WO2025049431A2

WO2025049431A2 - Control of flux toward 2-phenylethanol production

Info

Publication number: WO2025049431A2
Application number: PCT/US2024/043971
Authority: WO
Inventors: Karolina Z. KALBARCZYK; Stefan G. JENKINS; Amanda R. Reider Apel
Original assignee: Inscripta Inc
Current assignee: Inscripta Inc
Priority date: 2023-08-28
Filing date: 2024-08-27
Publication date: 2025-03-06
Anticipated expiration: 2026-02-28
Also published as: WO2025049431A3

Abstract

The present disclosure relates to microorganism(s) comprising modifications of genes in the shikimate pathway. The disclosure also relates to methods for modulating flux into shikimate pathway within a microorganism by selectively modulating gene expression of its components. Microorganism(s) of the disclosure are useful for the production of, e.g., 2-phenylethanol (2-PE), bakuchiol, p-coumaric acid, and its metabolites.

Description

Attorney Docket No. ISN-00725 CONTROL OF FLUX TOWARD 2-PHENYLETHANOL PRODUCTION CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the benefit of priority to U.S. Provisional Application Serial No.63/535,064, filed August 28, 2023, the content of which is incorporated herein by reference in its entirety. Sequence Listing The present application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 21, 2024, is named “ISN-00725_SL.xml”, and is 27,693 bytes in size. Field 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. The shikimate pathway links metabolism of carbohydrates to biosynthesis of aromatic compounds. In a sequence of metabolic steps, phosphoenolpyruvate and erythrose 4-phosphate are converted to chorismate, the precursor of the aromatic amino acids and many aromatic secondary metabolites. Once such product of the shikimate pathway is p-coumaric acid, which itself can be used as a precursor for synthesis of other molecules. Whilst microorganisms can be manipulated to produce industrial chemicals and fuels from organic substrates, there is little research on how modifying microorganisms to produce industrially useful compound(s) affects the biology of the microorganisms. The instant application relates to microorganisms modified to produce industrially useful compounds. The instant application also relates to methods for modulating flux into the shikimate pathway of microorganisms with select modifications, thus modulating production output of desired molecules in an industrial microbiology setting. Summary This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the 1 FH12463106.4 Attorney Docket No. ISN-00725 scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims. In some aspects, the present disclosure relates to microorganisms that have been modified to increase flux into the shikimate pathway to increase p-coumaric acid production, a direct precursor of bakuchiol. In some aspects, the present disclosure also relates to the characterization of various molecular pathways affected by the increased flux into the shikimate pathway, including the production of by-products of the shikimate pathway (e.g., 2- PE). In some aspects the disclosure provides a fungal cell producing bakuchiol comprising a modified phenylpyruvate decarboxylase (aro10) nucleic acid sequence. In other aspects, the disclosure provides a fungal cell producing bakuchiol comprising a modified branched-chain- 2-oxoacid decarboxylase (thi3) nucleic acid sequence. Yet in other aspects the disclosure provides a fungal cell comprising: i) a modified phenylpyruvate decarboxylase (aro10) nucleic acid sequence; and ii) a modified branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence. In some cases, a fungal cell described herein is a bakuchiol producing fugal cell. The bakuchiol can be produced from an endogenous p-coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. The modified aro10 nucleic acid sequence, the modified (thi3) nucleic acid sequence, or both can comprise a modification within an aro10 nucleic acid coding sequence, a modification within a thi3 nucleic acid coding sequence, or a modification within both sequences. Any of the aforementioned modifications can be in a promoter sequence, a coding sequence, or in a non-coding sequence of an aro10 nucleic acid sequence, a thi3 nucleic acid sequence, or a modification within both sequences. In certain cases, the modification is via a transgene independent of an ARO10 endogenous locus, a transgene independent of a THI3 endogenous locus, or a transgene independent of both loci. The modification can comprise an exogenous promoter driving expression of the ARO10 gene, the THI3 gene, or both. The modification can comprise an exogenous promoter reducing the expression of the ARO10 gene, the THI3 gene, or both. In some instances, the modified aro10 nucleic acid sequence, the modified thi3 nucleic acid sequence, or both can eliminate gene expression from one allele of the aro10 nucleic acid sequence (aro10-Δ1), one allele of 2 FH12463106.4 Attorney Docket No. ISN-00725 the (thi3-Δ1) nucleic acid sequence, or both in a haploid strain. In a diploid strain, the modified aro10 nucleic acid sequence, the modified thi3 nucleic acid sequence, or both can eliminate gene expression from one or both alleles. In some instances, the fungal cell does not produce 2-phenylethanol (2-PE). In some cases, the fungal cell is capable of providing a production titer of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 or 25 g/L of bakuchiol, e.g., at least 30 µg/L, at least 35 µg/L, at least 40 µg/L, at least 45 µg/L, at least 50 µg/L, at least 55 µg/L, at least 60 µg/L, or at least 65 µg/L of the bakuchiol. In some aspects, the fungal cell is a yeast cell. In some aspects the yeast cell is a Saccharomyces cerevisiae cell. In some aspects the disclosure provides a fungal cell comprising: i) a modification in a phenylpyruvate decarboxylase (aro10) nucleic acid sequence; and ii) a heterologous sequence encoding a bakuchiol synthase. In other aspects the disclosure provides a fungal cell comprising i) a modification in a branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence; and ii) a heterologous sequence encoding a bakuchiol synthase. Yet in other aspects the disclosure provides a fungal cell comprising: i) a modification in a phenylpyruvate decarboxylase (aro10) nucleic acid sequence; ii) a modification in a branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence; and iii) a heterologous sequence encoding a bakuchiol synthase. In certain instances the heterologous sequence encoding the bakuchiol synthase is a nucleic acid sequence derived from a plant selected from the group consisting of Psoralea corylifolia, P. grandulosa, P. drupaceae, Ulmus davidiana, Otholobium pubescens, Piper longum, and Aerva sangulnolenta Blum. In some cases the heterologous sequence encodes an amino acid sequence having at least 80%, at least 85%, at least 95%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. The bakuchiol can be produced from an endogenous p-coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. Any of the aforementioned modifications can be in a promoter sequence, a coding sequence, or in a non-coding sequence of an aro10 nucleic acid sequence, a thi3 nucleic acid sequence, or a modification within both sequences. In certain cases, the modification is via a transgene independent of an ARO10 endogenous locus, a transgene independent of a THI3 endogenous locus, or a transgene independent of both loci. The 3 FH12463106.4 Attorney Docket No. ISN-00725 modification can comprise an exogenous promoter driving expression of the ARO10 gene, the THI3 gene, or both. The modification can comprise an exogenous promoter reducing the expression of the ARO10 gene, the THI3 gene, or both. In some instances, the modification in the aro10 nucleic acid sequence, the modification in the thi3 nucleic acid sequence, or both can eliminate gene expression from one allele of the aro10 nucleic acid sequence (aro10-Δ1), one allele of the (thi3-Δ1) nucleic acid sequence, or both in a haploid strain. In a diploid strain, the modification in the aro10 nucleic acid sequence, the modification in the thi3 nucleic acid sequence, or both can eliminate gene expression from one or both alleles. In some instances, the fungal cell does not produce 2-phenylethanol (2-PE). In some cases, the fungal cell is capable of providing a production titer of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 or 25 g/L of bakuchiol, e.g., at least 30 g/L, at least 35 g/L, at least 40 g/L, at least 45 g/L, at least 50 g/L, at least 55 g/L, at least 60 g/L, or at least 65 g/L of the bakuchiol. In some aspects, the fungal cell is a yeast cell. In some aspects the yeast cell is a Saccharomyces cerevisiae cell. In some aspects the disclosure provides a method for reducing 2-phenylethanol (2-PE) production in a fungus by suppressing a gene from being expressed from i) a phenylpyruvate decarboxylase (aro10) nucleic acid sequence; and/or ii) an aro10 nucleic acid sequence and a branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence in the fungus. The fungus can be a bakuchiol producing fungus. In some aspects, the fungus comprises a heterologous sequence encoding a bakuchiol synthase, such as a nucleic acid sequence derived from a plant selected from the group consisting of Psoralea corylifolia, P. grandulosa, P. drupaceae, Ulmus davidiana, Otholobium pubescens, Piper longum, and Aerva sangulnolenta Blum. In some cases the heterologous sequence encodes an amino acid sequence having at least 80%, at least 85%, at least 95%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. The bakuchiol can be produced from an endogenous p- coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. In certain cases, no amounts of 2-PE are detectable via mass-spec in the fungus where expression of either i) aro10 nucleic acid sequence; or ii) aro10 and thi3 nucleic acid sequences are suppressed. Suppression of the aforementioned nucleic acid sequences can be via a nucleic 4 FH12463106.4 Attorney Docket No. ISN-00725 acid modification of a coding sequence, a non-coding sequence, or via a transgene. In some cases the fungus cell is a yeast cell, e.g., a Saccharomyces cerevisiae cell. In some aspects the disclosure relates to a method for increasing bakuchiol production and reducing 2-phenylethanol (2-PE) production in a yeast cell comprising: a) culturing the yeast under conditions for: suppressing gene expression of one or both of: i) a phenylpyruvate decarboxylase (ARO10) gene; and ii) a branched-chain-2-oxoacid decarboxylase (THI3) gene via a modification in one or both of a first aro10 nucleic acid target sequence and a first thi3 nucleic acid target sequence within the yeast; and increasing p-coumarate flux via a modification of at least one target shikimate nucleic acid sequence of one or more genes in the shikimate pathway; thereby increasing bakuchiol production and reducing 2-PE production from the fungus. The one or more genes in the shikimate pathway can be selected from the group consisting of ADH2, ADH4, ARO1, ARO2, ARO3, ARO4, ARO7, ARO8, ARO9, ARO10, FjTAL, HaTAL1, PHA2, TYR1, TRP1, TRP2, TRP3, TRP4, and TRP5. The modification of the at least one target shikimate nucleic acid sequence can be a knock-out in the one or more genes in the shikimate pathway that increases p-coumarate flux, a promoter swap in the one or more genes in the shikimate pathway that increases p-coumarate flux, or another similar suitable modification. The modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence can be within a coding region of the ARO10 gene or the THI3 gene, e.g., a knock-out of ARO10 (aro10-Δ1) and/or a knock-out of THI3 (thi3- Δ1). In some cases, the modification can be within a non-coding region of the ARO10 gene or the THI3 genes. In some aspects, production of the bakuchiol molecule in a aro10-Δ1 + thi3- Δ1 yeast is increased by at least 10%, at least 20%, or at least 30% as compared to production of the bakuchiol molecule in a fungus that does not have the aro10-Δ1 + thi3-Δ1 modifications. In some instances, production of 2-PE is eliminated in an aro10-Δ1 + thi3-Δ1 yeast (e.g., as detected by mass-spectrometry). In some instances the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is within a non-coding region of the ARO10 gene or the THI3 gene. In some instances the modification suppresses the promoter or another regulatory region of the ARO10 gene or the THI3 gene. In some cases, the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is via a transgene. In some instances the yeast is Saccharomyces cerevisiae. 5 FH12463106.4 Attorney Docket No. ISN-00725 In some aspects the disclosure provides a yeast comprising: a) one or more first modifications selected from: i) a modified phenylpyruvate decarboxylase (aro10) nucleic acid sequence; or ii) a modified aromatic amino acid aminotransferase 1 (aro8) nucleic acid sequence or a modified aromatic amino acid aminotransferase 2 (aro9) nucleic acid sequence; and b) one or more second modifications in a shikimate pathway gene for increasing p- coumarate flux whereby the yeast produces more 2-phenylethanol (2-PE) or a derivative thereof compared to a control yeast not having the one or more first modifications. The modification in the aro10 nucleic acid sequence can provide a modulation of a regulatory region of aro10 that substantially increases expression of ARO10, e.g., the modified aro10 nucleic acid sequence can be a promoter swap of the aro10 nucleic acid sequence. The modified phenylpyruvate decarboxylase (aro10) nucleic acid sequence can encode an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% identity to SEQ ID NO: 1. The modified aromatic amino acid aminotransferase 1 (aro8) nucleic acid sequence can encode an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% identity to SEQ ID NO: 3. The modified aromatic amino acid aminotransferase 2 (aro9) nucleic acid sequence can encode an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% identity to SEQ ID NO: 4. The modified aro8 or aro9 nucleic acid sequence(s) can be a knock-out of the ARO8 or ARO9 genes. The modified aro8 or aro9 nucleic acid sequence(s) can comprise a modulation of a regulatory region of ARO8 or ARO9 that substantially suppresses expression of one or both of ARO8 or ARO9 genes. The shikimate pathway gene for increasing p-coumarate flux can be selected from the group consisting of ADH2, ADH4, ARO1, ARO2, ARO3, ARO4, ARO7, ARO8, ARO9, ARO10, FjTAL, HaTAL1, PHA2, TYR1, TRP1, TRP2, TRP3, TRP4, and TRP5. The yeast can produce at least 5% more, at least 10% more, at least 15% more, at least 20% more, at least 25% more, or at least 30% more 2-phenylethanol (2-PE) or a derivative thereof compared to a control yeast not having the one or more first modifications. The yeast can be a bakuchiol producing yeast. The yeast can comprise a heterologous sequence encoding a bakuchiol synthase. The heterologous sequence encoding the bakuchiol synthase can be a nucleic acid sequence derived from a plant selected from the group consisting of Psoralea corylifolia, P. grandulosa, P. drupaceae, Ulmus davidiana, Otholobium pubescens, Piper longum, and Aerva sangulnolenta Blum, e.g., a heterologous sequence encoding an amino acid 6 FH12463106.4 Attorney Docket No. ISN-00725 sequence having at least 80%, at least 85%, at least 95%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. The bakuchiol synthase can produce bakuchiol from an endogenous p-coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. The yeast can produce at least 5% more, at least 10% more, at least 15% more, at least 20% more, at least 25% more, or at least 30% more 2-phenylethanol (2-PE) or a derivative thereof compared to a control yeast not having the modified aro10 nucleic acid sequence. In some cases the yeast is Saccharomyces cerevisiae. In some aspects, the disclosure provides a method for increasing production of 2- phenylethanol (2-PE) or a derivative thereof in a yeast comprising: culturing the yeast under conditions for: a) overexpressing a phenylpyruvate decarboxylase (aro10) nucleic acid sequence via a modification in a aro10 nucleic acid target sequence; and/or b) suppressing gene expression from one or both of an aromatic amino acid aminotransferase 1 (aro8) nucleic acid sequence or an aromatic amino acid aminotransferase 2 (aro9) nucleic acid sequence; thereby increasing production of 2-PE or a derivative thereof from the yeast. In some aspects, the modification of the aro10 nucleic acid sequence provides a modulation of a regulatory region of aro10 that substantially increases expression of aro10. In some aspects, the modification in the aro10 nucleic acid sequence is a promoter swap of the aro10 nucleic acid sequence. In some aspects the aro8 or aro9 nucleic acid sequence(s) are modified. In some aspects the modified aro8 or aro9 nucleic acid sequence(s) are a knock-out of the ARO8 or ARO9 genes. In some aspects, the modification of the aro8 or aro9 nucleic acid sequence(s) provide a modulation of a regulatory region of ARO8 or ARO9 that substantially suppresses expression of one or both of ARO8 or ARO9. In some aspects, the yeast is a bakuchiol producing yeast. In some aspects, the yeast comprises a heterologous sequence encoding a bakuchiol synthase. In some aspects, the heterologous sequence encoding the bakuchiol synthase is a nucleic acid sequence derived from a plant selected from the group consisting of Psoralea corylifolia, P. grandulosa, P. drupaceae, Ulmus davidiana, Otholobium pubescens, Piper longum, and Aerva sangulnolenta Blum. In some aspects, the heterologous sequence encodes an amino acid sequence having at least 80%, at least 85%, at least 95%, or 100% 7 FH12463106.4 Attorney Docket No. ISN-00725 identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.In some aspects, the bakuchiol synthase produces bakuchiol from an endogenous p-coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. In some aspects, the yeast produces at least 5% more, at least 10% more, at least 15% more, at least 20% more, or at least 25% more 2-PE or a derivative thereof compared to a yeast not having the modification in the aro10 nucleic acid target sequence. In some aspects, the yeast produces at least 5% more, at least 10% more, at least 15% more, at least 20% more, or at least 25% more 2-PE or a derivative thereof compared to a yeast not cultured under conditions for: a) overexpressing the phenylpyruvate decarboxylase (aro10) nucleic acid sequence via the modification in the aro10 nucleic acid target sequence; and/or b) suppressing gene expression from one or both of the aromatic amino acid aminotransferase 1 (aro8) nucleic acid sequence or the aromatic amino acid aminotransferase 2 (aro9) nucleic acid sequence. In some aspects the disclosure provides a method for increasing shikimate pathway flux in a yeast comprising: a) culturing the yeast under conditions for: suppressing gene expression of one or both of: i) a phenylpyruvate decarboxylase (aro10) nucleic acid sequence; and ii) a branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence via a modification in one or both of a first aro10 nucleic acid target sequence and a first thi3 nucleic acid target sequence within the yeast; thereby increasing shikimate pathway flux as determined by an increased p-coumaric acid production or p-coumaric acid derivative production compared to a yeast that does not comprise the modification. In some aspects, the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is within a coding region of one or both the ARO10 gene and the THI3 gene. In some aspects, the modification of the first aro10 nucleic acid sequence is a knock-out of ARO10 (aro10-Δ1) and the modification of the first thi3 nucleic acid sequence is a knock-out of THI3 (thi3-Δ1). In some instances, production of the p-coumaric acid or p-coumaric acid derivative in an aro10- Δ1 + thi3-Δ1 yeast is increased by at least 10%, at least 20%, or at least 30% as compared to production of the p-coumaric acid or p-coumaric acid derivative in a yeast that does not have the aro10-Δ1 + thi3-Δ1 modifications. In some instances, production of 2-PE is eliminated in 8 FH12463106.4 Attorney Docket No. ISN-00725 an aro10-Δ1 + thi3-Δ1 yeast (e.g., as detected by mass-spectrometry). In some instances the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is within a non-coding region of one or both the ARO10 gene and the THI3 gene. In such instances, the modification in the non-coding region substantially can suppress the expression of one or more of the aro10 nucleic acid sequence and the thi3 nucleic acid sequence. In some instances the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is within a non-coding region of one or both the ARO10 gene and the THI3 gene. In some instances, the modification suppresses the promoter or another regulatory region of one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence. In some instances, the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is via a transgene. In some instances, the yeast is Saccharomyces cerevisiae. In some aspects the disclosure provides a population of any one of the microorganism described herein, e.g., a population of modified fungal cells, a population of modified yeast cells. In some aspects, the disclosure provides a composition, e.g., a container comprising cryo- preserved cell populations of any one of the fungal cells described herein. It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below are provided as being part of the inventive subject matter disclosed herein and may be employed in suitable functional combinations to achieve the benefits described herein. Brief Description of Drawings The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which: Figure 1 (FIG. 1) depicts a schematic of the shikimate pathway and its intersection with the production of bakuchiol. 9 FH12463106.4 Attorney Docket No. ISN-00725 Figure 2 (FIG. 2) is a chart depicting detected amounts of 2-phenylethanol (mg/L), and OD600 from certain cell cultures, namely cultures from CEN.PK 113-7D, STR1510, STR1552, STR1611, STR1698, STR1765, STR1941, STR2004, STR2254 strains or strain pools. Figure 3 (FIG. 3) is a chart depicting detected amounts of 2-phenylethanol (mg/L), detected amounts of mevalonolactone (mg/L), detected amounts of bakuchiol (mg/L), and OD600 from certain cell cultures, namely cultures from strains STR2004 (S4403), STR2254 (S4878), STR2291 (S5064 and S5065), STR2292 (S5067 and S5068), STR2293 (S5067 and S5068), and STR2294 (S5069 and S5070). Figure 4 (FIG. 4) is a chart depicting detected amounts of 2-phenylethanol (mg/L) and bakuchiol (mg/L) from certain cell cultures, namely cultures from strains S4403, S4822, S4878 (aro10-Δ1), S4936 (aro10-Δ1), S5071 (adh2-Δ1), S5072 (adh2-Δ1), S5073 (adh4-Δ1), S5074 (adh4-Δ1), S5075 (aro8-Δ1), S5076 (aro8-Δ1), S5077 (aro9-Δ1), S5078 (aro9-Δ1), S5079 (adh2-Δ1), S5080 (adh2-Δ1), S5081 (adh4-Δ1), S5082 (adh4-Δ1), S5083 (aro8-Δ1), S5084 (aro8-Δ1), S5085 (aro9-Δ1), S5086 (aro9-Δ1), S5087 (aro3Δ::K222L), S5088 (aro9-Δ1), S5089 (aro9-Δ1), S5090 (FjTAL), S5091 (FjTAL), S5092 (HaTAL11), S5093 (HaTAL11), S5094 (aro3Δ::K222L), S5095 (aro3Δ::K222L), S5096 (aro10-Δ1 ARO9), S5097 (aro10-Δ1 ARO9), S5098 (aro10-Δ1 FJTAL), S5099 (aro10-Δ1 FJTAL), S5100 (aro10-Δ1 HaTAL1), S5101 (aro10-Δ1 HaTAL1), S5102 (aro10-Δ1 ECAROL), and S5103 (aro10-Δ1 ECAROL). Figure 5 (FIG.5) demonstrates 2-PE toxicity via a minimal inhibitory concentration assay. Strains producing bakuchiols (strains 3 to 5) show increased sensitivity to 2-PE relative to control strains (1 and 2). Figure 6 (FIG. 6) demonstrates knocking out ARO10 (in Strain 2287, B) eliminates 2-PE and more than doubles bakuchiol titers in tanks relative to a control strain with ARO10 (in Strain 2236, A). Figure 7 (FIG. 7) demonstrates certain yeast strains (e.g., Strain 2004) generate a substantial amount of 2-PE in fermentation (e.g., after 96 hours fermentation) allowing for the potential production of 2-PE as a main (intended) product. It should be understood that the drawings are not necessarily to scale (e.g., schematics). 10 FH12463106.4 Attorney Docket No. ISN-00725 Detailed Description Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in their entirety. Alleles created by recombinant DNA technology are named by use of the symbol for the gene that is altered, followed by a symbol to indicate the nature of the alteration: deletion (-Δ)( aro10-Δ1)); replacement (Δ::) (e.g., ARO3pΔ::K222L); promoter swap disruption (promoter > gene)(e.g., pGAL1>ARO10). As used herein, a “modification” or “modified” when used in the context of referring to a gene, a nucleic acid sequence, or a microorganism, means a change relative to a “parent” or reference gene/sequence/reference strain made by altering one or more nucleic acid sequences, via nucleic acid substitutions, deletions, or insertions. For the purposes of this disclosure, a variant may comprise a nucleic acid sequence that shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to about 100% sequence identity or homology with a reference or “parent” sequence. For purposes of this disclosure, a gene or a nucleic acid modification can be a change into an endogenous locus or it can be accomplished via a transgene. For purposes of this disclosure, the terms “variant” and “modified” and “modification” when used in the context of referring to a nucleic acid sequence, a gene, or a microorganism/strain are used interchangeably. As used herein, the term “production” or “bioproduction” is intended to mean production of a compound (e.g., bakuchiol, 2-PE, coumaric acid, aromatic amino acids, farnesene, farnesol, geosmin, geraniol, terpineol, limonene, myrcene, linalool, hinokitiol, pinene, cafestol, kahweol, cembrene, taxadiene, α-bisabolol, α-guaiene, bergamontene, and valencene) by way of biological or enzymatic synthesis (as opposed to chemical synthesis). In some 11 FH12463106.4 Attorney Docket No. ISN-00725 implementations, industrial manufacturing 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, 2-PE, coumaric acid, aromatic amino acids, farnesene, farnesol, geosmin, geraniol, terpineol, limonene, myrcene, linalool, hinokitiol, pinene, cafestol, kahweol, cembrene, taxadiene, α-bisabolol, α-guaiene, bergamontene, and valencene). A “promoter” refers to a nucleic acid sequence capable of initiating transcription of a gene (e.g., an ARO10 gene operably linked to a promoter). The term “operably linked” refers to a nucleic acid sequence placed into a functional relationship with another nucleic acid sequence, e.g., a promoter operably linked to a gene. The term “operably linked,” as used herein, has a meaning commonly known in the art. For example, a promoter is operably linked to a gene when that promoter is placed in a location that permits that promoter to initiate transcription of that gene. An enhancer is operably linked to a gene when that enhancer, when bound by an appropriate transcription factor, can regulate (e.g., enhance) expression of that gene. As used herein, the “coding sequence”, also known as the open reading frame (ORF), refers to a contiguous stretch of nucleotide triplets (codons) that can be translated into a specific amino acid sequence. The modification of a coding sequence can refer to a modification of an endogenous coding sequence, or to a coding sequence that is exogenously introduced into a cell. The “non-coding sequence” refers to stretches of DNA or RNA that do not code for protein sequences. Examples of non-coding sequences include introns, promoters, enhancers and silencers, long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and short interfering RNAs (siRNAs). The terms “target nucleic acid sequence” or “target sequence” and the like refer to any locus in vitro or in vivo, or in a nucleic acid (e.g., genome or episome) of a microorganim or population of microorganisms, in which a change of at least one nucleotide is desired using specific targeting. The cellular target sequence can be a genomic locus or extrachromosomal locus. 12 FH12463106.4 Attorney Docket No. ISN-00725 As used herein, the term “percent sequence identity” with respect to a reference nucleic acid or amino acid sequence is the percentage of nucleic acid bases or amino acid residues in a candidate sequence that are identical with the nucleic acid bases or amino acid residues in the reference sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods of sequence alignment are well known in the art. Optimal alignment of sequences can be conducted by methods described in Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; Pearson and Lipman, 1988, PNAS 85:2444, by computerized implementations of these algorithms. Alignments can be made using publicly available computer software such as BLASTp, BLASTn, BLAST-2, ALIGN or MegAlign Pro (DNASTAR) software. 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. 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%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±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- 11”. 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). 13 FH12463106.4 Attorney Docket No. ISN-00725 In an aspect, a microbial cell (e.g., a yeast or E. coli) producing bakuchiol is provided. See also US20180117082A1. In another aspect, a bakuchiol-producing microbial cell comprises one or more genome modifications modulating the expression or activity level of one or more genes selected from the group of ARO10, THI3, ARO8 and ARO9. In an aspect, a polypeptide product of the ARO10, THI3, ARO8 or ARO9 genes exhibit at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or 100% identity to SEQ ID NOs: 1, 2, 3, or 4. In another aspect, a knock-out or knock-down of one or more of ARO10, THI3, ARO8 and ARO9 is present. In an aspect, a bakuchiol-producing microbial cell (e.g., a yeast or E. coli) provided herein comprises one or more genetic modifications that reduce the production of 2-PE (i.e., 2-PE is eliminated or at a lower level in the presence of such genetic modification compared to a control strain without such genetic modification). In a further aspect, provided here is a bakuchiol-producing microbial cell that produces no detectable level of 2-PE or exhibits no or negligible 2-PE toxicity at a bakuchiol titer of at least 8, 9, 10, 12, 15, 18, 20, 22, 25, 28, 30, 35, 40 or 50 g/L. Shikimate Pathway Overall The shikimate pathway is a metabolic pathway found in plants, bacteria, fungi, and some parasites. It is responsible for the synthesis of certain aromatic compounds, including the amino acids phenylalanine, tyrosine, and tryptophan. These amino acids serve as building blocks for proteins and are also precursors for a wide range of secondary metabolites, such as lignin, flavonoids, alkaloids, and many others. The shikimate pathway consists of a series of enzymatic reactions that convert the simple sugar phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) into chorismate, a key intermediate. The shikimate pathway begins with the condensation of phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) catalyzed by the enzyme 3-deoxy-D- arabinoheptulosonate 7-phosphate synthase (DAHPS). This reaction produces a molecule called 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP). DAHP undergoes a series of enzymatic reactions to form chorismate. One of these steps is catalyzed by the enzyme 5- enolpyruvylshikimate 3-phosphate synthase (EPSP synthase). Chorismate is the central intermediate in the shikimate pathway. (see Figure 1 illustrating shikimate pathway in yeast). Chorismate serves as a branching point for the synthesis of phenylalanine, tyrosine, and 14 FH12463106.4 Attorney Docket No. ISN-00725 tryptophan. Further enzymatic reactions specific to each amino acid convert chorismate into the final aromatic amino acids. However, in the case of p-coumaric acid, “branch points” in the pathway can occur before or after the formation of these amino acids. In some cases, L-tyrosine can undergo decarboxylation, a chemical reaction that removes the carboxyl group (-COOH) from the amino acid molecule. This reaction is generally catalyzed by an enzyme with tyrosine decarboxylase activity. The decarboxylation of ^L- tyrosine leads to the formation of p-coumaric acid, the structure of which is derived from the carbon skeleton of L -tyrosine. Alternatively chorismate can be converted into p-coumaroyl- CoA through a series of enzymatic steps. First, chorismate can be converted into 4- hydroxybenzoate through the action of an enzyme with chorismate mutase activity. Next, 4- hydroxybenzoate can be converted into 4-coumaroyl-CoA through the actions of the enzymes 4-hydroxybenzoate-CoA ligase and 4-coumarate-CoA ligase. Lastly, 4-coumaroyl-CoA is converted into p-coumaric acid through the release of CoA by the enzyme thioesterase. This step frees p-coumaric acid from its CoA carrier, resulting in the production of the final product. Depending on the specific pathway or organism, further enzymatic reactions may occur to convert p-coumaric acid into other compounds. For example, in plants, p-coumaric acid can serve as a precursor for various secondary metabolites, such as flavonoids and lignin. These compounds play essential roles in plant structure, defense mechanisms, and various biological activities. In industrial microorganism(s), e.g., fungi and bacteria, p-coumaric acid can serve as a precursor for other important compounds, including compounds not endogenous to microorganism. Thus, modulation of activity of enzymes in the shikimate pathway̶ either by increasing their expression (e.g., via replacement of an endogenous promoter with a stronger promoter), decreasing their expression (e.g., via nucleic acid sequence modification), or knocking out one or more genes (e.g., via nucleic acid sequence modification)̶ provides a method for adjusting the flux into the pathway as to produce higher or lower quantities or a compound. Not to be bound by any particular theory, but one implementation of the modified microorganism(s) and methods described herein is based on one or more modifications of genes in the shikimate pathway for modulating flux within the shikimate pathway thereby skewing the pathway towards either increased or decreased production of its end products, by- 15 FH12463106.4 Attorney Docket No. ISN-00725 products, or metabolites. In certain instances these modifications are made within bakuchiol producing strains. In such strains, bakuchiol is being produced by a prenyltransferase enzyme that is native to plants and is heterologously introduced into a microorganism, preferably a fungi (e.g., yeast), although the disclosure contemplates that a bacteria may also be similarly modified. Production of bakuchiol likely occurs through a mechanism involving geranyl pyrophosphate (GPP), dimethylallyl pyrophosphate (DMAPP), isopentenyl pyrophosphate (IPP), and/or p-coumaric acid. As explained in further detail in the non-limiting working examples section herein, bakuchiol producing microorganisms (e.g. yeast) comprising a modification in one or more genes in the shikimate pathway, including, but not limited to genes in the shikimate pathway selected from, e.g. shikimate genes from yeast including ADH2, ADH4, ARO1, ARO2, ARO3, ARO4, ARO7, ARO8, ARO9, ARO10, FjTAL, HaTAL1, PHA2, TYR1, TRP1, TRP2, TRP3, TRP4, TRP5, were characterized with respects to amounts of p- coumaric acid, bakuchiol, 2-phenylethanol (2-PE), and mevalonolactone produced. In the course of the aforementioned characterization, the present disclosure identified certain “dials” within the shikimate pathway that can be “turned-on”, “turned-down”, or “turned-off”, in a manner that changes the output of e.g., 2-phenylethanol (2-PE), bakuchiol, or p-coumaric acid from the shikimate pathway. The disclosure provides certain microorganisms comprising modifications in one or more genes and its uses in modulating production of certain industrially useful compounds via modulation of flux within the shikimate pathway. In many instances, the microorganisms are bakuchiol producing microorganisms. Nonetheless, it is contemplated that certain modifications, e.g., ARO8 and/or ARO9 modifications, need not to occur in bakuchiol producing strains. In some aspects, the present disclosure relates to microorganism(s) comprising a heterologous sequence encoding a bakuchiol synthase, and modifications of genes within such microorganisms. It is contemplated that the sequence encoding the bakuchiol synthase can be a nucleic acid sequence derived from a plant selected from the group consisting of Psoralea corylifolia, P. grandulosa, P. drupaceae, Ulmus davidiana, Otholobium pubescens, Piper longum, and Aerva sangulnolenta Blum. Exemplary sequences include nucleic acid sequences encoding an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 16 FH12463106.4 Attorney Docket No. ISN-00725 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% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. It is contemplated that the bakuchiol is produced from an endogenous p- coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor, and that modifications in the one or more genes in the shikimate pathway modulate the flux within the shikimate pathway in a manner that either increases production of p-coumaric acid and its metabolites (e.g., flavonoids and lignin) or decreases its production. Microorganisms with Nucleic Acid Modifications in ARO10, THI3, ARO8 and ARO9 genes In some aspects, the disclosure contemplates microorganisms with modifications in nucleic acid sequence(s)̶ e.g., modifications for increasing or decreasing quantities of gene expression from a nucleic acid sequence(s)̶ that affect the flux within the shikimate pathway (e.g., the amount of a compound or by-product produced by the shikimate pathway by the microorganism). In some aspects, the disclosure contemplates microorganisms with modifications of an ARO10 gene, e.g., modifications for increasing or decreasing quantities of gene expression from an aro10 nucleic acid sequence within a microorganism. Such modifications can be implemented, e.g., for modulating the flux into the shikimate pathway thus producing higher or lower quantities of, e.g., 2-phenylethanol (2-PE), p-coumaric acid, and/or bakuchiol. The ARO10 gene is found in organisms that possess the shikimate pathway, including plants, bacteria, fungi, and some parasites. ARO10 is an enzyme involved in the shikimate pathway, specifically reported to be involved in the conversion of 3-dehydroquinate (3-DHQ) to 3- dehydroshikimate (3-DHS). This reaction is the sixth step in the shikimate pathway and is catalyzed by the enzyme 3-dehydroquinate dehydratase, which is encoded by the ARO10 gene. The reaction catalyzed by ARO10 is as follows: 3-dehydroquinate (3-DHQ) → 3-dehydroshikimate (3-DHS). 17 FH12463106.4 Attorney Docket No. ISN-00725 The conversion of 3-DHS to downstream intermediates continues until the formation of chorismate, which, as previously described, is a key branch point leading to the synthesis of various aromatic compounds. The product of the ARO10 gene is also speculated to play a functional role in reactions downstream of L-phenylalanine production. In some aspects, the disclosure provides microorganism(s) with a knockout of the ARO10 gene (aro10-Δ1) and its uses in methods for: a) eliminating 2-PE from being produced by the shikimate pathway; and/or b) increasing bakuchiol production. In some aspects, the disclosure provides microorganism(s) with a promoter swap of the ARO10 endogenous promoter (e.g., pGAL>ARO10) and its uses in methods for increasing 2-PE production as a by-product of the shikimate pathway. The present disclosure provides microorganism with modification(s) of the aro10 nucleic acid sequence for modulating flux into the shikimate pathway, thereby increasing or decreasing the production of, e.g., 2-PE, bakuchiol, and/or p-coumaric acid and its derivatives. In certain aspects, the disclosure provides a fungal cell producing bakuchiol comprising a modified phenylpyruvate decarboxylase (aro10) nucleic acid sequence. In certain aspects the disclosure provides a microorganism (e.g., a fungal cell) comprising: i) a modification in a phenylpyruvate decarboxylase (aro10) nucleic acid sequence; and ii) a heterologous sequence encoding a bakuchiol synthase. The modification can occur within a nucleic acid coding sequence, e.g., ARO10 gene knock-out. The modification can eliminate ARO10 gene expression by reducing or eliminating the functionality of an ARO10 gene promoter, regulatory sequence, enhancer, or the like. The modification can increase expression from the ARO10 gene, e.g., by replacing an endogenous ARO10 gene with a strong exogenous gene promoter (e.g., pGAL1>ARO10). The modified microorganism can be a haploid or a diploid microorganism. In some aspects, the disclosure contemplates microorganisms with modifications of an THI3 gene, e.g., modifications for increasing or decreasing quantities of gene expression from an thi3 nucleic acid sequence within a microorganism. The THI3 gene has not been reported to be directly associated with the shikimate pathway. Instead, THI3 is typically referred to as a gene involved in the biosynthesis of thiamine (vitamin B1) in yeast, thiamine biosynthesis involves a series of enzymatic reactions, including the conversion of 4-amino-5- hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) to 4-methyl-5-(beta- 18 FH12463106.4 Attorney Docket No. ISN-00725 hydroxyethyl)thiazole phosphate (THZ-P); a reaction catalyzed by the product of the THI3 gene, namely HMP kinase or HMP-P kinase, which catalyzes the phosphorylation of HMP-P to form THZ-P. The shikimate pathway, on the other hand, is a distinct metabolic pathway, albeit one that is also responsible for the synthesis of aromatic compounds. In some aspects, the disclosure provides microorganism(s) with a knockout of the THI3 gene (thi3-Δ1) and its uses in methods for increasing bakuchiol production from bakuchiol producing strains. In some aspects, the disclosure provides microorganim(s) with a promoter swap of the THI3 endogenous promoter (e.g., pGAL>THI3) and its uses in methods for increasing production of bakuchiol, p-coumaric acid, and its metabolites from the shikimate pathway. The present disclosure provides microorganism(s) with modification(s) of the thi3 nucleic acid sequence for modulating flux into the shikimate pathway, thereby increasing or decreasing the production of, e.g., 2-PE, bakuchiol, and/or p-coumaric acid and its derivatives. In certain aspects, the disclosure provides a modified microorganism (e.g., a modified fungal cell) producing bakuchiol comprising a modified branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence. In certain aspects the disclosure provides a modified microorganism (e.g., a modified fungal cell) comprising: i) a modification in a branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence; and ii) a heterologous sequence encoding a bakuchiol synthase. The modification can occur within a nucleic acid coding sequence, e.g., THI3 gene knock-out. The modification can eliminate THI3 gene expression by reducing or eliminating the functionality of an THI3 gene promoter, regulatory sequence, enhancer, or the like. The modification can increase expression from the THI3 gene, e.g., by replacing an endogenous THI3 gene with a strong exogenous gene promoter (e.g., pGAL1>THI3). The modified microorganism can be a haploid or a diploid microorganism. In some aspects, the disclosure contemplates microorganisms with modifications of one or more of i) a modified phenylpyruvate decarboxylase (aro10) nucleic acid sequence; and ii) a modified branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence. The disclosure provides that microorganisms comprising deleterious nucleic acid sequence modifications with regards to both ARO10 and THI3 genes product no detectable 2-PE. The disclosure provides that microorganisms comprising deleterious nucleic acid sequence modifications in bakuchiol producing strains produce significantly higher amounts of bakuchiol. In some aspects, production of a bakuchiol molecule in a aro10-Δ1 + thi3-Δ1 19 FH12463106.4 Attorney Docket No. ISN-00725 microorganism (e.g., a fungus such as yeast) is increased by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% as compared to production of the bakuchiol molecule in the microorganism that does not have the aro10-Δ1 + thi3-Δ1 modifications. In some aspects, production of 2-PE is eliminated in an aro10-Δ1 + thi3-Δ1 microorganism (e.g., a fungus such as yeast) as detected by mass-spectrometry. The modified microorganism can be a haploid or a diploid microorganism. In some aspects, the disclosure contemplates microorganisms with modifications of an ARO8 gene, e.g., modifications for increasing or decreasing quantities of gene expression from an aro8 nucleic acid sequence within a microorganism. ARO8 is an enzyme reported to play a role in the shikimate pathway, specifically in the conversion of 5-enolpyruvylshikimate 3- phosphate (EPSP) to chorismate. This reaction is the final step in the shikimate pathway and is catalyzed by the enzyme ARO8, also known as EPSP synthase. In yeast, the reaction catalyzed by ARO8 is as follows: EPSP → chorismite. Chorismate serves as a branching point for the biosynthesis of various aromatic compounds, including aromatic amino acids (phenylalanine, tyrosine, and tryptophan), as well as secondary metabolites such as flavonoids, lignin, and alkaloids. In some aspects, the disclosure contemplates microorganisms with modifications of an ARO9 gene, e.g., modifications for increasing or decreasing quantities of gene expression from an aro9 nucleic acid sequence within a microorganism. Conflicting functionalities are reported in the literature with regards to the product of the ARO9 gene: most papers describe ARO8 and ARO9 as two genes that carry out the same function. See, e.g., Iraqui I, Vissers S, Cartiaux M (1998) Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily. Mol Gen Genet 257, 238–248. Yet, other reports suggest that ARO8 must be upregulated to increase 2-PE flux while ARO9 should be eliminated (see Dai J, Xia H, Yang C and Chen X (2021) Sensing, uptake and catabolism of L-phenylalanine during 2-phenylethanol biosynthesis via the Ehrlich Pathway in Saccharomyces cerevisiae. Front. Microbiol. 12:601963); and yet other reports suggests the opposing outcome (see Hassing EJ, de Groot P, Marquenie V, Pronk J, Daran J 20 FH12463106.4 Attorney Docket No. ISN-00725 (2019) Connecting central carbon and aromatic amino acid metabolisms to improve de novo 2-phenylethanol production in Saccharomyces cerevisiae. Metabolic Engineering. Volume 56, December 2019, Pages 165-180. To resolve the conflicting reports, the disclosure characterized the function of ARO8 and ARO9 genes in the context of various bakuchiol producing strains. In some aspects, the disclosure contemplates microorganisms with modifications of one or more of i) a modified aromatic amino acid aminotransferase 1 (aro8) nucleic acid sequence or a modified aromatic amino acid aminotransferase 2 (aro9) nucleic acid sequence. The modified microorganism can be a haploid or a diploid microorganism. Microorganisms with Modifications in One or More of ADH2, ADH4, ARO1, ARO2, ARO3, ARO4, ARO7, FjTAL, HaTAL1, PHA2, TYR1, TRP1, TRP2, TRP3, TRP4, and TRP5. In some aspects, the disclosure contemplates microorganisms with modifications of an alcohol dehydrogenase 2 enzyme (adh2) nucleic acid sequence. Alcohol dehydrogenases are enzymes that catalyze the reversible conversion of alcohols to aldehydes or ketones, using NAD+ (nicotinamide adenine dinucleotide) or NADP+ (nicotinamide adenine dinucleotide phosphate) as cofactors. In yeast, alcohol dehydrogenases play a crucial role in the metabolism of ethanol (alcohol) produced during fermentation. In some aspects, the disclosure contemplates microorganisms with modifications of an alcohol dehydrogenase 4 enzyme (adh4) nucleic acid sequence. The ADH4 gene encodes an isoform of alcohol dehydrogenase that catalyze the conversion of alcohols to aldehydes or ketones using cofactors such as NAD+ (nicotinamide adenine dinucleotide) or NADP+ (nicotinamide adenine dinucleotide phosphate). In some aspects, the disclosure contemplates microorganisms with modifications of an DAHP synthase (aro1) nucleic acid sequence. The ARO1 gene encodes the enzyme 3-deoxy- D-arabino-heptulosonate-7-phosphate (DAHP) synthase in various organisms, including yeast and plants. The ARO1 gene encodes the enzyme DAHP synthase, which catalyzes the first step of the shikimate pathway. In this step, DAHP synthase condenses erythrose-4-phosphate and phosphoenolpyruvate to form 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP). 21 FH12463106.4 Attorney Docket No. ISN-00725 In some aspects, the disclosure contemplates microorganisms with modifications of 3- dehydroquinate synthase (aro2) nucleic acid sequence. The ARO2 gene encodes the enzyme 3-dehydroquinate synthase (DHQS), also known as dehydroquinate synthase. This enzyme catalyzes the conversion of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP), an intermediate compound in the shikimate pathway, into 3-dehydroquinate (DHQ). In some aspects, the disclosure contemplates microorganisms with modifications of a 3- dehydroquinate dehydratase (aro3) nucleic acid sequence. The ARO3 gene encodes the enzyme 3-dehydroquinate dehydratase, also known as dehydroquinate dehydratase. This enzyme catalyzes the conversion of 3-dehydroquinate (DHQ), an intermediate compound in the shikimate pathway, into 3-dehydroshikimate (DHS). In some aspects, the disclosure contemplates microorganisms with modifications of a 3- dehydroquinate synthase (aro4) nucleic acid sequence. The ARO4 gene encodes the enzyme 3-dehydroquinate synthase, also known as dehydroquinate synthase. This enzyme catalyzes the conversion of 3-dehydroshikimate (DHS), an intermediate compound in the shikimate pathway, into 3-dehydroquinate (DHQ). In some aspects, the disclosure contemplates microorganisms with modifications of a chorismate mutase (aro7) nucleic acid sequence. The ARO7 gene encodes the enzyme chorismate mutase, which catalyzes the conversion of chorismate to prephenate. Chorismate is a key intermediate in the shikimate pathway, and the conversion to prephenate is a pivotal step towards the production of aromatic amino acids. In some aspects, the disclosure contemplates microorganisms with modifications of a flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) nucleic acid sequence. L-tyrosine ammonia-lyase, TAL or Tyrase) is an enzyme in the natural phenols biosynthesis pathway. It transforms L-tyrosine into p-coumaric acid. In some aspects, the disclosure contemplates microorganisms with modifications of a Trehalose-6-phosphate synthase/phosphatase-like protein (HaTAL1) nucleic acid sequence, from the plant species Helianthus annuus, which is the scientific name for sunflower. Trehalose-6-phosphate synthase/phosphatase (TSP) is an enzyme with dual functionality, acting both as a synthase and a phosphatase. It plays a crucial role in the biosynthesis and metabolism of trehalose, a disaccharide sugar known for its protective role against various 22 FH12463106.4 Attorney Docket No. ISN-00725 stress conditions in plants. As a synthase, TSP catalyzes the formation of trehalose-6- phosphate from glucose-6-phosphate and UDP-glucose. As a phosphatase, it can convert trehalose-6-phosphate back to glucose-6-phosphate and trehalose. In some aspects, the disclosure contemplates microorganisms with modifications of a prephenate dehydratase (pha2) nucleic acid sequence. Prephenate dehydratase catalyzes the conversion of prephenate, an intermediate compound in the shikimate pathway, into phenylpyruvate. This reaction is essential for the biosynthesis of phenylalanine. In some aspects, the disclosure contemplates microorganisms with modifications of a tyrosine aminotransferase (tyr1) nucleic acid sequence. In yeast, tyrosine is metabolized through the tyrosine catabolic pathway to produce various compounds, including 4- hydroxyphenylpyruvate, which can further undergo additional reactions to be converted into other compounds. Tyrosine aminotransferase (encoded by the TYR1 gene) catalyzes the transfer of the amino group from tyrosine to α-ketoglutarate, resulting in the formation of 4- hydroxyphenylpyruvate and glutamate. This reaction is part of the catabolic pathway that breaks down tyrosine into intermediate metabolites. In some aspects, the disclosure contemplates microorganisms with modifications of a tyrosine aminotransferase (tyr2) nucleic acid sequence. In yeast, TRP2 refers to the gene that encodes for the enzyme anthranilate synthase, also known as tryptophan synthase. Anthranilate synthase is a key enzyme involved in the biosynthesis of the essential amino acid tryptophan. Anthranilate synthase (encoded by the TRP2 gene) is believed to be a multifunctional enzyme complex that catalyzes two consecutive reactions in the biosynthetic pathway of tryptophan. It converts chorismate, an intermediate in the shikimate pathway, into anthranilate in the first step. In the second step, it catalyzes the condensation of anthranilate with another molecule, indole, to form tryptophan. In some aspects, the disclosure contemplates microorganisms with modifications of a indole-3-glycerol phosphate synthase (IGPS), also known as tryptophan synthase, (tyr3) nucleic acid sequence. This enzyme converts indole-3-glycerol phosphate (IGP), an intermediate in the shikimate pathway, into indole and 1-(O-carboxyphenylamino)-1-deoxy- D-ribulose 5-phosphate (CDRP) in the first step. In the second step, it catalyzes the condensation of indole with serine and CDRP to form tryptophan. 23 FH12463106.4 Attorney Docket No. ISN-00725 In some aspects, the disclosure contemplates microorganisms with modifications of a anthranilate phosphoribosyltransferase (AnPRT) (tyr4) nucleic acid sequence. Anthranilate phosphoribosyltransferase (encoded by the TRP4 gene) catalyzes a crucial step in the tryptophan biosynthetic pathway. It converts anthranilate, an intermediate in tryptophan biosynthesis, into 5-phosphoribosyl anthranilate (PRA) by transferring a phosphoribosyl group from 5-phosphoribosyl-1-pyrophosphate (PRPP) to anthranilate. In some aspects, the disclosure contemplates microorganisms with modifications of a tryptophan synthase beta chain (tyr5) nucleic acid sequence. The tryptophan synthase complex is responsible for the final step in the biosynthesis of the essential amino acid tryptophan. A modified microorganism can comprise one or more of the modifications described herein, e.g., one or more modifications in genes selected from the group consisting of ADH2, ADH4, ARO1, ARO2, ARO3, ARO4, ARO7, ARO8, ARO9, ARO10, FjTAL, HaTAL1, PHA2, THI3, TYR1, TRP1, TRP2, TRP3, TRP4, and TRP5. The modified microorganism(s) comprising one or more of the modifications described herein can be haploid or diploid microorganism(s). It is contemplated that microorganisms of the disclosure include fungi (e.g., yeasts, including Saccharomyces cerevisiae). Methods for Reducing Shikimate Flux towards 2-PE production and Increasing Bakuchiol Production in Industrial Microorganisms One aspect of the disclosure relates to methods for modulating flux of the shikimate pathway by modifying genes that are directly or indirectly the shikimate pathway flux. In some aspects, the disclosure contemplates methods of reducing a previously unknown by-product of the shikimate pathway, 2-PE. In doing so, the disclosure contemplates that flux towards p- coumaric acid and its derivatives can be increased. In bakuchiol producing strains, the disclosure demonstrates that strategies for reducing 2-PE production can increase bakuchiol production in the microorganism, e.g., a fungal cell such as yeast. In some aspects, the disclosure provides a method for reducing 2-phenylethanol (2-PE) production in a microorganism (e.g., a fungal cell such as yeast) by suppressing a gene from being expressed from i) a phenylpyruvate decarboxylase (aro10) nucleic acid sequence; and/or ii) an aro10 nucleic acid sequence and a branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence in the microorganism (e.g., a fungal cell such as yeast). It is 24 FH12463106.4 Attorney Docket No. ISN-00725 contemplated that suppression of gene expression can be achieved via gene knock-out, modification of a coding sequence, modification of non-coding sequence, or via a transgene. In some aspects the disclosure provides a method for increasing bakuchiol production and reducing 2-phenylethanol (2-PE) production in a microorganism (e.g., a fungal cell such as yeast) cell comprising: a) culturing the microorganism under conditions for: suppressing gene expression of one or both of: i) a phenylpyruvate decarboxylase (ARO10) gene; and ii) a branched-chain-2-oxoacid decarboxylase (THI3) gene via a modification in one or both of a first aro10 nucleic acid target sequence and a first thi3 nucleic acid target sequence within the microorganism; and increasing p-coumarate flux via a modification of at least one target shikimate nucleic acid sequence of one or more genes in the shikimate pathway; thereby increasing bakuchiol production and reducing 2-PE production from the microorganism. In certain microorganism(s) described herein, production of the bakuchiol can be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40%, as compared to a strain that does not have the modification. See, e.g., Fig3 and Fig 4. In some cases, the microorganism is capable of providing a production titer of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 or 25 g/L of bakuchiol, e.g., at least 30 g/L, at least 35 g/L, at least 40 g/L, at least 45 g/L, at least 50 g/L, at least 55 g/L, at least 60 g/L, or at least 65 g/L of the bakuchiol. In many instances, the microorganism is a fungus (e.g., yeast). In certain microorganism(s) described herein, production of the 2-PE can be decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40%, as compared to a strain that does not have the modification. See, e.g., Fig3 and Fig 4. In certain instances, no amounts of 2-PE are produced by the microorganism(s) with these modification as detected by mass-spec. In some instances, the microorganism(s) for increasing the production of bakuchiol while decreasing (or eliminating) 2-PE production are fungal cells producing bakuchiol that comprise a modification in one or both of i) a phenylpyruvate decarboxylase (aro10) nucleic acid sequence and/or ii) a modification in a branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence. Such bakuchiol producing cells may comprise heterologous sequences encoding a bakuchiol synthase. 25 FH12463106.4 Attorney Docket No. ISN-00725 In some implementations, the microorganism comprising select modifications produces 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 µg/L, at least about 0.6 µg/L, at least about 0.7 µg/L, at least about 0.8 µg/L, at least about 0.9 µg/L, at least about 1.0 µg/L, at least about 1.1 µg/L, at least about 1.2 µg/L, at least about 1.3 µg/L, at least about 1.4 µg/L, at least about 1.5 µg/L, at least about 1.6 µg/L, at least about 1.7 µg/L, at least about 1.8 µg/L, at least about 1.9 µg/L, at least about 2.0 µg/L, at least about 2.1 µg/L, at least about 2.2 µg/L, at least about 2.3 µg/L, at least about 2.4 µg/L, at least about 2.5 µg/L, at least about 3.0 µg/L, at least about 4.0 µg/L, at least about 5.0 µg/L, at least about 10.0 µg/L, at least about 15.0 µg/L, at least about 20.0 µg/L, at least about 25.0 µg/L, at least about 30.0 µg/L, at least about 35.0 µg/L, at least about 40.0 µg/L, at least about 45.0 µg/L, at least about 50.0 µg/L, at least 100.0 µg/L, at least about 150.0 µg/L, at least about 200.0 µg/L, at least about 250.0 µg/L, at least about 300.0 µg/L, at least about 350.0 µg/L, at least about 400.0 µg/L, at least about 450.0 µg/L, at least about 500.0 µg/L, at least about 600.0 µg/L, at least about 700.0 µg/L, at least about 800.0 µg/L, at least about 900.0 µg/L, at least about 1.00 mg/L, at least about 1.25 mg/L, at least about 1.50 mg/L, at least about 1.75 mg/L, at least about 2.00 mg/L, at least about 2.25 mg/L, at least about 2.50 mg/L, at least about 2.75 mg/L, at least about 3.00 mg/L, at least about 3.25 mg/L, at least about 3.50 mg/L, at least about 3.75 mg/L, at least about 4.00 mg/L, at least about 4.00 mg/L, at least about 4.25 mg/L, at least about 4.50 mg/L, at least about 4.75 mg/L, at least about 5.00 mg/L or more of bakuchiol within at least about 48 hours of culture as compared to a microorganism not having the modification. Methods for Modulating Flux of the Shikimate Pathway Towards Increased Production of 2-PE and its Downstream Products One aspect of the disclosure relates to methods for modulating flux of the shikimate pathway by increasing shikimate flux towards 2-PE production. The instant disclosure provides the first reporting of 2-PE as a by-product of the shikimate pathway in bakuchiol producing strains. In some aspects, the disclosure contemplates methods of increasing production of a previously unknown by-product of the shikimate pathway, 2-PE, as well as its metabolites in a microorganism, e.g., a fungal cell such as yeast. In some aspects, the disclosure provides a microorganism (e.g., a fungal cell such as yeast) comprising: one or more first modifications selected from: a modified phenylpyruvate 26 FH12463106.4 Attorney Docket No. ISN-00725 decarboxylase (aro10) nucleic acid sequence; or a modified aromatic amino acid aminotransferase 1 (aro8) nucleic acid sequence or a modified aromatic amino acid aminotransferase 2 (aro9) nucleic acid sequence; and one or more second modifications in a shikimate pathway gene for increasing p-coumarate flux whereby the microorganism produces more 2-phenylethanol (2-PE) or a derivative thereof compared to a control microorganism not having the one or more first modifications. The modified aro10 nucleic acid sequence can be for substantially increasing expression of ARO10 gene, thereby increasing flux through increased ARO10 activity. It is contemplated that one such modification is a promoter swap of an endogenous ARO10 promoter with an exogenous promoter that is stronger, e.g., a pGAL1 promoter. See. e.g., FIG.3. In certain cases, the modified aro8 or aro9 nucleic acid sequence(s) can be knock-outs of the ARO8 or ARO9 genes; however the disclosure also contemplates other modifications to regulatory region(s) of ARO8 or ARO9 that substantially suppresses expression of one or both of ARO8 or ARO9 genes. In certain cases, the disclosure contemplates that increase 2-PE production can be achieved by increasing flux towards p-coumaric production. In some aspects, the disclosure contemplates modifications to genes in the shikimate pathway gene for increasing p-coumarate flux, including, but not-limited to, genes selected from the group consisting of ADH2, ADH4, ARO1, ARO2, ARO3, ARO4, ARO7, ARO8, ARO9, ARO10, FjTAL, HaTAL1, PHA2, TYR1, TRP1, TRP2, TRP3, TRP4, and TRP5. In certain microorganism described herein, production of the 2-PE and its derivatives can be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40%, as compared to a strain that does not have the modification in the ARO10 gene. See, e.g., Fig.2̶ Fig.4. In some cases, the microorganism is capable of providing a production titer of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 or 25 g/L of 2-PE and its derivatives, e.g., at least 30 g/L, at least 35 g/L, at least 40 g/L, at least 45 g/L, at least 50 g/L, at least 55 g/L, at least 60 g/L, or at least 65 g/L of 2-PE and its derivatives. In many instances, the microorganism is a fungal cell (e.g., a yeast). 27 FH12463106.4 Attorney Docket No. ISN-00725 Methods for Modulating Flux of the Shikimate Pathway Towards Increased Production of p-coumaric acid and its Derivatives In some aspects, the disclosure contemplates methods and modified microorganism for increased shikimate pathway flux towards anything downstream of phenylalanine, such as p- coumaric acid or p-coumaric acid derivatives. In some instances, the disclosure provides methods for increasing shikimate pathway flux in a microorganism (e.g., a fungal cell such as yeast) comprising: a) culturing the microorganism under conditions for: suppressing gene expression of one or both of: i) a phenylpyruvate decarboxylase (aro10) nucleic acid sequence; and ii) a branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence via a modification in one or both of a first aro10 nucleic acid target sequence and a first thi3 nucleic acid target sequence within the microorganism; a thereby increasing shikimate pathway flux as determined by an increased p-coumaric acid production or p-coumaric acid derivative production compared to a microorganism that does not comprise the modifications. In such instances, production of the p-coumaric acid or p-coumaric acid derivative in an aro10-Δ1 + thi3-Δ1 microorganism can be increased by at least 10%, at least 20%, or at least 30% as compared to production of the p-coumaric acid or p-coumaric acid derivative in a microorganism that does not have the aro10-Δ1 + thi3-Δ1 modifications. In some cases, production of 2-PE is eliminated in an aro10-Δ1 + thi3-Δ1 microorganism as detected by mass- spectrometry. The microorganism can be a Saccharomyces cerevisiae yeast. Bakuchiol-Producing Proteins and Nucleic Acids Not to be bound by particular theory, but one implementation of the methods described herein is based on bakuchiol being produced by a previously unknown prenyltransferase enzyme through a mechanism involving geranyl pyrophosphate (GPP), dimethylallyl pyrophosphate (DMAPP), isopentenyl pyrophosphate (IPP), or a combination thereof, and p- coumaric acid, in microorganisms. In such microorganism, particularly microorganism having heterologous sequences encoding bakuchiol, the flux of the shikimate pathway was observed to be altered. Moreover, previously unreported by-products of the shikimate pathway, namely 2-PE, were identified in bakuchiol producing microorganism. The present disclosure further provides additional example putative prenyltranferase enzymes capable of converting p-coumaric acid and GPP/DMAPP/IPP into bakuchiol. Thus, 28 FH12463106.4 Attorney Docket No. ISN-00725 the present disclosure provides bakuchiol-producing enzymes 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 NOs: 5-21. Table 1: Sequences Gene or Variant SEQ ID No.

29 FH12463106.4 Attorney Docket No. ISN-00725 BAK36(T1) G71D; S108L; T162H; P185V; V199G; P205L; L206Y; W^{209S L226M L234 I274L M287V V312W F318R}

Exemplary Embodiments The following list provides exemplary embodiments. Embodiment 1. A fungal cell producing bakuchiol comprising a modified phenylpyruvate decarboxylase (aro10) nucleic acid sequence. Embodiment 2. The fungal cell of Embodiment 1, wherein the modified aro10 nucleic acid sequence comprises a modification within an aro10 nucleic acid coding sequence. Embodiment 3. The fungal cell of Embodiment 1, wherein the modified aro10 nucleic acid sequence is within an aro10 nucleic acid non-coding sequence. Embodiment 4. The fungal cell of Embodiment 1, wherein the modified aro10 nucleic acid sequence comprises a modification via a transgene independent of the aro10 endogenous locus. Embodiment 5. The fungal cell of Embodiment 1, wherein the modified aro10 nucleic acid sequence is within an aro10 promoter sequence. Embodiment 6. The fungus of Embodiment 5, wherein the modified aro10 nucleic acid sequence comprises an exogenous promoter driving expression of the endogenous ARO10 gene. Embodiment 7. The fungus of Embodiment 6, wherein the exogenous promoter reduces the expression of the endogenous ARO10 gene. 30 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 8. The fungal cell of Embodiment 1, wherein the modified aro10 nucleic acid sequence eliminates gene expression from one allele of the aro10 nucleic acid sequence (aro10-Δ1) in a haploid strain. Embodiment 9. The fungal cell of Embodiment 8, wherein the modified aro10 nucleic acid sequence eliminates gene expression from both alleles of the aro10 nucleic acid sequence (aro10-Δ1) in a diploid strain. Embodiment 10. The fungal cell of Embodiment 8, wherein the modified aro10 nucleic acid sequence eliminates gene expression from one allele of the aro10 nucleic acid sequence (aro10-Δ1) in a diploid strain. Embodiment 11. The fungal cell of Embodiment 1, wherein the bakuchiol is produced from an endogenous p-coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. Embodiment 12. The fungal cell of Embodiment 1, wherein the fungal cell does not produce 2-phenylethanol (2-PE). Embodiment 13. The fungal cell of Embodiment 1, wherein the fungal cell is capable of providing a production titer of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 or 25 g/L of bakuchiol. Embodiment 14. A population of fungal cells of Embodiment 1, wherein the population of fungal cells yields at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 or 25 g/L of bakuchiol. Embodiment 15. A population of fungal cells of Embodiment 1, wherein the population of fungal cells yields at least 5% more bakuchiol compared to a strain not comprising the modified aro10 nucleic acid sequence at a same density. Embodiment 16. The fungal cell of Embodiment 1, wherein the fungal cell is a yeast cell. Embodiment 17. The fungal cell of Embodiment 16, wherein the yeast cell is a Saccharomyces cerevisiae cell. 31 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 18. A composition comprising a population of fungal cells of any one of Embodiments 1 - 17 in a container or package. Embodiment 19. A fungal cell comprising: i) a modification in a phenylpyruvate decarboxylase (aro10) nucleic acid sequence; and ii) a heterologous sequence encoding a bakuchiol synthase. Embodiment 20. The fungal cell of Embodiment 19, wherein the heterologous sequence encoding the bakuchiol synthase is a nucleic acid sequence derived from a plant selected from the group consisting of Psoralea corylifolia, P. grandulosa, P. drupaceae, Ulmus davidiana, Otholobium pubescens, Piper longum, and Aerva sangulnolenta Blum. Embodiment 21. The fungal cell of Embodiment 20, wherein the heterologous sequence encodes an amino acid sequence having at least 80%, at least 85%, at least 95%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. Embodiment 22. The fungal cell of Embodiment 19, wherein the modification in the aro10 nucleic acid sequence is within an aro10 nucleic acid coding sequence. Embodiment 23. The fungal cell of Embodiment 19, wherein the modification in the aro10 nucleic acid sequence is within an aro10 nucleic acid non-coding sequence. Embodiment 24. The fungal cell of Embodiment 19, wherein the modification in the aro10 nucleic acid sequence comprises a modification via a transgene independent of the ARO10 endogenous locus. Embodiment 25. The fungal cell of Embodiment 19, wherein the modification in the aro10 nucleic acid sequence is within an aro10 promoter sequence. Embodiment 26. The fungal cell of Embodiment 25, wherein the modified aro10 nucleic acid sequence comprises an exogenous promoter driving expression of the endogenous aro10 gene. Embodiment 27. The fungal cell of Embodiment 26, wherein the exogenous promoter reduces the expression of the endogenous aro10 gene. 32 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 28. The fungal cell of Embodiment 19, wherein the aro10 nucleic acid sequence modification eliminates gene expression from one allele of the aro10 nucleic acid sequence (aro10-Δ1) in a haploid strain. Embodiment 29. The fungal cell of Embodiment 28, wherein the aro10 nucleic acid sequence modification eliminates gene expression from both alleles of the aro10 nucleic acid sequence (aro10-Δ1) in a diploid strain. Embodiment 30. The fungal cell of Embodiment 28, wherein the aro10 nucleic acid sequence modification eliminates gene expression from one allele of the aro10 nucleic acid sequence (aro10-Δ1) in a diploid strain. Embodiment 31. The fungal cell of Embodiment 19, wherein the bakuchiol is produced from an endogenous p-coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. Embodiment 32. The fungal cell of Embodiment 19, wherein the fungal cell does not produce 2-phenylethanol (2-PE). Embodiment 33. The fungal cell of Embodiment 19, wherein the fungal cell is capable of providing a production titer of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 or 25 g/L of bakuchiol. Embodiment 34. A population of fungal cells of Embodiment 19, wherein the population of fungal cells yields greater than 52 mg/L of bakuchiol. Embodiment 35. A population of fungal cells of Embodiment 19, wherein the population of fungal cells yields at least 5% more bakuchiol compared to a strain not comprising the modified aro10 nucleic acid sequence at a same density. Embodiment 36. The fungal cell of Embodiment 19, wherein the fungal cell is a yeast cell. Embodiment 37. The fungal cell of Embodiment 36, wherein the yeast cell is a Saccharomyces cerevisiae cell. Embodiment 38. A composition comprising a population of fungal cells of any one of Embodiments 19 - 37, in a container or package. 33 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 39. A fungal cell producing bakuchiol comprising a modified branched- chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence. Embodiment 40. The fungal cell of Embodiment 39, wherein the modified thi3 nucleic acid sequence comprises a modification within a thi3 nucleic acid coding sequence. Embodiment 41. The fungal cell of Embodiment 39, wherein the modified thi3 nucleic acid sequence is within a thi3 nucleic acid non-coding sequence. Embodiment 42. The fungal cell of Embodiment 39, wherein the modified thi3 nucleic acid sequence comprises a modification via a transgene independent of the THI3 endogenous locus. Embodiment 43. The fungal cell of Embodiment 39, wherein the modified thi3 nucleic acid sequence is within a thi3 promoter sequence. Embodiment 44. The fungal cell of Embodiment 43, wherein the modified thi3 nucleic acid sequence comprises an exogenous promoter driving expression of the endogenous thi3 gene. Embodiment 45. The fungal cell of Embodiment 44, wherein the exogenous promoter reduces the expression of the endogenous thi3 gene. Embodiment 46. The fungal cell of Embodiment 39, wherein the modified thi3 nucleic acid sequence eliminates gene expression from one allele of the thi3 nucleic acid sequence (thi3-Δ1) in a haploid strain. Embodiment 47. The fungal cell of Embodiment 46, wherein the modified thi3 nucleic acid sequence eliminates gene expression from both alleles of the thi3 nucleic acid sequence (thi3-Δ1) in a diploid strain. Embodiment 48. The fungal cell of Embodiment 46, wherein the modified thi3 nucleic acid sequence eliminates gene expression from one allele of the thi3 nucleic acid sequence (thi3-Δ1) in a diploid strain. Embodiment 49. The fungal cell of Embodiment 39, wherein the bakuchiol is produced from an endogenous p-coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. 34 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 50. The fungal cell of Embodiment 39, wherein the fungal cell is capable of providing a production titer of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 or 25 g/L of bakuchiol. Embodiment 51. A population of fungal cells of Embodiment 39, wherein the population of fungal cells yields greater than 60 mg/L of bakuchiol at a density of 0.4 measured by OD600. Embodiment 52. A population of fungal cells of Embodiment 39, wherein the population of fungal cells yields at least 5% more bakuchiol compared to a strain not comprising the modified thi3 nucleic acid sequence at a same density. Embodiment 53. The fungal cell of Embodiment 39, wherein the fungal cell is a yeast cell. Embodiment 54. The fungal cell of Embodiment 53, wherein the yeast cell is a Saccharomyces cerevisiae cell. Embodiment 55. A composition comprising a population of fungal cells of any one of Embodiments 39 - 54 in a container or package. Embodiment 56. A fungal cell comprising: i) a modification in a branched-chain-2- oxoacid decarboxylase (thi3) nucleic acid sequence; and ii) a heterologous sequence encoding a bakuchiol synthase. Embodiment 57. The fungal cell of Embodiment 56, wherein the heterologous sequence encoding the bakuchiol synthase is a nucleic acid sequence derived from a plant selected from the group consisting of Psoralea corylifolia, P. grandulosa, P. drupaceae, Ulmus davidiana, Otholobium pubescens, Piper longum, and Aerva sangulnolenta Blum. Embodiment 58. The fungal cell of Embodiment 56, wherein the heterologous sequence encodes an amino acid sequence having at least 80%, at least 85%, at least 95%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21. 35 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 59. The fungal cell of Embodiment 56, wherein the modification in the thi3 nucleic acid sequence is within a thi3 nucleic acid coding sequence. Embodiment 60. The fungal cell of Embodiment 56, wherein the modification in the thi3 nucleic acid sequence is within a thi3 nucleic acid non-coding sequence. Embodiment 61. The fungal cell of Embodiment 56, wherein the modification in the thi3 nucleic acid sequence comprises a modification via a transgene independent of the THI3 endogenous locus. Embodiment 62. The fungal cell of Embodiment 56, wherein the modification in the thi3 nucleic acid sequence is within a THI3 promoter sequence. Embodiment 63. The fungal cell of Embodiment 62, wherein the modification in the thi3 nucleic acid sequence comprises an exogenous promoter driving expression of the endogenous THI3 gene. Embodiment 64. The fungal cell of Embodiment 63, wherein the exogenous promoter reduces the expression of the endogenous THI3 gene. Embodiment 65. The fungal cell of Embodiment 56, wherein the thi3 nucleic acid sequence modification eliminates gene expression from one allele of the thi3 nucleic acid sequence (thi3-Δ1) in a haploid strain. Embodiment 66. The fungal cell of Embodiment 65, wherein the thi3 nucleic acid sequence modification eliminates gene expression from both alleles of the thi3 nucleic acid sequence (thi3-Δ1) in a diploid strain. Embodiment 67. The fungal cell of Embodiment 65, wherein the thi3 nucleic acid sequence modification eliminates gene expression from one allele of the thi3 nucleic acid sequence (thi3-Δ1) in a diploid strain. Embodiment 68. The fungal cell of Embodiment 56, wherein the bakuchiol is produced from an endogenous p-coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. 36 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 69. The fungal cell of Embodiment 56, wherein the fungal cell produces a substantially similar amount of 2-phenylethanol (2-PE) as a cell that does not comprise the modification in the thi3 nucleic acid sequence. Embodiment 70. The fungal cell of Embodiment 56, wherein the fungal cell is capable of providing a production titer of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 or 25 g/L of bakuchiol. Embodiment 71. A population of fungal cells of Embodiment 56, wherein the population of fungal cells yields greater than 60 mg/L of bakuchiol. Embodiment 72. A population of fungal cells of Embodiment 56, wherein the population of fungal cells yields at least 5% more bakuchiol compared to a strain not comprising the modified thi3 nucleic acid sequence at a same density. Embodiment 73. The fungal cell of Embodiment 56, wherein the fungal cell is a yeast cell. Embodiment 74. The fungal cell of Embodiment 73, wherein the yeast cell is a Saccharomyces cerevisiae cell. Embodiment 75. A composition comprising a population of fungal cells of any one of Embodiments 56 - 74, in a container or package. Embodiment 76. A fungal cell comprising: i) a modified phenylpyruvate decarboxylase (aro10) nucleic acid sequence; and ii) a modified branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence. Embodiment 77. The fungal cell of Embodiment 76, wherein the fungal cell is a bakuchiol producing fugal cell. Embodiment 78. The fungal cell of Embodiment 77, wherein the bakuchiol is produced from an endogenous p-coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. Embodiment 79. The fungal cell of Embodiment 76, wherein the modified aro10 nucleic acid sequence, the modified (thi3) nucleic acid sequence, or both comprise a 37 FH12463106.4 Attorney Docket No. ISN-00725 modification within an aro10 nucleic acid coding sequence, a modification within a thi3 nucleic acid coding sequence, or a modification within both sequences. Embodiment 80. The fungal cell of Embodiment 76, wherein the modified aro10 nucleic acid sequence, the modified thi3 nucleic acid sequence, or both comprise a modification within an aro10 nucleic acid non-coding sequence, a modification within a thi3 nucleic acid non-coding sequence, or a modification within both sequences. Embodiment 81. The fungal cell of Embodiment 76, wherein the modified aro10 nucleic acid sequence, the modified thi3 nucleic acid sequence, or both comprise a modification via a transgene independent of an ARO10 endogenous locus, a transgene independent of a THI3 endogenous locus, or a transgene independent of both loci. Embodiment 82. The fungal cell of Embodiment 76, the modified aro10 nucleic acid sequence, the modified thi3 nucleic acid sequence, or both comprise a modification within an aro10 nucleic acid promoter sequence, a modification within a thi3 nucleic acid promoter sequence, or a modification within both promoter sequences. Embodiment 83. The fungal cell of Embodiment 82, the modified aro10 nucleic acid sequence, the modified thi3 nucleic acid sequence, or both comprise an exogenous promoter driving expression of the ARO10 gene, the THI3 gene, or both. Embodiment 84. The fungal cell of Embodiment 83, wherein the exogenous promoter reduces the expression of the ARO10 gene, the THI3 gene, or both. Embodiment 85. The fungal cell of Embodiment 76, wherein the modified aro10 nucleic acid sequence, the modified thi3 nucleic acid sequence, or both eliminate gene expression from one allele of the aro10 nucleic acid sequence (aro10-Δ1), one allele of the (thi3-Δ1) nucleic acid sequence, or both in a haploid strain. Embodiment 86. The fungal cell of Embodiment 85, wherein the modified aro10 nucleic acid sequence, the modified thi3 nucleic acid sequence, or both eliminate gene expression from both alleles of the aro10 nucleic acid sequence (aro10-Δ1), one allele of the (thi3-Δ1) nucleic acid sequence, or both in a diploid strain. Embodiment 87. The fungal cell of Embodiment 85, wherein the modified aro10 nucleic acid sequence, the modified thi3 nucleic acid sequence, or both eliminate gene 38 FH12463106.4 Attorney Docket No. ISN-00725 expression from one allele of the aro10 nucleic acid sequence (aro10-Δ1), one allele of the (thi3-Δ1) nucleic acid sequence, or both in a diploid strain. Embodiment 88. The fungal cell of Embodiment 76, wherein the fungal cell does not produce 2-phenylethanol (2-PE). Embodiment 89. The fungal cell of Embodiment 76, wherein the fungal cell produces at least 65 µg/L of the bakuchiol. Embodiment 90. A population of fungal cells of Embodiment 76, wherein the population of fungal cells yields greater than 65 mg/L of bakuchiol at a density of 0.4 measured by OD600. Embodiment 91. A population of fungal cells of Embodiment 76, wherein the population of fungal cells yields at least 5% more bakuchiol compared to a strain not comprising the modified aro10 nucleic acid sequence and the modified thi3 nucleic acid sequence at a same density. Embodiment 92. The fungal cell of Embodiment 76, wherein the fungal cell is a yeast cell. Embodiment 93. The fungal cell of Embodiment 92, wherein the yeast cell is a Saccharomyces cerevisiae cell. Embodiment 94. A composition comprising a population of fungal cells of any one of Embodiments 76 - 93 in a container or package. Embodiment 95. A method for reducing 2-phenylethanol (2-PE) production in a fungus by suppressing a gene from being expressed from i) a phenylpyruvate decarboxylase (aro10) nucleic acid sequence; and/or ii) an aro10 nucleic acid sequence and a branched-chain- 2-oxoacid decarboxylase (thi3) nucleic acid sequence in the fungus. Embodiment 96. The method of Embodiment 95, whereby the fungus is a bakuchiol producing fungus. Embodiment 97. The method of Embodiment 95, whereby the fungus comprises a heterologous sequence encoding a bakuchiol synthase. 39 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 98. The method of Embodiment 97, wherein the heterologous sequence encoding the bakuchiol synthase is a nucleic acid sequence derived from a plant selected from the group consisting of Psoralea corylifolia, P. grandulosa, P. drupaceae, Ulmus davidiana, Otholobium pubescens, Piper longum, and Aerva sangulnolenta Blum. Embodiment 99. The method of Embodiment 97, wherein the heterologous sequence encodes an amino acid sequence having at least 80%, at least 85%, at least 95%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21. Embodiment 100. The method of Embodiment 97, wherein the bakuchiol synthase produces bakuchiol from an endogenous p-coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. Embodiment 101. The method of Embodiment 95, whereby no amounts of 2-PE are detectable via mass-spec in the fungus where expression of either i) aro10 nucleic acid sequence; or ii) aro10 and thi3 nucleic acid sequences are suppressed. Embodiment 102. The method of Embodiment 95, whereby the gene expression of i) the aro10 nucleic acid sequence or ii) the aro10 nucleic acid sequence and the thi3 nucleic acid sequence is suppressed via a nucleic acid modification of the coding sequence. Embodiment 103. The method of Embodiment 95, whereby the gene expression of i) the aro10 nucleic acid sequence or ii) the aro10 nucleic acid sequence and the thi3 nucleic acid sequence is suppressed via a nucleic acid modification of the non-coding sequence. Embodiment 104. The method of Embodiment 95, whereby the gene expression of i) the aro10 nucleic acid sequence or ii) the aro10 nucleic acid sequence and the thi3 nucleic acid sequence is suppressed via a transgene. Embodiment 105. The method of Embodiment 95, wherein the fungus cell is a yeast cell. Embodiment 106. The method of Embodiment 95, wherein the yeast cell is a Saccharomyces cerevisiae cell. 40 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 107. A method for increasing bakuchiol production and reducing 2- phenylethanol (2-PE) production in a yeast cell comprising: culturing the yeast under conditions for: suppressing gene expression of one or both of: i) a phenylpyruvate decarboxylase (ARO10) gene; and ii) a branched-chain-2-oxoacid decarboxylase (THI3) gene via a modification in one or both of a first aro10 nucleic acid target sequence and a first thi3 nucleic acid target sequence within the yeast; and increasing p-coumarate flux via a modification of at least one target shikimate nucleic acid sequence of one or more genes in the shikimate pathway; thereby increasing bakuchiol production and reducing 2-PE production from the fungus. Embodiment 108. The method of Embodiment 107, wherein the one or more genes in the shikimate pathway are selected from the group consisting of ADH2, ADH4, ARO1, ARO2, ARO3, ARO4, ARO7, ARO8, ARO9, ARO10, FjTAL, HaTAL1, PHA2, TYR1, TRP1, TRP2, TRP3, TRP4, and TRP5. Embodiment 109. The method of Embodiment 108, wherein the modification of the at least one target shikimate nucleic acid sequence is a knock-out in the one or more genes in the shikimate pathway that increases p-coumarate flux. Embodiment 110. The method of Embodiment 108, wherein the modification of the at least one target shikimate nucleic acid sequence is a promoter swap in the one or more genes in the shikimate pathway that increases p-coumarate flux. Embodiment 111. The method of Embodiment 107, wherein the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is within a coding region of the ARO10 gene or the THI3 gene. Embodiment 112. The method of Embodiment 107, wherein the modification of the first aro10 nucleic acid sequence is a knock-out of ARO10 (aro10-Δ1) and the modification of the first thi3 nucleic acid sequence is a knock-out of THI3 (thi3-Δ1). Embodiment 113. The method of Embodiment 112, whereby production of the bakuchiol molecule in a aro10-Δ1 + thi3-Δ1 yeast is increased by at least 10%, at least 20%, 41 FH12463106.4 Attorney Docket No. ISN-00725 or at least 30% as compared to expression of the bakuchiol molecule in a fungus that does not have the aro10-Δ1 + thi3-Δ1 modifications. Embodiment 114. The method of Embodiment 112, whereby production of 2-PE is eliminated in an aro10-Δ1 + thi3-Δ1 yeast as detected by mass-spectrometry. Embodiment 115. The method of Embodiment 107, wherein the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is within a non-coding region of the ARO10 gene or the THI3 gene. Embodiment 116. The method of Embodiment 115, wherein the modification suppresses the promoter or another regulatory region of the ARO10 gene or the THI3 gene. Embodiment 117. The method of Embodiment 107, wherein the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is via a transgene. Embodiment 118. The method of Embodiment 107, wherein the yeast is Saccharomyces cerevisiae. Embodiment 119. A yeast comprising: a) one or more first modifications selected from: i) a modified phenylpyruvate decarboxylase (aro10) nucleic acid sequence; or ii) a modified aromatic amino acid aminotransferase 1 (aro8) nucleic acid sequence or a modified aromatic amino acid aminotransferase 2 (aro9) nucleic acid sequence; and b) one or more second modifications in a shikimate pathway gene for increasing p-coumarate flux whereby the yeast produces more 2-phenylethanol (2-PE) or a derivative thereof compared to a control yeast not having the one or more first modifications. Embodiment 120. The yeast of Embodiment 119, wherein the modified aro10 nucleic acid sequence is a modulation of a regulatory region of aro10 that substantially increases expression of ARO10. 42 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 121. The yeast of Embodiment 120, wherein the modified aro10 nucleic acid sequence is a promoter swap of the aro10 nucleic acid sequence. Embodiment 122. The yeast of Embodiment 119, wherein the modified aro8 or aro9 nucleic acid sequence(s) are a knock-out of the ARO8 or ARO9 genes. Embodiment 123. The yeast of Embodiment 122, wherein the modified aro8 or aro9 nucleic acid sequence(s) comprise a modulation of a regulatory region of ARO8 or ARO9 that substantially suppresses expression of one or both of ARO8 or ARO9 genes. Embodiment 124. The yeast of Embodiment 119, wherein the shikimate pathway gene for increasing p-coumarate flux are selected from the group consisting of ADH2, ADH4, ARO1, ARO2, ARO3, ARO4, ARO7, ARO8, ARO9, ARO10, FjTAL, HaTAL1, PHA2, TYR1, TRP1, TRP2, TRP3, TRP4, and TRP5. Embodiment 125. The yeast of Embodiment 119, whereby the yeast produces at least 5% more, at least 10% more, at least 15% more, at least 20% more, at least 25% more, or at least 30% more 2-PE or a derivative thereof compared to a control yeast not having the modified aro10 nucleic acid sequence. Embodiment 126. The yeast of Embodiment 119, wherein the yeast is a bakuchiol producing yeast. Embodiment 127. The yeast of Embodiment 119, wherein the yeast comprises a heterologous sequence encoding a bakuchiol synthase. Embodiment 128. The yeast of Embodiment 127, wherein the heterologous sequence encoding the bakuchiol synthase is a nucleic acid sequence derived from a plant selected from the group consisting of Psoralea corylifolia, P. grandulosa, P. drupaceae, Ulmus davidiana, Otholobium pubescens, Piper longum, and Aerva sangulnolenta Blum. Embodiment 129. The yeast of Embodiment 127, wherein the heterologous sequence encodes an amino acid sequence having at least 80%, at least 85%, at least 95%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21. 43 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 130. The yeast of Embodiment 127, wherein the bakuchiol synthase produces bakuchiol from an endogenous p-coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. Embodiment 131. The yeast of Embodiment 119, whereby the yeast produces at least 5% more, at least 10% more, at least 15% more, at least 20% more, at least 25% more, or at least 30% more bakuchiol or a derivative thereof compared to a control yeast not having the modified aro10 nucleic acid sequence. Embodiment 132. The yeast of Embodiment 119, wherein the yeast is Saccharomyces cerevisiae. Embodiment 133. A method for increasing production of 2-phenylethanol (2-PE) or a derivative thereof in a yeast comprising: culturing the yeast under conditions for: a) overexpressing a phenylpyruvate decarboxylase (aro10) nucleic acid sequence via a modification in a aro10 nucleic acid target sequence; and/or b) suppressing gene expression from one or both of an aromatic amino acid aminotransferase 1 (aro8) nucleic acid sequence or an aromatic amino acid aminotransferase 2 (aro9) nucleic acid sequence; thereby increasing production of 2-PE or a derivative thereof from the yeast. Embodiment 134. The method of Embodiment 133, wherein the modified aro10 nucleic acid sequence is a modulation of a regulatory region of aro10 that substantially increases expression of aro10. Embodiment 135. The method of Embodiment 134, wherein the modified aro10 nucleic acid sequence is a promoter swap of the aro10 nucleic acid sequence. Embodiment 136. The method of Embodiment 133, wherein the modified aro8 or aro9 nucleic acid sequence(s) are a knock-out of the ARO8 or ARO9 genes. Embodiment 137. The method of Embodiment 133, wherein the modified aro8 or aro9 nucleic acid sequence(s) comprise a modulation of a regulatory region of ARO8 or ARO9 that substantially suppresses expression of one or both of ARO8 or ARO9. 44 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 138. The method of Embodiment 133, whereby the yeast is a bakuchiol producing yeast. Embodiment 139. The method of Embodiment 133, whereby the yeast comprises a heterologous sequence encoding a bakuchiol synthase. Embodiment 140. The method of Embodiment 139, wherein the heterologous sequence encoding the bakuchiol synthase is a nucleic acid sequence derived from a plant selected from the group consisting of Psoralea corylifolia, P. grandulosa, P. drupaceae, Ulmus davidiana, Otholobium pubescens, Piper longum, and Aerva sangulnolenta Blum. Embodiment 141. The method of Embodiment 139, wherein the heterologous sequence encodes an amino acid sequence having at least 80%, at least 85%, at least 95%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21. Embodiment 142. The method of Embodiment 139, wherein the bakuchiol synthase produces bakuchiol from an endogenous p-coumaric acid precursor and an endogenous geranyl pyrophosphate (GPP) precursor. Embodiment 143. The method of Embodiment 133, whereby the yeast produces at least 5% more, at least 10% more, at least 15% more, at least 20% more, or at least 25% more 2-PE or a derivative thereof compared to a yeast not having the modification in the aro10 nucleic acid target sequence. Embodiment 144. A method for increasing shikimate pathway flux in a yeast comprising: culturing the yeast under conditions for: suppressing gene expression of one or both of: i) a phenylpyruvate decarboxylase (aro10) nucleic acid sequence; and ii) a branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence via a modification in one or both of a first aro10 nucleic acid target sequence and a first thi3 nucleic acid target sequence within the yeast; 45 FH12463106.4 Attorney Docket No. ISN-00725 thereby increasing shikimate pathway flux as determined by an increased p-coumaric acid production or p-coumaric acid derivative production compared to a yeast that does not comprise the modifications. Embodiment 145. The method of Embodiment 144, wherein the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is within a coding region of one or both the ARO10 gene and the THI3 gene. Embodiment 146. The method of Embodiment 145, wherein the modification of the first aro10 nucleic acid sequence is a knock-out of ARO10 (aro10-Δ1) and the modification of the first thi3 nucleic acid sequence is a knock-out of THI3 (thi3-Δ1). Embodiment 147. The method of Embodiment 144, whereby production of the p- coumaric acid or p-coumaric acid derivative in an aro10-Δ1 + thi3-Δ1 yeast is increased by at least 10%, at least 20%, or at least 30% as compared to expression of the bakuchiol molecule in a fungus that does not have the aro10-Δ1 + thi3-Δ1 modifications. Embodiment 148. The method of Embodiment 144, whereby production of 2-PE is eliminated in an aro10-Δ1 + thi3-Δ1 yeast as detected by mass-spectrometry. Embodiment 149. The method of Embodiment 144, wherein the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is within a non-coding region of one or both the ARO10 gene and the THI3 gene. Embodiment 150. The method of Embodiment 149, wherein the modification in the non-coding region substantially suppresses the expression of one or more of the aro10 nucleic acid sequence and the thi3 nucleic acid sequence. Embodiment 151. The method of Embodiment 144, wherein the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is within a non-coding region of one or both the ARO10 gene and the THI3 gene. Embodiment 152. The method of Embodiment 151, wherein the modification suppresses the promoter or another regulatory region of one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence. 46 FH12463106.4 Attorney Docket No. ISN-00725 Embodiment 153. The method of Embodiment 144, wherein the modification in one or both of the first aro10 nucleic acid target sequence and the first thi3 nucleic acid target sequence is via a transgene. Embodiment 154. The method of Embodiment 144, wherein the yeast is Saccharomyces cerevisiae. Examples Example 1 – Identification of 2-phenylethanol (2-PE) as a by-product of the shikimate pathway in a bakuchiol producing strain. Modified Saccharomyces cerevisiae strains were made to express a heterologous nucleic acid sequence encoding a bakuchiol synthase. It was previously discovered that bakuchiol could be produced in yeast by using a heterologous enzyme via two distinct precursors: p- coumaric acid and a geranyl pyrophosphate (GPP) precursor. Said bakuchiol synthase is not endogenous to yeast species. Bakuchiol is formed when the carboxylic acid group in p- coumaric acid is replaced by the geranyl group from the geranyl pyrophosphate. The identified bakuchiol synthase catalyzes the reaction in fungus (e.g., yeast) thereby generating bakuchiol in model organism(s) previously unable to produce detectable amounts of bakuchiol. The chemical reaction is illustrated below. 47 FH12463106.4 Attorney Docket No. ISN-00725

Because p-coumaric acid is a precursor of bakuchiol, it was hypothesized that increasing flux into the shikimate pathway to increase p-coumaric acid production could lead to increased yields of bakuchiol itself in bakuchiol producing fungus strains. To test this hypothesis in an exemplary fungus, a series of Saccharomyces strains were created with various types of modifications in genes within the Shikimate pathway (which produces p-coumaric acid). Figure 1 is a schematic illustrating the shikimate pathway, the terpene pathway, and the intersection where the bakuchiol synthase produces bakuchiol from p-coumaric acid and GPP. Figure 1 underscores select genes in the Shikimate pathway that produced unexpected outcomes when altered in a bakuchiol producing strain. TABLE 2: Selected strains and their genetic modifications. Strain*; ** Gene Modified Modification d d

48 FH12463106.4 Attorney Docket No. ISN-00725 Strain*; ** Gene Modified Modification STR2292 ARO10 THI3 ARO10 knockout (aro10-Δ1) and THI3 knock-out (thi3-Δ1) er a d d d d 4 5 d d d d ol

49 FH12463106.4 Attorney Docket No. ISN-00725 Strain*; ** Gene Modified Modification STR2236 Bakuchiol producing base strain a

** all strains further comprise a heterologous gene encoding a bakuchiol synthase. During these efforts, a by-product of the shikimate pathway; namely 2-phenylethanol (2- PE) was observed in certain strains.

no other group has reported biologically producing bakuchiol in yeast, there has been no prior reported precedent of 2-PE production while trying to produce bakuchiol. Some literature report attempts to engineer S. cerevisiae only for increasing 2-PE production. Moreover, no attempt was reported in a bakuchiol producing strain. See (Kim, B., Cho, B.-R. and Hahn, J.-S. (2014), Metabolic engineering of Saccharomyces cerevisiae for the production of 2-phenylethanol via Ehrlich pathway. Biotechnol. Bioeng., 111: 115-124). Without being bound to any scientific theory, the 2-PE byproduct production can consume and even competes for valuable shikimate pathway flux and thus reduce such flux available for bakuchiol production. As such, gene modifications are pursued with an aim to reduce 2-PE. The gene modifications pursued include promoter swaps and gene knockouts, for assessing the impact of various overexpressions and knockouts (KOs). As depicted in Figure 2, knocking out ARO10 eliminates 2-PE production and slightly increases bakuchiol production (see bar graphs for STR1552 and STR2254). The X-axis corresponds to strains or strain pool used in the experiment. The Y-axis shows the amount of 50 FH12463106.4 Attorney Docket No. ISN-00725 2-phenylethanol [mg/L] in comparison to the OD₆₀₀ of the culture. In strains where the endogenous promoter of the ARO10 gene was replaced with an inducible pGAL promoter (pGAL1>ARO10) Applicant observed a significant increase in 2-PE production and a significant reduction in bakuchiol. See Figure 3. The X-axis of figure 3 corresponds to strains or strain pool used in the experiment. The Y-axis depicts the density of each culture (OD600), the amount of bakuchiol produced by each strain (mg/L), the amount of 2-PE produced (mg/L), and the amount of a control product produced, mevalonolactone (mg/L). The amounts produced for each tested product from a strain without the gene modifications (“parent strain”) is shown as a comparator. As shown in Figure 3, knocking out THI3 (thi3-Δ1) has no impact on 2-PE production and slightly increases bakuchiol production. Swapping the promoter in front of THI3 with pGAL1 promoter, thus creating a strain with a chimeric, inducible promoter driving the expression of THI3, has no impact on 2-PE production and significantly increases bakuchiol production See Figure 3, under the label pGAL1>THI3. Knocking out both targets (aro10-Δ1 thi3-Δ1) eliminates 2-PE production and increases bakuchiol production. See Figure 3, aro10-Δ1 thi3-Δ1. In summary, a series of Saccharomyces strains were generated with modifications in genes for efficiently controlling production of 2-PE, including (1) strains that successfully eliminate the production of 2-PE for a high bakuchiol flux strain; and (2) strains that successfully increase the production of 2-PE resulting in a high 2-PE flux strain (see Table 2 with a representative list of Saccharomyces strains generated). It is contemplated that reduced 2-PE flux can be used for increased production of bakuchiol. It is also contemplated that reduced 2-PE for increased shikimate pathway flux can be used to increase flux towards compounds downstream of phenylalanine, such as p-coumaric acid or p-coumaric acid derivatives. Example 2 – Modulating Production of 2-phenylethanol (2-PE) in a bakuchiol producing strain. Modified Saccharomyces cerevisiae strains were generated to express a heterologous nucleic acid sequence encoding a bakuchiol synthase, along with various other gene modifications (e.g., gene knock-outs and promoter swaps). Briefly, bakuchiol producing strains were further engineered with knock-out modifications of several genes in the shikimate 51 FH12463106.4 Attorney Docket No. ISN-00725 pathway, including knockouts of ARO8 and ARO9. See Figure 1, underscoring ARO8, ARO9, and ARO10 in the shikimate pathway. Surprisingly, at least in bakuchiol producing strains, the impact of aro8 and aro9 deletions was remarkably different from what had been taught in the art. Some literature suggests, for example that ARO8 and ARO9 are genes that carry out the same function. See, e.g., Iraqui I, Vissers S, Cartiaux M (1998) Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily. Mol Gen Genet 257, 238–248. Yet, other reports suggest that ARO8 must be upregulated to increase 2-PE flux while ARO9 should be eliminated (see Dai J, Xia H, Yang C and Chen X (2021) Sensing, uptake and catabolism of L-phenylalanine during 2-phenylethanol biosynthesis via the Ehrlich Pathway in Saccharomyces cerevisiae. Front. Microbiol. 12:601963); and yet other reports suggests the opposing outcome (see Hassing EJ, de Groot P, Marquenie V, Pronk J, Daran J (2019) Connecting central carbon and aromatic amino acid metabolisms to improve de novo 2-phenylethanol production in Saccharomyces cerevisiae. Metabolic Engineering. Volume 56, December 2019, Pages 165-180. As illustrated in Figure 4, in a bakuchiol producing strain, knock out of either ARO8 (aro8- Δ1) or ARO9 (aro9-Δ1) increases 2-PE production. Significant distinctions are observed with regards to the production of 2-PE and bakuchiol: aro8-Δ1 leads to a significant increase in 2- PE and a reduction of bakuchiol production; while aro9-Δ1 leads to a slight (but clear) increase in 2-PE and an increase in bakuchiol production. Without being bound to any specific theory, the unique nature of the tested strains with a strong bakuchiol synthase pulling on shikimate flux can be a contributor of the high 2-PE production. These bakuchiol-producing aro8-Δ1 and/or aro9-Δ1 strains effectively increase 2-PE flux for 2-PE production in Saccharomyces strains. It is contemplated that increased 2-PE flux can be used for production of another compound further downstream of 2-PE. Example 3 – Modulating Production of bakuchiol in Saccharomyces strains with Modifications in Genes in the Shikimate Pathway Saccharomyces cerevisiae strains were generated expressing a heterologous nucleic acid sequence encoding a bakuchiol synthase and various gene modifications (e.g., gene knock-outs and promoter swaps). The Saccharomyces strains are described in the above section. As 52 FH12463106.4 Attorney Docket No. ISN-00725 depicted in Figure 3, knocking out both ARO10 and THI3 genes (aro10-Δ1 thi3-Δ1) eliminates 2-PE production and increases bakuchiol production. See Figure 3, aro10-Δ1 thi3-Δ1. Example 4 –Bakuchiol producing strains are sensitized to 2-PE toxicity which can be overcome by ARO10 removal. Modified S. cerevisiae strains that produce bakuchiol exhibit a higher sensitivity to 2-PE toxicity as observed in a minimal inhibitory concentration (MIC) assay (Figure 5). In tanks, as 2-PE accumulates, bakuchiol titers start to decline. Without being bound to any specific theory, this toxicity may be due to the suppression of nutrient transport by 2-PE. Bakuchiol-producing strains exhibit lower bakuchiol titers when fed with amino acids. Consistently, ARO10 is induced by aromatic amino acids. Knocking out ARO10 also addresses the 2-PE toxicity issue and allows for the doubling of bakuchiol titers in tanks (Figure 6). Example 5 – The production of 2-PE via fermentation. 2-PE is a sought-after ingredient and widely used in the food, cosmetic, and fragrance industries. Certain S. cerevisiae strains are also used to produce 2-PE via formation (Figure 7). 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. 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. 53 FH12463106.4

Claims

Attorney Docket No. ISN-00725 CLAIMS WHAT IS CLAIMED IS: 1. A fungal cell producing bakuchiol comprising one or both of (i) a modified phenylpyruvate decarboxylase (aro10) nucleic acid sequence and (ii) a modified branched-chain-2-oxoacid decarboxylase (thi3) nucleic acid sequence. 2. The fungal cell of claim 1, wherein the modified aro10 nucleic acid sequence comprises a modification within an aro10 coding sequence, and wherein the modified thi3 nucleic acid sequence comprises a modification within a thi3 coding sequence. 3. The fungal cell of claim 1, wherein the modified aro10 nucleic acid sequence is within an aro10 non-coding sequence, and wherein the modified thi3 nucleic acid sequence comprises a modification within a thi3 non-coding sequence. 4. The fungal cell of claim 1, wherein the modified aro10 or thi3 nucleic acid sequence comprises a modification via a transgene independent of the aro10 or thi3 endogenous locus, respectively. 5. The fungal cell of claim 1, wherein the modified aro10 or thi3 nucleic acid sequence is within an aro10 or thi3 promoter sequence, respectively. 6. The fungus of claim 5, wherein the modified aro10 or thi3 nucleic acid sequence comprises an exogenous promoter driving expression of the endogenous ARO10 or THI3 gene, respectively. 7. The fungus of claim 6, wherein the exogenous promoter reduces the expression of the endogenous ARO10 or THI3 gene. 8. The fungal cell of claim 1, wherein the modified aro10 or thi3 nucleic acid sequence eliminates gene expression from one allele of the aro10 nucleic acid sequence (aro10-Δ1) or one allele of the thi3 nucleic acid sequence (thi3-Δ1), respectively. 9. The fungal cell of claim 1, wherein the fungal cell does not produce 2-phenylethanol (2- PE). 10. The fungal cell of claim 1, wherein the fungal cell is capable of providing a production titer of at least 6 g/L of bakuchiol. 11. A population of fungal cells of claim 1, wherein the population of fungal cells yields at least 9 g/L of bakuchiol. 54 FH12463106.4 Attorney Docket No. ISN-00725 12. A population of fungal cells of claim 1, wherein the population of fungal cells yields at least 5% more bakuchiol compared to a strain not comprising the modified aro10 nucleic acid sequence at a same density. 13. The fungal cell of claim 1, wherein the fungal cell is a yeast cell. 14. The fungal cell of claim 13, wherein the yeast cell is a Saccharomyces cerevisiae cell. 15. A composition comprising a population of fungal cells of claim 1 in a container or package. 16. A yeast comprising: a) one or more first modifications selected from: i) a modified phenylpyruvate decarboxylase (aro10) nucleic acid sequence; or ii) a modified aromatic amino acid aminotransferase 1 (aro8) nucleic acid sequence or a modified aromatic amino acid aminotransferase 2 (aro9) nucleic acid sequence; and b) one or more second modifications in a shikimate pathway gene for increasing p- coumarate flux; whereby the yeast produces more 2-phenylethanol (2-PE) or a derivative thereof compared to a control yeast not having the one or more first modifications. 17. The yeast of claim 16, wherein the modified aro10 nucleic acid sequence is a modulation of a regulatory region of aro10 that substantially increases expression of ARO10. 18. The yeast of claim 16, wherein the modified phenylpyruvate decarboxylase (aro10) nucleic acid sequence encodes an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% identity to SEQ ID NO: 1; wherein the modified aromatic amino acid aminotransferase 1 (aro8) nucleic acid sequence encodes an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% identity to SEQ ID NO: 3; and wherein the modified aromatic amino acid aminotransferase 2 (aro9) nucleic acid sequence encodes an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% identity to SEQ ID NO: 4. 19. A method for increasing production of 2-phenylethanol (2-PE) or a derivative thereof in a yeast comprising: culturing the yeast under conditions for: a) overexpressing a phenylpyruvate decarboxylase (aro10) nucleic acid sequence via a modification in an aro10 nucleic acid target sequence; and/or 55 FH12463106.4 Attorney Docket No. ISN-00725 b) suppressing gene expression from one or both of an aromatic amino acid aminotransferase 1 (aro8) nucleic acid sequence or an aromatic amino acid aminotransferase 2 (aro9) nucleic acid sequence; thereby increasing production of 2-PE or a derivative thereof from the yeast. 20. The method of claim 19, wherein the modified aro10 nucleic acid sequence is a modulation of a regulatory region of aro10 that substantially increases expression of aro10. 56 FH12463106.4