Patent Application
20230029027
The present disclosure is generally related to the biosynthesis of organic compounds, such as cannabinoids, using recombinant enzymes, such as recombinant aromatic prenyltransferases.
The contents of the text file named “REBI_002_00US_SeqList_ST25.txt”, which was created on Apr. 12, 2019 and is 1.19 megabytes in size, are hereby incorporated by reference in its entirety.
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. Further, there is also a need for the synthesis of a diverse group of chemical compounds including not limited to cannabinoids using similar synthetic biology approaches.
The disclosure provides recombinant polypeptides comprising an amino acid sequence with at least 80% identity to the amino acid sequence of a prenyltransferase, wherein the recombinant polypeptide comprises at least one amino acid substitution compared to the amino acid sequence of the prenyltransferase, wherein said recombinant polypeptide converts a substrate and a prenyl donor to at least one prenylated product, and wherein the recombinant polypeptide produces a ratio of an amount of the at least one prenylated product to an amount of total prenylated products that is higher than the prenyltransferase under the same condition.
In some aspects, the recombinant polypeptide comprises an amino acid sequence with at least 95% identity to the amino acid sequence of the prenyltransferase. In some aspects, the amino acid sequence has at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of the prenyltransferase. In some aspects, 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 the amino acid sequence of the prenyltransferase.
In some aspects, the prenyltransferase is selected from the group consisting of ORF2, HypSc, PB002, PB005, PB064, PB065, and Atapt (interchangeably referred to herein as “PBJ”). In some aspects, the prenyl donor is selected from Dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl pyrophosphate (GGPP), or any combination thereof. In some aspects, the prenyl donor is not a naturally occurring donor of the prenyltransferase. In some aspects, the substrate is selected from olivetolic acid (OA), divarinolic acid (DVA), olivetol (0), divarinol (DV), orsellinic acid (ORA), dihydroxybenzoic acid (DHBA), apigenin, naringenin and resveratrol. In some aspects, the substrate is not a naturally occurring substrate of the prenyltransferase.
In some aspects, the at least one prenylated product comprises a prenyl group attached to any position on an aromatic ring of the substrate. In some aspects, the at least one prenylated product is selected from the group consisting of UNK1, UNK2, UNK3, RBI-08, 5-DOA, RBI-05, RBI-06, 4-O-GOA, RBI-02 (CBGA—cannabigerolic acid), RBI-04 (5-GOA), UNK4, RBI-56, UNK5, RBI-14 (CBFA), RBI-16 (5-FOA), RBI-24, RBI-28, RBI-26 (CBGVA—cannabigerovarinic acid), RBI-27, RBI-38, RBI-39, RBI-09, RBI-10, RBI-03 (5-GO), RBI-20, RBI-01 (CBG—cannabigerol), RBI-15, RBI-34, RBI-32, RBI-33, RBI-07, RBI-29, RBI-30, RBI-12, and RBI-11.
In some aspects, the prenyltransferase is ORF2. In some aspects, the substrate is OA and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO; 2-O; 4-O; 3-C; 5-C; or 5-C and 3-C on the aromatic ring of OA. In some aspects, the at least one prenylated product comprises UNK1, UNK2, UNK3, RBI-08, RBI-17, or RBI-18.
In some aspects, the substrate is OA and the prenyl donor is GPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO; 2-O; 4-O; 3-C; 5-C; or 3-C and 5-C on the aromatic ring of OA. In some aspects, the at least one prenylated product comprises RBI-05, RBI-06, UNK-4, RBI-02 (CBGA), RBI-04 (5-GOA) or RBI-07.
In some aspects, the substrate is OA and the prenyl donor is FPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from 2-O; 4-O; 3-C; and 5-C on the aromatic ring of OA. In some aspects, the at least one prenylated product comprises RBI-56, UNK5, RBI-14 (CBFA), or RBI-16 (5-FOA).
In some aspects, the substrate is DVA and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO; 2-O; 4-O; 3-C; and 5-C on the aromatic ring of DVA.
In some aspects, the substrate is DVA and the prenyl donor is GPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO; 2-O; 4-O; 3-C; 5-C; 3-C and 5-C; or 5-C and 2-O on the aromatic ring of DVA. In some aspects, the at least one prenylated product comprises RBI-24, RBI-28, UNK11, RBI-26, RBI-27, RBI-29, or RBI-30.
In some aspects, the substrate is DVA and the prenyl donor is FPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO; 2-O; 4-O; 3-C; and 5-C on the aromatic ring of DVA. In some aspects, the at least one prenylated product comprises UNK12, UNK13, UNK14, RBI-38, or RBI-39.
In some aspects, the substrate is O and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from 1-C/5-C; 2-O/4-O; or 3-C on the aromatic ring of O. In some aspects, the at least one prenylated product comprises RBI-10, UNK16, or RBI-09.
In some aspects, the prenyltransferase is HypSc. In some aspects, the substrate is O and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from 1-C/5-C; 2-O/4-O; or 3-C on the aromatic ring of O. In some aspects, the at least one prenylated product comprises RBI-10, UNK16 or RBI-09.
In some aspects, the prenyltransferase is PB005. In some aspects, the substrate is 0 and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from 1-C/5-C; 2-O/4-O; 3-C; 1-C and 5-C; or 1-C and 3-C on the aromatic ring of 0. In some aspects, the at least one prenylated product comprises RBI-10, UNK16, RBI-09, RBI-11 or RBI-12.
In some aspects, the substrate is O and the prenyl donor is GPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from 1-C/5-C; 2-O/4-O; or 3-C on the aromatic ring of O. In some aspects, the at least one prenylated product comprises RBI-20, RBI-01 (CBG), or RBI-03 (5-GO).
In some aspects, the substrate is O and the prenyl donor is FPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from 1-C/5-C; 2-O/4-O; 4-O/2-O; or 3-C on the aromatic ring of 0. In some aspects, the at least one prenylated product comprises RBI-15, UNK18 or UNK19.
In some aspects, the substrate is DV and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from 1-C/5-C; 2-O/4-O; or 3-C on the aromatic ring of DV. In some aspects, the at least one prenylated product comprises UNK54, UNK55 or UNK56.
In some aspects, the substrate is ORA and the prenyl donor is GPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO, 2-O, 4-O, 3-C, 5-C, or 5-C and 3-C on the aromatic ring of ORA.
In some aspects, the substrate is ORA and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO, 2-O, or 5-C on the aromatic ring of ORA.
In some aspects, the substrate is ORA and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO, 2-O, or 4-O on the aromatic ring of ORA.
In some aspects, the substrate is ORA and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO, or 3-C on the aromatic ring of ORA.
In some aspects, the prenyltransferase is PB064. In some aspects, the substrate is ORA and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO, 2-O or 3-C on the aromatic ring of ORA.
In some aspects, the prenyltransferase is PB065. In some aspects, the substrate is ORA and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO, or 2-O on the aromatic ring of ORA.
In some aspects, the prenyltransferase is PB002. In some aspects, the substrate is ORA and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position CO on the aromatic ring of ORA.
In some aspects, the prenyltransferase is Atapt. In some aspects, the substrate is ORA and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position 4-O on the aromatic ring of ORA.
In some aspects, the substrate is ORA and the prenyl donor is FPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO, 2-O, 4-O, 3-C, or 5-C on the aromatic ring of ORA.
In some aspects, the substrate is DHBA and the prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from CO, 2-O, 4-O, 3-C, or 5-C on the aromatic ring of DHBA.
In some aspects, the substrate is DV and the prenyl donor is GPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to positions 5-C and 1-C; or 3-C and 5-C on the aromatic ring of DV. In some aspects, the at least one prenylated product comprises RBI-36, or UNK35.
In some aspects, the substrate is OA and the prenyl donor is GPP, DMAPP or both. In some aspects, the at least one prenylated product comprises a prenyl group attached to positions 5-C and 3-C; or CO and 3-C on the aromatic ring of OA.
In some aspects, the substrate is OA and the prenyl donor is GPP, FPP or both. In some aspects, the at least one prenylated product comprises a prenyl group attached to positions 5-C and 3-C on the aromatic ring of OA.
In some aspects, the substrate is O and the prenyl donor is GPP, FPP or both. In some aspects, the at least one prenylated product comprises a prenyl group attached to positions 5-C and 3-C on the aromatic ring of O.
In some aspects, the substrate is apigenin and the prenyl donor is GPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from C-13; C-15; C-3; C-12; C-16; C-9; or C-5 on the aromatic ring of apigenin. In some aspects, the at least one prenylated product comprises UNK47, UNK48, UNK49, UNK50, or UNK51. In some aspects, the substrate is naringenin and the prenyl donor is GPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from C-3; or C-5 on the aromatic ring of naringenin. In some aspects, the at least one prenylated product comprises RBI-41 or RBI-42. In some aspects, the substrate is resveratrol and the prenyl donor is GPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from C-11; C-13; C-3; C-10; C-14; or C-1/5 on the aromatic ring of resveratrol. In some aspects, the at least one prenylated product comprises RBI-48 or RBI-49.
In some aspects, the substrate comprises olivetolic acid (OA), divarinolic acid (DVA), olivetol (0), 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,3S)-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 aspects, the substrate is a prenylated molecule. In some aspects, the prenylated molecule is selected from the group consisting of UNK1, UNK2, UNK3, RBI-08, 5-DOA, RBI-05, RBI-06, 4-O-GOA, RBI-02 (CBGA), RBI-04 (5-GOA), UNK4, RBI-56, UNK5, RBI-14 (CBFA), RBI-16 (5-FOA), RBI-24, RBI-28, RBI-26, RBI-27, RBI-38, RBI-39, RBI-09, RBI-10, RBI-03 (5-GO), RBI-20, RBI-01 (CBG), RBI-15, RBI-34, RBI-32, RBI-33, RBI-07, RBI-29, RBI-30, RBI-12, and RBI-11.
In some aspects, the amino acid sequence of ORF2 comprises SEQ ID NO: 1, and the at least one amino acid substitution comprises at least one amino acid substitution in SEQ ID NO: 1 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 aspects, the 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 aspects, the amino acid sequence of ORF2 comprises SEQ ID NO: 1, and the 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 aspects, the amino acid sequence of ORF2 comprises SEQ ID NO: 1, and the 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;
(b) A53T and 5214R; 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 aspects, the at least one prenylated product comprises UNK6, UNK7, UNK8, UNK9, or UNK10. In some aspects, the at least one prenylated product comprises UNK20, UNK21, UNK22, UNK23, UNK24, or UNK59. In some aspects, the at least one prenylated product comprises UNK25, UNK26, or UNK29. In some aspects, the at least one prenylated product comprises UNK25, UNK26 or UNK27. In some aspects, the at least one prenylated product comprises UNK25 or UNK28. In some aspects, the at least one prenylated product comprises UNK25, UNK26 or UNK28. In some aspects, the at least one prenylated product comprises UNK25 or UNK26. In some aspects, the at least one prenylated product comprises UNK25. In some aspects, the at least one prenylated product comprises UNK27. In some aspects, the at least one prenylated product comprises UNK30, UNK31, UNK32, UNK33, or UNK34. In some aspects, the at least one prenylated product comprises UNK36, UNK38, or RBI-22. In some aspects, the at least one prenylated product comprises UNK42. In some aspects, the at least one prenylated product comprises UNK46.
In some aspects, the substrate is DV and the prenyl donor is GPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from 3-C, 1-C, or 5-C on the aromatic ring of DV. In some aspects, the at least one prenylated product comprises RBI-32 or RBI-33.
In some aspects, the substrate is OA and the prenyl donor is GGPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from 3-C, or 5-C on the aromatic ring of OA. In some aspects, the at least one prenylated product comprises UNK60 or UNK61.
In some aspects, the substrate is ORA and the prenyl donor is GGPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from 3-C, or 5-C on the aromatic ring of ORA. In some aspects, the at least one prenylated product comprises UNK62 or UNK63.
In some aspects, the substrate is DVA and the prenyl donor is GGPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to a position selected from 3-C, or 5-C on the aromatic ring of DVA. In some aspects, the at least one prenylated product comprises UNK64 or UNK65.
The disclosure further provides nucleic acid molecules, comprising a nucleotide sequence encoding any one of the recombinant polypeptides disclosed herein, or a codon degenerate nucleotide sequence thereof. In some aspects, the nucleotide sequence comprises at least 500, 600, 700, 800, or 900 nucleotides. In some aspects, the nucleic acid molecule is isolated and purified.
The disclosure provides a cell vector, construct or expression system comprising any one of the nucleic acid molecules disclosed herein; and a cell, comprising any one of the cell vectors, constructs or expression systems disclosed herein. In some aspects, the cell is a bacteria, yeast, insect, mammalian, fungi, vascular plant, or non-vascular plant cell. In some aspects, the cell is a microalgae cell. In some aspects, the cell is an E. coli cell.
The disclosure provides a plant, comprising any one of the cells disclosed herein. In some aspects, the plant is a terrestrial plant.
The disclosure provides methods of producing at least one prenylated product, comprising, contacting any one of the recombinant polypeptides disclosed herein with a substrate and a prenyl donor, thereby producing at least one prenylated product. In some aspects, the recombinant polypeptide is the recombinant polypeptide of any one of claims 13, 16, 19, 22, 24, 27, 30, 34, 38, 41, 44, 47, 50, 52, 54, 56, 59, 62, 65, 68, 70, 72, 74, 77, 79, and 81.
The disclosure provides methods of producing at least one prenylated product, comprising, a) contacting a first recombinant polypeptide with a substrate and a first prenyl donor, wherein the first recombinant polypeptide is any of the recombinant polypeptides disclosed herein, thereby producing a first prenylated product; and b) contacting the first prenylated product and a second prenyl donor with a second recombinant polypeptide, thereby producing a second prenylated product. In some aspects, the first recombinant polypeptide and the second recombinant polypeptide are selected from the recombinant polypeptide of any one of claims 13, 16, 19, 22, 24, 27, 30, 34, 38, 41, 44, 47, 50, 52, 54, 56, 59, 62, 65, 68, 70, 72, 74, 77, 79, and 81.
In some aspects, the first recombinant polypeptide is the same as the second recombinant polypeptide. In some aspects, the first recombinant polypeptide is different from the second recombinant polypeptide. In some aspects, the first prenyl donor is the same as the second prenyl donor. In some aspects, the first prenyl donor is different from the second prenyl donor. In some aspects, the first prenylated product is the same as the second prenylated product. In some aspects, the first prenylated product is different from the second prenylated product.
In some aspects, (a) the first recombinant polypeptide is a recombinant polypeptide wherein the prenyltransferase is ORF2, and the second recombinant polypeptide is a recombinant polypeptide wherein the prenyltransferase is PB005; or the first recombinant polypeptide is a recombinant polypeptide wherein the prenyltransferase is PB005 and the second recombinant polypeptide is a recombinant polypeptide wherein the prenyltransferase is ORF2; (b) the first prenyl donor is GPP and the second prenyl donor is DMAPP; or the first prenyl donor is DMAPP, and the second prenyl donor is GPP; and (c) the substrate is O. In some aspects, the first prenylated product or the second prenylated product comprises a prenyl group attached to positions of 5-C and 3-C; 5-C and 1-C; and 5-C, 1-C and 3-C on the aromatic ring of 0.
In some aspects, (a) the first recombinant polypeptide is a recombinant polypeptide wherein the prenyltransferase is ORF2, and the second recombinant polypeptide is a recombinant polypeptide wherein the prenyltransferase is PB005; or the first recombinant polypeptide is a recombinant polypeptide wherein the prenyltransferase is PB005 and the second recombinant polypeptide is a recombinant polypeptide wherein the prenyltransferase is ORF2; (b) the first prenyl donor is FPP and the second prenyl donor is DMAPP; or the first prenyl donor is DMAPP, and the second prenyl donor is FPP; and (c) the substrate is O. In some aspects, the first prenylated product or the second prenylated product comprises a prenyl group attached to positions 5-C and 3-C; or 5-C and 1-C on the aromatic ring of O.
In some aspects, the second recombinant polypeptide is a cyclase. In some aspects, the cyclase comprises cannabidiolic acid synthase (CBDAS) or tetrahydrocannabinolic acid synthase (THCAS). Further details on CBDAS and THCAS are provided in “Cannabidiolic—acid synthase, the chemotype—determining enzyme in the fiber—type Cannabis sativa” Taura et al., Volume 581, Issue 16, Jun. 26, 2007, Pages 2929-2934; and “The Gene Controlling Marijuana Psychoactivity. Molecular Cloning and Heterologous Expression of Al-Tetrahydrocannabinolic acid synthase from Cannabis sativa L.” Sirikantaramas et al. The Journal of Biological Chemistry, Vol. 279, No. 38, Issue of September 17, pp. 39767-39774, 2004, respectively, each of which is incorporated herein by reference in their entireties for all purposes.
In some aspects, the cyclase is derived from a plant belonging to the Rhododendron genus and wherein the cyclase cyclizes an FPP moiety. In some aspects, the cyclase is Daurichromenic Acid Synthase (DCAS). Further details on DCAS is provided in “Identification and Characterization of Daurichromenic Acid Synthase Active in Anti-HIV Biosynthesis” Iijima et al. Plant Physiology August 2017, 174 (4) 2213-2230, the contents of which are incorporated herein by reference in its entirety.
In some aspects, the secondary enzyme is a methyltransferase. In some cases, the methyltransferase is a histone methyltransferase, N-terminal methyltransferase, DNA/RNA methyltransferase, natural product methyltransferase, or non-SAM dependent methyltransferases.
In some aspects, the at least one prenylated product comprises UNK40, UNK41, UNK66 or UNK67. In some aspects, the at least one prenylated product comprises UNK44 or UNK45.
In some aspects, the first recombinant polypeptide is PB005, and the second recombinant polypeptide is HypSc; or the first recombinant polypeptide is HypSc, and the second recombinant polypeptide is PB005. In some aspects, the substrate is DV; and the first prenyl donor and the second prenyl donor is DMAPP. In some aspects, the at least one prenylated product comprises a prenyl group attached to positions of 5C and 3C; or 5C and 1C on the aromatic ring of DV. In some aspects, the at least one prenylated product comprises UNK57 or UNK58.
The disclosure further provides compositions comprising the at least one prenylated product produced by any one of the methods disclosed herein. The disclosure also provides compositions comprising the first prenylated product and/or the second prenylated product produced by any one of the methods disclosed herein.
The disclosure provides a composition comprising a prenylated product, wherein the prenylated product comprises a substitution by a prenyl donor on an aromatic ring of a substrate, wherein the substrate is selected from the group consisting of olivetolic acid (OA), divarinolic acid (DVA), olivetol (0), divarinol (DV), orsellinic acid (ORA), dihydroxybenzoic acid (DHBA), apigenin, naringenin and resveratrol.
In some aspects, the prenyl donor is selected from the group consisting of DMAPP, GPP, FPP, GGPP, and any combination thereof. In some aspects, the prenylated product is selected from any of the prenylated products in Table C. In some aspects, the prenylated product is selected from the group consisting of UNK1, UNK2, UNK3, RBI-08, RBI-17, RBI-05, RBI-06, UNK4, RBI-02 (CBGA), RBI-04 (5-GOA), RBI-56, UNK5, RBI-14 (CBFA), RBI-16 (5-FOA), UNK6, UNK7, UNK8, UNK9, UNK10, RBI-24, RBI-28, UNK11, RBI-26 (CBGVA), RBI-27, UNK12, UNK13, UNK14, RBI-38, RBI-39, RBI-10, UNK16, RBI-09, RBI-10, UNK16, RBI-09, RBI-10, UNK16, RBI-09, RBI-10, RBI-03 (5-GO), RBI-20, RBI-01 (CBG), RBI-03 (5-GO), RBI-15, UNK18, UNK19, RBI-15, UNK54, UNK55, UNK56, UNK54, UNK20, UNK21, UNK22, UNK23, UNK24, UNK25, UNK26, UNK27, UNK28, UNK29, RBI-32, RBI-33, UNK30, UNK31, UNK32, UNK33, UNK34, UNK60, UNK61, UNK62, UNK63, UNK64, UNK65, RBI-07, RBI-29, RBI-30, RBI-36, UNK35, UNK36, RBI-22, UNK38, RBI-18, UNK40, UNK41, UNK42, RBI-12, RBI-11, UNK44, UNK45, UNK46, UNK57, UNK58, UNK59, UNK66, and UNK67. In some aspects, the prenylated product is selected from the group consisting of RBI-01, RBI-02, RBI-03, RBI-04, RBI-05, RBI-07, RBI-08, RBI-09, RBI-10, RBI-11, and RBI-12. In some aspects, the prenylated product is RBI-29 or UNK59.
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.
As used herein, the code names refer to the chemical compounds described in the specification and drawing of the present application. For example, the code name “RBI-24” refers to the chemical compound (E)-3,7-dimethylocta-2,6-dien-1-yl 2,4-dihydroxy-6-propylbenzoate, the chemical structure of which is shown in
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.
Further, other prenyltransferase homologues of ORF2 include HypSc, PB002, PB005, PB064, PB065, and Atapt.
This disclosure provides prenyltransferase mutants, engineered by the inventors to produce produces a ratio of an amount of at least one prenylated product to an amount of total prenylated products that is higher than that produced by the WT prenyltransferase under the same conditions. The disclosure also provides prenyltransferase mutants which have been engineered to catalyze reactions using a desired substrate and/or a desired donor and to produce higher amounts of a desired product, as compared to the WT prenyltransferase under the same conditions.
The production of cannabinoids at large industrial scale is made possible using microalgae and dark fermentation. Engineering into the chloroplast of the microalgae offers unique compartmentalization and environment. The Cannabis plant genes express in this single cell plant system and have the post-translational modifications. This dark fermentation process allows one to drive cell densities beyond 100 g/per liter and has been scaled to 10,000 L.
The disclosure provides recombinant polypeptides comprising an amino acid sequence with at least about 70% identity to the amino acid sequence of WT prenyltransferase. 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 prenyltransferase. In some aspects, the mutant recombinant polypeptides (interchangeably used with “recombinant polypeptides”) disclosed herein may comprise a modification at one or more amino acids, as compared to the WT prenyltransferase sequence. In some aspects, the mutant recombinant 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 prenyltransferase sequence.
In some aspects, the prenyltransferase is selected from the group consisting of ORF2, HypSc, PB002, PB005, PB064, PB065, and Atapt. The amino acid sequence of ORF2 is set forth in SEQ ID NO: 1. The amino acid sequence of PB005 is set forth in SEQ ID NO: 602. The amino acid sequence of PBJ or Atapt is set forth in SEQ ID NO: 604.
In some aspects, the prenyltransferase belongs to the ABBA family of prenyltransferases. In some aspects, the prenyltransferase comprises a protein fold with a central barrel comprising ten anti-parallel β-strands surrounded by α-helices giving rise to a repeated α-β-β-α (or “ABBA”) motif. Further details of this family and examples of prenyltransferases that may be used are provided in “The ABBA family of aromatic prenyltransferases: broadening natural product diversity” Tello et al. Cell. Mol. Life Sci. 65 (2008) 1459-1463, the contents of which are incorporated herein by reference in its entirety for all purposes.
In some aspects, the prenyltransferase is ORF2 comprising an amino acid sequence set forth in SEQ ID NO: 1. In some aspects, mutant recombinant 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 recombinant polypeptides disclosed herein catalyze a reaction using at least one prenyl donor. In some aspects, the at least one prenyl donor is DMAPP, GPP, FPP, or any combination thereof.
In some aspects, the mutant recombinant polypeptide uses a donor that is not a naturally occurring donor of the WT prenyltransferase. A “naturally-occurring donor” as used herein, refers to the donor that is used by the WT prenyltransferase to catalyze a prenylation reaction in nature (such as, in the organism that the WT prenyltransferase is found in nature). For instance, a naturally occurring donor of WT ORF2 is GPP; the disclosure provides ORF2 mutants that are able to use donors other than GPP (such as FPP) in the prenylation reaction.
In some aspects, the mutant recombinant polypeptides disclosed herein catalyze a reaction using any known substrate of a prenyltransferase such as ORF2, HypSc, PB002, PB005, PB064, PB065, and Atapt. In some aspects, the substrate is selected from the group consisting of OA, DVA, O, DV, ORA, DHBA, apigenin, naringenin and resveratrol.
In some aspects, the mutant recombinant polypeptide uses a substrate that is not a naturally occurring substrate of the WT prenyltransferase. A “naturally-occurring substrate” as used herein, refers to a substrate that is used by the WT prenyltransferase to catalyze a prenylation reaction in nature (such as, in the organism that the WT prenyltransferase is found in nature). For instance, a naturally occurring substrate of WT ORF2 is 1,3,6,8-tetrahydroxynaphthalene (THN); the disclosure provides ORF2 mutants that are able to use substrates other than THN (such as OA, apigenin, etc) in the prenylation reaction. Further details are provided in “Structural basis for the promiscuous biosynthetic prenylation of aromatic natural products” Kuzuyama et al., Nature volume 435, pages 983-987 (2005), the contents of which are incorporated by reference in its entirety.
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 recombinant polypeptides disclosed herein produce a higher amount of total nMol of prenylated products than the WT prenyltransferase. In some aspects, the mutant recombinant 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 prenyltransferase.
In some aspects, the mutant recombinant polypeptides disclosed herein have an enzymatic activity higher than WT prenyltransferase. In some aspects, the mutant recombinant 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 prenyltransferase.
The inventors have discovered a ratcheting mechanism of Orf2 mutants at Q161 and S214. WT enzyme contains an active site Q161 and 5214 which both form a weak hydrogen bond with the carboxylate of olivetolic acid, resulting in a 1:5 ratio CBGA:5GOA. Mutagenesis at position Q161 to Q161H, creating a more permanent hydrogen bond donor results in almost 100% CBGA production. Mutation to Q161P loses the hydrogen bond donor, as well as modifying the secondary structure at this position. Here the olivetolic acid flips its binding position within the active site, resulting in 97% 5GOA. Similarly 5214, which sits opposite in the pocket, can be mutated to S214H, which can also hydrogen bond to olivetolic acid carboxylate and also results in almost 100% CBGA production. Mutated to S214V also flips its binding position, resulting in 90% 5GOA. See
The inventors have also discovered a ratcheting mechanism of Orf2 mutants at Q295. The Q295 can interact with both the hydrocarbon tail of olivetolic acid, as well as the hydrophobic terminus of the GPP substrate. Mutation Q295 to Q295F enhances these hydrophobic interations, leading to 98% CBGA. Alternatively mutating to Q295H forms a protonated residue, which can destabilize the hydrocarbon tail, resulting in the substrate ratcheting binding orientation. The resulting hydrogen bond with the carboxylate of olivetolic acid stabilizes the flipped binding orientation, resulting in 90% 5GOA. See
The disclosure provides isolated or purified polynucleotides that encode any one of the recombinant 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 SEQ ID NO: 301 (ORF2); SEQ ID NO: 601 (PB005) and SEQ ID NO: 603 (PBJ).
The disclosure provides a vector comprising any one of the recombinant 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 recombinant polypeptides 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 recombinant polynucleotides 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 recombinant polynucleotides disclosed herein may be done in cell-free systems.
The disclosure provides methods of producing any one of the recombinant polynucleotides disclosed herein, comprising culturing the host cell comprising any one of the vectors disclosed herein, in a medium permitting expression of the recombinant polynucleotide, and isolating or purifying the recombinant polynucleotide from the host cell.
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 Varaints.
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. Construction of Synthesized Prenyltransferase Enzymes.
DNA plasmids encoding aromatic prenyltransferase enzymes (APTs) were ordered and delivered in the background of the T5 expression vector pD441-SR from DNA2.0 (now ATUM, catalog pD441-SR).
C. Expression and Purification of Proteins from the Synthesized Orf2 Gene Library of Orf2 Variants and Prenyltransferase Enzymes.
DNA plasmids containing each of the Orf2 variants or prenyltransferase enzymes 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” or “APTs” was individually inoculated into 2 milliliters LB media with 50 micrograms 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 (0D600) 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 and Aromatic Prenytransferase Enzymes for Protein Activity and Phenotypes.
The library of Orf2 variants and APTs 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 enzymes 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, Orf2 variant protein, or APT. These reactions were incubated for 16 hours at 30° C.
The prenylated products obtained from the various reactions described in these Examples is summarized in Table C below.
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. Clones that exhibited improved function relative to the wild type enzyme were subjected to “breakdown” analysis. “Breakdown” analysis involves creating all possible combinations of double mutations and all single combinations from the parental tripleton yielding 6 unique variant enzymes from a single parental tripleton. “Breakdown” variants were used to identify residues for site saturation where all 19 other amino acids were substituted at a single position.
The wild type Orf2 prenylation reaction using OA as substrate and DMAPP as donor produces 5 products as detected by HPLC. The respective retention times of these products are approximately 3.9, 5.44, 5.57, 6.29, and 6.66 minutes.
Table 1 provides a summary of the prenylation products produced from OA and DMAPP, their retention times, and the hypothesized prenylation site on OA.
Table 2 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce prenylated products using Olivetolic Acid (OA) as substrate and Dimethylallyl pyrophosphate (DMAPP) as donor. Table 2 lists the mutations within each of the mutants analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
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. Clones that exhibited improved function relative to the wild type enzyme were subjected to “breakdown” analysis. “Breakdown” analysis involves creating all possible combinations of double mutations and all single combinations from the parental tripleton yielding 6 unique variant enzymes from a single parental tripleton. “Breakdown” variants were used to identify residues for site saturation where all 19 other amino acids were substituted at a single position.
The wild type Orf2 prenylation reaction using OA as substrate and GPP as donor produces 6 products as detected by HPLC. The respective retention times of these products are approximately 6.14, 7.03 [CBGA], 7.27 [5-GOA], 8.17, 8.77, and 11.6 minutes.
Table 3 provides a summary of the prenylation products produced from OA and GPP, their retention times, and the hypothesized prenylation site on OA.
Table 4 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce prenylated products using OA as substrate and GPP as donor. Table 4 lists the mutations within each of the mutants analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
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. Clones that exhibited improved function relative to the wild type enzyme were subjected to “breakdown” analysis. “Breakdown” analysis involves creating all possible combinations of double mutations and all single combinations from the parental tripleton yielding 6 unique variant enzymes from a single parental tripleton. “Breakdown” variants were used to identify residues for site saturation where all 19 other amino acids were substituted at a single position.
The wild type Orf2 prenylation reaction using OA as substrate and FPP as donor produces 4 products as detected by HPLC. The respective retention times of these products are approximately 8.4 [CBFA], 8.8 [5-FOA], 9.9, and 11.1 minutes.
Table 5 provides a summary of the prenylation products produced from OA and FPP, their retention times, and the hypothesized prenylation site on OA.
Table 6 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce prenylated products using OA as substrate and FPP as donor. Table 6 lists the mutations within each of the mutants analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
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. Clones that exhibited improved function relative to the wild type enzyme were subjected to “breakdown” analysis. “Breakdown” analysis involves creating all possible combinations of double mutations and all single combinations from the parental tripleton yielding 6 unique variant enzymes from a single parental tripleton. “Breakdown” variants were used to identify residues for site saturation where all 19 other amino acids were substituted at a single position.
The wild type Orf2 prenylation reaction using O as substrate and GPP as donor produces 3 products as detected by HPLC. The respective retention times of these products are approximately 7.095 [CBG], 7.745 [5-GO], and 8.563 minutes.
Table 7A provides a summary of the prenylation products produced from O and GPP, their retention times, and the hypothesized prenylation site on O.
Tables 7B-7D provide NMR data of proton and carbon chemical shifts for CBG with (a) HSQC, (b) HMBC correlation and (c) final carbon and proton NMR assignments. The carbon and proton NMR assignments for CBG are shown in
Table 8 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce prenylated products using O as substrate and GPP as donor. Table 8 lists the mutations within each of the mutants analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
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. Clones that exhibited improved function relative to the wild type enzyme were subjected to “breakdown” analysis. “Breakdown” analysis involves creating all possible combinations of double mutations and all single combinations from the parental tripleton yielding 6 unique variant enzymes from a single parental tripleton. “Breakdown” variants were used to identify residues for site saturation where all 19 other amino acids were substituted at a single position.
The wild type Orf2 prenylation reaction using DVA as substrate and GPP as donor produces 6 products as detected by HPLC. The respective retention times of these products are approximately 5.28, 6.39, 6.46, 7.31, 7.85, and 10.79 minutes.
Table 9A provides a summary of the prenylation products produced from DVA and GPP, their retention times, and the hypothesized prenylation site on DVA.
Tables 9B-9D provide NMR data of proton and carbon chemical shifts for CBGVA with (a) HSQC, (b) HMBC correlation and (c) final carbon and proton NMR assignments (the HMBC “Proton list” column in all NMR assignment tables displays protons which are J-Coupled to and within 1-4 carbons of the corresponding carbon in the row). The carbon and proton NMR assignments for CBGVA are shown in
Tables 9E-9G provide NMR data of proton and carbon chemical shifts for RBI-29 with (a) HSQC, (b) HMBC correlation and (c) final carbon and proton NMR assignments. The carbon and proton NMR assignments for RBI-29 are shown in
Table 10 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce prenylated products using DVA as substrate and GPP as donor. Table 10 lists the mutations within each of the mutants analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
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. Clones that exhibited improved function relative to the wild type enzyme were subjected to “breakdown” analysis. “Breakdown” analysis involves creating all possible combinations of double mutations and all single combinations from the parental tripleton yielding 6 unique variant enzymes from a single parental tripleton. “Breakdown” variants were used to identify residues for site saturation where all 19 other amino acids were substituted at a single position.
The wild type Orf2 prenylation reaction using DVA as substrate and FPP as donor produces 5 products as detected by HPLC. The respective retention times of these products are approximately 7.05, 7.84, 8.03, 8.24, and 9.72 minutes.
Table 11 provides a summary of the prenylation products produced from DVA and FPP, their retention times, and the hypothesized prenylation site on DVA.
Table 12 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce prenylated products using DVA as substrate and FPP as donor. Table 12 lists the mutations within each of the mutants analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
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. Clones that exhibited improved function relative to the wild type enzyme were subjected to “breakdown” analysis. “Breakdown” analysis involves creating all possible combinations of double mutations and all single combinations from the parental tripleton yielding 6 unique variant enzymes from a single parental tripleton. “Breakdown” variants were used to identify residues for site saturation where all 19 other amino acids were substituted at a single position. A subset of Orf2 Mutant enzymes were screened for prenylation when using Orsillenic Acid (ORA) as substrate and GPP as donor.
The wild type Orf2 prenylation reaction using ORA as substrate and GPP as donor produces 6 products as detected by HPLC. The respective retention times of these products are approximately 4.6, 5.7, 5.83, 6.35, 7.26, and 9.26 minutes.
Table 13A provides a summary of the prenylation products produced from ORA and GPP, their retention times, and the hypothesized prenylation site on ORA.
Tables 13B-13D provide NMR data of proton and carbon chemical shifts for UNK59 with (a) HSQC, (b) HMBC correlation and (c) final carbon and proton NMR assignments. The carbon and proton NMR assignments for UNK59 are shown in
Table 14 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce prenylated products using ORA as substrate and GPP as donor. Table 14 lists the mutations within each of the mutants analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
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. Clones that exhibited improved function relative to the wild type enzyme were subjected to “breakdown” analysis. “Breakdown” analysis involves creating all possible combinations of double mutations and all single combinations from the parental tripleton yielding 6 unique variant enzymes from a single parental tripleton. “Breakdown” variants were used to identify residues for site saturation where all 19 other amino acids were substituted at a single position. A subset of Orf2 Mutant enzymes were screened for prenylation when using Apigenin as substrate and GPP as donor.
The wild type Orf2 prenylation reaction using Apigenin as substrate and GPP as donor produces 5 products as detected by HPLC. The respective retention times of these products are approximately 5.84, 6.77, 7.36, 7.68, and 8.19 minutes.
Table 15 provides a summary of the prenylation products produced from Apigenin and GPP, their retention times, and the hypothesized prenylation site on Apigenin.
Table 16 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce prenylated products using Apigenin as substrate and GPP as donor. Table 16 lists the mutations within each of the mutants analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
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. Clones that exhibited improved function relative to the wild type enzyme were subjected to “breakdown” analysis. “Breakdown” analysis involves creating all possible combinations of double mutations and all single combinations from the parental tripleton yielding 6 unique variant enzymes from a single parental tripleton. “Breakdown” variants were used to identify residues for site saturation where all 19 other amino acids were substituted at a single position. A subset of Orf2 Mutant enzymes were screened for prenylation when using Naringenin as substrate and GPP as donor.
The wild type Orf2 prenylation reaction using Naringenin as substrate and GPP as donor produces 2 products as detected by HPLC. The respective retention times of these products are approximately 6.86 and 7.49 minutes.
Table 17 provides a summary of the prenylation products produced from Naringenin and GPP, their retention times, and the hypothesized prenylation site on Naringenin.
Table 18 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce prenylated products using Naringenin as substrate and GPP as donor. Table 18 lists the mutations within each of the mutants analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
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. Clones that exhibited improved function relative to the wild type enzyme were subjected to “breakdown” analysis. “Breakdown” analysis involves creating all possible combinations of double mutations and all single combinations from the parental tripleton yielding 6 unique variant enzymes from a single parental tripleton. “Breakdown” variants were used to identify residues for site saturation where all 19 other amino acids were substituted at a single position. A subset of Orf2 Mutant enzymes were screened for prenylation when using Reservatrol as substrate and GPP as donor.
The wild type Orf2 prenylation reaction using Reservatrol as substrate and GPP as donor produces 4 products as detected by HPLC. The respective retention times of these products are approximately 5.15, 5.87, 7.3, and 8.44 minutes.
Table 19 provides a summary of the prenylation products produced from Reservatrol and GPP, their retention times, and the hypothesized prenylation site on Reservatrol.
Table 20 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce prenylated products using Reservatrol as substrate and GPP as donor. Table 20 lists the mutations within each of the mutants analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
Aromatic Prenyltransferase Enzymes were ordered, expressed, purified, and screened for prenylation as described in Example 1.
The prenylation reaction using ORA as substrate and DMAPP as donor produces 5 products as detected by HPLC. The respective retention times of these products are approximately 2.5, 2.77, 2.89, 4.78, and 4.96 minutes.
Table 21 provides a summary of the prenylation products produced from ORA and DMAPP, their retention times, and the hypothesized prenylation site on ORA.
Table 22 provides a summary of the analysis performed on the enzymatic activity of the APT enzymes to produce prenylated products using ORA as substrate and DMAPP as donor. Table 22 lists the APTs analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
Aromatic Prenyltransferase Enzymes were ordered, expressed, purified, and screened for prenylation as described in Example 1.
The prenylation reaction using DV as substrate and DMAPP as donor produces 5 products as detected by HPLC. The respective retention times of these products are approximately 4.04, 4.65, 5.26, 6.83, and 7.06 minutes.
Table 23 provides a summary of the prenylation products produced from DV and DMAPP, their retention times, and the hypothesized prenylation site on DV.
Table 24 provides a summary of the analysis performed on the enzymatic activity of the aromatic prenyltransferase enzymes to produce prenylated products using DV as substrate and DMAPP as donor. Table 24 lists the APTs analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
Aromatic Prenyltransferase Enzymes were ordered, expressed, purified, and screened for prenylation as described in Example 1.
The prenylation reaction using DV as substrate and GPP as donor produces 2 products as detected by HPLC. The respective retention times of these products are approximately 6.37 and 6.88 minutes.
Table 25 provides a summary of the prenylation products produced from DV and GPP, their retention times, and the hypothesized prenylation site on DV.
Table 26 provides a summary of the analysis performed on the enzymatic activity of the aromatic prenyltransferase enzymes to produce prenylated products using DV as substrate and GPP as donor. Table 26 lists the APTs analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
Aromatic Prenyltransferase Enzymes were ordered, expressed, purified, and screened for prenylation as described in Example 1.
The prenylation reaction using DVA as substrate and DMAPP as donor produces 4 products as detected by HPLC. The respective retention times of these products are approximately 4.21, 4.29, 4.84, and 5.55 minutes.
Table 27 provides a summary of the prenylation products produced from DVA and DMAPP, their retention times, and the hypothesized prenylation site on DVA.
Table 28 provides a summary of the analysis performed on the enzymatic activity of the aromatic prenyltransferase enzymes to produce prenylated products using DVA as substrate and DMAPP as donor. Table 26 lists the APTs analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
Aromatic Prenyltransferase Enzymes were ordered, expressed, purified, and screened for prenylation as described in Example 1.
The prenylation reaction using O as substrate and DMAPP as donor produces 5 products as detected by HPLC. The respective retention times of these products are approximately 5.46, 6.04, 6.98, 7.65, and 7.91 minutes.
Table 29 provides a summary of the prenylation products produced from O and DMAPP, their retention times, and the hypothesized prenylation site on O.
Table 30-a provides a summary of the analysis performed on the enzymatic activity of the aromatic prenyltransferase enzymes to produce prenylated products using O as substrate and DMAPP as donor. Table 30-a lists APTs analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
The amount of each prenylation product was measured by HPLC.
CBGA produced from an aromatic prenyltransferase reaction with OA and GPP and ORF2 or Orf2 variants as described in Example 3 was purified and used as a substrate in a subsequent aromatic prenyltransferase reaction with Orf2 or Orf2 variants and DMAPP as the donor. The prenylation reaction was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 4 millimolar DMAPP, 100 millimolar HEPES buffer at a pH of 7.5, 2 millimolar CBGA, and 40 micrograms Orf2 variant protein. These reactions were incubated for 16 hours at 30° C.
The prenylation reaction using CBGA as substrate and DMAPP as donor produced a product as detected by HPLC with a retention time of approximately 9.095 minutes.
Table 30-b provides a summary of the prenylation product produced from CBGA and DMAPP, the retention times, and the hypothesized prenylation site on CBGA.
RBI-04 (5-GOA) produced from an aromatic prenyltransferase reaction with OA and GPP using Orf2 or Orf2 variants as the prenyltransferase as described in Example 3 was purified and used as a substrate in a subsequent aromatic prenyltransferase reaction using Orf2 or Orf2 variants as the prenyltransferase. The prenylation reaction was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 4 millimolar DMAPP, 100 millimolar HEPES buffer at a pH of 7.5, 2 millimolar CBGA, and 40 micrograms Orf2 variant protein. These reactions were incubated for 16 hours at 30° C.
The prenylation reaction using RBI-04 (5-GOA) as substrate and DMAPP as donor produced a product as detected by HPLC with a retention time of approximately 9.088 minutes.
Table 31 provides a summary of the prenylation product produced from RBI-04 (5-GOA) and DMAPP, the retention times and the hypothesized prenylation site on RBI-04 (5-GOA).
RBI-04 (5-GOA) produced from an aromatic prenyltransferase reaction with OA and GPP using Orf2 or Orf2 variants as the prenyltransferase as described in Example 3 was purified and used as a substrate in a subsequent aromatic prenyltransferase reaction using Orf2 or Orf2 variants as the prenyltransferase. The prenylation reaction was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 4 millimolar FPP, 100 millimolar HEPES buffer at a pH of 7.5, 2 millimolar RBI-04 (5-GOA), and 40 micrograms Orf2 variant protein. These reactions were incubated for 16 hours at 30° C.
The prenylation reaction using RBI-04 (5-GOA) as substrate and FPP as donor produced a product as detected by HPLC with a retention time of approximately 16.59 minutes.
Table 32 provides a summary of the prenylation product produced from RBI-04 (5-GOA) and FPP, the retention times and the hypothesized prenylation site on RBI-04 (5-GOA).
RBI-04 (5-GOA) produced from an aromatic prenyltransferase reaction with OA and GPP using Orf2 or Orf2 variants as the prenyltransferase as described in Example 3 was purified and used as a substrate in a subsequent aromatic prenyltransferase reaction using Orf2 or Orf2 variants as the prenyltransferase. The prenylation reaction was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 2 millimolar GPP, 100 millimolar HEPES buffer at a pH of 7.5, 2 millimolar RBI-04 (5-GOA), and 20 micrograms Orf2 variant protein. These reactions were incubated for 16 hours at 30° C.
The prenylation reaction using RBI-04 (5-GOA) as substrate and GPP as donor produced a product as detected by HPLC with a retention time of approximately 11.6 minutes.
Table 33 provides a summary of the prenylation product produced from RBI-04 (5-GOA) and GPP, the retention times and the hypothesized prenylation site on RBI-04 (5-GOA).
RBI-08 produced from an aromatic prenyltransferase reaction with OA and DMAPP using Orf2 or Orf2 variants as the prenyltransferase as described in Example 2 was purified and used as a substrate in a subsequent aromatic prenyltransferase reaction using Orf2 or Orf2 variants as the prenyltransferase. The prenylation reaction was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 4 millimolar DMAPP, 100 millimolar HEPES buffer at a pH of 7.5, 1 millimolar RBI-08, and 40 micrograms Orf2 variant protein. These reactions were incubated for 16 hours at 30° C.
The prenylation reaction using RBI-08 as substrate and DMAPP as donor produced a product as detected by HPLC with a retention time of approximately 7.55 minutes.
Table 34 provides a summary of the prenylation product produced from RBI-08 and DMAPP, the retention times and the hypothesized prenylation site on RBI-08.
RBI-08 produced from an aromatic prenyltransferase reaction with OA and DMAPP using Orf2 or Orf2 variants as the prenyltransferase as described in Example 2 was purified and used as a substrate in a subsequent aromatic prenyltransferase reaction using Orf2 or Orf2 variants as the prenyltransferase The prenylation reaction was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 4 millimolar GPP, 100 millimolar HEPES buffer at a pH of 7.5, 2 millimolar RBI-08, and 40 micrograms Orf2 variant protein. These reactions were incubated for 16 hours at 30° C.
The prenylation reaction using RBI-08 as substrate and GPP as donor produced 2 products as detected by HPLC with retention times of approximately 8.22 and 9.1 minutes.
Table 35 provides a summary of the prenylation products produced from RBI-08 and GPP, the retention times and the hypothesized prenylation sites on RBI-08.
RBI-09 produced from an aromatic prenyltransferase reaction with 0 and DMAPP as described in Example 16 was purified and used as a substrate in a subsequent aromatic prenyltransferase reaction using Orf2 or Orf2 variants and GPP as the donor. The first prenyltransferase reaction can include any of the prenyltransferases listed in Example 16. The prenylation reaction was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 4 millimolar GPP, 100 millimolar HEPES buffer at a pH of 7.5, 2 millimolar RBI-09, and 40 micrograms Orf2 variant protein. These reactions were incubated for 16 hours at 30° C.
The prenylation reaction using RBI-09 as substrate and GPP as donor produced a product as detected by HPLC with a retention time of approximately 9.26 minutes.
Table 36 provides a summary of the prenylation product produced from RBI-09 and GPP, the retention times and the hypothesized prenylation sites on RBI-09.
RBI-010 produced from an aromatic prenyltransferase reaction with 0 and DMAPP as described in Example 16 was purified and used as a substrate in a subsequent aromatic prenyltransferase reaction using PB-005 or PB-006 as the prenyltransferase and DMAPP as the donor. The prenylation reaction was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 2 millimolar DMAPP, 100 millimolar HEPES buffer at a pH of 7.5, 2 millimolar RBI-10, and 20 micrograms APT protein. Two APT enzymes were tested. These reactions were incubated for 16 hours at 30° C.
The prenylation reaction using RBI-10 as substrate and DMAPP as donor produced 2 product as detected by HPLC with a retention times of approximately 7.65 and 7.91 minutes.
Table 37 provides a summary of the prenylation products produced from RBI-10 and DMAPP, the retention times and the hypothesized prenylation sites on RBI-10.
RBI-010 produced from an aromatic prenyltransferase reaction with 0 and DMAPP as described in Example 16 was purified and used as a substrate in a subsequent aromatic prenyltransferase reaction using PB-005 or Orf2 variants as the prenyltransferase and FPP as the donor. The prenylation reaction was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 4 millimolar FPP, 100 millimolar HEPES buffer at a pH of 7.5, 2 millimolar RBI-10, and 40 micrograms APT protein. Two APT enzymes were tested. These reactions were incubated for 16 hours at 30° C.
The prenylation reaction using RBI-10 as substrate and FPP as donor produced 2 products as detected by HPLC with a retention times of approximately 11.8 and 12.9 minutes.
Table 38 provides a summary of the prenylation products produced from RBI-10 and FPP, the retention times and the hypothesized prenylation sites on RBI-10.
RBI-010 produced from an aromatic prenyltransferase reaction with 0 and DMAPP as described in Example 16 was purified and used as a substrate in a subsequent aromatic prenyltransferase reaction using Orf2 variants as the prenyltransferase and GPP as the donor. The prenylation reaction was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 4 millimolar GPP, 100 millimolar HEPES buffer at a pH of 7.5, 2 millimolar RBI-10, and 40 micrograms Orf2 Variant protein. These reactions were incubated for 16 hours at 30° C.
The prenylation reaction using RBI-10 as substrate and GPP as donor produced 2 products as detected by HPLC with a retention times of approximately 9.2 and 9.7 minutes.
Table 39 provides a summary of the prenylation products produced from RBI-10 and GPP, the retention times and the hypothesized prenylation sites on RBI-10.
RBI-12 produced from an aromatic prenyltransferase reaction as described in Example 16 (1 reactions) or Example 24 (2 sequential reactions) was purified and used as a substrate in a subsequent aromatic prenyltransferase reaction using Orf2 variants as the prenyltransferase and GPP as the donor. The prenylation reaction was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 4 millimolar GPP, 100 millimolar HEPES buffer at a pH of 7.5, 2 millimolar RBI-12, and 40 micrograms Orf2 Variant protein. These reactions were incubated for 16 hours at 30° C.
The prenylation reaction using RBI-12 as substrate and GPP as donor produced a product as detected by HPLC with a retention time of approximately 11.27 minutes.
Table 40 provides a summary of the prenylation products produced from RBI-12 and GPP, the retention times and the hypothesized prenylation sites on RBI-12.
RBI-03 produced from an aromatic prenyltransferase reaction with 0 as substrate and GPP as donor as described in Example 5 was purified and used as a substrate in a subsequent aromatic prenyltransferase reaction with PB-005 as the prenyltransferase and GPP as the donor. The prenylation reaction was performed in a volume of 20 microliters and contained 20 millimolar magnesium chloride (MgCl2), 4 millimolar DMAPP, 100 millimolar HEPES buffer at a pH of 7.5, 2 millimolar RBI-03, and 40 micrograms APT enzyme. These reactions were incubated for 16 hours at 30° C.
The prenylation reaction using RBI-03 as substrate and DMAPP as donor produced 2 products as detected by HPLC with retention times of approximately 9.3 and 9.7 minutes.
Table 41 provides a summary of the prenylation products produced from RBI-03 and DMAPP, the retention times and the hypothesized prenylation sites on RBI-03.
Aromatic Prenyltransferase Enzymes were ordered, expressed, purified, and screened for prenylation as described in Example 1.
The prenylation reaction using O as substrate and FPP as donor produces 3 products as detected by HPLC. The respective retention times of these products are approximately 8.52, 9.57, and 10.94 minutes.
Table 42 provides a summary of the prenylation products produced from O and FPP, their retention times, and the hypothesized prenylation site on O.
Table 43 provides a summary of the analysis performed on the enzymatic activity of the aromatic prenyltransferase enzymes to produce prenylated products using O as substrate and FPP as donor. Table 43 lists APTs analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
Aromatic Prenyltransferase Enzymes were ordered, expressed, purified, and screened for prenylation as described in Example 1.
The prenylation reaction using ORA as substrate and FPP as donor produces 3 products as detected by HPLC. The respective retention times of these products are approximately 7.44, 7.98, and 8.96 minutes.
Table 44 provides a summary of the prenylation products produced from ORA and FPP, their retention times, and the hypothesized prenylation site on ORA.
Table 45 provides a summary of the analysis performed on the enzymatic activity of the aromatic prenyltransferase enzymes to produce prenylated products using ORA as substrate and FPP as donor. Table 45 lists APTs analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
Aromatic Prenyltransferase Enzymes were ordered, expressed, purified, and screened for prenylation as described in Example 1.
The prenylation reaction using OA as substrate and GGPP as donor produces 2 products as detected by HPLC. The respective retention times of these products are approximately 10.29 and 11.18 minutes.
Table 46 provides a summary of the prenylation products produced from OA and GGPP, their retention times, and the hypothesized prenylation site on OA.
Table 47 provides a summary of the analysis performed on the enzymatic activity of the aromatic prenyltransferase enzymes to produce prenylated products using OA as substrate and GGPP as donor. Table 47 lists APTs analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
Aromatic Prenyltransferase Enzymes were ordered, expressed, purified, and screened for prenylation as described in Example 1.
The prenylation reaction using ORA as substrate and GGPP as donor produces 2 products as detected by HPLC. The respective retention times of these products are approximately 8.98 and 9.06 minutes.
Table 48 provides a summary of the prenylation products produced from ORA and GGPP, their retention times, and the hypothesized prenylation site on ORA.
Table 49 provides a summary of the analysis performed on the enzymatic activity of the aromatic prenyltransferase enzymes to produce prenylated products using ORA as substrate and GGPP as donor. Table 49 lists APTs analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
Aromatic Prenyltransferase Enzymes were ordered, expressed, purified, and screened for prenylation as described in Example 1.
The prenylation reaction using DVA as substrate and GGPP as donor produces 2 products as detected by HPLC. The respective retention times of these products are approximately 9.48 and 9.87 minutes.
Table 50 provides a summary of the prenylation products produced from DVA and GGPP, their retention times, and the hypothesized prenylation site on DVA.
Table 51 provides a summary of the analysis performed on the enzymatic activity of the aromatic prenyltransferase enzymes to produce prenylated products using DVA as substrate and GGPP as donor. Table 51 lists APTs analyzed as well mAU*min areas from the HPLC analysis of the reaction products.
Table 52 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce CBFA and 5-FOA using Olivetolic Acid (OA) as substrate and FPP as donor. Table 52 lists the mutations within each of the tripleton mutants as well the nMol of CBFA produced, nMol of 5-FOA produced, total prenylated products produced (nMol of CBFA+5-FOA), % CBFA within total prenylated products (nMol of CBFA/[nMol of CBFA+5-FOA]), % enzymatic activity (total prenylated products produced by a mutant/total prenylated products produced by wild-type ORF2), CBFA production (% CBFA among total prenylated products*% enzymatic activity), and %5-FOA within prenylated products (nMol of 5-FOA/[nMol of CBFA+5-FOA]) for each of the ORF2 variants.
The amount of CBFA or 5-FOA (in nMols) generated by each of the ORF2 triple mutant clones was measured using HPLC.
While the CBFA production potential analysis shown in
Based on the analysis performed in
For the singleton and doubleton mutants resulting from the breakdown of triple mutants—H03, A04, C06, CO5, A09, H02, D04, G12, F09, A05, D11 and E09—the total amount of prenylated products (and the respective proportion of CBFA and 5-FOA); and % CBFA within the prenylated products was calculated.
In a similar manner, the triple mutants, H03, C06, A05 and G12, will also be subjected to “breakdown” analysis. Further, the singleton and double mutants resulting from the breakdown of H03, C06, A05 and G12, will be analyzed to determine the total amount of prenylated products (and the respective proportion of CBFA and 5-FOA); and % CBFA 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 FPP as donor. Based on this analysis, the amino acid sites listed in Table 55 were selected for targeted amino acid site saturation mutagenesis.
Site saturated mutagenesis was done for Q295, Q161, and S214 by replacing the wild type residue with each of the other 19 standard amino acids. The amount of total prenylated products, the CBFA 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 the other amino acid residues targeted for site saturation listed in Table 55; and the amount of total prenylated products and the CBFA 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 CBFA 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 FPP 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; % enzymatic activity, % CBFA, and CBFA production potential. The analysis of the stacking mutants shows that multiple stacking mutants have significantly higher % enzymatic activity, % CBFA, and CBFA production potential, compared to the WT ORF2 or either singleton substitution variant on its own, 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 OA and FPP, as compared to WT ORF2.
For instance, ORF2 double mutants—S214R-Q295F; S177W-Q295A; A53T-Q295F; and Q161S-Q295L have synergistically enhanced CBFA 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 additional triple mutants and planned site saturation mutagenesis experiments described above. These stacking mutants will further be analyzed to determine their % enzymatic activity, % CBFA, %5-FOA and CBFA production potential.
Table 60 provides a summary of the analysis performed on the enzymatic activity of the ORF2 variants to produce CBGA and 5-DOA using Olivetolic Acid (OA) as substrate and DMAPP as donor. Table 60 lists the mutations within each of the tripleton mutants as well the nMol of 3-DOA produced, nMol of 5-DOA produced, total prenylated products produced (nMol of 3-DOA+5-DOA), %3-DOA within total prenylated products (nMol of 3-DOA/[nMol of 3-DOA+5-DOA]), % enzymatic activity (total prenylated products produced by a mutant/total prenylated products produced by wild-type ORF2), 3-DOA production (%3-DOA among total prenylated products*% enzymatic activity), and %5-DOA within prenylated products (nMol of 5-DOA/[nMol of 3-DOA+5-DOA]) for each of the ORF2 variants.
The amount of 3-DOA or 5-DOA (in nMols) generated by each of the ORF2 triple mutant clones was measured using HPLC.
While the 3-DOA production potential analysis shown in
Based on the analysis performed in
Breakdown analysis for these triple mutants will be performed as described above in Example 34. The singleton and double mutants resulting from the breakdown of these mutants will be analyzed to determine the total amount of prenylated products (and the respective proportion of 5-DOA and 3-DOA); and %3-DOA within the prenylated products produced by these mutants.
Further, based on the analysis of the breakdown mutants, amino acid sites will be selected for targeted amino acid site saturation mutagenesis, as described above in Example 34; and mutants that have significantly higher 3-DOA production potential and/or the total amount of prenylated products, as compared to WT ORF2, will be identified. Finally, ORF2 stacking mutants that carry different novel combinations of the mutations identified by the analysis as being important for ORF2's enzymatic activity will be generated. These stacking mutants will further be analyzed to determine their % enzymatic activity, %3-DOA, %5-DOA and 3-DOA production potential.
The Proton NMR signals of selected compound were obtained in DMSO at 600 MHz and the proton NMR assignments of these compounds were shown in
This application claims the benefit of U.S. Provisional Application No. 62/833,449, filed Apr. 12, 2019, which application is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/027955 | 4/13/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62833449 | Apr 2019 | US |
