WO2019090286A2 - Production of valerenic acid in fungal cells - Google Patents

Abstract

Provided are fungal cells, e.g., yeast cells, that express heterologous enzymes on the biosynthesis pathway for valerenic acid and produce high levels of valerena-4,7(11)-diene and/or valerenic acid.

Description

PRODUCTION OF VALERENIC ACID IN FUNGAL CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of USSN 62/582,201, filed on

November 6, 2017, which is incorporated herein by reference in its entirety for all purposes. STATEMENT OF GOVERNMENTAL SUPPORT

[0002] This invention was made with government support under 1442724 awarded by the National Science Foundation. The government has certain rights in the invention.

BACKGROUND

[0003] The popularity of herbal medicines is exemplified by a $60 billion industry worldwide, and 20% of the total drug market (Kirk et al. (2014) Adv. Chronic Kidney Dis. 21(4): 377-384). Valerian, a medicinal preparation of Valeriana officinalis rhizome, root, and stolon extracts, is one such herbal medicine, and has been valued for its anxiolytic and sedative properties for millennia (Houghton (1988) J. Ethnopharmacol. 22(2): 121-142; Eadie (2004) Epilepsia, 45(11): 1338-1343). Valerian is currently 'generally recognized as safe' by the US Food and Drug Administration, and has been approved as a natural sleep aid in several countries (Kumar (2006) Phytother. Res. 20(12): 1023-1035). While V. officinalis extracts contain many bioactive compounds such as phenolic acids, valepotriates, lignans, flavonoids, amino acids, alkaloids, and tannins, the compound responsible for valerian's well-known activities is the sesquiterpenoid valerenic acid (Hritcu et al. (2016) Prevalence of use of herbal medicines and complementary and alternative medicine in Europe. In: Grosso, C. ed. Herbal medicine in depression. Cham: Springer International Publishing, pp. 135-181; Trauner e/ a/. (2008) Planta Medica, 74(1): 19-24; Takemoto et al. (2009) J. Nat. Med.

63(4): 380-385) (Fig. 1, panel A). Interestingly, valerenic acid has been shown to be produced in only two related genera, Valeriana and Centranthus, primarily in the root and inflorescence tissues (Hassan et al. (2008) Asian J. Plant Sci. 7(2): 195-200). Until recently, the mechanism for valerenic acid activity was unknown. In vivo studies in mice showed that valerenic acid allosterically modulates GAB AA receptor activity leading to sedative or anxiolytic effects (Becker et al. (2014) BMC Comp Alternativ. Med. 14: 267; Benke et al. (2009) Neuropharmacol. 56(1): 174-181; Trauner e/ a/. (2008) Planta Medica, 74(1): 19-24). Interestingly, studies showed that V. officinalis root extracts contain another naturally occurring valerenic acid-derived sesquiterpenoid, acetoxyvalerenic acid, that diminishes the anxiolytic effects of valerenic acid by binding to identical sites (Felgentreff et al. (2012) Phytomed. 19(13): 1216-1222; Khom, et al. (2007) Neuropharmacol. 53(1): 178-187; Benke et al. (2009) Neuropharmacol. 56(1): 174-181) (Fig. 1, panel A). Indeed, studies have shown that extracts with high valerenic acid:acetoxyvalerenic acid ratios have more pronounced sedative effects (Felgentreff et al. supra.). Effective use of valerian is hampered by inaccurate dosage guidelines and highly variable acetoxyvalerenic acid content, which often makes up a significant amount of crude root extract (Becker et al. supra., Felgentreff et al. supra.). Indeed, synthesis of valerenic acid is a source of isolated valerenic acid but suffers from low yields (ca. 6%) after many chemical conversion steps starting from (R)-pulegone (Kopp et al. (2009) Synlett. (11): 1769-1772). Microbial production of valerenic acid promises isolation of valerian's active compound in the absence of antagonistic

acetoxyvalerenic acid, lowers production costs, and does not suffer from inconsistent composition nor variable yields of plant derived valerenic acid which is the current source of this herbal medicine. [0004] The biosynthetic pathway for valerenic acid has not been fully known, but is thought to proceed from central carbon metabolism through the mevalonate pathway and farnesyl pyrophosphate (FPP), then cyclized by a sesquiterpene synthase, valerena-4,7(l 1)- diene synthase (VDS), to form valerena-4,7(l l)-diene, and likely decorated by one or more P450s, acyltransferases, and other modifying enzymes. Previous studies have found that the highest concentration of valerenic acid is localized to V. officinalis root tissues, and an expression study showed VDS transcripts were almost exclusively expressed in the root relative to other tissues (Yeo et al. (2013) J. Biol. Chem., 288(5): 3163-3173). Several Asteraceae P450s in the CYP71D clade have been shown to oxidize a primary carbon on a sesquiterpene hydrocarbon substrate. These include the Artemisia annua AaCYP71 AVI (which converts amorpha-4,11-diene to artemisinic acid, a precursor to the anti -malarial drug artemisinin), various germacrene A oxidases (GAOs) (which convert germacrene A to germacra-l(10),4, l l(13)-trien-12-oic acid, the precursor to the anti-cancer compound costunolide (Nguyen et al. (2010) J. Biol. Chem. 285(22): 16588-16598; Ro et al. (2006) Nature, 440(7086): 940-943; Ikezawa et a/. (2011) J. Biol. Chem. 286(24): 21601-21611), and Thapsia garganica TgCYP76AE2 (which catalyzes analogous oxidations on epikunzeaol to form epidihydrocostunolide (Andersen et al. (2017) Plant Physiol.11 '4(1): 56-72). Due to the high similarity of other Asteraceae sesquiterpenoid and the valerenic acid biosynthetic pathways, previous studies have postulated that a P450 may catalyze conversion of valerenadiene into valerenic acid (Fig. 1, panel A).

[0005] Thus far, attempts at identifying the full biosynthetic pathway for producing the drug valerenic acid or reconstituting this activity in a heterologous host have failed (Ricigliano et al. (2016) Phytochem. 125: 43-53).

SUMMARY

[0006] Herein, we demonstrate the use of yeast as an expression platform for the production of valerenic acid. As demonstrated herein, we engineered a yeast chassis for the production of the valerenic acid precursor valerenadiene at a titer of about 140 mg/L. Then, we used phylogenetic and expression analysis to identify a root-upregulated V. officinalis valerenadiene oxidase, VoCYP71DJl, and use dehydrogenases to produce a yeast strain capable of generating valerenic acid at about 4 mg/L.

[0007] Accordingly, various embodiments contemplated herein may include, but need not be limited to, one or more of the following: [0008] Embodiment 1 : A fungal cell that produces at least about 30 mg, e.g., at least about 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or more, valerena-4,7(l l)-diene per liter of culture.

[0009] Embodiment 2: The fungal cell of embodiment 1, comprising a

polynucleotide encoding a fusion protein comprising maltose binding protein (MBP)- valerena-4,7(l l)-diene synthase (VDS)- farnesyl diphosphate (FPP) synthase (ERG20) integrated into the genome of the fungal cell.

[0010] Embodiment 3 : The fungal cell of any one of embodiments 1 to 2, comprising two, three, four or more copies of the polynucleotide encoding a fusion protein comprising MBP-VDS-ERG20 integrated into its genome. [0011] Embodiment 4: The fungal cell of any one of embodiments 1 to 3, wherein the fusion protein comprising MBP-VDS-ERG20 has at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4.

[0012] Embodiment 5: A fungal cell that produces detectable levels of valerenic acid. In various embodiments, the fungal cell produces at least about 4 mg valerenic acid per liter of culture. [0013] Embodiment 6: A fungal cell comprising heterologous enzymes in the biosynthetic pathway of valerenic acid, wherein the fungal cell produces valerenic acid.

[0014] Embodiment 7: The fungal cell of any one of embodiments 5 to 6, comprising one or more polynucleotides encoding heterologous enzymes in the valerenic acid

biosynthesis pathway stably integrated into the genome of the cell.

[0015] Embodiment 8: The fungal cell of embodiment 7, wherein the one or more polynucleotides are integrated in the genome at the uridine auxotrophy (URA3) locus.

[0016] Embodiment 9: The fungal cell of any one of embodiments 7 to 8, wherein the one or more polynucleotides are operably linked to and expressed under the control of a galactose inducible promoter.

[0017] Embodiment 10: The fungal cell of any one of embodiments 1 to 9, wherein the fungal cell is transformed with heterologous polynucleotides encoding (i) a valerenadiene synthase (VDS), (ii) a Valeriana officinalis P450 VoCYP71DJl or homolog thereof that functions as a valerenadiene oxidase, (iii) an alcohol dehydrogenase and (iv) an aldehyde dehydrogenase.

[0018] Embodiment 11 : A transformed fungal cell comprising one or more heterologous polynucleotides integrated into the fungal cell's genome, wherein the one or more heterologous polynucleotides encode a valerenadiene synthase (VDS), that converts farnesyl pyrophosphate (FPP) to valerenadiene, wherein the fungal cell produces valerenic acid.

[0019] Embodiment 12: The fungal cell of embodiment 11, wherein the heterologous polynucleotides encode a valerenadiene oxidase comprising at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6 {Valeriana officinalis P450 VoCYP71DJl) and that converts valerenadiene to valerenic alcohol.

[0020] Embodiment 13 : The fungal cell of any one of embodiments 11 to 12, wherein the heterologous polynucleotides encode an alcohol dehydrogenase (ADH) that converts valerenic alcohol to valerenic aldehyde.

[0021] Embodiment 14: The fungal cell of any one of embodiments 11 to 13, an aldehyde dehydrogenase (ALDH) that converts valerenic aldehyde to valerenic acid. [0022] Embodiment 15: The fungal cell of any one of embodiments 5 to 14, wherein one or more polynucleotides encoding the heterologous enzymes comprise codon bias for improved expression in the fungal cell.

[0023] Embodiment 16: The fungal cell of any one of embodiments 5 to 15, wherein the fungal cell is transformed with polynucleotides encoding Valeriana officinalis P450 VoCYP71DJl, Artemisia annua alcohol dehydrogenase (AaADHl) and annua aldehyde dehydrogenase (AaALDHl).

[0024] Embodiment 17: The fungal cell of any one of embodiments 5 to 16, wherein the fungal cell is transformed with a fusion protein comprising MBP-VDS-ERG20. [0025] Embodiment 18: The fungal cell of any one of embodiments 6 to 17, wherein the fungal cell produces at least about 4 mg valerenic acid per liter of culture.

[0026] Embodiment 19: The fungal cell of any one of embodiments 1 to 18, wherein the fungal cell is a yeast cell, e.g., a Saccharomyces cell, a Saccharomyces cerevisiae cell.

[0027] Embodiment 20: The fungal cell of embodiment 19, wherein the yeast cell is from a genus selected from the group consisting of Saccharomyces, Pichia, Kluyveromyces, Candida, Aspergillus, Trichoderma, Chrysosporium, Fusarium and Neurospora.

[0028] Embodiment 21 : A fungal cell culture comprising a population of fungal cells as set forth in any one of embodiments 1 to 20.

[0029] Embodiment 22: A fungal cell culture comprising a population of fungal cells that produce detectable levels of valerenic acid. In various embodiments, the fungal cell culture produces at least about 4 mg valerenic acid per liter of culture.

[0030] Embodiment 23 : The fungal cell culture of embodiment 22, wherein the culture does not contain detectable or is devoid of hydroxyvalerenic acid and/or

acetoxyvalerenic acid. In some embodiments, the culture comprises detectable amounts of hydroxyvalerenic acid and/or acetoxyvalerenic acid.

[0031] Embodiment 24: A fungal cell culture that produces at least about 30 mg, e.g., at least about 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or more, valerena-4,7(l l)-diene per liter of culture.

[0032] Embodiment 25: A bioreactor comprising a fungal cell culture of any one of embodiments 21 to 24. [0033] Embodiment 26: The bioreactor of embodiment 25, comprising a culture volume in the range of 0.5L, 1L, 2L, 3L, 4L, 5L, 10L, 25L, 50L, 75L, 100L, 250L, 500L, 1000L, or more.

[0034] Embodiment 27: A method of producing valerenic acid comprising culturing a population of fungal cells of any one of embodiments 5 to 20 under conditions sufficient for the fungal cells to produce valerenic acid.

[0035] Embodiment 28: The method of embodiment 27, wherein the population of fungal cells is cultured in a medium comprising galactose.

[0036] Embodiment 29: The method of any one of embodiments 27 to 28, wherein the population of fungal cells is cultured in a medium lacking uridine.

[0037] Embodiment 30: The method of any one of embodiments 27 to 29, further comprising lysing the cells and/oror extracting with a solvent to isolate valerenic acid.

[0038] Embodiment 31 : The method of embodiment 30, wherein the valerenic acid is substantially or entirely free of hydroxyvalerenic acid and/or acetoxyvalerenic acid. [0039] Embodiment 32: Valerenic acid produced from a fungal cell of any one of embodiments 1 to 20. In various embodiments, the valerenic acid is formulated in a pharmaceutical composition or a nutraceutical composition.

[0040] Embodiment 33 : Valerenic acid of embodiment 32, wherein the valerenic acid is substantially or entirely free of hydroxyvalerenic acid and/or acetoxyvalerenic acid. [0041] Embodiment 34: A fusion protein comprising maltose binding protein (MBP)- valerena-4,7(l l)-diene synthase (VDS)- farnesyl diphosphate (FPP) synthase (ERG20).

[0042] Embodiment 35: The fusion protein of claim 34, wherein the MBP-VDS-

ERG20 fusion protein comprises at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4.

[0043] Embodiment 36: A polynucleotide encoding the MBP-VDS-ERG20 fusion protein of claim 34.

[0044] Embodiment 37: The polynucleotide of claim 36, wherein the polynucleotide comprises at least about 80%> sequence identity, e.g., at least about 85%>, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3. [0045] Embodiment 38: A fungal cell comprising the polynucleotide of claim 36.

[0046] Embodiment 39: An isolated and/or recombinant polynucleotide encoding a valerenadiene oxidase comprising at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6 (Valeriana officinalis P450 VoCYP71DJl) and that converts valerenadiene to valerenic alcohol.

[0047] Embodiment 40: A fungal cell comprising a heterologous polynucleotide encoding a valerenadiene oxidase comprising at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6 (Valeriana officinalis P450 VoCYP71DJl) and that converts valerenadiene to valerenic alcohol.

[0048] Embodiment 41 : An isolated and/or recombinant polynucleotide encoding a

P450 enzyme comprising at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 8, 10, 12, 14 or 16, wherein the P450 enzyme is enzymatically active.

[0049] Embodiment 42: A fungal cell comprising a heterologous polynucleotide encoding a P450 comprising at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8, 10, 12, 14 or 16 , wherein the P450 enzyme is enzymatically active. DEFINITIONS

[0050] The terms "isoprenoid," "isoprenoid compound," "terpene," "terpene compound," "terpenoid," and "terpenoid compound" are used interchangeably herein.

Isoprenoid compounds are made up various numbers of so-called isoprene (C5) units. The number of C-atoms present in the isoprenoids is typically evenly divisible by five (e.g., C5, CIO, C15, C20, C25, C30 and C40). Irregular isoprenoids and polyterpenes have been reported, and are also included in the definition of "isoprenoid." Isoprenoid compounds include, but are not limited to, monoterpenes, sesquiterpenes, triterpenes, polyterpenes, and diterpenes.

[0051] As used herein, the term "prenyl diphosphate" is used interchangeably with "prenyl pyrophosphate," and includes monoprenyl diphosphates having a single prenyl group (e.g. , IPP and DMAPP), as well as polyprenyl diphosphates that include two or more prenyl groups. Monoprenyl diphosphates include isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP).

[0052] As used herein, the term "terpene synthase" refers to any enzyme that enzymatically modifies IPP, DMAPP, or a polyprenyl pyrophosphate, such that a terpenoid compound is produced. The term "terpene synthase" includes enzymes that catalyze the conversion of a prenyl diphosphate into an isoprenoid.

[0053] The word "pyrophosphate" is used interchangeably herein with "diphosphate."

Thus, e.g., the terms "prenyl diphosphate" and "prenyl pyrophosphate" are interchangeable; the terms "isopentenyl pyrophosphate" and "isopentenyl diphosphate" are interchangeable; the terms "farnesyl diphosphate" and "farnesyl pyrophosphate" are interchangeable; etc.

[0054] The term "mevalonate pathway" or "MEV pathway" is used herein to refer to the biosynthetic pathway that converts acetyl-CoA to IPP. The mevalonate pathway comprises enzymes that catalyze the following steps: (a) condensing two molecules of acetyl- CoA to acetoacetyl-CoA; (b) condensing acetoacetyl-CoA with acetyl-CoA to form HMG- CoA; (c) converting HMG-CoA to mevalonate; (d) phosphorylating mevalonate to mevalonate 5-phosphate; (e) converting mevalonate 5-phosphate to mevalonate 5- pyrophosphate; and (f) converting mevalonate 5 -pyrophosphate to isopentenyl

pyrophosphate. See, e.g., Figure 1 of U.S. Patent No. 8,828,684.

[0055] As used herein, the term "prenyl transferase" is used interchangeably with the terms "isoprenyl diphosphate synthase" and "polyprenyl synthase" {e.g., "GPP synthase,"

"FPP synthase," "OPP synthase," etc.) to refer to an enzyme that catalyzes the consecutive Γ- 4 condensation of isopentenyl diphosphate with allylic primer substrates, resulting in the formation of prenyl diphosphates of various chain lengths.

[0056] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a nucleotide polymer of any length, including, but not limited to ribonucleotides and deoxynucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non- natural, or derivatized nucleotide bases. These terms include known types of nucleic acid sequence modifications, for example, substitution of one or more of the naturally occurring nucleotides with an analog; internucleotide modifications, such as, for example, those with uncharged linkages {e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g.,

aminoalklyphosphoramidates, aminoalkylphosphotriesters); those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those with intercalators (e.g., acridine, psoralen, etc.); and those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.). As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC-IUB Commission of Biochemical Nomenclature (Biochem. 9:4022, 1970).

[0057] The term "operably linked" refers to a nucleic acid sequence placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Additionally, in order to be operably linked it is not necessary that two sequences be immediately adjacent to one another.

[0058] The terms "DNA regulatory sequences," "control elements," and "regulatory elements," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate expression of a coding sequence and/or production of an encoded polypeptide in a host cell.

[0059] The terms "expression vector" or "vector" refer to a compound and/or composition that transduces, transforms, or infects a host microorganism, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell, or in a manner not native to the cell. An "expression vector" typically contains a sequence of nucleic acids (ordinarily RNA or DNA) to be expressed by the host microorganism. Optionally, the expression vector also comprises materials to aid in achieving entry of the nucleic acid into the host microorganism, such as a virus, liposome, protein coating, or the like. The expression vectors contemplated for use herein include those into which a nucleic acid sequence can be inserted, along with any preferred or required operational elements. Further, the expression vector is desirably one that can be transferred into a host microorganism and replicated therein. In certain embodiments suitable expression vectors include plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of a nucleic acid sequence introduced into the vector. Such plasmids, as well as other expression vectors, are well known to those of ordinary skill in the art.

[0060] Expression cassettes may be prepared comprising a transcription initiation or transcriptional control region(s) (e.g., a promoter), the coding region for the protein of interest, and a transcriptional termination region. Transcriptional control regions include those that provide for expression (e.g., including without limitation over-expression, constitutive expression and inducible expression) of the protein of interest in the genetically modified host cell.

[0061] The terms "optional" or "optionally" as used herein mean that the

subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0062] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include naturally occurring and non-naturally occurring amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0063] The term "naturally-occurring" as used herein as applied to a nucleic acid, an amino acid, a cell, or an organism, refers to a nucleic acid, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is naturally occurring.

[0064] The term "heterologous nucleic acid," as used herein, refers to a nucleic acid wherein at least one of the following is true: (a) the nucleic acid is foreign ("exogenous") to (i.e., not naturally found in) a given host microorganism or host cell; (b) the nucleic acid comprises a nucleotide sequence that is naturally found in (e.g., is "endogenous to") a given host microorganism or host cell (e.g., the nucleic acid comprises a nucleotide sequence endogenous to the host microorganism or host cell); however, in the context of a heterologous nucleic acid, the same nucleotide sequence as found endogenously is produced in an unnatural (e.g., greater than expected or greater than naturally found) amount in the cell, or a nucleic acid comprising a nucleotide sequence that differs in sequence from the endogenous nucleotide sequence but encodes the same protein (having the same or substantially the same amino acid sequence) as found endogenously is produced in an unnatural (e.g., greater than expected or greater than naturally found) amount in the cell; (c) the nucleic acid comprises two or more nucleotide sequences that are not found in the same relationship to each other in nature, e.g., the nucleic acid is recombinant. An example of a heterologous nucleic acid is a nucleotide sequence encoding a Caprifoliaceae enzyme in the valerenic acid biosynthesis pathway operably linked to a transcriptional control element (e.g., a promoter) to which an endogenous (naturally-occurring) Caprifoliaceae enzyme coding sequence is not normally operably linked. Another example of a heterologous nucleic acid a high copy number plasmid comprising a nucleotide sequence encoding an Caprifoliaceae enzyme in the valerenic acid biosynthesis pathway. Another example of a heterologous nucleic acid is a nucleic acid encoding a Caprifoliaceae enzyme in the valerenic acid biosynthesis pathway, where a fungal host cell that does not normally produce the

Caprifoliaceae enzyme is genetically modified with the nucleic acid encoding the

Caprifoliaceae enzyme; because Caprifoliaceae enzyme-encoding nucleic acids are not naturally found in fungal host cells, the nucleic acid is heterologous to the genetically modified fungal host cell.

[0065] As used herein, the term "recombinant" refers to a compound or composition produced by human intervention.

[0066] By "construct" is meant a recombinant nucleic acid, generally recombinant

DNA. [0067] The term "transformation" is used interchangeably herein with "genetic modification" and refers to a permanent or transient genetic change induced in a cell following introduction of new nucleic acid (i.e., DNA exogenous to the cell). Genetic change ("modification") can be accomplished either by incorporation of the new DNA into the genome of the host cell, or by transient or stable maintenance of the new DNA as an episomal element. Where the cell is a eukaryotic cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. In prokaryotic cells, permanent changes can be introduced or integrated into the chromosome or via extrachromosomal elements such as plasmids and expression vectors, which may contain one or more selectable markers to aid in their maintenance in the recombinant host cell.

[0068] A "host cell" as used herein refers to a cell in which a nucleic acid has been modified and/or into which has been introduced a nucleic acid. In certain embodiments host cells include cells in which a vector can be propagated and/or in which its DNA can be expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. In certain embodiments, host cells can be transgenic, in that they include nucleic acid molecules that have been introduced into the cell, such as a nucleic acid molecule encoding a one or more components of a pathway for the biosynthesis of valerenic acid, e.g., as described herein.

[0069] As used herein the term "isolated" is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the

polynucleotide, the polypeptide, or the cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

[0070] The term "conservative amino acid substitution" refers to the

interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains consists of cysteine and methionine. Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine- arginine, alanine-valine, and asparagine-glutamine.

[0071] "Synthetic nucleic acids" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments which are then enzymatically assembled to construct the entire gene. "Chemically synthesized," as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. The nucleotide sequence of the nucleic acids can be modified for improved expression based on introducing into the encoding polynucleotide the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

[0072] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity to a reference sequence (e.g., SEQ ID NOs: 1-22), sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in

Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif, USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0073] It is to be understood that the detailed description provided herein is exemplary and explanatory only and are not restrictive of any subject matter claimed. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. [0074] As used herein and in the appended claims, the singular forms "a," "and," and

"the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a genetically modified host cell" includes a plurality of such genetically modified host cells and reference to "the isoprenoid compound" includes reference to one or more isoprenoid compounds and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1, panel A shows important valerenic acid derivatives and related compounds. Valerenic acid, hydroxyvalerenic acid and acetoxyvalerenic acid are the three most abundant valerenic acids in V. officinalis. Valerenic acid and valerena-4,7(l l)-diene are reported to have sedative effects, while acetoxyvalerenic acid has antagonistic activities. Figure 1, panel B shows P450 activity in Asteraceae, Apiaceae,and Caprifoliaceae sesquiterpenoid drug pathways. Sesquiterpenes are produced in all plant species from MEV pathway-derived FPP by sesquiterpene synthases. Subsequently, P450 (CYP) activity is responsible for sesquiterpene acid formation in characterized biosynthetic pathways from various Asteraceae. Artemisia annua alcohol dehydrogenase (AaADH) and A. annua aldehyde dehydrogenase (AaALDH) have been shown to catalyze the conversion of artemisinic alcohol to artemisinic acid alongside P450 activity in the artemisinin pathway. Functionally characterized P450s are shown in blue.

[0076] Figure 2 illustrates that valerenadiene titer improved with VDS tagging strategies. Confocal microscopy of yeast cells expressing VDS-GFP shows GFP aggregation. ERG20 appeared to improve VDS solubility and provide increased flux through the pathway by increasing the supply of FPP. Cultures were extracted after 48 hours and analyzed for valerenadiene production by GC-MS. MBP-VDS-ERG20 narrowly outperformed both MBP- VDS-GFP and MBP-VDS-ERG20-GFP in production of valerenadiene. This VDS variant was integrated two additional times to produce the high titer valerenadiene strain, JWy608. Data represent the averages of three replicate cultures; error bars show s.d.

[0077] Figure 3 illustrates that GFP tagging of VDS indicates protein insolubility.

VDS was fused to either GFP or with two additional protein tags, MBP on the N-terminal and ERG20 on the C-terminal (3-tag VDS). The 3-tag VDS showed improved cytosolic expression.

[0078] Figure 4 illustrates heat map of candidate P450s involved in valerenic acid biosynthesis upregulated in the root. Sesquiterpene biosynthetic precursor enzymes upstream of valerenadiene are highly expressed in all tissues, while VDS is almost exclusively expressed in root tissue. P450s were selected by high expression in the root of V. officinalis; note, VoCYP71DJl (valerenadiene oxidase) shares a similar expression profile among tissue types with VDS. Expression values in log2 FPKM (fragments per Kilobase of transcript per million fragments mapped) were used, negative values were set to zero. Expression values shown represent the different developmental tissues.

[0079] Figure 5 illustrates a phylogenetic tree of V. officinalis P450s and related terpene-modifying P450s. Note, Germacrene A oxidase P450s from Asteraceae species clade with many of the V. officinalis candidates. The neighbor-joining tree was generated using MAFFT and RAxML. The numbers indicate the bootstrap value (%) from 100 replications. The scale bar shows the amino acid substitution ratio. GuCYP88D6, Glycyrrhiza uralensis β- amyrin-11 -oxidase (AB433179), was used as the outgroup. V. officinalis CYP proteins identified in this study are marked with arrowheads. The closed arrowhead indicates VoCYP71DJl having the activity of valerenic alcohol synthesis and the open arrowheads indicate CYPs incapable of producing oxidized valerenadiene in this study.

[0080] Figure 6 illustrates Gblocks curated alignment for VoCYPs and related

Asteraceae P450s. VoCYP714A33 (SEQ ID NO:23), VoCYP81Q107 (SEQ ID NO:24), VoCYP71DJl (SEQ ID NO:25), VoCYP71D510 (SEQ ID NO:26), VoCYP71D511 (SEQ ID NO:27), VoCYP71BE87 (SEQ ID NO:28), VoCYP71 AVI (SEQ ID NO:29), VoCYP71 AV2 (SEQ ID NO:30), VoCYP71AV7 (SEQ ID NO:31), VoCYP71AV8 (SEQ ID NO:32),

VoCYP71 AV9 (SEQ ID NO:33), VoCYP71BL2 (SEQ ID NO:34), VoCYP71BL5 (SEQ ID NO:35), VoCYP71 AE2 (SEQ ID NO:36), VoCYP71D442 (SEQ ID NO:37).

[0081] Figure 7 illustrates that VoCYP7 ID Jl produces oxidized valerenadiene. GC-

MS chromatograms of yeast extracts of JWy608 and JWy614. The addition of VoCYP71DJl produces a new peak (Peak 1), likely valerenic alcohol, with a mass of 220m/z (RT = 7.8 min).

[0082] Figure 8 illustrates a heatmap of Valeriana officinalis dehydrogenase expression. AaADHl and AaALDHl were BLASTed against the V. officinalis

transcriptome. Expression profiles for hits were analyzed in Excel. Select dehydrogenases are highlighted in yellow; VoADHl = voa_locus_40753_iso_l_len_634_ver_2, VoALDHl = voa_locus_940_iso_l_len_1560_ver_2. VoADHl and VoALDHl were selected on basis of homology for use in this work. [0083] Figure 9 illustrates in vivo production of valerenic acid by V. officinalis P450s and dehydrogenases in yeast. GC analysis identifies sesquiterpenoid products of extracts of yeast cultures with integrated candidate genes. Co-expression of A. annua dehydrogenases improved production of valerenic acid to about 4 mg/L. VoCYP71DJl was co-integrated with A. annua CPR. Ethyl acetate-extractable fractions were derivatized and analyzed by GC-MS in extracted ion mode (m/z 248). Mass spectra of valerenic acid (retention time 13.83 min, detected as methyl ester) relative to authentic standard is shown. Valerenic acid denoted with asterisk.

[0084] Figure 10 illustrates co-expression of V officinalis P450 candidates in

VoCYP71DJl expressing strain. JWy615. Valerenic acid levels were determined [arbitrary units (AU] and normalized to internal standard trans-caryophyllene standard. Evaluation of the data shows expression of P450 candidates with VoCYP71DJl did not increase valerenic acid production. Data represent the averages of three replicate cultures; error bars show s.d.

[0085] Figure 11, panels A-B, illustrates GCMS spectra of (panel A) valerena- 4,7(1 l)-diene from yeast culture and (panel B) underivatized valerenic acid standard.

Underivatized valerenic acid was difficult to detect at low concentrations and exhibited broad peaks when analyzed on GC.

[0086] Figure 12 illustrates GCMS profiles of transiently expressed valerenic acid pathway enzymes in Nicotiana benthamiana. We decided to use yeast as an expression platform, as our preliminary studies and other studies have encountered difficulties using N. benthamiana as a heterologous host for oxidized sesquiterpene production likely due to endogenous activities such as glycosylation (van Herpen et al. (2010) Plos One, 5(12):

el4222). Plasmids expressing Arabidopsis thaliana HMGR (AtHMGR, P14891),

Arabidopsis thaliana FPPS (AtFPPS, M 124151.3), Valeriana officinalis VDS, and select V. officinalis P450s were transformed into Agrobacteria tumefaciens and co-infiltrated into young N. benthamiana leaves. Valerena-4,7(1 l)-diene (Peak 2, retention time: 9.47 min), denoted by an asterisk, was produced at variable levels, but no detectable valerenic acid was formed. Dodecane (Peak 1, retention time: 4.63 min, 50mg/L) was used as an internal standard. For GC analysis, a different program was used; an initial temperature of 100°C was held for 1 min, followed by ramping to 250°C at a rate of 15°C/min to 250°C, followed by ramping to 300°C at a rate of 30°C/min, and then held at 300°C for 3 min. DETAILED DESCRIPTION

Introduction

[0087] Valeriana officinalis (Valerian) root extracts have been used by European and

Asian cultures for millennia for their anxiolytic and sedative properties. However, the efficacy of these extracts suffers from variable yields and composition, making these extracts a prime candidate for microbial production. Recently, valerenic acid, a C15 sesquiterpenoid, was identified as the active compound that modulates the GABAA channel. Although the first committed step, valerena-4,7(l l)-diene synthase, has been identified and described, the complete valerenic acid biosynthetic pathway remains to be elucidated. Sequence homology and tissue-specific expression profiles of V. officinalis putative P450s led to the discovery of a V. officinalis valerena-4,7(l l)-diene oxidase, VoCYP71DJl, which required co-expression with a V. officinalis alcohol dehydrogenase and aldehyde dehydrogenase to complete valerenic acid biosynthesis in yeast. Further, we demonstrated the stable integration of all pathway enzymes in yeast, resulting in the production of 140mg/L of valerena-4,7(l l)-diene and 4 mg/L of valerenic acid in millititer plates. These findings showcase Saccharomyces cerevisiae's potential as an expression platform for facilitating multiply-oxidized medicinal terpenoid pathway discovery, possibly paving the way for scale up and FDA approval of valerenic acid and other active compounds from plant-derived herbal medicines.

[0088] These findings demonstrate the usefulness of fungal host cells (e.g., yeast, e.g., Saccharomyces cerevisiae) as an expression platform for facilitating multiply-oxidized medicinal terpenoid pathway discovery, paving the way for scale up and FDA approval of valerenic acid and other active compounds from plant-derived herbal medicines. The fungal host cells described herein are useful for industrial production of valerenic acid at low cost as well as applications as base strains for producing valerenic acid derivatives and additional gene discovery. The fungal host cells described herein provide a less costly source of valerenic acid that can replace plant-derived valerenic acid, which is highly variable in composition and quality. This method uses a previously undescribed enzyme, VoCYP71DJl, to complete the valerenic acid pathway in yeast. Further, the engineered fungal host cells can be fermented in large tanks to produce valerenic acid and related compounds. [0089] Accordingly, provided are genetically modified fungal host cells that produce high levels of valerenadiene and/or valerenic acid. In various embodiments, the genetically modified fungal cells provided herein can comprise one or more copies of a polynucleotide encoding a maltose binding protein (MBP)- valerena-4,7(l l)-diene synthase (VDS)- farnesyl diphosphate (FPP) synthase (ERG20) fusion protein allowing for substantially increased production levels of valerenadiene, e.g., at least about 30 mg, e.g., at least about 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or more, valerena- 4,7(1 l)-diene per liter of culture. In various embodiments, the genetically modified fungal cells provided herein can comprise the full biosynthetic pathway for valerenic acid, allowing for commercially useful production levels of valerenic acid, e.g., at least about 4 mg, or more, valerenic acid per liter of culture. Further, methods are provided for the production of high levels of valerenadiene and/or valerenic acid in a genetically modified fungal host cell. The methods generally involve culturing a subject genetically modified host cell under conditions that promote production of high levels of an isoprenoid or isoprenoid precursor compound.

[0090] The valerenic biosynthesis pathway are described herein and valerenic acid derivatives and related compounds are depicted schematically in Figure 1. This pathway naturally occurs in cells of V. officinalis plants and can be reconstructed in a genetically modified fungal host cell transformed with heterologous polynucleotides encoding heterologous enzymes from Asteraceae plants. In various embodiments, the fungal cell is transformed with (i) a valerenadiene synthase (VDS), that converts farnesyl pyrophosphate (FPP) to valerenadiene, wherein the fungal cell produces valerenic acid; (ii) a valerenadiene oxidase that converts valerenadiene to valerenic alcohol; (iii) an alcohol dehydrogenase (ADH) that converts valerenic alcohol to valerenic aldehyde; and (iv) an aldehyde

dehydrogenase (ALDH) that converts valerenic aldehyde to valerenic acid.

Maltose Binding Protein (MBP)- valerena-4.,7(ll)-diene synthase (VPS)- farnesyl diphosphate (FPP) synthase (ERG20) Fusion Protein

[0091] In certain embodiments, a maltose binding protein (MBP)- valerena-4,7(l 1)- diene synthase (VDS)-farnesyl diphosphate (FPP) synthase (ERG20) fusion protein is provided. In various embodiments, the fusion protein comprises a VDS in the fusion protein is from an Asteraceae, Apiaceae, or Caprifoliaceae plant, e.g., from a Valeriana, Thapsia, Barnadesia or Artemisia plant, e.g., from a Valeriana officinalis, Thapsia gar ganica, Barnadesia spinosa, or Artemisia annua plant. In various embodiments, the VDS in the fusion protein is from Valeriana officinalis. In various embodiments, the VDS in the fusion protein has at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, and converts farnesyl pyrophosphate (FPP) to valerenadiene. In various embodiments, the VDS in the fusion protein is encoded by a polynucleotide having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: l, and encodes an enzyme that converts farnesyl pyrophosphate (FPP) to valerenadiene. In various embodiments, the MBP-VDS-ERG20 fusion protein has at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4, and converts farnesyl pyrophosphate (FPP) to valerenadiene. In various embodiments, the MBP-VDS-ERG20 fusion protein is encoded by a polynucleotide having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4, and encodes and enzyme that converts farnesyl pyrophosphate (FPP) to valerenadiene.

Fungal Cells Modified to Produce High Levels of Valerenadiene and/or Valerenic Acid

[0092] In certain embodiments, genetically modified fungal host cells are provided that comprise one or more genetic modifications that provide for increased production of valerenadiene, valerenic acid, and related derivatives (e.g., hydroxyvalerenic acid). In various embodiments, compared to a control host cell not genetically modified as described herein, a subject genetically modified host cell exhibits one or more of the following characteristics: increased production levels of valerenadiene and/or increased activity levels of one or more valerenic acid biosynthesis pathway enzymes allowing for production of valerenic acid by the host cell. In various embodiments, the fungal host cell is transformed with one or more copies of one or more heterologous polynucleotides encoding enzymes in the valerenic acid biosynthesis pathway, including one or more of (i) a valerenadiene synthase (VDS), that converts farnesyl pyrophosphate (FPP) to valerenadiene, wherein the fungal cell produces valerenic acid; (ii) a valerenadiene oxidase that converts valerenadiene to valerenic alcohol; (iii) an alcohol dehydrogenase (ADH) that converts valerenic alcohol to valerenic aldehyde; and (iv) an aldehyde dehydrogenase (ALDH) that converts valerenic aldehyde to valerenic acid. In certain embodiments the enzymes can be from an Asteraceae plant, e.g., from a Valeriana, Thapsia, Barnadesia or Artemisia plant, e.g., from a Valeriana officinalis, Thapsia garganica, Barnadesia spinosa Artemisia annua plant. In various embodiments, the fungal cell is further transformed with a polynucleotide encoding a cytochrome P450 reductase (CPR). In various embodiments, one or more copies of one or more polynucleotides encoding one or more heterologous enzymes in the valerenic acid biosynthesis pathway are integrated into the genome of the fungal host cell. In various embodiments, the one or more polynucleotides are integrated into the fungal host cell's genome at the uridine auxotrophy (URA3) locus. In various embodiments, the one or more polynucleotides are operably linked to and expressed under the control of a galactose inducible promoter. [0093] It was demonstrated that increased production levels of valerenadiene and/or increased activity levels of one or more valerenic acid biosynthesis pathway enzymes increases valerenadiene production by a subject genetically modified host cell and allows for production of valerenic acid by the host cell. Thus, in some embodiments, a subject genetically modified host cell exhibits increases in valerenadiene and/or valerenic acid production, where valerenadiene or valerenic acid production is increased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%>, at least about 35%o, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30- fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100- fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1000-fold, or more, in the genetically modified host cell, compared to the level of valerenadiene and/or valerenic acid produced in a control host cell that is not genetically modified as described herein. Valerenadiene and/or valerenic acid production can be readily determined using well-known analytical methods, e.g., gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, ion chromatography-mass spectrometry, pulsed amperometric detection, UV-VIS spectrometry, and the like.

[0094] In some embodiments, a subject genetically modified host cell provides for enhanced production of valerenadiene and/or valerenic acid per cell, e.g., the amount of valerenadiene and/or valerenic acid compound produced using a subject method is at least about 10%o, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%o, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 500-fold, or 1000-fold, or more, higher than the amount of the valerenadiene and/or valerenic acid produced by a host cell that is not genetically modified by the subject methods, on a per cell basis. In certain embodiments the number of cells can be measured by measuring dry cell weight or measuring optical density of the cell culture.

[0095] In other embodiments, a subject genetically modified host cell provides for enhanced production of valerenadiene and/or valerenic acid per unit volume of cell culture, e.g., the amount of valerenadiene and/or valerenic acid produced using a subject genetically modified host cell is at least about 10%, at least about 15%, at least about 20%, at least about 25%), at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%), at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 500-fold, or 1000-fold, or more, higher than the amount of the valerenadiene and/or valerenic acid produced by a host cell that is not genetically modified by the subject methods, on a per unit volume of cell culture basis. In various embodiments, methods evaluating valerenadiene and/or valerenic acid production on a per unit volume of cell culture basis can be normalized by measuring dry cell weight or measuring optical density of the unit volume of cell culture.

[0096] In some embodiments, the genetically modified fungal host cells described herein produce at least about 30 mg, e.g., at least about 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or more, valerena-4,7(l l)-diene per liter of culture. In some embodiments, the genetically modified fungal host cells described herein produce at least about 4 mg valerenic acid per liter of culture.

[0097] The subject methods can be used in a variety of different kinds of fungal host cells. Host fungal cells are, in many embodiments, unicellular organisms, or are grown in culture as single cells. Suitable fungal host cells for producing valerenadiene and/or valerenic acid include yeast cells, including without limitation, e.g., a genus selected from the group consisting of Saccharomyces, Pichia, Kluyveromyces, Candida, Aspergillus,

Trichoderma, Chrysosporium, Fusarium and Neurospora. Suitable yeast host cells include, but are not limited to, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa, and the like. In a particular embodiment, the yeast cell is Saccharomyces cerevisiae. In various embodiments, the host cell is a yeast cell capable of producing FPP. Such yeast cells are described, e.g., in Reider Apel, et al. Nucleic Acids Res (2017) 45;496-508 and Nakano, et al, Plant Biotechnology (2012) 29: 185-189. In various embodiments, the one or more of the heterologous polynucleotides encoding the heterologous enzymes are modified such that the nucleotide sequence reflects the codon preference for the particular host cell. For example, the nucleotide sequence will in some embodiments be modified for yeast codon preference. See, e.g., Bennetzen and Hall (1982) J. Biol. Chem. 257(6): 3026-3031; and kazusa.or.jp/codon.

Enzyme that Converts farnesyl pyrophosphate (FPP) to Valerenadiene

[0098] In one aspect, provided is a transformed fungal cell comprising one or more copies of one or more heterologous polynucleotides integrated into the fungal cell's genome, wherein the one or more heterologous polynucleotides encode a valerenadiene synthase (VDS), or variant or truncation thereof, that converts farnesyl pyrophosphate (FPP) to valerenadiene. In various embodiments, the fungal cell produces at least about 30 mg, e.g., at least about 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or more, valerena-4,7(l l)-diene per liter of culture. In various embodiments, the fungal cell produces at least about 4 mg valerenic acid per liter of culture. [0099] In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a heterologous polynucleotide encoding a VDS from an Asteraceae plant, e.g., from a Valeriana, Thapsia, Barnadesia or Artemisia plant, e.g., from a Valeriana officinalis, Thapsia garganica, Barnadesia spinosa Artemisia annua plant. In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a heterologous polynucleotide encoding a VDS, the VDS enzyme having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, and converts FPP to valerenadiene. In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a heterologous polynucleotide encoding a VDS, the polynucleotide having at least about 80% sequence identity, e.g., at least about

85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, and the encoded VDS converts FPP to valerenadiene. [0100] In various embodiments, the VDS is part of a MBP-VDS-ERG20 fusion protein, as described above and herein. In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a polynucleotide encoding a MBP-VDS-ERG20 fusion protein, the fusion protein having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4, and converts FPP to valerenadiene. In various embodiments, the fungal cell is transformed with a polynucleotide encoding a MBP-VDS-ERG20 fusion protein, the polynucleotide having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 3, and the encoded VDS enzyme converts FPP to valerenadiene. In various embodiments, the fungal cell comprises two, three, four, or more, copies of the polynucleotide encoding a MBP-VDS-ERG20 fusion protein integrated into its genome.

Enzyme that Converts Valerenadiene to Valerenic Alcohol

[0101] In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a heterologous polynucleotide encoding

valerenadiene oxidase, or variant or truncation thereof, that converts valerenadiene to valerenic alcohol. In various embodiments, the valerenadiene oxidase is a V. officinalis P450 enzyme, e.g., VoCYP71DJl, which functions as a valerenadiene oxidase. In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a polynucleotide encoding a valerenadiene oxidase, the enzyme having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6 (e.g., VoCYP71DJl), and wherein the valerenadiene oxidase converts valerenadiene to valerenic alcohol. In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a polynucleotide encoding a valerenadiene oxidase, the polynucleotide having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5 (e.g., VoCYP71DJl), wherein the polynucleotide encodes an enzyme that converts valerenadiene to valerenic alcohol. Enzyme that Converts Valerenic Alcohol to Valerenic Aldehyde

[0102] In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a heterologous polynucleotide encoding an alcohol dehydrogenase (ADH), or variant or truncation thereof, that converts valerenic alcohol to valerenic aldehyde. In various embodiments, the ADH is a Valeriana officinalis or Artemisia annua ADH. In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a polynucleotide encoding an ADH, the enzyme having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20 (V.

officinalis) or GenBank Accession Nos. ADK56099.1 A. annua) or AEI16475.1 A. annua), and wherein the ADH converts alcohol to valerenic aldehyde. In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a polynucleotide encoding an ADH, the polynucleotide having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19 (V. officinalis) or GenBank Accession No.

GU253890.1 (A. annua) or JF910157.1 (A. annua), and wherein the polynucleotide encodes an ADH that converts valerenic alcohol to valerenic aldehyde.

Enzyme that Converts Valerenic Aldehyde to Valerenic Acid

[0103] In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a heterologous polynucleotide encoding an aldehyde dehydrogenase (ALDH), or variant or truncation thereof, that converts valerenic aldehyde to valerenic acid. In various embodiments, the ALDH is a Valeriana officinalis or Artemisia annua ALDH. In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a polynucleotide encoding an ALDH, the enzyme having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18 (V.

officinalis) or GenBank Accession No. ACR61719.1 (A. annua), and wherein the ALDH converts aldehyde to valerenic acid. In various embodiments, the fungal cell is transformed with one or more copies, e.g., two, three or four copies, of a polynucleotide encoding an ALDH, the polynucleotide having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17 (V. officinalis) or GenBank Accession No. FJ809784.1 (A. annua), and wherein the polynucleotide encodes an ALDH that converts valerenic aldehyde to valerenic acid.

Additional Enzymes For Use in the Production of Valerenadiene and/or

Valerenic Acid in a Fungal Host Cell

[0104] The fungal host cells may additionally be transformed with a cytochrome

P450 reductase (CPR). In various embodiments, the fungal cell is further transformed with one or more copies, e.g., two, three or four copies, of a polynucleotide encoding a

cytochrome P450 reductase (CPR). In various embodiments, the fungal cell is transformed with a polynucleotide encoding a CPR , the enzyme having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 22. In various embodiments, the fungal cell is transformed with a polynucleotide encoding a CPR , the polynucleotide having at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 21. Fungal Host Cell Cultures

[0105] Further provided is a fungal cell culture comprising a population of fungal cells, as described above and herein. Generally, the fungal cells in a population of fungal cells in a culture are genetically identical or substantially genetically identical. In various embodiments, the fungal cell culture produces at least about 30 mg, e.g., at least about 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or more, valerena- 4,7(1 l)-diene per liter of culture, as described above and herein. In various embodiments, the fungal cell culture produces at least about 4 mg per liter of culture of valerenic acid. In various embodiments, the population of fungal cells is cultured in a medium comprising galactose. In various embodiments, the population of fungal cells is cultured in a medium lacking uridine.

[0106] In a further aspect, provided is a bioreactor comprising a fungal cell culture as described above and herein. In various embodiments, the bioreactor comprises a culture volume of at least about 0.5L, 1L, 2L, 3L, 4L, 5L, 10L, 25L, 50L, 75L, 100L, 250L, 500L, 1000L, or more. Method of Making Fungal Host Cells That Produce High Levels of valerena-4.,7(ll)- diene and/or Valerenic Acid

[0107] The genetically modified fungal host cell described herein can be generated using standard methods known to those skilled in the art. In some embodiments, a heterologous nucleic acid comprising a nucleotide sequence encoding an enzyme in the valerenic acid biosynthesis pathway is introduced into a host cell and replaces all or a part of an endogenous gene, e.g., via homologous recombination, e.g., via Cas9-aided homologous recombination, described in Reider Apel, et al. (2107) Nucleic Acids Res. 45: 496-508. In some embodiments, a heterologous nucleic acid a valerenic acid biosynthesis pathway enzyme is introduced into a parent host cell, and the heterologous nucleic acid comprises nucleic acid sequences that recombine with an endogenous nucleic acid {e.g., orotidine-5'- phosphate decarboxylase (URA3)). In some embodiments, the heterologous nucleic acid comprises a promoter that provides for regulated transcription, e.g., under the control of a galactose inducible promoter. [0108] To generate a genetically modified host cell, one or more nucleic acids comprising nucleotide sequences encoding one or more gene products is introduced stably or transiently into a host cell, using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, DEAE-dextran mediated transfection, liposome-mediated transfection, heat shock in the presence of lithium acetate, and the like. For stable transformation, a nucleic acid will generally further include a selectable marker, e.g., any of several well-known selectable markers such as neomycin resistance, ampicillin resistance, tetracycline resistance, chloramphenicol resistance, kanamycin resistance, and the like.

[0109] In many embodiments, the nucleic acid with which the host cell is genetically modified is an expression vector that includes a nucleic acid comprising a nucleotide sequence that encodes an enzyme in the valerenic acid biosynthesis pathway. Suitable expression vectors include, but are not limited to, baculovirus vectors, yeast plasmids {e.g., available from New England Biolabs, Clontech or ThermoFisher), yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as yeast). Thus, for example, a nucleic acid encoding a gene product(s) is included in any one of a variety of expression vectors for expressing the gene product(s). Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences. [0110] The nucleotide sequence in the expression vector can be operably linked to an appropriate expression control sequence(s) (promoter) to direct synthesis of the encoded gene product. Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see, e.g., Bitter et al. (1987) et/?. Enzymol. 153 : 516-544).

[0111] In addition, the expression vectors will in many embodiments contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture. [0112] Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the S. cerevisiae TRP1 gene, etc ; and a promoter derived from a highly-expressed gene to direct transcription of the gene product-encoding sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3 -phosphogly cerate kinase (PGK), a -factor, acid phosphatase, or heat shock proteins, among others.

[0113] In many embodiments, a genetically modified host cell is genetically modified with a nucleic acid that includes a nucleotide sequence encoding a gene product, where the nucleotide sequence encoding the gene product is operably linked to a constitutive promoter. In yeast, a number of vectors containing constitutive or inducible promoters may be used. See, Yeast Metabolic Engineering: Methods and Protocols (Methods in Molecular Biology) 2014th Ed., Valeria Mapelli (Editor), Humana Press; Synthetic Biology and Metabolic Engineering in Plants and Microbes Parts A: Metabolism in Microbes, Volumes 575-576 (Methods in Enzymol ogy) 1st Edition, 2016, Sarah E O'Connor (Editor), Academic Press, Biotechnology of Yeasts and Filamentous Fungi, 1st ed. 2017 Edition, by Andriy A. Sibirny (Editor), Springer. Illustrative constitutive promoters of use in yeast expression systems include without limitation, e.g., pCyc (Medium) Promoter, pAdh (Strong) Promoter, pSte5 (Weak) Promoter, yeast ADHl promoter, eye 100 promoter, cyc70 promoter, cyc43 promoter, cyc28 promoter, cycl6 promoter, pPGKl, pCYC Yeast Promoter, Yeast GPD (TDH3) Promoter, yeast mid-length ADHl promoter, Yeast CLB 1 promoter region, G2/M cell cycle specific. Illustrative positively regulated promoters of use in yeast expression systems include, e.g., yeast GALl promoter, Gall Promoter, A-Cell Promoter MFAl (RtL), A-Cell Promoter MFA2, Alpha-Cell Promoter Ste3, URA3 Promoter from S. cerevisiae, Yeast FIG1 promoter, yeast EN02 promoter, Partial DLD Promoter from Kluyveromyces lactis, JEN1 Promoter from Kluyveromyces lactis, Zif268-HIV binding sites + TEF constitutive yeast promoter, Zif268-HIV bs + LexA bs + mCYC promoter , mCYC promoter plus LexA binding sites, mCYC promoter plus Zif268-HIV binding sites, pGALl+ w/XhoI sites, A-Cell Promoter STE2 (backwards), Alpha-Cell Promoter MF (ALPHA) 1, Alpha-Cell Promoter MF(ALPHA)2, Cup-1 Heavy metal sensor. Illustrative repressible promoters that find use in yeast expression systems include without limitation, e.g., yeast fet3 promoter, yeast anbl promoter, MET 25 Promoter, yeast suc2 promoter , pFigl (Inducible) Promoter , pGall . See, e.g., parts.igem.org/Yeast.

[0114] In various embodiments, vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome, e.g., Cas9-gRNA pCut plasmids, available from addgene.org).

Methods of Producing Valerenic Acid

[0115] In certain embodiments, methods of producing valerenadiene, valerenic acid, and derivatives thereof (e.g., hydroxyvalerenic acid) are provided. The methods generally involve culturing a population of genetically modified fungal host cells, as described above and herein, in a suitable medium and under conditions such that the population of genetically modified cells produce valerenadiene and/or valerenic acid. In various embodiments, the methods can further comprise isolating the valerenadiene and/or valerenic acid from the cell and/or from the culture medium. [0116] In general, a subject genetically modified host cell is cultured in a suitable medium (e.g., Luria-Bertoni broth, optionally supplemented with one or more additional agents, such as an inducer (e.g., where one or more nucleotide sequences encoding a gene product is under the control of an inducible promoter)). In various embodiments, one or more of the polynucleotides encoding the heterologous enzymes are expressed under the control of a positively regulated galactose promoter, and the culture medium comprises a sufficient concentration of galactose to induce expression of the heterologous enzymes. In various embodiments, one or more of the polynucleotides encoding the heterologous enzymes is integrated into the genome of the fungal host cell at the uridine auxotrophy (URA3) locus, and the culture medium does not comprise uridine. [0117] In some embodiments, a subject genetically modified host cell is cultured in a suitable medium; and the culture medium is overlaid with an organic solvent, e.g., dodecane, forming an organic layer. The valerenadiene and/or valerenic acid compounds produced by the genetically modified host cell partitions into the organic layer, from which it can be purified. In some embodiments, where one or more gene product-encoding nucleotide sequence is operably linked to an inducible promoter, an inducer is added to the culture medium; and, after a suitable time, the valerenadiene and/or valerenic acid is isolated from the organic layer overlaid on the culture medium.

[0118] In some embodiments, the valerenadiene and/or valerenic acid will be separated from other products which may be present in the organic layer. Separation of the valerenadiene and/or valerenic acid from other products that may be present in the organic layer can be readily achieved using, e.g., standard chromatographic techniques. [0119] In some embodiments, the valerenadiene and/or valerenic acid is purified or isolated, e.g., at least about 40% purified or isolated, at least about 50% purified or isolated, at least about 60% purified or isolated, at least about 70% purified or isolated, at least about 80%) purified or isolated, at least about 90% purified or isolated, at least about 95% purified or isolated, at least about 98% purified or isolated, or more than 98% purified or isolated, where "pure" in the context of a valerenadiene and/or valerenic acid refers to valerenadiene and/or valerenic acid that is free from other compounds, contaminants, etc., substantially or entirely free of hydroxyvalerenic acid and/or acetoxyvalerenic acid.

Valerenic Acid Produced According to the Present Methods

[0120] Further provided is valerenadiene and/or valerenic acid produced from a fungal cell or fungal cell culture, as described above and herein. Further provided is a pharmaceutical composition or a nutraceutical composition comprising valerenic acid produced according to the methods described above and herein.

Kits

[0121] In certain embodiments, kits are provided that contain one or more of the vectors and/or cells described herein. In various embodiments, the kits comprise genetically modified fungal cells capable of producing produces at least about 30 mg, e.g., at least about 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or more, valerena-4,7(l l)-diene per liter of culture, as described above and herein. In various embodiments, the kits comprise genetically modified fungal cells capable of producing at least about 4 mg valerenic acid per liter of culture, as described above and herein. The cells or population of cells can be contained in one or more vials or ampoules in the kit. In various embodiments, the cells or population of cells are frozen, freeze-dried or lyophilized. In various embodiments, the cells or population of cells are provided on agar.

EXAMPLES

[0122] The following examples are offered to illustrate, but not to limit the claimed subject matter.

Example 1

De Novo Synthesis of Valerenic Acid in Saccharomyces cerevisiae Materials and methods.

[0123] Strain construction. The parent Saccharomyces cerevisiae strain used for all engineering was GTy23 { erg9 : : KanMX PC TR3 -ERG9 leu2-3, 112 : : Hi s3 MX6 PG AL 1 - ERG19/PGAL 10-ERG8 ura3-52: :URA3_PGALl-mvaS(Al 10G)/PGAL10-mvaE(CO) his3Al : :hphMX4_PGALl-ERG12/PGAL10-IDIl } previously used by our lab (Reider Apel et al. (2016) Nucleic Acids Res. 45(1): 496-508). The integration cassettes for all subsequent strains (Table 1) were created using the software tools CASdesigner (casdesigner.jbei.org) and DIVA (diva.jbei.org) and integrated using the previously reported, cloning-free methodology via Cas9-aided homologous recombination (Reider Apel et al. supra.).

Integration cassettes containing 1-kb flanking homology regions targeting a chosen genomic locus were constructed by PCR amplifying donor DNA fragments using primers generated by CASdesigner, then co-transformed with a Cas9-gRNA plasmid (pCut) targeting the chosen genomic locus. CASdesigner primers provide 30-60 nt of inter-fragment homology allowing 1-5 separate fragments to assemble via homologous recombination in vivo. pCuts targeting genomic loci were assembled in vivo from a linear backbone and a linear PCR fragment containing the new gRNA sequence, as described previously {Id.). The new gRNA sequence for the URA3 locus was chosen using DNA2.0 (www.dna20.com/eCommerce/cas9/input). To generate donor DNA fragments, native sequences {e.g., chromosomal homology regions, promoters) were amplified from CEN.PK2-1C genomic DNA, while heterologous sequences {e.g., P450 coding sequences, see, sequence listing, herein) were amplified from synthetic gene blocks having codon bias for improved expression in S. cerevisiae. All genes integrated in this study were expressed under galactose inducible promoters. Table 1. Yeast strains used in this study.

[0124] All PCRs used Phusion Hot Start II DNA polymerase

(www.thermofisher.com, cat. F549L). The following touchdown PCR cycling conditions were used for all PCRs: 1 cycle of 98°C for 15 sec; 25 cycles of 98°C for 10 sec, 65°C for 30 sec (dropping 1 degree each cycle after the first cycle), 72°C for 30 sec, and then 25 cycles of 98°C for 10 sec, 50°C for 30 sec, 72°C for 30 sec. Transformations were performed via heat- shock using ~ 200 ng pCut, about 1 μg donor DNA per sample, and 20 min heat shock at 42°C, then plated all cells on selective agarose plates (Gietz and Woods 2002). For assembling a pCut targeting a new site by homologous recombination, we used 200 ng linear pCut backbone and 500 ng of a 1- kb fragment containing the gRNA sequence, as described (Reider Apel et al. 2017). For multi-site integrations, we used 200 ng total linear pCut backbone, and the same amounts of gRNA fragment and donor DNA for each site as we would have for a single integration. Colonies were screened by PCR directed at the target locus, and for integrations, one representative colony was sequenced. Three to four biological replicates were analyzed for each strain. [0125] Synthetic genes and oligonucleotides. Oligonucleotides and synthetic genes were commercially synthesized (Integrated DNA Technologies, Inc.). Sequences having codon bias were designed based on the IDT online tool. Sequences of synthetic genes can be found in the sequence listing. P450 ORF predictions for gene synthesis were selected from the publically available transcriptome at Medicinal Plant Genomics Resource

(http://medicinalplantgenomics.msu.edu). Previously described synthetic, polynucleotides encoding Artemisia annua ADHl (JQ582842.1) and ALDHl (JQ609276.1) and having codon bias for improved expression in yeast were used in this study (Paddon et al. (2013) Nature, 496(7446): 528-532).

[0126] Culture and fermentation conditions. Selective agar plates used for transformations were purchased from Teknova (www.teknova.com, cat. C3080). Liquid selective medium used to grow transformants contained 0.2% (w/v) complete supplement mixture (CSM) lacking uracil (www.sunrisescience.com, cat. 1004-100), 0.67% yeast nitrogen base (www.difco.com, cat. 291920), and 2% dextrose. Nonselective medium contained 1% yeast extract, 2% peptone (Difco cat. 288620 and 211677, respectively), and either 2% dextrose (YPD) or 2% galactose and 0.2% dextrose (YPG). Nonselective agar YPD plates were purchased from Teknova (cat. Y100). Cultures were grown in plastic 96-deep well plates (www.vwr.com, cat. 29445-166) and glass test tubes for strain maintenance, while 2 ml of medium in 24-deep well plastic plates (CWR cat. 89080-534) were used for all production runs. Production cultures were spiked with 100 mg/L trans-caryophyllene (sigma cat. C9653) as an internal standard. Plastic plates were covered with AERASEAL™ film (www.excelscientific.com, cat. BS-25) and shaken at 800 rpm in a Multitron shaker

(www.infors-ht.com, model AJ185). Production runs were cultured for 48 hr in 2 ml of YPG before terpenoid extraction for analysis. Glass tubes were shaken at 200 rpm. All strains were grown at 30°C.

[0127] Confocal microscopy . To visualize GFP expression of tagged VDS variants in yeast strains, strains were grown in 5 ml YPD overnight, then back-diluted 1 : 100 into the same medium and grown 3-6 h at 200 rpm and 30 °C. Then, 1 ml of culture volume was centrifuged at 21,952 x g on a table-top centrifuge, washed with lx water, and 1 μΐ of the cell pellet was imaged using a Zeiss LSM 710 confocal system mounted on a Zeiss inverted microscope (www.zeiss.com) with a 63 A~ objective and processed using Zeiss Zen software.

[0128] Identification of differentially expressed P450s. Previously reported assembled transcripts and expression abundance estimations were retrieved from the

Medicinal Plant Genomics Resource (http://medicinalplantgenomics.msu.edu) and Plantrans DB (//lifecenter. sgst.cn/plantransdb). To select P450s for functional testing, published transcriptomes of V. officinalis were mined using a profile hidden Markov model search (HMMER) (Finn, et al. (2011) Nucleic Acids Res. 39(Web Server issue): W29-37).

Correlation of expression profiles was calculated by the Pearson product-moment correlation coefficient of log2 FPKM, with all log2 FPKM values less than zero were set to zero.

Subsequent heatmaps were generated using Multiple Experiment Viewer Software (MeV) version 4.5 (Howe et al. (2010) MeV: MultiExperiment Viewer. In: Ochs, M. F., Casagrande, J. T., and Davuluri, R. V. eds. Biomedical informatics for cancer research. Boston, MA: Springer US, pp. 267-277). DNA sequences identified in this work are deposited in

GenBank.

[0129] Phylogenetic analysis. Phylogenetic analysis was performed using the entire predicted amino acid sequences of V. officinalis P450 family proteins and related terpene- modifying P450 proteins from the GenBank database. Fourteen P450s with homology to V. officinalis candidates with known roles in terpenoid biosynthetic pathways were used in the analysis. The following accessions were used: Valeriana officinalis VoCYP71D442

(ALU63882.1), Santalum album SaCYP76F40 (AHB33947.1), Santalum album

SaCYP76F39 (AHB33940.1), Catharanthus roseus CrCYP76B6 (CAC80883), Thapsia garganica TgCYP76AE2 (AQY76213.1), Lactuca sativa LsCYP71BL2 (AEI59780.1), Barnadesia spinosa BsCYPl Ί AV7 (D5JBX1.1), Tanacetum cinerariifolium TcCYP71AV2 (AGO03789.1), Artemisia annua AaCYP71 AVI (Q1PS23.1), Cynara cardunculus var. Scolymus CcCYP71BL5 (AIA09038.1), Cynara cardunculus var. Scolymus CcCYP71AV9 (AIA09035.1), Cichorium intybus CiCYP71AV8 (E1B2Z9.1), Arabidopsis thaliana

AtCYP714Al (NP_001332750.1), and Arabidopsis thaliana AtCYP714A2

( P 001331286.1). Sequence alignments were generated on the basis of comparison of the amino acid sequences using the MAFFT L-INS-i algorithm with default parameters.

Alignments for each partition were generated using the default settings (gap opening penalty = 1.53 and offset value = 0.00) (Katoh et al. (2002) Nucleic Acids Res. 30(14): 3059-3066). A consistent alignment was selected using TrimAl, with the parameter automatedl (Capella- Gutierrez et al. (2009) Bioinformatics, 25(15): 1972-1973). Maximum likelihood analyses were conducted with RAxML v.7.2.8 (Stamatakis (2014) Bioinformatics 30(9): 1312-1313). Twenty randomized starting trees were generated with which the initial rearrangement setting and the number of distinct rate categories were determined. The best-known likelihood tree was found by performing 1000 repetitions for each of the amino acid datasets. One thousand non-parametric bootstrap replications were then performed using the bootstrap algorithm. The resulting tree was visualized using FigTree. The scale bar of 0.2 indicates a 20% change and each number shown next to the branches is the number of replicate trees in which the related taxa clustered in the bootstrap test. [0130] Metabolite quantification using GC-MS. Yeast cultures were grown in 2 mL of YPgal with 0.25μΜ CuS04 (Sigma cat. 209201) in 24-deep well plastic plates for 48 hours. For GC-MS analysis of valerenadiene, the cultures were overlaid with 0.4mL dodecane (spiked with internal standard), which was pipetted into 1.5mL Eppendorf tubes and spun at 21,952 x g for 1 minute. The resulting organic phase was removed and transferred to GC vials and mixed with EtOAc. For GC-MS analysis of valerenic acid, the cultures were extracted 1 : 1 with EtOAc spiked with 50 mg/L trans-caryophyllene by shaking with 100[iL of glass beads for 8 minutes at a frequency of 28 Hz in a Retsch mixer mill MM 400, then centrifuged at 21,952 x g for 1 minute. The resulting organic phase was removed and dried down at 54°C in vacuum, resuspended in 41 [iL EtOAc, 4 [iL of 40% v/v tetrabutylammonium hydroxide (TBAH) solution (Sigma cat. 86854). Then, 5 [iL of iodomethane (Sigma cat. 18504) was added to the sample, and the mixture was agitated by vortex for 10 s. An aliquot of the sample (1 μί) was injected into a cyclosil B column (J&W Scientific) operating at a He flow rate of 1 mL/min on GC-MS (GC model 6890, MS model 5973 Inert, Agilent). An initial temperature of 120°C was held for 3 min, followed by ramping to 250°C at a rate of 20°C/min, and then held at 250°C for another 3 min. The total flow was set to 8.3 mL/min and helium flow was set to 1 mL/min. All production

measurements were performed in biological triplicates or quadruplicates. A caryophyllene standard (Sigma cat. C9653) and a valerenic acid standard (Sigma cat. 51964) containing known concentrations of the internal standard were used to determine titers of valerenadiene and valerenic acid, respectively.

Results and Discusson.

[0131] Engineering production of valerenadiene precursor in yeast. First, we engineered a yeast strain to produce the valerenic acid precursor, valerenadiene, by expressing chromosomally integrated copies of valerenadiene synthase (VDS), which converts FPP to valerenadiene. Previous studies have produced low titers of valerenadiene in yeast (about 1 mg/L) from unstable, high-copy plasmids (Yeo et al. (2013) J. Biol. Chem., 288(5): 3163-3173; Pyle e/ a/. (2012) FEBS J. 279(17): 3136-3146). We used a previously developed mevalonate overproducing strain, GTy23, in which all enzymes converting acetyl- CoA to GPP are overexpressed from galactose-inducible promoters. After, we integrated a single copy of VDS, but noted low production, about 3 mg/L, consistent with other non- modified sesquiterpene synthases (Figure 2) (Son et al. (2014) Biochem. J. 463(2): 239-248; Beekwilder et al. (2014) Plant Biotechnol. J. 12(2): 174-182). Many studies have shown diterpene and sesquiterpene synthases suffer from cytosolic insolubility or instability when expressed in yeast (Ignea et al. (2015) Metabolic Engineer. 27: 65-75; Reider Apel et al. (2016) Nucleic Acids Res. 45(1): 496-508). Thus, we employed protein tagging strategies to visualize insoluble protein and to improve protein solubility. A single integration of a VDS- GFP tagged variant showed severe protein aggregation (Fig. 2, Fig. 3, top). We proceeded to test other recently described protein tag variations. We tested several combinations of an N- terminal MBP tag, a C-terminal GFP tag, and a C-terminal ERG20 tag (ERG20 being the enzyme catalyzing the formation of FPP). The best VDS variant was MBP-VDS-ERG20, which resulted in substantial titer improvements to -30 mg/L, almost an order of magnitude increase over untagged VDS. Integrating three additional copies of MBP-VDS-ERG20 led to strain JWy608, which produced a titer of -140 mg/L valerenadiene (Fig. 2). This strain was used for all P450 functional testing.

[0132] Identifying putative valerenadiene oxidase genes from V. officinalis. To select

P450s for functional testing, we mined the published transcriptomes of V. officinalis using a profile hidden Markov model to search for all P450s (Yeo et al. (2013) J. Biol. Chem., 288(5): 3163-3173; Pyle et a/. (2012) FEBS J. 279(17): 3136-3146; Finn, et al. (2011) Nucleic Acids Res. 39(Web Server issue): W29-37). Subsequently, we used isoform expression data generated by a previous study to identify likely candidates involved in valerenic acid biosynthesis based on root tissue preferential expression, and their correlation with the expression pattern of VDS in the tissue types (Yeo et al. (2013) J. Biol. Chem., 288(5): 3163-3173). However, we also identified two P450s, VoCYP81Q107 and

VoCYP71D510, that had constitutive expression over all tissue types, similar to the expression of upstream isoprenoid biosynthetic gene homologs for HMGR, IDI, and FPPS (Fig. 4). Of the candidates, the expression profile of VoCYP71DJl shared the highest similarity to the expression profile of VDS, with no expression in the leaf or callus, low expression in the stem, and the highest expression in the root (Fig. 4).

[0133] We made a phylogenetic tree of all putative V. officinalis P450s and included other functionally tested related P450s that oxidize sesquiterpenes (Fig. 5). We noticed that all of these upregulated P450s had homology to known terpene modifying enzymes, with the exception of VoCYP81Q107. VoCYP714A33 has homology to gibberellin oxidases (Nomura et al. (2013) Plant & Cell Physiol. 54(11): 1837-1851), such as AtCYP714Al and

AtCYP714Al, while VoCYP71BE87 shares homology with the Vitis vinifera P450

VvCYP71BE5 responsible for the formation of the sesquiterpenoid (-)-rotundone, a component of wine flavor (Takase et al. (2016) J Exp. Bot. 67(3): 787-798). VoCYP81Q107 has homology to P450s involved in the sesamin biosynthetic pathway (Hata et al. (2010) Plant Sci. 178(6): 510-516). Four of the six identified P450 candidates were classified as CYP71D P450s, consistent with the classification of other Asteraceae sesquiterpene oxidases that have been found to catalyze the oxidation of a primary carbon on sesquiterpenes forming the respective acids (Nguyen et al. (2010) J. Biol. Chem. 285(22): 16588-16598; Ro et al. (2006) Nature, 440(7086): 940-943; Andersen et al. (2017) Plant Physiol.174(1): 56-72; Ikezawa et a/. (2011) J. Biol. Chem. 286(24): 21601-21611; Nelson et al. (201 1) Plant J. 66(1): 194-211). Because amorphadiene oxidase AaCYP71 AVI catalyzes three successive oxidations on amorphadiene, similar to the oxidations seen in our target pathway, we expected high homology of AaCYP71AVl to our candidates. Of the selected putative V. officinalis P450s in the CYP71D family, VoCYP71D510 and VoCYP71D511 share approximately 51% homology with AaCYP71AVl, while VoCYP71DJl and

VoCYP71BE87 share approximately 44% homology with AaCYP71 AVI at the protein sequence level (Fig. 6). VoCYP71D510 and VoCYP71D511 share high homology to VoCYP71D442, previously identified as a possible V. officinalis P450 candidate involved in valerenic acid biosynthesis, but this enzyme did not co-express with VDS or upstream enzymes and did not produce oxidized sesquiterpenes (Ricigliano et al. (2016) Phytochem. 125: 43-53).

[0134] Functional identification of V. officinalis P450s acting on valerenadiene . To test the candidate P450s for activity, we co-integrated a single copy of each candidate with A. annua cytochrome P450 reductase, AaCPRl, into our high valerenadiene producer strain, JWy608. Unfortunately, no valerenic acid was produced by any of the candidate P450s.

However, we detected the production of trace amounts of hydroxylated valerenadiene by one of our candidate P450s, VoCYP71DJl (Fig. 1, panel A, Fig. 7). We were surprised to find that none of the other candidates functioned as a valerenadiene oxidase, despite sequence similarity and upregulated expression in the root.

[0135] Expression of alcohol and aldehyde dehydrogenases forms valerenic acid. Several Asteraceae pathways produce respective sesquiterpenoid acids in two steps, requiring only a synthase and a P450. However, several of these pathways and those of other terpenoids show increased production of the final product by overexpressing alcohol and aldehyde dehydrogenases, including the artemisinic acid, jolkinol C, zerumbone, and germacra-l(10),4, l l(13)-trien-12-oic acid pathways (Okamoto et al. (20\ \) FEBSJ. 278(16): 2892-2900; de Kraker et al. (2001) Plant Physiol. 125(4): 1930-1940; Luo et al. (2016) Proc. Natl. Acad. Sci. USA, 113(34): 5082-5089; Paddon et al. (2013) Nature, 496(7446): 528- 532).

[0136] We mined the V. officinalis transcriptome for genes with homology to A.

annua dehydrogenases and found VoADHl and VoALDHl . VoADHl had poor expression in the root transcriptome, while a VoALDHl was highly expressed in the root and stem tissues (Fig. 8). VoADH and VoALDH were co-integrated into our yeast strain expressing VoCYP71DJl, JWy614, resulting in trace amounts of valerenic acid, as determined by the mass spectrum and retention time relative to an authentic valerenic acid standard (Fig. 9).

[0137] Due to the low titer of valerenic acid in this strain, we surmised that another P450 candidate could be responsible for the conversion of valerenic alcohol to valerenic acid. We individually integrated all other P450 candidates into the valerenic acid base strain, JWy615, but failed to see improved valerenic acid titer (Fig. 10). Additionally, hydroxyvalerenic acid (Fig. 1, panel A), a valerenic acid derivative, was also not detected in these strains expressing additional P450 candidates.

[0138] To further improve titer, we integrated the previously published A. annua dehydrogenases AaADH and AaALDH, used to improve artemisinic acid titer in yeast, surmising that they might have activity on valerenic alcohol and valerenic aldehyde substrates. Co-integration of A. annua AaADHl and AaALDHl in JWy614 increased valerenic acid titer (Fig. 9); thus, although the VoADHl and VoALDHl identified in this study formed valerenic acid in yeast, it is possible that one or both of these enzymes are not actually responsible for the formation of valerenic acid in the native plant, which is consistent with their expression profiles. Our final strain, JWy627, produces 4 mg/L of valerenic acid. These results show that the techniques that led to the successful microbial production of artemisinin can be applied to other terpenoid pathways.

Summary and conclusions.

[0139] We have engineered a yeast chassis for the production of the sedative valerenic acid by engineering the production of the backbone valerenadiene, identified novel P450 oxidizing valerenadiene, and overexpressed these two genes, and completed the valerenic acid biosynthetic pathway in yeast. Valerenadiene and valerenic acid were produced at 140 mg/L and 4 mg/L, respectively. Phylogenetic and expression analyses were used to identify a valerenadiene oxidase, VoCYP71DJl . Further, expression of an ADH and ALDH were required to produce a yeast strain capable of generating valerenic acid.

Microbially produced valerenic acid may allow for more accurate studies of this drug, as plant derived material contains many bioactive compounds, including acetoxyvalerenic acid, a compound with antagonistic effects of valerenic acid. The gene testing strategy used in this study could prove valuable for gene discovery in other medicinally useful Asteraceae sesquiterpenoid pathways. These findings also illustrate that closely related P450s have been fine-tuned by evolutionary pressure for specialized metabolism. Additional strain engineering can improve valerenic acid titers for industrial applications.

[0140] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

INFORMAL SEQUENCE LISTING

SEQ ID No: 1 - nucleic acid sequence encoding valerena- , 7 (11) -diene synthase (VDS) , having codon bias for improved expression in yeast

ATGTCTGAGAGCTGTCTTTCATTTTCAAGTCCACCTCCCACAAAGAAAAATATTCAAGAGCC CGTAAGGCCGAATGCAAAGTTTCACAAAAGTGTCTGGGGCAACCACTTTTTAAAGTACGCCT CTAATCCAGAACAAATCGACTACGACGCTGACGAACAACATGAACAATTGAAGGAGGAGCTG AGGAAAAAGCTGGTGGTGAATGTAACTAACGAAAGGGTGGAGGAACAACTAAAGCTTATAGA CGCTATCCAGCGTTTGGGCGTAGCATATCATTTTCAACGTGAGATCGATGCAGTCCTGAATA ATCTGTTACTATTCAGGTCCAACAAGGACTCTGATGATATTTATATGGTGTCTCTGAGATTC AGACTTTTAAGACAACAAGGGCACAATGTTTCCTGCTCAGTATTTGAGAAATTTAAGAATAT AGATGGCAGATTCAAGGACTCCCTAAGAGATGATGTAAGAGGGTTGCTGTCACTATATGAGG CAACCCATATGAGGGTTCACAAAGAGGATATCTTAGAAGAAGCGTTAGAATTTACCATCTAT GAACTGGAGCAAGTTGTTAAGTTGTCCTCTAACGACACCTTACTTGCTTCCGAGGTGATTCA TGCGCTGAATATGCCTATCCGTAAGGGGTTGACGAGGATTGAAGCGAGGCACTTTATCAGTG TTTATCAACATGACAAAAGCCATGATGAGACCCTGCTTAAATTTTCTAAAATTGACTTTAAC ATGCTGCAAAAGTTGCATCAACGTGAGTTGGCAGACTTAACTATCTGGTGGGAAAAATTGAA CGTGGCTGAAAAGATGCCATACGCGAGAGATAGATTCGTGGAATGTTATTTTTGGGGGCTTG GTGTATACTTTGAGCCTCAATATTCCAGAGCTCGTAAAATGTTTGTAAAGGTGATAAATTTA ACTTCCCTTATTGATGACACGTATGATTCATATGGTACATTCGATGAATTGGATTTATTCAC AGATGCGGTTAAGAGGTGGAACGTAAATGAAACTGACAAATTGCCGGAGTATATGCGTCCAT TGTTTATGGAGCTTCTTAATGTTTATAACGCGATGGAGGAGGAGCTAAAGGAGGAGGGGGTT AGCTACAGAGTCGAGTACGCAAAACAATCTATGATACAGATAGTGACTGCATACAACGATGA GGCTATCTGGTACCACAACGGTTATGTCCCTACGTTTGACGAATATCTTAAAGTTGCCCTGA TCAGCTGCGGCTACATGTTATTGTCCACTATAAGCTTTGTCGGCATGGGGGTCACCACAGTC ACGAAACCCGCCTTTGACTGGGTAACAAATAACCCACTAATCCTAATTGCCTCATGTACAAT TAACAGACTGGCGGACGACAAGGTGGGGCACGAGCTAGAGCAGGAGAGAGGGCATGTTGCTT CAGGGGTGGAGTGCTATATGAAACATAATAATGCAACGAAACAGGAAGTGGTAATCGAATTT AACAAGAGAATTAGTAACGCTTGGAAAGACATCAATCAAGAGTGCTTGCACCCACTACCAGT ACCGCTTCACTTGGTAGTCCGTCCCTTATATCTAGCGTGCTTCATGAACGTATTTTATAAAG ATGAAGATTGGTATACCCATAGCAACACGCAGATGAAAGAGTGCATCAATTCTTTGTTAGTT GAAAGCGTCCCTTATTAA

SEQ ID No: 2 - amino acid sequence of valerena- , 7 (11) -diene synthase (VDS) (NCBI Reference Sequence No. J9R5V .1 ; has 99% sequence identity to Valeriana officinalis sesquiterpene synthase 1 NCBI Reference Sequence ID: AGB05610.1)

MSESCLSFSSPPPTKKNIQEPVRPNAKFHKSVWGNHFLKYASNPEQIDYDADEQHEQLKE ELRKKLWNVTNERVEEQLKLIDAIQRLGVAYHFQREIDAVLNNLLLFRSNKDSDDIYMV SLRFRLLRQQGHNVSCSVFEKFKNIDGRFKDSLRDDVRGLLSLYEATHMRVHKEDILEEA LEFTIYELEQWKLSSNDTLLASEVIHALNMPIRKGLTRIEARHFISVYQHDKSHDETLL KFSKIDFNMLQKLHQRELADLTIWWEKLNVAEKMPYARDRFVECYFWGLGVYFEPQYSRA RKMFVKVINLTSLIDDTYDSYGTFDELDLFTDAVKRWNVNETDKLPEYMRPLFMELLNVY NAMEEELKEEGVSYRVEYAKQSMIQIVTAYNDEAIWYHNGYVPTFDEYLKVALISCGYML LSTISFVGMGVTTVTKPAFDWVTNNPLILIASCTINRLADDKVGHELEQERGHVASGVEC YMKHNNATKQEWIEFNKRISNAWKDINQECLHPLPVPLHLWRPLYLACFMNVFYKDED WYTHSNTQMKECINSLLVESVPY* SEQ ID No: 3 - nucleic acid sequence encoding maltose binding protein (MBP) -valerena- , 7 (11) -diene synthase (VDS) - farnesyl diphosphate (FPP) synthase (ERG20) fusion protein, having codon bias for improved expression in yeast, MBP and ERG20 tags underlined

ATGAAGATTGAAGAAGGTAAGTTGGTTATCTGGATTAACGGTGACAAGGGTTACAACGGTTT GGCTGAAGTTGGTAAGAAATTTGAAAAAGATACCGGTATCAAGGTCACTGTTGAACACCCAG ACAAGTTGGAAGAAAAGTTTCCACAAGTTGCTGCCACTGGTGATGGTCCAGACATTATCTTC TGGGCTCATGACAGATTCGGTGGTTACGCCCAATCCGGTTTGTTAGCCGAGATCACCCCAGA TAAGGCTTTTCAAGATAAGTTGTATCCATTCACTTGGGATGCCGTCAGATACAACGGTAAGT TAATCGCCTACCCAATTGCTGTTGAAGCTTTGTCTTTGATCTACAATAAGGACTTGTTACCT AACCCACCAAAGACCTGGGAAGAAATCCCAGCTTTAGATAAGGAGTTAAAAGCTAAGGGTAA GTCCGCTTTGATGTTTAACTTGCAAGAACCATACTTCACTTGGCCATTGATCGCTGCTGATG GTGGTTACGCTTTTAAGTATGAAAACGGTAAATACGACATTAAGGATGTCGGTGTCGACAAT GCTGGTGCTAAGGCCGGTTTAACTTTCTTAGTCGATTTGATTAAGAATAAACATATGAATGC TGACACTGATTACTCTATTGCTGAAGCTGCTTTCAACAAGGGTGAAACCGCTATGACTATTA ACGGTCCATGGGCCTGGTCTAACATTGATACCTCTAAAGTCAACTACGGTGTCACCGTCTTG CCAACTTTTAAGGGTCAACCATCTAAGCCATTCGTCGGTGTCTTGTCTGCCGGTATTAACGC TGCCTCTCCAAATAAGGAATTGGCCAAGGAATTCTTAGAAAACTACTTGTTAACCGATGAAG GTTTAGAGGCCGTTAACAAGGATAAGCCATTAGGTGCTGTTGCTTTGAAGTCTTACGAAGAA GAGTTGGCTAAGGATCCAAGAATTGCTGCTACTATGGAAAACGCTCAAAAGGGTGAAATTAT GCCAAACATCCCACAAATGTCTGCTTTCTGGTACGCTGTTCGTACCGCCGTCATTAATGCCG CTTCTGGTCGTCAAACTGTTGATGAAGCCTTGAAGGACGCTCAAACCAGAATTACTAAGGGA GGTGGTGGAGGTGGATCTGAATCTTGCCTTTCATTCTCTTCTCCGCCGCCTACTAAAAAGAA CATACAAGAGCCTGTTAGACCCAATGCCAAGTTTCATAAGTCTGTGTGGGGGAATCATTTTC TGAAATATGCCTCTAATCCTGAGCAAATTGACTACGACGCGGACGAACAGCATGAACAACTA AAGGAAGAATTAAGGAAAAAACTGGTAGTAAACGTAACAAATGAACGTGTTGAAGAGCAGCT GAAACTAATCGATGCCATTCAACGTCTTGGCGTCGCGTACCATTTCCAAAGGGAGATTGATG CGGTCCTAAATAACCTTCTGTTATTTCGTAGCAATAAGGACTCCGACGATATCTACATGGTG TCTCTGCGTTTTCGTCTACTGCGTCAGCAGGGCCACAATGTTAGTTGCAGCGTCTTCGAAAA GTTCAAGAACATCGATGGTAGGTTTAAGGACAGCCTGCGTGACGACGTACGTGGTTTGCTGA GTTTATATGAAGCCACACATATGCGTGTACATAAGGAGGACATACTGGAAGAAGCACTAGAA TTCACTATCTACGAGCTAGAACAAGTTGTTAAACTTTCATCTAATGACACACTATTGGCCAG TGAAGTTATTCACGCTCTGAATATGCCTATACGTAAGGGCTTGACTAGAATCGAGGCCAGAC ACTTCATATCTGTTTACCAGCATGATAAGAGCCATGACGAGACATTGCTAAAGTTCAGTAAA ATAGACTTCAACATGCTTCAAAAACTACACCAGAGGGAGCTGGCCGACTTAACGATATGGTG GGAAAAGCTAAATGTGGCGGAGAAAATGCCGTACGCGCGTGATCGTTTCGTCGAATGCTACT TTTGGGGATTGGGGGTTTACTTCGAACCTCAGTATTCCAGAGCTAGAAAAATGTTTGTGAAA GTTATCAACCTTACAAGCCTTATCGATGACACGTACGACAGCTACGGTACATTCGACGAACT GGACTTATTCACGGATGCCGTTAAGAGATGGAACGTGAATGAGACTGACAAACTACCTGAGT ACATGAGGCCACTATTCATGGAGTTGCTGAACGTCTACAATGCAATGGAGGAGGAATTAAAG GAAGAGGGAGTGAGTTACAGAGTCGAATACGCTAAGCAATCTATGATCCAGATTGTCACAGC ATACAATGACGAGGCTATATGGTACCATAATGGCTATGTTCCCACCTTCGATGAGTATTTGA AAGTAGCTCTAATCAGCTGTGGGTATATGCTTCTAAGCACAATCTCCTTCGTCGGCATGGGC GTAACAACCGTAACCAAGCCTGCGTTCGACTGGGTTACTAATAACCCTTTAATCTTGATTGC CTCATGTACCATCAATCGTCTGGCTGATGACAAAGTCGGCCATGAACTAGAGCAAGAGAGAG GTCACGTCGCTTCCGGCGTTGAGTGTTATATGAAGCACAATAATGCAACGAAGCAAGAAGTA GTGATAGAGTTTAACAAGCGTATCAGCAATGCATGGAAAGATATCAACCAGGAGTGTCTTCA TCCGTTGCCTGTCCCCCTACACCTGGTAGTCCGTCCGCTATATTTGGCGTGCTTCATGAATG TTTTTTACAAAGATGAGGACTGGTATACACATTCTAATACACAAATGAAAGAATGTATTAAC AGCTTATTGGTAGAAAGTGTCCCCTATGGTAGCGGTAGCGGCAGCGCTTCAGAAAAAGAAAT TAGGAGAGAGAGATTCTTGAACGTTTTCCCTAAATTAGTAGAGGAATTGAACGCATCGCTTT TGGCTTACGGTATGCCTAAGGAAGCATGTGACTGGTATGCCCACTCATTGAACTACAACACT CCAGGCGGTAAGCTAAATAGAGGTTTGTCCGTTGTGGACACGTATGCTATTCTCTCCAACAA GACCGTTGAACAATTGGGGCAAGAAGAATACGAAAAGGTTGCCATTCTAGGTTGGTGCATTG AGTTGTTGCAGGCTTACTTCTTGGTCGCCGATGATATGATGGACAAGTCCATTACCAGAAGA GGCCAACCATGTTGGTACAAGGTTCCTGAAGTTGGGGAAATTGCCATCAATGACGCATTCAT GTTAGAGGCTGCTATCTACAAGCTTTTGAAATCTCACTTCAGAAACGAAAAATACTACATAG ATATCACCGAATTGTTCCATGAGGTCACCTTCCAAACCGAATTGGGCCAATTGATGGACTTA ATCACTGCACCTGAAGACAAAGTCGACTTGAGTAAGTTCTCCCTAAAGAAGCACTCCTTCAT AGTTACTTTCAAGACTGCTTACTATTCTTTCTACTTGCCTGTCGCATTGGCCATGTACGTTG CCGGTATCACGGATGAAAAGGATTTGAAACAAGCCAGAGATGTCTTGATTCCATTGGGTGAA TACTTCCAAATTCAAGATGACTACTTAGACTGCTTCGGTACCCCAGAACAGATCGGTAAGAT CGGTACAGATATCCAAGATAACAAATGTTCTTGGGTAATCAACAAGGCATTGGAACTTGCTT CCGCAGAACAAAGAAAGACTTTAGACGAAAATTACGGTAAGAAGGACTCAGTCGCAGAAGCC AAATGCAAAAAGATTTTCAATGACTTGAAAATTGAACAGCTATACCACGAATATGAAGAGTC TATTGCCAAGGATTTGAAGGCCAAAATTTCTCAGGTCGATGAGTCTCGTGGCTTCAAAGCTG ATGTCTTAACTGCGTTCTTGAACAAAGTTTACAAGAGAAGCAAATAA

SEQ ID No: 4 - amino acid sequence encoding MBP-valerena- 4 , 7 (11) -diene synthase (VDS) -ERG20 fusion protein, MBP and ERG20 tags underlined

MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDI I F WAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLP NPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDN AGAKAGLTFLVDLIKNKHMNADTDYS IAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVL PTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEE ELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKG GGGGGSESCLSFSSPPPTKKNIQEPVRPNAKFHKSVWGNHFLKYASNPEQIDYDADEQHEQL KEELRKKLWNVTNERVEEQLKLIDAIQRLGVAYHFQREIDAVLNNLLLFRSNKDSDDIYMV SLRFRLLRQQGHNVSCSVFEKFKNIDGRFKDSLRDDVRGLLSLYEATHMRVHKEDILEEALE FTIYELEQWKLSSNDTLLASEVIHALNMPIRKGLTRIEARHFISVYQHDKSHDETLLKFSK IDFNMLQKLHQRELADLTIWWEKLNVAEKMPYARDRFVECYFWGLGVYFEPQYSRARKMFVK VINLTSLIDDTYDSYGTFDELDLFTDAVKRWNVNETDKLPEYMRPLFMELLNVYNAMEEELK EEGVSYRVEYAKQSMIQIVTAYNDEAIWYHNGYVPTFDEYLKVALISCGYMLLSTISFVGMG VTTVTKPAFDWVTNNPLILIASCTINRLADDKVGHELEQERGHVASGVECYMKHNNATKQEV VIEFNKRISNAWKDINQECLHPLPVPLHLWRPLYLACFMNVFYKDEDWYTHSNTQMKECIN SLLVESVPYGSGSGSASEKEIRRERFLNVFPKLVEELNASLLAYGMPKEACDWYAHSLNYNT PGGKLNRGLSWDTYAILSNKTVEQLGQEEYEKVAILGWCIELLQAYFLVADDMMDKSITRR GQPCWYKVPEVGEIAINDAFMLEAAIYKLLKSHFRNEKYYIDITELFHEVTFQTELGQLMDL ITAPEDKVDLSKFSLKKHSFIVTFKTAYYSFYLPVALAMYVAGITDEKDLKQARDVLIPLGE YFQIQDDYLDCFGTPEQIGKIGTDIQDNKCSWVINKALELASAEQRKTLDENYGKKDSVAEA KCKKI FNDLKIEQLYHEYEES IAKDLKAKISQVDESRGFKADVLTAFLNKVYKRSK* SEQ ID No: 5 - nucleic acid sequence encoding Valeriana officinalis VOCYP71DJ1 valerenadiene oxidase, having codon bias for improved expression in yeast ATGATTATGGAGGATCTTAACTTCTCAATTATACACCCAATACTACTAATTTTCGTGGCATT CGTCATTATTGAGAACGTGAGAAAAAACAAAAAGGCGGTACGTAGACCGCCCGGCCCGTGGC AATTCCCGCTAATTGGAAATATGCACAATTTATTTGGTTCATCCCCGCACAGAGTACTGCGT GATCTAAGTAATAAATACGGGCCCATCATGTTGCTGCGTTTAGGGACAGTACCTACACTTGT GGTATCCTCTGCTGAGTTAGCTGAAGAGATACTGAAAATCAGAGGTGTTGAGTTTGCGGATA GGCCACACATTCTAGCAGCTGACATAGTCATCTATAATAGCACCGACATACTGTTTTCACCG TATGGCGATCATTGGCGTCAAATGAGGAAGGTCTGTGCAATGGAGCTTCTTAGTACTAGAAA AGTACAGTCCTTCCGTTCTATCAGGGAAGAGGAGGTACTAAATTTACTTCAACTAATATCTT CATCTTGTGCAAGCGGGTCCGCGTTGAACTTGACTAAACTACTATTTTCCTTTACTTACACA GTTATCACACGTGTGACGTTTGGTGAGAAGTGGACCGAACAACCCGAAAAATTCAGCTCCCT TTTAAGTGAACTAGTCGTGCTTTTTTCTGGCTTTAACATAGCTGATATGTACCCGAGCGTCA AGTTTATTCAAGGCGCGGGAGGGTTCAGGGCGAGAGCAGAGAAGGTCCACCAAAGGATGGAT GAGACGTTCACCAACATAATCATGAAACAACGTGAAAAAAACAAGGCAACAAGTACCGGAGA GTCCAACCAGGAACACCTGATTAACGTTCTATTACGTGTTCAGAAACATGCAGCTGGAACCG AGAATCCATTCACTGATGACAGCATCAAAGGCGTTCTACTAGATATTTTTAATGGTGGCAGT GAAACTAGCTCCACTACGATGGAGTGGGCGATGGCTGAGTTGATTAAAAACCCAAGGGCAAT GGAACGTGCGCAAACAGAGTTAAGACAAGCTTTCAGCGGGAAAGGAAATGTAGAGGAAACAG GACTTGATAAGCTGAAGTATTTTCACTGTATTATAAAAGAGACAATGAGATTGCACCCTCCG TTTCCACTGATGGTCCCACGTCAGAATAGGCATGAATGCGAGATAAATGGGTACATCATTCC GGCCAAGACTAAAGTATTAGTGAACGGATGGGCTATCAGTAGAAACCCCAAATACTGGGGAC CCGACGCCGACGTCTTTAAGCCGGAGAGATTCCTGGATAACAGGACTACCCATGACTACAAA GGGACCAACTCTGAATATATACCCTTCGGCGCTGGAAAGAGGATCTGTCCAGGGACTACATT CGCTTTGGCGGCTGTAGAGCTACCCCTGGCGCAGCTTTTATACCATTTCGACTGGAATTTAG CAACTGGCCTACAGAATAAAGAGCTGGAAATGACCGAACAATTCGGCATTGTGGTAAGGAAG AAATGCAACTTACACCTAAACCCCCTACCTTACAGCGGTTCCTTCCTAGATAACTAA

SEQ ID No: 6 - amino acid sequence of Valeriana officinalis VOCYP71DJ1 valerenadiene oxidase

MIMEDLNFS I IHPILLI FVAFVI IENVRKNKKAVRRPPGPWQFPLIGNMHNLFGSSPHRV LRDLSNKYGPIMLLRLGTVPTLWSSAELAEEILKIRGVEFADRPHILAADIVIYNSTDI LFSPYGDHWRQMRKVCAMELLSTRKVQSFRS IREEEVLNLLQLISSSCASGSALNLTKLL FSFTYTVITRVTFGEKWTEQPEKFSSLLSELWLFSGFNIADMYPSVKFIQGAGGFRARA EKVHQRMDETFTNI IMKQREKNKATSTGESNQEHLINVLLRVQKHAAGTENPFTDDS IKG VLLDI FNGGSETSSTTMEWAMAELIKNPRAMERAQTELRQAFSGKGNVEETGLDKLKYFH CI IKETMRLHPPFPLMVPRQNRHECEINGYI IPAKTKVLVNGWAISRNPKYWGPDADVFK PERFLDNRTTHDYKGTNSEYIPFGAGKRICPGTTFALAAVELPLAQLLYHFDWNLATGLQ NKELEMTEQFGIWRKKCNLHLNPLPYSGSFLDN* SEQ ID No: 7 - nucleic acid sequence encoding Valeriana officinalis VOCYP71D510 (a V. officinalis P450 enzyme in the CYP71D family) , having codon bias for improved expression in yeast

ATGGACTCATTTACCATAATCCTTATCAACGTTGTACCAGTTTTACTGATTTTCCTACTGTT TCGTAGATGGAAAAGTGCTAAAGCTGTCAATCTTCCGCCAGGTCCCCCCAAGCTACCATTGA TAGGCAGTCTTCTGCACATGGGTCAGTTGCCGCATAGAAGCTTGAAGGAGCTGGCGGGAAAG TACGGACCTCTAATGCACATTCAGTTAGGAGAAATTAGCGCAATTGTAGTGTCATCTCCAAG AGTTGCTAAACTAGTGACAAAAACGCACGACCTAAGCTTTGCGTCAAGACCAGTTATCTTGG CAAGTGAGATAGTTGGATATCATAACACTGACATCGCATTTGCCCCATATGGTGACTACTGG CGTCAAATGCGTAAAATAGCCACGTTAGAGTTATTGTCTGCGAAAAAAGTGAGGTCCTTCTG TTCTATCAGAGAGGAGGAGGCAAAAAATTTGATTGAATCTATACATTCTACCAGTGGGAAGT CATTCGATCTAACCGAAAAGGTTTTCTCACTTACAAACAACGTAATCTGCAGAGCGACCTTT GGCGATAGGTATAAGGACCAAGATTATTTGATAAAGATCCTTAAACAAGTAGTGAATTTAGC TGGAGGGTTTGACGTAGCTGACCTATTCCCCAGCTTGAAGCTACTTCACTTAGCGACAGGAA TGAGGCCAAAACTTGAGAATCTAAGGAAGGATCTAGATAGAATATTTGATGAAATTATCAAT GAGCGTACCAAGAGGTTGAAAAACGGCACAAATACGCATGAAGACAACGAAGATATCGTTGA TGTTCTAGTTAGGCTGAAGGAATCCGGGGCGCTAGAGTTCCCTATCACCCAAAACAACATTA AGGCGGTAATACTAGATATGTTCTTAGCCGGATCCGACACGTCCTCAACAACTATCGAATGG GCTATGGCAGAAATGATGCGTAATCCAAGAGTTTTGGAAAAGGCACAGGCGGAGCTTCGTCA GGCTATGAACGGTAAAAAGGTAATAGAAGAATCAGATATTAAGGAAACAGGGTCATACTTCA AGTTAGTAATTAAAGAGACATTGAGAATGCATCCGCCTGTCGCGCTTCTTCTACCAAGGGAA TGTCGTGAGGAGTGCGAAATCGATGGCTACACCATCCCAGTGAAGACAAAGGTAATGGTCAA CGCTTGGGCGATTGGGAGGGATCCTGAGTATTGGAAAGACGCTGACAGCTTTTATCCCGAGC GTTTCGAGAACTCTGATGTAGACTATTTGGGGAGTAACTACGAATTTATCCCCTTTGGAAGT GGGCGTAGGATATGTCCTGGCATGACCTTTGGACTGGCGAACGTCGAGTTGCCACTAGCGAA TTTGCTTTACCATTTCGATTGGAAACTGCCCTCAGGTATGAAACCGGAAGATGTGGACATGA CCGAGGACTTTGGGGCTTCTCTTAGAATAAAAAACAACCTTCACGTTGTGGCGACGAGGTAC AGCTACTCCTCTTAG

SEQ ID No: 8 - amino acid sequence of Valeriana officinalis VoCYP71D510 (a V. officinalis P450 enzyme in the CYP71D family)

MDSFTIILINWPVLLIFLLFRRWKSAKAVNLPPGPPKLPLIGSLLHMGQLPHRSLKELA GKYGPLMHIQLGEISAIWSSPRVAKLVTKTHDLSFASRPVILASEIVGYHNTDIAFAPY GDYWRQMRKIATLELLSAKKVRSFCSIREEEAKNLIESIHSTSGKSFDLTEKVFSLTNNV ICRATFGDRYKDQDYLIKILKQWNLAGGFDVADLFPSLKLLHLATGMRPKLENLRKDLD RI FDEI INERTKRLKNGTNTHEDNEDIVDVLVRLKESGALEFPITQNNIKAVILDMFLAG SDTSSTTIEWAMAEMMRNPRVLEKAQAELRQAMNGKKVIEESDIKETGSYFKLVIKETLR MHPPVALLLPRECREECEIDGYTIPVKTKVMVNAWAIGRDPEYWKDADSFYPERFENSDV DYLGSNYEFIPFGSGRRICPGMTFGLANVELPLANLLYHFDWKLPSGMKPEDVDMTEDFG ASLRIKNNLHWATRYSYSS* SEQ ID No: 9 - nucleic acid sequence encoding Valeriana officinalis VOCYP71D511 (a V. officinalis P450 enzyme in the CYP71D family) , having codon bias for improved expression in yeast

ATGGACTTCATTGCCATAGCTCTTCCCTCAGTGGCGTTCCTGTTCTTTTTGTTGAAATTAGT CAGGAAATTGCGTTCCCCGAAAAGATTACCACCTGGGCCTTGGAAACTGCCATTGATCGGGT CCCTTTTGCATATGGCCGGACCATTACCACACAGAACATTGAAAGATCTAGCTGAGAAGTAC GGCCCTTTAATGCATTTACAATTAGGAGAGATCTCTGCAATTGTGGTCTCTAGCCCGGAGAT GGTAAATGAATTTATGAAAACACACGATATCGCTTTTGCTAGTAGGCCGCCCGTTTTAGCGA TCGAGATTGTTGCTTACAATAGAGATGATATAGCGTTCGCACCATATGGTGATTACTGGAGA CAAATGCGTAAGATCGCTACATTAGAACTACTTAGTGTAAAGAAGGTGGGTAGCTTCTCTTC TATCCGTGAGAAAGAAGTGCACAATCTTGTTGAGTCAATCTCCAGCTCAGGATCAATCATCC CGATAGACATGACAGAAAAGTTATTTGGTCTTATTAGCAGCGTCGCAGCTAGGGCATCATTC GGTAACAAATGTAAAGACCAAGACAGTTTCCTAGAGTTAACAAATGAGATTATCTCTTTAGC AGGTGGATTTAACATTTTCGACCTTTTTCCGTCTTTTAAACTTCTGCACAGGCTGACAGGAA TGAGGCAGAAATTTGAGATGATGCATCAAAAGGTTGACCAAGTTTTTGAGAACATTATTAAA GATCATATACAAGAAAGAGCCGATGATACCCACGGGAACGACCACACGGAAGATCTATTAGA CGTTCTGCTACGTCTAAAGGACGAAGGCCTTGAGTTTCCTATAACTTACACCAACGTCAAAG CGGTTATACTTAATGCTTTCTCCGGAGGCTCCGACACGTCCAGCACCACTATAGAGTGGGCG ATGACGGAGTTGATGAGGAATCCTCGTGTAATGGAAAAAGCGCAAGCAGACTTAAGAGAAGC ACTGAAGGGAAAACAGGTAGTCAACGAGAATGATATTAAAGACTTACCGTATTTGAAGTTGG TTATGAAAGAAACCATGCGTCTTCATACACCTCTACCACTACTGGTGCCTAGAGAGTGCCGT CAGGAAGTAGAAATTGACGGGTACACAATAACAGTAGGTACTAAGATCATTATAAATGCTTG GGCTATTGCGAGGGACCCCCAGTATTGGAAAGATCCGGAATCTTTTTACCCGGAGAGGTTCG AGGACGGTACGGTGGACTTTAAAGGTTCTAACTACGAGTTTATCCCGTTCGGCTCTGGAAGA AGGATGTGCCCAGGCATTGCTTTTGCACTAGCTACAGTCGAGTTACCACTGGCCAATTTACT ATATCATTTCGATTGGAAGCTACCAAACGAAATGAAACCCGAGGACTTGGACACTAACGAAT TTTTCGGCGCAACAGTCAAAAAACTTAACCACCTGTGTCTTATCGCCACTAGGAGGACTCCC ACTCTATAA

SEQ ID No: 10 - amino acid sequence of Valeriana officinalis VoCYP71D511 (a V. officinalis P450 enzyme in the CYP71D family)

MDFIAIALPSVAFLFFLLKLVRKLRSPKRLPPGPWKLPLIGSLLHMAGPLPHRTLKDLAE KYGPLMHLQLGEISAIVVSSPEMVNEFMKTHDIAFASRPPVLAIEIVAYNRDDIAFAPYG DYWRQMRKIATLELLSVKKVGSFSS IREKEVHNLVESISSSGSIIPIDMTEKLFGLISSV AARASFGNKCKDQDSFLELTNE11SLAGGFNI FDLFPSFKLLHRLTGMRQKFEMMHQKVD QVFENI IKDHIQERADDTHGNDHTEDLLDVLLRLKDEGLEFPITYTNVKAVILNAFSGGS DTSSTTIEWAMTELMRNPRVMEKAQADLREALKGKQVVNENDIKDLPYLKLVMKETMRLH TPLPLLVPRECRQEVEIDGYTITVGTKI I INAWAIARDPQYWKDPESFYPERFEDGTVDF KGSNYEFIPFGSGRRMCPGIAFALATVELPLANLLYHFDWKLPNEMKPEDLDTNEFFGAT VKKLNHLCLIATRRTPTL* SEQ ID No: 11 - nucleic acid sequence encoding Valeriana officinalis VOCYP81Q107 (an enzyme having homology to P450s involved in the sesamin biosynthetic pathway) , having codon bias for improved expression in yeast

ATGCTCCGAAAATTGTACGATGGTGGTGAGTCATCATCTAAGGTTGAATTGAAAACCACACT CTCTGAGCTAACATTTAACATCATATTGAGGATGATTGCCGGAAAAAGATATTTTGGGGAAG ATGTGAAAGAGAACGAGGAGGCTGTTCGGTTTAGAAGTTTGATAAAGGAGATTGTTAAGTAC GGCGGAGCATCGAATCCCGGAGATTTCTTGCCGATTCTTGGGTGGTTTGATTATGGCGGGTT TCAGAAGAATCTGACGAGGATCGGTAAGCAGATGGACGGTTTGTTGCAGGGATTAATCGAAG AGCATAGACGTGAGAAGAATAAAAATACGATGGTGGATCATCTTCTTTCCCTTCAAGAATCA GAACCGGAATATTACACCGATGAAATCATCAAAGGCCTAATGATTGTGATGGTAACCGCCGG AACAGACACATCGTCAGTGACAGTCGAATGGGCAATGTCGCTATTACTCAACCACCCCGAAA TACTAAAGAAAGCACGAGCCGAAATCGATAAAGAAGTCGGAGAAAGCCGTTTAGTAGACGAA CCGGATCTCCCAAAACTACCTTATCTCCAAAACATAATTCTCGAAACACTCAGAATGTTCCC ATCGGCACCACTACTAATCCCGCACGAGTCGTCAGAGGATTTCAAGCTTGGAGAATACGATG TACCAAAAGGAACAATCGTTTTAATCAATGCATGGGCTATACATCGGGATCCGAACGTATGG GATGATCCGACGAGTTTTAATCCCGATAGATTCAATGATTTTAATACTAGTAATACTAGTAC TGTCGGTGGAGCGGTAATGGTGGCGAATAGTAAGTTGTTACCTTTTGGGATGGGACGGAGGC AGTGCCCAGGATCGGGTTTGGCTCAACGGATGGTCGGGTTAGCGTTGGCTTCGATGATACAG TGTTTTGATTGGGAAAGAGTTAGTGACGGGTTAGTCGATTTAGCCGAAGGACTCGGAGTTAC AATGCCAAAAGCTGAGCCGCTCGAGGCTGTGTGTACACCGCGGGTGTTCGCGCGGAATCTTC TATTTGGATGA SEQ ID No: 12 - amino acid sequence of Valeriana officinalis VoCYP81Q107 (an enzyme having homology to P450s involved in the sesamin biosynthetic pathway)

MLRKLYDGGESSSKVELKTTLSELTFNIILRMIAGKRYFGEDVKENEEAVRFRSLIKEIV KYGGASNPGDFLPILGWFDYGGFQKNLTRIGKQMDGLLQGLIEEHRREKNKNTMVDHLLS LQESEPEYYTDEI IKGLMI\ VTAGTDTSSVTVEWAMSLLLNHPEILKKARAEIDKEVGE SRLVDEPDLPKLPYLQNI ILETLRMFPSAPLLIPHESSEDFKLGEYDVPKGTIVLINAWA IHRDPNVWDDPTSFNPDRFNDFNTSNTSTVGGA\/MVANSKLLPFGMGRRQCPGSGLAQRM VGLALASMIQCFDWERVSDGLVDLAEGLGVTMPKAEPLEAVCTPRVFARNLLFG*

SEQ ID No: 13 - nucleic acid sequence encoding Valeriana officinalis VOCYP714A33 (a V. officinalis P450 enzyme having homology to gibberellin oxidases) , having codon bias for improved expression in yeast

ATGAGATTGAAGCTAACAAGACAAGGCATAAAAGGTCCAAAGCCTCATTTTATGTATGGAAA TGTTCCTCAGATGCAGAAAATCCAATCTGCCGCTGTAGAATCTGGTAGCTGCAACCATGGCG AAATCATCGCGCACGATTACACTTGTGCCCTCTTCCCATATTTCGAACAATGGAGAAAACAA TACGGTTTAGTATACACATACTCAACTGGGAACAAACAGCATTTGTATATAACCAAAGCCGA ATTAGTCAAGGAGATGAATCAGTCTGGATTAGGGAAGCCTTCTTATATTACCAAGAGACTAG CTCCTCTGCTTGGCAATGGCATTTTAAGATCAAATGGCCATCTCTGGGCCCAACAGAGAAAA ATTGTTGCCCCTGAATTCTTCATGGATAAAGTCAAGGGTATGCTGGGTCTGATGTTGGAATC AACACAGCCACTGATTAAGAAATGGGAAGAATCAATTGAAAGCCAAGGTGGAAAAATAGCTG AGATTAGAATTGATCAAGATTTCAGGGGCGTCTCTGCTGACGTCATCTCAAGAACTTGCTTT GGAACTTCTTACTCCAAAGGCAAACTCATTTTCTCCAAGCTTAGAACTCTTCAACACACCTT CTCTTCTGGAGGTTTCCTTTTTACTCTTCCTACATTCGGATTTCTTGCGAGAAAGAACCACA AGGAGATCAAGAATTTGGAGAAAGAGATAGACACGTTAATCTGGGATGCAGTTAAAGAACGG CAAAGAGAGTGTTTAGAGAAATCATCTTCAGAGAAGGATCTTTTACAGATGTTATTAGAAGG AGCCATGAATGATGAATGTCTAGGAGCAGAATCGTCAAAATCATTCATCGTTGATAATTGCA AAAACATTTATTTCGCCGGCCATGAAGCAACCGCCGTCGCAGCTTCATGGTCCATAATGTTG CTTGCTTTGCATCCAGAATGGCAGTCTCACATTCGAGAAGAAATGTCTCAAGTTTCCAATAA TGGAATCCTAGATTCAGATTCCCTATCCAAAATGAAAACTGTAACGATGGTGATCCAAGAAG CGTTGCGATTATATCCACCAGCAGCATTTGTGTCGAGAGAAGCATTTGCAGAAACAAAAATA GGAAATATTAAGATTCCAAAAGGGGTATGCATATGGACATTAATACCCACGCTGCATCGTGA TCCTGATAATTGGGGATTAGATTCTAATGAATTTAAACCAGAGAGATTCGCTAATGGAGTAT CAAATGCTTGCAAGATTCCACAAGCTTACGTTCCATTTGGGCTTGGTCCTAGGCTGTGTTTA GGGCGAAATTTCGCTATGGTTCAGCTGAAAGTTGTTTTATCTCTAATCATATCCAAGTTCAA ATTCTCCTTGTCGCCGAATTACAAGCACTCGCCTCATTATAAAATGATCGTCGAACCTGGGA ATGGAGTCAATATCTTGATTCAAAAGATTTAA SEQ ID No: 14 - amino acid sequence of Valeriana officinalis VOCYP714A33 (a putative V. officinalis P450 enzyme having homology to gibberellin oxidases)

MRLKLTRQGIKGPKPHFMYGNVPQMQKIQSAAVESGSCNHGEI IAHDYTCALFPYFEQWR KQYGLVYTYSTGNKQHLYITKAELVKEMNQSGLGKPSYITKRLAPLLGNGILRSNGHLWA QQRKIVAPEFFMDKVKGMLGLMLESTQPLIKKWEESIESQGGKIAEIRIDQDFRGVSADV ISRTCFGTSYSKGKLI FSKLRTLQHTFSSGGFLFTLPTFGFLARKNHKEIKNLEKEIDTL IWDAVKERQRECLEKSSSEKDLLQMLLEGAMNDECLGAESSKSFIVDNCKNIYFAGHEAT AVAASWS IMLLALHPEWQSHIREEMSQVSNNGILDSDSLSKMKTVTMVIQEALRLYPPAA FVSREAFAETKIGNIKIPKGVCIWTLIPTLHRDPDNWGLDSNEFKPERFANGVSNACKIP QAYVPFGLGPRLCLGRNFAMVQLKWLSLIISKFKFSLSPNYKHSPHYKMIVEPGNGVNI LIQKI*

SEQ ID No: 15 - nucleic acid sequence encoding Valeriana officinalis VOCYP71BE87 (shares homology with the Vitis vinifera P450 WCYP71BE5 responsible for the formation of the sesquiterpenoid (-) -rotundone) , having codon bias for improved expression in yeast ATGGTATGGTTTTTACTGAAAGACAGGACTTTCCTATTGCAGAAATTTTTGTGGTATGATAG TACTTCTATCGCCTTCAGCTCTTACGGGGACTACTGGCGTCAGCTAAGGAAGATTTGTACGA TGAACCTATTGAGTACAAAGCGTGTGGAACAGCTGAGAAGCATTAGGGAGGAGGAAGCTTTG AACCTAGTTAGGAGAATATCCACAAACGGCGATTCTTTGCCATTTAATCTAAGCAAGGCTAT TTTTAATTTGACCAGCACGGTCACGAGTCGTGCGGCGTTCGGGAATAAAAACAAAGACCAAG AAGAATTCGAGGTAGTTCTGGACCAAGTTCTGAAGGCGCTGGGCGGTTTTAACATAGGCGAC ATGTACCCGAAAGCTAAATTACTTCATAAGATAACCGGGGCGCGTGCCTCCATGAACAAGAT ACAGAAGAGAGTGGATAGAATTTTACAAAACATCTTAGTCGACCACCGTAATAGGAAGCAGG AATCTTTAACAGATGATTACGAGGATCTAGTGGACGTACTACTTAGAATCCAAAATGAAGAC CAACTTCAATTCCCAGTAACTGATAACTGTATAAAAGCAGTCATCTTAGACGTGTTCGGTGG TGGGAGCGAAACAAGTAGCGCTGCTACTGAGTGGGCAATGAGTGAAATGGTGAAGAATCCCC ACATCATGAAAAGGGCTCAAGCTGAAGTTCGTAAAGTTTTCGATGAGAAAAGAAATGTAGAT GAGACCGGCTTGGGGGAGCTTAAATATCTGCAGTGTGTAATAAAAGAGACCCTGAGGCTGCA TCCACCGTTGCCATTACTGGTTCCCAGAGAGAATAGTGCCGAGTGTGAAGTTAACGGATTTC TGATTCCAGCGAACTGTAAAGTTATCATAAATGCGTGGGCGATCTCCCGTGATCCTAAGTAC TGGGTGGATGCAGAGATTTTCAAACCCGAAAGATTTATGGATAATAGTATTGATTACCAGGG GACGAACTTTGGATACATACCATTCGGCGCTGGTAGGAGGATTTGTCCGGGAATGTCCTTCG GTATGGCGAACATCGAACTTCCCCTGGCCCAGCTATTATTCCACTTCAACTGGAAGCTACCT AACGAGTCAAATCAGGAGGAGATCGACATGACAGAAGAGTTCGGAATCTCAGTAAGGCGTAA AAACCATCTAAACTTGATCCCCGTCCTGTACCATAGATCAGACTTTATAGTGTAA SEQ ID No: 16 - amino acid sequence of Valeriana officinalis VOCYP71BE87 (shares homology with the Vitis vinifera P450 WCYP71BE5 responsible for the formation of the

sesquiterpenoid (-) -rotundone) MVWFLLKDRTFLLQKFLWYDSTS IAFSSYGDYWRQLRKICTMNLLSTKRVEQLRS IREEE ALNLVRRISTNGDSLPFNLSKAI FNLTSTVTSRAAFGNKNKDQEEFEVVLDQVLKALGGF NIGDMYPKAKLLHKITGARASMNKIQKRVDRILQNILVDHRNRKQESLTDDYEDLVDVLL RIQNEDQLQFPVTDNCIKAVILDVFGGGSETSSAATEWAMSEMVKNPHIMKRAQAEVRKV FDEKRNVDETGLGELKYLQCVIKETLRLHPPLPLLVPRENSAECEVNGFLIPANCKVI IN AWAISRDPKYWVDAEI FKPERFMDNS IDYQGTNFGYIPFGAGRRICPGMSFGMANIELPL AQLLFHFNWKLPNESNQEEIDMTEEFGISVRRKNHLNLIPVLYHRSDFIV*

SEQ ID No: 17 - nucleic acid sequence encoding Valeriana officinalis aldehyde dehydrogenase (VoALDHl) , having codon bias for improved expression in yeast

ATGGCCGGGGATAGCAACGGATCTCTAGATAGTTTTGTCAAGATCCCTGACATTAAATTTAC TAAATTATTTATAAACGGTGAATTTATTGACTCAATTAGCGGATCAACATTTGAAACTATTG ACCCCCGTACAGGAGAAGTCATTACTAGAGTAGCTGAAGGCAGAAAAGAGGATATTGATTTA GCGGTCAAGGCAGCAAGGAATGCATTCGACCATGGACCGTGGCCTAGGTTCAGCGGAAGCGC GCGTGGTAAGATTATGATGAAATTTGCCAATCTGGTAGACGAGAATGCTGAAGAACTTGCGA TCTTAGATACCATCGATGGGGGCAAGTTATTTGGGATAGGTAAGGGACACGATATACCACAA GCTGCAGAATGTCTTAGATATTATGCAGGAGCCGCCGATAAAATCCACGGAGAAACACTAAA GATGTCATCTGAATTTCAGGCCTACACACTGAAGGAACCCGTAGGGGTTGTCGGTCATATCA TACCTTGGAACTTTCCTTCTCAAATGTTCCTTATGAAGGTAGGGCCGGCTTTGGCAGCAGGA TGTACTATGGTGGTTAAACCTGCCGAGCAGACTCCGCTTAGCGCTCTATTCTACGCGCATCT AGCTAAGTTGGCAGGGGTTCCGGATGGGGTCATAAACGTCGTAACAGGTTTCGGTGGTACCG CCGGAAGTGCCATTTCTAGTCACATGGACATTGACATGGTTAGCTTTACAGGCTCAACTGAG GTAGGCAGGCTAGTTATGCAAGCCGCAGCTCTGAGTAACCTGAAGCCAGTATCACTTGAGTT GGGCGGTAAAAGCCCACTAATGATTTTCGACGACGCGGATGTGGACAAAGCGGTTGACTTAG CTCTGCTAGGATCTTTGTATAATAAAGGGGAAATATGCGTAGCGGGAACCAGGATCTTCGTG CAGGAGGGTATTTACGACAAATTTCTGGAGAAACTGGCAGTGGGGATAAAGACGTGGGTCGT AGGGGACCCCTTCCATCCTAGCACTAGGCAAGGTCCGCAAGTCGATAAGAAACAATATGAAA AGGTTTTATCTTACATAGAGCATGGGAAGACCGAGGGCGCAACCCTGTTTGCTGGAGGAAAT CCATGTGGAAAGAAGGGGTACTTCATTGAACCCACCATTTTCACGGACGTAAAAGACCACAT GAAAATCGCCAAAGAAGAAATCTTTGGTCCGGTGATGAGTGTATTCAAGTTTAAGACCGTTG AGGAGGGAATTGAAAGAGCAAACGCAACGAAGTACGGCCTTGCGGCCGGGATCGTAACCAAC AATCTGAATATCGCCAACACTGTTAGTAGATCCATAAGGGCGGGCGTCATATGGATTAACTG TTATTTTGCGTTTGATAGAGACTGTCCATACGGAGGGTATAAACAGAGCGGTTTCGGCAGAG ACCTTGGAATGGATGCGCTTCATAAATATCTGCATGTGAAGGCTGTCGCAACCCCAATATAC AACTCCCCGTGGCTATAA SEQ ID No: 18 - amino acid sequence of Valeriana officinalis aldehyde dehydrogenase (VoALDHl)

MAGDSNGSLDSFVKIPDIKFTKLFINGEFIDSISGSTFETIDPRTGEVITRVAEGRKEDI DLAVKAARNAFDHGPWPRFSGSARGKIMMKFANLVDENAEELAILDTIDGGKLFGIGKGH DIPQAAECLRYYAGAADKIHGETLKMSSEFQAYTLKEPVGWGHIIPWNFPSQMFLMKVG PALAAGCTMWKPAEQTPLSALFYAHLAKLAGVPDGVINWTGFGGTAGSAISSHMDIDM VSFTGSTEVGRLVMQAAALSNLKPVSLELGGKSPLMI FDDADVDKAVDLALLGSLYNKGE ICVAGTRIFVQEGIYDKFLEKLAVGIKTWWGDPFHPSTRQGPQVDKKQYEKVLSYIEHG KTEGATLFAGGNPCGKKGYFIEPTIFTDVKDHMKIAKEEIFGPVMSVFKFKTVEEGIERA NATKYGLAAGIVTNNLNIANTVSRS IRAGVIWINCYFAFDRDCPYGGYKQSGFGRDLGMD ALHKYLHVKAVATPIYNSPWL*

SEQ ID No: 19 - nucleic acid sequence encoding Valeriana officinalis alcohol dehydrogenase (VoADHl) , having codon bias for improved expression in yeast

ATGACCAAGAGTTCCGGTGAGGTAATCAGTTGCAAAGCCGCCGTTATATACAAATCTGGAGA GCCTGCAAAAGTTGAAGAGATTAGAGTGGACCCACCGAAATCATCAGAGGTAAGAATAAAGA TGTTGTATGCATCTCTATGCCATACGGATATTCTATGCTGTAACGGACTACCCGTCCCTTTA TTTCCGCGTATACCTGGCCATGAGGGAGTTGGCGTAGTCGAATCAGCGGGTGAGGACGTGAA AGATGTCAAGGAGGGTGACATCGTGATGCCACTGTATCTTGGAGAATGTGGGGAGTGCCTTA ACTGTTCATCCGGGAAAACAAATTTATGTCACAAGTATCCACTTGACTTTAGCGGTGTTCTA CCCAGCGATGGGACCAGTCGTATGTCCGTCGCTAAGTCAGGCGAAAAAATTTTCCATCATTT CAGTTGTTCCACATGGTCCGAGTACGTCGTGATAGAGTCTAGTTATGTCGTAAAAGTCGACT CTCGTCTGCCCCTACCACACGCCTCCTTTCTGGCGTGCGGGTTTACCACCGGATACGGTGCT GCATGGAAAGAAGCTGATATCCCCAAGGGAAGTACGGTAGCAGTGTTAGGGCTGGGCGCGGT GGGTTTAGGTGTAGTTGCTGGCGCGCGTTCACAAGGTGCCAGTCGTATAATTGGAGTTGACA TCAACGACAAAAAAAAAGCCAAAGCTGAGATATTTGGAGTTACTGAGTTTCTTAATCCGAAA CAGTTGGGTAAAAGTGCTTCCGAAAGCATCAAAGACGTTACTGGCGGCCTGGGCGTGGACTA TTGCTTTGAGTGTACCGGAGTACCGGCTTTATTAAATGAGGCTGTAGACGCTTCAAAGATTG GATTAGGGACTATCGTCATGATCGGAGCCGGAATGGAAACCTCTGGTGTTATCAATTACATT CCTCTTCTATGTGGAAGAAAGCTGATCGGCTCTATCTATGGTGGAGTTCGTATAAGGTCCGA CCTACCCTTGATTATCGAAAAATGCATAAATAAAGAAATTCCACTTAATGAGTTACAAACTC ACGAAGTGTCTTTGGAGGGCATAAACGATGCATTCGGTATGCTGAAGCAGCCAGACTGTGTG AAAATAGTAATCAAGTTTGAACAGAAGTGA

SEQ ID No: 20 - amino acid sequence of Valeriana officinalis alcohol dehydrogenase (VoADHl)

MTKSSGEVISCKAAVIYKSGEPAKVEEIRVDPPKSSEVRIKMLYASLCHTDILCCNGLPV PLFPRIPGHEGVGWESAGEDVKDVKEGDIVMPLYLGECGECLNCSSGKTNLCHKYPLDF SGVLPSDGTSRMSVAKSGEKIFHHFSCSTWSEYWIESSYWKVDSRLPLPHASFLACGF TTGYGAAWKEADIPKGSTVAVLGLGAVGLGWAGARSQGASRIIGVDINDKKKAKAEIFG VTEFLNPKQLGKSASESIKDVTGGLGVDYCFECTGVPALLNEAVDASKIGLGTIVMIGAG METSGVINYIPLLCGRKLIGS IYGGVRIRSDLPLI IEKCINKEIPLNELQTHEVSLEGIN DAFGMLKQPDCVKIVIKFEQK* SEQ ID No: 21 - nucleic acid sequence encoding Valeriana officinalis cytochrome P450 reductase, (VoCPRl) , having codon bias for improved expression in yeast ATGGACACGAACAGCGATCTATTGAGGTCCATCGAGAGTTATCTGGGCGTAAGTATCTCTAG CAACACACTTGTCCTTATTGCGACGACTTCAGTTGCTATTGTCGTCGGGCTTTTGGTCTTTG TTTGGAAGAAAAGCAGCGGTGGGTCCAAAGAATTTAAGCCGGTTGTCTTGCCTAAATCACCC ACAGTCGAAGACGACGAGGACGAGGCCGAGGCCCCTCCGGGGAAGACAAAACTAAGCATTTT CTTTGGTACTCAAACTGGTACTGCGGAGGGATTCGCTAAGGCGCTTGCCGACGAAATTAAAG CAAAGTATGAGAAGGCTATTGTCAAGGTGATCGATTTAGACGACTATGCAGCCGATGATGAC GTATACGAGGAGAAATTAAAAAAAGAGACCTTGGCCTTCTTTATGGTTGCAACATATGGTGA CGGGGAACCCACCGACAACGCTGCAAGATTTTACAAGTGGTTCTCTGAGGGTCAAGACAGAG GTACTTGGCTTCAGCAATTAACTTATGGGGTCTTTGCACTTGGCAACAGACAATATGAACAT TTCAACAAGATTGGGAAAGTAATAGATGATCAGTTGGTTGAGCAGGGGGCCAAGAGGCTAGT GCCAATTGGCCTTGGAGACGATGATCAGTGCATCGAGGACGATTTTGCCGCCTGGAAGGAGC TACTTTGGCCTGAATTAGACCAGCTGTTAAGAGACGAGGATGACACAAGCACCGTAGCCACA CCTTACACAGCGGTAATTCCGGAATATAGAGTTGTAATACATGATCCCGATACAACGACGTC CGATGATATGAACCTTCACGTCCCAAACGGTAATGTTAGTTTTGACATTCACCACCCTTGTA GAGCCAATGTTGCAGTCCAGAGAGAGCTTCATAAGCCGGAGAGCGACCGTTCTTGCATACAT TTAGAGTTCGACATAAGCGGTACCGGCATCACCTACGAGACAGGGGATCACGTAGGAGTGTT TGCCGAGAATTGCGAAGAGACAGTTGAGGAGGCGGCGAAGCTGTTGGGACAACCGCTAGACA TGTTGTTTTCCATCCATACAGATAAGGATGATGGCTCCTCACAGGGCAGCTCTTTACCACCG CCCTTCCCAGGGCCGTGCACCCTTCGTACTGCCCTGGCAAGGTATGCGGACGTGCTGAACCC TCCTAGGAAAGCTGCATTAGTGGCACTAGCAGCCCATGCGACTGAACCGGCAGAAGCAGAGA GGTTGAAGTTCCTGTCTAGCCCTCAGGGTAAAGATGAATACTCACAGTGGGTGTTAGGCTCC CAAAGAAGTTTGCTAGAGGTCATGGCTGAATTCCCTAGTGCGAAACCTCCCATTGGAGTCTT CTTCGCTGCTGTCGCGCCACGTCTACCGCCCCGTTACTATTCTATCTCATCCTCCCCTAGGT ACGCCGGTGACCGTGTACACGTGACTTGTGCCCTGGTTTATGGACCAAGTCCCACCGGAAGG ATCCATAAGGGGGTATGCAGTACCTGGATGAAGAACGCGGTACCTTTGGGCAAATCTGACGA CTGTTCATGGGCGCCCATTTTCATCAGGACGAGCAATTTCAAATTACCCGCCGATCCAAGCG TCCCGATAATAATGGTCGGACCAGGGACTGGATTAGCGCCGTTCCGTGGATTCCTGCAAGAA CGTCTTTCTCTGAAGGAGGAGGGTGCCCAGTTGGGTCCCGCCCTACTGTTTTTCGGTTGCAG GAACAGGAGGATGGATTTTATATATGAAGAGGAACTGAACAATTTTGTCGACGAAGGCGTGA TTTCAGAATTGATTGTTGCATTTTCTCGTGAGGGCCCCACCAAAGAATACGTCCAACACAAA ATCATAGAGAAGGCTGCGGATATTTGGAGTCTTATAAGCGAGGGAGCTTATTTATATGTGTG TGGTGATGCGAAGGGTATGGCTAGAGACGTCCACCGTACCTTACACACAGTAGTTCAAGAAC AGGAGAAAGTTGATAGTACTAAGGCTGAAGCTATTGTGAAGAAGTTACAAATGGATGGACGT TATCTTAGGGACGTATGGTAA

SEQ ID No: 22 - amino acid sequence of Valeriana officinalis cytochrome P 50 reductase, (VoCPRl)

MDTNSDLLRSIESYLGVSISSNTLVLIATTSVAIWGLLVFVWKKSSGGSKEFKPWLPK SPTVEDDEDEAEAPPGKTKLSIFFGTQTGTAEGFAKALADEIKAKYEKAIVKVIDLDDYA ADDDVYEEKLKKETLAFFMVATYGDGEPTDNAARFYKWFSEGQDRGTWLQQLTYGVFALG NRQYEHFNKIGKVIDDQLVEQGAKRLVPIGLGDDDQCIEDDFAAWKELLWPELDQLLRDE DDTSTVATPYTAVIPEYRWIHDPDTTTSDDMNLHVPNGNVSFDIHHPCRANVAVQRELH KPESDRSCIHLEFDISGTGITYETGDHVGVFAENCEETVEEAAKLLGQPLDMLFSIHTDK DDGSSQGSSLPPPFPGPCTLRTALARYADVLNPPRKAALVALAAHATEPAEAERLKFLSS PQGKDEYSQWVLGSQRSLLE\/MAEFPSAKPPIGVFFAAVAPRLPPRYYSISSSPRYAGDR VHVTCALVYGPSPTGRIHKGVCSTWMKNAVPLGKSDDCSWAPIFIRTSNFKLPADPSVPI IMVGPGTGLAPFRGFLQERLSLKEEGAQLGPALLFFGCRNRRMDFIYEEELNNFVDEGVI SELIVAFSREGPTKEYVQHKIIEKAADIWSLISEGAYLYVCGDAKGMARDVHRTLHTWQ EQEKVDSTKAEAIVKKLQMDGRYLRDVW*

Claims

CLAIMS What is claimed is:

1. A fungal cell that produces at least about 30 mg, e.g. , at least about 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or more, valerena-4,7(l l)-diene per liter of culture.

2. The fungal cell of claim 1, comprising a polynucleotide encoding a fusion protein comprising maltose binding protein (MBP)-valerena-4,7(l l)-diene synthase (VDS)- farnesyl diphosphate (FPP) synthase (ERG20) integrated into the genome of the fungal cell.

3. The fungal cell of any one of claims 1 to 2, comprising two, three, four or more copies of the polynucleotide encoding a fusion protein comprising MBP-VDS- ERG20 integrated into its genome.

4. The fungal cell of any one of claims 1 to 3, wherein the fusion protein comprising MBP-VDS-ERG20 has at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4.

5. A fungal cell that produces detectable levels of valerenic acid in in vitro culture.

6. The fungal cell of claim 5, wherein the fungal cell produces at least about 4 mg valerenic acid per liter of culture.

7. A fungal cell comprising heterologous enzymes in the biosynthetic pathway of valerenic acid, wherein the fungal cell produces valerenic acid.

8. The fungal cell of any one of claims 5 to 7, comprising one or more polynucleotides encoding heterologous enzymes in the valerenic acid biosynthesis pathway stably integrated into the genome of the cell.

9. The fungal cell of claim 8, wherein the one or more polynucleotides are integrated in the genome at the uridine auxotrophy (URA3) locus.

10. The fungal cell of any one of claims 8 to 9, wherein the one or more polynucleotides are operably linked to and expressed under the control of a galactose inducible promoter.

11. The fungal cell of any one of claims 1 to 10, wherein the fungal cell is transformed with heterologous polynucleotides encoding (i) a valerenadiene synthase (VDS), (ii) a Valeriana officinalis P450 VoCYP71DJl or homolog thereof that functions as a valerenadiene oxidase, (iii) an alcohol dehydrogenase and (iv) an aldehyde dehydrogenase.

12. A transformed fungal cell comprising one or more heterologous polynucleotides integrated into the fungal cell's genome, wherein the one or more

heterologous polynucleotides encode a valerenadiene synthase (VDS), that converts farnesyl pyrophosphate (FPP) to valerenadiene, wherein the fungal cell produces valerenic acid.

13. The fungal cell of claim 12, wherein the heterologous polynucleotides encode a valerenadiene oxidase comprising at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6 (Valeriana officinalis P450 VoCYP71DJl) and that converts valerenadiene to valerenic alcohol.

14. The fungal cell of any one of claims 12 to 13, wherein the heterologous polynucleotides encode an alcohol dehydrogenase (ADH) that converts valerenic alcohol to valerenic aldehyde.

15. The fungal cell of any one of claims 12 to 14, an aldehyde dehydrogenase (ALDH) that converts valerenic aldehyde to valerenic acid.

16. The fungal cell of any one of claims 7 to 15, wherein one or more polynucleotides encoding the heterologous enzymes comprise codon bias for improved expression in the fungal cell.

17. The fungal cell of any one of claims 7 to 16, wherein the fungal cell is transformed with polynucleotides encoding Valeriana officinalis P450 VoCYP71DJl, Artemisia annua alcohol dehydrogenase (AaADHl) and annua aldehyde dehydrogenase (AaALDHl).

18. The fungal cell of any one of claims 7 to 17, wherein the fungal cell is transformed with a fusion protein comprising MBP-VDS-ERG20.

19. The fungal cell of any one of claims 7 to 18, wherein the fungal cell produces at least about 4 mg valerenic acid per liter of culture.

20. The fungal cell of any one of claims 1 to 19, wherein the fungal cell is a yeast cell.

21. The fungal cell of claim 20, wherein the yeast cell is from a genus selected from the group consisting of Saccharomyces, Pichia, Kluyveromyces, Candida, Aspergillus, Trichoderma, Chrysosporhim, Fusarium and Neurospora.

22. A fungal cell culture comprising a population of fungal cells as set forth in any one of claims 1 to 21.

23. A fungal cell culture comprising a population of fungal cells that produce detectable levels of valerenic acid.

24. The fungal cell culture of claim 23, wherein population of fungal cells produce at least about 4 mg valerenic acid per liter of culture.

25. The fungal cell culture of claim 23, wherein the culture does not contain detectable or is devoid of hydroxyvalerenic acid and/or acetoxyvalerenic acid.

26. The fungal cell culture of claim 23, wherein the culture comprises detectable amounts of hydroxyvalerenic acid and/or acetoxyvalerenic acid.

27. A fungal cell culture that produces at least about 30 mg {e.g., at least about 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or more) valerena-4,7(l l)-diene per liter of culture.

28. A bioreactor comprising a fungal cell culture of any one of claims 22 to 27.

29. The bioreactor of claim 28, comprising a culture volume in the range of 0.5L, 1L, 2L, 3L, 4L, 5L, 10L, 25L, 50L, 75L, 100L, 250L, 500L, 1000L, or more.

30. A method of producing valerenic acid, said method comprising cultunng a population of fungal cells of any one of claims 7 to 21 under conditions sufficient for the fungal cells to produce valerenic acid.

31. The method of claim 30, wherein the population of fungal cells is cultured in a medium comprising galactose.

32. The method of any one of claims 30 to 31, wherein the population of fungal cells is cultured in a medium lacking uridine.

33. The method of any one of claims 30 to 32, further comprising lysing the cells and/or extracting with a solvent to isolate valerenic acid.

34. The method of claim 33, wherein the valerenic acid is substantially or entirely free of hydroxyvalerenic acid and/or acetoxyvalerenic acid.

35. Valerenic acid produced from a fungal cell of any one of claims 1 to

21.

36. Valerenic acid of claim 35, wherein the valerenic acid is substantially or entirely free of hydroxyvalerenic acid and/or acetoxyvalerenic acid.

37. A pharmaceutical composition or a nutraceutical comprising the valerenic acid of any one of claims 35 to 36.

38. A fusion protein comprising maltose binding protein (MBP)-valerena- 4,7(1 l)-diene synthase (VDS)- farnesyl diphosphate (FPP) synthase (ERG20).

39. The fusion protein of claim 38, wherein the MBP-VDS-ERG20 fusion protein comprises at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4.

40. A polynucleotide encoding the MBP-VDS-ERG20 fusion protein of claim 38.

41. The polynucleotide of claim 40, wherein the polynucleotide comprises at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3.

42. A fungal cell comprising the polynucleotide of claim 40.

43. An isolated and/or recombinant polynucleotide encoding a valerenadiene oxidase comprising at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6 (Valeriana officinalis P450 VoCYP71DJl) and that converts valerenadiene to valerenic alcohol.

44. A fungal cell comprising a heterologous polynucleotide encoding a valerenadiene oxidase comprising at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6 (Valeriana officinalis P450 VoCYP71DJl) and that converts valerenadiene to valerenic alcohol.

45. An isolated and/or recombinant polynucleotide encoding a P450 enzyme comprising at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 8, 10, 12, 14 or 16, wherein the P450 enzyme is enzymatically active.

46. A fungal cell comprising a heterologous polynucleotide encoding a

P450 comprising at least about 80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8, 10, 12, 14 or 16 , wherein the P450 enzyme is enzymatically active.