Enzymes and methods for the synthesis of bakuchiol and derivatives

Abstract

Disclosed herein are prenyltransferases capable of producing Bakuchiol or analogs thereof from geranyl pyrophosphate (GPP) and cinnamic acid, coumaric acid, caffeic acid, and / or ferulic acid. Also disclosed herein are engineered cells which express a prenyltransferase capable of producing bakuchiol or analogs thereof and method of using the prenyltransferase and cells for producing bakuchiol or analogs thereof.

Description

[0001] ENZYMES AND METHODS FOR THE SYNTHESIS OF BAKUCHIOL AND

[0002] DERIVATIVES

[0003] RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 524,506, filed on June 30, 2023, and U.S. Provisional Application No. 63 / 525,294, filed on July 6, 2023, the entire teachings of which are incorporated herein by reference.

[0005] BACKGROUND OF THE INVENTION

[0006] The Psoralea corylifolia plant has been used in Asia, China and India, as a medicinal herb for the treatment of a variety of conditions, including cardiotonic, vasodilator, antitumor, antibacterial and cytotoxic conditions and, more specifically, for the treatment of skin conditions such as alopecia, leukoderma, leprosy, psoriasis and eczema.(l) The plant produces a multitude of chemicals primarily concentrated in the seed and fruit, with about 90 compounds isolated and characterized thus far. (2) One of the most abundant chemicals is Bakuchiol, which accumulates to ~6 % w / w in dried seeds of the plant. Other compounds found in lower concentrations in the seed include 3-hydroxy-bakuchiol and certain coumarins such as psoralidin, psoralen, isopsoralen and angelicin.(3) Being the most abundant compound in the plant’s seeds, bakuchiol constitutes both the most studied and the only commercially available product isolated from the seeds of Psoralea corylifolia. Bakuchiol has been shown to have many of the clinical effects that have been associated with the plant, specifically its activity in skin conditions (anti- aging, anti-pigmentation, acne, etc.). As a result, it is used as a retinol alternative in various creams and other skin care products. (4, 5, 6) Other bakuchiol analogs described herein are poorly available due to the lack of a ready source for their isolation but may also have beneficial properties.

[0007] Currently, Bakuchiol is commercially available only from plant extracts, resulting in low availability and a correspondingly high price tag (>$2000 / Kg). In addition, Bakuchiol is co-extracted with other plant compounds, such as phototoxic psoralen and isopsoralen that must be removed because they can cause skin irritation and dermatitis if present in cosmetic skin formulations. (7) Thus, an alternative low- cost method for the production of a less toxic and high purity bakuchiol-containing composition is required.

[0008] SUMMARY OF THE INVENTION

[0009] Described herein are enzymes capable of prenylating hydroxycinnamic acids to efficiently obtain bakuchiol and one or more analogs thereof. These enzymes can be expressed in engineered cells to efficiently produce bakuchiol and analogs thereof when fermented under the correct conditions. Also disclosed are methods for providing an alternative source of bakuchiol and its derivatives. Specifically, fermentation methods for cost-effectively producing a high yield of bakuchiol which is substantially free from the toxic compounds found in extracts from Psoralea corylifolia are disclosed. These and other aspects of the invention will be disclosed in more detail herein.

[0010] Some aspects of the present disclosure are directed to a mutant membranebound prenyltransferase comprising an amino acid sequence with at least 85% identity to the amino acid sequence of SEQ ID NOS. 35, 2 or 1, having at least one amino acid modification at a position selected from the group consisting of 78, 99, 123, 282, and 328 of SEQ ID NO: 35, 2 or 1, wherein the mutant membrane - prenyltransferase is capable of producing bakuchiol or analogs thereof from geranyl pyrophosphate (GPP) and at least one of cinnamic acid, coumaric acid, caffeic acid, and ferulic acid. In some embodiments, the mutant membrane-bound prenyltransferase further comprises at least one additional amino acid modification at a position selected from the group consisting of 106, 111, 209, 323, 344, 385, and 400 of SEQ ID NO: 35, 2 or 1. In some embodiments, the bakuchiol analog is selected from the group consisting of dehydrobakuchiol, 3-hydroxy-Bakuchiol, and 3- methoxy -bakuchiol.

[0011] In some embodiments, the membrane -bound prenyltransferase comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1. In some embodiments, the membrane-bound prenyltransferase comprises an amino acid sequence with at least 97% identify to the amino acid sequence of SEQ ID NO: 35, 2, or 1. 6. In some embodiments, the mutant membrane-bound prenyltransferase comprises an amino acid sequence with at least

[0012] 4891 -1065-4412, v. 1 90% identity to the amino acid sequence of SEQ ID NO: 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34. In some embodiments, the mutant membrane-bound prenyltransferase comprises an amino acid sequence with between 98.5% and 99.8% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1.

[0013] In some embodiments, the mutant membrane-bound prenyltransferase (MPT) transfers geranyl pyrophosphate (GPP) to cinnamic acid, coumaric acid, caffeic acid, and / or ferulic acid to produce dehydrobakuchiol, bakuchiol, 3-hydroxy-bakuchiol, and / or 3-methoxy-bakuchiol, respectively, with a higher efficiency compared to either of enzymes MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2)). In some embodiments, the higher efficiency of transferring GPP to cinnamic acid, coumaric acid, caffeic acid, and / or ferulic acid comprises at least 1.5 times higher efficiency compared to either of enzymes MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2).

[0014] In some embodiments, the at least one amino acid modification relative to SEQ ID NO: 35, 2, or 1 comprises a substitution, deletion, or insertion. In some embodiments, the mutant MPT further comprises one or more substitutions, deletions, and / or insertions at one or more amino acid positions corresponding to positions R121, V124, N165-N172, D176-K184, Y229-Y236, K246-F251, C255-R260, Y298- D306 I310-G317, and Y391-W395 of SEQ ID NO: 35, 2, or 1. In some embodiments, the mutant MPT comprises an amino terminal truncation comprising deletion of between 1 and 73 amino acids corresponding to amino acids in positions 2-74 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In some embodiments, the mutant MPT comprises an amino acid sequence with at least 95% identity to an amino acid sequence selected from SEQ ID NO: 6-10, 13, and 23-33. 33 and 44-46.

[0015] Other aspects of the present disclosure are directed to an engineered cell that expresses a membrane -bound prenyltransferase as disclosed herein, wherein the cell is capable of producing bakuchiol and / or analogs thereof in the presence of geranyl pyrophosphate (GPP) and at least one of cinnamic acid, coumaric acid, caffeic acid, and ferulic acid. In some embodiments, the cell has been engineered to provide greater flux of GPP through the GPP pathway. In some embodiments, the cell has been engineered to provide greater flux of tyrosine or phenylalanine through the respective aromatic amino acid synthesis pathways. In some embodiments, the

[0016] 4891 -1065-4412, v. 1 greater flux through an aromatic amino acid synthesis pathway is achieved by overexpressing an Aro4 enzyme (EC 2.5.1.54) comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 4. In some embodiments, the greater flux through an aromatic amino acid synthesis pathway is achieved by expressing a feedback insensitive Aro4 enzyme having a sequence with at least 95% identity to SEQ ID NO: 5.

[0017] In some embodiments, the cell is a yeast cell, an algal cell, or a bacterial cell. In some embodiments, the yeast cell is Saccharomyces, Pichia, or Yarrowia.

[0018] In some embodiments, the cell is capable of synthesizing coumaric acid, caffeic acid, and / or ferulic acid when tyrosine is present in the cell through endogenous production. In some embodiments, the cell is capable of producing cinnamic acid, coumaric acid, caffeic acid, or ferulic acid when phenylalanine is present in the cell through endogenous production.

[0019] In some embodiments, production of bakuchiol or analogs thereof in the cell is dependent on or is enhanced by supplementation of cinnamic acid, coumaric acid, and / or ferulic acid within the fermentation medium in which the cell is grown.

[0020] In some embodiments, the cell is capable of converting one or more of cinnamic acid to coumaric acid, coumaric acid to caffeic acid, and caffeic acid to ferulic acid. In some embodiments, the cell expresses phenylalanine ammonia lyase and / or tyrosine ammonia lyase of the family EC 4.3.1.24 and / or family EC 4.3.1.25. In some embodiments, the cell expresses cinnamate 3-hydroxylase. In some embodiments, the cinnamate 3-hydroxylase is a p450 enzyme of the CYP73A family or family EC 1.14.14.91. In some embodiments, the cell expresses caffeic acid O- methyltransferase of the family EC 2.1.1.68

[0021] In some embodiments, the cell is capable of converting bakuchiol to 3- hydroxy-bakuchiol, and / or 3-hydroxy-bakuchiol to 3-methoxy-bakuchiol. In some embodiments, the conversion of bakuchiol to 3-hydroxybakuchiol is catalyzed by a P450 hydroxylase of the family CYP98A. In some embodiments, the conversion of 3-hydroxybakuchiol to 3-methoxybakuchiol is catalyzed by a methyltransferase of the family EC 2.1.1.68 or EC 2.1.1.42.

[0022] 4891 -1065-4412, v. 1 Some aspects of the present disclosure are directed to a method of producing bakuchiol by fermenting the cell as disclosed herein in the presence of one or more of glucose, glycerol, cinnamic acid, coumaric acid, caffeic acid, and ferulic acid.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The foregoing and other objects and advantages will be apparent upon consideration of the following detailed description, taken in conjunction with the accompanying drawings.

[0025] FIG. 1 depicts the activities of some prenyltransferases discovered from Psoralea corylifolia.

[0026] FIG. 2 depicts the pathways involved in synthesizing bakuchiol and analogs thereof in an engineered cell.

[0027] FIG. 3 is a ribbon model depicting the structure of prenyltransferase MPT95 with geranyl pyrophosphate (GPP) and coumaric acid docked within the active site.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] Given the high price of commercially available bakuchiol and the difficulty involved in isolating and purifying bakuchiol from natural sources and toxic compounds found therewith, the inventors set out to identify, isolate, characterize and mutagenize prenyltransferases from the Psoralea corylifolia plant capable of condensing geranyl pyrophosphate (GPP) with hydroxycinnamic acids to provide bakuchiol and derivatives thereof. Further, engineered cells were designed to produce bakuchiol and analogs thereof through fermentation of the engineered cells with a carbon source. Engineered cells that can make bakuchiol and analogs thereof with supplemented cinnamic acid, coumaric acid, caffeic acid, and / or ferulic acid are also described. Thus, the inventors herein disclose methods for providing an alternative source for purified bakuchiol and its derivatives which avoid the problems with the prior art. Specifically, fermentation methods for cost-effectively producing high yield of bakuchiol which is free from the toxic compounds found in extracts from Psoralea corylifolia are disclosed.

[0030] 4891 -1065-4412, v. 1 Some aspects of the present disclosure are directed to a membrane-bound prenyltransferase (MPT) comprising an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1, wherein the membrane-prenyltransferase is capable of producing bakuchiol or analogs thereof from geranyl pyrophosphate (GPP) and at least one of cinnamic acid, coumaric acid, caffeic acid, and ferulic acid. In some embodiments, the MPT comprises an amino acid sequence with at least 75, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.25%, 90.5%, 90.75%, 91%, 91.25%, 91.5%, 91.75%, 92%, 92.25%, 92.5%, 92.75%, 93%, 93.25%, 93.5%, 93.75%, 94%, 94.25%, 94.5%, 94.75%, 95%, 95.25%, 95.5%, 95.75%, 96%, 96.25%, 96.5%, 96.75%, 97%, 97.25%, 97.5%, 97.75%, 98%, 98.25%, 98.5%, 98.75%, 99%, 99.25%, 99.5%, 99.75%, or 100% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1. In some embodiments, the MPT comprises SEQ ID NO: 35, 2, or 1. In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In some embodiments, the MPT comprises an amino acid sequence with less than 100%, 99.75%, 99.5%, 99.25%, 99%, 98.75%, 98.5%, 98.25%, 98%, 97.75%, 97.5%, 97.25%, 97%, 96.75%, 96.5%, 96.25%, 96%, 95.75%, 95.5%, 95.25%, 95%, 94.75%, 94.5%, 94.25%, 94%, 93.75%, 93.5%, 93.25%, 93%, 92.75%, 92.5%, 92.25%, 92%, 91.75%, 91.5%, 91.25%, 91%, 90.75%, 90.5%, 90.25%, 90%, 89%, 88%, 87%, 86%, or 85% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1.

[0031] In some embodiments, the mutant membrane-bound prenyltransferase comprises an amino acid sequence with at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 35, 2, or 1. The at least one amino acid modification can be any amino acid modification, or any combinations of amino acid modifications disclosed herein. In some embodiments, the at least one amino acid modification is at least one modification at a position comprising position 78, 99, 123, 282 or 328 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In some embodiments, the at least one amino acid modification is at least one modification at a position comprising position 106, 111, 209, 323, 344, 385, and 400 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In some embodiments, the at least one amino acid modification is a deletion. In some embodiments, the at least

[0032] 4891 -1065-4412, v. 1 one amino acid modification is an insertion. In some embodiments, the at least one amino acid modification is a substitution. In certain embodiments, the at least one amino acid substitution is selected from W78C, H99N, L106W, R111Q, H123Y, M209L, M282L, M323L, E328K, G344A, E385S, E385M, or I400V. In certain embodiments, the substitution is a conservative substitution.

[0033] In some embodiments, the at least one amino acid modification comprises one, two, three, four, five, six, seven, eight, nine, ten, or eleven, amino acid modifications at positions comprising positions 78, 99, 106, 111, 123, 209, 282, 323, 344, 385, or 400 of the amino acid sequence of SEQ ID NO: 35, of SEQ ID NO: 2, or of SEQ ID NO: 1. In certain embodiments, the at least one amino acid modification comprises amino acid modifications at positions 78, 99, 106, 111, 209, 282, 323, 328, 344, and 400 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In certain embodiments, the at least one amino acid modification comprises amino acid modifications at positions 106, 111, 209, 282, 323, and 328 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In certain embodiments, the at least one amino acid modification comprises amino acid modifications at positions 209, 282, 323, 328, 344, and 400 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In certain embodiments, the at least one amino acid modification comprises amino acid modifications at positions 78, 209, 282, 323, 328, and 344 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In certain embodiments, the at least one amino acid modification comprises amino acid modifications at positions 78, 99, 106, 111, 209, 323, 328, and 344 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In certain embodiments, the at least one amino acid modification comprises amino acid modifications at positions 78, 99, 106, 111, 209, 282, 323, 328 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In certain embodiments, the at least one amino acid modification comprises amino acid modifications at positions 78, 99, 106, 111, 209, 282, 323, 328, 344 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In certain embodiments, the at least one amino acid modification comprises amino acid modifications at positions 99, 106, 111, 209, 282, 323, 328, 344 of the amino acid sequence of SEQ ID NO: 35, 2, or 1.

[0034] In some embodiments, the amino acid modification comprises a substitution, deletion, or insertion at one or more amino acid positions corresponding to positions R121, V124, N165-N172, D176-K184, Y229-Y236, K246-F251, C255-R260, Y298-

[0035] 4891 -1065-4412, v. 1 D306 I310-G317, and Y391-W395 of SEQ ID NO: 35, SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the at least one amino acid modification comprises 1 to 61 amino acid modifications. In some embodiments, the at least one amino acid modification comprises deletion of 39 to 73 amino acids in the N-terminal domain to create an N-truncated membrane -bound prenyltransferase. In some embodiments the at least one amino acid modification comprises a substitution, deletion, or insertion at one or more amino acids corresponding to positions R121, V124, N165-N172, D176- K184, Y229-Y236, K246-F251, C255-R260, Y298-D306 I310-G317, and Y391- W395 of SEQ ID NO: 1 or SEQ ID NO: 2 and deletion of 39 to 73 amino acids in the N-terminal domain of SEQ ID NO: 35, SEQ ID NO: 1 or SEQ ID NO: 2.

[0036] In certain aspects, the prenyltransferase has been mutated such that it transfers geranyl pyrophosphate (GPP) to coumaric acid to produce bakuchiol with a higher efficiency compared to either of enzymes MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2). In some embodiments, the MPT which transfers geranyl pyrophosphate (GPP) to coumaric acid to produce bakuchiol with a higher efficiency does so with an efficiency that is 1.1 times more efficient to 1,000 times more efficient or more when compared to the efficiency of MPT85 and / or MPT94. Specifically the higher efficiency constitutes an efficiency 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 8 times 10 times, 15 times, 20 times, 25 times, 35 times, 50 times, 75 times, 100 times, 200 times, 400 times, 600 times, 800 times, 1000 times, 2000 times or more when compared to the enzymes MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2).

[0037] In some embodiments, the membrane -bound prenyltransferase comprises an amino acid sequence with at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 35, and transfers geranyl pyrophosphate (GPP) to caffeic acid to produce 3-hydroxybakuchiol with at a higher efficiency compared to either of enzymes MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2). In some embodiments, the MPT that has been mutated to transfer geranyl pyrophosphate (GPP) to caffeic acid to produce 3-hydroxybakuchiol with a higher efficiency, does so with an efficiency that is 1.1 times more efficient to 1,000 times more efficient or more when compared to the efficiency of MPT85 and / or MPT94. Specifically the higher efficiency constitutes an efficiency 1.2 times, 1.3

[0038] 4891 -1065-4412, v. 1 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 8 times 10 times, 15 times, 20 times, 25 times, 35 times, 50 times, 75 times, 100 times, 200 times, 400 times, 600 times, 800 times, 1000 times, 2000 times or more when compared to the enzymes MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2).

[0039] As described above, the mutant membrane-bound prenyltransferase comprises at least one amino acid modification, such as at least one amino acid modification compared to a wild-type membrane-bound prenyltransferase. The at least one amino acid modification can be any amino acid modification, or combinations of any amino acid modifications disclosed herein. In some embodiments, the amino acid modification comprises a substitution, deletion, or insertion at one or more amino acid positions corresponding to positions W78, H99, L106, Ri l l, R121, H123, V124, N165, 1166, Y167, T168, A169, G I70, 1171, N172, D176, 1177, E178, 1179, D180, K181, 1182, N183, K184, M209, Y229, F230, V231, L232, G233, T234, V235, Y236, K246, R247, Y248, P249, A250, F251, C255, F256, F257, 1258, 1259, R260, M282, Y298, V299, 1300, 1301, A302, F303, F304, K305, D306, 1310, E311, G312, D313, K314, E315, H316, G317, M323, E328, G344, E385, Y391, M392, F393, M394, W395, or 1400 of SEQ ID NO: 35, SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the at least one amino acid modification comprises 1 to 62 amino acid modifications. In some embodiments, the at least one amino acid modification comprises deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

[0040] 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,

[0041] 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,

[0042] 67, 68, 69, 70, 71, 72, or 73 amino acids in the N-terminal domain to create an N- truncated membrane -bound prenyltransferase. In some embodiments, the at least one amino acid modification comprises deletion of amino acids 2-4, 2-5, 2-6, 2-7, 2-8, 2- 9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2- 24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-33, 2-34, 2-35, 2-36, 2-37, 2-38, 2-39, 2-40, 2-41, 2-42, 2-43, 2-44, 2-45 from the N-terminal domain to create an N- truncated membrane-bound prenyltransferase. In some embodiments the at least one amino acid modification comprises a substitution, deletion, or insertion at one or more amino acids corresponding to positions W78, H99, L106, Ri l l, R121, H123, V124,

[0043] 4891 -1065-4412, v. 1 N165, 1166, Y167, T168, A169, G170, 1171, N172, D176, 1177, E178, 1179, D180, K181, 1182, N183, K184, M209, Y229, F230, V231, L232, G233, T234, V235, Y236, K246, R247, Y248, P249, A250, F251, C255, F256, F257, 1258, 1259, R260, M282, Y298, V299, 1300, 1301, A302, F303, F304, K305, D306, 1310, E311, G312, D313, K314, E315, H316, G317, M323, E328, G344, E385, Y391, M392, F393, M394, W395, or 1400 of SEQ ID NO: 35, SEQ ID NO: 2 or SEQ ID NO: 1 and deletion of 1 to 6, 7 to 12, 13 to 26, 27 to 36, 37 to 40, or 39 to 73 amino acids in the N-terminal domain of SEQ ID NO: 1 or SEQ ID NO: 2

[0044] 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 proline; 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.

[0045] In some embodiments, the at least one amino acid modification compared to MPT85 (SEQ ID NO: 1) comprises a deletion, insertion or substitution at one or more amino acid positions corresponding to 78, 99, 106, 111, 121, 123, 124, 165, 166, 167, 168, 169, 170, 171, 172, 176, 177, 178, 179, 180, 181, 182, 183, 184, 209, 229, 230,

[0046] 4891 -1065-4412, v. 1 231, 232, 233, 234, 235, 236, 246, 247, 248, 249, 250, 251, 255, 256, 257, 258, 259,

[0047] 260, 282, 298, 299, 300, 301, 302, 303, 304, 305, 306, 310, 311, 312, 313, 314, 315,

[0048] 316, 317, 323, 328, 344, 385, 391, 392, 393, 394, 395, or 400 of SEQ ID NO: 1. In some embodiments, the MPT comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty, fifty-one, fifty-two, fifty-three, fifty-four, fifty-five, fifty-six, fifty-seven, fifty-eight, fifty-nine, sixty, sixty-one, sixty-two, sixty-three, sixty-four, sixty-five, sixty-six, sixty-seven, sixty-eight, sixty-nine, seventy, seventy-one, seventy-two or all seventy-three of the amino acid modifications. In some embodiments, the MPT further comprises a truncation (e.g., 1-73 amino acids) at the N terminus.

[0049] In some embodiments, the membrane-bound prenyltransferase (MPT) comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 1 (MPT85). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 1 (MPT85). In some embodiments, the MPT comprises a functional fragment of SEQ ID NO: 1 (MPT85). In some embodiments, the functional fragment of SEQ ID NO: 1 (MPT85) has at least the first

[0050] 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

[0051] 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,

[0052] 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73 amino acids deleted from the amino terminus.

[0053] In some embodiments, the at least one amino acid modification compared to MPT94 (SEQ ID NO: 2) comprises a deletion, insertion or substitution at one or more amino acid positions corresponding to 78, 99, 106, 111, 121, 123, 124, 165, 166, 167,

[0054] 168, 169, 170, 171, 172, 176, 177, 178, 179, 180, 181, 182, 183, 184, 209, 229, 230,

[0055] 231, 232, 233, 234, 235, 236, 246, 247, 248, 249, 250, 251, 255, 256, 257, 258, 259,

[0056] 260, 282, 298, 299, 300, 301, 302, 303, 304, 305, 306, 310, 311, 312, 313, 314, 315,

[0057] 4891 -1065-4412, v. 1 316, 317, 323, 328, 344, 385, 391, 392, 393, 394, 395, or 400 of SEQ ID NO: 2. In some embodiments, the MPT comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty, fifty-one, fifty-two, fifty-three, fifty-four, fifty-five, fifty-six, fifty-seven, fifty-eight, fifty-nine, sixty, sixty-one, sixty-two, sixty-three, sixty-four, sixty-five, sixty-six, sixty-seven, sixty-eight, sixty-nine, seventy, seventy-one, seventy-two or all seventy-three of the amino acid modifications. In some embodiments, the MPT further comprises a truncation (e.g., 1-73 amino acids) at the N terminus.

[0058] In some embodiments, the membrane-bound prenyltransferase (MPT) comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 2 (MPT94). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 2 (MPT94). In some embodiments, the MPT comprises a functional fragment of SEQ ID NO: 2 (MPT94). In some embodiments, the functional fragment of SEQ ID NO: 2 (MPT94) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

[0059] 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,

[0060] 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73 amino acids deleted from the amino terminus. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 7 (MPT94.2). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 7 (MPT94.2). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 7. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%,

[0061] 4891 -1065-4412, v. 1 99.5%, 99.9%, or 100% identity to SEQ ID NO: 8 (MPT94.3). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 8 (MPT94.3). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 9 (MPT94.4). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 9 (MPT94.4). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 10 (MPT94.5). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 10 (MPT94.5). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 13 (MPT94.8). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 13 (MPT94.8). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 13.

[0062] In some embodiments, the membrane-bound prenyltransferase (MPT) comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 14 (MPT94.15). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 14 (MPT94.15). In some embodiments, the MPT comprises a functional fragment of SEQ ID NO: 14 (MPT94.15). In some embodiments, the functional fragment of SEQ ID NO: 14 (MPT94.15) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

[0063] 4891 -1065-4412, v. 1 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73 amino acids deleted from the amino terminus. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 23 (MPT94.15t). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 23 (MPT94.15t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO:

[0064] 23.

[0065] In some embodiments, the membrane-bound prenyltransferase (MPT) comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 15 (MPT94.16). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 15 (MPT94.16). In some embodiments, the MPT comprises a functional fragment of SEQ ID NO: 15 (MPT94.16). In some embodiments, the functional fragment of SEQ ID NO: 15 (MPT94.16) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73 amino acids deleted from the amino terminus. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 24 (MPT94.16t). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 24 (MPT94.16t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO:

[0066] 24.

[0067] In some embodiments, the membrane-bound prenyltransferase (MPT) comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 16 (MPT94.17). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 16 (MPT94.17). In some embodiments, the MPT comprises a functional fragment of SEQ ID NO: 16

[0068] 4891 -1065-4412, v. 1 (MPT94.17). In some embodiments, the functional fragment of SEQ ID NO: 16 (MPT94.17) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

[0069] 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,

[0070] 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

[0071] 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73 amino acids deleted from the amino terminus. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 25 (MPT94.17t). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 25 (MPT94.17t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO:

[0072] 25.

[0073] In some embodiments, the membrane-bound prenyltransferase (MPT) comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 17 (MPT94.18). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 17 (MPT94.18). In some embodiments, the MPT comprises a functional fragment of SEQ ID NO: 17 (MPT94.18). In some embodiments, the functional fragment of SEQ ID NO: 17 (MPT94.18) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

[0074] 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,

[0075] 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

[0076] 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73 amino acids deleted from the amino terminus. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 26 (MPT94.18t). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 26 (MPT94.18t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO:

[0077] 26.

[0078] In some embodiments, the membrane-bound prenyltransferase (MPT) comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%,

[0079] 4891 -1065-4412, v. 1 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 18 (MPT94.19). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 18 (MPT94.19). In some embodiments, the MPT comprises a functional fragment of SEQ ID NO: 18 (MPT94.19). In some embodiments, the functional fragment of SEQ ID NO: 18 (MPT94.19) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73 amino acids deleted from the amino terminus. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 27 (MPT94.19t). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 27 (MPT94.19t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 27.

[0080] In some embodiments, the membrane-bound prenyltransferase (MPT) comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 19 (MPT94.20). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 19 (MPT94.20). In some embodiments, the MPT comprises a functional fragment of SEQ ID NO: 19 (MPT94.20). In some embodiments, the functional fragment of SEQ ID NO: 19 (MPT94.20) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73 amino acids deleted from the amino terminus. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 28 (MPT94.20t). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 28 (MPT94.20t).

[0081] 4891 -1065-4412, v. 1 In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 28.

[0082] In some embodiments, the membrane-bound prenyltransferase (MPT) comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 20 (MPT94.21). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 20 (MPT94.21). In some embodiments, the MPT comprises a functional fragment of SEQ ID NO: 20 (MPT94.21). In some embodiments, the functional fragment of SEQ ID NO: 20 (MPT94.21) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73 amino acids deleted from the amino terminus. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 29 (MPT94.21t). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 29 (MPT94.21t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 29.

[0083] In some embodiments, the membrane-bound prenyltransferase (MPT) comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 21 (MPT94.22). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 21 (MPT94.22). In some embodiments, the MPT comprises a functional fragment of SEQ ID NO: 21 (MPT94.22). In some embodiments, the functional fragment of SEQ ID NO: 21 (MPT94.22) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73 amino acids deleted from the amino terminus. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%,

[0084] 4891 -1065-4412, v. 1 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 30 (MPT94.22t). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 30 (MPT94.22t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO:

[0085] 30.

[0086] In some embodiments, the membrane-bound prenyltransferase (MPT) comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 22 (MPT94.23). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 22 (MPT94.23). In some embodiments, the MPT comprises a functional fragment of SEQ ID NO: 22 (MPT94.23). In some embodiments, the functional fragment of SEQ ID NO: 22 (MPT94.23) has at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73 amino acids deleted from the amino terminus. In some embodiments, the membrane-bound prenyltransferase (MPT) functional fragment comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO: 31 (MPT94.23t). In some embodiments, the MPT comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 31 (MPT94.23t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO:

[0087] 31.

[0088] “Identity” refers to the extent to which the sequence of two or more nucleic acids or polypeptides is the same. In some embodiments, percent identity between a sequence of interest and a second sequence over a window of evaluation, e.g., over the length of the sequence of interest, may be computed by aligning the sequences, determining the number of residues (nucleotides or amino acids) within the window of evaluation that are opposite an identical residue allowing the introduction of gaps to maximize identity, dividing by the total number of residues of the sequence of interest or the second sequence (whichever is greater) that fall within the window, and multiplying by 100. When computing the number of identical residues needed to

[0089] 4891 -1065-4412, v. 1 achieve a particular percent identity, fractions are to be rounded to the nearest whole number. Percent identity can be calculated with the use of a variety of computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., generate alignments and provide percent identity between sequences of interest. The algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:22264-2268, 1990) modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993 is incorporated into the NBLAST and XBLAST programs of Altschul et al. (Altschul, et al., J. Mol. Biol. 215:403-410, 1990). To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Altschul, et al. Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs may be used. A PAM250 or BLOSUM62 matrix may be used. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI). See the Web site having URL ncbi.nlm.nih.gov for these programs. In a specific embodiment, percent identity is calculated using BLAST2 with default parameters as provided by the NCBI.

[0090] Fusion Proteins

[0091] Some aspects of the present disclosure are directed to a fusion protein comprising a polypeptide having geranyl diphosphate (GPP) synthase activity and a polypeptide having prenyltransferase activity. As used herein, "GPP synthase activity" is the ability to catalyze the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to geranyl pyrophosphate. As used herein, " prenyltransferase activity" is the ability to catalyze the transfer of a prenyl group from one compound (donor) to another (acceptor).

[0092] Some aspects of the present disclosure are directed to a fusion protein comprising a polypeptide having Geranyl diphosphate synthase activity and a polypeptide having prenyltransferase activity. The polypeptide having prenyltransferase activity is not limited, so long as the polypeptide is capable of prenylating cinnamic acid, coumaric acid, caffeic acid, and / or ferulic acid. In some embodiments, the polypeptide having prenyltransferase activity comprises a polypeptide sequence having at least 85%, 87%, 90%, 92%, 93%, 94%, 95%, 95.5%,

[0093] 4891 -1065-4412, v. 1 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.3%, 99.5%, 99.7%, 99.8% or 99.9% identity to the polypeptide sequence of MPT85 (SEQ ID NO: 1), or MPT94 (SEQ ID NO: 2).

[0094] In some embodiments, the polypeptide having prenyltransferase activity has at least one amino acid modification compared to MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2). The amino acid modification may be a deletion, substitution or insertion. When the amino acid modification is a deletion, it may comprise the deletion of a single amino acid residue, or multiple amino acid residues, so long as the modified polypeptide retains the ability to prenylate a bakuchiol precursor. In some embodiments, the deletion involves a 1-6, 7-12, 13-26, 27-36, 1-39, 1-61, 1-73, or 1- 103 amino acid deletion on the n-terminus of the polypeptide and thus provides a truncated prenyltransferase. In some embodiments, a portion or the entirety of the N- terminal sequence of the polypeptide having prenyltransferase activity can be replaced by an amino acid sequence which increases the expression, folding and / or activity of the polypeptide.

[0095] In other embodiments, the amino acid modification comprises a deletion, substitution or insertion at one or more amino acid positions comprising W78, H99, L106, Ri l l, R121, H123, V124, N165, 1166, Y167, T168, A169, G I70, 1171, N172, D176, 1177, E178, 1179, D180, K181, 1182, N183, K184, M209, Y229, F230, V231, L232, G233, T234, V235, Y236, K246, R247, Y248, P249, A250, F251, C255, F256, F257, 1258, 1259, R260, M282, Y298, V299, 1300, 1301, A302, F303, F304, K305, D306, 1310, E311, G312, D313, K314, E315, H316, G317, M323, E328, G344, E385, Y391, M392, F393, M394, W395, and / or 1400 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0096] In some embodiments, the polypeptide having GPP synthase activity has an amino acid sequence with greater than 90% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the polypeptide having GPP synthase activity has an amino acid sequence with greater than 90%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, 99.9% or 100% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the polypeptide having GPP synthase activity can be a farnesyl pyrophosphate synthase. In some embodiments, this farnesyl pyrophosphate synthase is Yarrowia

[0097] 4891 -1065-4412, v. 1 Erg20. In some embodiments, the polypeptide with GPP synthase activity is UniProt # Q6C6W3 or the F88W.N119W double mutant.

[0098] In some embodiments, the polypeptide having prenyltransferase activity has improved activity for prenylating cinnamic acids with GPP, as compared to the same unfused prenyltransferase. In some embodiments, the polypeptide having prenyltransferase activity has at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or higher activity for condensing GPP with cinnamic acids as compared to the same unfused prenyltransferase. In other embodiments, the fusion protein provides enhanced yield of bakuchiol or analogs thereof over yield provided by an unfused prenyltransferase.

[0099] In some embodiments, the fusion protein further comprises a linker polypeptide between the polypeptide having Geranyl diphosphate synthase activity (GPS) and the polypeptide having prenyltransferase activity. The linker is not limited and may be any suitable linker. For example, a linker can be a short polypeptide (e.g., 5-52 amino acids). Often a linker is composed of small amino acid residues such as serine, glycine, and / or alanine, but can also contain charged amino acids such as lysine, arginine, aspartic acid or glutamic acid. A heterologous domain could comprise a transmembrane domain, a secretion signal domain, etc. In some embodiments, the linker is a polypeptide. In some embodiments, the polypeptide is 5 to 52 amino acids in length.

[0100] In some embodiments the geranyl diphosphate is fused to the N-terminus of the prenyl transferase.

[0101] Cells and cell culture

[0102] The cell is not limited and may be any suitable cell for expression. In some embodiments, the cell may be a microorganism or a plant. In some embodiments, the microorganism is a bacteria (e.g., E. coli), an algae, or a yeast. In some embodiments, the yeast is an oleaginous yeast (e.g., a Yarrowia lipolytica strain). In some embodiments, the bacteria is Escherichia coli.

[0103] Suitable cells may include, but are not limited to, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia

[0104] 4891 -1065-4412, v. 1 opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha (now known as Pichia angusta), Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus, Schizosaccharomyces pompe, Dekkera bruxellensis, Arxula adeninivorans, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas reinhardtii, Yarrowia lipolytica and the like. In some embodiments, the cell is a protease-deficient strain of Saccharomyces cerevisiae. In some embodiments, the cell is a eukaryotic cell other than a plant cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a plant cell, where the plant cell is one that does not normally produce bakuchiol or analogs thereof. In some embodiments, the cell is Saccharomyces cerevisiae. In some embodiments, the cell is Yarrowia lipolytica. In some embodiments, the cell disclosed herein is cultured in vitro.

[0105] In some embodiments, the cell is a prokaryotic cell. Suitable prokaryotic cells may include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus sp., Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al, (1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302. Examples of Salmonella strains which can be employed may include, but are not limited to, Salmonella typhi and S. typhimurium. Suitable Shigella strains may include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Typically, the laboratory strain is one that is non-pathogenic. Non-limiting examples of other suitable bacteria may include, but are not limited to, Bacillus subtilis, Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodo spirillum rubrum, Rhodococcus sp., and the like.

[0106] In some embodiments, the cell has been engineered to provide greater flux of GPP through the GPP pathway. The method for obtaining increased flux of GPP through the GPP pathway is not particularly limited, and may include methods disclosed herein or known in the art. For example, increased flux of GPP through the pathway may be achieved by increasing the expression of enzymes involved in the 1-

[0107] 4891 -1065-4412, v. 1 deoxy-d-xylulose-5-phosphate (DXP) pathway. Increased expression of DXP synthase, DXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP- ME) synthetase, CDP-ME kinase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEDCDP) synthase, 4-hydroxy-3-methylbut-2-l-yl diphosphate synthase (Gcpe), or

[0108] 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB). Additionally or alternatively, increased flux of GPP through the pathway may be achieved by increasing the expression of enzymes involved in the mevalonate pathway (MVA). For example increased expression of acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate-5 -kinase, phosphomevalonate kinase, mevalonate-

[0109] 5-phosphate decarboxylase, isopentenyl phosphate kinase, mevalonate pyrophosphate decarboxylase, and / or isopentenyl pyrophosphate isomerase.

[0110] In some embodiments, the cell has been engineered to provide greater flux of tyrosine through the tyrosine biosynthesis pathway. In some embodiments, the cell has been engineered to provide greater flux of phenylalanine through the phenylalanine biosynthesis pathway. In some embodiments, the cell has been engineered to provide greater flux of both tyrosine and phenylalanine through the respective aromatic amino acid biosynthesis pathways. To increase the flux to Tyr and Phe, certain enzymes of the aromatic pathway will be overexpressed such as the first enzyme of this pathway, DAHP synthase (EC 2.5.1.54) specifically Aro4 as the wild type sequence (Uniprot Q6CCS4) or a feedback insensitive (to Tyrosine) mutant Aro4k221 L. Other enzymes in the aromatic amino acid pathway that will be upregulated or be overexpressed include the pentafunctional DHAP to 5-enolpyruvate shikimate- 3-phosphate (e.g Arol-Q6ClX5), chorismite synthase (e.g Q6C8Q1), chorismite mutase (e.g Q6C5J7), prephenate dehydrogenase (e.g Q6C1B7), and hydroxyphenylpyruvate aminotransferase.

[0111] In some embodiments, the greater flux through an aromatic amino acid synthesis pathway is achieved by overexpressing an Aro4 enzyme (EC 2.5.1.54). In some embodiments, the Aro4 enzyme comprises an amino acid sequence having at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% identity to SEQ ID NO:4. In some embodiments, the greater flux through an aromatic amino acid synthesis pathway is achieved by expressing a feedback insensitive Aro4 enzyme. In some embodiments,

[0112] 4891 -1065-4412, v. 1 the feedback insensitive Aro4 enzyme has a sequence with at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% identity to SEQ ID NO: 5.

[0113] In some embodiments, the cell has been modified to knockdown expression of one or more genes that produce unwanted byproducts in the biosynthesis pathways leading to the production of bakuchiol and analogs thereof. In some embodiments the cell has been modified to knockout one or more genes that produce unwanted byproducts in the bakuchiol biosynthesis pathway. It will be appreciated that the strategy to select knockdown or knockout of any one or more genes will depend on whether the gene-encoded product is critical for the survival of the cell, and the resulting increase in bakuchiol yield obtained by the gene knockdown or knockout.

[0114] In some embodiments, the cell is a yeast cell, an algal cell, or a bacterial cell. In some embodiments, the yeast cell is Saccharomyces, Pichia, or Yarrowia.

[0115] In some embodiments, the cell is capable of synthesizing coumaric acid, caffeic acid, and / or ferulic acid when tyrosine is present in the cell through endogenous production. In some embodiments, the cell is capable of producing cinnamic acid, coumaric acid, caffeic acid, or ferulic acid when phenylalanine is present in the cell through endogenous production.

[0116] In some embodiments, production of bakuchiol or analogs thereof in the cell is dependent on, or is enhanced by supplementation of cinnamic acid, coumaric acid, and / or ferulic acid within the fermentation medium in which the cell is grown.

[0117] In some embodiments, the cell is capable of converting one or more of cinnamic acid to coumaric acid, coumaric acid to caffeic acid, and caffeic acid to ferulic acid. In some embodiments, the cell expresses phenylalanine ammonia lyase and / or tyrosine ammonia lyase of the family EC 4.3.1.24 and / or family EC 4.3.1.25. In some embodiments, the cell expresses cinnamate 3-hydroxylase. In some embodiments, the cinnamate 3-hydroxylase is a p450 enzyme of the CYP73A family or family EC 1.14.14.91. In some embodiments, the cell expresses caffeic acid O- methyltransferase of the family EC 2.1.1.68

[0118] In some embodiments, the cell is capable of converting bakuchiol to 3- hydroxy-bakuchiol, and / or 3-hydroxy-bakuchiol to 3-methoxy-bakuchiol. In some embodiments, the conversion of bakuchiol to 3-hydroxybakuchiol is catalyzed by a

[0119] 4891 -1065-4412, v. 1 P450 hydroxylase of the family CYP98A. In some embodiments, the conversion of 3-hydroxybakuchiol to 3-methoxybakuchiol is catalyzed by a methyltransferase of the family EC 2.1.1.68 or EC 2.1.1.42.

[0120] An expression vector or vectors can be constructed to include exogenous nucleotide sequences coding for the membrane-bound prenyltransferase described herein operably linked to expression control sequences functional in the cell. Expression vectors applicable include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be coexpressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

[0121] The term “exogenous” is intended to mean that the referenced molecule or the referenced activity is introduced into the cell. The molecule can be introduced, for

[0122] 4891 -1065-4412, v. 1 example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the cell. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in the cell. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term “heterologous” refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid can utilize either or both a heterologous or homologous encoding nucleic acid.

[0123] In some embodiments, the cell expressing the prenyltransferase is capable of making bakuchiol or analogs thereof in the presence of a carbon source and, optionally, cinnamic acid, coumaric acid, caffeic acid and / or ferulic acid. Exemplary carbon sources include sugar carbons such as sucrose, glucose, mannitol, galactose, fructose, mannose, isomaltose, xylose, pannose, maltose, arabinose, cellobiose and 3-, 4-, or 5- oligomers thereof. Other carbon sources include alcohol carbon sources such as, ethanol, glycerol. Other carbon sources may contain a combination of the above carbon sources such as, for example, gluco se / mannitol or glucose / ethanol. Other carbon sources include acid and esters such as acetate or formate, or fatty acids having four to twenty-two carbon atoms or fatty acid esters thereof. Other carbon sources can include renewal feedstocks and biomass. Exemplary renewal feedstocks include cellulosic biomass, hemicellulosic biomass and lignin feedstocks. Mixed carbon sources can also be used, such as a fatty acid and a sugar as described herein.

[0124] Depending on the cell, the appropriate culture medium may be used. For example, descriptions of various culture media may be found in “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington

[0125] 4891 -1065-4412, v. 1 D.C., USA, 1981). As used here, “medium” as it relates to the growth source refers to the starting medium be it in a solid or liquid form. “Cultured medium”, on the other hand and as used here refers to medium (e.g. liquid medium) containing microbes that have been fermentatively grown and can include other cellular biomass. The medium generally includes one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0126] The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large-scale culture procedures. Useful yields of the products can be obtained under aerobic culture conditions. An exemplary growth condition for achieving, one or more bakuchiol or analog products includes aerobic culture or fermentation conditions. In certain embodiments, the microbial organism can be sustained, cultured or fermented under aerobic conditions.

[0127] Substantially aerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 5% and 100% of saturation. The percent of dissolved oxygen can be maintained by, for example, sparging air, pure oxygen or a mixture of air and oxygen.

[0128] The culture conditions can be scaled up and grown continuously for manufacturing bakuchiol products. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of bakuchiol products. Generally, and as with non-continuous culture procedures, the continuous and / or near- continuous production of bakuchiol products will include culturing a bakuchiol producing organism on sufficient nutrients and medium to sustain and / or nearly sustain growth in an exponential phase. Continuous culture under such conditions can include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, the desired microorganism can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and / or near- continuous culture conditions also can include all time intervals in between these

[0129] 4891 -1065-4412, v. 1 exemplary periods. It is further understood that the time of culturing the microbial organism is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.

[0130] Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of bakuchiol and bakuchiol analog products can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

[0131] In some embodiments, the methods further comprise a step of purifying or isolating the bakuchiol or analogs thereof from the culture. Methods of isolation are not limited and may be any suitable method known in the art. Purification methods include, for example, extraction procedures (e.g., using supercritical carbon dioxide, ethanol or mixtures of these two), as well as methods that include continuous liquidliquid extraction, pervaporation, evaporation, filtration, membrane filtration (including reverse osmosis, nanofiltration, ultrafiltration, and microfiltration), membrane filtration with diafiltration, membrane separation, reverse osmosis, electrodialysis, distillation, extractive distillation, reactive distillation, azeotropic distillation, crystallization and recrystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, carbon adsorption, hydrogenation, and ultrafiltration or centrifugal partition chromatography (CPC).

[0132] In some embodiments, the cells are grown in stirred tank fermenters with feed supplementation (sugars with or without organic acids) where the dissolved oxygen, temperature, and pH are be controlled according to the optimal growth and production process. In some embodiments, aqueous non-miscible organic solvents are supplemented to dissolve added organic acids or extract the bakuchiol products as they are being synthesized. In some embodiments, these solvents may include, but are not limited to, isopropyl myristate (IPM), diisobutyl adipate, Bis(2-ethylhexyl) adipate, decane, dodecane, hexadecane or anther organic solvent with logP>5. The later number (logP) is defined as the log of a compound’ s partition between water and octanol and is a standard parameter of a compound's hydrophobicity (the larger the

[0133] 4891 -1065-4412, v. 1 logP the less soluble in water). Depending on the fermentation process, the products can be isolated and purified using different methods.

[0134] In some embodiments, the targeted bakuchiol or analog thereof precipitates together with the cell biomass after centrifugation, or is isolated in the solids after water removal using spray drying or other methods that remove water (i.e lyophilization, ultrafiltration etc). In one embodiment, an aqueous miscible organic solvent (ethanol, acetonitrile, etc.) is added to the bakuchiol containing cell pellet to dissolve the products. In some embodiments, a simple filtration, ultrafiltration or centrifugation can remove the cells and the aqueous / organic media evaporated to dryness or to a small volume from which the bakuchiol product will precipitate or crystalize. Alternatively, the bakuchiol containing cell pellet can be extracted with an aqueous immiscible organic solvent (ethyl acetate, heptane, decane, etc.) or supercritical carbon dioxide (with 0-10% Ethanol) to extract the bakuchiol. Evaporation of the organic solvent and a possible recrystallization will produce pure bakuchiol or analog thereof. If the bakuchiol is not extracted by the previous methods and are trapped inside the cell, cells lysis may be required prior to extraction methods described above. In some embodiments, cells are disrupted using mechanical methods or by suspension in appropriate lysis buffers from which the bakuchiol can be extracted with an organic aqueous immiscible solvent (ethyl acetate, hexane, decane, methylene chloride, etc.). In other embodiments, cells may be suspended in an organic solvent (ethanol, methanol, methylene chloride, etc.) that extracts the bakuchiol from the cells.

[0135] In some embodiments, an organic solvent is required during growth that is separated at the end of the fermentation. Back extraction with alkaline aqueous solvent or a different organic solvent with low boiling point and high polarity (ethanol, acetonitrile, etc.) will remove the bakuchiol. Isolation can then involve a simple pH shift if water is used, or an evaporation if organic solvents are used. In both cases, a recrystallization step may be required at the end to improve purity of the product.

[0136] EXAMPLES

[0137] Example 1: Screening of prenyltransferases with coumaric and caffeic feeds

[0138] 4891 -1065-4412, v. 1 Plasmids expressing each gene were transformed into K lipolytica strain SB 1334. This strain is engineered to produce increased amounts of GPP and contains an Erg20.A28 mutation, by overexpression of ERG20.F88W.N119W (SEQ ID NO: 36) and the ERG20.A28 allele (SEQ ID NO: 40), and disruption of the endogenous Erg20 as described in co-owned PCT Application No. PCT / US2022 / 046926, incorporated by reference in its entirety. Transformants were plated on YPD (10 g / L yeast extract, 20 g / L peptone, 20 g / L dextrose) media agar plates with 1 mg / mL hygromycin and grown for 48 h at 30 °C. Colonies were picked into 0.5 mL YDCM medium (yeast nitrogen base + nitrogen, 6.71 g / L; Casamino acids, 10 g / L; Dextrose monohydrate, 66 g / L; MES hydrate 19.5, g / L; pH adjusted to 6.5 with KOH) containing 1 mg / mL hygromycin in 96 well blocks (2.0 mL / well). These precultures were grown for 24 h at 30 °C with 1000 rpm in a high-speed shaker before 2 pL from each well was used to inoculate assay cultures of 0.5 mL YDCM medium containing 1 mg / mL hygromycin in 96 well blocks (2.0 mL / well). The assay cultures were grown for 24 h at 30 °C with 1000 rpm in a high-speed shaker before they were fed 10 mM p-coumaric acid or caffeic acid. After an additional 24 h, the cultures were quenched with equal parts EtOH containing the internal standard (0.2 mg / mL of 3,5- Diisopropyl-2-hydroxybenzoic acid CAS# 2215-21-6), centrifuged, and clarified solutions were then analyzed by HPLC-MS.

[0139] Table 1: Initial screen of prenyltransferases with coumaric or caffeic acid feeds. Product concentrations in pM. Assays were carried out with replicates of n=7.

[0140] Example 2: Synthesis of bakuchiol from glucose

[0141] To generate a strain that is able to produce bakuchiol from sugar, strain SB3503 was created by integrating bakuchiol prenyltransferase, MPT94.0, and the GPP synthase, ERG20.L88W.N119W, into the genome of SB2728. The later strain produces coumaric acid from glucose using AtPAL, a phenylalanine ammonia lyase,

[0142] 4891 -1065-4412, v. 1 and a cytochrome P450 system consisting of AtC4H (CYP73A5), a cytochrome P450 from the CYP73A subfamily (EC 1.14.14.91), and its accessory protein, AtATR2, a cytochrome P450 reductase, all from Arabidopsis thaliana.

[0143] To assess bakuchiol production, 16 independent transformants were assessed for bakuchiol titers in 96-well plate fermentation. 0.5 mL YDCM medium were inoculated with each transformant in 96 well blocks (2.0 mL / well). These precultures were grown for 24 h at 30 °C with 1000 rpm in a high-speed shaker before 2 pL from each well was used to inoculate assay cultures of fresh 0.5 mL YDCM medium in 96 well blocks (2.0 mL / well). The assay cultures were grown for 48 h at 30 °C with 1000 rpm in a high-speed shaker then quenched with equal parts EtOH containing the internal standard (0.2 mg / mL of 3,5-Diisopropyl-2-hydroxybenzoic acid CAS# 2215-21-6), centrifuged, and clarified solutions were then analyzed by HPLC-MS. These transformants were able to produce bakuchiol (26 to 313 pM) from sugar while the parent that did not carry MPT94.0 and the ERG20.E88W.N119W did not produce any bakuchiol.

[0144] Table 2: Bakuchiol production from sugar. Top five bakuchiol titers (pM) obtained from individual transformants of SB3503. No bakuchiol was detected with the parent strain, SB2728.

[0145] Example 3: Synthesis of 3-hydroxy bakuchiol from glucose

[0146] 4891 -1065-4412, v. 1 For the synthesis of 3-hydroxybakuchiol from glucose the strain developed in Experiment 2 will be used as background. In this strain, enzymes that can convert bakuchiol to 3-hydroxybakuchiol will be included. Such enzymes include P450 monooxygenases from the CYP98A subfamily (EC 1.14.14.96). Specifically, AtC3H, a mutant CYP98A85 from Amborella trichopoda (NCBI: XP_011625941.1), with increased activity on bakuchiol is expressed in the strain. In addition, another pathway working in parallel can be engineered consisting of a coumarate 3-hydroxylase and an engineered MPT85 or MP94, as described here, with increased activity on caffeic acid to 3-hydroxy caffeic acid.

[0147] Example 4: Synthesis of 3-methoxy bakuchiol from glucose

[0148] For the synthesis of 3-methoxybakuchiol, the previous optimized strain for making 3-hydroxybakuchiol will be used. In this strain a methyl-transferase will be included to convert 3-hydroxybakuchiol to 3-methoxybakuchiol. For this step enzymes in 2.1.1.42 or 2.1.1.68 family will be used. Specifically, an engineered methyltransferase 0MT9 (Uniprot: I1I1F5; (SEQ ID NO: 42)) will be used.

[0149] Example 5: Synthesis of bakuchiol and 3-hydroxy bakuchiol with coumaric and caffeic acid supplementation

[0150] Bakuchiol and 3-hydroxybakuchiol can be synthesized in a strain that has an upregulated pathway for GPP biosynthesis as background as described in Example 2. In this organism MPT85 or MPT94 optimized for coumaric or caffeic acid activity will be expressed. The cells will be grown in a carbon source (glucose, glycerol etc) and will be fed coumaric or caffeic acid.

[0151] Example 6: N-terminal truncations of MPT94

[0152] Sequence analysis of MPT94 predicted that the first 85 amino acids constitute a signal peptide. However, MPT94.1 (SEQ ID NO: 6) with amino acids 2-85 removed (preserving the start codon) was inactive. A model of the three-dimensional structure

[0153] 4891 -1065-4412, v. 1 of MPT94 predicted secondary structural elements in N-terminal region of the protein so several truncations based on these structural elements (MPT94.2-8, SEQ ID NO: 7- 13) were screened.

[0154] Plasmids expressing each gene were transformed into Y. lipolytica strain SB 1334. Transformants were plated on YPD media agar plates with 1 mg / mL hygromycin and grown for 48 h at 30 °C. Colonies were picked into 0.5 mL YDCM medium containing 1 mg / mL hygromycin in 96 well blocks (2.0 mL / well). These precultures were grown for 24 h at 30 °C with 1000 rpm in a high-speed shaker before 2 pL from each well was used to inoculate assay cultures of 0.5 mL YDCM medium containing 1 mg / mL hygromycin in 96 well blocks (2.0 mL / well). The assay cultures were grown for 24 h at 30 °C with 1000 rpm in a high-speed shaker before they were fed 3 mM p-coumaric acid. After an additional 24 h, the cultures were quenched with equal parts EtOH containing the internal standard, centrifuged, and clarified solutions were then analyzed by HPLC-MS.

[0155] Table 3: Screen of MPT94 and N-terminal truncations with coumaric acid feed. Product concentrations in pM. Assays were carried out with replicates of n=8.

[0156] As shown in Table 3, MPT94 activity increases with more residues removed from the N-terminus for 6, 12, 26, and 36 residues removed (MPT94.2-MPT94.5). However, MPT94.6 and MPT94.7, with 62 and 70 residues removed, respectively, were inactive, showing that there is a limit to how many residues can be removed from the N-terminus before abolishing enzyme activity.

[0157] MPT94.8, with 39 residues removed, was screened under the same conditions as those above.

[0158] 4891 -1065-4412, v. 1 Table 4: Screen of MPT94 and N-terminal truncations with coumaric or caffeic acid feed. Product concentrations in |jM. Assays were carried out with replicates of n=4.

[0159] Example 7: Additional prenyltransferase sequences from plant genomes

[0160] MPT94.15-MPT94.22 (SEQ ID NO: 14-21) discovery. N-terminal truncated versions (MPT94.15t-MPT94.22t, SEQ ID NO: 23-30) were constructed with residues 2-37 removed (the same residues removed in MPT94.5, which produced the highest concentration of bakuchiol in Table 3).

[0161] Plasmids expressing each gene were transformed into Y. lipolytica strain SB3185. Transformants were plated on YPD media agar plates with 1 mg / mL hygromycin and grown for 72 h at 30 °C. Colonies were picked into 0.5 mL YDCM medium containing 1 mg / mL hygromycin in 96 well blocks (2.0 mL / well). These precultures were grown for 24 h at 30 °C with 1000 rpm in a high-speed shaker before 5 pL from each well was used to inoculate assay cultures of 0.5 mL YDCM medium containing 1 mg / mL hygromycin in 96 well blocks (2.0 mL / well). The assay cultures were grown for 24 h at 30 °C with 1000 rpm in a high-speed shaker before they were fed 3 mM p-coumaric acid and 1% oleic acid. After an additional 24 h, the cultures were quenched with equal parts EtOH containing the internal standard, centrifuged, and clarified solutions were then analyzed by HPLC-MS.

[0162] Table 5: Screen of MPT94.5 and isoforms with coumaric acid feed. Product concentrations in pM. Assays were carried out with replicates of n=8. Residue numbering corresponds to that of full length MPT94.

[0163] 4891 -1065-4412, v. 1

[0164] As shown in Table 5, MPT94.22t produced the highest concentration of bakuchiol. Analysis of the mutations in MPT94.15t-MPT94.22t and their activity suggested that the mutation C78W should further improve the activity of MPT94.22t and so MPT94.23t was screened under the same conditions as above.

[0165] Table 6: Screen of MPT94.5 and isoforms with coumaric acid feed. Product concentrations in pM. Assays were carried out with replicates of n=4-8. Residue numbering corresponds to that of full length MPT94.

[0166] Example 8: Mutagenesis of MPT94.22t

[0167] Positions H123 and E385 (residue numbering corresponds to that of full length MPT94.22) in MPT94.22t were selected for saturation mutagenesis based on examination of a model of the protein’s three-dimensional structure.

[0168] Plasmids expressing MPT94.22t with NNS degenerate codons at the targeted positions were transformed into Y. lipolytica strain SB3185. Transformants were plated on YPD media agar plates with 1 mg / mL hygromycin and grown for 48 h at 30 °C. Eighty colonies of each position were picked into 0.5 mL YDCM medium containing 1 mg / mL hygromycin in 96 well blocks (2.0 mL / well). These precultures

[0169] 4891 -1065-4412, v. 1 were grown for 24 h at 30 °C with 1000 rpm in a high-speed shaker before 2 pL from each well was used to inoculate assay cultures of 0.5 mL YDCM medium containing 1 mg / mL hygromycin in 96 well blocks (2.0 mL / well). The assay cultures were grown for 24 h at 30 °C with 1000 rpm in a high-speed shaker before they were fed 3 mM p-coumaric acid and 1% oleic acid. After an additional 24 h, the cultures were quenched with equal parts EtOH containing the internal standard, centrifuged, and clarified solutions were then analyzed by HPLC-MS. Cultures with >10% increased bakuchiol concentration compared to MPT94.22t were selected for sequencing and rescreening in replicate under the same conditions as above.

[0170] Table 7: Rescreen of MPT94.22t mutants with coumaric acid feed. Product concentrations in pM. Assays were carried out with replicates of n=8. Residue numbering corresponds to that of full length MPT94.22.

[0171] Example 9: Bakuchiol synthesis in 2L fermenter

[0172] To evaluate the productivity of MPT94 and validate the small scale results, strain SB3706 was created by integrating a truncated Yarrowia HMGR synthase into SB3503 (described in Example 2). This strain can make coumaric acid and GPP from glucose and MPT94 converts them to bakuchiol.

[0173] A 2L fermenter containing 800 mL of broth (10 g / L ammonium sulfate, 20 g / L casamino acids, 3.4 g / L yeast nitrogen base) was inoculated with a fresh culture of SB3706 (initial GD600=0.3) and the cells grew at pH 5.5, 30 °C and 20% dissolved oxygen. Culture was supplemented with 3g / L / hr (dry weight) dextrose for 5 days and accumulated 11.7 mM (1.9 g / L) of coumaric acid and 5.45 mM (1.39 g / L) of bakuchiol.

[0174] 4891 -1065-4412, v. 1 REFERENCES

[0175] 1. Alam F., et al Phytotherapy Research 2018, 32, 597-615.

[0176] 2. Zhang, X. et all, The American Journal of Chinese Medicine, 2016, 44(1), 35- 60. 3. Alam F. et al Phytotherapy Research, 2018, 32, 597-615.

[0177] 4. Jafernik, K. et al Nat Prod Res 2020, 5828-5842.

[0178] 5. Chaudhuri, RK, Bojanowski K Int J Cosmetic Sci 2014, 36, 221.

[0179] 6. Draelos ZD, et al Journal of Drugs in Dermatology, 2020, 19(12), 1181.

[0180] 7. Afzal, N, Sivanami RK J. Cosmetic Dermatology, 2023, 00, 1-2.

[0181] 4891 -1065-4412, v. 1 LISTING OF AMINO ACID SEQUENCES

[0182] MPT85 (SEQ ID NO: 1)

[0183] MASMFLGSLPLASSVNYIGRITRSKNCTESYHATSYITNASSNKTEKIKHEYAN

[0184] MRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETL

[0185] WGSIKQFCDAFYRFSRPHVIIGTAVNIIVMSSLALEKSSDISPKFFIGLFQVIVTI

[0186] LSMNIYTAGINQLTDIEIDKINKPYLPLASGEYSYKTGVTIITLCAILSLGVGWI

[0187] VGSPALFWSSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAFF

[0188] LHIQTHVFKRPMMISKPVMFGTAFMSFFYVIIAFFKDIPDIEGDKDHGVKSLT

[0189] MRLGQERVFWICVSLLLTGYGAAIVVGATSSFLWCKLITVSGHALLASIFWN

[0190] RAKSVDLKSHQEITSLYMFMWKLFYIEYFIIPLMR

[0191] MPT94 (SEQ ID NO: 2)

[0192] MASMFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYA

[0193] NMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPET

[0194] LLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILTT

[0195] IPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAIMSLLVGWV

[0196] VGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAFF

[0197] LHIQTHVFKRPMMIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTM

[0198] RLGQERVFWICVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNR

[0199] ANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0200] Y1ERG20 (WT) (SEQ ID NO: 3)

[0201] MSKAKFESVFPRISEELVQLLRDEGLPQDAVQWFSDSLQYNCVGGKLNRGLS

[0202] VVDTYQLLTGKKELDDEEYYRLALLGWLIELLQAFFLVSDDIMDESKTRRGQ

[0203] PCWYLKPKVGMIAINDAFMLESGIYILLKKHFRQEKYYIDLVELFHDISFKTEL

[0204] GQLVDLLTAPEDEVDLNRFSLDKHSFIVRYKTAYYSFYLPVVLAMYVAGITN

[0205] PKDLQQAMDVLIPLGEYFQVQDDYLDNFGDPEFIGKIGTDIQDNKCSWLVNK

[0206] ALQKATPEQRQILEDNYGVKDKSKELVIKKLYDDMKIEQDYLDYEEEVVGDI

[0207] KKKIEQVDESRGFKKEVLNAFLAKIYKRQK*

[0208] Y1ARO4 (WT) (SEQ ID NO: 4)

[0209] MSRSSSPNASSAEDVRILGYDPLLAPALLQTEVASTKNARETVSKGRKDSIDVI

[0210] TGKSDKLLCIVGPCSLHDPKAAMEYAQRLKELSDKLSGELVIVMRAYLEKPR

[0211] TTVGWKGLINDPDMDESFNINKGLRLSRKVFCDLTDLGLPIASEMLDTISPQF

[0212] LADLLSLGAIGARTTESQLHRELASGLSFPVGFKNGTDGTLGVAVDAVQAAS

[0213] HPHHFMGVTKQGVAAITTTKGNENCFIILRGGKKGTNYDAESVAECKKATES

[0214] MLMVDCSHGNSNKDYRNQPKVSKAVAEQVAAGEKKIIGVMIESNIHEGNQK

[0215] VPKEGPSALKYGVSITDACVSWETTVDMLTELANAVKERRNKN

[0216] 4891 -1065-4412, v. 1 Y1ARO4_K221L (SEQ ID NO: 5)

[0217] MSRSSSPNASSAEDVRILGYDPLLAPALLQTEVASTKNARETVSKGRKDSIDVI

[0218] TGKSDKLLCIVGPCSLHDPKAAMEYAQRLKELSDKLSGELVIVMRAYLEKPR

[0219] TTVGWKGLINDPDMDESFNINKGLRLSRKVFCDLTDLGLPIASEMLDTISPQF

[0220] LADLLSLGAIGARTTESQLHRELASGLSFPVGFKNGTDGTLGVAVDAVQAAS

[0221] HPHHFMGVTLQGVAAITTTKGNENCFIILRGGKKGTNYDAESVAECKKATES

[0222] MLMVDCSHGNSNKDYRNQPKVSKAVAEQVAAGEKKIIGVMIESNIHEGNQK

[0223] VPKEGPSALKYGVSITDACVSWETTVDMLTELANAVKERRNKN

[0224] MPT94.1 (SEQ ID NO: 6)

[0225] MAASGESLESHEAQHHTPETLLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLA

[0226] LENSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFS

[0227] VRTGVIIITLCAIMSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRY

[0228] PAFAALCFFIIRGLMFHVAFFLHIQTHVFKRPMMIPKSVMFGTAFMSFFYVIIA

[0229] LFKDIPDIEGDKEHGVKSLTMRLGQERVFWICVSLLLMAYGGAIVVGATSSFL

[0230] WSKLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLI

[0231] R

[0232] MPT94.2 (SEQ ID NO: 7)

[0233] MSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYANMRHR

[0234] QHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLLGSIK

[0235] RFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILTTIPMNL

[0236] YASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAIMSLLVGWVVGSPA

[0237] LFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAFFLHIQTH

[0238] VFKRPMMIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTMRLGQE

[0239] RVFWICVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNRANSVD

[0240] LKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0241] MPT94.3 (SEQ ID NO: 8)

[0242] MSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYANMRHRQHNLKH

[0243] NYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLLGSIKRFCDAF

[0244] YRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILTTIPMNLYASGLN

[0245] QLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAIMSLLVGWVVGSPALFWTSF

[0246] AYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAFFLHIQTHVFKRP

[0247] 4891 -1065-4412, v. 1 MMIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTMRLGQERVFWI CVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNRANSVDLKSHE EITSLYMFMWKLFYIEYLMIPLIR

[0248] MPT94.4 (SEQ ID NO: 9)

[0249] MTESYHATSYITTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCED

[0250] WARNFVVNAASGESLESHEAQHHTPETLLGSIKRFCDAFYRFSRPHVITGTAI NIIVMSSLALENSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPY LPLASGEFSVRTGVIIITLCAIMSLLVGWVVGSPALFWTSFAYFVLGTVYTINL

[0251] PLLRWKRYPAFAALCFFIIRGLMFHVAFFLHIQTHVFKRPMMIPKSVMFGTAF MSFFYVIIALFKDIPDIEGDKEHGVKSLTMRLGQERVFWICVSLLLMAYGGAI VVGATSSFLWSKLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLF

[0252] YIEYLMIPLIR

[0253] MPT94.5 (SEQ ID NO: 10)

[0254] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNA

[0255] ASGESLESHEAQHHTPETLLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALE

[0256] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0257] TGVIIITLCAIMSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPA

[0258] FAALCFFIIRGLMFHVAFFLHIQTHVFKRPMMIPKSVMFGTAFMSFFYVIIALF

[0259] KDIPDIEGDKEHGVKSLTMRLGQERVFWICVSLLLMAYGGAIVVGATSSFLW

[0260] SKLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0261] MPT94.6 (SEQ ID NO: 11)

[0262] MHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLLGSIKRFCD

[0263] AFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILTTIPMNLYASG

[0264] LNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAIMSLLVGWVVGSPALFWT

[0265] SFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAFFLHIQTHVFKR

[0266] PMMIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTMRLGQERVFW

[0267] ICVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNRANSVDLKSHE

[0268] EITSLYMFMWKLFYIEYLMIPLIR

[0269] MPT94.7 (SEQ ID NO: 12)

[0270] MVSTCEDWARNFVVNAASGESLESHEAQHHTPETLLGSIKRFCDAFYRFSRP

[0271] HVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEI

[0272] 4891 -1065-4412, v. 1 DKINKPYLPLASGEFSVRTGVIIITLCAIMSLLVGWVVGSPALFWTSFAYFVLG

[0273] TVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAFFLHIQTHVFKRPMMIPKSV

[0274] MFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTMRLGQERVFWICVSLLLM

[0275] AYGGAIVVGATSSFLWSKLITVSGHALLASIFWNRANSVDLKSHEEITSLYMF

[0276] MWKLFYIEYLMIPLIR

[0277] MPT94.8 (SEQ ID NO: 13)

[0278] MSWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASG

[0279] ESLESHEAQHHTPETLLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALENSS

[0280] DISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGV

[0281] IIITLCAIMSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAA

[0282] LCFFIIRGLMFHVAFFLHIQTHVFKRPMMIPKSVMFGTAFMSFFYVIIALFKDIP

[0283] DIEGDKEHGVKSLTMRLGQERVFWICVSLLLMAYGGAIVVGATSSFLWSKLI

[0284] TVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0285] MPT94.15 (SEQ ID NO: 14)

[0286] MASMFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYA

[0287] NMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNAASGESLESHEAQNHTPET

[0288] LWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILT

[0289] TIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAILSLLVGW

[0290] VVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAF

[0291] FLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTL

[0292] RLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWSKLITVSGHALLASIFWNR

[0293] ANSVDLKSHEEITSLYMFMWKLFYVEYLMIPLIR

[0294] MPT94.16 (SEQ ID NO: 15)

[0295] MASMFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYA

[0296] NMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPET

[0297] LWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILT

[0298] TIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAILSLLVGW

[0299] VVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAF

[0300] FLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTL

[0301] RLGQKRVFWICVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNR

[0302] ANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0303] 4891 -1065-4412, v. 1 MPT94.17 (SEQ ID NO: 16)

[0304] MASMFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYA

[0305] NMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPET

[0306] LLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILTT

[0307] IPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAILSLLVGWV

[0308] VGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAFF

[0309] LHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTL

[0310] RLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWSKLITVSGHALLASIFWNR

[0311] ANSVDLKSHEEITSLYMFMWKLFYVEYLMIPLIR

[0312] MPT94.18 (SEQ ID NO: 17)

[0313] MASMFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYA

[0314] NMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNAASGESLESHEAQHHTPET

[0315] LLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILTT

[0316] IPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAILSLLVGWV

[0317] VGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAFF

[0318] LHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTL

[0319] RLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWSKLITVSGHALLASIFWNR

[0320] ANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0321] MPT94.19 (SEQ ID NO: 18)

[0322] MASMFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYA

[0323] NMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNAASGESLESHEAQNHTPET

[0324] LWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILT

[0325] TIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAILSLLVGW

[0326] VVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAF

[0327] FLHIQTHVFKRPMMIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLT

[0328] LRLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWSKLITVSGHALLASIFWN

[0329] RANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0330] MPT94.20 (SEQ ID NO: 19)

[0331] MASMFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYA

[0332] NMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNAASGESLESHEAQNHTPET

[0333] LWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILT

[0334] TIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAILSLLVGW

[0335] VVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAF

[0336] 4891 -1065-4412, v. 1 FLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTL

[0337] RLGQKRVFWICVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNR

[0338] ANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0339] MPT94.21 (SEQ ID NO: 20)

[0340] MASMFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYA

[0341] NMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNAASGESLESHEAQNHTPET

[0342] LWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILT

[0343] TIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAIMSLLVGW

[0344] VVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAF

[0345] FLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTL

[0346] RLGQKRVFWICVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNR

[0347] ANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0348] MPT94.22 (SEQ ID NO: 21)

[0349] MASMFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYA

[0350] NMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNAASGESLESHEAQNHTPET

[0351] LWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILT

[0352] TIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAILSLLVGW

[0353] VVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAF

[0354] FLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTL

[0355] RLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWSKLITVSGHALLASIFWNR

[0356] ANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0357] MPT94.23 (SEQ ID NO: 22)

[0358] MASMFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYA

[0359] NMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQNHTPET

[0360] LWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILT

[0361] TIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAILSLLVGW

[0362] VVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAF

[0363] FLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTL

[0364] RLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWSKLITVSGHALLASIFWNR

[0365] ANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0366] 4891 -1065-4412, v. 1 MPT94.15t (SEQ ID NO: 23)

[0367] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNA

[0368] ASGESLESHEAQNHTPETLWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALE

[0369] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0370] TGVIIITLCAILSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAF

[0371] AALCFFIIRGLMFHVAFFLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFK

[0372] DIPDIEGDKEHGVKSLTLRLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWS

[0373] KLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYVEYLMIPLIR

[0374] MPT94.16t (SEQ ID NO: 24)

[0375] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNA

[0376] ASGESLESHEAQHHTPETLWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALE

[0377] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0378] TGVIIITLCAILSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAF

[0379] AALCFFIIRGLMFHVAFFLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFK

[0380] DIPDIEGDKEHGVKSLTLRLGQKRVFWICVSLLLMAYGGAIVVGATSSFLWS

[0381] KLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0382] MPT94.17t (SEQ ID NO: 25)

[0383] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNA

[0384] ASGESLESHEAQHHTPETLLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALE

[0385] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0386] TGVIIITLCAILSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAF

[0387] AALCFFIIRGLMFHVAFFLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFK

[0388] DIPDIEGDKEHGVKSLTLRLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWS

[0389] KLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYVEYLMIPLIR

[0390] MPT94.18t (SEQ ID NO: 26)

[0391] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNA

[0392] ASGESLESHEAQHHTPETLLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALE

[0393] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0394] TGVIIITLCAILSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAF

[0395] AALCFFIIRGLMFHVAFFLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFK

[0396] DIPDIEGDKEHGVKSLTLRLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWS

[0397] KLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0398] 4891 -1065-4412, v. 1 MPT94.19t (SEQ ID NO: 27)

[0399] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNA

[0400] ASGESLESHEAQNHTPETLWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALE

[0401] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0402] TGVIIITLCAILSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAF

[0403] AALCFFIIRGLMFHVAFFLHIQTHVFKRPMMIPKSVMFGTAFMSFFYVIIALFK

[0404] DIPDIEGDKEHGVKSLTLRLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWS

[0405] KLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0406] MPT94.20t (SEQ ID NO: 28)

[0407] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNA

[0408] ASGESLESHEAQNHTPETLWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALE

[0409] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0410] TGVIIITLCAILSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAF

[0411] AALCFFIIRGLMFHVAFFLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFK

[0412] DIPDIEGDKEHGVKSLTLRLGQKRVFWICVSLLLMAYGGAIVVGATSSFLWS

[0413] KLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0414] MPT94.2U (SEQ ID NO: 29)

[0415] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNA

[0416] ASGESLESHEAQNHTPETLWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALE

[0417] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0418] TGVIIITLCAIMSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPA

[0419] FAALCFFIIRGLMFHVAFFLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFK

[0420] DIPDIEGDKEHGVKSLTLRLGQKRVFWICVSLLLMAYGGAIVVGATSSFLWS

[0421] KLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0422] MPT94.22t (SEQ ID NO: 30)

[0423] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNA

[0424] ASGESLESHEAQNHTPETLWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALE

[0425] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0426] TGVIIITLCAILSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAF

[0427] AALCFFIIRGLMFHVAFFLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFK

[0428] DIPDIEGDKEHGVKSLTLRLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWS

[0429] KLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0430] 4891 -1065-4412, v. 1 MPT94.23t (SEQ ID NO: 31)

[0431] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNA

[0432] ASGESLESHEAQNHTPETLWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALE

[0433] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0434] TGVIIITLCAILSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAF

[0435] AALCFFIIRGLMFHVAFFLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFK

[0436] DIPDIEGDKEHGVKSLTLRLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWS

[0437] KLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0438] MPT94.24 (SEQ ID NO: 32)

[0439] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNA

[0440] ASGESLESHEAQNHTPETLWGSIKQFCDAFYRFSRPYVITGTAINIIVMSSLALE

[0441] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0442] TGVIIITLCAILSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAF

[0443] AALCFFIIRGLMFHVAFFLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFK

[0444] DIPDIEGDKEHGVKSLTLRLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWS KLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR

[0445] MPT94.25 (SEQ ID NO: 33)

[0446] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNA

[0447] ASGESLESHEAQNHTPETLWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALE

[0448] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0449] TGVIIITLCAILSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAF

[0450] AALCFFIIRGLMFHVAFFLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFK

[0451] DIPDIEGDKEHGVKSLTLRLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWS KLITVSGHALLASIFWNRANSVDLKSHSEITSLYMFMWKLFYIEYLMIPLIR

[0452] MPT94.26 (SEQ ID NO: 34)

[0453] MTTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDCARNFVVNA

[0454] ASGESLESHEAQNHTPETLWGSIKQFCDAFYRFSRPHVITGTAINIIVMSSLALE

[0455] NSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVR

[0456] TGVIIITLCAILSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAF

[0457] AALCFFIIRGLMFHVAFFLHIQTHVFKRPMLIPKSVMFGTAFMSFFYVIIALFK

[0458] DIPDIEGDKEHGVKSLTLRLGQKRVFWICVSLLLMAYGAAIVVGATSSFLWS KLITVSGHALLASIFWNRANSVDLKSHMEITSLYMFMWKLFYIEYLMIPLIR

[0459] 4891 -1065-4412, v. 1 MPT85_94 Consensus (SEQ ID NO: 35)

[0460] MASMFLGSLPLASSXNYIGRXTRSKXCTESYHATSYITXASXNKTEKIXHEYA

[0461] NMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPET

[0462] LXGSIKXFCDAFYRFSRPHVIXGTAXNIIVMSSLALEXSSDISXKFFIGLFQXIX

[0463] TXXXMNXYXXGXNQLTDIEIDKINKPYLPLASGEXSXXTGVXIITLCAIXSLX

[0464] VGWXVGSPALFWXSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMF

[0465] HVAFFLHIQTHVFKRPMMIXKXVMFGTAFMSFFYVIIAXFKDIPDIEGDKXHG

[0466] VKSLTMRLGQERVFWICVSLLLXXYGXAIVVGATSSFLWXKLITVSGHALLA

[0467] SIFWNRAXSVDLKSHXEITSLYMFMWKLFYIEYXXIPLXR

[0468] ERG20.F88W.N119W (GPS 1.1) (SEQ ID NO:36)

[0469] MSKAKFESVFPRISEELVQLLRDEGLPQDAVQWFSDSLQYNCVGGKLNRGLS VVDTYQEETGKKEEDDEEYYREAEEGWEIEEEQAFWEVSDDIMDESKTRRG QPCWYEKPKVGMIAIWDAFMEESGIYIEEKKHFRQEKYYIDEVEEFHDISFKT

[0470] EEGQEVDEETAPEDEVDENRFSEDKHSFIVRYKTAYYSFYEPVVEAMYVAGI TNPKDEQQAMDVEIPEGEYFQVQDDYEDNFGDPEFIGKIGTDIQDNKCSWEV

[0471] NKAEQKATPEQRQIEEDNYGVKDKSKEEVIKKEYDDMKIEQDYEDYEEEVV GDIKKKIEQVDESRGFKKEVENAFEAKIYKRQK

[0472] AtATR2 (SEQ ID NO: 37)

[0473] MSSSSSSSTSMIDEMAAIIKGEPVIVSDPANASAYESVAAEESSMEIENRQFAMI VTTSIAVEIGCIVMEVWRRSGSGNSKRVEPEKPEVIKPREEEIDDGRKKVTIFFG

[0474] TQTGTAEGFAKAEGEEAKARYEKTRFKIVDEDDYAADDDEYEEKEKKEDVAF FFEATYGDGEPTDNAARFYKWFTEGNDRGEWEKNEKYGVFGEGNRQYEHF NKVAKVVDDIEVEQGAQREVQVGEGDDDQCIEDDFTAWREAEWPEEDTIERE

[0475] EGDTAVATPYTAAVEEYRVSIHDSEDAKFNDINMANGNGYTVFDAQHPYKAN VAVKREEHTPESDRSCIHEEFDIAGSGETYETGDHVGVECDNESETVDEAEREE DMSPDTYFSEHAEKEDGTPISSSEPPPFPPCNERTAETRYACEESSPKKSAEVAE

[0476] AAHASDPTEAEREKHEASPAGKDEYSKWVVESQRSEEEVMAEFPSAKPPEGV FFAGVAPREQPRFYSISSSPKIAETRIHVTCAEVYEKMPTGRIHKGVCSTWMKN AVPYEKSENCSSAPIFVRQSNFKEPSDSKVPIIMIGPGTGEAPFRGFEQEREAEV ESGVEEGPSVEFFGCRNRRMDFIYEEEEQRFVESGAEAEESVAFSREGPTKEY

[0477] VQHKMMDKASDIWNMISQGAYEYVCGDAKGMARDVHRSEHTIAQEQGSMD STKAEGFVKNEQTSGRYERDVW

[0478] AtPAE (SEQ ID NO: 38)

[0479] MDQIEAMLCGGGEKTKVAVTTKTLADPLNWGLAADQMKGSHLDEVKKMVE

[0480] EYRRPVVNLGGETLTIGQVAAISTVGGSVKVELAETSRAGVKASSDWVMESM NKGTDSYGVTTGFGATSHRRTKNGTALQTELIRFLNAGIFGNTKETCHTLPQS ATRAAMLVRVNTLLQGYSGIRFEILEAITSLLNHNISPSLPLRGTITASGDLVPLS YIAGLLTGRPNSKATGPDGESLTAKEAFEKAGISTGFFDLQPKEGLALVNGTAV GSGMASMVLFEANVQAVLAEVLSAIFAEVMSGKPEFTDHLTHRLKHHPGQIE AAAIMEHILDGSSYMKLAQKVHEMDPLQKPKQDRYALRTSPQWLGPQIEVIR

[0481] 4891 -1065-4412, v. 1 QATKSIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKL

[0482] MFAQFSELVNDFYNNGLPSNLTASSNPSLDYGFKGAEIAMASYCSELQYLANP

[0483] VTSHVQSAEQHNQDVNSEGEISSRKTSEAVDIEKEMSTTFEVGICQAVDERHEE

[0484] ENLRQTVKNTVSQVAKKVLTTGINGELHPSRFCEKDLLKVVDREQVFTYVDD

[0485] PCSATYPLMQRLRQVIVDHALSNGETEKNAVTSIFQKIGAFEEELKAVLPKEVE

[0486] AARAAYGNGTAPIPNRIKECRSYPLYRFVREELGTKLLTGEKVVSPGEEFDKVF

[0487] TAMCEGKLIDPLMDCLKEWNGAPIPIC

[0488] AtC4H (CYP73A5) (SEQ ID NO: 39)

[0489] MDLLLLEKSLIAVFVAVILATVISKLRGKKLKLPPGPIPIPIFGNWLQVGDDLNH

[0490] RNLVDYAKKFGDLFLLRMGQRNLVVVSSPDLTKEVLLTQGVEFGSRTRNVVF

[0491] DIFTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQNREGWEFEAASV

[0492] VEDVKKNPDSATKGIVLRKRLQLMMYNNMFRIMFDRRFESEDDPLFLRLKAL

[0493] NGERSRLAQSFEYNYGDFIPILRPFLRGYLKICQDVKDRRIALFKKYFVDERK

[0494] QIASSKPTGSEGLKCAIDHILEAEQKGEINEDNVLYIVENINVAAIETTLWSIEW

[0495] GIAELVNHPEIQSKLRNELDTVLGPGVQVTEPDLHKLPYLQAVVKETLRLRMA

[0496] IPLLVPHMNLHDAKLAGYDIPAESKILVNAWWLANNPNSWKKPEEFRPERFFE

[0497] EESHVEANGNDFRYVPFGVGRRSCPGIILALPILGITIGRMVQNFELLPPPGQSK

[0498] VDTSEKGGQFSLHILNHSIIVMKPRNC

[0499] Y1.ERG20.A28 (GPS 1.2) (SEQ ID NO: 40)

[0500] MSKAKFESVFPRISEELVQLLRDEGLPQDAVQWFSDSLQYNCVGGKLNRGLS

[0501] VVDTYQLLTGKKELDDEEYYRLALLGWLIELLQAFLSDDIMDESKTRRGQPC

[0502] WYLKPKVGMIAINDAFMLESGIYILLKKHFRQEKYYIDLVELFHDISFKTELG

[0503] QLVDLLTAPEDEVDLNRFSLDKHSFIVRYKTAYYSFYLPVVLAMYVAGITNP

[0504] KDLQQAMDVLIPLGEYFQVQDDYLDNFGDPEFIGKIGTDIQDNKCSWLVNKA

[0505] LQKATPEQRQILEDNYGVKDKSKELVIKKLYDDMKIEQDYLDYEEEVVGDIK

[0506] KKIEQVDESRGFKKEVLNAFLAKIYKRQK*

[0507] AtC3H (CYP98A85) (SEQ ID NO: 41)

[0508] MESLFLLAFSLLALIISVKLLYSKRFKLPPGPRPWPLFGNLHEIEPVRFRCFAKW

[0509] AERYGPIMSVWIGGSLNVIVSSPELAREVLKEQDQHLANRHRTRSAAKFSREG

[0510] TDLIWADYGPHYVKVRKLCTLELFSVKRLEALRAIREEEVSAMVESLYTDCK

[0511] GKSEERLVLRTYLSVVTFNHITRLVFGKRFINSKGEMEEQGKEFKDIVATGNEL

[0512] SASLSIAEHLPWLQSLFPLEVEAFDKHWDRRDRLTRTIMEEHTKARLESGSEQ

[0513] QHFVGALLSLRDEYDLSDDTVTGLLWDMIQAGMDTIAITCEWGMAELIRNPQ

[0514] VQAKAQEELDRVIGDKRAMTESDFSQLPYLRCIAKESLRLHPPTPLMLPHRAS

[0515] KHIKLGGYDVPKGSNVHVNAWAIARHPDTWKDPTVFRPERFLEDDVDMKGQ

[0516] DFRLLPFGSGRRICPGATLGTYLLQLMLGRMLHGFRWTTIDASSIDMSEDPGL

[0517] VAFMTTPLVAVATPRLPSDLYSCQSMKV

[0518] 4891 -1065-4412, v. 1 0MT9 (SEQ ID NO: 42)

[0519] MGSTAADMAATADEEACMFALQLASSSILPMTLKNAIELGLLDTLVQASGKSL

[0520] TPAEVAAKLPSSSNPAAPDMVDRMLRLLASYGVVSCAVEEGENGKLSRRYAA

[0521] APVCKWLTPNEDGVSMAALALMNQDKVLMESWYYLKDAVLDGGIPFNKAY

[0522] GMSAFEYHGTDPRFNRVFNEGMKNHSIIITKKLLDLYPGFEGLGTLVDVGGGV

[0523] GATVGAIVARHPAIKGINFDLPHVISEGIPFPGVTHVGGDMFQKVPSGDAILMK

[0524] WILHDWSDAHCATLLKNCYDALPAHGKVVIVECILPVNPEATPKAQGVFHVD

[0525] MIMLAHNPGGKERYEREFEELARGAGFTGVKATYIYANAWAIEFTK

[0526] 4891 -1065-4412, v. 1

Claims

CLAIMSWhat is claimed is:

1. A mutant membrane-bound prenyltransferase (MPT) comprising an amino acid sequence with at least 85% identity to the amino acid sequence of SEQ ID NO. 35, 2 or 1, having at least one amino acid modification at a position selected from the group consisting of 78, 99, 123, 282, and 328 of SEQ ID NO: 35, 2 or 1, wherein the mutant MPT is capable of producing bakuchiol or analogs thereof from geranyl pyrophosphate (GPP) and at least one of cinnamic acid, coumaric acid, caffeic acid, and ferulic acid.

2. The mutant membrane-bound prenyltransferase of claim 1, further comprising at least one additional amino acid modification at a position selected from the group consisting of 106, 111, 209, 323, 344, 385, and 400 of SEQ ID NO: 35, 2 or 1.

3. The mutant membrane-bound prenyltransferase of claim 1, wherein the bakuchiol analog is selected from the group consisting of dehydrobakuchiol, 3-hydroxy-Bakuchiol, and 3-methoxy-bakuchiol.

4. The mutant membrane-bound prenyltransferase of any one of claims 1-3, wherein the mutant MPT comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1.

5. The mutant membrane-bound prenyltransferase of any one of claims 1-3, wherein the mutant MPT comprises an amino acid sequence with at least 97% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1.

6. The mutant membrane-bound prenyltransferase of any one of claims 1-3, wherein the mutant MPT comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.

7. The mutant membrane-bound prenyltransferase of any one of claims 1-3, wherein the MPT has an amino acid sequence which comprises an amino acid sequence with between 98.5% and 99.8% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1.4891 -1065-4412, v.

18. The mutant membrane-bound prenyltransferase (MPT) of claim 1, wherein the mutant MPT transfers geranyl pyrophosphate (GPP) to cinnamic acid, coumaric acid, caffeic acid, and / or ferulic acid to produce dehydrobakuchiol, bakuchiol, 3-hydroxy-bakuchiol, and / or 3-methoxy- bakuchiol, respectively, with a higher efficiency compared to either of enzymes MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2).

9. The mutant membrane-bound prenyltransferase of claim 7, wherein the higher efficiency of transferring GPP to cinnamic acid, coumaric acid, caffeic acid, and / or ferulic acid comprises at least 1.5 times higher efficiency compared to either of enzymes MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2).

10. The mutant membrane-bound prenyltransferase of any one of claims 1-3, wherein the at least one amino acid modification and at least one additional amino acid modification are individually selected from substitutions, deletions or insertions.

11. The mutant membrane -bound prenyltransferase of any one of claims 1-3, further comprising one or more substitutions, deletions, and / or insertions at a position selected from R 121, V124, N165-N172, D176-K184, Y229- Y236, K246-F251, C255-R260, Y298-D306, 1310-G317, and Y391-W395 of SEQ ID NO: 35, 2, or 1.

12. The mutant membrane-bound prenyltransferase of any one of claims 1-3, wherein the mutant MPT comprises an amino terminal truncation comprising deletion of between 1 and 73 amino acids corresponding to amino acids in positions 2-74 of the amino acid sequence of SEQ ID NO: 35, 2 or 1.

13. The mutant membrane-bound prenyltransferase of claim 12, wherein the mutant MPT comprises an amino acid sequence with at least 90% identity to an amino acid sequence selected from SEQ ID NO: 6-10, 13, and 23-33. 33 and 44-46.

14. An engineered cell that expresses the mutant membrane-bound prenyltransferase of any one of claims 1-13 or an MPT comprising an amino acid sequence of MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO:4891 -1065-4412, v. 12), wherein the cell is capable of producing bakuchiol and / or analogs thereof in the presence of geranyl pyrophosphate (GPP) and at least one of cinnamic acid, coumaric acid, caffeic acid, and ferulic acid.

15. The cell of claim 14, wherein the cell has been engineered to provide greater flux of GPP through the GPP pathway.

16. The cell of claim 14 or 15, wherein the cell has been engineered to provide greater flux of tyrosine or phenylalanine through the respective aromatic amino acid synthesis pathways.

17. The cell of claim 16, wherein the greater flux through an aromatic amino acid synthesis pathway is achieved by overexpressing an Aro4 enzyme (EC 2.5.1.54) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:4.

18. The cell of claim 16, wherein the greater flux through an aromatic amino acid synthesis pathway is achieved by expressing a feedback insensitive Aro4 enzyme having a sequence with at least 95% identity to SEQ ID NO: 5.

19. The cell of claim 14 or 15, wherein the cell is a yeast cell, an algal cell, or a bacterial cell.

20. The cell of claim 19, wherein the yeast cell is Saccharomyces, Pichia, or Yarrowia.

21. The cell of claim 14 or 15, wherein the cell is capable of synthesizing cinnamic acid, coumaric acid, caffeic acid, and / or ferulic acid when phenylalanine and / or tyrosine is present in the cell through endogenous production.

22. The cell of claim 14 or 15, wherein production of bakuchiol or analogs thereof in the cell is dependent on or is enhanced by supplementation of cinnamic acid, coumaric acid, caffeic acid, and / or ferulic acid within the fermentation medium in which the cell is grown.

23. The cell of claim 21, wherein the cell is capable of converting one or more of cinnamic acid to coumaric acid, coumaric acid to caffeic acid, and caffeic acid to ferulic acid.4891 -1065-4412, v.

124. The cell of claim 21, wherein the cell expresses phenylalanine ammonia lyase and / or tyrosine ammonia lyase of the family EC 4.3.1.24 and / or the family EC 4.3.1.25.

25. The cell of claim 24, wherein the cell expresses cinnamate 3-hydroxylase.

26. The cell of claim 25, wherein the cinnamate 3-hydroxylase is a p450 enzyme of the CYP73A family or family EC 1.14.14.91.

27. The cell of claim 24, wherein the cell expresses caffeic acid O- methyltransferase of the family EC 2.1.1.68.

28. The cell of claim 21, wherein the cell is capable of converting bakuchiol to 3-hydroxy-bakuchiol, and / or 3-hydroxy-bakuchiol to 3-methoxy- bakuchiol.

29. The cell of claim 28, wherein the conversion of bakuchiol to 3- hydroxybakuchiol is catalyzed by a P450 hydroxylase of the family CYP98A.

30. The cell of claim 28, wherein the conversion of 3-hydroxybakuchiol to 3- methoxybakuchiol is catalyzed by a methyltransferase of the family EC 2.1.1.68 or EC 2.1.1.42.

31. A method of producing bakuchiol by fermenting the cell of any one of claims 14-30 in the presence of one or more of glucose, glycerol, cinnamic acid, coumaric acid, caffeic acid, and ferulic acid.4891 -1065-4412, v. 1

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