NL2031273B1

NL2031273B1 - Bioproduction of bakuchiol

Info

Publication number: NL2031273B1
Application number: NL2031273A
Authority: NL
Inventors: Reider Apel Amanda; Kumar Abhinav
Original assignee: Sestina Bio Llc
Priority date: 2022-03-04
Filing date: 2022-03-14
Publication date: 2023-09-11

Abstract

The present disclosure relates to synthetic biology and, in particular the bioproduction of bakuchiol. The present disclosure provides examples of the proteins that produce bakuchiol through a mechanism involving GPP/DMAPP/IPP and p—coumaric acid. Based on the identification of these proteins, the present disclosure provides examples of nucleic acids encoding the disclosed proteins, transgenic cells that produce bakuchiol, methods of producing bakuchiol, bioproduction batches of bakuchiol, and methods of detecting bakuchiol.

Description

BIOPRODUCTION OF BAKUCHIOL

Background

[0001] The following discussion is merely provided to aid the reader in understanding the disclosure and is not admitted to describe or constitute prior art thereto.

[0002] Bakuchiol 1s terpenoid produced by a number of plants, including Psoralea corylifolia, P. grandulosa, P. drupaceae, Ulmus davidiana, Otholobium pubescens, Piper longum, and Aerva sangulnolenta Blum. However, the enzyme(s) and thus the exact synthesis process responsible for producing bakuchiol are unknown.

[0003] Bakuchiol is utilized for a variety of commercial, pharmaceutical, and cosmetic purposes due to 1ts reported activities, such as anti-wrinkle, anti-acne, anti-inflammatory, anti- osteoporosis, ant-oxidant, anti-biofilm, and anti-cancer effects. Bakuchiol is also used in traditional Chinese medicine.

[0004] Though there have been previous attempts to chemically synthesize bakuchiol, the resulting synthesis schemes were difficult and inefficient. Instead, most bakuchiol used today is extracted from plants via laborious and expensive extraction procedures. To date, the lack of identification of the enzyme(s) responsible for producing bakuchiol has hindered bioproduction of bakuchiol in, for example, a transgenic host or bioreactor.

Summary

[0005] The present disclosure provides examples generally related to synthetic biology and, in particular, the bioproduction of bakuchiol. Some examples provided herein may be employed to overcome the pre-existing challenges and may have the benefit of identifying the enzyme(s) responsible for producing bakuchiol and for the development of efficient bioproduction of bakuchiol.

[0006] The present disclosure provides examples of the proteins that produce bakuchiol through a mechanism involving GPP/DMAPP/IPP and p-coumaric acid. Based on the identification of these proteins, the present disclosure provides examples of nucleic acids encoding the disclosed proteins, transgenic cells that produce bakuchiol, methods of producing bakuchiol, bioproduction batches of bakuchiol, and methods of detecting bakuchiol.

[0007] In one aspect, the disclosure provides transgenic cells, comprising a transgene encoding a transgenic protein comprising an amino acid sequence with at least about 65% identity to:

MHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETL

WGSIKQFCDAFYRFSRPHVIIGTAVNIIVMSSLALEKSSDISPKFFIGLFQVIVTILSMNIYT

AGINQLTDIEIDKINKPYLPLASGEYSYKTGVTHTLCAILSLGVGWIVGSPPLFWSNFAYF

VLGTVYSIDLPLMRWKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRPMMIPKPVMEF

GTAFMSFFY VIIAFFKDIPDIEGDKDHGVKSLTMRLGQKRVFWICVSLLLTGYGAAIVVG

ATSSFLWCKLITVSGHALLASIFWNRAKSVDLKSHQEITSLYMFMWKLFYAEYFIPLMR

(SEQ ID NO: 1; BAK28), or

MASMFLGSLPLASSVNYIGRITRSKNCTESYHATSYITNASSNKTEKIKHEYANMRHRQH

NLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLWGSIKQFCDAFYR

FSRPHVHGTAVNIIVMSSLALEKSSDISPKFFIGLFQVIVTILSMNIYTAGINQLTDIEIDKIN

KPYLPLASGEYSYKTGVTHTLCAILSLGVGWIVGSPPLFWSNFAYFVLGTVYSIDLPLMR

WKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRPMMIPKSVMFGTAFMSFFYVIIAFF

KDIPDIEGDKDHGVKSLTMRLGQERVFWICVSLLLTGYGAAIVVGATSSFLWCKLITVSG

HALLASIFWNRAKSVDLKSHOEITSLYMFMWKLFYAEYFIPLMR (SEQ ID NO: 2;

BAK36); wherein the transgenic protein catalyzes the production of bakuchiol, exhibits prenyltransferase activity, or both.

[0008] The transgenic protein can comprise an amino acid sequence with at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 1 or

SEQ ID NO: 2.

[0009] The transgenic cell can be prokaryotic. For example, the transgenic cell can be selected from Escherichia coli (E. coli), an Acinetobacter species, a Pseudomonas species, a

Streptomyces species, and a Mycobacterium species.

[0010] The transgenic cell can be eukaryotic. For example, the transgenic cell can be selected from Saccharomyces cerevisiae (S. cerevisiae) or other yeast species, a filamentous fungi, an algae, and an amoeba. In some implementations, the filamentous fungi can be selected from an

Aspergillus species and a Trichoderma species. In some implementations, the amoeba can be

Dictyostelium discoideum. In some implementations, the algae can be selected from

Botryococcus braunii, Chlorella sp. Crypthecodinium cohnit, Cylindrotheca sp, Nitzschia sp.,

Phaeodactylum tricornutum, Schizochytrium sp., and Tetraselmis suecia.

[0011] Bakuchiol can be produced when the transgenic cell is cultured in the presence of p- coumaric acid and (i) geranyl pyrophosphate (GPP), (ii) dimethylallyl pyrophosphate (DMAPP), (iii) isopentenyl pyrophosphate (IPP), or any combination of (i)-(iii).

[0012] The transgenic cell may produce at least about 0.1 pg/L, at least about 0.2 pg/L, at least about 0.3 pg/L, at least about 0.4 pg/L, at least about 0.5 ug/L, at least about 0.6 pg/L, at least about 0.7 pg/L, at least about 0.8 ug/L, at least about 0.9 pg/L, at least about 1.0 pg/L, at least about 1.1 pg/L, at least about 1.2 ug/L, at least about 1.3 pg/L, at least about 1.4 pg/L, at least about 1.5 pg/L, at least about 1.6 ng/L, at least about 1.7 pg/L, at least about 1.8 pg/L, at least about 1.9 ug/L, at least about 2.0 pg/L, at least about 2.1 pg/L, at least about 2.2 pg/L, at least about 2.3 pg/L, at least about 2.4 ug/L, at least about 2.5 pg/L, or more of bakuchiol within at least about 48 hours when cultured in the presence of p-coumaric acid and (i) geranyl pyrophosphate (GPP), (ii) dimethylallyl pyrophosphate (DMAPP), (iii) isopentenyl pyrophosphate (IPP), or any combination of (1)-(iii).

[0013] In some implementations, the transgene is integrated into the transgenic cell’s genome. In some implementations, the transgene is not integrated into the transgenic cell’s genome.

[0014] In some implementations, expression of the transgene can be driven by a GAL 1 promoter. In some implementations, expression of the transgene is driven by an inducible promoter.

[0015] The transgenic protein can have at least about 90% identity to SEQ ID NO: 1 or SEQ ID

NO: 2. The transgenic protein can comprise SEQ ID NO: 1 or SEQ ID NO: 2. The transgenic protein can consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0016] In another aspect, the present disclosure provides methods of producing bakuchiol, comprising culturing a transgenic cell disclosed here {e.g., described in the foregoing aspect) in a culture medium and in the presence of p-coumaric acid and (i) geranyl pyrophosphate (GPP), (ii)

dimethylallyl pyrophosphate (DMAPP), (iii) isopentenyl pyrophosphate (IPP), or any combination of (i)-(iii).

[0017] The culture medium can further comprise about 3% w/v maltodexrin, about 0.2% w/v glucose, alpha-amylase, or any combination thereof.

[0018] In some implementations, the culture medium can comprises at least about 1.50 mM p- coumaric acid, at least about 1.75 mM p-coumaric acid, at least about 2.00 p-coumaric acid, at least about 2.25 mM p-coumaric acid, at least about 2.50 mM p-coumaric acid, at least about 2.75 mM p-coumaric acid, at least about 3.00 p-coumaric acid, at least about 3.25 mM p- coumaric acid, at least about 3.50 mM p-coumaric acid, at least about 3.75 mM p-coumaric acid, atleast about 4.00 p-coumaric acid or more. In some implementations, the culture medium does not comprise exogenous p-coumaric acid, GPP, DMAPP, IPP, or a combination thereof.

[0019] In some implementations, at least about 0.1 ug/L, at least about 0.2 pg/L, at least about 0.3 pg/L, at least about 0.4 ug/L, at least about 0.5 pg/L, at least about 0.6 pg/L, at least about 0.7 ug/L, at least about 0.8 ug/L, at least about 0.9 pg/L, at least about 1.0 ug/L, at least about 1.1 ug/L, at least about 1.2 pg/L, at least about 1.3 pg/L. at least about 1.4 pg/L, at least about 1.5 ng/L, at least about 1.6 pg/L, at least about 1.7 pg/L, at least about 1.8 pg/L, at least about 1.9 pg/L, at least about 2.0 ug/L, at least about 2.1 pg/L, at least about 2.2 pg/L, at least about 2.3 ug/L, at least about 2.4 ug/L, at least about 2.5 pg/L, or more of bakuchiol within at least about 48 hours of culture.

[0020] In another aspect, the present disclosure provides bioproduction batches of bakuchiol, wherein the bakuchiol has a chemical purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, and no single impurity of greater than about 1%.

[0021] The bakuchiol can be in the form of an oil or in an aqueous solution. The concentration of bakuchiol in the batch can be at least about 0.1 pg/L, at least about 0.2 pg/L, at least about 0.3 pg/L, at least about 0.4 pg/L, at least about 0.5 ug/L, at least about 0.6 ug/L, at least about 0.7 pg/L, at least about 0.8 pg/L, at least about 0.9 ug/L, at least about 1.0 ug/L, at least about 1.1 pg/L, at least about 1.2 pg/L, at least about 1.3 ug/L, at least about 1.4 ng/L, at least about 1.5 ug/L. at least about 1.6 ug/L, at least about 1.7 pg/L, at least about 1.8 pg/L. at least about 1.9 pg/L, at least about 2.0 pg/L, at least about 2.1 pg/L, at least about 2.2 pg/L, at least about 2.3 pg/L, at least about 2.4 ng/L, at least about 2.5 pg/L, or more.

[0022] A bioproduction batch of bakuchiol of the present disclosure can be produced by the disclosed bioproduction methods (e.g., the methods of the forgoing aspect). 5 [0023] In another aspect, the present disclosure provides isolated proteins comprising an amino acid sequence with at least about 65% identity to:

MHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETL

WGSIKQFCDAFYRFSRPHVHIGTAVNIVMSSLALEKSSDISPKFEFIGLFQVIVTILSMNIYT

AGINQLTDIEIDKINKPYLPLASGEYSYKTGVTHTLCAILSLGVGWIVGSPPLFWSNFAYF

VLGTVYSIDLPLMRWKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRPMMIPKPVMF

GTAFMSFFY VIIAFFKDIPDIEGDKDHGVKSLTMRLGQKRVFWICVSLLLTGYGAAIVVG

ATSSFLWCKLITVSGHALLASIFWNRAKSVDLKSHQEITSLYMFMWKLFYAEYFIIPLMR

(SEQ ID NO: 1; BAK28), or

MASMFLGSLPLASSVNYIGRITRSKNCTESYHATSYITNASSNKTEKIKHEYANMRHRQH

NLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLWGSIKQFCDAFYR

FSRPHVHGTAVNIJVMSSLALEKSSDISPKFFIGLFQVIVTILSMNIYTAGINQLTDIEIDKIN

KPYLPLASGEYSYKTGVTITLCAILSLGVGWIVGSPPLFWSNFAYFVLGTVYSIDLPLMR

WKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRPMMIPKSVMFGTAFMSFFY VIIAFF

KDIPDIEGDKDHGVKSLTMRLGQERVFWICVSLLLTGYGAAIVVGATSSFLWCKLITVSG

HALLASIFWNRAKSVDLKSHQEITSLYMFMWKLFYAEYFIIPLMR (SEQ ID NO: 2;

BAK36).

[0024] The amino acid sequence of the isolated proteins can comprise at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, at least about 99%, or 100% identity with SEQ ID NO: 1 or

SEQ ID NO: 2. The amino acid sequence can have at least 90% identity to SEQ ID NO: 1 or

SEQ ID NO: 2. The amino acid sequence can comprise SEQ ID NO: 1 or SEQ ID NO: 2. The amino acid sequence can consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0025] The isolated protein can exhibit prenyltransferase activity, the isolated protein can catalyze the production of bakuchiol, or both.

[0026] In another aspect, the present disclosure provides nucleic acids comprising a nucleic acid sequence encoding any isolated protein disclosed herein (e.g., the isolated proteins of the foregoing aspect).

[0027] In another aspect, the present disclosure provides isolated host cells that produces an isolated protein disclosed herein or that comprises a nucleic acid disclosed herein (e.g., the isolated proteins or nucleic acids of the foregoing aspects).

[0028] The foregoing general description and following detailed description are examples and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.

[0029] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below are contemplated as being part of the inventive subject matter disclosed herein and may be employed in any combination to achieve the benefits described herein.

Brief Description of Drawings

[0030] Figure 1 (FIG. 1) shows, in one implementation, that enzymes BAK28 (SEQ ID NO: 1) and BAK36 (SEQ ID NO: 2) specifically produce bakuchiol.

[0031] Figure 2 (FIG. 2) shows, in one implementation, quantification of the bakuchiol produced by BAK28 and BAK36. The five BAK28 bio replicates have similar titer around 1.0 ug/L. The three BAK36 bio replicates have similar around 0.1 ug /L

[0032] Figure 3 (FIG. 3) shows, in one implementation, the p-coumaric acid involved for bakuchiol production, and increasing p-coumaric acid can augment production of bakuchiol. For example, in BAK28, production peaked at 2.0 ug/L at a p-coumaric acid concentration of 209 mg/L. 10033] Figure 4 (FIG. 4) shows. in one implementation, a sequence alignment of BAK28 and

BAK36.

[0034] Figure 5 (FIG. 5) shows, in one implementation, the predicted transmembrane regions of

BAK28 and BAK36.

[0035] Figure 6 (FIG. 6) shows, in one implementation, a flowchart of the process used to prepare engineered S. cerevisiae expressing putative bakuchiol-producing proteins.

[0036] Figure 7 (FIG. 7) shows, in one implementation, the results of a Blast search for homologs of BAK28 and BAK36 in the NCBI database.

Detailed Description

[0037] Bakuchiol is known to be produced by several plant species, including Psoralea corylifolia, Piper Longum and Ulmus davidiana. However, the complete biosynthetic pathway to produce the compound is previously unknown prior to this application. The present disclosure provides examples that identify prenyliransterase enzymes that may be used to produce bakuchiol through a mechanism involving p-coumaric acid and at least one of geranyl pyrophosphate (GPP), dimethylallyl pyrophosphate (DMAPP), isopentenyl pyrophosphate (IPP), or a combination thereof. This discovery may enable bioproduction of bakuchiol. Thus, the present disclosure provides example methods of producing bakuchiol in a bioreactor or fermenter, which may displace the need for difficult, inefficient synthesis or costly cultivation and extraction of bakuchiol-producing plants. Further, the present disclosure provides example methods of detecting and quantifying bakuchiol via mass spectrometry, which can be useful for quality control of bakuchiol batches produced not only by the disclosed bioproduction methods, but by pre-existing methods as well.

I. Definitions

[0038] It is to be understood that the disclosed compositions and methods are not limited to the particular implementations described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular implementations only, and is not intended to be limiting. The scope of the present technology will be limited only by the appended claims.

[0039] As used herein, certain terms may have the following defined meanings. As used in the specification and claims, the singular form “a,” “an” and “the” include singular and plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a single cell as well as a plurality of cells, including mixtures thereof.

[0040] As used herein, “about” means the recited quantity exactly and small variations within a limited range encompassing plus or minus 10% of the recited quantity. In other words, the limited range encompassed can include 10%, 29%, +8%, £7%, £6%, £5%, +4%, 13%, +2%, +1%, 20.5%, £0.2%, +0. 1%, £0.05%, or smaller, as well as the recited value itself. Thus, by way of example, “about 10” should be understood to mean “10” and a range no larger than “9-117.

[0041] As used herein, the term “bioproduction” is intended to mean production of a compound (e.g., bakuchiol) by way of biological or enzymatic synthesis (as opposed to chemical synthesis).

In some implementations, bioproduction may be performed by a transgenic organism or microbe that has been engineered to express enzymes involved in the biological synthesis of a compound of interest (e.g., bakuchiol).

[0042] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the composition or method. “Consisting of” shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps.

Examples and implementations defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).

[0043] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0044] For the purpose of the description, a phrase in the form “A/B” or in the form “A and/or

B” means (A), (B), or (A and B).

IL. Bakuchiol

[0045] Bakuchiol is a phenolic compound having a single hydroxyl group on the aromatic ring and an unsaturated hydrocarbon chain. It has been isolated from the seeds of Psoralea. corvlifolia. The chemical structure of bakuchiol is provided below i ns ™ \ 3 !

HO ’ . In one implementation, the bakuchiol chemical structure 1s also presented as

Ms i

Horen RC

[0046] Bakuchiol has been reported as possessing antibacterial activity, anti-inflammatory activity, anti-cancer activity, anti-oxidant activity, and other beneficial properties. As a result, it may be used in supplements, cosmetics, and other consumer products, and it may be employed for pharmaceutical use. However, there are currently a number of limitations associated with the use of this compound due primarily to its low concentration in natural sources, as well as the presence of co-existing toxic components. One of the main problems related to the use of bakuchiol compositions isolated from plants in the Psoralea genus is the presence of psoralens, such as psoralen and isopsoralen, which are associated with a number of health risks.

Additionally, pre-existing methods of chemically synthesizing bakuchiol or extracting it from plants are generally inefficient and resource intensive.

[0047] The presently disclosed proteins and methods make it possible to bioproduce bakuchiol, thus addressing the limitations of pre-existing chemical and extraction-based methodologies.

TI. Bakuchiol-Producing Proteins and Nucleic Acids

[0048] Not to be bound by particular theory, but one implementation of the methods described herein is based on bakuchiol being produced by an unknown prenyltransferase enzyme through a mechanism involving geranyl pyrophosphate (GPP), dimethylallyl pyrophosphate (DMAPP), isopentenyl pyrophosphate (IPP), or a combination thereof, and p-coumaric acid. As explained in further detail in the Non-limiting Working Examples section herein, transcriptome analysis was performed on three known bakuchiol producers (Psoralea coryiifolia, Piper Longum and Ulmus davidiana), genes encoding putative prenyltranferase enzymes (i.e., “BAK genes”) were identified, and all putative prenyltranferase enzymes (BAK genes) were integrated into S. cerevisiae so that the resulting strains could be screened. Of the 196 putative enzymes analyzed, only two proteins were identified that produced bakuchiol when expressed in S. cerevisiae:

MHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETL

WGSIKQFCDAFYRFSRPHVHGTAVNIVMSSLALEKSSDISPKFFIGLFQVIVTILSMNIYT

AGINQLTDIEIDKINKPYLPLASGEYSYKTGVTHTLCAILSLGVGWIVGSPPLFWSNFAYF

VLGTVYSIDLPLMRWKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRPMMIPKPVMEF

GTAFMSFFY VIIAFFKDIPDIEGDKDHGVKSLTMRLGQKRVFWICVSLLLTGYGAAIVVG

ATSSFLWCKLITVSGHALLASIFWNRAKSVDLKSHQEITSLYMFMWKLFYAEYFHPLMR

(SEQ ID NO: 1; referred to herein as “BAK28”), and

MASMFLGSLPLASSVNYIGRITRSKNCTESYHATSYITNASSNKTEKIKHEYANMRHRQH

NLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLWGSIKQFCDAFYR

FSRPHVIGTAVNIJVMSSLALEKSSDISPKFFIGLFQVIVTILSMNIYTAGINQLTDIEIDKIN

KPYLPLASGEYSYKTGVTHTLCAILSLGVGWIVGSPPLFWSNFAYFVLGTVYSIDLPLMR

WKSHPALAALFFFVIRGLTEHVGEFLHLQTHVFKRPMMIPKSVMFGTAFMSFFYVILAFF

KDIPDIEGDKDHGVKSLTMRLGQERVFWICVSLLLTGYGAAIVVGATSSFLWCKLITVSG

HALLASIFWNRAKSVDLKSHQEITSLYMFMWKLFYAEYFIIPLMR (SEQ ID NO: 2; referred to herein as “BAK36”).

[0049] The present disclosure further provides additional example putative prenyltranferase enzymes capable of converting p-coumaric acid and GPP/DMAPP/IPP into bakuchiol. Thus, the present disclosure provides bakuchiol-producing proteins that have at least about 65% - e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1.

Similarly, the present disclosure provides bakuchiol-producing proteins that have at least about 65% - e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID

NO: 2. In some implementations, the bakuchiol-producing protein may be share at least about 90% identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some implementations, the bakuchiol- producing protein may be share at least about 95% identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some implementations, the bakuchiol-producing protein may be share at least about 99% identity with SEQ ID NO: 1 or SEQ ID NO: 2. Thus, this disclosure contemplates and encompasses proteins with varying degrees of sequence identity compared to SEQ ID NOs: 1 and 2, so long as the protein exhibits prenyltranferase activity, is able to produce bakuchiol, or both.

[0050] SEQ ID NO: 1 and SEQ ID NO: 2 are structurally similar and share similar amino acid sequences. SEQ ID NO: 1 1s missing a 49 amino acid segments at its N termini that is present in

SEQ ID NO: 2. Aside from this truncation, there are only two other amino acid substitutions across the length of the protein sequences. This indicates that SEQ ID NOs: 1 and 2 may represent splice variants of the same gene, and further shows in one implementation that the minimum domain involved for activity may be less than the entire 409 amino acid sequence of

SEQ ID NO: 2, and a protein that is longer than the 361 amino acid sequence of SEQ ID NO: 1 may be active as well. Accordingly, the present disclosure contemplates protein sequences that are the same length, longer, or shorter than SEQ ID NO: 1 or SEQ ID NO: 2.

[0051] For example, a bakuchiol-producing protein of the present disclosure may comprise SEQ

ID NO: 1 (i.e, it is 361 amino acids or longer). In some implementations, a bakuchiol-producing protein of the present disclosure may comprise 361 amino acids that have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID

NO: 1. In some implementations, a bakuchiol-producing protein of the present disclosure may consist of SEQ ID NO: 1. In some implementations, a bakuchiol-producing protein of the present disclosure may consist of 361 amino acids that have at least about 65% - e.g., at least about 70%, atleast about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 929%, at least about 93%, at least about 94%, at least about 95%, at least about

96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1. In some implementations, a bakuchiol-producing protein of the present disclosure may comprise about 365, about 370, about 375, about 380, about 385, about 390, about 395, about 400, about 405, about 410, about 415, about 420, about 425, about 430, about 435, about 440, or about 450 amino acids, wherein at least about 361 amino acids of the protein have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1. In some implementations, a bakuchiol-producing protein of the present disclosure may comprise about 365 to about 700 amino acids, about 365 to about 650 amino acids, about 365 to about 600 amino acids, about 365 to about 550 amino acids, about 365 to about 500 amino acids, about 365 to about 450 amino acids, about 365 to about 400 amino acids, about 375 to about 700 amino acids, about 375 to about 650 amino acids, about 375 to about 600 amino acids, about 375 to about 550 amino acids, about 375 to about 500 amino acids, about 375 to about 450 amino acids, about 375 to about 400 amino acids, about 385 to about 700 amino acids, about 385 to about 650 amino acids, about 385 to about 600 amino acids, about 385 to about 550 amino acids, about 385 to about 500 amino acids, about 385 to about 450 amino acids, about 385 to about 400 amino acids, about 395 to about 700 amino acids, about 395 to about 650 amino acids, about 395 to about 600 amino acids, about 395 to about 550 amino acids, about 395 to about 500 amino acids, about 395 to about 450 amino acids, or about 395 to about 400 amino acids, wherein at least about 361 amino acids of the protein have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1.

[0052] Similarly, a bakuchiol-producing protein of the present disclosure may comprise SEQ ID

NO: 2 (i.e, it is 409 amino acids or longer). In some implementations, a bakuchiol-producing protein of the present disclosure may comprise 409 amino acids that have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about

90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID

NO: 2. In some implementations, a bakuchiol-producing protein of the present disclosure may consist of SEQ ID NO: 2. In some implementations, a bakuchiol-producing protein of the present disclosure may consist of 409 amino acids that have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 2. In some implementations, a bakuchiol-producing protein of the present disclosure may comprise about 410, about 415, about 420, about 425, about 430, about 435, about 440, or about 450 amino acids, wherein at least about 409 amino acids of the protein have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 2. In some implementations, a bakuchiol-producing protein of the present disclosure may comprise about 410 to about 700 amino acids, about 410 to about 650 amino acids, about 410 to about 600 amino acids, about 410 to about 550 amino acids, about 410 to about 500 amino acids, about 410 to about 450 amino acids, about 420 to about 700 amino acids, about 420 to about 650 amino acids, about 420 to about 600 amino acids, about 420 to about 550 amino acids, about 420 to about 500 amino acids, about 420 to about 450 amino acids, about 430 to about 700 amino acids, about 430 to about 650 amino acids, about 430 to about 600 amino acids, about 430 to about 550 amino acids, about 430 to about 500 amino acids, about 430 to about 450 amino acids, about 440 to about 700 amino acids, about 440 to about 650 amino acids, about 440 to about 600 amino acids, about 440 to about 550 amino acids, about 440 to about 500 amino acids, or about 440 to about 450 amino acids, wherein at least about 409 amino acids of the protein have at least about 65% - eg, at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%,

or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID

NO: 2.

[0053] Bakuchiol-producing proteins described herein include also those that are shorter than

SEQ ID NO: 1 or SEQ ID NO: 2. A bakuchiol-producing protein may be less than 409 or less than 361 amino acids in length, so long as the protein has a catalytic domain that has at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with

SEQ ID NO: 1 or SEQ ID NO: 2. In some implementations at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, or at least about 350 amino acids of the protein can have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: { or SEQ ID NO: 2, so long as the protein exhibits prenyltransferase activity, is able to catalyze bakuchiol production, or both.

[0054] Indeed, for the purposes of this disclosure, any of the disclosed proteins is considered a “bakuchiol-producing protein” if the protein exhibits prenyltransferase activity, catalyzes the production of bakuchiol, or both.

[0055] For the purposes of this disclosure, all of the foregoing proteins can be isolated, in a form in which the protein is essentially free of other proteins, contaminants, or macromolecules (e.g., nucleic acids, lipids, etc).

[0056] The present disclosure also provides nucleic acids comprising a nucleic acid sequence encoding any one of the proteins disclosed herein. Those skilled in the art understand that a nucleic acid sequence can be designed/determined based on a known amino acid sequence as a result of known codon specificity. Thus, in some implementations, the nucleic acid may comprise a nucleic acid sequence encoding SEQ ID NO:1, SEQ ID NO: 2, or a protein that has at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1 or SEQ ID NO: 2, so long as the protein exhibits prenyltransferase activity, is able to catalyze bakuchiol production, or both.

[0057] Because the disclosed proteins may be of particular value when used in the context of transgenic expression in a microbial chassis, the nucleic acid sequence encoding any one of the disclosed proteins may be codon-optimized for a given expression system. For example, the nucleic acid sequence may be codon-optimized for expression is a yeast system, such as S. cerevisiae. Alternatively, the nucleic acid sequence may be codon-optimized for expression is a prokaryotic system, such as E. coli.

[0058] The disclosed nucleic acids that encode a bakuchiol-producing protein can be incorporated into an expression vector or expression cassette. The nucleic acid can be transduced or transformed into a transgenic cell such that the nucleic acid sequence encoding the bakuchiol- producing protein is integrated into the genome of the host cell or transgenic cell, Alternatively, the nucleic acid sequence encoding the bakuchiol-producing protein may be expressed without integration into the host genome {e.g., in the form of a plasmid). For those implementations in which genome integration is desired, any suitable methods of integration can be used, including but not limited to Cas-based systems (e.g., Cas9, Cas12, etc.), homologous recombination, gene gun, conjugation protocols, lambda red, etc.

[0059] An expression cassette or vector for expressing the nucleic acid sequence encoding the bakuchiol-producing protein may comprise a promoter and a terminator. Known promoters that can be used are well established in the art, and include but are not limited to GAL 1, TEF2, TEFI,

TDH3, ENO2, CCW12, EF-1a promoter, CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-1. In some implementations, an inducible or repressible promoter, such as GALI, GAL2, GAL7, GAL10, CUPL, MET3,

MET17, or MET25, may be used. Inducible promoters operably link the expression of a target gene (e.g., the nucleic acid sequence encoding a bakuchiol-producing protein) to a specific signal or a particular biotic or abiotic factor. Types of inducible promoters that may be utilized in the disclosed include, but are not limited to, chemically-inducible promoters (i.e., antibiotics, steroids, metals, etc.), light-inducible promoters, heat-inducible promoters, and hypoxia- inducible promoters. Transcription terminators that may be used are also known in the art {see

Bittner et al., Methods in Enzymol. 153:516-544 (1987)), and include but are not limited to

GAT2, Rho-dependent terminators, Rho-independent terminators, poly-A sequences, and the like (see Curran et al., Metab. Eng., 19:88-97 (2013)).

[0060] For the purposes of the present disclosure, any of the foregoing proteins can be expressed in a host cell or transgenic cell and any of the foregoing nucleic acids may incorporated into a host cell or transgenic cell in order to produce bakuchiol according to the disclosed methods.

IV. Host Cells and Transgenic Cells

[0061] Bioproduction of bakuchiol can rely on a host cell that expresses a bakuchiol-producing protein as disclosed herein or a transgenic cell that expresses a bakuchiol-producing protein as disclosed herein. A host cell may or may not natively express the bakuchiol-producing protein. A transgenic cell does not natively express the bakuchiol-producing protein.

[0062] The present disclosure provides an isolated host cell or a transgenic cell that expresses any of the disclosed bakuchiol-producing proteins. In one aspect, the present disclosure provides a transgenic cell that comprises a transgene encoding any of the disclosed bakuchiol-producing proteins. In some implementations, the isolated host cell or transgenic cell may comprise express a bakuchiol-producing protein that has at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1. In some implementations, the isolated host cell or transgenic cell may comprise express a bakuchiol-producing protein that has at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with

SEQ ID NO: 2. In some implementations, the bakuchiol-producing protein expressed by the isolated host cell or transgenic cell may be share at least 90% identity with SEQ ID NO: 1 or

SEQ ID NO: 2. In some implementations, the bakuchiol-producing protein expressed by the isolated host cell or transgenic cell may be share at least 95% identity with SEQ ID NO: 1 or

SEQ ID NO: 2. In some implementations, the bakuchiol-producing protein expressed by the isolated host cell or transgenic cell may be share at least 99% identity with SEQ ID NO: 1 or

SEQ ID NO: 2. Thus, this disclosure contemplates and encompasses expression of proteins with varying degrees of sequence identity compared to SEQ ID NOs: 1 and 2, so long as the protein exhibits prenyltransferase activity, catalyzes the production of bakuchiol, or both.

[0063] In some implementations, an isolated host cell or transgenic cell of the present disclosure can express a bakuchiol-producing protein comprising SEQ ID NO: 1 (i.e, it is 361 amino acids or longer). In some implementations, an isolated host cell or transgenic cell of the present disclosure can express a bakuchiol-producing protein comprising 361 amino acids that have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1. In some implementations, an isolated host cell or transgenic cell of the present disclosure can express a bakuchiol-producing protein consisting of SEQ ID NO: 1. In some implementations, an isolated host cell or transgenic cell of the present disclosure can express a bakuchiol-producing protein consisting of 361 amino acids that have at least about 65% - eg, at least about 70%. at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID

NO: 1. In some implementations, an isolated host cell or transgenic cell of the present disclosure can express a bakuchiol-producing protein comprising about 365, about 370, about 375, about 380, about 385, about 390, about 395, about 400, about 405, about 410, about 415, about 420, about 425, about 430, about 435, about 440, or about 450 amino acids, wherein at least about 361 amino acids of the protein have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1. Varying degrees of sequence identity and coverage are acceptable, so long as the protein exhibits prenyltransferase activity, catalyzes the production of bakuchiol, or both.

[0064] In some implementations, an isolated host cell or transgenic cell of the present disclosure can express a bakuchiol-producing protein comprising SEQ ID NO: 2 (i.e., it is 409 amino acids or longer). In some implementations, an isolated host cell or transgenic cell of the present disclosure can express a bakuchiol-producing protein comprising 409 amino acids that have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about

IO 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 2. In some implementations, an isolated host cell or transgenic cell of the present disclosure can express a bakuchiol-producing protein consisting of SEQ ID NO: 2. In some implementations, an isolated host cell or transgenic cell of the present disclosure can express a bakuchiol-producing protein consisting of 409 amino acids that have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID

NO: 2. In some implementations, an isolated host cell or transgenic cell of the present disclosure can express a bakuchiol-producing protein comprising about 410, about 415, about 420, about 425, about 430, about 435, about 440, or about 450 amino acids, wherein at least about 409 amino acids of the protein have at least about 65% - e.g., at least about 70%, at least about 75% atleast about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 2. Varying degrees of sequence identity and coverage are acceptable, so long as the protein exhibits prenyltransferase activity, catalyzes the production of bakuchiol, or both.

[0065] In some implementations, an isolated host cell or transgenic cell of the present disclosure can express a bakuchiol-producing protein that is shorter than SEQ ID NO: 1 or SEQ ID NO: 2.

For example, the expressed bakuchiol-producing protein may be less than 409 or less than 361 amino acids in length, so long as the protein has a catalytic domain that has at least about 65% - eg, at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID

NO: 1 or SEQ ID NO: 2. In some implementations at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, or at least about 350 amino acids of the protein can have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1 or SEQ ID NO: 2. Varying degrees of sequence identity and coverage are acceptable, so long as the protein exhibits prenyltransferase activity, catalyzes the production of bakuchiol, or both.

[0066] Various prokaryotic and eukaryotic expression systems are commonly used for bioproduction, though factors including the growth conditions, type of fermenter utilized, toxicity (if any) of the product. and other metabolic considerations of the microbe producing the product of interest may be employed to select a suitable system. Thus, in some implementations, a host cell or a transgenic cell suitable for expressing the disclosed bakuchiol-producing proteins may be a prokaryote. In in some implementations, a host cell or a transgenic cell suitable for expressing the disclosed bakuchiol-producing proteins may be a eukaryote.

[0067] In some implementations, the isolated host cell or transgenic cell is a prokaryote. Model prokaryotic systems that may be utilized as a transgenic cell include but are not limited to

Escherichia coli (E. coli), an Acinetobacter species, a Pseudomonas species, a Streptomyces species, and a Mycobacterium species. Additional suitable prokaryotic expression systems include, but are not limited to, Klebsiella, Lactococcus, Mannheimia, Corynebacterium, Vibrio, and Bacillis.

[0068] In some implementations, the isolated host cell or transgenic cell is a eukaryote. Model eukaryotic systems that may be utilized as a transgenic cell include but are not limited to

Saccharomyces cerevisiae (S. cerevisiae) or other yeast species; a filamentous fungi, optionally selected from an Aspergillus species and a Trichoderma species; an algae, optionally selected from Botryococcus braunii, Chlorella sp., Crypthecodinium cohnii, Cylindrotheca sp, Nitzschia sp. Phaeodactylum tricornutum, Schizochytrium sp., and Tetraselmis suecia; and an amoeba, which is optionally Dictyostelium discoideum. Additional suitable eukaryotic expression systems include, but are not limited to, Pichia pastoris, Yarrowia lipolvtica, Kluyveromyces marxianus,

Rhodosporidium toruloides. Aspergillus (oryzae, nidulans, niger), Trichoderma reesei, and

Penicillium chrysogenum.

[0069] In some implementations, for the isolated host cells and transgenic cells of the present disclosure bakuchiol 1s produced when the cell is cultured in the presence of p-coumaric acid and (1) geranyl pyrophosphate (GPP), (ii) dimethylallyl pyrophosphate (DMAPP), (iii) isopentenyl pyrophosphate (IPP), or any combination of (i)-(iii) thereof. The amount of bakuchiol produced may vary. For example, an isolated host cell or a transgenic cell of the present disclosure may produce at least about 0.1 ug/L - e.g., at least about 0.2 pg/L, at least about 0.3 pg/L, at least about 0.4 ug/L, at least about 0.5 pg/L, at least about 0.6 pg/L, at least about 0.7 pg/L, at least about 0.8 pg/L, at least about 0.9 ug/L, at least about 1.0 pg/L, at least about 1.1 pg/L, at least about 1.2 pg/L, at least about 1.3 ug/L, at least about 1.4 pg/L, at least about 1.5 pg/L, at least about 1.6 ug/L, at least about 1.7 pg/L, at least about 1.8 pg/L, at least about 1.9 pg/L, at least about 2.0 pg/L, at least about 2.1 ng/L, at least about 2.2 pg/L, at least about 2.3 ug/L, at least about 2.4 ng/L, at least about 2.5 pg/L, or more, of bakuchiol within at least about 48 hours of culture. Longer or shorter periods of culture time are also possible. For the purposes of the disclosed compositions and methods, it is understood that in some implementations p-coumaric acid, GPP, DMAPP, IPP, or all or a combination thereof may be produced endogenously by the host cell or transgenic cell, and do not require exogenous addition into, for example, the cell culture medium. In some implementations, exogenous p-coumaric acid, GPP, DMAPP, IPP, or all or a combination thereof may be added to the culture medium.

[0070] As noted above, in implementations involving a transgenic cell (e.g., S. cerevisiae or E. coli), the transgenic cell will comprise a transgene encoding the bakuchiol-producing protein, and the transgene can be integrated into the transgenic cell’s genome. The transgene may be integrated within an expression cassette that appropriately drives expression of the bakuchiol- producing protein. For those implementations in which genome integration of the transgene is preferred or desired, known methods of integration can be used, including but not limited to Cas- based systems (e.g., Cas9, Cas12, etc.), homologous recombination, gene gun, conjugation protocols, lambda red, etc. Alternatively, in some implementations, the transgene may not be integrated into the genome, and instead may express the bakuchiol-producing protein from, for example, a plasmid or similar vector.

[0071] An expression cassette or vector for expressing the transgene may comprise a promoter and a terminator. Suitable promoters that can be used may include but are not limited to GALI,

TEF2, TEFL, TDH3, ENO2, CCW12, EF-1a promoter, CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-1. In some implementations, the promoter is GAL1. In some implementations, an inducible or repressible promoter, such as GAL1, GAL2, GAL7, GAL10, CUP1, MET3, MET17, or MET25, may be used. Inducible promoters operably link the expression of a target gene (e.g., the nucleic acid sequence encoding a bakuchiol-producing protein) to a specific signal or a particular biotic or abiotic factor. Types of inducible promoters that may be utilized in the disclosed include, but are not limited to, chemically-inducible promoters (i.e. antibiotics, steroids, metals, etc.), light- inducible promoters, heat-inducible promoters, and hypoxia-inducible promoters. Transcription terminators that may be used are also known in the art (see Bittner et al., Methods in Enzymol. 153:516-544 (1987)), and include but are not limited to GAT2, Rho-dependent terminators, Rho- independent terminators, poly-A sequences, and the like. In some implementations, the terminator is GAT2.

V. Methods of Bioproduction and Batches Produced Therefrom

[0072] The identification, isolation, and characterization of previously unknown bakuchiol- producing prenyltransterase enzymes allows, for the first time, methods of bioproduction of bakuchiol. Thus, the present disclosure provides methods of producing bakuchiol, comprising culturing an isolated host cell or a transgenic cell disclosed herein in a culture medium and in the presence of p-coumaric acid and geranyl pyrophosphate (GPP), dimethylallyl pyrophosphate (DMAPP), isopentenyl pyrophosphate (IPP), or any combination of GPP, DMAPP, and IPP. For the purposes of the disclosed methods, it is understood that in some implementations p-coumaric acid, GPP, DMAPP, IPP, or all or a combination thereof may be produced endogenously by the host cell or transgenic cell, and do not require exogenous addition into, for example, the cell culture medium. In some implementations, exogenous p-coumaric acid, GPP, DMAPP, IPP, or all or a combination thereof may be added to the culture medium.

[0073] In some implementations, the methods comprise culturing a transgenic cell (e.g., S. cerevisiae or E. coli) comprising a transgene that encodes a bakuchiol-producing protein that has at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at

IO least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1; or at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 2. In some implementations, the bakuchiol-producing protein expressed by the transgenic cell may be share at least 90% identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some implementations, the bakuchiol-producing protein expressed by the transgenic cell may be share at least 95% identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some implementations, the bakuchiol-producing protein expressed by the transgenic cell may be share at least 99% identity with SEQ ID NO: 1 or SEQ ID NO: 2. Thus, the protein may possess varying degrees of sequence identity compared to SEQ ID NOs: 1 and 2, so long as the protein exhibits prenyltransferase activity, catalyzes the production of bakuchiol, or both.

[0074] In some implementations, the methods comprise culturing a transgenic cell comprising a transgene encoding a bakuchiol-producing protein comprising SEQ ID NO: 1 (i.e, it is 361 amino acids or longer). In some implementations, the methods comprise culturing a transgenic cell comprising a transgene encoding a bakuchiol-producing protein comprising 361 amino acids that have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%. at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1. In some implementations, the methods comprise culturing a transgenic cell comprising a transgene encoding a bakuchiol-producing protein consisting of

SEQ ID NO: 1. In some implementations, the methods comprise culturing a transgenic cell comprising a transgene encoding a bakuchiol-producing protein consisting of 361 amino acids that have at least about 65% - eg, at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 949%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1. In some implementations, the methods comprise culturing a transgenic cell comprising a transgene encoding a bakuchiol-producing protein comprising about 365, about 370, about 375, about 380, about 385, about 390, about 395, about 400, about 403, about 410, about 415, about 420, about 425, about 430, about 435, about 440, or about 450 amino acids, wherein at least about 361 amino acids of the protein have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1.

Varying degrees of sequence identity and coverage may be employed, so long as the protein exhibits prenyltransferase activity, catalyzes the production of bakuchiol, or both.

[0075] In some implementations, the methods comprise culturing a transgenic cell comprising a transgene encoding a bakuchiol-producmg protein comprising SEQ ID NO: 2 (i.e, it is 409 amino acids or longer). In some implementations, the methods comprise culturing a transgenic cell comprising a transgene encoding a bakuchiol-producing protein comprising 409 amino acids that have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 949%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 2. In some implementations, the methods comprise culturing a transgenic cell comprising a transgene encoding a bakuchiol-producing protein consisting of

SEQ ID NO: 2. In some implementations, the methods comprise culturing a transgenic cell comprising a transgene encoding a bakuchiol-producing protein consisting of 409 amino acids that have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%. at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 2. In some implementations, the methods comprise culturing a transgenic cell comprising a transgene encoding a bakuchiol-producing protein comprising about 410, about 415, about 420, about 425, about 430, about 435, about 440, or about 450 amino acids, wherein at least about 409 amino acids of the protein have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%. or any values in between any of the two aforementioned values, identity with SEQ ID NO: 2. Varying degrees of sequence identity and coverage may be employed, so long as the protein exhibits prenyltransferase activity, catalyzes the production of bakuchiol, or both.

[0076] In some implementations, the methods comprise culturing a transgenic cell comprising a transgene encoding a bakuchiol-producmg protein that is shorter than SEQ ID NO: 1 or SEQ ID

NO: 2. For example, the expressed bakuchiol-producing protein may be less than 409 or less than 361 amino acids in length, so long as the protein has a catalytic domain that has at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with

SEQ ID NO: 1 or SEQ ID NO: 2. In some implementations at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, or at least about 350 amino acids of the protein can have at least about 65% - e.g., at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%. at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, or any values in between any of the two aforementioned values, identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0077] Various prokaryotic and eukaryotic expression systems can be utilized for the disclosed methods. In some implementations, the transgenic cell used in the methods may be a prokaryote, including but are not limited to Escherichia coli (E. coli), an Acinetobacter species, a

Pseudomonas species, a Streptomyces species, and a Mycobacterium species. Additionally suitable prokaryotic expression systems include, but are not limited to, Klebsiella, Lactococcus,

Mannheimia, Corynebacterium, Vibrio, and Bacillis. In in some implementations, the transgenic cell used in the methods may be a eukaryote, including but are not limited to Saccharomyces cerevisiae (S. cerevisiae) or other yeast species; a filamentous fungi, optionally selected from an

Aspergillus species and a Trichoderma species; an algae, optionally selected from Botryococcus braunii, Chlorella sp., Crypthecodinium cohnii, Cylindrotheca sp., Nitzschia sp., Phaeodactylum tricornutum, Schizochytrium sp., and Tetraselmis suecia; and an amoeba, which is optionally

Dictyostelium discoideum. Additional suitable eukaryotic expression systems include, but are not limited to, Pichia pastoris, Yarrowia lipolytica, Kluyveromyces marxianus, Rhodosporidium toruloides. Aspergillus (oryzae, nidulans, niger), Trichoderma reesei, and Penicillium chrysogenum.

[0078] The disclosed methods can be carried out in a bioproduction reactor, fermentation tank, culture flask, or other suitable containers for bioproduction. Various different culture mediums can be selected based on the particular transgenic species used and the growth conditions, among other things. In some implementations, minimal culture medium may be supplemented as needed to optimize growth and production of a given transgenic cell type. For example, in some implementations, such as those utilizing transgenic S. cerevisiae, the culture medium may comprise about 3% w/v maltodexrin, about 0.2% w/v glucose, alpha-amylase, or any combination thereof.

[0079] As discussed above, and without being bound by any particular theory, it is believed that bioproduction of bakuchiol is catalyzed through a mechanism involved p-coumaric acid and geranyl pyrophosphate (GPP), dimethylallyl pyrophosphate (DMAPP), isopentenyl pyrophosphate (IPP), or any combination of GPP, DMAPP, and IPP. Thus, in some implementations the culture medium used for the disclosed methods may optionally include some p-coumaric acid to supplement that which is endogenously produced by a given transgenic cell or host cell. Indeed, In some implementations p-coumaric acid may be produced endogenously by the host cell or transgenic cell and the culture medium is not supplemented. In some implementations, the culture medium may comprise at least about 1.50 mM p-coumaric acid - e.g. at least about 1.75 mM p-coumaric acid, at least about 2.00 p-coumaric acid, at least about 2.25 mM p-coumaric acid, at least about 2.50 mM p-coumaric acid, at least about 2.75 mM p-coumaric acid, at least about 3.00 p-coumaric acid, at least about 3.25 mM p-coumaric acid, at least about 3.50 mM p-coumaric acid, at least about 3.75 mM p-coumaric acid, at least about 4.00 p-coumaric acid, or more.

[0080] The disclosed methods are the first to achieve production of bakuchiol in by a transgenic organism. These methods of bioproduction may be further optimized and developed to increase yield. For example, in some implementations, the disclosed methods may produce at least about 0.1 pg/L, at least about 0.2 pg/L, at least about 0.3 pg/L, at least about 0.4 pg/L, at least about 0.5 ug/L, at least about 0.6 ng/L, at least about 0.7 ug/L, at least about 0.8 ug/L, at least about 09 pg/L, at least about 1.0 pg/L, at least about 1.1 pg/L, at least about 1.2 pg/L, at least about 1.3 pg/L, at least about 1.4 ug/L, at least about 1.5 pg/L, at least about 1.6 pg/L, at least about 1.7 ug/L, at least about 1.8 ug/L, at least about 1.9 pg/L, at least about 2.0 pg/L, at least about 2.1 ug/L, at least about 2.2 ng/L, at least about 2.3 pg/L, at least about 2.4 pg/L, at least about 2.5 pg/L, at least about 3.0 pg/L, at least about 3.5 ug/L, at least about 4.0 ug/L, at least about 4.5 ng/L, at least about 5.0 ug/L, at least about 5.5 pg/L, at least about 6.0 pg/L, at least about 6.5 pg/L, at least about 7.0 ug/L, at least about 7.5 pg/L, at least about 8.0 pg/L, at least about 8.5 ug/L, at least about 9.0 ug/L, at least about 9.5 pg/L, at least about 10.0 pg/L, at least about 20 pg/L, at least about 30 pg/L, at least about 40 ug/L, at least about 50 pg/L, at least about 75 pg/L, at least about 100 pg/L, or more of bakuchiol within at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 36 hours, or at least about 48 hours of culture. In some implementations, the disclosed methods may produce at least about 0.1 pg/L, at least about 0.2 ng/L, at least about 0.3 pg/L, at least about 0.4 pg/L, at least about 0.5 pg/L, at least about 0.6 pg/L, at least about 0.7 ug/L, at least about 0.8 pg/L, at least about 0.9 pg/L, at least about 1.0 pg/L, at least about 1.1 ug/L, at least about 1.2 pg/L, at least about 1.3 pg/L, at least about 1.4 ug/L, at least about 1.5 pg/L, at least about 1.6 pg/L, at least about 1.7 pg/L, at least about 1.8 pg/L, at least about 1.9 pg/L, at least about 2.0 pg/L, at least about

2.1 ng/L, at least about 2.2 pg/L, at least about 2.3 ug/L, at least about 2.4 ng/L, at least about 2.5 pg/L, at least about 3.0 ug/L, at least about 3.5 pg/L, at least about 4.0 pg/L, at least about 4.5 pg/L, at least about 5.0 ug/L, at least about 5.5 pg/L, at least about 6.0 ug/L, at least about 6.5 ug/L, at least about 7.0 ng/L, at least about 7.5 pg/L, at least about 8.0 ug/L, at least about 85 ug/L, at least about 9.0 ug/L, at least about 9.5 pg/L, at least about 10.0 pg/L, at least about 20 ug/L, at least about 30 pg/L, at least about 40 pg/L, at least about 50 ug/L, at least about 75 pg/L, at least about 100 ug/L, or more of bakuchiol within about 6 hours of culture or less, about 12 hours of culture or less, about 18 hours of culture or less, about 24 hours of culture or less, about 36 hours of culture or less, or about 48 hours of culture or less.

[0081] The disclosed methods are the first to provide a process of bioproducing bakuchiol in batches that can be used for commercial consumption. This, the present disclosure provides batches of bakuchiol produced by the methods disclosed herein. A bioproduction batch of bakuchiol may have a chemical purity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. or any values in between any of the two aforementioned values,, and no single impurity of greater than 1%, no greater than about 0.5%, or greater than about 0.1%. The level of impurities in a given batch of bakuchiol can be determined by high-performance liquid chromatography (HPLC) and other suitable techniques.

[0082] The physical state of the bakuchiol batch can vary as need and depending on the stage of the production process, and the disclosed batches may be solid or liquid. Liquid batches of bakuchiol may be in the form of a non-aqueous solution, such as an oil, an organic solvent, or an aqueous solution. The concentration of bakuchiol in a liquid batch (e.g., in an oil or aqueous solution) may be at least about 0.1 pg/L, at least about 0.2 ng/L, at least about 0.3 ug/L, at least about 0.4 pg/L, at least about 0.5 ug/L, at least about 0.6 pg/L, at least about 0.7 pg/L, at least about 0.8 pg/L, at least about 0.9 ug/L, at least about 1.0 pg/L, at least about 1.1 pg/L, at least about 1.2 pg/L, at least about 1.3 ng/L, at least about 1.4 pg/L, at least about 1.5 pg/L, at least about 1.6 ug/L, at least about 1.7 pg/L, at least about 1.8 ug/L, at least about 1.9 pg/L, at least about 2.0 pg/L, at least about 2.1 ug/L, at least about 2.2 ug/L, at least about 2.3 pg/L, at least about 2.4 pg/L, at least about 2.5 ug/L, at least about 3.0 pg/L, at least about 3.5 pg/L, at least about 4.0 ug/L, at least about 4.5 pg/L, at least about 5.0 pg/L, at least about 5.5 pg/L, at least about 6.0 ug/L, at least about 6.5 pg/L, at least about 7.0 pg/L, at least about 7.5 pg/L, at least about 8.0 ug/L, at least about 8.5 ug/L, at least about 9.0 pg/L, at least about 9.5 pg/L, at least about 10.0 ug/L, at least about 20 ug/L, at least about 30 pg/L, at least about 40 pg/L, at least about 50 pg/L, at least about 75 pg/L, at least about 100 pg/L, or more.

VL Detection and Quantitation of Bakuchiol

[0083] The present disclosure provides methods of detecting bakuchiol and methods of quantifying bakuchiol using analytic techniques, including mass spectrometry. These methods may be useful for quality control of bakuchiol production by the disclosed bioproduction methods and any other known techniques of bakuchiol synthesis, extraction, or isolation.

[0084] In one implementation, bakuchiol can be detected by liquid chromatography mass spectrometry (LCMS) using, for example, an Agilent 1290 UHPLC and a 6460 triple-quadrupole mass spectrometer. Quantitation and compound identity can be determined by using an external standard curve of an authentic sample of bakuchiol.

[0085] Aqueous samples of bakuchiol can be diluted with isopropyl alcohol. In one implementation, the additional of isopropyl alcohol is not a purification process, per se, and the sample remains a 1-phase solution. However, the isopropyl alcohol may be extracting bakuchiol from hydrophobic surfaces such as lab ware and cellular membranes. The isopropyl alcohol may also help to clean the sample by precipitating proteins and other interfering material.

[0086] Beyond the addition of isopropyl alcohol, additional optional preparation processes include, but are not limited to extracting bakuchiol from the sample and centrifuging the sample to obtain a bakuchiol-containing supernatant.

[0087] Samples can be separated on a Waters BEH 50 mm x 2.1 mM column, heated to 70°C, using water and acetonitrile mobile phases with a flow rate of 0.5 mL/min. The gradient may comprise of the following: 0 minutes 0%B, 1 minutes 99% B, 2 minutes 99% B, and 2.1 minutes 0% B. The gradient can utilize a linear ramp for transitions, and the process can be about 3 minutes long —e.g., about 2 minutes, about 2.5 minutes about 3 minutes, about 3.5 minutes, or about 4 minutes. A specific MRM can be used to detect bakuchiol in the mass spectrometry with an ESI source in the negative ion mode: Parent 255.2 m/z (unit), Product 172.1 m/z, Fragmenter 120V, Collision Energy 20 V, Cell Accelerator Voltage 5V with a 300 ms dwell time. Optical detection can also conducted at 260 nm with a 0.5 s response time.

[0088] Beyond this implementation, the present disclosure provides methods for determining an amount of bakuchiol in a sample by mass spectrometry, the method comprising: (1) ionizing bakuchiol from the sample to generate one or more ions detectable by mass spectrometry; (ii) determining an amount of bakuchiol ions by multiple reaction or high resolution accurate mass mass spectrometry; and (iii) relating the amount of bakuchiol 10ns to the amount of bakuchiol 1n the sample, wherein a limit of detection of the method for bakuchiol is between about 0.001 pg/L and 0.0001 pg/L.

[0089] Various methods of ionization are known and can be utilized. For example, ionizing can comprise atmospheric pressure chemical ionization (APCI), electrospray ionization (ESD), or, if paired with gas chromatography, electron impact (EI) ionization Both APCI and ESI can be performed in negative ionization mode or positive ionization mode.

[0090] In some implementations, when using ESI in negative ion mode, the one or more ions (e.g., daughter ions after collision activation) may comprise an ion with a mass to charge ratio (m/z) of 172.1+0.5.

[0091] Prior to ionization, various methods of chromatography can be performs to isolate the bakuchiol and increase the sensitivity and selectivity of the mass spectroscopy. The chromatography may be liquid chromatography (LC) or gas chromatography (GC).prior to ionizing, the sample is subjected to liquid chromatography. Exemplary forms of LC that can be utilized include, but are not limited to, high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), ultra high performance liquid chromatography (UHPLC), and supercritical fluid chromatography (SFC).

[0092] As discussed above, optional preparation processes that may be performed prior to ionizing, include diluting the sample with an alcohol (e.g., isopropyl alcohol), extracting the bakuchiol from the sample, centrifuging the sample to obtain the supernatant, or a combination thereof.

Non-Limiting Working Examples

[0093] The following examples are given to illustrate the present disclosure. It should be understood, however, that the disclosure is not to be limited to the specific conditions or details described in these examples.

L Example 1 - Identification of Bakuchiol-Producing Proteins and Bioproduction in

Yeast

[0094] To identify bakuchiol-producing enzymes, a transcriptome analysis was performed on

RNA-SEQ datasets of known bakuchiol producers, Psoralea corylifolia, Piper Longum and

Ulmus davidiana. The transcriptomes were assembled and searched for prentyltranferase enzymes using both a homology-based and PFAM HMM-based approaches. The homology- based search used four candidate enzymes with known GPP or DM APP prenyltranferase activity. In addition, the three transcriptomes were searched for known terpene synthase containing motifs, DDxxD, NSE/DTE, and RRx(8)W.

[0095] To assemble the transcriptomes adaptors and low-quality bases were trimmed out at the ends from the fastq files using trim-galore (bioinformatics .babraham.ac.uk/projects/trim_galore).

Reads were assembled as described in Grabherr et al., Nature biotechnology, 2011, 2907), 644~ 652 (dot.org/ 10. 1038/nbt, 1883; 1.0, "Trinity”). Gene expression levels and assembly statistics were calculated as described in Haas et al, Nature protocols, 2013, (8), 1494-1512 {doi.org/10.1038/nprot.2013.084). The transcriptomes were assessed for quality, and open reading frames (ORFs) were identified and translated into protein sequences.

[0096] A homology sear was performed for candidate prenyltransferases using the following query candidates:

CIPT1_lemon A0A077K8G3jCGT1A_CITLI Coumarin 8-geranyltransferase 1, chloroplastic

OS=Citrus limon;

FuPT1 BOYAZ2IBOYAZ2_ASPFC Prenyltransferase FmPT1 OS=Neosartorya fumigata (strain CEA 10);

PGT1 Q8W405|[PGT1_LITER 4-hydroxybenzoate geranyltransferase 1 OS=Lithospermum erythrorhizon; and

OGT Morus alba oxyresveratrol geranyltransferase (OGT).

[0097] Profile based searches for PFAM families were performed using the methods described in

Wheeler et al., Bioinformatics, 2013, 29: 2487-2489. The PFAM families are listed below.

PF00432 (Prenyltransferase);

PF01239 (Protein prenyltransferase alpha subunit repeat);

PF01397 (Terpene synthase, N-terminal domain);

PF03936 (Terpene synthase family, metal binding domain);

PF06330 (Trichodiene synthase); and

PF19086 (Terpene synthase family 2, C-terminal metal binding).

[0098] Sequences matching “prenyl transferase” in their annotations in Uniprot database were also searched.

[0099] Blast searches were performed using e-value cutoff of 1E-30 and query coverage of 80%.

[0100] Hmm searches were performed using e-value cutoff of 1E-15.

[0101] All search hits were annotated using Blast against the public nr database.

Prenyltransferase hit results were trimmed to a total of 196 polypeptides, removing transcripts: under 200 amino acids in length, over 700 amino acids in length, close isoforms, 3" trancation or sequences without start codon and stop codons, and under 0.3 TPM (transcripts per million).

[0102] All putative prenyltranferase enzymes (referred to herein as “BAK genes”) were integrated into S. cerevisiae via standard LiAc chemical transformation methodologies using a

Casl2-based system for directed nuclease-guided genomic integration. The BAK genes were expressed from the GAL80 locus, driven by a GAL! promoter and GAT2 terminator.

[0103] Resulting strams were grown and assayed at 30°C in 96 mid-well plates with 3% w/v maltodextrin, 0.2% glucose defined medium {modified from Westfall 2012) with alpha-amylase for 24-48 hours, before transfer to the same medium with and 1.5-3mM p-Coumaric Acid for 48 hours.

[0104] Bakuchiol was detected by LCMS using an Agilent 1290 UHPLC and a 6460 triple- quadrupole mass spectrometer. Quantitation and compound identity were determined by using an external standard curve of an authentic sample of bakuchiol. Briefly, microfermentation samples were diluted with isopropyl alcohol, extracted, centrifuged, and then the supernatant was transferred into an appropriate vial or plate. Samples were separated on a Waters BEH 50 mm x 2.1 mM column, heated to 70°C, using water and acetonitrile mobile phases with a flow rate of 0.5 mL/min. The gradient consisted of the following steps: 0 minutes 0%B, 1 minutes 99% B, 2 minutes 99% B, and 2.1 minutes 0% B. The gradient used a linear ramp for all transitions, and the method was 3 minutes long. A specific MRM was used to detect bakuchiol in the mass spectrometry with an EST source in the negative ion mode: Parent 255.2 m/z (unit), Product 172.1 m/z, Fragmenter 120V, Collision Energy 20 V, Cell Accelerator Voltage 5V with a 300 ms dwell time. Optical detection was also conducted at 260 nm with a 0.5 s response time.

[0105] Two strains, expressing BAK28 (SEQ ID NO: 1) and BAK36 (SEQ ID NO: 2) showed specific production of Bakuchiol that was not detected in the other samples or in the negative control (FIG. 1 - top 2 lines). The putative bakuchiol peak retained shape across standard addition with the expected area change (Figure 1- third line) and showed the expected MS/MS spectra (FIG. I- right panel), further confirming the identity of the peak. The amount of bakuchiol produced was measured to be -lug/L for BAK28 and ~0.1ug/L for BAK36 (FIG. 2).

The addition of p-coumaric acid to the mediums was required for the formation of bakuchiol, further supporting the proposed mechanism (FIG. 3, zero p-coumaric acid). In addition, titration of p-coumaric acid showed that 3mM or just over 200mg/L doubled the production of bakuchiol under the current strain conditions (FIG. 3).

[0106] Sequence alignment and TOPCONS transmembrane analysis of the two polypeptides shows that BAK28 1s a truncation of BAK36 with two additional amino acid changes and that both are predicted to have several transmembrane regions (FIG. 4, FIG. 5; BAK SEQS tabl; topcons.cbr.su.se/pred/).

[0107] To further optimize the polypeptide sequence present in the three plant transcriptomes, the sequence of BAK36 was used to search for similar polypeptides using e-value cut off of le- 10. Unique hits from all transcriptomes were combined and annotated, using blast against the public nr database (but run locally). 178 hits were returned and all fifteen sequences with TPM levels above ten were ordered for further analysis (BAK SEQS tab2). Each of these fifteen sequences will be transduced into S. cerevisiae as described above and screened for bakuchiol production.

[0108] A flow chart summarizing the foregoing strain engineering is provided in FIG. 6. As shown in FIG. 7, Blast analysis of SEQ ID NOs: 1 and 2 (i.e., BAK28 and BAK36, respectively) had no close homologs in the NCBI database, indicated that the disclosed proteins have not been previously discovered. % ok kok ok

[0109] It should be appreciated that all combinations of the disclosed concepts are contemplated as being part of the inventive subject matter disclosed herein and may be employed in any combination to achieve the benefits described herein.

[0110] The present technology is not to be limited in terms of the particular implementations described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art.

Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. Itis also to be understood that the terminology used herein is for the purpose of describing particular implementations only, and is not intended to be limiting.

Clauses

I. A transgenic cell, comprising a transgene encoding a transgenic protein comprising an amino acid sequence with at least about 65% identity to:

MHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHH

TPETLWGSIKQFCDAFYRFSRPHVIGTAVNIVMSSLALEKSSDISPKFFIGLFQVI

VTILSMNIYTAGINQLTDIEIDKINKPYLPLASGEYSYKTGVTHTLCAILSLGVGWI

VGSPPLFWSNFAYFVLGTVYSIDLPLMRWKSHPALAALFFFVIRGLTFHVGFFLH

LQTHVFKRPMMIPKPVMFGTAFMSFFYVHAFFKDIPDIEGDKDHGVKSLTMRLG

QKRVFWICVSLLLTGYGAAIVVGATSSFLWCKLITVSGHALLASIFWNRAKSVDL

KSHQEITSLYMFMWKLFYAEYFIIPLMR (SEQ ID NO: 1; BAK28), or

MASMFLGSLPLASSVNYIGRITRSKNCTESYHATSYITNASSNKTEKIKHEYANM

RHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLWGSI

KQFCDAFYRFSRPHVHGTAVNIVMSSLALEKSSDISPKFFIGLFQVIVTILSMNIYT

AGINQLTDIEIDKINKPYLPLASGEYSYKTGVTUTLCAILSLGVGWIVGSPPLFWS

NFAYFVLGTVYSIDLPLMRWKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRP

MMIPKSVMFEGTAFMSFFY VIIAFFKDIPDIEGDKDHGVKSLTMRLGQERVFWICV

SLLLTGYGAAIVVGATSSFLWCKLITVSGHALLASIFWNRAKSVDLKSHQEITSLY

MFMWKLFYAEYFIPLMR (SEQ ID NO: 2; BAK30); wherein the transgenic protein catalyzes the production of bakuchiol, exhibits prenyltransferase activity, or both. 2. The transgenic cell of clause 1, wherein the transgenic protein comprises an amino acid sequence with at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 1 or SEQ ID NO: 2. 3. The transgenic cell of clause 1 or 2, wherein the transgenic cell is prokaryotic. 4. The transgenic cell of any one of clauses 1-3, wherein the transgenic cell is selected from

Escherichia coli (E. coli), an Acinetobacter species, a Pseudomonas species, a

Streptomyces species, and a Mycobacterium species. 5. The transgenic cell of clause 1 or 2, wherein the transgenic cell is eukaryotic.

6. The transgenic cell of any one of clauses 1, 2, or 5, wherein the transgenic cell is selected from Saccharomyces cerevisiae (S. cerevisiae) or other yeast species, a filamentous fungi, an algae, and an amoeba. 7. The transgenic cell of clause 6, wherein the filamentous fungi is selected from an

Aspergillus species and a Trichoderma species. 8. The transgenic cell of clause 6, wherein the amoeba is Dictyostelium discoideum. 9. The transgenic cell of clause 6, wherein the algae 1s selected from Botryococcus braunii,

Chlorella sp., Crypthecodinium cohnii, Cylindrotheca sp., Nitzschia sp., Phaeodactylum tricornutum, Schizochytrium sp., and Tetraselmis suecia. 10. The transgenic cell of any one of clauses 1-9, wherein bakuchiol is produced when the transgenic cell is cultured in presence of p-coumaric acid and (i) geranyl pyrophosphate (GPP), (ii) dimethylallyl pyrophosphate (DMAPP), (iii) isopentenyl pyrophosphate (IPP), or any combination of (1)-(iii). 11. The transgenic cell of any one of clauses 1-10, wherein the transgenic cell produces at least about 0.1 pg/L, at least about 0.2 pg/L, at least about 0.3 pg/L, at least about 0.4 pg/L, at least about 0.5 pg/L, at least about 0.6 pg/L, at least about 0.7 pg/L, at least about 0.8 pg/L, at least about 0.9 pg/L, at least about 1.0 pg/L, at least about 1.1 pg/L, at least about 1.2 pg/L, at least about 1.3 pg/L, at least about 1.4 pg/L, at least about 1.5 pg/L, at least about 1.6 pg/L, at least about 1.7 ug/L, at least about 1.8 ug/L, at least about 1.9 ug/L, at least about 2.0 ng/L, at least about 2.1 pg/L, at least about 2.2 ug/L, at least about 2.3 pg/L, at least about 2.4 pg/L, at least about 2.5 ug/L, or more of bakuchiol within at least about 48 hours when cultured in the presence of p-coumaric acid and (i) geranyl pyrophosphate (GPP), (ii) dimethylallyl pyrophosphate (DMAPP), (iii) isopentenyl pyrophosphate (IPP), or any combination of (i)-(iii). 12. The transgenic cell of any one of clauses 1-11, wherein the transgene is integrated into the transgenic cell’s genome. 13. The transgenic cell of any one of clauses 1-11, wherein the transgene is not integrated into the transgenic cell’s genome.

14. The transgenic cell of any one of clauses 1-13, wherein expression of the transgene is driven by a GAL 1 promoter. 15. The transgenic cell of any one of clauses 1-13, wherein expression of the transgene is driven by an inducible promoter. 16. The transgenic cell of any one of clauses 1-15, the transgenic protein has at least about 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2. 17. The transgenic cell of any one of clauses 1-16, wherein the transgenic protein comprises

SEQ ID NO: 1 or SEQ ID NO: 2. 18. The transgenic cell of any one of clauses 1-16, wherein the transgenic protein consists of

SEQ ID NO: 1 or SEQ ID NO: 2. 19. A method of producing bakuchiol, comprising culturing the transgenic cell according to one of clauses 1-18 in a culture medium and in the presence of p-coumaric acid and (i) geranyl pyrophosphate (GPP), (ii) dimethylallyl pyrophosphate (DMAPP), (iii) isopentenyl pyrophosphate (IPP), or any combination of (i)-(ii1). 20. The method of clause 19, wherein the culture medium further comprises about 3% w/v maltodexrin, about 0.2% w/v glucose, alpha-amylase, or any combination thereof. 21. The method of clause 19 or 20, wherein the culture medium comprises at least about 1.50 mM p-coumaric acid, at least about 1.75 mM p-coumaric acid, at least about 2.00 p- coumaric acid, at least about 2.25 mM p-coumaric acid, at least about 2.50 mM p- coumaric acid, at least about 2.75 mM p-coumaric acid, at least about 3.00 p-coumaric acid, at least about 3.25 mM p-coumaric acid, at least about 3.50 mM p-coumaric acid, at least about 3.75 mM p-coumaric acid, at least about 4.00 p-coumaric acid or more. 22. The method of any one of clauses 19-20, wherein the culture medium does not comprise exogenous p-coumaric acid, GPP, DMAPP, IPP, or any combination thereof. 23. The method of any one of clauses 19-22, wherein at least about 0.1 pg/L, at least about 0.2 pg/L, at least about 0.3 pg/L, at least about 0.4 pg/L, at least about 0.5 pg/L, at least about 0.6 ug/L, at least about 0.7 ng/L, at least about 0.8 pg/L, at least about 0.9 ug/L, at least about 1.0 pg/L, at least about 1.1 pg/L, at least about 1.2 pg/L, at least about 1.3 ug/L, at least about 1.4 pg/L, at least about 1.5 pg/L, at least about 1.6 pg/L, at least about 1.7 pg/L, at least about 1.8 pg/L, at least about 1.9 pg/L, at least about 2.0 pg/L, at least about 2.1 pg/L, at least about 2.2 ug/L, at least about 2.3 pg/L, at least about 2.4 pg/L, at least about 2.5 pg/L, or more of bakuchiol within at least about 48 hours of culture. 24 A bioproduction batch of bakuchiol, wherein the bakuchiol has a chemical purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 04%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, and no single impurity of greater than about 1%. 25. The bioproduction batch of bakuchiol of clause 24, wherein the bakuchiol is in an oil. 26. The bioproduction batch of bakuchiol of clause 24, wherein the bakuchiol is in an aqueous solution. 27. The bioproduction batch of bakuchiol of clause 25 or 26, wherein the concentration of bakuchiol is at least about 0.1 ug/L, at least about 0.2 ug/L, at least about 0.3 pg/L, at least about 0.4 pg/L, at least about 0.5 pg/L, at least about 0.6 pg/L, at least about 0.7 pg/L, at least about 0.8 pg/L, at least about 0.9 ug/L, at least about 1.0 pg/L, at least about 1.1 pg/L, at least about 1.2 pg/L, at least about 1.3 pg/L, at least about 1.4 pg/L, at least about 1.5 pg/L, at least about 1.6 pg/L, at least about 1.7 pg/L, at least about 1.8 ug/L, at least about 1.9 pg/L, at least about 2.0 pg/L, at least about 2.1 ug/L, at least about 2.2 ug/L, at least about 2.3 pg/L, at least about 2.4 ug/L, at least about 2.5 pg/L, or more. 28. The bioproduction batch of bakuchiol of any one of clauses 24-27, wherein the bakuchiol is produced by the method according to any one of clauses 19-23. 29. An isolated protein comprising an amino acid sequence with at least about 65% identity to:

MHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHH

TPETLWGSIKQFCDAFYRFSRPHVIGTAVNITVMSSLALEKSSDISPKFFIGLFQVI

VTILSMNIYTAGINQLTDIEIDKINKPYLPLASGEYSYKTGVTHTLCAILSLGVGWI

VGSPPLFWSNFAYFVLGTVYSIDLPLMRWKSHPALAALFFFVIRGLTFHVGFFLH

LQTHVFKRPMMIPKPVMFGTAFMSFFY VHAFFKDIPDIEGDKDHGVKSLTMRLG

QKRVFWICVSLLLTGYGAAIVVGATSSFLWCKLITVSGHALLASIFWNRAKSVDL

KSHQEITSLYMFMWKLFYAEYFIPLMR (SEQ ID NO: i; BAK28), or

MASMFLGSLPLASSVNYIGRITRSKNCTESYHATSYITNASSNKTEKIKHEYANM

RHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLWGSI

KQFCDAFYRFSRPHVIIGTAVNIIVMSSLALEKSSDISPKFFIGLFQVIVTILSMNIYT

AGINQLTDIEIDKINKPYLPLASGEYSYKTGVTUTLCAILSLGVGWIVGSPPLFWS

NFAYFVLGTVYSIDLPLMRWKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRP

MMIPKSVMFEGTAFMSFFY VIIAFFKDIPDIEGDKDHGVKSLTMRLGQERVFWICV

SLLLTGYGAAIVVGATSSFLWCKLITVSGHALLASIFWNRAKSVDLKSHQEITSLY

MFMWKLFYAEYFHPLMR (SEQ ID NO: 2; BAK36). 30. The isolated protein of clause 29, wherein the amino acid sequence comprises at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2. 31. The isolated protein of clause 29 or 30, wherein the amino acid sequence has at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2. 32. The isolated protein of clause 29, wherein the amino acid sequence comprises Or consists of SEQ ID NO: 1 or SEQ ID NO: 2. 33. The isolated protein of clause 29, wherein the amino acid sequence consists of SEQ ID

NO: 1 or SEQ ID NO: 2. 34. The isolated protein of any one of clauses 29-33, wherein the isolated protein exhibits prenyltransferase activity. 35. The isolated protein of any one of clauses 29-34, wherein the isolated protein catalyzes the production of bakuchiol. 36. A nucleic acid comprising a nucleic acid sequence encoding the isolated protein of any one of clauses 29-35. 37. An isolated host cell that produces the isolated protein of any one of clauses 29-35 or that comprises the nucleic acid of clause 36.

38. A bakuchiol-producing enzyme as disclosed herein. 39. A transgenic cell capable of producing bakuchiol as disclosed herein. 40. A method of producing bakuchiol as disclosed herein. 41. Use of an isolated protein according to any of the clauses 29 to 35 for the production of bakuchiol. 42, Use of a nucleic acid according to clause 36 for the production of bakuchiol. 43. Use of a transgenic cell according to any one of the clauses 1 to 18 for the production of bakuchiol. 44. Use of an isolated host cell according to clause 37 for the production of bakuchiol. 45. An expression cassette or vector for expressing the nucleic acid according to clause 36. 46. Bakuchiol obtainable by the method according to any one of the clauses 18 to 23. 47. A transgene encoding a transgenic protein comprising an amino acid sequence with at least about 65% identity to:

MHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHH

TPETLWGSIKQFCDAFYRFSRPHVHIGTAVNIVMSSLALEKSSDISPKFFIGLFQVI

VTILSMNIYTAGINQLTDIEIDKINKPYLPLASGEYSYKTGVTHTLCAILSLGVGWI

VGSPPLFWSNFAYFVLGTVYSIDLPLMRWKSHPALAALFFFVIRGLTFHVGFFLH

LQTHVFKRPMMIPKPVMFGTAFMSFFYVHAFFKDIPDIEGDKDHGVKSLTMRLG

QKRVFWICVSLLLTGYGAAIVVGATSSFLWCKLITVSGHALLASIFWNRAKSVDL

KSHQEITSLYMFMWKLFYAEYFIPLMR (SEQ ID NO: 1; BAK28), or

MASMFLGSLPLASSVNYIGRITRSKNCTESYHATSYITNASSNKTEKIKHEYANM

RHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLWGSI

KQFCDAFYRFSRPHVIIGTAVNIIVMSSLALEKSSDISPKFFIGLEQVIVTILSMNIYT

AGINQLTDIEIDKINKPYLPLASGEYSYKTGVTHTLCAILSLGVGWIVGSPPLFWS

NFAYFVLGTVYSIDLPLMRWKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRP

MMIPKSVMFGTAFMSFFY VIIAFFKDIPDIEGDKDHGVKSLTMRLGQERVFWICV

SLLLTGYGAAIVVGATSSFLWCKLITVSGHALLASIFWNRAKSVDLKSHQEITSLY

MFMWKLFYAEYFIIPLMR (SEQ ID NO: 2; BAK36);

wherein the transgenic protein catalyzes the production of bakuchiol, exhibits prenyltransferase activity, or both. 48. Use of a transgene according to clause 47 for integration of said transgene in a genome of a cell, wherein genome integration is preferably achieved by one or more methods selected from the group of Cas-based systems (e.g., Cas9, Casl2, etc.), homologous recombination, gene gun, conjugation protocols, and lambda red.

2031273SEQ.txtUSB

SEQUENCE LISTING

<110> Sestina Bio, LLC <120> BIOPRODUCTION OF BAKUCHIOL <130> P186782NL00 <160> 2 <170> BiSSAP 1.3.6 <210> 1 <211> 361 <212> PRT <213> Artificial Sequence <220> <223> BAK28 <400> 1

Met His Glu Tyr Ala Asn Met Arg His Arg Gln His Asn Leu Lys His 1 5 10 15

Asn Tyr Gly Gly Ile Glu Gly Val Ser Thr Cys Glu Asp Trp Ala Arg

Asn Phe Val Val Asn Ala Ala Ser Gly Glu Ser Leu Glu Ser His Glu

Ala Gln His His Thr Pro Glu Thr Leu Trp Gly Ser Ile Lys Gln Phe 60

Cys Asp Ala Phe Tyr Arg Phe Ser Arg Pro His Val Ile Ile Gly Thr 65 70 75 80

Ala Val Asn Ile Ile Val Met Ser Ser Leu Ala Leu Glu Lys Ser Ser 85 90 95

Asp Ile Ser Pro Lys Phe Phe Ile Gly Leu Phe Gln Val Ile Val Thr 100 105 110

Ile Leu Ser Met Asn Ile Tyr Thr Ala Gly Ile Asn Gln Leu Thr Asp 115 120 125

Ile Glu Ile Asp Lys Ile Asn Lys Pro Tyr Leu Pro Leu Ala Ser Gly 130 135 140

Glu Tyr Ser Tyr Lys Thr Gly Val Thr Ile Ile Thr Leu Cys Ala Ile 145 150 155 160

Leu Ser Leu Gly Val Gly Trp Ile Val Gly Ser Pro Pro Leu Phe Trp 165 170 175

Ser Asn Phe Ala Tyr Phe Val Leu Gly Thr Val Tyr Ser Ile Asp Leu 180 185 190

Pro Leu Met Arg Trp Lys Ser His Pro Ala Leu Ala Ala Leu Phe Phe 195 200 205

Phe Val Ile Arg Gly Leu Thr Phe His Val Gly Phe Phe Leu His Leu 210 215 220

Gln Thr His Val Phe Lys Arg Pro Met Met Ile Pro Lys Pro Val Met 225 230 235 240

Pagina 1

2031273SEQ.txtUSB

Phe Gly Thr Ala Phe Met Ser Phe Phe Tyr Val Ile Ile Ala Phe Phe 245 250 255

Lys Asp Ile Pro Asp Ile Glu Gly Asp Lys Asp His Gly Val Lys Ser 260 265 270

Leu Thr Met Arg Leu Gly Gln Lys Arg Val Phe Trp Ile Cys Val Ser 275 280 285

Leu Leu Leu Thr Gly Tyr Gly Ala Ala Ile Val Val Gly Ala Thr Ser 290 295 300

Ser Phe Leu Trp Cys Lys Leu Ile Thr Val Ser Gly His Ala Leu Leu 305 310 315 320

Ala Ser Ile Phe Trp Asn Arg Ala Lys Ser Val Asp Leu Lys Ser His 325 330 335

Gln Glu Ile Thr Ser Leu Tyr Met Phe Met Trp Lys Leu Phe Tyr Ala 340 345 350

Glu Tyr Phe Ile Ile Pro Leu Met Arg 355 360 <210> 2 <211> 409 <212> PRT <213> Artificial Sequence <220> <223> BAK36 <400> 2

Met Ala Ser Met Phe Leu Gly Ser Leu Pro Leu Ala Ser Ser Val Asn 1 5 10 15

Tyr Ile Gly Arg Ile Thr Arg Ser Lys Asn Cys Thr Glu Ser Tyr His

Ala Thr Ser Tyr Ile Thr Asn Ala Ser Ser Asn Lys Thr Glu Lys Ile

Lys His Glu Tyr Ala Asn Met Arg His Arg Gln His Asn Leu Lys His 60

Asn Tyr Gly Gly Ile Glu Gly Val Ser Thr Cys Glu Asp Trp Ala Arg 65 70 75 80

Asn Phe Val Val Asn Ala Ala Ser Gly Glu Ser Leu Glu Ser His Glu 85 90 95

Ala Gln His His Thr Pro Glu Thr Leu Trp Gly Ser Ile Lys Gln Phe 100 105 110

Cys Asp Ala Phe Tyr Arg Phe Ser Arg Pro His Val Ile Ile Gly Thr 115 120 125

Ala Val Asn Ile Ile Val Met Ser Ser Leu Ala Leu Glu Lys Ser Ser 130 135 140

Asp Ile Ser Pro Lys Phe Phe Ile Gly Leu Phe Gln Val Ile Val Thr 145 150 155 160

Ile Leu Ser Met Asn Ile Tyr Thr Ala Gly Ile Asn Gln Leu Thr Asp 165 170 175

Ile Glu Ile Asp Lys Ile Asn Lys Pro Tyr Leu Pro Leu Ala Ser Gly 180 185 190

Glu Tyr Ser Tyr Lys Thr Gly Val Thr Ile Ile Thr Leu Cys Ala Ile

Pagina 2

2031273SEQ.txtUSB 195 200 205

Leu Ser Leu Gly Val Gly Trp Ile Val Gly Ser Pro Pro Leu Phe Trp 210 215 220

Ser Asn Phe Ala Tyr Phe Val Leu Gly Thr Val Tyr Ser Ile Asp Leu 225 230 235 240

Pro Leu Met Arg Trp Lys Ser His Pro Ala Leu Ala Ala Leu Phe Phe 245 250 255

Phe Val Ile Arg Gly Leu Thr Phe His Val Gly Phe Phe Leu His Leu 260 265 270

Gln Thr His Val Phe Lys Arg Pro Met Met Ile Pro Lys Ser Val Met 275 280 285

Phe Gly Thr Ala Phe Met Ser Phe Phe Tyr Val Ile Ile Ala Phe Phe 290 295 300

Lys Asp Ile Pro Asp Ile Glu Gly Asp Lys Asp His Gly Val Lys Ser 305 310 315 320

Leu Thr Met Arg Leu Gly Gln Glu Arg Val Phe Trp Ile Cys Val Ser 325 330 335

Leu Leu Leu Thr Gly Tyr Gly Ala Ala Ile Val Val Gly Ala Thr Ser 340 345 350

Ser Phe Leu Trp Cys Lys Leu Ile Thr Val Ser Gly His Ala Leu Leu 355 360 365

Ala Ser Ile Phe Trp Asn Arg Ala Lys Ser Val Asp Leu Lys Ser His 370 375 380

Gln Glu Ile Thr Ser Leu Tyr Met Phe Met Trp Lys Leu Phe Tyr Ala 385 390 395 400

Glu Tyr Phe Ile Ile Pro Leu Met Arg 405

Pagina 3

Claims

Conclusions

I. Transgenic cell containing a transgene encoding a transgenic protein comprising an amino acid sequence with at least approximately 65% identity to: MHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTP ETLWGSIKQFCDAFYRFSRPHVIIGTAVNIIVMSSLALEKSSDISPKFFIGLFQVIVTILSMNI YTAGINQLTDIEIDKINKPYLPLASGEYSY KTGVTHTLCAILSLGVGWIVGSPPLFWSNFA YFVLGTVYSIDLPLMRWKSHPALAALFFFVIRGLTFHV GFFLHLQTHVFKRPMMIPKPV MFGTAFMSFFY VIIAFFKDIPDIEGDKDHGVKSLTMRLGQKRVFWICVSLLLTGYGAAIV VGATSSFLWCKLITVSGHALLASIFWNRAKSVDLKSHQEITSLYMFMWKLFY AEYFITPL MR (SEQ ID NO: 1; BAK28) FYRFSRPHVIIGTAVNIIVMSSLALEKSSDISPKFFIGLFQVIVTILSMNIYTAGINQLTDIEID KINKPYLPLASGEYSYKTGVTHTLCAILSLGVGWIVGSPPLFWSNFAYFVLGTVYSIDLPL MRWKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRPMMIPKSVMFGTAFMSFFY VIIA FFKDIPDIEGDKDHGVKSLTMRL GQERVFWICVSLLLTGYGAAIVVGATSSFLWCKLITV SGHALLASIFWNRAKSVDLKSHQEITSLYMFMWKLFYAEYFIIPLMR (SEQ ID NO: 2; BAK36); wherein the transgenic protein catalyzes bakuchiol production, exhibits prenyltransferase activity, or both.

The transgenic cell of claim 1, wherein the transgenic protein comprises an amino acid sequence of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2.

A transgenic cell according to claim 1 or 2, wherein the transgenic cell is prokaryotic.

Transgenic cell according to any one of claims 1-3, wherein the transgenic cell is selected from Escherichia coli (E. coli), an Acinetobacter species, a Pseudomonas species, a Streptomyces species and a Mycobacterium species.

The transgenic cell of claim 1 or 2, wherein the transgenic cell is eukaryotic.

Transgenic cell according to any one of claims 1, 2 or 5, wherein the transgenic cell is selected from Saccharomyces cerevisiae (S. cerevisiae) or other yeast species, a filamentous fungus, an alga and an amoeba.

7. Transgenic cell according to claim 6, wherein the filamentous fungi is selected from an Aspergillus species and a Trichoderma species.

The transgenic cell of claim 6, wherein the amoeba is Dictyostelium discoideum.

Transgenic cell according to claim 6, wherein the algae is selected from Botryococcus braunii, Chlorella sp., Crypthecodinium cohnit, Cylindrotheca sp., Nitzschia sp., Phaeodactylum tricornutum, Schizochytrium sp. and Tetraselmis suecia.

A transgenic cell according to any one of claims 1 to 9, wherein bakuchiol is produced when the transgenic cell is grown in the presence of p-coumaric acid and (i) geranyl pyrophosphate (GPP), (ii) dimethylallyl pyrophosphate (DMAPP), (iii) isopentenyl pyrophosphate (IPP), or any combination of (i)-{iii).

The transgenic cell of any one of claims 1 to 10, wherein the transgenic cell is at least about 0.1 pg/L, at least about 0.2 pg/L. produces at least about 0.3 pg/L, at least about 0.4 pg/L. , at least about 0.5 pg/L, at least about 0.6 ug/L, at least about 0.7 pg/L, at least about 0.8 pg/L, at least about 0.9 ug/L , at least about 1.0 pg/L, at least about 1.1 pg/L, at least about 1.2 pg/L, at least about 1.3 ug/L, at least about 1.4 pg/ L, at least about 1.5 pg/L, at least about 1.6 pg/L, at least about 1.7 ug/L, at least about 1.8 ug/L, at least about 1.9 ng/ L, at least about 2.0 pg/L, at least about 2.1 ug/L, at least about 2.2 pg/L, at least about 2.3 ug/L, at least about 2.4 ug/ L, at least about 2.5 µg/L, or more bakuchiol within at least about 48 hours when grown in the presence of p-coumaric acid and (i) geranyl pyrophostat (GPP), { i1) dimethylallyl pyrophosphate (DMAPP), (iii) isopentenyl pyrophosphate (IPP), or any combination of (1)-(iii).

A transgenic cell according to any one of claims 1 to 11, wherein the transgene is integrated into the genome of the transgenic cell.

A transgenic cell according to any one of claims 1 to 11, wherein the transgene is not integrated into the genome of the transgenic cell.

A transgenic cell according to any one of claims 1 to 13, wherein expression of the transgene is driven by a GAL1 promoter.

A transgenic cell according to any one of claims 1 to 13, wherein expression of the transgene is driven by an inducible promoter.

The transgenic cell of any one of claims 1 to 15, wherein the transgenic protein has at least about 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2.

A transgenic cell according to any one of claims 1 to 16, wherein the transgenic protein comprises SEQ ID NO: 1 or SEQ ID NO: 2,

A transgenic cell according to any one of claims 1 to 16, wherein the transgenic protein consists of SEQ ID NO: 1 or SEQ ID NO: 2.

A method for producing bakuchiol, comprising culturing the transgenic cell according to any one of claims 1 to 18 in a culture medium and in the presence of p-coumaric acid and (i) geranyl pyrophosphate (GPP), (ii) dimethylallyl pyrophosphate (DMAPP) , (iii) 1sopentenylpyrophosphate (IPP), or any combination of (1)-(ii1).

The method of claim 19, wherein the culture medium further comprises about 3% w/v maltodexrin, about 0.2% w/v glucose, alpha-amylase or a combination thereof.

The method of claim 19 or 20, wherein the culture medium contains at least about 1.50 mM p-coumaric acid, at least about 1.75 mM p-coumaric acid, at least about

2.00 p-coumaric acid, at least about 2.25 mM p-coumaric acid, at least about 2.50 mM p-coumaric acid, at least about 2.75 mM p-coumaric acid, at least about 3.00 p-coumaric acid, at least about 3.25 mM p-coumaric acid, at least about 3.50 mM p-coumaric acid, at least about 3.75 mM p-coumaric acid, at least about 4.00 p-coumaric acid or more.

A method according to any one of claims 19-20, wherein the culture medium does not contain exogenous p-coumaric acid, GPP, DMAPP, IPP or a combination thereof.

The method of any one of claims 19 to 22, wherein at least about 0.1 µg/L, at least about 0.2 µg/L, at least about 0.3 µg/L, at least about 0.4 µg /L, at least about 0.5 pg/L, at least about 0.6 pg/L, at least about 0.7 ug/L, at least about 0.8 pg/L, at least about 0.9 pg /L, at least about 1.0 ug/L, at least about 1.1 pg/L, at least about 1.2 pg/L, at least about 1.3 ug/L, at least about 1.4 pg /L, at least about 1.5 pg/L, at least about 1.6 ng/L, at least about 1.7 pg/L, at least about 1.8 ug/L, at least about 1.9 ug /L, at least about 2.0 pg/L, at least about 2.1 pg/L, at least about 2.2 ug/L, at least about 2.3 ug/L, at least about 2.4 ug /l, at least about 2.5 µg/l, or more bakuchiol within at least about 48 hours of culture.

24. A bioproduction batch of bakuchiol, wherein the bakuchiol has a chemical purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% , at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100%, and no impurities greater than about 1%.

The bioproduction batch of bakuchiol according to claim 24, wherein the bakuchiol is in an oil.

The bioproduction batch of bakuchiol according to claim 24, wherein the bakuchiol is in an aqueous solution.

The bioproduction batch of bakuchiol according to claim 25 or 26, wherein the concentration of bakuchiol is at least about 0.1 pg/L, at least about 0.2 ug/L, at least about 0.3 pg/L, at least about is 0.4 pg/L. , at least about 0.5 pg/L, at least about 0.6 ug/L, at least about 0.7 pg/L. at least about 0.8 ug/L, at least about 0.9 ng/L, at least about 1.0 pg/L, at least about 1.1 pg/L, at least about 1.2 pg/L , at least about 1.3 ug/L, at least about 1.4 ug/L, at least about 1.5 pg/L, at least about 1.6 ng/L, at least about 1.7 ug/L , at least about 1.8 pg/L, at least about 1.9 ng/L, at least about 2.0 pg/L, at least about 2.1 ug/L, at least about 2.2 ug/L . at least about 2.3 µg/L, at least about 2.4 ng/L, at least about 2.5 µg/L, or more.

A bioproduction batch of bakuchiol according to any one of claims 24-27, wherein the bakuchiol is produced by the method according to any one of claims 19-23.

29. An isolated protein comprising an amino acid sequence with at least approximately 65% identity to: MHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTP ETLWGSIKQFCDAFYRFSRPHVIIGTAVNIVMSSLALEKSSDISPKFFIGLFQVIVTILSMNI YTAGINQLTDIEIDKINKPYLPLASGEYSYKTGVTHTLCAILSLGVGW IVGSPPLFWSNFA YFVLGTVYSIDLPLMRWKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRPMMIPKPV MFGTAFMSFFY VIAFFKDIPDIEGDKDHGVKSLTMRLGQKRVFWICVSLLLTGYGAATV VGATSSFLWCKLITVSGHALLASIFWNRAKSVDLKSHQEITSLYMFMWKLFYAEYFIPL MR (SEQ ID NO: 1; BAK28) or MASMFLGSLPLASSVNYIGRITRSKNCTESYHATSYITNASSNKTEKIKHEYANMRH RQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLWGSIKQFCDA FYRFSRPHVIIGTAVNIIVMSSLALEKSSDISPKFFIGLFQVIVTILSMNIYTAGINQLTDIEID KINKPYLPLASGEYSYKTGVT HTLCAILSLGVGWIVGSPPLFWSNFAYFVLGTVYSIDLPL MRWKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRPMMIPKSVMFGTAFMSFFY VIIA FFKDIPDIEGDKDHGVKSLTMRLGQERVFWICVSLLLTGYGAAIVVGATSSFLWCKLITV SGHALLASIFWNRAKSVDLKSHQEITSLYMFMWKLFYAEYFIPL MR (SEQ ID NO: 2 ; BAK36).

The isolated protein of claim 29, wherein the amino acid sequence comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 1 or SEQ ID NO: 2 includes.

The isolated protein of claim 29 or 30, wherein the amino acid sequence has at least 90% identity with SEQ ID NO: 1 or SEQ ID NO: 2.

The isolated protein of claim 29, wherein the amino acid sequence comprises or consists of SEQ ID NO: 1 or SEQ ID NO: 2.

The isolated protein of claim 29, wherein the amino acid sequence consists of SEQ ID NO: | or SEQ ID NO: 2.

The isolated protein of any one of claims 29 to 33, wherein the isolated protein exhibits prenyltransferase activity.

The isolated protein of any one of claims 29 to 34, wherein the isolated protein catalyzes the production of bakuchiol.

A nucleic acid comprising a nucleic acid sequence encoding the isolated protein of any one of claims 29-35.

An isolated host cell producing the isolated protein of any one of claims 29- or comprising the nucleic acid of claim 36.

38. A bakuchiol-producing enzyme as described herein.

39. A transgenic cell capable of producing bakuchiol as described herein.

40. A method for producing bakuchiol as described herein.

Use of an isolated protein according to any one of claims 29 to 35 for the production of bakuchiol.

Use of a nucleic acid according to claim 36 for the production of bakuchiol.

Use of a transgenic cell according to any one of claims 1 to 18 for the production of bakuchiol.

Use of an isolated host cell according to claim 37 for the production of bakuchiol.

An expression cassette or vector for expressing the nucleic acid of claim 36.

46. Bakuchiol obtainable by the method according to any one of claims 18 to 18

23.

47. A transgene encoding a transgenic protein comprising an amino acid sequence with at least approximately 65% identity with: MHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTP ETLWGSIKQFCDAFYRFSRPHVIGTAVNIVMSSLALEKSSDISPKFFIGLFQVIVTILSMNI YTAGINQLTDIEIDKINKPYLPLASGEYSYKTGVTHT LCAILSLGVGWIVGSPPLFWSNFA YFVLGTVYSIDLPLMRWKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRPMMIPKPV MFGTAFMSFFY VHAFFKDIPDIEGDKDHGVKSLTMRLGQKRVFWICVSLLLTGYGAAIV VGATSSFLWCKLITVSGHALLASIFWNRAKSVDLKSHQEITSLYMFMWKLFYAEYFIPL MR (SEQ ID NO: 1; BAK28) or MASMFLGSLPLASSVNYIGRITRSKNCTESYHATSYITNASSNKTEKIKHEYANMRH RQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLWGSIKQFCDA FYRFSRPHVIIGTAVNIIVMSSLALEKSSDISPKFFIGLFQVIVTILSMNIYTAGINQLTDIEID KINKPYLP LASGEYSYKTGVTHTLCAILSLGVGWIVGSPPLFWSNFAYFVLGTVYSIDLPL MRWKSHPALAALFFFVIRGLTFHVGFFLHLQTHVFKRPMMIPKSVMFGTAFMSFFY VIIA FFKDIPDIEGDKDHGVKSLTMRLGQERVFWICVSLLLTGYGAAIVVGATSSFLWCKLITV

SGHALLASIFWNRAKSVDLKSHQEITSLYMFMWKLFYAEYFIIPLMR (SEQ ID NO: 2; BAK36); wherein the transgenic protein catalyzes bakuchiol production, exhibits prenyltransferase activity, or both.

Use of a transgene according to claim 47 for integration of said transgene into a genome of a cell, wherein genome integration is preferably achieved by one or more methods selected from the group of Cas-based systems (e.g. Cas9, Cas12, etc. .), homologous recombination, gene gun, conjugation protocols and lambda ed.