Patent Application
20210301267
The contents of the text file named “REBI_001_01US_SeqList_ST25.txt”, which was created on Nov. 12, 2019 and is 1,190 megabytes in size, are hereby incorporated by reference in its entirety.
The present disclosure is generally related to the biosynthesis of organic compounds, such as cannabinoids, using recombinant enzymes.
Cannabinoids include a group of more than 100 chemical compounds mainly found in the plant Cannabis sativa L. Due to the unique interaction of cannabinoids with the human endocannabinoid system, many of these compounds are potential therapeutic agents for the treatment of several medical conditions. For instance, the psychoactive compound Δ9-tetrahydrocannabinol (Δ9-THC) has been used in the treatment of pain and other medical conditions. Several synthetic Cannabis-based preparations have been used in the USA, Canada and other countries as an authorized treatment for nausea and vomiting in cancer chemotherapy, appetite loss in acquired immune deficiency syndrome and symptomatic relief of neuropathic pain in multiple sclerosis.
Cannabinoids are terpenophenolic compounds, produced from fatty acids and isoprenoid precursors as part of the secondary metabolism of Cannabis. The main cannabinoids produced by Cannabis are Δ9-tetrahydrocannabidiol (THC), cannabidiol (CBD) and cannabinol (CBN), followed by cannabigerol (CBG), cannabichromene (CBC) and other minor constituents. Currently, Δ9-THC and CBD are either extracted from the plant or chemically synthesized. However, agricultural production of cannabinoids faces challenges such as plant susceptibility to climate and diseases, low content of less-abundant cannabinoids, and need for extraction of cannabinoids by chemical processing. Furthermore, chemical synthesis of cannabinoids has failed to be a cost-effective alternative mainly because of complex synthesis leading to high production cost and low yields.
Therefore, there is a pressing need for biotechnology-based synthetic biology approaches which can enable the synthesis of high-quality cannabinoids in a cost-effective and environmentally friendly manner.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% identity to SEQ ID NO: 1, wherein said amino acid sequence comprises at least one amino acid substitution to SEQ ID NO: 1, wherein said at least one amino acid substitution does not comprise an alanine substitution at a position selected from the group consisting of amino acid positions 47, 64, 110, 121, 123, 126, 161, 175, 177, 214, 216, 288, 294, and 295.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% identity to SEQ ID NO: 1, wherein said amino acid sequence comprises at least one amino acid substitution to SEQ ID NO: 1, wherein at least one of said at least one amino acid substitution to SEQ ID NO: 1 is located on a position chosen from the group consisting of amino acid positions 1-46, 48-63, 65-109, 111-120, 122, 124, 125, 127-160, 162-174, 176, 178-213, 215, 217-287, 289-293, and 296-307.
In some cases, the amino acid sequence has at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some cases, the at least one amino acid substitution comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions to SEQ ID NO: 1. In some cases, said at least one of said at least one amino acid substitution is located on a position chosen from the group consisting of amino acid positions 17, 25, 38, 49, 53, 106, 108, 112, 118, 119, 121, 123, 161, 162, 166, 173, 174, 177, 205, 209, 213, 214, 216, 219, 227, 228, 230, 232, 271, 274, 283, 286, 288, 294, 295, and 298. In some cases, said at least one of said at least one amino acid substitution is located on a position chosen from the group consisting of amino acid positions 17, 25, 38, 49, 53, 106, 108, 112, 118, 119, 162, 166, 173, 174, 205, 209, 213, 219, 227, 228, 230, 232, 271, 274, 283, 286, 288, and 298.
In some cases, said at least one of said at least one amino acid substitution is chosen from the group consisting of A17T, C25V, Q38G, V49A, V49L, V49S, A53C, A53D, A53E, A53F, A53G, A53H, A53I, A53K, A53L, A53M, A53N, A53P, A53Q, A53R, A53S, A53T, A53V, A53W, A53Y, M106E, A108G, E112D, E112G, K118N, K118Q, K119A, K119D, Y121W, F123A, F123H, F123W, Q161A, Q161C, Q161D, Q161E, Q161F, Q161G, Q161H, Q161I, Q161K, Q161L, Q161M, Q161N, Q161P, Q161R, Q161S, Q161T, Q161V, Q161W, Q161Y, M162A, M162F, D166E, N173D, L174V, S177E, S177W, S177Y, G205L, G205M, C209G, F213M, S214A, S214C, S214D, S214E, S214F, S214G, S214H, S214I, S214K, S214L, S214M, S214N, S214P, S214Q, S214R, S214T, S214V, S214W, S214Y, Y216A, L219F, D227E, R228E, R228Q, C230N, C230S, A232S, V271E, L274V, Y283L, G286E, Y288A, Y288C, Y288D, Y288E, Y288F, Y288G, Y288H, Y288I, Y288K, Y288L, Y288M, Y288N, Y288P, Y288Q, Y288R, Y288S, Y288T, Y288V, Y288W, V294A, V294F, V294N, Q295A, Q295C, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295K, Q295L, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, L298A, L298Q, and L298W.
In some cases, said recombinant polypeptide converts olivetolic acid (OA) and geranyl diphosphate (GPP) to cannabigerolic acid (CBGA) and 5-geranyl olivetolate (5-GOA), and wherein said recombinant polypeptide produces a ratio of an amount of CBGA to a combined amount of CBGA and 5-GOA that is higher than wild-type ORF2 enzyme under the same condition. In some cases, said ratio is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295F, Q295L, Q295V, Q295I, Q295M, Q295E, Q295C, Q295W, Q295G, Q295T, Q295P, Q295S, Q295D, Q295N, Q295K, and Q295Y. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 139-143; 145-150; and 153-157. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161H, Q161Y, Q161R, Q161D, Q161F, Q161K, Q161M, Q161E, Q161L, Q161S, Q161T, Q161N, and Q161I. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 120-122; 124-129; 132-134; and 137. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214H, S214R, S214Q, S214E, S214K, S214M, S214I, S214L, S214N, S214D, S214Y, S214F, and S214T. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 160-162; 164-169; 171, 172, 174, and 177. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Y288F, Y288P, Y288A, Y288L, Y288C, Y288I, Y288M, Y288G, Y288T, Y288S, Y288W, Y288D, Y288E, Y288N, Y288K, Y288R, and Y288H. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 98-110; 112-114; and 116.
In some cases, said at least one amino acid substitution to SEQ ID NO: 1 comprises two or more amino acid substitutions to SEQ ID NO: 1 selected from the group consisting of
(a) A17T, C25V, Q38G, V49A, V49L, V49S, A53C, A53D, A53E, A53F, A53G, A53H, A53I, A53K, A53L, A53M, A53N, A53P, A53Q, A53R, A53S, A53T, A53V, A53W, A53Y, M106E, A108G, E112D, E112G, K118N, K118Q, K119A, K119D, Y121W, F123A, F123H, F123W, Q161A, Q161C, Q161D, Q161E, Q161F, Q161G, Q161H, Q161I, Q161K, Q161L, Q161M, Q161N, Q161P, Q161R, Q161S, Q161T, Q161V, Q161W, Q161Y, M162A, M162F, D166E, N173D, L174V, S177E, S177W, S177Y, G205L, G205M, C209G, F213M, S214A, S214C, S214D, S214E, S214F, S214G, S214H, S214I, S214K, S214L, S214M, S214N, S214P, S214Q, S214R, S214T, S214V, S214W, S214Y, Y216A, L219F, D227E, R228E, R228Q, C230N, C230S, A232S, V271E, L274V, Y283L, G286E, Y288A, Y288C, Y288D, Y288E, Y288F, Y288G, Y288H, Y288I, Y288K, Y288L, Y288M, Y288N, Y288P, Y288Q, Y288R, Y288S, Y288T, Y288V, Y288W, V294A, V294F, V294N, Q295A, Q295C, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295K, Q295L, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, L298A, L298Q, and L298W; or
(b) A53T and S214R; S177W and Q295A; S214R and Q295F; Q161S and 5214R; S177W and 5214R; Q161S and Q295L; Q161S and Q295F; V49A and 5214R; A53T and Q295F; Q161S and S177W; Q161S, V294A and Q295W; A53T, Q161S and Q295W; A53T and S177W; A53T, Q161S, V294A and Q295W; A53T, V294A and Q295A; V49A and Q295L; A53T, Q161S, V294N and Q295W; A53T and Q295A; Q161S, V294A and Q295A; A53T and Q295W; A53T, V294A and Q295W; A53T, Q161S and Q295A; A53T, Q161S, V294A and Q295A; and A53T, Q161S, V294N and Q295A. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 275-294; and 296-299.
In some cases, said recombinant polypeptide converts olivetolic acid (OA) and geranyl diphosphate (GPP) to cannabigerolic acid (CBGA) and 5-geranyl olivetolate (5-GOA), and wherein said recombinant polypeptide produces a ratio of an amount of 5-GOA to a combined amount of CBGA and 5-GOA that is higher than wild-type ORF2 enzyme under the same condition. In some cases, said ratio is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295H and Q295R. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 144 and 152. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161W, Q161G, Q161V, Q161C, and Q161P. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 118, 119, 123, 130, 131, 135, and 136. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214V, S214C, S214W, S214P, S214A, and S214G. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 159, 163, 170, 175 and 176. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of A53T and Q161S; A53T, Q161S and V294A; A53T, Q161S and V294N; A53T and V294A; and V49A and Q161S. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 272-274, 295, and 300.
In some cases, said recombinant polypeptide converts olivetolic acid (OA) and geranyl diphosphate (GPP) to cannabigerolic acid (CBGA) and 5-geranyl olivetolate (5-GOA), and wherein said recombinant polypeptide produces a combined amount of CBGA and 5-GOA that is higher than wild-type ORF2 enzyme under the same condition. In some cases, said combined amount is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295H, Q295F, Q295W, Q295M, Q295D, Q295L, and Q295Y. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 140, 142, 144, 147, 148, 156 and 157. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161H, Q161Y, Q161L, Q161R, Q161C, Q161T, Q161S, Q161F, Q161I, and Q161P. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 119, 122, 124, 125, 127, 130, 132-134, and 137. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214G and S214H. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 163 and 164. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Y288L, Y288P, Y288I, Y288A, Y288F, Y288C, Y288M, and Y288T. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 98, 99, 102, 105, 107, 108, 110, and 114.
In some cases, said at least one amino acid substitution to SEQ ID NO: 1 comprises two or more amino acid substitutions selected from the group consisting of:
(a) A17T, C25V, Q38G, V49A, V49L, V49S, A53C, A53D, A53E, A53F, A53G, A53H, A53I, A53K, A53L, A53M, A53N, A53P, A53Q, A53R, A53S, A53T, A53V, A53W, A53Y, M106E, A108G, E112D, E112G, K118N, K118Q, K119A, K119D, Y121W, F123A, F123H, F123W, Q161A, Q161C, Q161D, Q161E, Q161F, Q161G, Q161H, Q161I, Q161K, Q161L, Q161M, Q161N, Q161P, Q161R, Q161S, Q161T, Q161V, Q161W, Q161Y, M162A, M162F, D166E, N173D, L174V, S177E, S177W, S177Y, G205L, G205M, C209G, F213M, S214A, S214C, S214D, S214E, S214F, S214G, S214H, S214I, S214K, S214L, S214M, S214N, S214P, S214Q, S214R, S214T, S214V, S214W, S214Y, Y216A, L219F, D227E, R228E, R228Q, C230N, C230S, A232S, V271E, L274V, Y283L, G286E, Y288A, Y288C, Y288D, Y288E, Y288F, Y288G, Y288H, Y288I, Y288K, Y288L, Y288M, Y288N, Y288P, Y288Q, Y288R, Y288S, Y288T, Y288V, Y288W, V294A, V294F, V294N, Q295A, Q295C, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295K, Q295L, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, L298A, L298Q, and L298W; or
(b) A53T and V294A; A53T, Q161S and V294A; A53T, Q161S and V294N; Q161S, V294A and Q295A; A53T, Q161S and Q295A; A53T, Q161S, V294A and Q295A; A53T, Q161S, V294N and Q295A; A53T and Q295W; Q161S, V294A and Q295W; A53T, Q161S and Q295W; A53T, V294A and Q295W; A53T, Q161S, V294A and Q295W; A53T, Q161S, V294N and Q295W; S177W and Q295A; A53T and S177W; A53T and Q295F; A53T and 5214R; A53T and Q161S; Q161S and Q295F; Q161S and Q295L; and S214R and Q295F. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 272-274, 276, 277, 279-287; 290, 293-297, and 299.
In some cases, said recombinant polypeptide converts olivetolic acid (OA) and geranyl diphosphate (GPP) to cannabigerolic acid (CBGA) and 5-geranyl olivetolate (5-GOA), and wherein said recombinant polypeptide has a CBGA production potential that is higher than wild-type ORF2 enzyme under the same condition. In some cases, said CBGA production potential is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295F, Q295M, Q295W, Q295L, Q295I, Q295C, Q295G, Q295V, Q295D, and Q295H. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 139, 140, 142-145, 147, 148, 155 and 156. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161H, Q161Y, Q161R, Q161F, Q161L, Q161S, Q161T, Q161I, Q161M, and Q161K. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 122, 124-128, 132-134, and 137. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214H, S214Q, and S214R. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 164, 171 and 172. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Y288L, Y288P, Y288I, Y288A, Y288F, Y288C, Y288M, and Y288T. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 98, 99, 102, 105, 107, 108, 110 and 114. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 comprises two or more amino acid substitutions selected from the group consisting of:
(a) A17T, C25V, Q38G, V49A, V49L, V49S, A53C, A53D, A53E, A53F, A53G, A53H, A53I, A53K, A53L, A53M, A53N, A53P, A53Q, A53R, A53S, A53T, A53V, A53W, A53Y, M106E, A108G, E112D, E112G, K118N, K118Q, K119A, K119D, Y121W, F123A, F123H, F123W, Q161A, Q161C, Q161D, Q161E, Q161F, Q161G, Q161H, Q161I, Q161K, Q161L, Q161M, Q161N, Q161P, Q161R, Q161S, Q161T, Q161V, Q161W, Q161Y, M162A, M162F, D166E, N173D, L174V, S177E, S177W, S177Y, G205L, G205M, C209G, F213M, S214A, S214C, S214D, S214E, S214F, S214G, S214H, 52141, S214K, S214L, S214M, S214N, S214P, S214Q, S214R, S214T, S214V, S214W, S214Y, Y216A, L219F, D227E, R228E, R228Q, C230N, C230S, A232S, V271E, L274V, Y283L, G286E, Y288A, Y288C, Y288D, Y288E, Y288F, Y288G, Y288H, Y288I, Y288K, Y288L, Y288M, Y288N, Y288P, Y288Q, Y288R, Y288S, Y288T, Y288V, Y288W, V294A, V294F, V294N, Q295A, Q295C, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295K, Q295L, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, L298A, L298Q, and L298W; or
(b) A53T_Q161S_V294A; A53T_Q161S_V294N; A53T_Q295A; Q161S_V294A_Q295A; A53T_Q161S_Q295A; A53T_V294A_Q295A; A53T_Q161S_V294A_Q295A; A53T_Q161S_V294N_Q295A; A53T_Q295W; Q161S_V294A_Q295W; A53T_Q161S_Q295W; A53T_V294A_Q295W; A53T_Q161S_V294A_Q295W; A53T_Q161S_V294N_Q295W; S177W_Q295A; S177W_S214R; Q161S_S177W; A53T_S177W; V49A_S214R; A53T_Q295F; A53T_S214R; A53T_Q161S; Q161S_Q295F; Q161S_Q295L; Q161S_S214R; and S214R_Q295F. In some cases, the recombinant polypeptide of any one of claims Error! Reference source not found.-Error! Reference source not found., wherein said amino acid sequence is selected from the group consisting of SEQ ID NOs: 273-290, and 292-299.
In some cases, said recombinant polypeptide converts olivetolic acid (OA) and geranyl diphosphate (GPP) to cannabigerolic acid (CBGA) and 5-geranyl olivetolate (5-GOA), and wherein said recombinant polypeptide has a 5-GOA production potential that is higher than wild-type ORF2 enzyme under the same condition. In some cases, said 5-GOA production potential is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295H and Q295Y. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 144 and 157. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161C, Q161L, Q161T, Q161P, Q161I, and Q161S. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 119, 125, 127, 130, 133 and 134. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is S214G. In some cases, said amino acid sequence is SEQ ID NO: 163. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of A53T_V294A; V49A_Q295L; and V49A_Q161S. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 272, 291, and 300.
In some cases, said recombinant polypeptide converts DVA and geranyl diphosphate (GPP) to 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 condition. In some cases, said ratio is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295F, Q295I, Q295W, Q295V, Q295M, Q295L, Q295E, Q295H, Q295P, Q295C, Q295T and Q295Y. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 139, 141, 142, 144, 145, 147, 148, 150, 154, 155, 156, and 157. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161H, Q161Y, Q161R, Q161K, Q161E, Q161S, Q161D, Q161F, Q161T, Q161G, and Q161P. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 120, 121, 122, 123, 124, 126, 130, 132, 133, 134, and 137. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214R, S214H, S214K, S214E, S214Q, S214L, S214M, S214Y, 52141, S214F, S214D, S214N, S214G, S214T, and S214V. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 171, 172, 174, 175, and 177. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Y288F, Y288I, Y288L, Y288M, Y288P, Y288T, Y288W, Y288C, and Y288G. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 99, 102, 103, 105, 107, 108, 110, 114, and 116.
In some cases, said at least one amino acid substitution to SEQ ID NO: 1 comprises two or more amino acid substitution to SEQ ID NO: 1 selected from the group consisting of:
(a) A17T, C25V, Q38G, V49A, V49L, V49S, A53C, A53D, A53E, A53F, A53G, A53H, A53I, A53K, A53L, A53M, A53N, A53P, A53Q, A53R, A53S, A53T, A53V, A53W, A53Y, M106E, A108G, E112D, E112G, K118N, K118Q, K119A, K119D, Y121W, F123A, F123H, F123W, Q161A, Q161C, Q161D, Q161E, Q161F, Q161G, Q161H, Q161I, Q161K, Q161L, Q161M, Q161N, Q161P, Q161R, Q161S, Q161T, Q161V, Q161W, Q161Y, M162A, M162F, D166E, N173D, L174V, S177E, S177W, S177Y, G205L, G205M, C209G, F213M, S214A, S214C, S214D, S214E, S214F, S214G, S214H, 52141, S214K, S214L, S214M, S214N, S214P, S214Q, S214R, S214T, S214V, S214W, S214Y, Y216A, L219F, D227E, R228E, R228Q, C230N, C230S, A232S, V271E, L274V, Y283L, G286E, Y288A, Y288C, Y288D, Y288E, Y288F, Y288G, Y288H, Y288I, Y288K, Y288L, Y288M, Y288N, Y288P, Y288Q, Y288R, Y288S, Y288T, Y288V, Y288W, V294A, V294F, V294N, Q295A, Q295C, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295K, Q295L, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, L298A, L298Q, and L298W; or
(b) A53T_S214R, Q161S_Q295L, S177W_Q295A, Q161S_Q295F, A53T_Q295F, S214R_Q295F, Q161S_S177W, Q16S_S214R, A53T_Q161S, A53T_V294A, A53T_S177W, A53T_Q161S_V294A_Q295W, A53T_V294A_Q295W, A53T_Q161S_V294N_Q295W, A53T_Q161S_V294A, A53T_Q295W, A53T_Q161S_V294N, Q161S_V294A_Q295A, A53T_Q161S_V294A_Q295A, A53T_Q161S_Q295W, A53T_Q161S_V294N_Q295A, A53T_Q161S_Q295A, A53T_Q295A, S177W_S214R, A53T_V294A_Q295A, Q161S_V294A_Q295W_V49A_S214R, and V49A_Q295L. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 275-294; and 296-299.
In some cases, said recombinant polypeptide converts DVA and geranyl diphosphate (GPP) to CBGVA and 5-GDVA, and wherein said recombinant polypeptide produces a ratio of an amount of 5-GDVA to a combined amount of CBGVA and 5-GDVA that is higher than wild-type ORF2 enzyme under the same condition. In some cases, said ratio is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295G, Q295D, Q295S, Q295N, Q295K, and Q295R. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 140, 143, 146, 149, 153, and 153. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161V, Q161L, Q161I, Q161M, Q161W, and Q161C. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 119, 125, 127, 128, 135, and 136. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214W, S214P, and S214C. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 159, 170, and 176. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Y288H, Y288S, Y288N, Y288K, Y288D, Y288R, and Y288E. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 100, 101, 104, 106, 109, 112, and 113.
In some cases, said recombinant polypeptide converts DVA and geranyl diphosphate (GPP) to CBGVA and 5-GDVA, and wherein said recombinant polypeptide produces a combined amount of CBGVA and 5-GDVA that is higher than wild-type ORF2 enzyme under the same condition. In some cases, said combined amount is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295D, Q295F, and Q295Y. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 140, 142, and 157. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161H, Q161C, Q161M, Q161R, Q161F, Q161T, Q161S, Q161Y, Q161L, and Q161I. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 119, 122, 124, 125, 127, 128, 132, 133, 134, and 137. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214R and S214H. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 164 and 172. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Y288F, Y288I, Y288L, Y288M, Y288P, Y288T, and Y288W. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 102, 105, 107, 108, 110, 114, and 116.
In some cases, said at least one amino acid substitution to SEQ ID NO: 1 comprises two or more amino acid substitutions selected from the group consisting of:
(a) A17T, C25V, Q38G, V49A, V49L, V49S, A53C, A53D, A53E, A53F, A53G, A53H, A53I, A53K, A53L, A53M, A53N, A53P, A53Q, A53R, A53S, A53T, A53V, A53W, A53Y, M106E, A108G, E112D, E112G, K118N, K118Q, K119A, K119D, Y121W, F123A, F123H, F123W, Q161A, Q161C, Q161D, Q161E, Q161F, Q161G, Q161H, Q161I, Q161K, Q161L, Q161M, Q161N, Q161P, Q161R, Q161S, Q161T, Q161V, Q161W, Q161Y, M162A, M162F, D166E, N173D, L174V, S177E, S177W, S177Y, G205L, G205M, C209G, F213M, S214A, S214C, S214D, S214E, S214F, S214G, S214H, 52141, S214K, S214L, S214M, S214N, S214P, S214Q, S214R, S214T, S214V, S214W, S214Y, Y216A, L219F, D227E, R228E, R228Q, C230N, C230S, A232S, V271E, L274V, Y283L, G286E, Y288A, Y288C, Y288D, Y288E, Y288F, Y288G, Y288H, Y288I, Y288K, Y288L, Y288M, Y288N, Y288P, Y288Q, Y288R, Y288S, Y288T, Y288V, Y288W, V294A, V294F, V294N, Q295A, Q295C, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295K, Q295L, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, L298A, L298Q, and L298W; or
(b) A53T_S214R, Q161S_Q295L, S177W_Q295A, Q161S_Q295F, A53T_Q295F, S214R_Q295F, Q161S_S177W, Q161S_S214R, A53T_Q161S, and A53T_V294A. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 224, 272, 287, 289, 293, 294, 296, 297, 298, and 299.
In some cases, said recombinant polypeptide converts DVA and geranyl diphosphate (GPP) to 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. In some cases, said CBGVA production potential is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295F, Q295W, Q295H, Q295I, Q295M, Q295L, Q295Y, and Q295D. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 140, 142, 144, 145, 147, 148, 156, and 157. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161H, Q161R, Q161Y, Q161S, Q161F, Q161K, and Q161T. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 122, 124, 126, 132, 133, 134, and 137. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214R, S214H, S214D, S214E, S214N, S214K, S214Q, and S214T. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 160, 161, 164, 166, 169, 171, 172, and 174. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Y288F, Y288I, Y288L, Y288M, Y288P, Y288T, Y288W, Y288C, and Y288G. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 99, 102, 103, 105, 107, 108, 110, 114, and 116.
In some cases, said at least one amino acid substitution to SEQ ID NO: 1 comprises two or more amino acid substitutions selected from the group consisting of:
(a) A17T, C25V, Q38G, V49A, V49L, V49S, A53C, A53D, A53E, A53F, A53G, A53H, A53I, A53K, A53L, A53M, A53N, A53P, A53Q, A53R, A53S, A53T, A53V, A53W, A53Y, M106E, A108G, E112D, E112G, K118N, K118Q, K119A, K119D, Y121W, F123A, F123H, F123W, Q161A, Q161C, Q161D, Q161E, Q161F, Q161G, Q161H, Q161I, Q161K, Q161L, Q161M, Q161N, Q161P, Q161R, Q161S, Q161T, Q161V, Q161W, Q161Y, M162A, M162F, D166E, N173D, L174V, S177E, S177W, S177Y, G205L, G205M, C209G, F213M, S214A, S214C, S214D, S214E, S214F, S214G, S214H, S214I, S214K, S214L, S214M, S214N, S214P, S214Q, S214R, S214T, S214V, S214W, S214Y, Y216A, L219F, D227E, R228E, R228Q, C230N, C230S, A232S, V271E, L274V, Y283L, G286E, Y288A, Y288C, Y288D, Y288E, Y288F, Y288G, Y288H, Y288I, Y288K, Y288L, Y288M, Y288N, Y288P, Y288Q, Y288R, Y288S, Y288T, Y288V, Y288W, V294A, V294F, V294N, Q295A, Q295C, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295K, Q295L, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, L298A, L298Q, and L298W; or
(b) A53T_S214R, S177W_Q295A, S214R_Q295F, Q16S_S214R, S177W_S214R, Q161S_Q295L, Q161S_Q295F, V49A_S214R, A53T_Q295F, Q161S_S177W, Q161S_V294A_Q295A, A53T_Q161S_Q295W, A53T_S177W, A53T_V294A_Q295W, A53T_Q295W, Q161S_V294A_Q295W, V49A_Q295L, A53T_Q161S_V294A_Q295W A53T_Q161S_V294N_Q295A, A53T_Q161S_Q295A, A53T_Q161S_V294A_Q295A A53T_Q161S_V294N_Q295W, A53T_Q161S, A53T_Q161S_V294A, A53T_Q161S_V294N, and A53T_V294A_Q295A. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 272-274, 276-277, and 279-300.
In some cases, said recombinant polypeptide converts DVA and geranyl diphosphate (GPP) to CBGVA and 5-GDVA, and wherein said recombinant polypeptide has a 5-GDVA production potential that is higher than wild-type ORF2 enzyme under the same condition. In some cases, said 5-GDVA production potential is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295D and Q295Y. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 140 and 157. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161C, Q161M, Q161T, Q161L, Q161F, and Q161I. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 119, 122, 125, 127, 128, and 134. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 comprises two or more amino acid substitutions.
In some cases, said two or more amino acid substitutions are selected from the group consisting of: A17T, C25V, Q38G, V49A, V49L, V49S, A53C, A53D, A53E, A53F, A53G, A53H, A53I, A53K, A53L, A53M, A53N, A53P, A53Q, A53R, A53S, A53T, A53V, A53W, A53Y, M106E, A108G, E112D, E112G, K118N, K118Q, K119A, K119D, Y121W, F123A, F123H, F123W, Q161A, Q161C, Q161D, Q161E, Q161F, Q161G, Q161H, Q161I, Q161K, Q161L, Q161M, Q161N, Q161P, Q161R, Q161S, Q161T, Q161V, Q161W, Q161Y, M162A, M162F, D166E, N173D, L174V, S177E, S177W, S177Y, G205L, G205M, C209G, F213M, S214A, S214C, S214D, S214E, S214F, S214G, S214H, 52141, S214K, S214L, S214M, S214N, S214P, S214Q, S214R, S214T, S214V, S214W, S214Y, Y216A, L219F, D227E, R228E, R228Q, C230N, C230S, A232S, V271E, L274V, Y283L, G286E, Y288A, Y288C, Y288D, Y288E, Y288F, Y288G, Y288H, Y288I, Y288K, Y288L, Y288M, Y288N, Y288P, Y288Q, Y288R, Y288S, Y288T, Y288V, Y288W, V294A, V294F, V294N, Q295A, Q295C, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295K, Q295L, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, L298A, L298Q, and L298W.
In some cases, said two or more amino acid substitutions are selected from the group consisting of: Q161S_Q295F, A53T_Q295F, S214R_Q295F, Q161S_S177W, Q16S_S214R, A53T_S177W, A53T_V294A_Q295W, A53T_Q161S_V294A_Q295W, A53T_Q295W, Q161S_V294A_Q295A, A53T_Q161S_V294N_Q295W, A53T_Q161S, A53T_Q161S_Q295W, S177W_S214R, A53T_Q161S_V294A_Q295A, A53T_Q161S_V294N_Q295A, A53T_Q161S_Q295A, A53T_Q161S_V294A, Q161S_V294A_Q295W, and A53T_Q161S_V294N. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 273, 274, 276, 277, 279-290, and 293-300.
In some cases, said recombinant polypeptide converts Olivetol (O) and geranyl diphosphate (GPP) to 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 condition. In some cases, said ratio is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295G, Q295M, Q295H, Q295F, Q295C, Q295L, and Q295S. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 139, 142, 143, 144, 147, 148, and 153. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161F, Q161Y, Q161L, Q161H, Q161M, Q161I, Q161C, Q161S, Q161T, Q161D, Q161G, Q161W, and Q161N. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 127, 128, 129, 133, 134, 136, and 137. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214Q, S214H, S214E, S214N, S214F, S214L, S214I, S214M, S214K, and S214D. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 160, 161, 162, 164, 165, 166, 167, 168, 169, and 171. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Y288F, Y288L, Y288I, Y288M, Y288T, Y288C, Y288A, Y288W, Y288P, Y288S, Y288G, and Y288R. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 98, 99, 102, 103, 105, 107, 108, and 109-117.
In some cases, said at least one amino acid substitution to SEQ ID NO: 1 comprises two or more amino acid substitution to SEQ ID NO: 1 selected from the group consisting of:
(a) A17T, C25V, Q38G, V49A, V49L, V49S, A53C, A53D, A53E, A53F, A53G, A53H, A53I, A53K, A53L, A53M, A53N, A53P, A53Q, A53R, A53S, A53T, A53V, A53W, A53Y, M106E, A108G, E112D, E112G, K118N, K118Q, K119A, K119D, Y121W, F123A, F123H, F123W, Q161A, Q161C, Q161D, Q161E, Q161F, Q161G, Q161H, Q161I, Q161K, Q161L, Q161M, Q161N, Q161P, Q161R, Q161S, Q161T, Q161V, Q161W, Q161Y, M162A, M162F, D166E, N173D, L174V, S177E, S177W, S177Y, G205L, G205M, C209G, F213M, S214A, S214C, S214D, S214E, S214F, S214G, S214H, 52141, S214K, 5214L, S214M, S214N, S214P, S214Q, S214R, S214T, S214V, S214W, S214Y, Y216A, L219F, D227E, R228E, R228Q, C230N, C230S, A232S, V271E, L274V, Y283L, G286E, Y288A, Y288C, Y288D, Y288E, Y288F, Y288G, Y288H, Y288I, Y288K, Y288L, Y288M, Y288N, Y288P, Y288Q, Y288R, Y288S, Y288T, Y288V, Y288W, V294A, V294F, V294N, Q295A, Q295C, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295K, Q295L, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, L298A, L298Q, and L298W; or
(b) A53T, Q161S, V294A, and Q295A; A53T, Q161S, and Q295A; A53T, Q161S, and V294A; A53T, Q161S, V294N, and Q295A; A53T, Q161S, and V294N; Q161S, V294A, and Q295A; A53T and V294A; A53T, Q161S, and Q295W; A53T, V294A, and Q295A; A53T, Q161S, V294N, and Q295W; A53T, Q161S, V294A, and Q295W; Q161S and Q295L; A53T and Q161S; Q161S, V294A, and Q295W; Q161S and Q295F; A53T and Q295W; A53T and Q295A; A53T, V294A, and Q295W; A53T and Q295F; A53T and S177W; S177W and Q295A; Q161S and S177W; S214R and Q295F; A53T and 5214R; Q165 and 5214R; Q161S and Q295L; A53T and Q161S; Q161S and Q295F; A53T and Q295F; and A53T and V294A. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 275-294; and 296-299.
In some cases, said recombinant polypeptide converts O and geranyl diphosphate (GPP) to CBG and 5-GO, and wherein said recombinant polypeptide produces a ratio of an amount of 5-GO to a combined amount of CBG and 5-GO that is higher than wild-type ORF2 enzyme under the same condition. In some cases, said ratio is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295Y, Q295N, Q295D, Q295W, Q295T, Q295V, Q295P, Q295I, Q295R, Q295K, and Q295E. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 140, 141, 145, 146, 149, 150, 152, 154, 155, 156, and 157. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161R, Q161K, and Q161P. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 126, 130, and 132. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214W, S214V, S214T, S214R, S214C, S214P, and S214G. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 159, 163, 170, 172, 174, 175, and 176. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Y288N, Y288H, Y288D, Y288K, and Y288E. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 100, 101, 104, 106, and 109. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of V49A and Q295L; V49A and S214R; and S177W and S214R. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 288, 291, and 292.
In some cases, said recombinant polypeptide converts O and geranyl diphosphate (GPP) to CBG and 5-GO, and wherein said recombinant polypeptide produces a combined amount of CBG and 5-GO that is higher than wild-type ORF2 enzyme under the same condition. In some cases, said combined amount is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295H, Q295M, Q295G, Q295F, Q295W, and Q295D. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 140, 142, 143, 144, 148, and 156. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161F, Q161Y, Q161L, Q161H, Q161I, Q161M, Q161C, Q161T, Q161S, Q161D, Q161G, Q161R, Q161N, and Q161W. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 127, 128, 129, 132, 133, 134, 136, and 137. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214Q, S214H, S214E, S214N, S214F, S214I, S214L, and S214M. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 161, 162, 164, 165, 167, 168, 169, and 171. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Y288F, Y288L, Y288I, Y288M, Y288T, Y288C, and Y288P. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 99, 102, 105, 107, 108, 110, and 114.
In some cases, said at least one amino acid substitution to SEQ ID NO: 1 comprises two or more amino acid substitutions selected from the group consisting of:
(a) A17T, C25V, Q38G, V49A, V49L, V49S, A53C, A53D, A53E, A53F, A53G, A53H, A53I, A53K, A53L, A53M, A53N, A53P, A53Q, A53R, A53S, A53T, A53V, A53W, A53Y, M106E, A108G, E112D, E112G, K118N, K118Q, K119A, K119D, Y121W, F123A, F123H, F123W, Q161A, Q161C, Q161D, Q161E, Q161F, Q161G, Q161H, Q161I, Q161K, Q161L, Q161M, Q161N, Q161P, Q161R, Q161S, Q161T, Q161V, Q161W, Q161Y, M162A, M162F, D166E, N173D, L174V, S177E, S177W, S177Y, G205L, G205M, C209G, F213M, S214A, S214C, S214D, S214E, S214F, S214G, S214H, S214I, S214K, S214L, S214M, S214N, S214P, S214Q, S214R, S214T, S214V, S214W, S214Y, Y216A, L219F, D227E, R228E, R228Q, C230N, C230S, A232S, V271E, L274V, Y283L, G286E, Y288A, Y288C, Y288D, Y288E, Y288F, Y288G, Y288H, Y288I, Y288K, Y288L, Y288M, Y288N, Y288P, Y288Q, Y288R, Y288S, Y288T, Y288V, Y288W, V294A, V294F, V294N, Q295A, Q295C, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295K, Q295L, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, L298A, L298Q, and L298W; or
(b) Q161S and Q295L; A53T and Q161S; Q161S and Q295F; A53T, Q161S, V294A, and Q295A; A53T, Q161S, and Q295A; A53T and Q295F; A53T, Q161S, and V294A; A53T, Q161S, V294N, and Q295A; A53T, Q161S, and V294N; Q161S, V294A, and Q295A; A53T and V294A; A53T, Q161S, and Q295W; A53T, V294A, and Q295A; A53T, Q161S, V294N, and Q295W; A53T, Q161S, V294A, and Q295W; Q161S, V294A, and Q295W; A53T and S177W; A53T and Q295W; A53T and Q295W; A53T and Q295A; A53T, V294A, and Q295W; S177W and Q295A; and Q161S and S177W.
In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 214, 224, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 289, 290, 293, 296, and 297. In some cases, said recombinant polypeptide converts O and geranyl diphosphate (GPP) to 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. In some cases, said CBGVA production potential is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295H, Q295M, Q295G, Q295F, Q295W, and Q295C. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 139, 142, 143, 144, 148, and 156. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161F, Q161Y, Q161L, Q161H, Q161M, Q161I, Q161C, Q161S, Q161T, Q161D, Q161G, and Q161W. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 127, 128, 133, 134, 136, and 137. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of S214Q, S214H, S214E, S214N, S214F, 52141, S214L, and S214M. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 161, 162, 164, 165, 167, 168, 169, and 171. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Y288F, Y288L, Y288I, Y288M, Y288T, Y288C, and Y288W. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 99, 102, 105, 107, 108, 114, and 116.
In some cases, said at least one amino acid substitution to SEQ ID NO: 1 comprises two or more amino acid substitutions selected from the group consisting of:
(a) A17T, C25V, Q38G, V49A, V49L, V49S, A53C, A53D, A53E, A53F, A53G, A53H, A53I, A53K, A53L, A53M, A53N, A53P, A53Q, A53R, A53S, A53T, A53V, A53W, A53Y, M106E, A108G, E112D, E112G, K118N, K118Q, K119A, K119D, Y121W, F123A, F123H, F123W, Q161A, Q161C, Q161D, Q161E, Q161F, Q161G, Q161H, Q161I, Q161K, Q161L, Q161M, Q161N, Q161P, Q161R, Q161S, Q161T, Q161V, Q161W, Q161Y, M162A, M162F, D166E, N173D, L174V, S177E, S177W, S177Y, G205L, G205M, C209G, F213M, S214A, S214C, S214D, S214E, S214F, S214G, S214H, 52141, S214K, S214L, S214M, S214N, S214P, S214Q, S214R, S214T, S214V, S214W, S214Y, Y216A, L219F, D227E, R228E, R228Q, C230N, C230S, A232S, V271E, L274V, Y283L, G286E, Y288A, Y288C, Y288D, Y288E, Y288F, Y288G, Y288H, Y288I, Y288K, Y288L, Y288M, Y288N, Y288P, Y288Q, Y288R, Y288S, Y288T, Y288V, Y288W, V294A, V294F, V294N, Q295A, Q295C, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295K, Q295L, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, L298A, L298Q, and L298W; or
(b) A53T_Q161S_V294A_Q295W, A53T_Q161S_V294A, Q161S_Q295L, A53T_Q295F, A53T_Q295A, A53T_Q161S_V294N, Q161S_Q295F, A53T_Q161S_Q295W, A53T_Q161S, A53T_S177W, Q161S_V294A_Q295W, A53T_V294A, A53T_V294A_Q295W, S177W_Q295A, Q161S_S177W, and A53T_S214R.
In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 224, 272, 273, 275, 280, 282, 283, 284, 285, 287, 289, 290, 293, 294, 296, and 297. In some cases, said recombinant polypeptide converts O and geranyl diphosphate (GPP) to CBG and 5-GO, and wherein said recombinant polypeptide has a 5-GO production potential that is higher than wild-type ORF2 enzyme under the same condition. In some cases, said 5-GO production potential is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% higher than wild-type ORF2 enzyme under the same condition. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q295H, Q295W, and Q295F. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 142, 144, and 156. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of Q161L, Q161Y, Q161F, Q161I, Q161C, Q161M, Q161T, Q161H, Q161S, Q161R, Q161G, Q161N, and Q161D. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 127, 128, 129, 132, 133, 134, and 137. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is S214E, 52141, S214T, S214F, S214M, S214L, S214G, S214N, S214V, S214C, S214D, and S214Y. In some cases, said amino acid sequence is SEQ ID NO: 159, 160, 161, 162, 163, 165, 167, 168, 169, 174, 175, and 177. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is Y288F and Y288L. In some cases, said amino acid sequence is SEQ ID NO: 102 and 107.
In another aspect, the disclosure provides recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts olivetolic acid (OA) and geranyl diphosphate (GPP) to cannabigerolic acid (CBGA) and 5-geranyl olivetolate (5-GOA), and wherein said recombinant polypeptide produces a ratio of an amount of CBGA to a combined amount of CBGA and 5-GOA that is higher than wild-type ORF2 enzyme under the same condition.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts olivetolic acid (OA) and geranyl diphosphate (GPP) to cannabigerolic acid (CBGA) and 5-geranyl olivetolate (5-GOA), and wherein said recombinant polypeptide produces a ratio of an amount of 5-GOA to a combined amount of CBGA and 5-GOA that is higher than wild-type ORF2 enzyme under the same condition.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts olivetolic acid (OA) and geranyl diphosphate (GPP) to cannabigerolic acid (CBGA) and 5-geranyl olivetolate (5-GOA), and wherein said recombinant polypeptide produces a combined amount of CBGA and 5-GOA that is higher than wild-type ORF2 enzyme under the same condition.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts olivetolic acid (OA) and geranyl diphosphate (GPP) to cannabigerolic acid (CBGA) and 5-geranyl olivetolate (5-GOA), and wherein said recombinant polypeptide has a CBGA production potential that is higher than wild-type ORF2 enzyme under the same condition.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts olivetolic acid (OA) and geranyl diphosphate (GPP) to cannabigerolic acid (CBGA) and 5-geranyl olivetolate (5-GOA), and wherein said recombinant polypeptide has a 5-GOA production potential that is higher than wild-type ORF2 enzyme under the same condition.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts DVA and geranyl diphosphate (GPP) to 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 condition.
In another aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts DVA and geranyl diphosphate (GPP) to CBGVA and 5-GDVA, and wherein said recombinant polypeptide produces a ratio of an amount of 5-GDVA to a combined amount of CBGVA and 5-GDVA that is higher than wild-type ORF2 enzyme under the same condition.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts DVA and geranyl diphosphate (GPP) to CBGVA and 5-GDVA, and wherein said recombinant polypeptide produces a combined amount of CBGVA and 5-GDVA that is higher than wild-type ORF2 enzyme under the same condition.
In another aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts DVA and geranyl diphosphate (GPP) to 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.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts DVA and geranyl diphosphate (GPP) to CBGVA and 5-GDVA, and wherein said recombinant polypeptide has a 5-GDVA production potential that is higher than wild-type ORF2 enzyme under the same condition.
In another aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts Olivetol (O) and geranyl diphosphate (GPP) to 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 condition.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts Olivetol (O) and geranyl diphosphate (GPP) to CBG and 5-GO, and wherein said recombinant polypeptide produces a ratio of an amount of 5-GO to a combined amount of CBG and 5-GO that is higher than wild-type ORF2 enzyme under the same condition.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts Olivetol (O) and geranyl diphosphate (GPP) to CBG and 5-GO, and wherein said recombinant polypeptide produces a combined amount of CBG and 5-GO that is higher than wild-type ORF2 enzyme under the same condition.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts Olivetol (O) and geranyl diphosphate (GPP) to 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.
In one aspect, the disclosure provides a recombinant polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, wherein said recombinant polypeptide converts Olivetol (O) and geranyl diphosphate (GPP) to CBG and 5-GO, and wherein said recombinant polypeptide has a 5-GO production potential that is higher than wild-type ORF2 enzyme under the same condition.
In some cases, said amino acid sequence has at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some cases, said at least one amino acid substitution comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions to SEQ ID NO: 1. In some cases, said at least one amino acid substitution is located on a position chosen from the group consisting of 1-46, 48-63, 65-109, 111-120, 122, 124, 125, 127-160, 162-174, 176, 178-213, 215, 217-287, 289-293, and 296-307. In some cases, said amino acid sequence comprises said at least one amino acid substitution to SEQ ID NO: 1 at a position selected from the group consisting of A17, C25, Q38, V49, A53, M106, A108, E112, K118, K119, Y121, F123, Q161, M162, D166, N173, L174, S177, G205, C209, F213, S214, Y216, L219, D227, R228, C230, A232, V271, L274, Y283, G286, Y288, V294, Q295, and L298. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of A17T, C25V, Q38G, V49A, V49L, V49S, A53C, A53D, A53E, A53F, A53G, A53H, A53I, A53K, A53L, A53M, A53N, A53P, A53Q, A53R, A53S, A53T, A53V, A53W, A53Y, M106E, A108G, E112D, E112G, K118N, K118Q, K119A, K119D, Y121W, F123A, F123H, F123W, Q161A, Q161C, Q161D, Q161E, Q161F, Q161G, Q161H, Q161I, Q161K, Q161L, Q161M, Q161N, Q161P, Q161R, Q161S, Q161T, Q161V, Q161W, Q161Y, M162A, M162F, D166E, N173D, L174V, S177E, S177W, S177Y, G205L, G205M, C209G, F213M, S214A, S214C, S214D, S214E, S214F, S214G, S214H, S214I, S214K, S214L, S214M, S214N, S214P, S214Q, S214R, S214T, S214V, S214W, S214Y, Y216A, L219F, D227E, R228E, R228Q, C230N, C230S, A232S, V271E, L274V, Y283L, G286E, Y288A, Y288C, Y288D, Y288E, Y288F, Y288G, Y288H, Y288I, Y288K, Y288L, Y288M, Y288N, Y288P, Y288Q, Y288R, Y288S, Y288T, Y288V, Y288W, V294A, V294F, V294N, Q295A, Q295C, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295K, Q295L, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, L298A, L298Q, and L298W. In some cases, the recombinant polypeptide comprises any one of the recombinant polypeptides disclosed herein.
In some aspects, the disclosure provides a nucleic acid molecule, comprising a nucleotide sequence encoding any one of the recombinant polypeptides disclosed herein, or a codon degenerate nucleotide sequence thereof.
In one aspect, the disclosure provides a nucleic acid molecule, comprising a nucleotide sequence with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1-198 or a codon degenerate nucleotide sequence thereof. In some cases, the nucleotide sequence comprises at least 500, 600, 700, 800, or 900 nucleotides. In some cases, said nucleic acid molecule is isolated and purified.
In one aspect, the disclosure provides a cell vector, construct or expression system comprising any or more of the nucleic acid molecules disclosed herein.
In one aspect, the disclosure provides a cell, comprising said cell vector, construct or expression system disclosed herein. In some cases, said cell is a bacteria, yeast, insect, mammalian, fungi, vascular plant, or non-vascular plant cell. In some cases, said cell is a microalgae cell. In some cases, said cell is an E. coli cell.
In one aspect, the disclosure provides a plant comprising any cell disclosed herein. In some cases, the plant is a terrestrial plant.
In one aspect, the disclosure provides a method of producing a prenylation product, comprising: contacting said recombinant polypeptide of any one of claims 1-198 with a substrate and a donor, thereby producing said prenylation product. In some cases, said substrate comprises olivetolic acid (OA), divarinolic acid (DVA), olivetol (O), resveratrol, piceattanol and related stilbenes, naringenin, apigenin and related flavanones and flavones, respectively, Isoliquiritigenin, 2′-O-methylisoliquiritigenin and related chalcones, catechins and epi-catechins of all possible stereoisomers, biphenyl compounds such as 3,5-dihydroxy-biphenyl, benzophenones such as phlorobenzophenone, isoflavones such as biochanin A, genistein, daidzein, 2,4-dihydroxybenzoic acid, 1,3-benzenediol, 2,4-dihydroxy-6-methylbenzoic acid; 1,3-Dihydroxy-5-methylbenzene; 2,4-Dihydroxy-6-aethyl-benzoesaeure; 5-ethylbenzene-1,3-diol 2,4-dihydroxy-6-propylbenzoic acid; 5-propylbenzene-1,3-diol; 2-butyl-4,6-dihydroxybenzoic acid; 5-butylbenzene-1,3-diol; 2,4-dihydroxy-6-pentyl-benzoic acid; 5-pentylbenzene-1,3-diol; 5-hexylbenzene-1,3-diol; 2-heptyl-4,6-dihydroxy-benzoic acid; 5-heptylbenzene-1,3-diol; 5-Dodecylbenzene-1,3-diol; 5-nonadecylbenzene-1,3-diol; 1,3-Benzenediol; 3,4′,5-Trihydroxystilbene; 4′5-Tetrahydroxystilbene; 1,2-Diphenylethylene; 2-Phenylbenzopyran-4-one; 2-Phenylchroman-4-one; 1,3-benzenediol; 5,7,4′-Trihydroxyflavone; (E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one; 4,4′-dihydroxy-2′-methoxychalcone; 1,3-Diphenylpropenone; (2R,3 S)-2-(3,4-Dihydroxyphenyl)chroman-3,5,7-triol; (2R,3R)-2-(3,4-Dihydroxyphenyl)-3,5,7-chromanetriol; Phenylbenzene; 5-Phenylresorcinol; diphenylmethanone; 3-phenyl-4H-chromen-4-one; 5,7-Dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one; 4′,5,7-Trihydroxyisoflavone; 4′,7-Dihydroxyisoflavone; 4-Hydroxy-6-methyl-2H-pyran-2-one; 1,6-DHN; or any combination thereof. In some cases, said substrate comprises olivetolic acid (OA), olivetol (O), or divarinic acid (DVA). In some cases, said donor comprises dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), or farnesyl pyrophosphate (FPP). In some cases, said substrate comprises olivetolic acid (OA) and said donor comprises geranyl pyrophosphate (GPP), and wherein said prenylation product comprises CBGA, 5-GOA, or a prenylated product on any other position on an aromatic ring of said substrate.
In some cases, said prenylation product comprises CBGA and 5-GOA, and wherein said recombinant polypeptide produces a ratio of an amount of CBGA to a combined amount of CBGA and 5-GOA that is higher than wild-type ORF2 enzyme under the same acid condition. In some cases, said prenylation product comprises CBGA and 5-GOA, and wherein said recombinant polypeptide produces a ratio of an amount of 5-GOA to a combined amount of CBGA and 5-GOA that is higher than wild-type ORF2 enzyme under the same acid condition. In some cases, said prenylation product comprises CBGA and 5-GOA, and wherein said recombinant polypeptide produces a combined amount of CBGA and 5-GOA that is higher than wild-type ORF2 enzyme under the same acid condition. In some cases, said prenylation product comprises CBGA and 5-GOA, and wherein said recombinant polypeptide has a CBGA production potential that is higher than wild-type ORF2 enzyme under the same acid condition.
In some cases, said acid condition has a pH of less than 7. In some cases, the method of claim 219, wherein said acid condition has a pH of less than 6. In some cases, said acid condition has a pH of about 5.3. In some cases, said substrate comprises olivetol (O) and said donor comprises geranyl pyrophosphate (GPP), and wherein said prenylation product comprises CBG, 5-GO or a prenylated product on any other position on an aromatic ring of said substrate. In some cases, said substrate comprises divarinic acid (DVA) and said donor comprises geranyl pyrophosphate (GPP), and wherein said prenylation product comprises CBGVA, 5-GDVA or a prenylated product on any other position on an aromatic ring of said substrate.
In some cases, said contacting comprises contacting said substrate and said donor with said recombinant polypeptide in a cell. In some cases, said cell is a bacteria, yeast, insect, mammalian, fungi, microalgae, or plant cell. In some cases, said contacting comprises contacting said substrate and said donor with said recombinant polypeptide in a cell-free reaction. In some cases, said contacting comprises contacting said substrate and said donor with said recombinant polypeptide in a reaction, wherein said recombinant polypeptide is secreted by a cell into a media before the contacting.
In yet another aspect, the disclosure provides a composition, comprising a prenylation product and any one recombinant polypeptide disclosed herein. In some cases, said prenylation product comprises CBGA, CBG, CBGVA or any combination thereof. In some cases, said prenylation product comprises 5-GOA, 5-GO. In some cases, said prenylation product comprises 5-GDVA.
In one aspect, the disclosure provides a method of screening for an ORF2 mutant, comprising: providing two or more ORF2 mutants; contacting each of said two or more ORF2 mutants with a substrate and a donor, thereby producing at least two prenylation products under the same condition; measuring a ratio of an amount of one of said at least two prenylation products to a combined amount of said at least two prenylation products for each of said two or more ORF2 mutants; and identifying said ORF2 mutant with the highest ratio among said two or more ORF2 mutants. In some cases, each of said two or more ORF2 mutants comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1. In some cases, said two or more ORF2 mutants comprises different amino acid sequences. In some cases, each of said amino acid sequences comprises at least one amino acid substitution to SEQ ID NO: 1. In some cases, each of said at least one amino acid substitution is located at a different amino acid position. In some cases, said at least one amino acid substitution is located on a position chosen from the group consisting of amino acid positions 1-46, 48-63, 65-109, 111-120, 122, 124, 125, 127-160, 162-174, 176, 178-213, 215, 217-287, 289-293, and 296-307. In some cases, said at least one amino acid substitution is located on a position chosen from the group consisting of amino acid positions 17, 25, 38, 49, 53, 106, 108, 112, 118, 119, 121, 123, 161, 162, 166, 173, 174, 177, 205, 209, 213, 214, 216, 219, 227, 228, 230, 232, 271, 274, 283, 286, 288, 294, 295, and 298.
In some cases, said substrate comprises olivetolic acid (OA), divarinolic acid (DVA), olivetol (O), resveratrol, piceattanol and related stilbenes, naringenin, apigenin and related flavanones and flavones, respectively, Isoliquiritigenin, 2′-O-methylisoliquiritigenin and related chalcones, catechins and epi-catechins of all possible stereoisomers, biphenyl compounds such as 3,5-dihydroxy-biphenyl, benzophenones such as phlorobenzophenone, isoflavones such as biochanin A, genistein, daidzein, 2,4-dihydroxybenzoic acid, 1,3-benzenediol, 2,4-dihydroxy-6-methylbenzoic acid; 1,3-Dihydroxy-5-methylbenzene; 2,4-Dihydroxy-6-aethyl-benzoesaeure; 5-ethylbenzene-1,3-diol 2,4-dihydroxy-6-propylbenzoic acid; 5-propylbenzene-1,3-diol; 2-butyl-4,6-dihydroxybenzoic acid; 5-butylbenzene-1,3-diol; 2,4-dihydroxy-6-pentyl-benzoic acid; 5-pentylbenzene-1,3-diol; 5-hexylbenzene-1,3-diol; 2-heptyl-4,6-dihydroxy-benzoic acid; 5-heptylbenzene-1,3-diol; 5-Dodecylbenzene-1,3-diol; 5-nonadecylbenzene-1,3-diol; 1,3-Benzenediol; 3,4′,5-Trihydroxystilbene; 4'S-Tetrahydroxystilbene; 1,2-Diphenylethylene; 2-Phenylbenzopyran-4-one; 2-Phenylchroman-4-one; 1,3-benzenediol; 5,7,4′-Trihydroxyflavone; (E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one; 4,4′-dihydroxy-2′-methoxychalcone; 1,3-Diphenylpropenone; (2R,3 S)-2-(3,4-Dihydroxyphenyl)chroman-3,5,7-triol; (2R,3R)-2-(3,4-Dihydroxyphenyl)-3,5,7-chromanetriol; Phenylbenzene; 5-Phenylresorcinol; diphenylmethanone; 3-phenyl-4H-chromen-4-one; 5,7-Dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one; 4′,5,7-Trihydroxyisoflavone; 4′,7-Dihydroxyisoflavone; 4-Hydroxy-6-methyl-2H-pyran-2-one; 1,6-DHN; or any combination thereof.
In some cases, said substrate comprises olivetolic acid (OA), olivetol (O), or divarinic acid (DVA). In some cases, said donor comprises dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), or farnesyl pyrophosphate (FPP). In some cases, said substrate comprises olivetolic acid (OA) and said donor comprises geranyl pyrophosphate (GPP), and wherein said at least two prenylation products comprises CBGA and 5-GOA. In some cases, said substrate comprises olivetol (O) and said donor comprises geranyl pyrophosphate (GPP), and wherein said at least two prenylation products comprises CBG and 5-GO. In some cases, said substrate comprises divarinic acid (DVA) and said donor comprises geranyl pyrophosphate (GPP), and said at least two prenylation products comprises CBGVA and 5-GDVA.
In one aspect, the disclosure provides methods of making a prenyltransferase mutant, comprising: aligning a crystal structure of a prenyltransferase with a crystal structure of wild-type ORF2 enzyme; identifying an amino acid position on said prenyltransferase corresponding to any one of amino acid positions 17, 25, 38, 49, 53, 106, 108, 112, 118, 119, 162, 166, 173, 174, 205, 209, 213, 219, 227, 228, 230, 232, 271, 274, 283, 286, 288, and 298 on said wild-type ORF2 enzyme, based on alignment of said crystal structures; and substitute said amino acid position on said prenyltransferase with a different amino acid, thereby generating said prenyltransferase mutant.
In one aspect, the disclosure provides a method of making a prenyltransferase mutant, comprising: aligning an amino acid sequence of a prenyltransferase with an amino acid sequence of wild-type ORF2 enzyme; identifying an amino acid position on said prenyltransferase corresponding to any one of amino acid positions 17, 25, 38, 49, 53, 106, 108, 112, 118, 119, 162, 166, 173, 174, 205, 209, 213, 219, 227, 228, 230, 232, 271, 274, 283, 286, 288, and 298 on said wild-type ORF2 enzyme, based on alignment of said amino acid sequences; and substitute said amino acid position on said prenyltransferase with a different amino acid, thereby generating said prenyltransferase mutant.
In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of A53T, A53I, A53V, A53L, A53M, A53K, A53S, A53C, and A53Q. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 179, 185, 186, 187, 188, 191, 193, 194, and 195. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of A53L, A53K, A53V, A53M, A53Q, A53T, A53S, A53C, and A53I. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 179, 185, 186, 187, 188, 191, 193, 194, and 195. In some cases, said at least one amino acid substitution to SEQ ID NO: 1 is selected from the group consisting of A53L, A53T, A53V, A53I, A53M, A53Q, A53K, and A53S. In some cases, said amino acid sequence is selected from the group consisting of SEQ ID NOs: 185, 186, 187, 188, 191, 193, 194, and 195.
As used herein, and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a protein” can refer to one protein or to mixtures of such protein, and reference to “the method” includes reference to equivalent steps and/or processes known to those skilled in the art, and so forth.
As used herein, the term “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%. For example, “about 100” encompasses 90 and 110.
The term “wild type”, abbreviated as “WT”, is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene, protein, or characteristic as it occurs in nature as distinguished from mutant or variant forms. For example, a WT protein is the typical form of that protein as it occurs in nature.
The term “mutant protein” is a term of the art understood by skilled persons and refers to a protein that is distinguished from the WT form of the protein on the basis of the presence of amino acid modifications, such as, for example, amino acid substitutions, insertions and/or deletions.
Amino acid modifications may be amino acid substitutions, amino acid deletions and/or amino acid insertions. Amino acid substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions. A conservative replacement (also called a conservative mutation, a conservative substitution or a conservative variation) is an amino acid replacement in a protein that changes a given amino acid to 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 by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another; or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. Other illustrative examples of conservative substitutions include the changes of: 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 praline; 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.
Amino acid substitution, interchangeably referred to as amino acid replacement, at a specific position on the protein sequence is denoted herein in the following manner: “one letter code of the WT amino acid residue-amino acid position-one letter code of the amino acid residue that replaces this WT residue”. For example, an ORF2 polypeptide which is a Q295F mutant refers to an ORF2 polypeptide in which the wild type residue at the 295th amino acid position (Q or glutamine) is replaced with F or phenylalanine. Some mutants have more than one amino acid substitutions, for example, mutant L174V_S177E refers to an ORF2 polypeptide in which the wild type residue at the 174th amino acid position (L or leucine) is replaced with V or valine; and the wild type residue at the 177th amino acid position (S or serine) is replaced with E or glutamic acid.
The modified peptides can be chemically synthesized, or the isolated gene can be site-directed mutagenized, or a synthetic gene can be synthesized and expressed in bacteria, yeast, baculovirus, tissue culture, and the like.
As used herein, “total prenylated products” produced refers to the sum of nMols of the various prenylated products produced by an enzyme in a set period of time. For instance, when OA is used as a substrate and GPP is used as a donor, then the “total prenylated products” refers to a sum of the nMol of CBGA and the nMol of 5-GOA produced by the prenyltranferase enzyme ORF2 in a set period of time.
As used herein, “% prenylated product 1” within total prenylated products is calculated using the equation: nMol of prenylated product 1/[nMol of total prenylated products]. For example, “% CBGA” is calculated using the equation: nMol of CBGA/[nMol of CBGA+5-GOA]. Also, as an example, “%5-GOA” within prenylated products is calculated using the equation: nMol of 5-GOA/[nMol of CBGA+5-GOA].
As used herein, % enzymatic activity of an ORF2 mutant is calculated using the equation: total prenylated products produced by a mutant/total prenylated products produced by wild-type ORF2. For example, wild-type ORF2 has 100% enzyme activity.
As used herein, the production or production potential of a prenylated product 1 is calculated using the formula: % product 1 among total prenylated products * % enzymatic activity. For example, “CBGA production potential” (used interchangeably with “CBGA production”) is calculated using the equation: % CBGA among total prenylated products * % enzymatic activity. Also, as an example, “5-GOA production potential” (used interchangeably with “5-GOA production”) is calculated using the equation: %5-GOA among total prenylated products * enzymatic activity.
A “vector” is used to transfer genetic material into a target cell. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, adenoviruses, lentiviruses, and adeno-associated viruses). In embodiments, a viral vector may be replication incompetent. Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of components, e.g. nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e. the entire reference sequence or a smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100. Comparison of sequences to determine percent identity can be accomplished by a number of well-known methods, including for example by using mathematical algorithms, such as, for example, those in the BLAST suite of sequence analysis programs.
The biosynthesis of cannabinoids often starts with the short-chain fatty acid, hexanoic acid. Initially, the fatty acid is converted to its coenzyme A (CoA) form by the activity of an acyl activating enzyme. Subsequently, olivetolic acid (OA) is biosynthesized by the action of a type III polyketide synthase (PKS), and, in some cases, a polyketide cyclase (olivetolic acid cyclase [OAC]).
A geranyl diphosphate:olivetolate geranyltransferase, named cannabigerolic acid synthase (CBGAS), is responsible for the C-alkylation by geranyl diphosphate (GPP) to CBGA. Subsequently, the monoterpene moiety of CBGA is often stereoselectively cyclized by three different enzymes cannabichromenic acid synthase (CBCAS), cannabidiolic acid synthase (CBDAS) and tetrahydrocannabinolic acid synthase (THCAS) to synthesize cannabichromenic acid (CBCA), cannabidiolic acid (CBDA) and Δ9-THCA, respectively.
The central precursor for cannabinoid biosynthesis, CBGA, is synthesized by the aromatic prenyltransferase CBGAS by the condensation of GPP and OA. In considering the biosynthesis of cannabinoids in a heterologous system, one major challenge is that CBGAS (e.g. CsPT1 and CsPT4) is an integral membrane protein, making high titer of functional expressed protein in E. coli and other heterologous systems unlikely. Besides the integral membrane prenyltransferases found in plants, soluble prenyltransferases are found in fungi and bacteria. For instance, Streptomyces sp. strain CL190 produces a soluble prenyltransferase NphB or ORF2, which is specific for GPP as a prenyl donor and exhibits broad substrate specificity towards aromatic substrates. When expressed in E. coli, ORF2 of SEQ ID NO:2 is as a 33 kDa soluble, monomeric protein having 307 residues. Further details about ORF2 and other aromatic prenyltransferases may be found in U.S. Pat. Nos. 7,361,483; 7,544,498; and 8,124,390, each of which is incorporated herein by reference in its entirety for all purposes.
ORF2 is a potential alternative to replace the native CBGAS in a biotechnological production of cannabinoids and other prenylated aromatic compounds. However, the wild type ORF2 enzyme produces a large amount of 5-geranyl olivetolate (5-GOA) and only a minor amount of CBGA, the latter of which is the desired product for cannabinoid biosynthesis.
This disclosure provides ORF2 mutants, engineered by the inventors to produce higher ratios of CBGA to 5-GOA (and other compounds), as compared to WT ORF2; as well as ORF2 mutants which have been engineered to catalyze reactions using a desired substrate and to produce higher amounts of a desired product.
The disclosure provides recombinant polypeptides comprising an amino acid sequence with at least about 70% identity to the amino acid sequence of WT ORF2 (set forth in SEQ ID NO: 1). In some aspects, the polypeptides disclosed herein may have a sequence identity of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% identity to the amino acid sequence of WT ORF2 (set forth in SEQ ID NO: 1). In some aspects, the mutant ORF2 polypeptides disclosed herein may comprise a modification at one or more amino acids, as compared to the WT ORF2 sequence. In some aspects, the mutant ORF2 polypeptides disclosed herein may comprise a modification at 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, or 36 amino acids, as compared to the WT ORF2 sequence.
In some aspects, mutant ORF2 polypeptides disclosed herein comprise a modification in one or more amino acid residues selected from the group consisting of the following amino acid residues, A17, C25, Q38, V49, A53, M106, A108, E112, K118, K119, Y121, F123, Q161, M162, D166, N173, L174, S177, G205, C209, F213, S214, Y216, L219, D227, R228, C230, A232, V271, L274, Y283, G286, Y288, V294, Q295, and L298 of the WT ORF2 polypeptide. For instance, the mutant ORF2 polypeptides disclosed herein may comprise an amino acid modification at 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, or 36 amino acids selected from the group consisting of the following amino acid residues, A17, C25, Q38, V49, A53, M106, A108, E112, K118, K119, Y121, F123, Q161, M162, D166, N173, L174, S177, G205, C209, F213, S214, Y216, L219, D227, R228, C230, A232, V271, L274, Y283, G286, Y288, V294, Q295, and L298 of the WT ORF2 polypeptide.
In some aspects, the mutant ORF2 polypeptides disclosed herein may comprise an amino acid substitution of at least one amino acid residue selected from the group consisting of A17, C25, Q38, V49, A53, M106, A108, E112, K118, K119, Y121, F123, Q161, M162, D166, N173, L174, S177, G205, C209, F213, S214, Y216, L219, D227, R228, C230, A232, V271, L274, Y283, G286, Y288, V294, Q295, and L298. For instance, the mutant ORF2 polypeptides disclosed herein may comprise an amino acid substitution of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, or 36 amino acids selected from the group consisting of A17, C25, Q38, V49, A53, M106, A108, E112, K118, K119, Y121, F123, Q161, M162, D166, N173, L174, S177, G205, C209, F213, S214, Y216, L219, D227, R228, C230, A232, V271, L274, Y283, G286, Y288, V294, Q295, and L298.
In some aspects, the mutant ORF2 polypeptides disclosed herein comprise an amino acid sequence comprising at least one amino acid substitution, as compared to the amino acid sequence of WT ORF2, wherein the at least one amino acid substitution does not comprise an alanine substitution on an amino acid residue selected from the group consisting of 47, 64, 110, 121, 123, 126, 161, 175, 177, 214, 216, 288, 294 and 295.
In some aspects, the mutant ORF2 polypeptides disclosed herein comprise an amino acid sequence comprising at least one amino acid substitution, as compared to the amino acid sequence of WT ORF2, wherein at least one amino acid substitution is at a position selected from the group consisting of 1-46, 48-63, 65-109, 111-120, 122, 124, 125, 127-160, 162-174, 176, 178-213, 215, 217-287, 289-293, 296-307, on WT-ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein comprise an amino acid sequence with at least about 70% identity (for instance, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% identity, inclusive of all values and subranges therebetween) to the amino acid sequence of SEQ ID Nos 2-300. In some aspects, the mutant ORF2 polypeptides disclosed herein comprise the amino acid sequence of SEQ ID Nos 2-300. In some aspects, the mutant ORF2 polypeptides disclosed herein consist of the amino acid sequence of SEQ ID Nos 2-300.
In some aspects, the mutant ORF2 polypeptides disclosed herein catalyze a reaction using GPP as a donor of the prenyl group. In some aspects, the mutant ORF2 polypeptides disclosed herein catalyze a reaction using any known substrate of ORF2, or a homologue thereof. In some aspects, the substrate is selected from the group consisting of olivetolic acid (OA), divarinolic acid (DVA) and olivetol (O). In some aspects, the substrate is any natural or synthetic phenolic acids with a 1,3-dihydroxyl motif, alternatively a resorcinol ring including but not limited to resveratrol, piceattanol and related stilbenes, naringenin, apigenin and related flavanones and flavones, respectively, Isoliquiritigenin, 2′-O-methylisoliquiritigenin and related chalcones, catechins and epi-catechins of all possible stereoisomers, biphenyl compounds such as 3,5-dihydroxy-biphenyl, benzophenones such as phlorobenzophenone, isoflavones such as biochanin A, genistein, and daidzein. For instance, the substrate may be any substrate listed in Tables A and B; and
In some aspects, the products of ORF2 prenylation may further serve as substrates for ORF2. Therefore, the substrate may also be any product of an ORF2 prenylation reaction.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a higher amount of total nMol of prenylated products than WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce an amount of total nMol of prenylated products that is about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than the amount of total nMol of prenylated products produced by WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein have an enzymatic activity higher than WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein have an activity that is about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than the enzymatic activity of WT ORF2.
In some aspects the mutant ORF2 polypeptides disclosed herein catalyze the reaction converting OA and GPP to CBGA and 5-GOA.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a higher amount of total nMol of prenylated products than WT ORF2, wherein the mutant ORF2 polypeptides use OA as substrate and GPP as donor; and the prenylated products are CBGA and 5-GOA. In some aspects, the mutant ORF2 polypeptides disclosed herein produce an amount of total nMol of prenylated products that is about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than the amount of total nMol of prenylated products produced by WT ORF2, wherein the prenylated products are CBGA and 5-GOA.
The ratio of CBGA to 5-GOA produced by WT ORF2 is about 20:80. In some aspects, the mutant ORF2 polypeptides disclosed herein produce a ratio of CBGA to 5-GOA that is higher than the ratio of CBGA to 5-GOA produced by WT ORF2 under the same conditions. In some aspects, the mutant ORF2 polypeptides disclosed herein produce a ratio of CBGA to 5-GOA that is lower than the ratio of CBGA to 5-GOA produced by WT ORF2 under the same conditions. In some embodiments, the ratio of CBGA to 5-GOA produced by the mutant ORF2 proteins of this disclosure may range from about 1:99 to up to 20:80; or more than about 20:80 to 99:1, for example about 5:95, about 10:90, about 15:85, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, or about 99:1, including all values and subranges that lie therebetween.
In some aspects, the mutant ORF2 polypeptides disclosed herein have an enzymatic activity higher than WT ORF2, wherein the mutant ORF2 polypeptides use OA as substrate and GPP as donor. In some aspects, the mutant ORF2 polypeptides disclosed herein have an activity that is about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than the enzymatic activity of WT ORF2, wherein the mutant ORF2 polypeptides use OA as substrate and GPP as donor.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a higher % of CBGA than the WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher % of CBGA than WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a lower % of CBGA than the WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce about 1% to about 100% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%), inclusive all the values and subranges that lie therebetween, lower % of CBGA than WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a higher % of 5-GOA than WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher % of 5-GOA than the WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a lower % of 5-GOA than WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce about 1% to about 100% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%), inclusive all the values and subranges that lie therebetween, lower % of 5-GOA than the WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein have a CBGA production potential that is higher than that of WT ORF2. As used herein, CBGA production potential of an enzyme is calculated by multiplying the % CBGA in total prenylated products by the % activity calculated when the mutant ORF-2 uses OA as substrate and GPP as donor. In some aspects, the mutant ORF2 polypeptides disclosed herein have a CBGA production potential that is 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than that of WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a lower % of 5-GOA than WT ORF2; and have a CBGA production potential that is higher than that of WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein have a 5-GOA production potential that is higher than that of WT ORF2. As used herein, 5-GOA production potential of an enzyme is calculated by multiplying the %5-GOA in total prenylated products by the % activity calculated when the mutant ORF-2 uses OA as substrate and GPP as donor. In some aspects, the mutant ORF2 polypeptides disclosed herein has a 5-GOA production potential that is 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than that of WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein catalyze the reaction of converting DVA and GPP to CBGVA and 5-GDVA.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a higher amount of total nMol of prenylated products than WT ORF2, wherein the mutant ORF2 polypeptides use DVA as substrate and GPP as donor; and the prenylated products are CBGVA and 5-GDVA. In some aspects, the mutant ORF2 polypeptides disclosed herein produce an amount of total nMol of prenylated products that is about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than the amount of total nMol of prenylated products produced by WT ORF2, wherein the prenylated products are CBGVA and 5-GDVA.
The ratio of CBGVA to 5-GDVA produced by WT ORF2 is about 16:84. In some aspects, the mutant ORF2 polypeptides disclosed herein produce a ratio of CBGVA to 5-GDVA that is higher than the ratio of CBGVA to 5-GDVA produced by WT ORF2 under the same conditions. In some aspects, the mutant ORF2 polypeptides disclosed herein produce a ratio of CBGVA to 5-GDVA that is lower than the ratio of CBGVA to 5-GDVA produced by WT ORF2 under the same conditions. In some embodiments, the ratio of CBGVA to 5-GDVA produced by the mutant ORF2 proteins of this disclosure may range from about 1:99 to up to about 16:84; or more than about 16:84 to about 99:1, for example about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, or about 99:1, including all values and subranges that lie therebetween.
In some aspects, the mutant ORF2 polypeptides disclosed herein have an enzymatic activity higher than WT ORF2, wherein the mutant ORF2 polypeptides use DVA as substrate and GPP as donor. In some aspects, the mutant ORF2 polypeptides disclosed herein have an activity that is about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than the enzymatic activity of WT ORF2, wherein the mutant ORF2 polypeptides use DVA as substrate and GPP as donor.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a higher % of CBGVA than the WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher % of CBGVA than WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a lower % of CBGVA than the WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce about 1% to about 100% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%), inclusive all the values and subranges that lie therebetween, lower % of CBGVA than WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a higher % of 5-GDVA than WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher % of 5-GDVA than the WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a lower % of 5-GDVA than WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce about 1% to about 100% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%), inclusive all the values and subranges that lie therebetween, lower % of 5-GDVA than the WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein have a CBGVA production potential is higher than that of WT ORF2. As used herein, CBGVA production potential of an enzyme is calculated by multiplying the % CBGVA in total prenylated products by the % activity calculated when the mutant ORF-2 uses DVA as substrate and GPP as donor. In some aspects, the mutant ORF2 polypeptides disclosed herein have a CBGVA production potential that is 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than that of WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a lower % of 5-GDVA than WT ORF2; and have a CBGVA production potential that is higher than that of WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein have a 5-GDVA production potential that is higher than that of WT ORF2. As used herein, 5-GDVA production potential of an enzyme is calculated by multiplying the %5-GDVA in total prenylated products by the % activity calculated when the mutant ORF-2 uses DVA as substrate and GPP as donor. In some aspects, the mutant ORF2 polypeptides disclosed herein has a 5-GDVA production potential that is 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than that of WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein catalyze the reaction of converting O and GPP to CBG and 5-GO. See
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a higher amount of total nMol of prenylated products than WT ORF2, wherein the mutant ORF2 polypeptides use O as substrate and GPP as donor; and the prenylated products are CBG and 5-GO. In some aspects, the mutant ORF2 polypeptides disclosed herein produce an amount of total nMol of prenylated products that is about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than the amount of total nMol of prenylated products produced by WT ORF2, wherein the prenylated products are CBG and 5-GO.
The ratio of CBG to 5-GO produced by WT ORF2 is about 62:38. In some aspects, the mutant ORF2 polypeptides disclosed herein produce a ratio of CBG to 5-GO that is higher than the ratio of CBG to 5-GO produced by WT ORF2 under the same conditions. In some aspects, the mutant ORF2 polypeptides disclosed herein produce a ratio of CBG to 5-GO that is lower than the ratio of CBG to 5-GO produced by WT ORF2 under the same conditions. In some embodiments, the ratio of CBG to 5-GO produced by the mutant ORF2 proteins of this disclosure may range from about 99:1 to up to about 62:38; or less than about 62:38 to about 1:99, for example about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, or about 99:1, including all values and subranges that lie therebetween.
In some aspects, the mutant ORF2 polypeptides disclosed herein have an enzymatic activity higher than WT ORF2, wherein the mutant ORF2 polypeptides use O as substrate and GPP as donor. In some aspects, the mutant ORF2 polypeptides disclosed herein have an activity that is about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than the enzymatic activity of WT ORF2, wherein the mutant ORF2 polypeptides use O as substrate and GPP as donor.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a higher % of CBG than the WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher % of CBG than WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a lower % of CBG than the WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce about 1% to about 100% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%), inclusive all the values and subranges that lie therebetween, lower % of CBG than WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a higher % of 5-GO than WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce about 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher % of 5-GO than the WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a lower % of 5-GO than WT ORF2. In some aspects, the mutant ORF2 polypeptides disclosed herein produce about 1% to about 100% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%), inclusive all the values and subranges that lie therebetween, lower % of 5-GO than the WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein have a CBG production potential is higher than that of WT ORF2. As used herein, CBG production potential of an enzyme is calculated by multiplying the % CBG in total prenylated products by the % activity calculated when the mutant ORF-2 uses O as substrate and GPP as donor. In some aspects, the mutant ORF2 polypeptides disclosed herein have a CBG production potential that is 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than that of WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein produce a lower % of 5-GO than WT ORF2; and have a CBG production potential that is higher than that of WT ORF2.
In some aspects, the mutant ORF2 polypeptides disclosed herein have a 5-GO production potential is higher than that of WT ORF2. As used herein, 5-GO production potential of an enzyme is calculated by multiplying the %5-GO in total prenylated products by the % activity calculated when the mutant ORF-2 uses O as substrate and GPP as donor. In some aspects, the mutant ORF2 polypeptides disclosed herein has a 5-GO production potential that is 1% to about 1000% (for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%), inclusive all the values and subranges that lie therebetween, higher than that of WT ORF2.
In some aspects, the ORF2 mutants disclosed herein have improved function at a pH range of about 3 to about 8, for instance, about 3.5, about 4, about 4.5 about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, including all values and subranges therebetween. In some aspects, the ORF2 mutants disclosed herein which have improved function at acidic pH may be used in combination with at least one other enzyme which has optimal function at acidic pH. In some aspects, the at least one enzyme is CBD synthase.
In some aspects, the mutant ORF2 polypeptides disclosed herein have improved enzymatic function (increased amount of prenylated products, increased % CBGA or 5-GOA produced, increased % activity and/or increased CBGA or 5-GOA production potential) at pH 5.3 compared to WT ORF2 at the same pH. WT ORF2 has little to no activity at this acidic pH. Therefore, in some aspects, the ORF2 mutants disclosed herein which have improved function at pH 5.3 may be used in situations where a low pH is needed to be maintained. For instance, CBD synthase has optimal pH at around 5.3. Therefore, the ORF2 mutants disclosed herein which have improved function at pH 5.3 may be used for carrying out single-pot reactions along with CBD synthase.
The disclosure provides isolated or purified polynucleotides that encode any one of the ORF2 mutant polypeptides disclosed herein. The disclosure provides polynucleotides comprising a nucleic acid sequence with at least about 80% identity (for instance, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%, and inclusive of all values and subranges therebetween) to the nucleic acid sequence set forth in [[all the DNA SEQ ID Nos.]].
The disclosure provides a vector comprising any one of the ORF2 mutant polynucleotide sequences disclosed herein.
The disclosure further provides a host cell comprising any one of the vectors disclosed herein; any one of the polynucleotides disclosed herein; or any one of the polynucleotides encoding the ORF2 mutants disclosed herein. Non-limiting examples of host cells include microbial host cells, such as, for example, bacteria, E. coli, yeast, microalgae; non-microbial hosts, such as, for example, insect cells, mammalian cell culture, plant cultures; and whole terrestrial plants. In some aspects, expression of any one of the vectors disclosed herein; any one of the polynucleotides disclosed herein; or any one of the polynucleotides encoding the ORF2 mutants disclosed herein may be done ex vivo or in vitro. In some aspects, expression of any one of the vectors disclosed herein; any one of the polynucleotides disclosed herein; or any one of the polynucleotides encoding the ORF2 mutants disclosed herein may be done in cell-free systems.
The disclosure provides methods of producing any one of the mutant ORF2 polypeptides disclosed herein, comprising culturing the host cell comprising any one of the vectors disclosed herein, in a medium permitting expression of the mutant ORF2 polypeptide, and isolating or purifying the mutant ORF2 polypeptide from the host cell.
The disclosure provides methods of producing CBGA in a host cell, comprising expressing a mutant ORF2 polypeptide, wherein the mutant ORF2 polypeptide has a CBGA production potential that is higher than that of WT ORF2, and wherein the mutant ORF2 polypeptide produces a lower % of 5-GOA than WT ORF2. In some aspects, the CBGA is produced ex vivo or in vitro. In some aspects, CBGA is produced in cell-free systems. In some aspects, the CBGA is produced in host cells, such as, for example, microbial host cells, such as, for example, bacteria, E. coli, yeast, microalgae; non-microbial hosts, such as, for example, insect cells, mammalian cells, plant cells; and whole terrestrial plants. In some aspects, the CBGA is produced in the medium of host cell culture.
The disclosure provides methods of producing 5-GOA in a host cell, comprising expressing a mutant ORF2 polypeptide, wherein the mutant ORF2 polypeptide has a 5-GOA production potential that is higher than that of WT ORF2, and wherein the mutant ORF2 polypeptide produces a lower % of CBGA than WT ORF2. In some aspects, the 5-GOA is produced ex vivo or in vitro. In some aspects, 5-GOA is produced in cell-free systems. In some aspects, the 5-GOA is produced in host cells, such as, for example, microbial host cells, such as, for example, bacteria, E. coli, yeast, microalgae; non-microbial hosts, such as, for example, insect cells, mammalian cells, plant cells; and whole terrestrial plants. In some aspects, the 5-GOA is produced in the medium of host cell culture.
The disclosure provides methods of producing CBGVA in a host cell, comprising expressing a mutant ORF2 polypeptide, wherein the mutant ORF2 polypeptide has a CBGVA production potential that is higher than that of WT ORF2, and wherein the mutant ORF2 polypeptide produces a lower % of 5-GDVA than WT ORF2. In some aspects, the CBGVA is produced ex vivo or in vitro. In some aspects, CBGVA is produced in cell-free systems. In some aspects, the CBGVA is produced in host cells, such as, for example, microbial host cells, such as, for example, bacteria, E. coli, yeast, microalgae; non-microbial hosts, such as, for example, insect cells, mammalian cells, plant cells; and whole terrestrial plants. In some aspects, the CBGVA is produced in the medium of host cell culture.
The disclosure provides methods of producing 5-GDVA in a host cell, comprising expressing a mutant ORF2 polypeptide, wherein the mutant ORF2 polypeptide has a 5-GDVA production potential that is higher than that of WT ORF2, and wherein the mutant ORF2 polypeptide produces a lower % of CBGVA than WT ORF2. In some aspects, the 5-GDVA is produced ex vivo or in vitro. In some aspects, 5-GDVA is produced in cell-free systems. In some aspects, the 5-GDVA is produced in host cells, such as, for example, microbial host cells, such as, for example, bacteria, E. coli, yeast, microalgae; non-microbial hosts, such as, for example, insect cells, mammalian cells, plant cells; and whole terrestrial plants. In some aspects, the 5-GDVA is produced in the medium of host cell culture.
The disclosure provides methods of producing CBG in a host cell, comprising expressing a mutant ORF2 polypeptide, wherein the mutant ORF2 polypeptide has a CBG production potential that is higher than that of WT ORF2, and wherein the mutant ORF2 polypeptide produces a lower % of 5-GO than WT ORF2. In some aspects, the CBG is produced ex vivo or in vitro. In some aspects, CBG is produced in cell-free systems. In some aspects, the CBG is produced in host cells, such as, for example, microbial host cells, such as, for example, bacteria, E. coli, yeast, microalgae; non-microbial hosts, such as, for example, insect cells, mammalian cells, plant cells; and whole terrestrial plants. In some aspects, the CBG is produced in the medium of host cell culture.
The disclosure provides methods of producing 5-GO in a host cell, comprising expressing a mutant ORF2 polypeptide, wherein the mutant ORF2 polypeptide has a 5-GO production potential that is higher than that of WT ORF2, and wherein the mutant ORF2 polypeptide produces a lower % of CBG than WT ORF2. In some aspects, the 5-GO is produced ex vivo or in vitro. In some aspects, 5-GO is produced in cell-free systems. In some aspects, the 5-GO is produced in host cells, such as, for example, microbial host cells, such as, for example, bacteria, E. coli, yeast, microalgae; non-microbial hosts, such as, for example, insect cells, mammalian cells, plant cells; and whole terrestrial plants. In some aspects, the 5-GO is produced in the medium of host cell culture.
It is to be understood that the description above as well as the examples that follow are intended to illustrate, and not limit, the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
All patents, patent applications, references, and journal articles cited in this disclosure are expressly incorporated herein by reference in their entireties for all purposes.
A. Construction of a Synthesized Gene Library of n=96 Orf2 Variants with Select Amino Acid Substitutions and Other Orf2 Variants.
DNA plasmids encoding the 96 “tripleton” variants of orf2 (orf2 variants) were ordered and delivered in the background of the T5 expression vector pD441-SR from DNA2.0 (now ATUM, catalog pD441-SR). The sequences for the 96 variants are described as SEQ ID NO: DNA_150247-DNA_150342. Each Orf2 variant contains a unique combination of three amino acid substitutions relative to the base construct (SEQ ID NO: DNA_consensus).
All variants aside from the tripleton parental variants were created using site directed mutagenesis with QuikChange II Site-Directed Mutagenesis Kit (Agilent catalog #200523). Standard manufacturer protocols were employed.
B. Expression and Purification of Proteins from the Synthesized Orf2 Gene Library of Orf2 Variants.
DNA plasmids containing each of the Orf2 variants were individually transformed into OneShot BL21(DE3) chemically competent E. coli cells (Invitrogen catalog C600003) according to the chemically competent cell transformation protocol provided by Invitrogen. This resulted in 96 individual E. coli cell lines, each containing one plasmid encoding an Orf2 variant.
To induce protein expression, individual cell lines encoding each of the “orf2 variants” was individually inoculated into 2 milliliters LB media with 50 micrrograms per milliliter of Kanamycin sulfate in 15 milliliter culture tubes and grown at 37 degrees Celsius for 16 hours with vigorous shaking. After 16 hours, each culture was diluted into 38 milliliters LB media with 50 micrograms per milliliter of Kanamycin sulfate for a total of 40 milliliters. The absorbance at 600 nm (OD600) was monitored until it reached a value of 0.6 absorbance units. When the OD600 reached a value of 0.6, then IPTG was added to each culture to a final concentration of 500 micrograms per milliliter, resulting in an “induced culture.” Each “induced culture” was grown at 20 degrees Celsius with vigorous shaking for 20 hours.
After the cultures were grown under protein induction conditions, the target protein was extracted following a standard protein purification protocol. Each “induced culture” was spun at 4,000G for 5 minutes. The supernatant was discarded, leaving only a cell pellet. Each individual cell pellet was resuspended in 25 milliliters of a solution containing 20 millimolar Tris-HCL, 500 millimolar sodium chloride, 5 millimolar imidazole, and 10% glycerol (“lysis buffer”), resulting in a “cell slurry.” To each individual “cell slurry”, 30 microliters of 25 units per microliter Benzonase (Millipore, Benzonase, catalog number 70664-1), as well as 300 microliters of phosphatase and protease inhibitor (Thermo-Fisher, Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free, catalog number 78441) was added. Each individual “cell slurry” was then subjected to 30 second pulses of sonication, 4 times each, for a total of 120 seconds, using the Fisher Scientific Sonic Dismembrator Model 500 under 30% amplitude conditions. In between each 30 second pulse of sonication, the “cell slurry” was placed on ice for 30 seconds. After sonication, each individual “cell slurry” was centrifuged for 45 minutes at 14,000 times gravity.
Protein purification columns (Bio-Rad, Econo-Pac Chromotography Columns, catalog number 7321010) were prepared by adding 1.5 milliliters His60 resin slurry (Takara, His60 nickel superflow resin, catalog number 635660). 5 milliliters deionized water was added to resin slurry, to agitate and rinse the resin. The columns were then uncapped and the resulting flow-through was discarded. Then, 5 milliliters deionized water was added a second time, and the resulting flow-through was discarded. Then, 10 milliliters “lysis buffer” was added to the resin, completely disturbing the resin bed, and the flow-through was discarded.
The protein purification columns were capped, and the supernatant from the “cell slurry” was added to the resin bed without disturbing the resin bed. The columns were uncapped, allowing the supernatant to pass over the resin bed. The resin was then washed 2 times with 10 milliliters of a solution containing 20 millimolar Tris-HCl, 500 millimolar sodium chloride, and 20 millimolar imidazole (“wash buffer”). The flow-through from the wash steps was discarded. The protein was then eluted off the column with 10 milliliters of a solution containing 20 millimolar Tris-HCl, 200 millimolar sodium chloride, and 250 millimolar imidazole. The eluted protein was collected and dialyzed overnight in 4 liters of a solution containing 200 millimolar Tris-HCl and 800 millimolar sodium chloride in 3.5-5.0 kilodalton dialysis tubing (Spectrum Labs, Spectra/Por dialysis tubing, catalog number 133198). After overnight dialysis, protein was concentrated to approximately 10 milligrams per milliliter using centrifugal protein filters (Millipore Amicon Ultra-15 Ultracel 10K, catalog number UFC901024).
C. Screening of the Orf2 Protein Variants for Protein Activity and Phenotypes.
The library of orf2 variants were screened for protein expression by western blot with an anti-HIS antibody (Cell Signaling Technologies, anti-his monoclonal antibody, catalog number 23655) according to the protocol provided by Cell Signaling Technologies for the antibody. The variants that had detectable levels of protein expression as determined by western blot were used in a prenylation assay.
Proteins that exhibited detectable expression by Western blot were assayed for prenylation activity using a substrate (e.g. olivetolic acid, olivetol, divarinic acid, etc.) and a donor molecule (e.g. GPP, FPP, DMAPP, etc.). Unless otherwise stated, each prenylation reaction assay was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 2 millimolar donor molecule (e.g. GPP), 100 millimolar HEPES buffer at a pH of 7.5, 2 millimolar substrate (e.g. olivetolic acid), and 20 micrograms Orf2 protein or Orf2 variant protein. These reactions were incubated for 16 hours at 30° C.
For those prenylation reactions carried out at pH 5.3, the same prenylation reaction assay protocol above was carried out with the exception of replacing HEPES buffer with sodium citrate at pH 5.3.
To assess prenylation activity by HPLC the prenylation products were extracted from the assay reaction with the following protocol: 40 microliters of ethyl acetate was added to each reaction and vortexed thoroughly. After vortexing, each reaction was centrifuged for 10 minutes at 14,000G. The top layer (“organic layer”) was collected. This was repeated twice. The collected organic layer was evaporated, and the resulting residue was resuspended in 40 microliters of 100% methanol. After resuspending in methanol, 40 microliters of 100% HPLC grade water was added to bring the final solution to 50% methanol. These will be referred to as the “variant reactions with GPP.”
The final 50% methanol solutions were run on a Thermo Fisher UltiMate 3000 UHPLC with an Acclaim RSLC 120 angstrom C18 column with a 4 millimeter Phenomonex Securityguard guard column (54 millimeter total column length).
The wild type Orf2 standard prenylation assay produces two products as determined by HPLC, with retention times of approximately 6.9 minutes (an “early product” which is cannabigerolic acid [CBGA]) and 7.2 minutes (a “late product” 5-GOA) in a ratio of −20% CBGA to 80% 5-GOA.
A rational design approach was used to generate a library of 96 ORF2 triple mutants in which each triple mutant carried amino acid substitutions at 3 of 36 selected residues following the methods described in Example 1. These triple mutants may be interchangeably referred to as tripleton variants or tripleton mutants. Each amino acid substitution was employed 3-5 times in the library. From 66 of the 96 clones each carrying a unique tripleton ORF2 variant, ORF2 mutant proteins were expressed and their activity was analyzed as described in Example 1.
Table 1 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce CBGA and 5-GOA using Olivetolic Acid (OA) as substrate and Geranyl pyrophosphate (GPP) as donor. Table 1 lists the mutations within each of the tripleton mutants as well the nMol of CBGA produced, nMol of 5-GOA produced, total prenylated products produced (nMol of CBGA+5-GOA), % CBGA within total prenylated products (nMol of CBGA/[nMol of CBGA+5-GOA]), % enzymatic activity (total prenylated products produced by a mutant/total prenylated products produced by wild-type ORF2), CBGA production (% CBGA among total prenylated products * % enzymatic activity), and %5-GOA within prenylated products (nMol of 5-GOA/[nMol of CBGA+5-GOA]) for each of the ORF2 variants.
The amount of CBGA or 5-GOA (in nMols) generated by each of the ORF2 triple mutant clones was measured using HPLC.
While the CBGA production potential analysis shown in
Based on the analysis performed in
For the singleton and doubleton mutants resulting from the breakdown of triple mutants—E09, H02, A04, D04, A09, C05, H011, F09, D011, and G05—the total amount of prenylated products (and the respective proportion of CBGA and 5-GOA); and % CBGA within the prenylated products was calculated.
In a similar manner, the triple mutants, G12, C06 and F08, will also be subjected to “breakdown” analysis. Further, the singleton and double mutants resulting from the breakdown of G12, C06 and FO will be analyzed to determine the total amount of prenylated products (and the respective proportion of CBGA and 5-GOA); and % CBGA within the prenylated products produced by these mutants, as described above.
This analysis provided important insights into which positions on ORF2, when mutated, are likely to give rise to significant effects on the enzymatic activity of ORF2 in the reaction using Olivetolic Acid (OA) as substrate and Geranyl pyrophosphate (GPP) as donor. Based on this analysis, the following amino acid sites were selected for targeted amino acid site saturation mutagenesis—M106, Q161, 5177, Q295, Y288, 5214, M162 and A232.
Site saturated mutagenesis was done for Q295, Q161, 5214 and Y288 by replacing the wild type residue with each of the other 19 standard amino acids. The amount of total prenylated products, the CBGA production potential and GOA production potential was measured for each of the site saturated mutants. These results are depicted in
Similarly, site saturated mutagenesis will also be completed for M106, and S177; and the amount of total prenylated products and the CBGA production potential will be measured for each of these site saturated mutants.
From the results described above, multiple mutations of Q295, Q161, Y288 and 5214 that have significantly higher CBGA production potential and/or the total amount of prenylated products, as compared to WT ORF2, were identified. Thus, the ORF2 mutants disclosed herein have unexpectedly superior enzymatic functions, in a reaction using OA as a substrate and GPP as donor, as compared to WT ORF2.
Finally, ORF2 stacking mutants, that carry different novel combinations of the mutations identified by our analysis as being important for ORF2's enzymatic activity, were analyzed to determine the total amount of prenylated products they produce (
For instance, ORF2 double mutants, A53T-S214R; Q161S-Q295L; Q161S-Q295F; S177W-Q295A have synergistically enhanced CBGA production potential and % activity as compared to either of the single mutants. See
More stacking mutants will be generated as described above, based on the breakdown analysis of triple mutants, G12, C06 and F08, and planned site saturation mutagenesis experiments described above. These stacking mutants will further be analyzed to determine their % enzymatic activity, % CBGA, %5-GOA and CBGA production potential.
Four triple mutants (D12 carrying A53T_E112D_G205M mutations; D6 carrying A53E_Q161A_V294N mutations; H9 carrying E112G_G205M_L298W mutations; and E9 carrying A53T_M106E_Q161S mutations) that had improved activity vs. the WT ORF2, based on
Site-saturated mutagenesis was done for A53 by replacing the wild type residue at this site with each of the 19 other standard amino acids. % of enzymatic activity as compared to WT ORF2 was measured. These results are depicted in
The site saturated mutants of Q161 described in Example 1 were also analyzed to determine their % activity in a reaction using OA as substrate and GPP as donor. These results are depicted in
From the results described above, multiple mutations of A53 and Q161 that have significantly higher % activity, as compared to WT ORF2, were identified. Thus, the ORF2 mutants disclosed herein have unexpectedly superior enzymatic functions, in a reaction using OA as a substrate and GPP as donor, as compared to WT ORF2.
Similarly, site saturated mutagenesis will also be completed for E112 and G205 as described above; and % of enzymatic activity, as compared to WT ORF2, will be measured for each of these mutants.
The library of tripleton mutants described in Example 1, was screened for the ability to produce CBGVA and 5-GDVA using DVA as substrate and Geranyl pyrophosphate (GPP) as donor; and this enzymatic activity was compared to that of the WT ORF2. Table 5 provides the summary of the analysis performed on the enzymatic activity of the ORF2 triple mutants to produce CBGVA and 5-GDVA using DVA as substrate and Geranyl pyrophosphate (GPP) as donor. Table 11 lists the mutations within each of the tripleton mutants as well the nMol CBGVA, nMol of 5-GDVA, total prenylated products, % CBGVA products among prenylated products, % enzymatic activity, CBGVA production potential and %5-GDVA among total prenylated products of each of the ORF2 triple mutants.
The amount of CBGVA or 5-GDVA (in nMols) generated by each of the ORF2 triple mutant clones was measured using HPLC.
While the CBGVA production potential analysis shown in
Based on the analysis performed in
For the singleton and doubleton mutants resulting from the breakdown of triple mutants—A09; H02; C05; E09; D04; H11; A04; G05; F09; and D11—the total amount of prenylated products (and the respective proportion of CBGVA and 5-GDVA); and % CBGVA within the prenylated products was calculated.
For the remaining triple mutants in Table 6, the breakdown of the triple mutants into singletons and doubletons; and the determination of the total amount of prenylated products and % CBGVA produced by these mutants will be determined.
This analysis provided important insights into which positions on ORF2, when mutated, are likely to give rise to significant effects on the enzymatic activity of ORF2 in the reaction using DVA as substrate and GPP as donor. Based on this analysis, the following amino acid sites were selected for targeted amino acid site saturation mutagenesis—V49, Q161, 5177, Y288, M106, Q295, 5214, M162 and A232.
Site saturated mutants of Q295, Q161, 5214 and Y288, described in Example 1, were analyzed to determine the amount of total prenylated products they produce; their respective CBGVA production potential; and 5-GDVA production potential using DVA as substrate and GPP as donor. These results are depicted in
In a similar manner, site saturated mutants of V49, 5177, and M106 will also be generated. Mutant ORF2 enzymatic function using DVA as substrate and GPP as donor for each of these site saturated mutants will be analyzed as described above.
From the results described above, multiple mutations of Q295, Q161, Y288 and 5214 that have significantly higher CBGVA production potential and/or the total amount of prenylated products, as compared to WT ORF2, were identified. Thus, the ORF2 mutants disclosed herein have unexpectedly superior enzymatic functions, in a reaction using DVA as a substrate and GPP as donor, as compared to WT ORF2.
Finally, ORF2 stacking mutants that carry different novel combinations of the mutations that our analysis identified as being important for ORF2's enzymatic activity were analyzed to obtain the total amount of prenylated products produced by each of the stacking mutants (
The analysis of the stacking mutants shows that multiple stacking mutants have significantly higher % enzymatic activity, % CBGVA, and CBGVA production potential, compared to the WT ORF2, thereby indicating that the ORF2 stacking mutants disclosed herein have synergistically enhanced effects compared to the individual single mutants. Thus, the ORF2 stacking mutants disclosed herein have unexpectedly superior enzymatic functions, in a reaction using DVA and GPP, as compared to WT ORF2.
For instance, ORF2 double mutants, A53T-S214R; Q161S-Q295L; Q161S-Q295F; S177W-Q295A; A53T-Q295F; and Q161S-S177W have synergistically enhanced CBGVA production potential and % activity as compared to either of the single mutants. See
More stacking mutants will be generated as described above, based on the planned breakdown analysis of triple mutants described above, and planned site saturation mutagenesis experiments described above. These stacking mutants will further be analyzed to determine their % enzymatic activity, % CBGVA, %5-GDVA and CBGVA production potential.
Four triple mutants (A09 carrying V49A_Q161S_V294A; E9 carrying A53T_M106E_Q161S; G12 carrying A17T_Q161W_A232S; and D12 carrying A53T_E112D_G205M) that had improved activity vs. the WT ORF2, based on
The % enzymatic activity when using DVA as substrate and GPP as donor was calculated for each of the single and double mutants derived from the breakdown analysis of A09 and E09.
In a similar fashion, breakdown analysis for the other two triple mutants—G12 and D12—will also be done; and the % activity for the mutants derived from the breakdown will be measured, as described above.
Based on the breakdown analysis of triple mutants A09 and E09: V49, Q161, A53 and Q295 were selected for performing site-saturated mutagenesis. % enzymatic activity (when using DVA as substrate and GPP as donor) of site-saturated mutants of A53, Q295 and Q161 described in Examples 1 and 2 was determined, and was compared to that of WT ORF2. These results are shown in
Similarly, site saturated mutants of V49 will also be generated; and the % activity of these mutants will be compared to that of the WT ORF2 in a reaction using DVA as substrate and GPP as donor.
From the results described above, multiple mutations of A53, Q295, and Q161 that have significantly higher % activity, as compared to WT ORF2, were identified. Thus, the ORF2 mutants disclosed herein have unexpectedly superior enzymatic functions, in a reaction using DVA as a substrate and GPP as donor, as compared to WT ORF2.
Finally, ORF2 stacking mutant clones that carry different novel combinations of the mutations that our analysis identified as being important for ORF2's % enzymatic activity were analyzed to measure their % enzymatic activity, as shown in
The analysis of the stacking mutants shows that multiple stacking mutants have significantly higher % enzymatic activity, compared to the WT ORF2, thereby indicating that the ORF2 stacking mutants disclosed herein have synergistically enhanced effects compared to the individual single mutants. Thus, the ORF2 stacking mutants disclosed herein have unexpectedly superior enzymatic functions, in a reaction using DVA and GPP, as compared to WT ORF2.
The library of tripleton mutants was screened for the ability to produce CBG and 5-GO using O as substrate and Geranyl pyrophosphate (GPP) as donor; and this enzymatic activity was compared to that of the WT ORF2. Table 22 provides the summary of the analysis performed on the enzymatic activity of the ORF2 triple mutants to produce CBG and 5-GO using O as substrate and Geranyl pyrophosphate (GPP) as donor. Table 9 lists the mutations within each of the tripleton mutants as well the nMol CBG, nMol of 5-GO, total prenylated products, % CBG products among prenylated products, % enzymatic activity, CBG production potential and %5-GO among total prenylated products of each of the ORF2 triple mutants.
The amount of CBG or 5-GO (in nMols) generated by each of the ORF2 triple mutant clones was measured using HPLC.
While the CBG production potential analysis shown in
Based on the analysis performed in
For the singleton and doubleton mutants resulting from the breakdown of triple mutants—E09; A09; A04; H11; H02; D04; C05; F09; D011; and G05—the total amount of prenylated products (and the respective proportion of CBG and 5-GO); and % CBG within the prenylated products was calculated.
The breakdown analysis of the remaining triple mutants listed in Table 23 will be performed in a similar manner as described above; and the total amount of prenylated products and % CBG produced using O as substrate and GPP as donor by the mutants derived from this breakdown will be measured.
The breakdown analysis of triple mutants—E09; A09; A04; H11; H02; D04; C05; F09; D011; and G05—provided important insights into which positions on ORF2, when mutated, are likely to give rise to significant effects on the enzymatic activity of ORF2 in the reaction using Olivetol (O) as substrate and Geranyl pyrophosphate (GPP) as donor. Based on this analysis, the following amino acid sites were selected for targeted amino acid site saturation mutagenesis—Q161, Q295, 5214, V294 M106, L174, A232, Y288 and M162. Site saturated mutagenesis was done for Q295, Q161, 5214 and Y288 by replacing the wild type residue with each of the other 19 standard amino acids. The amount of total prenylated products produced; CBG production potential; and 5-GO production potential were measured for Q295, Q161, 5214 and Y288 site saturated mutants, as depicted in
Similarly, site saturated mutagenesis will also be completed for M106, and V294; and the amount of total prenylated products produced; CBG production potential; and 5-GO production potential will be measured for each of these site saturated mutants.
From the results described above, multiple mutations of Q295, Q161 and 5214 that have significantly higher CBG or 5-GO production potential; and/or higher amount of prenylated products, as compared to WT ORF2, were identified. Thus, the ORF2 mutants disclosed herein have unexpectedly superior enzymatic functions, in a reaction using O as a substrate and GPP as donor, as compared to WT ORF2.
Finally, ORF2 stacking mutants, which carry different novel combinations of the mutations that our analysis identified as being important for ORF2's enzymatic activity were analyzed to determine the total amount of prenylated products produced by each of the stacking mutants (
For instance, ORF2 double mutants, Q161S-Q295L; A53T-Q161S; A53 T-V294A; Q161S-Q295F; and A53T-Q295F have synergistically enhanced CBGVA production potential and % activity as compared to either of the single mutants. See
In the future, more stacking mutants will be generated as described above, based on the breakdown analysis of remaining triple mutants listed in Table 10, and planned site saturation mutagenesis experiments described above. These stacking mutants will further be analyzed to determine their % enzymatic activity, % CBG, %5-GO and CBG production potential.
Six triple mutants (E09 carrying A53T_M106E_Q161S mutations; D12 carrying A53T_E112D_G205M; C06 carrying Q161A_M162F_Q295A; C11 carrying E112D_L219F_V294F; G12 carrying A17T_Q161W_A232S; and A09 carrying V49A_Q161S_V294A) had improved activity vs. the WT ORF2, based on
In a similar fashion, breakdown of the other triple mutants—D12, C06, C11 and G12—will be performed; and the % activity for the mutants derived from the breakdown of D12, C06, C11 and G12 will analyzed as described above.
From the analysis of the breakdown mutants of E09 and A09, the following sites were identified as target residues for site saturation: V294, Q161, and A53. Site-saturated mutants of Q161, and A53 described in Examples 1 and 2 were analyzed to determine their % enzymatic activity as compared to WT ORF2 using O as substrate and GPP as donor. These results are depicted in
Site-saturated mutagenesis will be done in the same manner for V294; and % activity will be measured for each of these site saturated mutants.
The library of tripleton mutants was screened for the ability to produce CBGA and 5-GOA using OA as substrate and Geranyl pyrophosphate (GPP) as donor at pH 5.3; and this enzymatic activity was compared to that of the WT ORF2. Table 30 provides the summary of the analysis performed on the enzymatic activity of the ORF2 triple mutants to produce CBG and 5-GO using O as substrate and Geranyl pyrophosphate (GPP) as donor.
The amount of CBGA and 5-GOA produced by each of the ORF2 triple mutants at pH 5.3 was measured using HPLC.
In the cluster analysis shown in
Based on the analysis performed in
Each of these triple mutants listed in Tables 31 and 32 will be broken down to their respective single mutants and double mutants; and all the ORF2 mutants thus generated will be analyzed for their activity at pH 5.3.
Finally, ORF2 mutants carrying point substitutions—Q161H, S214H, and Q295H mutations—were analyzed for their % activity and CBGA production potential at pH 5.3. The results surprisingly show that although WT ORF2 has little to no activity and little to no CBGA production potential at pH 5.3, Q161H and S214H have unexpectedly enhanced activity at this acidic pH. See
Table 34 lists all the amino acid sequences disclosed herein. Sequence information indicates the amino acid substitution of the sequence in comparison to the WT ORF2 (consensus ORF2).
Table 35 lists all the nucleic acid sequences disclosed herein. The protein sequence (WT or mutant ORF2) encoded by the respective nucleic acid sequence is provided in Column 1 of Table 35.
This application is a continuation of U.S. application Ser. No. 16/682,845, filed on Nov. 13, 2019, which is a continuation of the International Application No. PCT/US2019/023046, filed Mar. 19, 2019, which claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62/645,081, filed Mar. 19, 2018, the disclosure of each of which is incorporated herein by reference in their entireties for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 62645081 | Mar 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16682845 | Nov 2019 | US |
| Child | 17081724 | US | |
| Parent | PCT/US2019/023046 | Mar 2019 | US |
| Child | 16682845 | US |
