacting said recombinant polypeptide of  claim 31  with a substrate and a donor, thereby producing said prenylation product. 
     
     
         46 . The method of  claim 45 , wherein said substrate comprises divarinic acid (DVA) and said donor comprises geranyl pyrophosphate (GPP), and wherein said prenylation product comprises CBGVA, 5-GDVA, a prenylated product on any other position on an aromatic ring of said substrate, or any combination thereof. 
     
     
         47 . The method of  claim 46 , wherein said prenylation product comprises CBGVA and 5-GDVA, and wherein said recombinant polypeptide produces a ratio of an amount of CBGVA to a combined amount of CBGVA and 5-GDVA that is higher than wild-type ORF2 enzyme under the same acid condition; a combined amount of CBGVA and 5-GDVA that is higher than wild-type ORF2 enzyme under the same acid condition; or a combination thereof. 
     
     
         48 . A method of producing a prenylation product, comprising: contacting said recombinant polypeptide of  claim 38  with a substrate and a donor, thereby producing said prenylation product. 
     
     
         49 . The method of  claim 48 , wherein said substrate comprises olivetol (O) and said donor comprises geranyl pyrophosphate (GPP), and wherein said prenylation product comprises CBG, 5-GO, a prenylated product on any other position on an aromatic ring of said substrate, or any combination thereof. 
     
     
         50 . The method of  claim 49 , wherein said prenylation product comprises CBG and 5-GO, and wherein said recombinant polypeptide produces a ratio of an amount of CBG to a combined amount of CBG and 5-GO that is higher than wild-type ORF2 enzyme under the same acid condition; a combined amount of CBG and 5-GO that is higher than wild-type ORF2 enzyme under the same acid condition; or a combination thereof.