Enzymes and methods for the synthesis of bakuchiol and its derivatives

Abstract

A prenyltransferase capable of producing bakuchiol or its analogues from geranyl pyrophosphate (GPP) and cinnamic acid, coumaric acid, caffeic acid and / or ferulic acid is disclosed herein. Manipulated cells expressing a prenyltransferase capable of producing bakuchiol or its analogues, as well as methods of using the prenyltransferase and cells to produce bakuchiol or its analogues, are also disclosed herein.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 524,506, filed Jun. 30, 2023, and U.S. Provisional Patent Application No. 63 / 525,294, filed Jul. 6, 2023, the entire teachings of which are incorporated herein by reference.

Background Art

[0002] Background of the Invention The Psoralea corylifolia plant has been used in Asia, China, and India as a herb for the treatment of various conditions, including cardiotonic, vasodilatory, antitumor, antibacterial, and cytotoxic conditions, and more specifically, for the treatment of skin conditions such as alopecia, vitiligo, leprosy, psoriasis, and eczema (1). This plant produces a number of chemical substances that are mainly concentrated in the seeds and fruits, and approximately 90 compounds have been isolated and characterized to date (2). One of the most abundant chemical substances is bakuchiol, which accumulates up to about 6% w / w in the dry seeds of this plant. Other compounds found at lower concentrations in the seeds include 3-hydroxy-bakuchiol as well as certain coumarins, such as psoralidin, psoralen, isopsoralen, and angelicin (3). Since bakuchiol is the most abundant compound in the seeds of the plant, it constitutes the most studied and the only commercially available product isolated from the seeds of Psoralea corylifolia. Bakuchiol has been shown to have its activity in many of the clinical effects associated with the plant, specifically in skin conditions (anti-aging, anti-pigmentation, acne, etc.). As a result, bakuchiol is used as a retinol alternative in various creams and other skin care products (4, 5, 6). Other bakuchiol analogs described herein are difficult to obtain due to the lack of an immediate source for their isolation, but they may also have beneficial properties.

[0003] Currently, bakuchiol is commercially available only from plant extracts, resulting in low availability and correspondingly high price tags (higher than $2000 / kg). Furthermore, bakuchiol is co-extracted with other plant compounds, such as phototoxic psoralens and isoporalens, which must be removed because their presence in cosmetic skin formulations can cause skin irritation and dermatitis (7). Therefore, there is a need for alternative, low-cost methods for producing bakuchiol-containing compositions with low toxicity and high purity. [Overview of the Initiative] [Means for solving the problem]

[0004] Summary of the Invention Enzymes capable of prenylating hydroxycinnamic acid to efficiently obtain bakuchiol and one or more analogues thereof are described herein. These enzymes can be expressed in cells engineered to efficiently produce bakuchiol and its analogues when fermented under the correct conditions. Methods for providing alternative sources of bakuchiol and its derivatives are also disclosed. Specifically, a fermentation method is disclosed for cost-effectively producing high-yield bakuchiol substantially free of toxic compounds found in extracts from Psoralea corylifolia. These and other aspects of the present invention are disclosed herein in more detail.

[0005] Some aspects of this disclosure relate to a mutant membrane-bound prenyltransferase comprising an amino acid sequence having 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, and capable of producing bakuchiol or an analog 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 further 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-hydroxybakuchiol, and 3-methoxybakuchiol.

[0006] In some embodiments, the membrane-bound prenyltransferase contains an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1. In some embodiments, the membrane-bound prenyltransferase contains an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1. 6. In some embodiments, the mutant membrane-bound prenyltransferase contains an amino acid sequence having 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. In some embodiments, the mutant membrane-bound prenyltransferase contains an amino acid sequence having between 98.5% and 99.8% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1.

[0007] In some embodiments, the mutant membrane-bound prenyltransferase (MPT) transfers geranyl pyrophosphate (GPP) to cinnamic acid, coumaric acid, caffeic acid, and / or ferulic acid, producing dehydrobakuchiol, bakuchiol, 3-hydroxybakuchiol, and / or 3-methoxybakuchiol, respectively, with higher efficiency compared to either enzyme 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 includes at least a 1.5-fold higher efficiency compared to either enzyme MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2).

[0008] In some embodiments, at least one amino acid modification compared to SEQ ID NO: 35, 2, or 1 includes substitutions, deletions, or insertions. In some embodiments, the mutant MPT further includes 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 includes an amino-terminal shortening including a deletion between amino acids 1-73 corresponding to amino acids at positions 2-74 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In some embodiments, the mutant MPT includes an amino acid sequence having at least 95% identity to an amino acid sequence selected from SEQ ID NOs: 6-10, 13, 23-33, 33, and 44-46.

[0009] Other aspects of the present disclosure relate to engineered cells expressing the membrane-bound prenyltransferase disclosed herein, which are capable of producing bakuchiol and / or analogues 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 cells are engineered to provide a greater flow of GPP via the GPP pathway. In some embodiments, the cells are engineered to provide a greater flow of tyrosine or phenylalanine via their respective aromatic amino acid synthesis pathways. In some embodiments, the greater flow via the aromatic amino acid synthesis pathway is achieved by overexpressing the Aro4 enzyme (EC2.5.1.54) having an amino acid sequence having at least 95% identity to SEQ ID NO: 4. In some embodiments, the greater flow via the aromatic amino acid synthesis pathway is achieved by expressing the feedback-insensitive Aro4 enzyme having a sequence having at least 95% identity to SEQ ID NO: 5.

[0010] In some embodiments, the cells are yeast cells, algal cells, or bacterial cells. In some embodiments, the yeast cells are Saccharomyces, Pichia, or Yarrowia.

[0011] In some embodiments, cells can synthesize coumaric acid, caffeic acid, and / or ferulic acid if tyrosine is present in the cell via endogenous production. In some embodiments, cells can produce cinnamic acid, coumaric acid, caffeic acid, or ferulic acid if phenylalanine is present in the cell via endogenous production.

[0012] In some embodiments, the production of bakuchiol or its analogues in cells depends on, or is enhanced by, the supplementation of cinnamic acid, coumaric acid, and / or ferulic acid in the fermentation medium in which the cells are grown.

[0013] In some embodiments, cells are capable of converting cinnamic acid to coumarate, coumarate to caffeic acid, and caffeic acid to ferulic acid, one or more of these. In some embodiments, cells express phenylalanine ammonia lyase and / or tyrosine ammonia lyase of family EC4.3.1.24 and / or family EC4.3.1.25. In some embodiments, cells express cinnamic acid 3-hydroxylase. In some embodiments, cinnamic acid 3-hydroxylase is a p450 enzyme of family CYP73A or family EC1.14.14.91. In some embodiments, cells express caffeic acid O-methyltransferase of family EC2.1.1.68.

[0014] In some embodiments, cells are capable of converting bakuchiol to 3-hydroxybakuchiol and / or 3-hydroxybakuchiol to 3-methoxybakuchiol. In some embodiments, the conversion of bakuchiol to 3-hydroxybakuchiol is catalyzed by a P450 hydroxylase of family CYP98A. In some embodiments, the conversion of 3-hydroxybakuchiol to 3-methoxybakuchiol is catalyzed by a methyltransferase of family EC2.1.1.68 or EC2.1.1.42.

[0015] Some aspects of this disclosure relate to a method for producing bakuchiol by fermenting the cells disclosed herein in the presence of one or more of glucose, glycerol, cinnamic acid, coumaric acid, caffeic acid, and ferulic acid.

[0016] The aforementioned and other purposes and benefits will become clear when considering the following detailed description in conjunction with the attached drawings. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 shows the activity of several prenyltransferases discovered in Psoralea corylifolia. [Figure 2] Figure 2 shows the pathways involved in the synthesis of bakuchiol and its analogues in the manipulated cells. [Figure 3] Figure 3 is a ribbon model showing the structure of prenyltransferase MPT95, in which geranyl pyrophosphate (GPP) and coumarate are combined within the active site. [Modes for carrying out the invention]

[0018] Detailed description of the invention Given the high price of commercially available bakuchiol and the difficulties in isolating and purifying bakuchiol from natural sources and the toxic compounds found therein, the inventors attempt to identify, isolate, characterize, and mutagenesize a prenyltransferase from the plant Psoralea corylifolia capable of condensing geranyl pyrophosphate (GPP) with hydroxycinnamic acid in order to provide bakuchiol and its derivatives. Furthermore, engineered cells were designed to produce bakuchiol and its analogues via fermentation of engineered cells using a carbon source. Engineered cells capable of producing bakuchiol and its analogues using supplemented cinnamic acid, coumaric acid, caffeic acid, and / or ferulic acid are also described. Accordingly, the inventors disclose a method for providing an alternative source for purified bakuchiol and its derivatives that avoids the problems associated with the prior art. Specifically, a fermentation method for cost-effectively producing high-yield bakuchiol free from toxic compounds found in extracts derived from Psoralea corylifolia is disclosed.

[0019] Some aspects of this disclosure relate to a membrane-bound prenyltransferase (MPT) comprising an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 35, 2, or 1, which is capable of producing bakuchiol or an analog thereof from geranyl pyrophosphate (GPP) and at least one of cinnamic acid, coumaric acid, caffeic acid, and ferulic acid. In some embodiments, the MPT is 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%, It includes amino acid sequences having 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. In some embodiments, the MPT includes 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 is 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% relative to the amino acid sequence of SEQ ID NO: 35, 2, or 1. , containing amino acid sequences with identity of 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 less than 85%.

[0020] In some embodiments, the mutant membrane-bound prenyltransferase comprises an amino acid sequence having 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 may be any amino acid modification disclosed herein or any combination of amino acid modifications. In some embodiments, the at least one amino acid modification is at least one modification at a position including positions 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 including positions 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 one amino acid modification is an insertion. In some embodiments, the at least one amino acid modification is a substitution. In certain embodiments, 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.

[0021] In some embodiments, at least one amino acid modification includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid modifications at positions including 78, 99, 106, 111, 123, 209, 282, 323, 344, 385, or 400 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In certain embodiments, at least one amino acid modification includes 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, at least one amino acid modification includes 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, at least one amino acid modification includes 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, at least one amino acid modification includes 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, at least one amino acid modification includes 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, at least one amino acid modification includes amino acid modifications at positions 78, 99, 106, 111, 209, 282, 323 and 328 of the amino acid sequence of SEQ ID NO: 35, 2, or 1. In certain embodiments, at least one amino acid modification includes amino acid modifications at positions 78, 99, 106, 111, 209, 282, 323, 328, and 344 of the amino acid sequence of SEQ ID NO: 35, 2, or 1.

[0022] In some embodiments, the amino acid modifications include substitutions, deletions 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, SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, at least one amino acid modification includes 1 to 61 amino acid modifications. In some embodiments, at least one amino acid modification includes a deletion of 39 - 73 amino acids in the N - terminal domain to create an N - truncated membrane - bound prenyltransferase. In some embodiments, at least one amino acid modification includes substitutions, deletions or insertions 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 a deletion of 39 - 73 amino acids in the N - terminal domain of SEQ ID NO: 35, SEQ ID NO: 1 or SEQ ID NO: 2.

[0023] In certain embodiments, the prenyltransferase is mutated to transfer geranyl pyrophosphate (GPP) to coumaric acid to produce bactiol with higher efficiency compared to either enzyme MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2). In some embodiments, the MPT that transfers geranyl pyrophosphate (GPP) to coumaric acid to produce bactiol with higher efficiency does so with an efficiency 1.1 - fold more efficient to 1,000 - fold more efficient or more efficient than the efficiency of MPT85 and / or MPT94. Specifically, the higher efficiency constitutes an efficiency 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, 3 - fold, 4 - fold, 5 - fold, 8 - fold, 10 - fold, 15 - fold, 20 - fold, 25 - fold, 35 - fold, 50 - fold, 75 - fold, 100 - fold, 200 - fold, 400 - fold, 600 - fold, 800 - fold, 1000 - fold, 2000 - fold or higher than the efficiency of enzyme MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2) when compared.

[0024] In some embodiments, the membrane-bound prenyltransferase comprises an amino acid sequence having 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, transfers geranyl pyrophosphate (GPP) to chlorogenic acid, and produces 3-hydroxybactioll with higher efficiency compared to either of the enzymes MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2). In some embodiments, MPT mutated to transfer geranyl pyrophosphate (GPP) to chlorogenic acid and produce 3-hydroxybactioll with higher efficiency performs it at an efficiency 1.1-fold more efficient to 1,000-fold more efficient or more efficient than the efficiency of MPT85 and / or MPT94 when compared. Specifically, the higher efficiency comprises an efficiency of 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, 3-fold, 4-fold, 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, 25-fold, 35-fold, 50-fold, 75-fold, 100-fold, 200-fold, 400-fold, 600-fold, 800-fold, 1,000-fold, 2,000-fold or higher compared to the enzymes MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2).

[0025] As described above, the mutant membrane-bound prenyltransferase comprises at least one amino acid modification, for example, at least one amino acid modification compared to wild-type membrane-bound prenyltransferase. The at least one amino acid modification may be any amino acid modification disclosed herein or any combination of amino acid modifications. In some embodiments, amino acid modifications are made at the positions of SEQ ID NO: 35, SEQ ID NO: 1 or 2: W78, H99, L106, R111, R121, H123, V124, N165, I166, Y167, T168, A169, G170, I171, N172, D176, I177, E178, I179, D180, K181, I182, N183, K184, M209, Y229, F230, V231, L232, G233, T234, V235, Y236, K246, R247, Y248, P2 This includes substitutions, deletions, or insertions at one or more amino acid positions corresponding to 49, A250, F251, C255, F256, F257, I258, I259, R260, M282, Y298, V299, I300, I301, A302, F303, F304, K305, D306, I310, E311, G312, D313, K314, E315, H316, G317, M323, E328, G344, E385, Y391, M392, F393, M394, W395, or I400. In some embodiments, at least one amino acid modification includes 1 to 62 amino acid modifications. In some embodiments, at least one amino acid modification involves the deletion of 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, or 73 amino acids in the N-terminal domain to create an N-shortened membrane-bound prenyltransferase.In some embodiments, at least one amino acid modification involves the 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-shortened membrane-bound prenyltransferase. In some embodiments, at least one amino acid modification is at the positions of SEQ ID NO: 35, SEQ ID NO: 2, or SEQ ID NO: 1, W78, H99, L106, R111, R121, H123, V124, N165, I166, Y167, T168, A169, G170, I171, N172, D176, I177, E178, I179, D180, K181, I182, N183, K184, M209, Y229, F230, V231, L232, G233, T234, V235, Y236, K246, R247, Y248, P249, A250, F251, C255, F256, F257, I2 This includes substitutions, deletions, or insertions in one or more amino acids corresponding to 58, I259, R260, M282, Y298, V299, I300, I301, A302, F303, F304, K305, D306, I310, E311, G312, D313, K314, E315, H316, G317, M323, E328, G344, E385, Y391, M392, F393, M394, W395, or I400, as well as deletions of amino acids 1-6, 7-12, 13-26, 27-36, 37-40, or 39-73 in the N-terminal domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0026] Amino acid modifications can be amino acid substitutions, amino acid deletions, and / or amino acid insertions. Amino acid substitutions can be conservative or non-conservative. A conservative substitution (also called a conservative mutation, conservative substitution, or conservative variation) is an amino acid substitution in a protein that changes a given amino acid to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). As used herein, a “conservative variation” refers to the substitution of an amino acid residue with another biologically similar residue. Examples of conservative variations include the substitution of another hydrophobic residue with one hydrophobic residue, e.g., isoleucine, valine, leucine, or methionine; or the substitution of another polar residue with one polar residue, e.g., lysine to arginine, aspartic acid to glutamic acid, or asparagine to glutamine. Other exemplary examples of conservative substitutions include changes from alanine to serine; from arginine to lysine; from asparagine to glutamine or histidine; from aspartic acid to glutamic acid; from cysteine ​​to serine; from glutamine to asparagine; from glutamic acid to aspartic acid; from glycine to proline; from histidine to asparagine or glutamine; from isoleucine to leucine or valine; from leucine to valine or isoleucine; from lysine to arginine, glutamine or glutamic acid; from methionine to leucine or isoleucine; from phenylalanine to tyrosine, leucine or methionine; from serine to threonine; from threonine to serine; from tryptophan to tyrosine; from tyrosine to tryptophan or phenylalanine; and from valine to isoleucine or leucine.

[0027] In some embodiments, at least one amino acid modification compared to MPT85 (SEQ ID NO: 1) is 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, 231, 232, 233, 234, 235, 236, 246, 247, 2 This includes deletions, insertions, or substitutions at one or more amino acid positions corresponding to 48, 249, 250, 251, 255, 256, 257, 258, 259, 260, 282, 298, 299, 300, 301, 302, 303, 304, 305, 306, 310, 311, 312, 313, 314, 315, 316, 317, 323, 328, 344, 385, 391, 392, 393, 394, 395, or 400. In some embodiments, the MPT includes at least 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 or 73 amino acid modifications. In some embodiments, the MPT further includes an N-terminal truncation (e.g., 1 to 73 amino acids).

[0028] In some embodiments, the membrane-bound prenyltransferase (MPT) contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 1 (MPT85). In some embodiments, the MPT contains an amino acid sequence having at least 90% identity with SEQ ID NO: 1 (MPT85). In some embodiments, the MPT contains a functional fragment of SEQ ID NO: 1 (MPT85). In some embodiments, the functional fragment of Sequence ID No. 1 (MPT85) 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, or 73 amino acids deleted from the amino terminus.

[0029] In some embodiments, at least one amino acid modification compared to MPT94 (SEQ ID NO: 2) is 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, 231, 232, 233, 234, 235, 236, 246, 247, 2 This includes deletions, insertions, or substitutions at one or more amino acid positions corresponding to 48, 249, 250, 251, 255, 256, 257, 258, 259, 260, 282, 298, 299, 300, 301, 302, 303, 304, 305, 306, 310, 311, 312, 313, 314, 315, 316, 317, 323, 328, 344, 385, 391, 392, 393, 394, 395, or 400. In some embodiments, the MPT includes at least 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, or 73 amino acid modifications. In some embodiments, the MPT further includes shortenings at the N-terminus (e.g., 1 to 73 amino acids).

[0030] In some embodiments, the membrane-bound prenyltransferase (MPT) contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 2 (MPT94). In some embodiments, the MPT contains an amino acid sequence having at least 90% identity with SEQ ID NO: 2 (MPT94). In some embodiments, the MPT contains a functional fragment of SEQ ID NO: 2 (MPT94). In some embodiments, the functional fragment of Sequence 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, 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 7 (MPT94.2). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 8 (MPT94.3). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 9 (MPT94.4). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 10 (MPT94.5). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 13 (MPT94.8). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with SEQ ID NO: 13 (MPT94.8). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 13.

[0031] In some embodiments, the membrane-bound prenyltransferase (MPT) contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 14 (MPT94.15). In some embodiments, the MPT contains an amino acid sequence having at least 90% identity with SEQ ID NO: 14 (MPT94.15). In some embodiments, the MPT contains a functional fragment of SEQ ID NO: 14 (MPT94.15). In some embodiments, the functional fragment of Sequence 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, 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 23 (MPT94.15t). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with SEQ ID NO: 23 (MPT94.15t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 23.

[0032] In some embodiments, the membrane-bound prenyltransferase (MPT) contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 15 (MPT94.16). In some embodiments, the MPT contains an amino acid sequence having at least 90% identity with SEQ ID NO: 15 (MPT94.16). In some embodiments, the MPT contains a functional fragment of SEQ ID NO: 15 (MPT94.16). In some embodiments, the functional fragment of Sequence 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 24 (MPT94.16t). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with SEQ ID NO: 24 (MPT94.16t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 24.

[0033] In some embodiments, the membrane-bound prenyltransferase (MPT) contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 16 (MPT94.17). In some embodiments, the MPT contains an amino acid sequence having at least 90% identity with SEQ ID NO: 16 (MPT94.17). In some embodiments, the MPT contains a functional fragment of SEQ ID NO: 16 (MPT94.17). In some embodiments, the functional fragment of Sequence 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, 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 25 (MPT94.17t). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with SEQ ID NO: 25 (MPT94.17t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 25.

[0034] In some embodiments, the membrane-bound prenyltransferase (MPT) contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 17 (MPT94.18). In some embodiments, the MPT contains an amino acid sequence having at least 90% identity with SEQ ID NO: 17 (MPT94.18). In some embodiments, the MPT contains a functional fragment of SEQ ID NO: 17 (MPT94.18). In some embodiments, the functional fragment of Sequence 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, 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 26 (MPT94.18t). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with SEQ ID NO: 26 (MPT94.18t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 26.

[0035] In some embodiments, the membrane-bound prenyltransferase (MPT) contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 18 (MPT94.19). In some embodiments, the MPT contains an amino acid sequence having at least 90% identity with SEQ ID NO: 18 (MPT94.19). In some embodiments, the MPT contains a functional fragment of SEQ ID NO: 18 (MPT94.19). In some embodiments, the functional fragment of Sequence 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 27 (MPT94.19t). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with SEQ ID NO: 27 (MPT94.19t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 27.

[0036] In some embodiments, the membrane-bound prenyltransferase (MPT) contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 19 (MPT94.20). In some embodiments, the MPT contains an amino acid sequence having at least 90% identity with SEQ ID NO: 19 (MPT94.20). In some embodiments, the MPT contains a functional fragment of SEQ ID NO: 19 (MPT94.20). In some embodiments, the functional fragment of Sequence 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 28 (MPT94.20t). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with SEQ ID NO: 28 (MPT94.20t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 28.

[0037] In some embodiments, the membrane-bound prenyltransferase (MPT) contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 20 (MPT94.21). In some embodiments, the MPT contains an amino acid sequence having at least 90% identity with SEQ ID NO: 20 (MPT94.21). In some embodiments, the MPT contains a functional fragment of SEQ ID NO: 20 (MPT94.21). In some embodiments, the functional fragment of Sequence 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 29 (MPT94.21t). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with SEQ ID NO: 29 (MPT94.21t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 29.

[0038] In some embodiments, the membrane-bound prenyltransferase (MPT) contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 21 (MPT94.22). In some embodiments, the MPT contains an amino acid sequence having at least 90% identity with SEQ ID NO: 21 (MPT94.22). In some embodiments, the MPT contains a functional fragment of SEQ ID NO: 21 (MPT94.22). In some embodiments, the functional fragment of Sequence 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 30 (MPT94.22t). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with SEQ ID NO: 30 (MPT94.22t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 30.

[0039] In some embodiments, the membrane-bound prenyltransferase (MPT) contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 22 (MPT94.23). In some embodiments, the MPT contains an amino acid sequence having at least 90% identity with SEQ ID NO: 22 (MPT94.23). In some embodiments, the MPT contains a functional fragment of SEQ ID NO: 22 (MPT94.23). In some embodiments, the functional fragment of Sequence 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 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% identity with SEQ ID NO: 31 (MPT94.23t). In some embodiments, the MPT includes an amino acid sequence having at least 90% identity with SEQ ID NO: 31 (MPT94.23t). In some embodiments, the MPT consists of the amino acid sequence of SEQ ID NO: 31.

[0040] "Identity" refers to the degree to which the sequences of two or more nucleic acids or polypeptides are identical. In some embodiments, the percentage identity between a target sequence and a second sequence over an evaluation window, for example, over the length of the target sequence, can be calculated by aligning the sequences, allowing for the introduction of gaps to maximize identity, determining the number of residues (nucleotides or amino acids) opposite identical residues within the evaluation window, dividing by the total number of residues in the target sequence or the second sequence (whichever is larger) that fall within the window, and multiplying by 100. When calculating the number of identical residues required to achieve a particular percentage identity, fractions are rounded to the nearest integer. Percent identity can be calculated using various computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, and Gapped BLAST generate alignments and provide percentage identity between target sequences. The Karlin and Altschul algorithm (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:22264-2268, 1990), modified as seen in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993, is incorporated into Altschul et al.'s NBLAST and XBLAST programs (Altschul, et al., J. Mol. Biol. 215:403-410, 1990). For comparative purposes, gapped alignment is obtained using Gapped BLAST, as described by Altschul et al. (Altschul, et al. Nucleic Acids Res. 25: 3389-3402, 1997). When using the BLAST and Gapped BLAST programs, the default parameters for each program may be used. PAM250 or BLOSUM62 matrices may be used.Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI). For information on these programs, please refer to the website at URL ncbi.nlm.nih.gov. In specific embodiments, percent identity is calculated using BLAST2 with default parameters, as provided by NCBI.

[0041] Fusion protein Some aspects of this disclosure relate to fusion proteins comprising polypeptides having geranyl pyrophosphate (GPP) synthase activity and polypeptides having prenyltransferase activity. As used herein, “GPP synthase activity” is the ability to catalyze the condensation of dimethylallyl pyrophosphate and isopentenyl pyrophosphate 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 compound (acceptor).

[0042] Some aspects of this disclosure relate to fusion proteins comprising a polypeptide having geranyl diphosphate synthase activity and a polypeptide having prenyltransferase activity. The polypeptide having prenyltransferase activity is not limited as 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%, 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).

[0043] 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. If the amino acid modification is a deletion, it may include the deletion of a single amino acid residue or multiple amino acid residues, as long as the modified polypeptide retains the ability to prenylate bakuchiol precursors. In some embodiments, the deletion involves the deletion of amino acids 1-6, 7-12, 13-26, 27-36, 1-39, 1-61, 1-73, or 1-103 at the n-terminus of the polypeptide, thus providing a truncated prenyltransferase. In some embodiments, part or all of the N-terminal sequence of the polypeptide having prenyltransferase activity may be replaced by an amino acid sequence that increases polypeptide expression, folding, and / or activity.

[0044] In other embodiments, amino acid modifications include W78, H99, L106, R111, R121, H123, V124, N165, I166, Y167, T168, A169, G170, I171, N172, D176, I177, E178, I179, D180, K181, I182, N183, K184, M209, Y229, F230, V231, L232, G233, T234, V235, Y236, K246, R247, Y248, P249, A25 Includes deletions, substitutions, or insertions at one or more amino acid positions, including 0, F251, C255, F256, F257, I258, I259, R260, M282, Y298, V299, I300, I301, A302, F303, F304, K305, D306, I310, E311, G312, D313, K314, E315, H316, G317, M323, E328, G344, E385, Y391, M392, F393, M394, W395, and / or I400.

[0045] In some embodiments, the polypeptide having GPP synthase activity has an amino acid sequence that has more 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 that has more 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 may be farnesyl pyrophosphate synthase. In some embodiments, this farnesyl pyrophosphate synthase is Yarrowia Erg20. In some embodiments, the polypeptide having GPP synthase activity is UniProt# Q6C6W3 or F88W.N119W double mutant.

[0046] In some embodiments, polypeptides having prenyltransferase activity have improved activity in prenylating cinnamic acid with GPP compared to the same unfused prenyltransferase. In some embodiments, polypeptides having prenyltransferase activity have at least 1.1 times, 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, 2.5 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times or more activity in condensing GPP with cinnamic acid compared to the same unfused prenyltransferase. In other embodiments, the fusion protein provides bakuchiol or an analogue thereof in an enhanced yield exceeding the yield provided by the unfused prenyltransferase.

[0047] In some embodiments, the fusion protein further comprises a linker polypeptide between a polypeptide having geranyl diphosphate synthase activity (GPS) and a polypeptide having prenyltransferase activity. The linker is not limited and can be any suitable linker. For example, the linker may be a short polypeptide (e.g., 5-52 amino acids). Often, the linker consists of small amino acid residues, e.g., serine, glycine, and / or alanine, but may also contain charged amino acids, e.g., lysine, arginine, aspartic acid, or glutamic acid. Heterogeneous domains may include transmembrane domains, secretory signaling domains, etc. In some embodiments, the linker is a polypeptide. In some embodiments, the polypeptide is 5-52 amino acids in length.

[0048] In some embodiments, geranyl diphosphate is fused to the N-terminus of prenyltransferase.

[0049] Cells and cell cultures The cells are not limited and can be any suitable cells for expression. In some embodiments, the cells may be microorganisms or plants. In some embodiments, the microorganisms may be bacteria (e.g., E. coli), algae, or yeast. In some embodiments, the yeast may be an oily yeast (e.g., Yarrowia lipolytica strain). In some embodiments, the bacteria may be Escherichia coli.

[0050] Suitable cells include Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia 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 The cells may include, but are not limited to, niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas reinhardtii, and Yarrowia lipolytica. In some embodiments, the cells are protease-deficient strains of Saccharomyces cerevisiae. In some embodiments, the cells are eukaryotic cells other than plant cells. In some embodiments, the cells are plant cells. In some embodiments, the cells are plant cells that do not normally produce bakuchiol or its analogues. In some embodiments, the cells are Saccharomyces cerevisiae. In some embodiments, the cells are Yarrowia lipolytica. In some embodiments, the cells disclosed herein are cultured in vitro.

[0051] In some embodiments, the cells are prokaryotic cells. Suitable prokaryotic cells may include, but are not limited to, any of the various laboratory strains, such as Escherichia coli, Lactobacillus sp., Salmonella sp., and Shigella sp. See, for example, Carrier et al, (1992) J. Immunol. 148:1176-1181; U.S. Patent No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302. Examples of Salmonella strains that may be used 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, laboratory strains are non-pathogenic strains. Other suitable bacteria, though not limited to these, may include, but are not limited to, Bacillus subtilis, Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, and Rhodococcus sp.

[0052] In some embodiments, cells are manipulated to provide a greater flow of GPP through the GPP pathway. Methods for obtaining an increased flow of GPP through the GPP pathway are not particularly limited and may include methods disclosed herein or known in the art. For example, an increased flow of GPP through the pathway may be achieved by increasing the expression of enzymes involved in the 1-deoxy-d-xylulose-5-phosphate (DXP) pathway: increased expression of DXP synthase, DXP ​​reductisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) synthase, CDP-ME kinase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEDCDP) synthase, 4-hydroxy-3-methylbuta-2-1-yldiphosphate synthase (Gcpe), or 4-hydroxy-3-methylbuta-2-enyldiphosphate reductase (LytB). Furthermore, an increased flow 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-5-phosphate decarboxylase, isopentenyl phosphate kinase, mevalonate pyrophosphate decarboxylase, and / or isopentenyl pyrophosphate isomerase.

[0053] In some embodiments, cells are engineered to provide a greater flow of tyrosine via the tyrosine biosynthesis pathway. In some embodiments, cells are engineered to provide a greater flow of phenylalanine via the phenylalanine biosynthesis pathway. In some embodiments, cells are engineered to provide a greater flow of both tyrosine and phenylalanine via their respective aromatic amino acid biosynthesis pathways. To increase the flow to Tyr and Phe, certain enzymes in the aromatic pathway, for example, DAHP synthase (EC2.5.1.54), the first enzyme in this pathway, specifically Aro4 (Uniprot Q6CCS4) as the wild-type sequence or the feedback-insensitive mutant Aro4 (to tyrosine), are engineered. k221L However, it is overexpressed. Other enzymes in the aromatic amino acid pathway that are upregulated or overexpressed include pentafunctional DHAPs from 5-enolpyruvate shikimic acid-3-phosphate (e.g., Aro1-Q6C1X5), chorismite synthase (e.g., Q6C8Q1), chorismite mutase (e.g., Q6C5J7), prephenate dehydrogenase (e.g., Q6C1B7), and hydroxyphenylpyruvate aminotransferase.

[0054] In some embodiments, a larger flow via the aromatic amino acid synthesis pathway is achieved by overexpressing the Aro4 enzyme (EC2.5.1.54). In some embodiments, the Aro4 enzyme contains 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, a larger flow via the aromatic amino acid synthesis pathway is achieved by expressing a feedback-insensitive Aro4 enzyme. In some embodiments, the feedback-insensitive Aro4 enzyme contains a 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: 5.

[0055] In some embodiments, cells are modified to knock down the expression of one or more genes that produce undesirable byproducts in the biosynthetic pathway leading to the production of bakuchiol and its analogues. In some embodiments, cells are modified to knock out one or more genes that produce undesirable byproducts in the bakuchiol biosynthetic pathway. It is understood that the strategy for selecting which of the one or more genes to knock down or knock out depends on whether the gene-encoded product is important for cell survival and the resulting increase in bakuchiol yield obtained by the gene knockdown or knockout.

[0056] In some embodiments, the cells are yeast cells, algal cells, or bacterial cells. In some embodiments, the yeast cells are Saccharomyces, Pichia, or Yarrowia.

[0057] In some embodiments, cells can synthesize coumaric acid, caffeic acid, and / or ferulic acid if tyrosine is present in the cell via endogenous production. In some embodiments, cells can produce cinnamic acid, coumaric acid, caffeic acid, or ferulic acid if phenylalanine is present in the cell via endogenous production.

[0058] In some embodiments, the production of bakuchiol or its analogues in cells depends on, or is enhanced by, the supplementation of cinnamic acid, coumaric acid, and / or ferulic acid in the fermentation medium in which the cells are grown.

[0059] In some embodiments, cells are capable of converting cinnamic acid to coumarate, coumarate to caffeic acid, and caffeic acid to ferulic acid, one or more of these. In some embodiments, cells express phenylalanine ammonia lyase and / or tyrosine ammonia lyase of family EC4.3.1.24 and / or family EC4.3.1.25. In some embodiments, cells express cinnamic acid 3-hydroxylase. In some embodiments, cinnamic acid 3-hydroxylase is a p450 enzyme of family CYP73A or family EC1.14.14.91. In some embodiments, cells express caffeic acid O-methyltransferase of family EC2.1.1.68.

[0060] In some embodiments, cells are capable of converting bakuchiol to 3-hydroxybakuchiol and / or 3-hydroxybakuchiol to 3-methoxybakuchiol. In some embodiments, the conversion of bakuchiol to 3-hydroxybakuchiol is catalyzed by a P450 hydroxylase of family CYP98A. In some embodiments, the conversion of 3-hydroxybakuchiol to 3-methoxybakuchiol is catalyzed by a methyltransferase of family EC2.1.1.68 or EC2.1.1.42.

[0061] Expression vectors (one or more) may be constructed to contain an exogenous nucleotide sequence encoding a membrane-bound prenyltransferase described herein, operably linked to a functional expression regulatory sequence in cells. Applicable expression vectors include plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, for example, those containing a vector and selectable sequence or marker for stable integration into a host chromosome. Furthermore, expression vectors may also contain one or more selectable marker genes and appropriate expression regulatory sequences. Selectable marker genes may also include, for example, those that provide resistance to antibiotics or toxins, complement nutritional deficiencies, or supply essential nutrients not present in the culture medium. Expression regulatory sequences may include constitutive and inductive promoters, transcriptional enhancers, transcriptional terminators, etc., well known in the art. When two or more exogenous coding nucleic acids are to be co-expressed, both nucleic acids may be inserted, for example, in a single expression vector or in separate expression vectors. For expression in a single vector, the coding nucleic acid may be operationally ligated to one common expression regulatory sequence, or to different expression regulatory sequences, e.g., one inductive promoter and one constitutive promoter. Transformation of an exogenous nucleic acid sequence may be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, e.g., Northern blotting or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. It will be understood by those skilled in the art that the exogenous nucleic acid is expressed in an amount sufficient to produce the desired product, and it will be further understood that the expression level may be optimized to obtain sufficient expression using methods well known in the art and disclosed herein.

[0062] The term “exogenous” is intended to mean that the molecule or activity mentioned is introduced into the cell. The molecule may be introduced, for example, by integration into the host chromosome, or by introduction of coding nucleic acids into non-chromosomal genetic material, such as plasmids. Therefore, when the term is used in relation to the expression of coding nucleic acids, it refers to the introduction of coding nucleic acids into the cell in an expressible form. When used in relation to biosynthetic activity, the term refers to the activity introduced into the host. The source may be, for example, homogeneous or heterogeneous coding nucleic acids that express the activity mentioned after introduction into the cell. Therefore, the term “endogenous” refers to the molecule or activity mentioned that is present in the cell. Similarly, when the term is used in relation to the expression of coding nucleic acids, it refers to the expression of coding nucleic acids contained within a microbial organism. The term “heterogeneous” refers to a molecule or activity originating from a source other than the species mentioned, while “homogeneous” refers to a molecule or activity originating from the host microorganism. Therefore, exogenous expression of coding nucleic acids may utilize either heterogeneous or homogeneous coding nucleic acids, or both.

[0063] In some embodiments, cells expressing prenyltransferase can produce bakuchiol or analogues 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, e.g., sucrose, glucose, mannitol, galactose, fructose, mannose, isomaltose, xylose, pannose, maltose, arabinose, cellobiose, and their 3-, 4-, or 5-oligomers. Other carbon sources include alcohol carbon sources, e.g., ethanol, glycerol. Other carbon sources may include combinations of the above carbon sources, e.g., glucose / mannitol or glucose / ethanol. Other carbon sources include acids and esters, e.g., acetates or formates, or fatty acids having 4 to 22 carbon atoms or their fatty acid esters. Other carbon sources may include recycled feedstocks and biomass. Exemplary regenerative feedstocks include cellulose biomass, hemicellulose biomass, and lignin feedstocks. Mixed carbon sources, such as fatty acids and sugars as described herein, may also be used.

[0064] Depending on the cells, an appropriate culture medium may be used. For example, descriptions of various culture media can be found in the "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981). As used herein, "culture medium" refers to a starting medium, which may be in solid or liquid form with respect to the growth source. On the other hand, "cultured medium," as used herein, refers to a medium containing fermentatively grown microorganisms (microbes) (e.g., a liquid medium) and may contain other cellular biomass. Culture media generally contain one or more carbon sources, nitrogen sources, inorganic salts, vitamins, and / or trace elements.

[0065] Culture conditions may include, for example, liquid culture procedures as well as fermentation and other large-scale culture procedures. Useful yields of the product may be obtained under aerobic culture conditions. Exemplary growth conditions for achieving one or more bakuchiol or analog products include aerobic culture or fermentation conditions. In certain embodiments, microorganisms may be sustained, cultured or fermented under aerobic conditions.

[0066] Substantially aerobic conditions include, for example, cultivation, batch fermentation, or continuous fermentation where the dissolved oxygen concentration in the culture medium remains between 5% and 100% saturation. The percentage of dissolved oxygen can be maintained, for example, by spraying with air, pure oxygen, or a mixture of air and oxygen.

[0067] Culture conditions can be continuously scaled up and expanded to produce 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 bakuchiol products in commercial quantities. Generally, as with discontinuous culture procedures, continuous and / or nearly continuous production of bakuchiol products involves culturing bakuchiol-producing organisms on a medium with sufficient nutrients and nutrients to sustain and / or nearly sustain logarithmic growth. Continuous cultivation under such conditions may include, for example, periods of one, two, three, four, five, six, or seven days or longer. Furthermore, continuous cultivation may include periods of one, two, three, four, or five weeks or longer, and up to several months. Alternatively, the desired microorganism may be cultured for several hours if appropriate for a particular application. It should be understood that continuous and / or nearly continuous culture conditions may also include all time intervals between these exemplary periods. It should be further understood that the time spent culturing the microorganisms should be sufficient to produce a sufficient quantity of product for the desired purpose.

[0068] Fermentation procedures are well known in the field. Briefly, fermentation for the biosynthesis of bakuchiol and bakuchiol analog products can be used, for example, in 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 field.

[0069] In some embodiments, the method further includes the step of purifying or isolating bakuchiol or an analogue thereof from the culture. The isolation method is 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 a mixture of the two), as well as methods including continuous liquid-liquid extraction, pervaporation, evaporation, filtration, membrane filtration (including reverse osmosis, nanofiltration, ultrafiltration, and microfiltration), membrane filtration with dialysis, 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, as well as ultrafiltration or centrifugal partition chromatography (CPC).

[0070] In some embodiments, cells are grown in a stirred tank fermenter with feed supplementation (sugars with or without organic acids), during which dissolved oxygen, temperature, and pH are controlled according to the optimal growth and production process. In some embodiments, an aqueous, immiscible organic solvent is supplied to dissolve the added organic acids or to extract the bakuchiol product while 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 another organic solvent having logP > 5. The latter number (logP) is defined as the logarithm of the distribution of the compound between water and octanol and is a standard parameter of hydrophobicity of the compound (the larger the logP, the lower the solubility in water). Depending on the fermentation process, the product may be isolated and purified using different methods.

[0071] In some embodiments, targeted bakuchiol or its analogues are precipitated with the cellular biomass after centrifugation, or isolated as a solid after dehydration using spray drying or other methods of water removal (i.e., freeze-drying, ultrafiltration, etc.). In one embodiment, an aqueous miscible organic solvent (ethanol, acetonitrile, etc.) is added to the bakuchiol-containing cell pellet to dissolve the product. In some embodiments, simple filtration, ultrafiltration, or centrifugation may remove the cells, and the aqueous / organic medium may be evaporated until it dries or until it reaches a small volume from which the bakuchiol product precipitates or crystallizes. Alternatively, the bakuchiol-containing cell pellet may be extracted using 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 possible recrystallization produce pure bakuchiol or its analogues. If the bakuchiol is not extracted by the previous method and is trapped inside the cells, cell lysis may be required before the above extraction method. In some embodiments, cells are destroyed by mechanical means or by suspension in a suitable lysis buffer from which bakuchiol can be extracted using an organic aqueous immiscible solvent (such as ethyl acetate, hexane, decane, or methylene chloride). In other embodiments, cells may be suspended in an organic solvent (such as ethanol, methanol, or methylene chloride) from which bakuchiol can be extracted.

[0072] In some embodiments, an organic solvent is required during growth and separated at the end of fermentation. Back extraction using an alkaline aqueous solvent or a different organic solvent with a low boiling point and high polarity (e.g., ethanol, acetonitrile) is used to isolate bakuchiol. As a result, isolation may involve a simple pH shift if water is used, or evaporation if an organic solvent is used. In both cases, a recrystallization step may be required at the end to improve the purity of the product. [Examples]

[0073] (Example 1) Screening of prenyltransferases using coumaric and caffeic feeds Plasmids expressing each gene were transformed into Y. lipolytica strain SB1334. This strain was engineered to produce increased amounts of GPP by overexpression of the ERG20.F88W.N119W (SEQ ID NO: 36) and ERG20.A28 alleles (SEQ ID NO: 40), as well as by disruption of endogenous Erg20, and contains the Erg20.A28 mutation, as described in shared PCT application number PCT / US2022 / 046926, which is incorporated entirely by reference. Transformants were seeded on YPD (10 g / L yeast extract, 20 g / L peptone, 20 g / L dextrose) agar plates containing 1 mg / mL hygromycin and grown at 30°C for 48 hours. Colonies were collected in 0.5 mL of YDCM medium containing 1 mg / mL hygromycin from a 96-well block (2.0 mL / well) (yeast nitrogen base + nitrogen, 6.71 g / L; casamino acid, 10 g / L; dextrose monohydrate, 66 g / L; MES hydrate, 19.5 g / L; pH adjusted to 6.5 with KOH). These pre-cultures were grown in a high-speed shaker at 1000 rpm at 30°C for 24 hours. Then, 2 μL from each well was used to inoculate the assay cultures from 0.5 mL of YDCM medium containing 1 mg / mL hygromycin into a 96-well block (2.0 mL / well). The assay cultures were grown in a high-speed shaker at 1000 rpm at 30°C for 24 hours, and then supplied with 10 mM p-coumaric acid or caffeic acid. After another 24 hours, the culture was quenched with an equal volume of EtOH containing an internal standard (0.2 mg / mL of 3,5-diisopropyl-2-hydroxybenzoate CAS#2215-21-6), centrifuged, and the clarified solution was then analyzed by HPLC-MS. [Table 1]

[0074] (Example 2) Synthesis of bakuchiol from glucose To generate a strain capable of producing bakuchiol from sugar, strain SB3503 was created by incorporating bakuchiol prenyltransferase MPT94.0 and GPP synthase ERG20.F88W.N119W into the genome of SB2728. The latter strain produces coumarate from glucose using a cytochrome P450 system consisting of phenylalanine ammonia lyase AtPAL, all derived from Arabidopsis thaliana, and the cytochrome P450 AtC4H (CYP73A5) from the CYP73A subfamily (EC1.14.14.91) and its accessory protein, AtATR2 (cytochrome P450 reductase).

[0075] To evaluate bakuchiol production, 16 independent transformants were assessed for bakuchiol titer in 96-well plate fermentation. Each transformant was inoculated into 0.5 mL of YDCM medium in a 96-well block (2.0 mL / well). These pre-cultures were grown in a high-speed shaker at 1000 rpm at 30°C for 24 hours. Subsequently, 2 μL from each well was used to inoculate 0.5 mL of fresh YDCM medium into a 96-well block (2.0 mL / well) as an assay culture. The assay cultures were grown in a high-speed shaker at 1000 rpm at 30°C for 48 hours, then quenched with an equal volume of EtOH containing an internal standard (0.2 mg / mL of 3,5-diisopropyl-2-hydroxybenzoate CAS#2215-21-6), centrifuged, and the clarified solution was analyzed by HPLC-MS. These transformants were able to produce bakuchiol (26–313 μM) from sugar, whereas the parents that did not possess MPT94.0 and ERG20.F88W.N119W did not produce any bakuchiol at all. [Table 2]

[0076] (Example 3) Synthesis of 3-hydroxybactiol from glucose For the synthesis of 3-hydroxybakuchiol from glucose, the strain developed in Experiment 2 is used as the background. This strain contains enzymes capable of converting bakuchiol to 3-hydroxybakuchiol. Such enzymes include P450 monooxygenases from the CYP98A subfamily (EC1.14.14.96). Specifically, AtC3H (NCBI:XP_011625941.1), a mutant CYP98A85 from Amborella trichopoda with increased activity for bakuchiol, is expressed in the strain. Furthermore, another parallel-functioning pathway can be manipulated, consisting of coumarate 3-hydroxylase and the engineered MPT85 or MP94 described herein, with increased activity from caffeate to 3-hydroxycaffeate.

[0077] (Example 4) Synthesis of 3-methoxybactiol from glucose For the synthesis of 3-methoxybactiol, a strain previously optimized to produce 3-hydroxybactiol is used. This strain includes a methyltransferase to convert 3-hydroxybactiol to 3-methoxybactiol. For this step, an enzyme from the 2.1.1.42 or 2.1.1.68 family is used. Specifically, the engineered methyltransferase OMT9 (Uniprot:I1I1F5; (SEQ ID NO: 42)) is used.

[0078] (Example 5) Synthesis of bakuchiol and 3-hydroxybakuchiol using coumaric acid and caffeic acid supplementation Bakuchiol and 3-hydroxybakuchiol can be synthesized in a strain having an upregulated pathway for GPP biosynthesis as a background, as described in Example 2. In this organism, MPT85 or MPT94 optimized for coumarate or caffeate activity is expressed. The cells are grown in a carbon source (glucose, glycerol, etc.) to supply coumarate or caffeate.

[0079] (Example 6) N-terminus shortening of MPT94 Sequence analysis of MPT94 predicted that the first 85 amino acids constitute the signal peptide. However, MPT94.1 (SEQ ID NO: 6), with amino acids 2–85 removed (preserving the start codon), was inactive. A three-dimensional structural model of MPT94 predicted secondary structural elements in the N-terminal region of the protein, and therefore, several shortenings (MPT94.2–8, SEQ ID NOs: 7–13) based on these structural elements were screened.

[0080] Plasmids expressing each gene were transformed into Y. lipolytica strain SB1334. Transformants were seeded onto YPD agar plates containing 1 mg / mL hygromycin and grown at 30°C for 48 hours. Colonies were collected in 0.5 mL of YDCM medium containing 1 mg / mL hygromycin in a 96-well block (2.0 mL / well). These pre-cultures were grown in a high-speed shaker at 1000 rpm at 30°C for 24 hours. Subsequently, 2 μL from each well was used to inoculate the assay cultures in 0.5 mL of YDCM medium containing 1 mg / mL hygromycin into a 96-well block (2.0 mL / well). The assay cultures were grown in a high-speed shaker at 1000 rpm at 30°C for 24 hours, and then supplied with 3 mM p-coumarate. After another 24 hours, the culture was quenched with an equal volume of EtOH containing an internal standard, centrifuged, and the clarified solution was then analyzed by HPLC-MS. [Table 3]

[0081] As shown in Table 3, MPT94 activity increases as more residues are removed from the N-terminus, when 6, 12, 26, and 36 residues are removed (MPT94.2 to MPT94.5). However, MPT94.6 and MPT94.7, with 62 and 70 residues removed respectively, are inactive, indicating that there is a limit to how many residues can be removed from the N-terminus before the enzyme activity is neutralized.

[0082] MPT94.8, from which 39 residues were removed, was screened under the same conditions as above. [Table 4]

[0083] (Example 7) Further prenyltransferase sequences from plant genomes Discovery of MPT94.15~MPT94.22 (SEQ ID NOs. 14~21). N-terminal shortened versions (MPT94.15t~MPT94.22t, SEQ ID NOs. 23~30) were constructed with residues 2~37 (the same residues removed in MPT94.5, which produced the highest concentration of bakuchiol in Table 3) removed.

[0084] Plasmids expressing each gene were transformed into Y. lipolytica strain SB3185. Transformants were seeded onto YPD agar plates containing 1 mg / mL hygromycin and grown at 30°C for 72 hours. Colonies were collected in 0.5 mL of YDCM medium containing 1 mg / mL hygromycin in a 96-well block (2.0 mL / well). These pre-cultures were grown in a high-speed shaker at 1000 rpm at 30°C for 24 hours. Subsequently, 5 μL from each well was used to inoculate the assay cultures in 0.5 mL of YDCM medium containing 1 mg / mL hygromycin into a 96-well block (2.0 mL / well). The assay cultures were grown in a high-speed shaker at 1000 rpm at 30°C for 24 hours, and then supplied with 3 mM p-coumarate and 1% oleic acid. After another 24 hours, the culture was quenched with an equal volume of EtOH containing an internal standard, centrifuged, and the clarified solution was then analyzed by HPLC-MS. [Table 5-1] [Table 5-2]

[0085] As shown in Table 5, MPT94.22t produced the highest concentration of bakuchiol. Analysis of mutations and their activity in MPT94.15t to MPT94.22t suggested that the C78W mutation should further improve the activity of MPT94.22t, and therefore MPT94.23t was screened under the same conditions as above. [Table 6]

[0086] (Example 8) Mutagenesis of MPT94.22t Positions H123 and E385 in MPT94.22t (residue numbering corresponding to that of full-length MPT94.22) were selected for saturation mutagenesis based on testing of a three-dimensional structural model of the protein.

[0087] A plasmid expressing MPT94.22t with an NNS degenerate codon at a targeted location was used to transform Y. lipolytica strain SB3185. The transformants were seeded onto YPD agar plates containing 1 mg / mL hygromycin and grown at 30°C for 48 hours. Eighty colonies from each location were collected in 0.5 mL of YDCM medium containing 1 mg / mL hygromycin in a 96-well block (2.0 mL / well). These pre-cultures were grown in a high-speed shaker at 1000 rpm at 30°C for 24 hours. Then, 2 μL from each well was used to inoculate the assay cultures in 0.5 mL of YDCM medium containing 1 mg / mL hygromycin into a 96-well block (2.0 mL / well). The assay cultures were grown in a high-speed shaker at 1000 rpm at 30°C for 24 hours, and then supplied with 3 mM p-coumarate and 1% oleic acid. After another 24 hours, the cultures were quenched with an equal volume of EtOH containing an internal standard, centrifuged, and the clarified solution was analyzed by HPLC-MS. Cultures with bakuchiol concentrations >10% higher compared to MPT94.22t were selected for sequencing, replicated under the same conditions as above, and rescreened. [Table 7]

[0088] (Example 9) Bakuchiol synthesis in a 2L fermentation tank To evaluate the productivity of MPT94 and validate small-scale results, strain SB3706 was created by incorporating a shortened Yarrowia HMGR synthase into SB3503 (described in Example 2). This strain can produce coumarate and GPP from glucose, which MPT94 converts to bakuchiol.

[0089] Fresh SB3706 cultures (initial OD600=0.3) were inoculated into a 2L fermenter containing 800mL of broth (10g / L ammonium sulfate, 20g / L casamino acid, 3.4g / L yeast nitrogen base), and the cells were grown at pH 5.5, 30°C, and 20% dissolved oxygen. When the cultures were supplemented with 3g / L / hour (dry weight) of dextrose for 5 days, 11.7mM (1.9g / L) of coumaric acid and 5.45mM (1.39g / L) of bakuchiol accumulated.

[0090] References 1. Alam F., et al Phytotherapy Research 2018, 32, 597-615. 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. 4. Jafernik, K. et al Nat Prod Res 2020, 5828-5842. 5. Chaudhuri, RK, Bojanowski K Int J Cosmetic Sci 2014, 36, 221. 6. Draelos ZD, et al Journal of Drugs in Dermatology, 2020, 19(12), 1181. 7. Afzal, N, Sivanami RK J. Cosmetic Dermatology, 2023, 00, 1-2.

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Claims

1. A mutant membrane-bound prenyltransferase (MPT) comprising an amino acid sequence 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, and having at least 85% identity with the amino acid sequence of SEQ ID NO: 35, 2, or 1, wherein the mutant MPT is capable of producing bakuchiol or an analog 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 according to claim 1, further comprising at least one further 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 according to claim 1, wherein the bakuchiol analog is selected from the group consisting of dehydrobakuchiol, 3-hydroxybakuchiol, and 3-methoxybakuchiol.

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

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

6. A mutant membrane-bound prenyltransferase according to any one of claims 1 to 3, comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NOs: 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. A mutant membrane-bound prenyltransferase according to any one of claims 1 to 3, having an amino acid sequence that includes an amino acid sequence having 98.5% to 99.8% identity with the amino acid sequence of SEQ ID NO: 35, 2, or 1.

8. A mutant membrane-bound prenyltransferase (MPT) according to claim 1, which transfers geranyl pyrophosphate (GPP) to cinnamic acid, coumaric acid, caffeic acid and / or ferulic acid to produce dehydrobakuchiol, bakuchiol, 3-hydroxybakuchiol and / or 3-methoxybakuchiol, respectively, with higher efficiency compared to either enzyme MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2).

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

10. The mutant membrane-bound prenyltransferase according to any one of claims 1 to 3, wherein the at least one amino acid modification and the at least one further amino acid modification are individually selected from substitution, deletion or insertion.

11. A mutant membrane-bound prenyltransferase according to any one of claims 1 to 3, further comprising one or more substitutions, deletions and / or insertions at positions selected from 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.

12. A mutant membrane-bound prenyltransferase according to any one of claims 1 to 3, comprising an amino-terminal shortening including a deletion between amino acids 1 to 73 corresponding to amino acids at positions 2 to 74 of the amino acid sequence of SEQ ID NO: 35, 2, or 1.

13. A mutant membrane-bound prenyltransferase according to claim 12, comprising an amino acid sequence having at least 90% identity with an amino acid sequence selected from SEQ ID NOs: 6-10, 13 and 23-33, 33 and 44-46.

14. A manipulated cell expressing a mutant membrane-bound prenyltransferase according to any one of claims 1 to 13 or an MPT comprising the amino acid sequence of MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2), which is capable of producing bakuchiol and / or its analogues in the presence of geranyl pyrophosphate (GPP) and at least one of cinnamic acid, coumaric acid, caffeic acid, and ferulic acid.

15. The cell according to claim 14, which is manipulated to provide a greater flow of GPP through the GPP pathway.

16. The cell according to claim 14 or 15, which is engineered to provide a greater flow of tyrosine or phenylalanine through the respective aromatic amino acid synthesis pathway.

17. The cell according to claim 16, wherein the larger flow via the aromatic amino acid synthesis pathway is achieved by overexpressing the Aro4 enzyme (EC2.5.1.54) having an amino acid sequence having at least 95% identity with SEQ ID NO:

4.

18. The cell according to claim 16, wherein the larger flow via the aromatic amino acid synthesis pathway is achieved by expressing a feedback-insensitive Aro4 enzyme having a sequence having at least 95% identity with SEQ ID NO:

5.

19. The cell according to claim 14 or 15, which is a yeast cell, an algal cell, or a bacterial cell.

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

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

22. The cell according to claim 14 or 15, wherein the production of bakuchiol or an analogue of the cell depends on or is enhanced by the supplementation of cinnamic acid, coumaric acid, caffeic acid and / or ferulic acid in the fermentation medium in which the cell is grown.

23. The cell according to claim 21, which is capable of converting cinnamic acid to coumaric acid, converting coumaric acid to caffeic acid, and converting caffeic acid to ferulic acid, one or more of these.

24. The cell according to claim 21, expressing phenylalanine ammonia lyase and / or tyrosine ammonia lyase of family EC4.3.1.24 and / or family EC4.3.1.

25.

25. The cell according to claim 24, which expresses cinnamic acid 3-hydroxylase.

26. The cell according to claim 25, wherein the cinnamic acid 3-hydroxylase is a p450 enzyme of the CYP73A family or family EC1.14.14.

91.

27. The cell according to claim 24, expressing caffeate O-methyltransferase of family EC2.1.1.

68.

28. The cell according to claim 21, which is capable of converting bakuchiol to 3-hydroxybakuchiol and / or 3-hydroxybakuchiol to 3-methoxybakuchiol.

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

30. The cell according to claim 28, wherein the conversion of 3-hydroxybakuchiol to 3-methoxybakuchiol is catalyzed by a methyltransferase of family EC2.1.1.68 or EC2.1.1.

42.

31. A method for producing bakuchiol by fermenting the cells described in any one of claims 14 to 30 in the presence of one or more of glucose, glycerol, cinnamic acid, coumaric acid, caffeic acid, and ferulic acid.

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