Enzyme engineering to alter the functional repertoire of cannabinoid synthases

Abstract

Described herein are variant, novel cannabinoid synthases, nucleic acids encoding same, and various uses thereof.

What is claimed is: 
     
         1 . A variant cannabinoid synthase or an active fragment thereof comprising a non-naturally occurring amino acid sequence relative to a wild-type cannabinoid synthase or an active fragment thereof which acts on a substrate to produce an altered amount of a cannabinoid relative to an amount of the cannabinoid produced by the wild-type cannabinoid synthase or active fragment thereof. 
     
     
         2 . The variant cannabinoid synthase or active fragment thereof of  claim 1 , wherein the variant cannabinoid synthase is a variant cannabidiolic acid (CBDA) synthase comprising a non-naturally occurring amino acid sequence relative to a wild type CBDA synthase of SEQ ID NO: 1043. 
     
     
         3 . The variant cannabinoid synthase or active fragment thereof of  claim 1 , wherein the variant cannabinoid synthase is a variant cannabidiolic acid (CBDA) synthase comprising a non-naturally occurring amino acid sequence relative to a wild type consensus CBDA synthase of SEQ ID NO: 1046. 
     
     
         4 . The variant cannabinoid synthase or active fragment thereof of  claim 1 , wherein the variant cannabinoid synthase is a tetrahydrocannabinolic acid (THCA) synthase comprising a non-naturally occurring amino acid sequence relative to a wild type THCA synthase of SEQ ID NO: 1044. 
     
     
         5 . The variant cannabinoid synthase or active fragment thereof of  claim 1 , wherein the variant cannabinoid synthase is a tetrahydrocannabinolic acid (THCA) synthase comprising a non-naturally occurring amino acid sequence relative to a wild type consensus THCA synthase of SEQ ID NO: 1047. 
     
     
         6 . The variant cannabinoid synthase or active fragment thereof of  claim 1 , wherein the variant cannabinoid synthase is a cannabichromenic acid (CBCA) synthase comprising a non-naturally occurring amino acid sequence relative to a wild type CBCA synthase of SEQ ID NO: 1045. 
     
     
         7 . The variant cannabinoid synthase or active fragment thereof of  claim 1 , wherein the variant cannabinoid synthase is a cannabichromenic acid (CBCA) synthase comprising a non-naturally occurring amino acid sequence relative to a wild type consensus CBCA synthase of SEQ ID NO: 1048. 
     
     
         8 . The variant cannabinoid synthase or active fragment thereof of  claim 1 , wherein the cannabinoid is selected from the group consisting of: tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol (THC), tetrahydrocannabinvarin (THCV), cannabidiolic acid (CBDA), cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabichromevarin (CBCV), cannabichromenic acid (CBCA), and a combination thereof. 
     
     
         9 . The variant cannabinoid synthase or active fragment thereof of  claim 1 , wherein the substrate is a naturally occurring substrate. 
     
     
         10 . The variant cannabinoid synthase or active fragment thereof of  claim 9 , wherein the naturally occurring substrate is selected from the group consisting of cannabigerol (CBG), cannabigerolic acid (CBGA), cannbigerovarinic acid (GBGVA), and any homolog thereof. 
     
     
         11 . The variant cannabinoid synthase or active fragment thereof of  claim 1 , wherein the substrate is a non-naturally occurring substrate. 
     
     
         12 . The variant cannabinoid synthase or active fragment thereof of  claim 11 , wherein the non-naturally occurring substrate can comprise: a non-naturally occurring tail variant, a prenyl donor, or a combination thereof. 
     
     
         13 . The variant cannabinoid synthase or active fragment thereof of  claim 1 , wherein the altered amount of the cannabinoid produces a change in a proportion of a first cannabinoid to a second cannabinoid. 
     
     
         14 . The variant cannabinoid synthase or active fragment thereof of  claim 13 , wherein the variant cannabinoid synthase is a variant CBDA synthase. 
     
     
         15 . The variant cannabinoid synthase or active fragment thereof of  claim 14 , wherein the first cannabinoid is CBDA and the second cannabinoid is THCA. 
     
     
         16 . The variant cannabinoid synthase or active fragment thereof of  claim 15 , wherein a proportion of CBDA:THCA produced by a wild type CBDA synthase is about 95:5. 
     
     
         17 . The variant cannabinoid synthase or active fragment thereof of  claim 14 , wherein the non-naturally occurring amino acid sequence comprises an amino acid mutation at position 445 relative to a wild-type consensus CBDA synthase amino acid sequence set forth in SEQ ID NO: 1045. 
     
     
         18 . The variant cannabinoid synthase or active fragment thereof of  claim 17 , wherein the amino acid mutation at position 445 is selected from the group consisting of: I445M and I445L. 
     
     
         19 . The variant cannabinoid synthase or active fragment thereof of  claim 1 , wherein the altered amount of the cannabinoid is an increase or a decrease in a yield of the cannabinoid. 
     
     
         20 . The variant cannabinoid synthase or active fragment thereof of  claim 19 , wherein the yield is a nMol of the cannabinoid produced per milligram of the substrate. 
     
     
         21 . The variant cannabinoid synthase or active fragment thereof of  claim 19 , wherein the decrease in the yield is less than 100%, less than 200%, or less than 300% of a yield of the cannabinoid of the wild-type cannabinoid synthase or active fragment thereof. 
     
     
         22 . The variant cannabinoid synthase or active fragment thereof of  claim 19 , wherein the increase in the yield is more than 100%, more than 200%, or more than 300% of a yield of the cannabinoid of the wild-type cannabinoid synthase or active fragment thereof. 
     
     
         23 . The variant cannabinoid synthase or active fragment thereof of  claim 22 , wherein the variant cannabinoid synthase is a variant CBDA synthase. 
     
     
         24 . The variant cannabinoid synthase or active fragment thereof of  claim 23 , wherein the non-naturally occurring amino acid sequence comprises at least one amino acid mutation at position 69, 414, or 445 relative to a wild-type consensus CBDA synthase amino acid sequence set forth in SEQ ID NO: 1045. 
     
     
         25 . The variant cannabinoid synthase or active fragment thereof of  claim 24 , wherein the at least one amino acid mutation at position 69 is selected from the group consisting of: H69R, H69G, H69K, H69Q, H69A, and H69V. 
     
     
         26 . The variant cannabinoid synthase or active fragment thereof of  claim 24 , wherein the at least one amino acid mutation at position 414 is selected from the group consisting of: A414T, A414I, and A414V. 
     
     
         27 . The variant cannabinoid synthase or active fragment thereof of  claim 24 , wherein the at least one amino acid mutation at position 445 is 1445V. 
     
     
         28 . The variant cannabinoid synthase or active fragment thereof of  claim 23 , wherein the non-naturally occurring amino acid sequence comprises at least two amino acid mutations at positions selected from the group consisting of: 69, 180, 414, and 445 relative to a wild-type consensus CBDA synthase amino acid sequence set forth in SEQ ID NO: 1045. 
     
     
         29 . The variant cannabinoid synthase or active fragment thereof of  claim 28 , wherein one of the at least two amino acid mutations is at amino acid position 69. 
     
     
         30 . The variant cannabinoid synthase or active fragment thereof of  claim 29 , wherein one of the at least two amino acid mutations at amino acid position 69 is selected from the group consisting of H69A, H69C, H69D, H69E, H69F, H69G, H69I, H69K, H69L, H69M, H69N, H69P, H69Q, H69R, H69S, H69T, H69V, H69W, and H69Y. 
     
     
         31 . The variant cannabinoid synthase or active fragment thereof of  claim 29 , wherein one of the at least two amino acid mutations at amino acid position 69 is selected from the group consisting of H69K, H69Q, H69V, and H69G. 
     
     
         32 . The variant cannabinoid synthase or active fragment thereof of  claim 28 , wherein one of the at least two amino acid mutations is at amino acid position 180. 
     
     
         33 . The variant CBDA synthase or active fragment thereof of  claim 32 , wherein one of the at least two amino acid mutations at amino acid position 180 is selected from the group consisting of C180A, C180D, C180E, C180F, C180G, C180H, C180I, C180K, C180L, C180M, C180N, C180P, C180Q, C180R, C180S, C180T, C180V, C180W, and C180Y. 
     
     
         34 . The variant cannabinoid synthase or active fragment thereof of  claim 28 , wherein one of the at least two amino acid mutations is at amino acid position 414. 
     
     
         35 . The variant cannabinoid synthase or active fragment thereof of  claim 34 , wherein one of the at least two amino acid mutations at amino acid position 414 is selected from the group consisting of A414C, A414D, A414E, A414F, A414G, A414H, A414I, A414K, A414L, A414M, A414N, A414P, A414Q, A414R, A414S, A414T, A414V, A414W, and A414Y. 
     
     
         36 . The variant cannabinoid synthase or active fragment thereof of  claim 34 , wherein one of the at least two amino acid mutations at amino acid position 414 is selected from the group consisting of: A414V and A414I. 
     
     
         37 . The variant cannabinoid synthase or active fragment thereof of  claim 28 , wherein one of the at least two amino acid mutations is at amino acid position 445. 
     
     
         38 . The variant cannabinoid synthase or active fragment thereof of  claim 37 , wherein one of the at least two amino acid mutations at amino acid position 445 is selected from the group consisting of I445A, I445C, I445D, I445E, I445F, I445G, I445H, 1445K, I445L, I445M, I445N, I445P, I445Q, I445R, I445S, I445T, I445V, 1445W, and I445Y. 
     
     
         39 . The variant cannabinoid synthase or active fragment thereof of  claim 37 , wherein one of the at least two amino acid mutations at amino acid position 445 is I445M. 
     
     
         40 . The variant cannabinoid synthase or active fragment thereof of  claim 28 , wherein the at least two amino acid mutations are at a pair of positions selected from the group consisting of: 69/180, 69/414, 69/445, 180/414, 180/445, and 414/445. 
     
     
         41 . The variant cannabinoid synthase or active fragment thereof of  claim 28 , wherein the at least two amino acid mutations are selected from the group consisting of: A414V/H69K, A414V/H69Q, A414V/H69V, A414V/H69G, A414V/I44M, A414I/H69K, I445M/H69K, and I445M/H69Q. 
     
     
         42 . The variant cannabinoid synthase or active fragment thereof of  claim 1 , wherein the non-naturally occurring amino acid sequence comprises at least three amino acid mutations at positions selected from the group consisting of: 69, 180, 414, and 445 relative to a wild-type consensus CBDA synthase amino acid sequence set forth in SEQ ID NO: 1045. 
     
     
         43 . The variant cannabinoid synthase or active fragment thereof of  claim 42 , wherein the at least three amino acid mutations are at a triple of positions selected from the group consisting of: 69/180/441, 69/180/445, 69/414/445, and 180/414/445. 
     
     
         44 . The variant cannabinoid synthase or active fragment thereof of  claim 42 , wherein the at least three amino acid mutations are H69Q/A414V/I445M. 
     
     
         45 . A variant cannabidiolic acid (CBDA) synthase or active fragment thereof comprising an amino acid mutation at a position selected from the group consisting of: 69, 414, 180, and 445 relative to a wild-type consensus CBDA synthase set forth in SEQ ID NO: 1045. 
     
     
         46 . The variant CBDA synthase or active fragment thereof of  claim 45 , wherein a mutation at amino acid position 69 is selected from the group consisting of: H69R, H69G, H69K, H69Q, H69A, and H69V. 
     
     
         47 . The variant CBDA synthase or active fragment thereof of  claim 46 , wherein the mutation at amino acid position 69 results in the variant CBDA synthase producing an increase yield of a CBDA relative to a wild type CBDA synthase or active fragment thereof. 
     
     
         48 . The variant CBDA synthase or active fragment thereof of  claim 47 , wherein the yield of the variant or active fragment thereof is more than 100%, more than 200%, or more than 300% of a yield of CBDA of the wild-type CBDA synthase or active fragment thereof 
     
     
         49 . The variant CBDA synthase or active fragment thereof of  claim 48 , wherein the yield is a nMol of CBDA produced per milligram of a substrate. 
     
     
         50 . The variant CBDA synthase or active fragment thereof of  claim 49 , wherein the substrate is cannabigerolic acid (CBGA). 
     
     
         51 . The variant CBDA synthase or active fragment thereof of  claim 45 , wherein a mutation at amino acid position 414 is selected from the group consisting of: A414T, A414I, and A414V. 
     
     
         52 . The variant CBDA synthase or active fragment thereof of  claim 51 , wherein the mutation at amino acid position 414 results in the variant CBDA synthase producing an increase yield of a CBDA relative to a wild type CBDA synthase or active fragment thereof. 
     
     
         53 . The variant CBDA synthase or active fragment thereof of  claim 52 , wherein the yield of the variant or active fragment thereof is more than 100% or more than 200% of a yield of CBDA of the wild-type CBDA synthase or active fragment thereof. 
     
     
         54 . The variant CBDA synthase or active fragment thereof of  claim 53 , wherein the yield is a nMol of CBDA produced per milligram of a substrate. 
     
     
         55 . The variant CBDA synthase or active fragment thereof of  claim 54 , wherein the substrate is cannabigerolic acid (CBGA). 
     
     
         56 . The variant CBDA synthase or active fragment thereof of  claim 45 , wherein a mutation at amino acid position 445 is selected from the group consisting of: I445M, I445L, and 1445V. 
     
     
         57 . The variant CBDA synthase or active fragment thereof of  claim 56 , wherein the mutation 1445V results in the variant CBDA synthase producing an increase yield of a CBDA relative to a wild type CBDA synthase or active fragment thereof. 
     
     
         58 . The variant CBDA synthase or active fragment thereof of  claim 57 , wherein the yield of the variant or active fragment thereof is more than 100% of a yield of CBDA of the wild-type CBDA synthase or active fragment thereof 
     
     
         59 . The variant CBDA synthase or active fragment thereof of  claim 58 , wherein the yield is a nMol of CBDA produced per milligram of a substrate. 
     
     
         60 . The variant CBDA synthase or active fragment thereof of  claim 59 , wherein the substrate is cannabigerolic acid (CBGA). 
     
     
         61 . The variant CBDA synthase or active fragment thereof of  claim 56 , wherein the mutation I445L or I445M results in the variant CBDA synthase produces an increased proportion of cannabidiolic acid (CBDA): tetrahydrocannabinolic acid (THCA) relative to a wild type CBDA synthase or active fragment thereof 
     
     
         62 . The variant CBDA synthase or active fragment thereof of  claim 61 , wherein a proportion of CBDA:THCA produced by the wild type CBDA synthase or active fragment thereof is about 95:5. 
     
     
         63 . A variant cannabidiolic acid (CBDA) synthase or active fragment thereof comprising at least two amino acid mutations at amino acid positions selected from the group consisting of: 69, 180, 414, and 445, relative to a wild-type consensus CBDA synthase set forth in SEQ ID NO: 1045. 
     
     
         64 . The variant CBDA synthase or active fragment thereof of  claim 63 , wherein the at least two amino acid mutations result in the variant CBDA synthase producing an increase yield of a CBDA relative to a wild type CBDA synthase or active fragment thereof. 
     
     
         65 . The variant CBDA synthase or active fragment thereof of  claim 64 , wherein the yield of the variant or active fragment thereof is more than 100%, more than 200%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, or more than 800% of a yield of CBDA of the wild-type CBDA synthase or active fragment thereof 
     
     
         66 . The variant CBDA synthase or active fragment thereof of  claim 65 , wherein the yield is a nMol of CBDA produced per milligram of a substrate. 
     
     
         67 . The variant CBDA synthase or active fragment thereof of  claim 66 , wherein the substrate is cannabigerolic acid (CBGA). 
     
     
         68 . The variant CBDA synthase or active fragment thereof of  claim 63 , wherein the variant CBDA synthase or active fragment thereof produces an increased proportion of cannabidiolic acid (CBDA): tetrahydrocannabinolic acid (THCA) relative to the wild-type CBDA synthase or active fragment thereof 
     
     
         69 . The variant CBDA synthase or active fragment thereof of  claim 68 , wherein a proportion of CBDA:THCA produced by the wild type CBDA synthase or active fragment thereof is about 95:5. 
     
     
         70 . The variant CBDA synthase or active fragment thereof of  claim 63 , wherein one of the at least two amino acid mutations is at amino acid position 69. 
     
     
         71 . The variant CBDA synthase or active fragment thereof of  claim 70 , wherein one of the at least two amino acid mutations at amino acid position 69 is selected from the group consisting of H69A, H69C, H69D, H69E, H69F, H69G, H69I, H69K, H69L, H69M, H69N, H69P, H69Q, H69R, H69S, H69T, H69V, H69W, and H69Y. 
     
     
         72 . The variant CBDA synthase or active fragment thereof of  claim 70 , wherein one of the at least two amino acid mutations at amino acid position 69 is selected from the group consisting of H69K, H69Q, H69V, and H69G. 
     
     
         73 . The variant CBDA synthase or active fragment thereof of  claim 63 , wherein one of the at least two amino acid mutations is at amino acid position 180. 
     
     
         74 . The variant CBDA synthase or active fragment thereof of  claim 73 , wherein one of the at least two amino acid mutations at amino acid position 180 is selected from the group consisting of C180A, C180D, C180E, C180F, C180G, C180H, C180I, C180K, C180L, C180M, C180N, C180P, C180Q, C180R, C180S, C180T, C180V, C180W, and C180Y. 
     
     
         75 . The variant CBDA synthase or active fragment thereof of  claim 73 , wherein one of the at least two amino acid mutations is at amino acid position 414. 
     
     
         76 . The variant CBDA synthase or active fragment thereof of  claim 75 , wherein one of the at least two amino acid mutations at amino acid position 414 is selected from the group consisting of A414C, A414D, A414E, A414F, A414G, A414H, A414I, A414K, A414L, A414M, A414N, A414P, A414Q, A414R, A414S, A414T, A414V, A414W, and A414Y. 
     
     
         77 . The variant CBDA synthase or active fragment thereof of  claim 75 , wherein one of the at least two amino acid mutations at amino acid position 414 is selected from the group consisting of: A414V and A414I. 
     
     
         78 . The variant CBDA synthase or active fragment thereof of  claim 63 , wherein one of the at least two amino acid mutations is at amino acid position 445. 
     
     
         79 . The variant CBDA synthase or active fragment thereof of  claim 78 , wherein one of the at least two amino acid mutations at amino acid position 445 is selected from the group consisting of I445A, I445C, I445D, 1445E, I445F, I445G, I445H, 1445K, I445L, I445M, I445N, I445P, I445Q, I445R, I445S, I445T, I445V, 1445W, and I445Y. 
     
     
         80 . The variant CBDA synthase or active fragment thereof of  claim 78 , wherein one of the at least two amino acid mutations at amino acid position 445 is I445M. 
     
     
         81 . The variant CBDA synthase or active fragment thereof of  claim 63 , wherein the at least two amino acid mutations are selected from the group consisting of: A414V/H69K, A414V/H69Q, A414V/H69V, A414V/H69G, A414V/I44M, A414I/H69K, I445M/H69K, and I445M/H69Q. 
     
     
         82 . The variant CBDA synthase or active fragment thereof of  claim 63 , wherein the variant CBDA synthase comprises at least three amino acid mutations at amino acid positions selected from the group consisting of: 69, 180, 414, and 445, relative to a wild-type consensus CBDA synthase set forth in SEQ ID NO: 1045. 
     
     
         83 . The variant CBDA synthase or active fragment thereof of  claim 82 , wherein one of the at least three amino acid mutations is at amino acid position 69. 
     
     
         84 . The variant CBDA synthase or active fragment thereof of  claim 83 , wherein one of the at least three amino acid mutations at amino acid position 69 is selected from the group consisting of H69A, H69C, H69D, H69E, H69F, H69G, H69I, H69K, H69L, H69M, H69N, H69P, H69Q, H69R, H69S, H69T, H69V, H69W, and H69Y. 
     
     
         85 . The variant CBDA synthase or active fragment thereof of  claim 83 , wherein one of the at least three amino acid mutations at amino acid position 69 is selected from the group consisting of H69K, H69Q, H69V, and H69G. 
     
     
         86 . The variant CBDA synthase or active fragment thereof of  claim 82 , wherein one of the at least three amino acid mutations is at amino acid position 180. 
     
     
         87 . The variant CBDA synthase or active fragment thereof of  claim 86 , wherein one of the at least three amino acid mutations at amino acid position 180 is selected from the group consisting of C180A, C180D, C180E, C180F, C180G, C180H, C1801, C180K, C180L, C180M, C180N, C180P, C180Q, C180R, C1805, C180T, C180V, C180W, and C180Y. 
     
     
         88 . The variant CBDA synthase or active fragment thereof of  claim 82 , wherein one of the at least three amino acid mutations is at amino acid position 414. 
     
     
         89 . The variant CBDA synthase or active fragment thereof of  claim 88 , wherein one of the at least three amino acid mutations at amino acid position 414 is selected from the group consisting of A414C, A414D, A414E, A414F, A414G, A414H, A414I, A414K, A414L, A414M, A414N, A414P, A414Q, A414R, A414S, A414T, A414V, A414W, and A414Y. 
     
     
         90 . The variant CBDA synthase or active fragment thereof of  claim 88 , wherein one of the at least three amino acid mutations at amino acid position 414 is selected from the group consisting of: A414V and A414I. 
     
     
         91 . The variant CBDA synthase or active fragment thereof of  claim 82 , wherein one of the at least three amino acid mutations is at amino acid position 445. 
     
     
         92 . The variant CBDA synthase or active fragment thereof of  claim 91 , wherein one of the at least three amino acid mutations at amino acid position 445 is selected from the group consisting of I445A, I445C, I445D, I445E, I445F, I445G, I445H, I445K, I445L, I445M, I445N, I445P, I445Q, I445R, I445S, I445T, I445V, I445W, and I445Y. 
     
     
         93 . The variant CBDA synthase or active fragment thereof of  claim 91 , wherein one of the at least three amino acid mutations at amino acid position 445 is I445M. 
     
     
         94 . The variant CBDA synthase or active fragment thereof of  claim 82 , wherein the at least three amino acid mutations are H69Q, A414V, and I445M. 
     
     
         95 . A nucleic acid construct comprising a nucleic acid encoding the variant cannabinoid synthase or active fragment thereof of any one of  claims 1 - 94  operably linked to a promoter. 
     
     
         96 . A vector comprising the nucleic acid construct of  claim 95 . 
     
     
         97 . A microorganism comprising the nucleic acid construct of  claim 95 . 
     
     
         98 . The microorganism of  claim 97 , wherein the microorganism is a yeast or a bacteria. 
     
     
         99 . The microorganism of  claim 98 , wherein the yeast is a  Saccharomyces cerevisiae.    
     
     
         100 . The microorganism of  claim 98 , wherein the bacteria is an  Escherichia coli.    
     
     
         101 . A plant comprising the nucleic acid construct of  claim 95 . 
     
     
         102 . The plant of  claim 101 , wherein the plant is a vascular plant or a non-vascular plant. 
     
     
         103 . The plant of  claim 102 , wherein the vascular plant is a plant in the genus  Cannabis.    
     
     
         104 . The plant of  claim 103 , wherein the plant is genus  Cannabis  is selected from the group consisting of:  Cannabis satvia, Cannabis indica , and  Cannabis ruderalis.    
     
     
         105 . The plant of  claim 102 , wherein the non-vascular plant is a microalgae. 
     
     
         106 . A method of producing a cannabinoid, comprising: (i) contacting a cell with a nucleic acid encoding the variant cannabinoid synthase of any one of  claims 1 - 90 , (ii) expressing the variant cannabinoid synthase, and (iii) isolating a cannabinoid produced by the cell. 
     
     
         107 . The method of  claim 106 , wherein the cell is a plant cell or a microorganism cell. 
     
     
         108 . The method of  claim 106 , further comprising expanding the cell to produce a plurality of expanded cells. 
     
     
         109 . The method of  claim 107 , wherein the expanding occurs in a bioreactor. 
     
     
         110 . The method of  claim 107 , further comprising isolating and purifying the cannabinoid from the plurality of expanded cells. 
     
     
         111 . A method of producing a cannabinoid, comprising: contacting the variant cannabinoid synthase of any one of  claims 1 - 94  to a substrate of the variant cannabinoid synthase. 
     
     
         112 . The method of  claim 111 , wherein the contacting occurs ex vivo. 
     
     
         113 . The method of  claim 111 , wherein the substrate is a naturally occurring substrate. 
     
     
         114 . The method of  claim 113 , wherein the naturally occurring substrate is selected from the group consisting of cannabigerol (CBG), cannabigerolic acid (CBGA), cannbigerovarinic acid (GBGVA), and any homolog thereof 
     
     
         115 . The method of  claim 111 , wherein the substrate is a non-naturally occurring substrate. 
     
     
         116 . The method of  claim 115 , wherein the non-naturally occurring substrate can comprise:
 a non-naturally occurring tail variant, a prenyl donor, or a combination thereof.