CHIMERIC TERPENE SYNTHASES

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional

Application Serial No. 62/630,640, entitled“CHIMERIC TERPENE SYNTHASES” filed on February 14, 2018, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The disclosure relates to chimeric terpene synthases, methods for making chimeric terpene synthases, and methods for making terpenes using the same.

BACKGROUND

Terpenes are a diverse class of organic compounds built from five carbon building blocks and encompass at least 400 distinct structural families. Given their structural diversity, terpenes have numerous roles including acting as pheromones, anti-oxidants, and anti-microbial agents. Although terpene synthases produce terpenes in both prokaryotes and eukaryotes, the wide array of terpene isomers often hinder high yield extractions from naturally occurring sources.

Furthermore, the structural complexity of terpenes often limits de novo chemical synthesis.

SUMMARY

Aspects of the disclosure relate to chimeric terpene synthases comprising an amino acid sequence at least 90% identical to an amino acid selected from the group consisting of: SEQ ID NOs: 1-52. In some embodiments, the chimeric terpene synthase comprises an amino acid sequence at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid selected from the group consisting of: SEQ ID NOs: 1-52. In some embodiments, the chimeric terpene synthase comprises an amino acid sequence identical to an amino acid selected from the group consisting of: SEQ ID NOs: 1-52.

Further aspects of the disclosure relate to nucleic acid molecules encoding a chimeric terpene synthase described herein. In some embodiments, a nucleic acid molecule comprises a sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 67-118. In some embodiments, a nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 67-118.

Further aspects of the disclosure relate to vectors comprising a nucleic acid molecule described herein. In some embodiments, the vector is a viral vector, a vector for transient expression, or a vector for inducible expression. In some embodiments, the vector is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated vector, a galactose- inducible vector, or a doxycycline-inducible vector.

Further aspects of the disclosure relate to host cells comprising a nucleic acid described herein, or a vector described herein.

In some embodiments, the host cell is a fungal cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a Saccharomyces, Pichia, Kluyveromyces,

Hansenula, or Yarrowia cell. In some embodiments, the cell is a Saccharomyces cerevisiae cell.

In some embodiments, the host cell is a plant cell.

In some embodiments, the host cell is a bacteria cell.

Further aspects of the disclosure relate to nucleic acid molecules encoding a chimeric terpene synthase, wherein at least 10% of the nucleic acid molecule sequence, or the amino acid sequence, is derived from a rare or extinct plant. In some embodiments, at least 40% of the nucleic acid molecule sequence, or the amino acid sequence, is derived from a rare or extinct plant.

In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the nucleic acid molecule sequence, or the amino acid sequence, is derived from a rare or extinct plant. In some embodiments, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% of the nucleic acid molecule sequence, or the amino acid sequence, is derived from a rare or extinct plant.

In some embodiments, the chimeric terpene synthase is a chimeric sesquiterpene synthase. In some embodiments, the rare or extinct plant is selected from the group consisting of: Hibiscadelphus wilderianus, Leucadendron grandiflorum, Macrostylis villosa, Orbexilum stipulatum, Shorea cuspidate, and Wendlandia angustifolia.

Further aspects of the disclosure relate to nucleic acid molecules encoding a chimeric terpene synthase. In some embodiments, at least 10% of the nucleic acid molecule sequence, or the amino acid sequence is derived from a rare or extinct plant. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the nucleic acid molecule sequence is derived from a rare or extinct plant. In some embodiments, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% of the nucleic acid molecule sequence is derived from a rare or extinct plant.

In some embodiments, the nucleic acid molecule further comprises a TATA box sequence.

Further aspects of the disclosure relate to methods of producing one or more

sesquiterpenes, wherein the method comprises culturing a host cell described herein under conditions suitable for producing the one or more sesquiterpenes.

Further aspects of the disclosure relate to compositions comprising one or more sesquiterpenes produced by the methods described herein.

In one embodiment, at least one of the one or more sesquiterpenes is an aroma compound.

Further aspects of the disclosure relate to methods of producing a perfume, wherein the method comprises: culturing a host cell described herein under conditions suitable for producing the one or more sesquiterpenes; and extracting the one or more sesquiterpenes.

Each of the limitations of the compositions and methods described herein may encompass various described embodiments. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. The drawings are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

FIG. 1 is a series of pictures depicting structures of identified sesquiterpenes produced using sesquiterpene synthases (SQTSs) containing rare sequences from H. wilderianus.

FIG. 2 is a series of pictures depicting structures of identified sesquiterpenes produced using SQTSs containing rare sequences from L. grandiflorum. FIG. 3 is a series of pictures depicting structures of sesquiterpenes produced using SQTSs containing rare sequences from M. villosa.

FIG. 4 is a series of pictures depicting structures of sesquiterpenes produced using SQTSs containing rare sequences from O. stipulatum.

FIG. 5 is a series of pictures depicting structures of identified sesquiterpenes produced using SQTSs containing rare sequences from S. cuspidata.

FIG. 6 is a series of pictures depicting structures of identified sesquiterpenes produced using SQTSs containing rare sequences from W. angustifolia.

FIG. 7 is a graph showing chimera product distribution versus plant species. The chimeras are categorized based on the sesquiterpene produced in highest yield.

FIGS. 8A-8F include a series of pictures depicting species of rare plants. FIG. 8A depicts Hibiscadelphus wilderianus (from Radlkofer el al, New and Noteworthy Hawaiian Plants. Hawaiian Board of Agriculture and Forestry Botanical Bulletin. 1911;(1): 1-15). FIG. 8B depicts Leucadendron grandiflorum (from Salisbury el al, The Paradisus Londinensis or Coloured Figures of Plants Cultivated in the Vicinity of the Metropolis. 1805; (Volume 1, part 2): 105). FIG. 8C depicts Macrostylis villosa subsp. Villosa (from“Red List of South African Plants: Macrostylis villosa subsp. villosa,” 2007). FIG. 8D depicts Orbexilum stipulatum (from Short,“Orbexilum stipulatum collected at Falls of the Ohio,” 1840 from The Philadelphia Herbarium at the Academy of Natural Sciences). FIG. 8E depicts Shorea cuspidata (from“Kew Royal Botanical Gardens: Shorea cuspidata specimen K000700460,” 1962). FIG. 8F depicts Wendlandia angustifolia (from“Kew Royal Botanical Gardens: Wendlandia angustifolia K000030921,” collection date not recorded).

FIG. 9 is a series of pictures depicting selected gas chromatography-mass spectrometry (GC/MS) chromatograms from H. wilderianus chimera screening data (Table 4).

FIG. 10 is a series of pictures depicting selected GC/MS chromatograms from L.

grandiflorum chimera screening data (Table 5).

FIG. 11 is a series of pictures depicting selected GC/MS chromatograms from L.

grandiflorum chimera screening data (Table 5).

FIG. 12 is a series of pictures depicting selected GC/MS chromatograms from M. villosa chimera screening data (Table 6). FIG. 13 is a series of pictures depicting selected GC/MS chromatograms from S.

cuspidata chimera screening data (Table 8).

FIG. 14 is a series of pictures depicting selected GC/MS chromatograms from W.

angustifolia chimera screening data (Table 9).

FIG. 15 is a series of pictures depicting selected GC/MS chromatograms from W.

angustifolia chimera screening data (Table 9).

DETAILED DESCRIPTION

Although terpenes are widely used in the fragrance industry, purification of terpenes from natural sources and de novo chemical synthesis often have high production costs and low yield. This disclosure is premised, in part, on the unexpected finding that chimeric terpene synthases comprising a portion of a terpene synthase sequence from at least one rare or extinct plant can be leveraged to produce a diversity of sesquiterpenes. Accordingly, provided herein are chimeric terpene synthases, methods for making chimeric terpene synthases, and methods for making terpenes using the described chimeric terpene synthases. In some embodiments, the chimeric terpene synthases are chimeric sesquiterpene synthases.

This invention is not limited in its application to the details of construction and the arrangement of components set forth in the description. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Additionally, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of terms such as“including,”“comprising,”“having,”

“containing,”“involving,” and/or variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Chimeric Terpene Synthases

Aspects of the present disclosure relate to chimeric terpene synthases comprising fragments (e.g., sequences) from at least two terpene synthases, wherein at least one of the two or more terpene synthases is from a rare or extinct plant. For example, the sequence of a chimeric terpene synthase may comprise one or more fragments (e.g., one or more portions of the total sequence) from at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten terpene synthases. It should be appreciated that chimeric terpene synthases described herein can be synthetic. Accordingly, chimeric terpene synthases, including synthetic chimeric terpene synthases, described herein comprise sequences derived from more than one terpene synthase, wherein at least one of the terpene synthases is from a rare or extinct plant. In some embodiments, the chimeric terpene synthases are chimeric sesquiterpene synthases.

Terpene synthases are enzymes that catalyze the formation of terpenes from isoprenoid diphosphate substrates. At least two types of terpene synthases have been characterized: classic terpene synthases and isoprenyl diphosphate synthase-type terpene synthases. Classic terpene synthases are found in prokaryotes (e.g., bacteria) and in eukaryotes (e.g., plants, fungi and amoebae), while isoprenyl diphosphate synthase-type terpene synthases have been found in insects (see, e.g., Chen et al, Terpene synthase genes in eukaryotes beyond plants and fungi: Occurrence in social amoebae. Proc Natl Acad Sci U SA. 2016; 113(43): 12132- 12137, which is hereby incorporated by reference in its entirety for this purpose). Several highly conserved structural motifs have been reported in classic terpene synthases, including an aspartate-rich “DDxx(x)D/E” motif and a“NDxxSxxxD/E”(SEQ ID NO: 55) motif, which have both been implicated in coordinating substrate binding (see, e.g., Starks et al, Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase. Science. 1997 Sep

19;277(5333):1815-20; and Christianson et al, Unearthing the roots of the terpenome. Curr Opin Chem Biol. 2008 Apr;12(2):141-50, each of which is hereby incorporated by reference in its entirety for this purpose).

Terpene synthases may be classified by the type of terpenes they produce. As used herein, unless otherwise indicated, terpenes are organic compounds comprising isoprene ( i.e ., CsHs) units and derivatives thereof. For example, terpenes include pure hydrocarbons with the molecular formula (CsH ₈ ) _n , in which n represents the number of isoprene subunits. Terpenes also include oxygenated compounds (often referred to as terpenoids). Terpenes are structurally diverse compounds and, for example, may be cyclic (e.g., monocyclic, multi-cyclic, homocyclic and heterocyclic compounds) or acyclic (e.g., linear and branched compounds). In some embodiments, a terpene may have an odor. As used herein, an aroma compound refers to a compound that has an odor. Any methods known in the art, including mass spectrometry (e.g., gas chromatography-mass spectrometry (GC/MS, shown in Example 2 below), may be used to identify a terpene of interest. Terpene synthases may include, for example, monoterpene synthases, diterpene synthases, and sesquiterpene synthases. Certain non-limiting examples of monoterpene synthases and sesquiterpene synthases may be found, for example, in Degenhardt el al. ,

Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry . 2009 Oct-Nov;70(15-16): 1621-37, which is hereby incorporated by reference in its entirety for this purpose.

Monoterpene synthases catalyze the formation of 10-carbon monoterpenes. Generally, monoterpene synthases use geranyl diphosphate (GPP) as a substrate. Non-limiting examples of monoterpene synthases include Myrcene synthase (UniProtKb Identifier: 024474), (R)-limonene synthase (UniprotKB Identifier: Q2XSC6), (E)-beta-ocimene synthase (UniProtKB Identifier: Q5CD81) and Limonene synthase (UniProtKB Identifier: Q9FV72). Non-limiting examples of monoterpenes include, but are not limited to, limonene, sabinene, thujene, carene, borneol, eucalyptol and camphene.

Diterpene synthases promote the formation of 20-carbon diterpenes. Generally, diterpene synthases use geranylgeranyl diphosphate as a substrate. Non-limiting examples of diterpene synthases include cis-abienol synthase (UniProtKB identifier: H8ZM73), sclareol synthase (UniProtKB identifier: K4HYB0) and abietadiene synthase (Q38710). See, e.g., Gong et al., Diterpene synthases and their responsible cyclic natural products. Nat Prod Bioprospect.

2014;4(2):59-72, which is hereby incorporated by reference in its entirety for this purpose. Non limiting examples of diterpenes include, but are not limited to, cembrene and sclareol.

Sesquiterpene synthases catalyze the formation of 15-carbon sesquiterpenes. Generally, sesquiterpene synthases convert farnesyl diphosphate (FDP) into sesquiterpenes. Non-limiting examples of sesquiterpene synthases include (+)-delta-cadinene synthase (UniProtKB Identifier: Q9SAN0), UniProtKB Identifier: A0A067FTE8, Beta-eudesmol synthase (UniProtKB Identifier: B1B1U4), (+)-delta-cadinene synthase isozyme XC14 (UniProtKB Identifier: Q39760), (+)- delta-cadinene synthase isozyme XC1 (UniProtKB Identifier: Q39761), (+)-delta-cadinene synthase isozyme A (UniProtKB Identifier: Q43714), Sesquiterpene synthase 2 (UniProtKB Identifier: Q9FQ26), Putative delta-guaiene synthase (UniProtKB Identifier: A0A0A0QUT9), Delta-guaiene synthase 1 (UniProtKB Identifier: D0VMR6), Alpha-zingiberene synthase (UniProtKB Identifier: Q5SBP4), (Z)-gamma-bisabolene synthase 1 (UniProtKB Identifier: Q9T0J9), A0A067D5M4, Delta-elemene synthase (UniProtKB Identifier: A0A097ZIE0), ShoBecSQTSl, A0A068UHT0, terpene synthase (UniProtKB Identifier: G5CV47), A0A068VE40 and A0A068VI46.

In some embodiments, a sesquiterpene synthase is an alpha-guaiene synthase. As used herein, an alpha-guaiene synthase is capable of catalyzing the formation of alpha-guaiene. In some embodiments, an alpha-guaiene synthase uses (2E,6E)-farnesyl diphosphate as a substrate. Non-limiting examples of alpha-guaiene synthases include UniProtKB Identifier: D0VMR6, UniProtKB Identifier: D0VMR7, UniProtKB Identifier: D0VMR8, UniProtKB Identifier:

Q49SP3. As disclosed herein, an alpha-guaiene synthase may comprise a sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99%, including all values in between) identical to SEQ ID NO: 17, 22, or 29. In certain embodiments, an alpha-guaiene synthase comprises SEQ ID NO: 17, 22, or 29. In certain embodiments an alpha-guaiene synthase consists of SEQ ID NO: 17, 22, or 29.

As used herein, unless otherwise indicated, sesquiterpenes include sesquiterpene hydrocarbons and sesquiterpene alcohols (sesquiterpenols). Non-limiting examples of sesquiterpenes include but are not limited to, delta-cadinene, epi-cubenol, tau-cadinol, alpha- cadinol, gamma-selinene, 10-epi-gamma-eudesmol, gamma-eudesmol, alpha/beta-eudesmol, juniper camphor, 7-epi-alpha-eudesmol, cryptomeridiol isomer 1, cryptomeridiol isomer 2, cryptomeridiol isomer 3, humulene, alpha-guaiene, delta- guaiene, zingiberene, beta-bisabolene, beta-farnesene, beta-sesquiphellandrene, cubenol, alpha-bisabolol, alpha-curcumene, trans- nerolidol, gamma, bisabolene, beta-caryophyllene, trans-Sesquisabinene hydrate, delta-elemene, cis-eudesm-6-en-l l-ol, daucene, isodaucene, trans-bergamotene, alpha- zingiberene,

sesquisabinene hydrate, and 8-Isopropenyl-l, 5-dimethyl- 1,5-cyclodecadiene.

The present disclosure also encompasses chimeric terpene synthases that are multi functional (e.g., capable of producing more than one sesquiterpene). In some embodiments, a chimeric terpene synthase is capable of producing delta-cadinene and alpha-cadinol. In some embodiments, a chimeric terpene synthase is capable of producing delta-cadinene, tau-cadinol, and alpha-cadinol. In some embodiments, a chimeric terpene synthase is capable of producing alpha-guaiene and delta-guaiene. In some embodiments, the chimeric terpene synthase is capable of producing beta-caryophyllene and humulene. In some embodiments, a chimeric terpene synthase ( e.g ., a chimeric sesquiterpene synthase) of the present disclosure comprises an amino sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99%, including all values in between) identical to a sequence selected from the group consisting of SEQ ID NOs: 1-52. In some embodiments, the chimeric terpene synthase comprises an amino acid sequence provided in SEQ ID NOs: 1-52.

In some embodiments, a chimeric terpene synthase comprises one or more sequences provided in SEQ ID NOs: 119-357.

The term“sequence identity,” as known in the art, refers to a relationship between the sequences of two polypeptides or polynucleotides, as determined by sequence comparison (alignment). In the art, identity also means the degree of sequence relatedness between two sequences as determined by the number of matches between strings of two or more residues (e.g., nucleic acid or amino acid residues). Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g.,“algorithms”).

Identity of related polypeptides can be readily calculated by any of the methods known to one of ordinary skill in the art. The“percent identity” of two sequences (e.g., nucleic acid or amino acid sequences) may, for example, be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST ^® and XBLAST ^® programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990.

BLAST ^® protein searches can be performed, for example, with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST ^® can be utilized, for example, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST ^® and Gapped BLAST ^® programs, the default parameters of the respective programs (e.g., XBLAST ^® and NBLAST ^® ) can be used, or the parameters can be adjusted appropriately as would be understood by one of ordinary skill in the art.

Another local alignment technique which may be used, for example, is based on the Smith- Waterman algorithm (Smith, T.L. & Waterman, M.S. (1981)“Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197). A general global alignment technique which may be used, for example, is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970)“A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453), which is based on dynamic programming. More recently, a Fast Optimal Global Sequence Alignment Algorithm

(FOGSAA) was developed that purportedly produces global alignment of nucleic acid and amino acid sequences faster than other optimal global alignment methods, including the Needleman- Wunsch algorithm.

The present disclosure also encompasses compositions comprising one or more terpenes ( e.g ., sesquiterpenes) produced by any one of the chimeric terpene synthases (e.g., sesquiterpene synthases) described herein. In some embodiments, the composition comprises at least one terpene (e.g., sesquiterpene) that is an aroma compound. In some embodiments, the composition is a perfume (e.g., comprising a single fragrance or a mixture of fragrances). In some embodiments, the composition further comprises a fixative (i.e., stabilizer) to reduce volatility of the composition. Non-limiting examples include fixatives include resinoids (e.g., benzoin, olibanum, storax, labdanum, myrrh and tolu balsam) and benzyl benzoate. In some

embodiments, the composition further comprises ethyl alcohol. In some embodiments, the composition further comprises distilled water.

In certain embodiments, a terpene synthase (e.g., sesquiterpene synthase) of the present disclosure produces a terpene (e.g., sesquiterpene) composition that comprises at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at 70%, at least 80%, at least 90%, at least 95%, or 100% including any values in between of a particular terpene, such as a sesquiterpene. Non-limiting examples of sesquiterpenes include delta-cadinene, epi-cubenol, tau-cadinol, alpha-cadinol, gamma-selinene, 10-epi-gamma- eudesmol, gamma-eudesmol, alpha/beta-eudesmol, juniper camphor, 7-epi-alpha-eudesmol, cryptomeridiol isomer 1, cryptomeridiol isomer 2, cryptomeridiol isomer 3, humulene, alpha- guaiene, delta-guaiene, zingiberene, beta-bisabolene, beta-famesene, beta-sesquiphellandrene, cubenol, alpha-bisabolol, alpha-curcumene, trans-nerolidol, gamma, bisabolene, beta- caryophyllene, trans-Sesquisabinene hydrate, delta-elemene, cis-eudesm-6-en-l l-ol, daucene, isodaucene, trans-bergamotene, alpha-zingiberene, sesquisabinene hydrate, and 8-Isopropenyl- 1,5-dimethyl- 1,5-cyclodecadiene. As a non-limiting example, a terpene synthase may be heterologously expressed in a host cell, the sesquiterpenes produced by the recombinant host cell may be extracted, and the types of sesquiterpenes in the composition may be determined using gas chromatography-mass spectrometry. In some embodiments, a terpene synthase may be recombinantly expressed and is purified. In some embodiments, the sesquiterpenes produced by a purified terpene synthase may be extracted and the types of sesquiterpenes in the composition may be determined using gas chromatography-mass spectrometry.

In certain embodiments, an alpha-guaiene synthase is capable of producing a

sesquiterpene composition that comprises at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at 70%, at least 80%, at least 90%, at least 95%, or 100% including any values in between of alpha-guaiene. In some embodiments, an alpha-guaiene synthase is capable of producing a sesquiterpene composition that comprises between 1% to 10%, between 5% to 20%, between 15% to 20%, between 16% and 20%, between 17% and 20%, between 18% and 20%, between 19% and 20%, between 20% and 25%, between 20% and 24%, between 20% and 23%, between 20% and 22%, between 20% and 21%, between 20% and 30%, between 30% and 40%, between 40% and 50%, between 50% and 60%, between 60% and 70%, between 70% and 80% , between 80% and 90%, or between 90% and 100%, including any values in between alpha-guaiene.

Rare and extinct plants

At least one portion of the sequence of the chimeric terpene synthases disclosed herein is derived from a rare or extinct plant. As used herein, the term“rare plant” or“rare plants” encompasses plants that are uncommon, scarce, infrequently encountered, endangered (e.g., threatened), vulnerable, only available in private collections, not found in the endemic location, only available in cultivation, and/or extinct. In some embodiments, a rare plant is a plant that is infrequently encountered (e.g., only encountered in a few locations such as 1, 2, 3, 4, or 5 locations). In some embodiments, a rare plant is an extinct plant. As used herein, an extinct plant refers to a species of plant: having no living members; classified as having no living members; or predicted by one of ordinary skill in the art to have no living members. As a non limiting example, the International Union for Conservation of Nature (IUCN) Red list of Threatened Species may be used to determine the conservation status of a plant and identify rare plants. For example, plants classified as extinct, extinct in the wild, critically endangered, endangered, vulnerable, and near threatened on the IUCN Red List may be considered rare plants.

Non-limiting examples of rare plants include Leucadendron grandiflorum, Shorea cuspidata, Macrostylis villosa, Orbexilum stipulatum, Myrcia skeldingii, Nesiota Elliptica, Macrostylis villosa, Wendlandia angustofola, Erica Pyramidalis, Stenocarpus dumbeenis, Pradosia glaziovii, Crassula subulata, Hibiscadelphus wilderianus, and Erica foliacea.

In some embodiments, the rare plant may be Hibiscadelphus wilderianus. The

Hibiscadelphus genus belongs to the tribe Hibisceae (Malvaceae) and members of the genus often have petals that form a tubular structure in which the lower petals are often shorter than the upper three petals (see, e.g., Oppenheimer el al, A new species of Hibiscadelphus Rock

(Malvaceae, Hibisceae) from Maui, Hawaiian Islands; PhytoKeys, 2014;(39):65-75, which is hereby incorporated by reference in its entirety). The Hibiscadelphus genus is endemic to Hawaii and at least eight species have been described. Four of these species are extinct

(including Hibiscadelphus bombycinus, Hibiscadelphus crucibracteatus, Hibiscadelphus wilderianus, and Hibiscadelphus woodii ), two of these species only persist in cultivation

( Hibiscadelphus giffardianus and Hibiscadelphus hualalaiensis ), and two are extant in the wild ( Hibiscadelphus distans and Hibiscadelphus stellatus).

Hibiscadelphus wilderianus is an extinct tree species last observed at an elevation of 2,600 feet in 1910 on the lava fields of Auwahi on the island of Maui in Hawaii (see, e.g., Radlkofer et al, New and Noteworthy Hawaiian Plants; Hawaiian Board of Agriculture and Forestry Botanical Bulletin, 1911 ;(1): 1- 15;“The IUCN Red List of Threatened Species:

Hibiscadelphus wilderianus,” World Conservation Monitoring Centre, 1998, each of which is hereby incorporated by reference in its entirety). A description in Latin of Hibiscadelphus wilderianus can be found in the Radlkofer et al. original report. A photo of a tree branch with leaves and fruit was included in the original Radlkofer et al. report and is reproduced in FIG. 8A.

In some embodiments, the rare plant may be Leucadendron grandiflorum. Leucadendron is a dioecious genus that belongs to the Proteaceae family and is endemic to South Africa.

Species in the Leucadendron genus include evergreen shrubs and often have cone-shaped infructescences (seed heads). There are at least 80 species in the Leucadendron genus including L. album, L. arcuatum, L. argenteum, L. barkerae, L. bonum, L. brunioides, L. burchellii, L. cadens, L. chamelaea, L. cinereum, L. comosum, L. concavum, L. conicum, L. coniferum, L. cordatum, L. coriaceum, L. corymbosum, L. cryptocephalum, L. daphnoides, L. diemontianum,

L. discolor, L. dregei, L. dubium, L. elimense, L. ericifolium, L. eucalyptifolium, L. flexuosum, L. floridum, L. foedum, L. galpinii, L. gandogeri, L. glaberrimum, L. globosum, L. grandiflorum, L. gydoense, L. immoderatum, L. lanigerum, L. laureolum, L. laxum, L. levisanus, L. linifolium, L. loeriense, L. loranthifolium, L. macowanii, L. meridianum, L. meyerianum, L. microcephalum, L. modestum, L. muirii, L. nervosum, L. nitidum, L. nobile, L. olens, L. orientale, L. osbornei, L. platyspermum, L. pondoense, L. procerum, L. pubescens, L. pubibracteolatum, L. radiatum, L. remotum, L. roodii, L. rourkei, L. rubrum, L. salicifolium, L. salignum, L. sericeum, L. sessile, L. sheilae, L. singular, L. sorocephalodes, L. spirale, L. spissifolium, L. stellare, L. stelligerum, L. strobilinum, L. teretifolium, L. thymifolium, L. tinctum, L. tradouwense, L. uliginosum, L.

verticillatum, and L. xanthoconus.

Leucadendron grandiflorum is also known commonly as Wynberg Conebush and was last observed in 1806 in Clapham, South Africa. Recorded sightings of Leucadendron grandiflorum have occurred on Wynberg Mountain and this species may have existed on the south slopes of Wynberg hill on moister granite soils (see, e.g., T. Rebelo,“Wynberg Conebush - extinct for 200 years,” iSpot, 25 July 2015, which is hereby incorporated by reference in its entirety). Leucadendron grandiflorum has been described and depicted in Salisbury et al, The Paradisus Londinensis or Coloured Figures of Plants Cultivated in the Vicinity of the Metropolis. 1805; (Volume 1, part 2): 105; see www -dot- biodiversitylibrary.org-backslash- ia/mobot31753000575172#page/248/mode/lup, the contents of each of which is hereby incorporated by reference in its entirety. No modem collections of Leucadendron grandiflorum have been recorded, and it is considered that this species was likely scarce or extinct by the early 1800s (see, e.g., T. Rebelo,“Wynberg Conebush - extinct for 200 years,” iSpot, 25 July 2015; Catalogue of Life: Leucadendron grandiflorum (Salisb.) R. Br., 20 December 2017). Sister species include L. globosum and L. elimense. FIG. 8B depicts Leucadendron grandiflorum.

In some embodiments, the rare plant may be Macrostylis villosa. The Macrostylis genus belongs to the Rutaceae family and includes at least ten species (e.g., Macrostylis barbigera, Macrostylis cassiopoides, Macrostylis cauliflora, Macrostylis crassifolia, Macrostylis decipiens, Macrostylis hirta, Macrostylis ramulosa, Macrostylis squarrosa, Macrostylis tenuis, and

Macrostylis villosa).

There are two recognized subspecies of Macrostylis villosa , M. villosa (Thunb.) Sond. subsp. minor and M. villosa (Thunb.) Sond. subsp. villosa. M. villosa (Thunb.) Sond. subsp. minor is classified as extinct as its habitat was converted to agriculture and extensive searches have failed to relocate surviving plants. It was previously found on the Western Cape in South Africa and inhabited gravel and clay soil on slopes (see, e.g.,“Red List of South African Plants: Macrostylis villosa subsp. minor,” 2005, which is hereby incorporated by reference in its entirety). M. villosa (Thunb.) Sond. subsp. villosa is considered endangered due to population loss from urban expansion, foreign plant invasions and conversion of habitat to agriculture. A picture of M. villosa (Thunb.) Sond. subsp. villosa is reproduced in FIG. 8C (see, e.g.,“Red List of South African Plants: Macrostylis villosa subsp. villosa,” 2007, which is hereby incorporated by reference in its entirety).

In some embodiments, the rare plant may be Orbexilum stipulatum (Psoralea stipulata). Orbexilum belongs to the Fabaceae family and members of this genus often have characteristic pod walls that are rugose and free from hair. Orbexilum also may be distinguished by its “scarcely accrescent calyx” (see, e.g., Turner, Revision of the genus Orbexilum (Fabaceae:

Psoraleeae). Lundellia. 2008; (11): 1-7, which is hereby incorporated by reference in its entirety). Orbexilum species include O. chiapasanum, O. gracile, O. lupinellum, O. macrophyllum, O. melanocarpum, O. oliganthum, O. onobrychis, O. pedunculatum, O. simplex, O. stipulatum, and O. virgatum.

O. stipulatum, also known as the“Largestipule Leather-root” or as the“Falls-of-the- Ohio Scurfpea” was only found on Rock Island in Kentucky. The last recorded observation of O. stipulatum was in 1881, prior to resurfacing and flooding of this island. Despite many searches of similar habitats, including intensive searches in 1998, on both the Kentucky and Indiana shores of the Ohio River, this species has not been relocated. Therefore, this species has been classified as extinct (see, e.g., NatureServe Explorer: Orbexilum stipulatum - (Torr. &

Gray) Rydb., 2016 and Baskin el al. described above, which is each hereby incorporated by reference in its entirety).

O. stipulatum was a perennial herb and had leaves that were divided into 3 leaflets, each about 2 cm in length. The species had a persistent appendage at the base of the leaves and was also described as having a corolla tube that did not extend beyond the calyx. It is likely that this plant bloomed in late May to mid-June, but seeds have not been observed in nature (see e.g., “NatureServe Explorer: Orbexilum stipulatum - (Torr. & Gray) Rydb.,” 2016; and Raskin el al, Geographical origin of the specimens of Orbexilum stipulatum (T. & G.) Rydb. (Psoralea stipulata T. & G.). Castanea. 1986;(51): 207-210, each of which is hereby incorporated by reference in its entirety). A picture of O. stipulatum may be found in Short,“Orbexilum stipulatum collected at Falls of the Ohio,” 1840 from The Philadelphia Herbarium at the

Academy of Natural Sciences is reproduced in FIG. 8D.

In some embodiments, the rare plant may be Shorea cuspidata. Shorea is a genus in the Dipterocarpaceae family and includes many rainforest trees endemic to southeast Asia.

Many Shorea species are angiosperms (flowering plants). Non-limiting examples of Shorea species may include Shorea affinis, Shorea congestiflora, Shorea cordifolia, Shorea disticha, Shorea megistophylla, Shorea trapezifolia, Shorea zeylanica, Shorea acuminatissima, Shorea alutacea, Shorea angustifolia, Shorea bakoensis, Shorea balanocarpoides, Shorea chaiana, Shorea collaris, Shorea cuspidata, Shorea faguetiana, Shorea faguetioides, Shorea gibbosa, Shorea hopeifolia, Shorea iliasii, Shorea induplicata, Shorea kudatensis, Shorea laxa, Shorea longiflora, Shorea longisperma, Shorea macrobalanos, Shorea mujongensis, Shorea multiflora, Shorea obovoidea, Shorea patoiensis, Shorea peltata, Shorea polyandra, Shorea richetia, Shorea subcylindrica, Shorea tenuiramulosa, and Shorea xanthophylla.

S. cuspidata is a tree endemic to Malaysia that is currently classified as extinct on the IUCN Red Fist (“The IUCN Red Fist: Shorea cuspidata,” 1998, which is incorporated in its entirety by reference), although there have been a few recorded sightings of S. cuspidata subsequent to this classification in Bako National Park, Fambir National Park, and the

Semenggoh Arboretum (Ashton, Shorea cuspidata. Tree Flora of Sabah and Sarawek.

2004;(5):246-247; Fing et ah, Diversity of the tree flora in Semenggoh Arboretum, Sarawak, Borneo. Gardens’ Bulletin Singapore. 2012; (64): 139-169, which is each incorporated by reference in its entirety). Shorea cuspidata may be considered a rare plant. Shorea cuspidata has been characterized as a medium- sized tree with flowers second and pale lime-yellow petals (see, e.g., Ashton, Man. Dipt. Brun. 1968: f. 10, pi. 14 (stem-base)). A picture of a Shorea cuspidata specimen is reproduced in FIG. 8E (“Kew Royal Botanical Gardens: Shorea cuspidata specimen K000700460,” 1962, which is hereby incorporated by reference in its entirety). In some embodiments, the rare plant may be Wendlandia angustifolia. Wendlandia is a genus of flowering plants that belongs to the Rubiaceae family. Non-limiting examples of Wendlandia species may include Wendlandia aberrans, Wendlandia acuminata, Wendlandia amocana, Wendlandia andamanica, Wendlandia angustifolia, Wendlandia appendiculata, Wendlandia arabica, Wendlandia arborescens, Wendlandia augustini, Wendlandia

basistaminea, Wendlandia bicuspidata, Wendlandia bouvardioides, Wendlandia brachyantha, Wendlandia brevipaniculata, Wendlandia brevituba, and Wendlandia buddleacea.

W. angustifolia is a plant native to India that is currently classified as extinct in the IUCN Red List (see“The IUCN Red List: Wendlandia angustifolia,” 1998, which is hereby

incorporated by reference in its entirety). Subsequent to this classification, W. angustifolia was reportedly observed in Kalakkad Mundantharai Tiger Reserve in India (Viswanathan el al, Rediscovery of Wendlandia Angustifolia Wight Ex Hook.f. (Rubiaceae), from Tamil Nadu, a Species Presumed Extinct. Journal of The Bombay Natural History Society. 2000 97(2):311-313, which is hereby incorporated by reference in its entirety). W. angustifolia may be considered a rare plant. W. angustifolia has been described as a shrub or tree with ternately whorled and linear-lanceolate leaves (see, e.g., Viswanathan et al., Rediscovery of Wendlandia Angustifolia Wight Ex Hook.f. (Rubiaceae), from Tamil Nadu, a Species Presumed Extinct, Journal of The Bombay Natural History Society. 2000; 97(2):311-313, which is hereby incorporated by reference in its entirety). A picture of a specimen is reproduced in FIG. 8F (“Kew Royal Botanical Gardens: Wendlandia angustifolia K000030921,” collection date not recorded), which is hereby incorporated by reference in its entirety.

Methods of producing chimeric terpene synthases and terpenes

Also described herein are nucleic acid molecules encoding chimeric terpene synthases. In some embodiments, at least 10% (e.g., at least 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,

19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99%, including all values in between) of the nucleic acid molecule encoding such a chimeric terpene synthase may be derived from a rare or extinct plant.

In some instances, a nucleic acid molecule encoding a chimeric terpene synthase comprises a nucleotide sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99%, including all values in between) identical to a sequence selected from the group consisting of SEQ ID NOs: 67-118. In some instances, a nucleic acid molecule encoding a chimeric terpene synthase comprises a nucleotide sequence that is identical to a sequence selected from the group consisting of SEQ ID NOs: 67-118. In some instances, a nucleic acid molecule encoding a chimeric terpene synthase further comprises the nucleotide sequence TATA (TATA box sequence). In some instances, a nucleic acid molecule encoding a chimeric terpene synthase comprises the nucleotide sequence TATA (TATA box sequence) that is located N-terminal to a sequence selected from the group consisting of SEQ ID NOs: 67-118. In some instances, a nucleic acid molecule encoding a chimeric terpene synthase comprises a nucleotide sequence that encodes for a sequence set forth in SEQ ID NOs: 119-357.

In some embodiments, at least 10% (e.g., at least 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,

34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,

50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,

66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98%, 99%, or more than 99%, including all values in between) of the amino acid sequence of the chimeric terpene synthase (e.g., a chimeric sesquiterpene synthase) may be derived from a rare or extinct plant. In some instances, a chimeric terpene synthase comprises one or more sequences set forth in SEQ ID NOs: 119-357.

Also described herein are chimeric terpene synthases that are capable of producing alpha- guaiene. In some embodiments, at least 10% (e.g., at least 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,

33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,

49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,

81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,

97%, 98%, 99%, or more than 99%, including all values in between) of the nucleic acid molecule encoding such a chimeric terpene synthase may be derived from a rare or extinct plant.

In some embodiments, at least 10% (e.g., at least 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,

34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,

50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,

66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98%, 99%, or more than 99%, including all values in between) of the amino acid sequence of the chimeric terpene synthase that is capable of producing alpha-guaiene may be derived from a rare or extinct plant.

In some instances, construction of the chimeras may include sequence (e.g., nucleic acid sequence and/or amino acid sequence) alignments between at least two terpene synthases of interest. For example, sequence alignment analysis may be used to identify fragments (e.g., domains) of a particular terpene synthase to include in a chimeric terpene synthase. In some embodiments, the chimeric terpene synthase is a chimeric sesquiterpene synthase. Non-limiting examples of analyses may include the types described in the blastn-mapdamage and tblastn pipelines described in Example 2.

In some embodiments, a chimeric terpene synthase coding sequence comprises a mutation at 1, 2, 3, 4, 5, or more positions corresponding to a reference chimeric terpene synthase coding sequence. In some embodiments, the chimeric terpene synthase coding sequence comprises a mutation in 1, 2, 3, 4, 5, or more codons of the coding sequence relative to a reference chimeric terpene synthase coding sequence. As will be understood by one of ordinary skill in the art, a mutation within a codon may or may not change the amino acid that is encoded by the codon due to degeneracy of the genetic code. In some embodiments, the one or more mutations in the coding sequence do not alter the amino acid sequence of the chimeric terpene synthase relative to the amino acid sequence of a reference chimeric terpene synthase.

In some embodiments, the one or more mutations in a chimeric terpene synthase sequence alter the amino acid sequence of the chimeric terpene synthase relative to the amino acid sequence of a reference chimeric terpene synthase. In some embodiments, the one or more mutations alter the amino acid sequence of the chimeric terpene synthase relative to the amino acid sequence of a reference chimeric terpene synthase and alter (enhance or reduce) an activity of the chimeric terpene synthase relative to the reference chimeric terpene synthase.

The skilled artisan will also realize that mutations in a chimeric terpene synthase coding sequence may result in conservative amino acid substitutions to provide functionally equivalent variants of the foregoing polypeptides, e.g., variants that retain the activities of the polypeptides. As used herein, a“conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics or functional activity of the protein in which the amino acid substitution is made.

In some instances, an amino acid is characterized by its R group (see, e.g., Table 1). For example, an amino acid may comprise a nonpolar aliphatic R group, a positively charged R group, a negatively charged R group, a nonpolar aromatic R group, or a polar uncharged R group. Non-limiting examples of an amino acid comprising a nonpolar aliphatic R group include alanine, glycine, valine, leucine, methionine, and isoleucine. Non-limiting examples of an amino acid comprising a positively charged R group includes lysine, arginine, and histidine. Non limiting examples of an amino acid comprising a negatively charged R group include aspartic acid and glutamic acid. Non-limiting examples of an amino acid comprising a nonpolar, aromatic R group include phenylalanine, tyrosine, and tryptophan. Non-limiting examples of an amino acid comprising a polar uncharged R group include serine, threonine, cysteine, proline, asparagine, and glutamine.

Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et ak, eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et ak, eds., John Wiley & Sons, Inc., New York, 2010.

Non-limiting examples of functionally equivalent variants of polypeptides may include conservative amino acid substitutions in the amino acid sequences of proteins disclosed herein. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 residues can be changed when preparing variant polypeptides. In some embodiments, amino acids are replaced by conservative amino acid substitutions.

Table 1. Non-limiting Examples of Conservative Amino Acid Substitutions

Amino acid substitutions in the amino acid sequence of a polypeptide to produce a chimeric terpene synthase (e.g., chimeric sesquiterpene synthase) variant having a desired property and/or activity can be made by alteration of the coding sequence of the chimeric terpene synthase (e.g., chimeric sesquiterpene synthase). Similarly, conservative amino acid

substitutions in the amino acid sequence of a polypeptide to produce functionally equivalent variants of the polypeptide typically are made by alteration of the coding sequence of the chimeric terpene synthase (e.g., chimeric sesquiterpene synthase).

Mutations (e.g., substitutions) can be made in a nucleotide sequence by a variety of methods known to one of ordinary skill in the art. For example, mutations can be made by PCR- directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), or by chemical synthesis of a gene encoding a polypeptide.

Any suitable method, including circular permutation (Yu and Lutz, Trends Biotechnol. 2011 Jan;29(l): 18-25), may be used to produce variants. In circular permutation, the linear primary sequence of a polypeptide can be circularized (e.g., by joining the N-terminal and C- terminal ends of the sequence) and the polypeptide can be severed (“broken”) at a different location. Thus, the linear primary sequence of the new polypeptide may have low sequence identity (e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less or less than 5%, including all values in between) as determined by linear sequence alignment methods (e.g., Clustal Omega or BLAST). Topological analysis of the two proteins, however, may reveal that the tertiary structure of the two polypeptides is similar or dissimilar. Without being bound by a particular theory, a variant polypeptide created through circular permutation of a reference polypeptide and with a similar tertiary structure as the reference polypeptide can share similar functional characteristics (e.g., enzymatic activity, enzyme kinetics, substrate specificity or product specificity). In some instances, circular permutation may alter the secondary structure, tertiary structure or quaternary structure and produce an enzyme with different functional characteristics (e.g., increased or decreased enzymatic activity, different substrate specificity, or different product specificity).

See, e.g., Yu and Lutz, Trends Biotechnol. 2011 Jan;29(l): 18-25.

It should be appreciated that in a protein that has undergone circular permutation, the linear amino acid sequence of the protein would differ from a reference protein that has not undergone circular permutation. However, one of ordinary skill in the art would be able to readily determine which residues in the protein that has undergone circular permutation correspond to residues in the reference protein that has not undergone circular permutation by, for example, aligning the sequences and detecting conserved motifs, and/or by comparing the structures or predicted structures of the proteins, e.g., by homology modeling.

Aspects of the present disclosure relate to the recombinant expression of genes encoding enzymes, functional modifications and variants thereof, as well as uses relating thereto.

A nucleic acid encoding any of the chimeric terpene synthases described herein may be incorporated into any appropriate vector through any method known in the art. For example, the vector may be an expression vector, including but not limited to a viral vector ( e.g ., a lentiviral, retroviral, adenoviral, or adeno-associated viral vector), any vector suitable for transient expression, or any vector for inducible expression (e.g., a galactose-inducible or doxycycline- inducible vector). A non-limiting example of a vector for expression of a chimeric terpene synthase (e.g., a chimeric sesquiterpene synthase) is described in Example 2 below.

In some embodiments, a vector replicates autonomously in the cell. A vector can contain one or more endonuclease restriction sites that are cut by a restriction endonuclease to insert and ligate a nucleic acid containing a gene described herein to produce a recombinant vector that is able to replicate in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Cloning vectors include, but are not limited to: plasmids, fosmids, phagemids, vims genomes and artificial chromosomes. As used herein, the terms "expression vector" or "expression construct" refer to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell (e.g., microbe), such as a yeast cell. In some embodiments, the nucleic acid sequence of a gene described herein is inserted into a cloning vector such that it is operably joined to regulatory sequences and, in some embodiments, expressed as an RNA transcript. In some embodiments, the vector contains one or more markers, such as a selectable marker as described herein, to identify cells transformed or transfected with the recombinant vector.

In some embodiments, a vector is capable of integrating into the genome of a host cell.

A coding sequence and a regulatory sequence are said to be“operably joined” or “operably linked” when the coding sequence and the regulatory sequence are covalently linked and the expression or transcription of the coding sequence is under the influence or control of the regulatory sequence. If the coding sequence is to be translated into a functional protein, the coding sequence and the regulatory sequence are said to be operably joined or linked if induction of a promoter in the 5’ regulatory sequence transcribes the coding sequence and if the nature of the linkage between the coding sequence and the regulatory sequence does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the

corresponding RNA transcript to be translated into a protein. Thus, a promoter region is operably joined or linked to a coding sequence if the promoter region transcribes the coding sequence and the transcript can be translated into the protein or polypeptide of interest. In some embodiments, the nucleic acid encoding any of the proteins described herein is under the control of regulatory sequences ( e.g ., enhancer sequences). In some embodiments, a nucleic acid is expressed under the control of a promoter. The promoter can be a native promoter, e.g., the promoter of the gene in its endogenous context, which provides normal regulation of expression of the gene. Alternatively, a promoter can be a promoter that is different from the native promoter of the gene, e.g., the promoter is different from the promoter of the gene in its endogenous context. As used herein, a“heterologous promoter” or

“recombinant promoter” is a promoter that is not naturally or normally associated with or that does not naturally or normally control transcription of a DNA sequence to which it is operably joined or linked. In some embodiments, a nucleotide sequence is under the control of a heterologous promoter.

In some embodiments, the promoter is a eukaryotic promoter. Non-limiting examples of eukaryotic promoters include TDH3, PGK1, PKC1, TDH2, PYK1, TPI1, ATI, CMV, EFla, SV40, PGK1 (human or mouse), Ubc, human beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GAL1, GAL 10, TEF1, GDS, ADH1, CaMV35S, Ubi, HI, U6, as would be known to one of ordinary skill in the art (see, e.g., Addgene website: blog.addgene.org/plasmids-101-the- promoter-region). In some embodiments, the promoter is a prokaryotic promoter (e.g., bacteriophage or bacterial promoter). Non-limiting examples of bacteriophage promoters include Pis Icon, T3, T7, SP6, and PL. Non-limiting examples of bacterial promoters include Pbad, PmgrB, Ptrc2, Plac/ara, Ptac, Pm.

In some embodiments, the promoter is an inducible promoter. As used herein, an “inducible promoter” is a promoter controlled by the presence or absence of a molecule. Non limiting examples of inducible promoters include chemically-regulated promoters and physically-regulated promoters. For chemically-regulated promoters, the transcriptional activity can be regulated by one or more compounds, such as alcohol, tetracycline, galactose, a steroid, a metal, or other compounds. For physically-regulated promoters, transcriptional activity can be regulated by a phenomenon such as light or temperature. Non-limiting examples of tetracycline- regulated promoters include anhydrotetracycline (aTc)-responsive promoters and other tetracycline -responsive promoter systems (e.g., a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)). Non-limiting examples of steroid-regulated promoters include promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily. Non-limiting examples of metal-regulated promoters include promoters derived from metallothionein (proteins that bind and sequester metal ions) genes. Non-limiting examples of pathogenesis-regulated promoters include promoters induced by salicylic acid, ethylene or benzothiadiazole (BTH). Non-limiting examples of temperature/heat-inducible promoters include heat shock promoters. Non-limiting examples of light-regulated promoters include light responsive promoters from plant cells. In certain embodiments, the inducible promoter is a galactose-inducible promoter. In some embodiments, the inducible promoter is induced by one or more physiological conditions (e.g., pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, or concentration of one or more extrinsic or intrinsic inducing agents). Non-limiting examples of an extrinsic inducer or inducing agent include amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones or any combination thereof.

In some embodiments, the promoter is a constitutive promoter. As used herein, a “constitutive promoter” refers to an unregulated promoter that allows continuous transcription of a gene. Non-limiting examples of a constitutive promoter includes CPI, CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, Ac5, polyhedrin, TEF1, GDS, CaM35S, Ubi, HI, and U6.

Other inducible promoters or constitutive promoters known to one of ordinary skill in the art are also contemplated herein.

The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but generally include, as necessary, 5’ non-transcribed and 5’ non- translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. In particular, such 5’ non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined or linked gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences. The vectors disclosed herein may include 5' leader or signal sequences. The regulatory sequence may also include a terminator sequence. In some embodiments, a terminator sequence marks the end of a gene in DNA during transcription. The choice and design of one or more appropriate vectors suitable for inducing expression of one or more genes described herein in a heterologous organism is within the ability and discretion of one of ordinary skill in the art.

Expression vectors containing the necessary elements for expression are commercially available and known to one of ordinary skill in the art (see, e.g., Sambrook et ah, Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012).

Any suitable host cell may be used to produce any of the chimeric terpene synthases disclosed herein, including eukaryotic cells or prokaryotic cells. Suitable host cells include fungal cells (e.g., yeast cells) and bacteria cells (e.g., E. coli cells). Non-limiting examples of genera of yeast for expression include Saccharomyces (e.g., S. cerevisiae), Pichia,

Kluyveromyces (e.g., K. lactis), Hansenula and Yarrowia. In some embodiments, the yeast strain is an industrial polyploid yeast strain. Other non-limiting examples of fungal cells include cells obtained from Aspergillus spp., Penicillium spp., Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp.

The term“cell,” as used herein, may refer to a single cell or a population of cells, such as a population of cells belonging to the same cell line or strain. Use of the singular term“cell” should not be construed to refer explicitly to a single cell rather than a population of cells.

A vector encoding any of the chimeric terpene synthases (e.g., chimeric sesquiterpene synthases) described herein may be introduced into a suitable host cell using any method known in the art. Non-limiting examples of yeast transformation protocols are described in Example 2 below and in Gietz et al, Yeast transformation by the LiAc/SS Carrier DNA/PEG method.

Methods Mol Biol. 2006;313:107-20, which is hereby incorporated by reference in its entirety for this purpose. Host cells may be cultured under any conditions suitable as would be understood by one of ordinary skill in the art. For example, any media, temperature, and incubation conditions known in the art may be used. For host cells carrying an inducible vector, cells may be cultured with an appropriate inducible agent to promote expression.

Any of the cells disclosed herein can be cultured in media of any type (rich or minimal) and any composition prior to, during, and/or after contact and/or integration of a nucleic acid.

The conditions of the culture or culturing process can be optimized through routine

experimentation as understood by one of ordinary skill in the art. In some embodiments, the selected media is supplemented with various components. In some embodiments, the concentration and amount of a supplemental component is optimized. In some embodiments, other aspects of the media and growth conditions (e.g., pH, temperature, etc.) are optimized through routine experimentation. In some embodiments, the frequency that the media is supplemented with one or more supplemental components, and the amount of time that the cell is cultured is optimized.

Culturing of the cells described herein can be performed in culture vessels known and used in the art. In some embodiments, an aerated reaction vessel (e.g., a stirred tank reactor) is used to culture the cells. In some embodiments, a bioreactor or fermentor is used to culture the cell. Thus, in some embodiments, the cells are used in fermentation. As used herein, the terms “bioreactor” and“fermentor” are interchangeably used and refer to an enclosure, or partial enclosure, in which a biological, biochemical and/or chemical reaction takes place, involving a living organism or part of a living organism. A“large-scale bioreactor” or“industrial-scale bioreactor” is a bioreactor that is used to generate a product on a commercial or quasi commercial scale. Large scale bioreactors typically have volumes in the range of liters, hundreds of liters, thousands of liters, or more.

In some embodiments, a bioreactor comprises a cell (e.g., a yeast cell) or a cell culture (e.g., a yeast cell culture), such as a cell or cell culture described herein. In some embodiments, a bioreactor comprises a spore and/or a dormant cell type of an isolated microbe (e.g., a dormant cell in a dry state).

Non-limiting examples of bioreactors include: stirred tank fermentors, bioreactors agitated by rotating mixing devices, chemostats, bioreactors agitated by shaking devices, airlift fermentors, packed-bed reactors, fixed-bed reactors, fluidized bed bioreactors, bioreactors employing wave induced agitation, centrifugal bioreactors, roller bottles, and hollow fiber bioreactors, roller apparatuses (for example benchtop, cart-mounted, and/or automated varieties), vertically- stacked plates, spinner flasks, stirring or rocking flasks, shaken multi-well plates, MD bottles, T-flasks, Roux bottles, multiple- surface tissue culture propagators, modified fermentors, and coated beads (e.g., beads coated with serum proteins, nitrocellulose, or carboxymethyl cellulose to prevent cell attachment).

In some embodiments, the bioreactor includes a cell culture system where the cell (e.g., yeast cell) is in contact with moving liquids and/or gas bubbles. In some embodiments, the cell or cell culture is grown in suspension. In other embodiments, the cell or cell culture is attached to a solid phase carrier. Non-limiting examples of a carrier system includes microcarriers ( e.g ., polymer spheres, microbeads, and microdisks that can be porous or non-porous), cross-linked beads (e.g., dextran) charged with specific chemical groups (e.g., tertiary amine groups), 2D microcarriers including cells trapped in nonporous polymer fibers, 3D carriers (e.g., carrier fibers, hollow fibers, multicartridge reactors, and semi-permeable membranes that can comprising porous fibers), microcarriers having reduced ion exchange capacity, encapsulation cells, capillaries, and aggregates. In some embodiments, carriers are fabricated from materials such as dextran, gelatin, glass, or cellulose.

In some embodiments, industrial-scale processes are operated in continuous, semi- continuous or non-continuous modes. Non-limiting examples of operation modes are batch, fed batch, extended batch, repetitive batch, draw/fill, rotating-wall, spinning flask, and/or perfusion mode of operation. In some embodiments, a bioreactor allows continuous or semi-continuous replenishment of the substrate stock, for example a carbohydrate source and/or continuous or semi-continuous separation of the product, from the bioreactor.

In some embodiments, the bioreactor or fermentor includes a sensor and/or a control system to measure and/or adjust reaction parameters. Non-limiting examples of reaction parameters include biological parameters (e.g., growth rate, cell size, cell number, cell density, cell type, or cell state, etc.), chemical parameters (e.g., pH, redox-potential, concentration of reaction substrate and/or product, concentration of dissolved gases, such as oxygen concentration and CO2 concentration, nutrient concentrations, metabolite concentrations, concentration of an oligopeptide, concentration of an amino acid, concentration of a vitamin, concentration of a hormone, concentration of an additive, serum concentration, ionic strength, concentration of an ion, relative humidity, molarity, osmolarity, concentration of other chemicals, for example buffering agents, adjuvants, or reaction by-products), physical/mechanical parameters (e.g., density, conductivity, degree of agitation, pressure, and flow rate, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, conversion rate, as well as

thermodynamic parameters, such as temperature, light intensity/quality, etc.). Sensors to measure the parameters described herein are well known to one of ordinary skill in the relevant mechanical and electronic arts. Control systems to adjust the parameters in a bioreactor based on the inputs from a sensor described herein are well known to one of ordinary skill in the art in bioreactor engineering. Terpenes produced by any of the host cells disclosed herein may be extracted using any method known in the art. A non-limiting example of a method for sesquiterpene extraction is provided in Example 2. Any of the terpenes produced from the methods, compositions, or host cells described herein may be used in a suitable composition for topical application to, for example, skin, hair, clothing, or articles in a home (e.g., a perfume). As used herein, the term “perfume” is any fragrance formulation suitable for application to the hair, skin, or clothing of a person or an article in a home. This term includes, but is not limited to: an eau de cologne, eau de toilette, eau de parfum, perfume extract or extrait. In addition to comprising one or more terpenes of the application, such a perfume may include, for example, one or more natural oils, fixatives, emollients, or solvents.

Examples of natural oils which may be used in perfume formulations include, but are not limited to: amyris oil; angelica seed oil; angelica root oil; aniseed oil; valerian oil; basil oil; bay oil; mugwort oil; benzoin resin; bergamot oil; birch tar oil; bitter almond oil; savory oil; bucco- leaf oil; cabreuva oil; cade oil; calamus oil; camphor oil; cananga oil; cardamom oil; cascarilla oil; cassia oil; castoreum absolute; cedar-leaf oil; cedarwood oil; cistus oil; citronella oil; lemon oil; copaiba balsam oil; coriander oil; costus root oil; cumin oil; cypress oil; davana oil; dill oil; dillseed oil; elemi oil; tarragon oil; eucalyptus citriodora oil; eucalyptus oil; fennel oil; fir oil; galbanum oil; geranium oil; grapefruit oil; guaiac wood oil; gurjun balsam oil; helichrysum oil; ginger oil; iris root oil; calamus oil; blue chamomile oil; Roman chamomile oil; carrot-seed oil; cascarilla oil; pine-needle oil; spearmint oil; caraway oil; labdanum oil; lavandin oil; lavender oil; lemongrass oil; lovage oil; lime oil (e.g., distilled or pressed lime oil); linaloe oil: litsea cubeba oil; bay leaf oil; mace oil; marjoram oil; mandarin oil; massoi bark oil; ambrette oil; clary sage oil; myristica oil; myrrh oil; myrtle oil; clove leaf oil; clove flower oil; neroli oil; olibanum oil; opopanax oil; orange oil; origanum oil; palmar osa oil; patchouli oil; perilla oil; Peru balsam oil; parsley leaf oil; parsley seed oil; petitgrain oil; peppermint oil; pepper oil; pimento oil; pine oil; pennyroyal oil; rosewood oil; rose oil; rosemary oil; Dalmatian sage oil; Spanish sage oil; sandalwood oil; celery seed oil; spike lavender oil; Japanese aniseed oil; styrax oil; tagetes oil; fir-needle oil; tea-tree oil; turpentine oil; thyme oil; tuberose absolute; vanilla extract; violet leaf absolute; verbena oil; vetiver oil; juniper oil; wine-lees oil; wormwood oil; wintergreen oil; ylang oil; hyssop oil; civet absolute; cinnamon leaf oil; cinnamon bark oil; as well as fractions thereof or constituents isolated therefrom; and combinations thereof. Other examples of compounds which may be used in perfume formulations may include: wood moss absolute; beeswax absolute; cassia absolute; eau de brouts absolute; oakmoss absolute; galbanum resin; helichrysum absolute; iris root absolute; jasmine absolute; labdanum absolute; labdanum resin; lavandin absolute; lavender absolute; mimosa absolute; tincture of musk; myrrh absolute; olibanum absolute; orange blossom absolute; rose absolute; Tolu balsam; Tonka absolute; as well as fractions thereof or constituents isolated therefrom; and combinations thereof.

As used herein, the term“emollient” means a fatty or oleaginous substance which increases tissue moisture content (and may, for example, render skin softer and more pliable). Emollients for use with the instant compounds and methods may include any appropriate animal fats/oils, vegetable oils, and/or waxes. As a non-limiting set of examples, an emollient for use with the instant compositions and methods may be of natural or synthetic origin and may include: cold-pressed almond oil, jojoba oil, sunflower oil, olive oil, hazelnut oil, avocado oil, safflower oil, grapeseed oil, coconut oil, wheat germ oil, apricot kernel oil, natural waxes and “butters” ( e.g ., unrefined beeswax, shea butter, jojoba butter, and/or cocoa butter), Schercemol™ LL Ester, Schercemol™ 1818 Ester, butylene glycol, capric/caprylic triglyceride, ceteareth-20, one or more fatty alcohols (e.g., cetearyl alcohol, cetyl alcohol, and/or coconut fatty acids), one or more silicones (e.g., cyclomethicone, dimethicone, and/or cyclopentasiloxane), emulsifying wax, petroleum jelly, fatty acids, glyceryl stearate, hydrogenated oils, isopropyl myristate , mineral oil, octyl palmitate, paraffin, squalene, stearic acid, palmitoyl proline, or magnesium palmitoyl glutamate.

As used herein, the term“fixative” means a compound used to equalize the vapor pressures (and thus the volatilities) of one or more compounds in the perfume. As a non-limiting set of examples, a fixative for use with the compounds and perfumes described herein may be: dipropylene glycol, diethyl phthalate, Hedione®, Abalyn™ D-E Methyl Ester of Rosin, Jojoba (such as Floraesters K-100 Jojoba or Floraesters K-20W Jojoba), Sepicide LD, and/or Foralyn™ 5020-F CG Hydrogenated Rosinate.

As used herein, the term“solvent” is the diluent used to create a perfume. As a non limiting example, the solvent may be an alcohol (e.g., an ethyl alcohol), 1,2-hexanediol, 1,2- heptanediol, a neutral smelling oil (e.g., fractionated coconut oil or jojoba oil), or one or more volatile silicones. As a non-limiting example, Perfumers’ Alcohol (a type of ethyl alcohol) may be used. Perfumers’ Alcohol is prepared from 200 proof ethyl alcohol which may contain very small amounts of butyl alcohol, denatonium benzoate (Britex), and/or hexylene glycol. Various grades of Perfumers’ Alcohol are available including SDA 40B 200 Proof and SDA-B 200 proof.

Additional compounds or fragrance materials for use in the perfume composition according to the disclosure may include any compounds which are customarily used in the field.

The present invention is further illustrated by the following Examples, which in no way should be construed as limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

EXAMPLES

Example 1. Functional characterization of chimeric terpene synthases.

Genomic DNA from 12 extinct plant samples were sequenced (Table 2). Sesquiterpene synthase (SQTS) fragments were recovered from seven plants (Table 11), but gaps in the sequencing prevented reconstruction of full-length genes. A library comprising 2,738 terpene synthase chimeras (containing sequence from sesquiterpene synthases from extant plants to fill the sequence gaps) was screened. The expression of 52 SQTS chimeras (sequences provided in Table 10) from six rare plants (Table 2) led to the production of sesquiterpenes in the screening strain. Methods and materials for each of the procedures described in this Example may be found in Example 2.

Table 2. Rare Plants that were Sequenced (The plants from which functional sesquiterpene chimeras were reconstructed are shown bold face and underlined.)

The terpenes produced by the functional SQTS chimeras were identified initially based on gas chromatography-mass spectrometry (GC/MS) data. In some cases, authentic standards or essential oils containing characterized sesquiterpenes were available to confirm mass spectrum- and retention time-based identifications. In other cases, standards were not available and structural identifications were made based on mass spectral analysis alone. The different methods used to identify the structures are detailed in Table 3, and the specific methods used to identify each sesquiterpene are indicated in Tables 4-9. In some cases, products were identified only as“sesquiterpene” or“sesquiterpenol.” In one case, a mass spectrum was recovered but did not yield a match in the NIST/internal database. This sesquiterpenol was identified in the product tables as an“unidentified sesquiterpenol” and additional characterization may be used to determine its structure.

Fourteen SQTS chimeras derived from Hibiscadelphus wilderianus produced 1 or more sesquiterpenes (FIG. 1, Table 3). Seven SQTS chimeras derived from Leucadendron

grandiflorum also produced sesquiterpenes (FIG. 2, Table 5), as did six SQTS chimeras from Macrostylis villosa (FIG. 3, Table 6), two from O. stipulatum (FIG. 4, Table 7), six from Shorea cuspidata (FIG. 5, Table 8), and seventeen from Wendlandia angustifolia (FIG. 6, Table 9). The SQTSs were found to produce one to nine different terpenes. The product profiles of the plant SQTS chimeras were different when the functional SQTS chimeras were grouped by the terpenes produced in highest yield (FIG. 7). Delta-cadinene synthases were the most numerous group of functional chimeras at a total of 22 and were derived from four of the plants. 10 of the 14 of the synthases from H. wilderianus were of this variety. Alpha-cadinol was frequently detected as a minor product of the delta-cadinene synthases; however, three SQTS chimeras from S. cuspidata yielded more alpha-cadinol than delta-cadinene. These six SQTS chimeras derived S. cuspidata produced a very similar product mixture (Table 8, FIG. 13).

The screening of the 2,738-member chimeric sesquiterpene synthase library resulted in the successful expression of 52 functional chimeric sesquiterpene synthases (SQTSs). Fourteen synthases were derived from H. wilderianus, a tree which went extinct in Hawaii over 100 years ago. Cadinene, cadinol, and eudesmol-type sesquiterpenes were produced by these chimeras. A few active chimeras were also generated from O. stipulatum, a plant that went extinct in

Kentucky in the 1800s. Two guaienes and gamma-bisabolene were produced by these synthases. Seven functional SQTS chimeras were constructed from L. grandiflorum, a plant that went extinct over 200 years ago. Diverse sesquiterpene and sesquiterpenol structures were produced by these chimeras, along with those derived from three other plants.

Table 3. The six methods used to identify the sesquiterpenes produced by the sesquiterpene synthases.

Table 4. Functional sesquiterpene synthase chimeras derived from H. wilderianus sequences and their associated products.

¹ The structure identification ranking key is defined in Table 3, with lower numbers indicating a higher degree of confidence. ² The composition of total sesquiterpenes from each chimera was a rough estimate based on a common ion count (m/z 204.2). The ratio of metabolites may have been different in the production strains and it is possible other minor metabolites were detected when samples were prepared. Representative GC/MS chromatograms for the chimeras with bold font can be found in FIG. 9. ³ Co-eluted under these run conditions. The peak was partially resolved under longer run conditions, about 6/4 alpha/beta-eudesmol. Table 5. Functional sesquiterpene synthase chimeras derived from L. grandiflorum sequences and their associated products.

¹ The structure identification ranking key is defined in Table 3, with lower numbers indicating a higher degree of confidence.

² The composition of total sesquiterpenes from each chimera was a rough estimate based on a common ion count (m/z 204.2). The ratio of metabolites may have been different in the production strains and other minor metabolites may have been detected when samples were prepared. Representative GC/MS chromatograms for the chimeras with bold font can be found in FIG. 10 and FIG.l l.

³ Co-eluted under these run conditions. The peak was partially resolved under longer run conditions, about 6/4 alpha/beta-eudesmol.

Table 6. Functional sesquiterpene synthase chimeras derived from M. villosa sequences and their associated products.

¹ The structure identification ranking key is defined in Table 3, with lower numbers indicating a higher degree of confidence.

² The composition of total sesquiterpenes from each chimera was a rough estimate based on a common ion count (m/z 204.2). The ratio of metabolites may have been different in the production strains and other minor metabolites may have been detected when samples were prepared. Representative GC/MS chromatograms for the chimeras with bold font can be found in FIG. 12. Table 7. Functional sesquiterpene synthase chimeras derived from O. stipulatum sequences and their associated products.

¹ The structure identification ranking key is defined in Table 3, with lower numbers indicating a higher degree of confidence.

2 The composition of total sesquiterpenes from each chimera was a rough estimate based on a common ion count (m/z 204.2). The ratio of metabolites may have been different in the production strains and other minor metabolites may have been detected when samples were prepared.

Table 8. Functional sesquiterpene synthase chimeras derived from S. cuspidata sequences and their associated products.

¹ The structure identification ranking key is defined in Table 3, with lower numbers indicating a higher degree of confidence.

² The composition of total sesquiterpenes from each chimera was a rough estimate based on a common ion count (m/z 204.2). The ratio of metabolites may have been different in the production strains and it is possible other minor metabolites were detected when samples were prepared. Representative GC/MS chromatograms for the chimeras with bold font can be found in FIG. 13.

Table 9. Functional sesquiterpene synthase chimeras derived from W. angustifoHa sequences and their associated products.

¹ The structure identification ranking key is defined in Table 3, with lower numbers indicating a higher degree of confidence. ² The composition of total sesquiterpenes from each chimera was a rough estimate based on a common ion count (m/z 204.2). The ratio of metabolites may have been different in the production strains and it is possible other minor metabolites were detected when samples were prepared. Representative GC/MS chromatograms for the chimeras with bold font can be found in Appendix FIG. 14 and FIG. 15. ³ Co-eluted under these run conditions. The peak was partially resolved under longer run conditions, about 6/4 alpha/beta-eudesmol.

Table 10. Amino acid (AA) and nucleic acid sequences of sesquiterpene chimeras.

Table 11. Non-limiting examples of sequence fragment(s) derived from rare plants.

Example 2. Materials and methods for construction of terpene synthase chimeras.

Terpene synthases for Capture-seq and chimera scaffolding

Candidate sesquiterpene synthases (SQTSs) were designed by combining sequence fragments from rare flower genomes (Table 11) with“scaffold” SQTSs from sources including UniProt and GenBank.

For Capture-seq (targeted sequencing of terpene synthases), a subset of 5,171 terpene synthases (TPSs) were compiled from UniProt that had nucleotide sequences in EMBL/Genbank. Oligonucleotide chips were generated for enriching the flower DNA samples for TPS- homologous sequences, and then subjected first to Illumina sequencing. The Capture-seq libraries were also sequenced a second time at higher depth.

For SQTS chimera reconstruction, sequences closer to annotated SQTSs than annotated mono-, di-, or tri-terpene synthases were selected. This set of 1,521 putative SQTSs were used (in both nucleotide and peptide form) as query sequences for blastn and tblastn in the chimera construction pipeline below.

Chimera reconstruction

Two methods were used for constructing chimeric SQTSs: 1) the blastn-mapDamage pipeline, and 2) the tblastn pipeline. blastn-mapdamage pipeline

Generally, the blastn-mapdamage pipeline conservatively detects fragments with high nucleotide similarity to the scaffolds resulting in chimeric terpene synthases (e.g., chimeric sesquiterpene synthases) that are likely very close to the original enzyme sequences in the rare flowers. To detect mutations that may be artifacts of stereotypical rare DNA damage, barn- formatted Illumina read alignments were inputted into mapDamage software.

Specifically, the following steps were used to generate alignments of DNA fragments from each flower to various SQTS scaffolds: 1. Illumina reads (fastq files) from genomic capture-seq runs were combined and assembled by SPADES into longer contigs.

2. The 1521-set of SQTS scaffolds were used as queries in a blastn search with default parameters against the SPADES contigs. Relatively few scaffolds had hits, so all of the scaffolds with hits were chosen to serve as references for read alignment in the next step.

3. Combined reads from the sequencing runs were quality-trimmed (using bbduk) and pair-merged (using bbmerge) and aligned to chosen SQTS reference sequences using bwa mem. Results were reformatted to bam, sorted, and indexed.

4. mapDamage was run on the aligned reads. This resulted in a read alignment where SNPs resembling DNA damage were assigned low quality scores.

5. Read alignments were processed as follows: bases with quality < 25 were masked (changed to the reference); alignments were reformatted to fasta; SNPs with counts < 6 were masked; duplicate reads were removed; SNPs with frequency < 0.1 were masked; reads that were exact subsequences of other reads were removed; reads were translated in the frame of the reference; and subsequences were removed again. The quality and SNP frequency thresholds used for masking the alignment were determined empirically by looking at distributions of quality and SNP frequency.

6. Read alignments and SPADE contig alignments (after reference-frame translation) were combined and realigned using Clustal Omega. This was done because some contigs spanned regions of the scaffolds that the reads did not.

The alignments from the above steps were used to construct SQTS chimeras as follows:

1. The alignment was split into“independent subregions” such that each subregion did not contain any fragment (aligned read) overlapping with and differing from a fragment from another subregion (identical overlaps were allowed between subregions).

2. In each subregion, all possible combinations of“compatible fragments” were

enumerated. Compatible fragments were defined as fragments that either overlapped identically (and therefore could be merged into a longer fragment) or did not overlap at all (and, e.g., were assumed to come from the same haplotype). Fragment combinations were“max-coverage” - that is, contained as many compatible fragments as possible. Each max-coverage fragment combination was considered to be a possible reconstruction of that region of the alignment, and was merged into a superfragment (which may have contained gaps) and saved.

3. Superfragments from each subregion were downsampled to 90% or 95% identity using a custom, iterative algorithm, and all possible combinations of downsampled superfragments from different subregions were combined. Regions that were shorter than a certain threshold are downsampled to a single sequence. Each combination of superfragments was merged into the scaffold to generate a chimera sequence. The downsampling parameters were varied slightly varied according to the sample and scaffold to allow >1 but <100 chimeras to be constructed in each case.

After running the above pipeline on each sample, a total of 1136 chimeras were generated. A significant fraction of the chimeras were constructed purely from aligned reads.

A total of 652 sesquiterpene synthase chimeras were created using these methods. tblastn pipeline

Generally, the tblastn pipeline maximized the sensitivity of detecting fragments homologous to the SQTS scaffolds, and therefore cast a wide net for potentially usable sequences.

Specifically, the following steps were used to generate alignments of DNA fragments from each flower to various SQTS scaffolds:

1. The 1521-set of SQTS scaffolds were used as protein queries to tblastn to search all frames translations of the SPADES contigs (described above).

2. Hits (aligned contigs) were filtered to a minimum of 40% identity to the scaffold and a minimum length that depends on hit identity by a heuristic function. The filtering criteria were chosen by inspecting plots of hit length versus identity across all samples.

3. Downsampling scaffolds was performed by hierarchically clustering the scaffolds by the number of identical residues to each hit. The scaffold in each cluster with the greatest number of identities across all of its hits was kept for chimera reconstruction. Downsampling reduced the number of scaffolds by 20-fold. This step was skipped for samples in which fewer than 10 scaffolds have hits.

4. Certain scaffolds were always chosen as a cluster representation because they were previously identified as having activity and/or were known in the literature (even if another sequence had more identities to hits). These preferred scaffolds were not downsampled, and tblastn hits were kept for chimera construction.

5. The aligned portions of all contigs hitting a scaffold were realigned to the scaffold using Clustal Omega. Unaligned portions of contigs were discarded as likely representing introns. This alignment was then used for chimera construction.

6. Chimeras were constructed from aligned tblastn hits using the combinatorial

compatible fragments method described above without downsampling in subregions. Both“max-coverage” (as many as possible compatible fragments in each set) and “min-coverage” (only one compatible fragment in each set) chimeras were generated. The min-coverage chimeras may avoid combining fragments from unrelated sequences.

The tblastn pipeline yielded 10,114“max-coverage” chimeras and 2,624“min-coverage” chimeras. Certain max-coverage chimeras were downsampled to 95% identity by CD-HIT. This resulted in 388 sequences (382 after removing sequences with ambiguous amino acids). Certain max coverage chimeras were filtered to a minimum rare DNA content of 60% and downsampled to 90% identity. This resulted in 1320 sequences. Certain min-coverage chimeras were filtered to a minimum rare DNA content of 10% and downsampled to 95% identity by CD-HIT.

Encoding and synthesis order

Each enzyme was codon-optimized twice: once using a yeast expression-weighted codon table, and once using a yeast expression- weighted codon table after removing codons with <10% frequency. A different random number was used as the seed for each encoding. Encodings for different enzymes were completely independent - no specific procedure was used to preserve codons at residues inherited by chimeras from scaffolds.

Sequences encoding the chimeric enzymes were cloned into the pESC-URA3 screening vector, driven by pGALl and terminated by tCYCl. Chimera reconstruction aided by extant transcriptome

For one of the extinct flower species, Shorea cuspidata, transcriptome sequencing data was available on an extant relative Shorea beccariana. This made it possible to construct chimeras using SQTS scaffolds from a related flower. This was done in a 2-step process:

1. The S. beccariana (Sb) transcriptome data were assembled and mined for SQTS homologs. The data were downloaded from the data set SRR687302 from the NCBI SRA database. Assembly was done using Trinity, and ORFs were predicted via Transdecoder. BLAST was used to identify fragments homologous to a set of 1,500 curated SQTS sequences.

2. The identified Sb SQTSs or SQTS fragments were used as scaffold sequences in either the tblastn or blastn-mapDamage pipelines to reconstruct chimeras. If the scaffold was a fragment itself, it was in turn merged into the closest Uniprot- sourced SQTS sequence to generate a full-length chimera.

Screening Strain and Sesquiterpene Synthase Transformation

The chimeric sesquiterpene synthases were transformed into high copy pESC-URA3- derived expression vectors under the control of the galactose-inducible P(gall) promoter (Sikorski et al, A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(l):19-27, which is hereby incorporated by reference in its entirety for this purpose).

These vectors were transformed into a haploid Saccharomyces cerevisiae CEN.PK2 strain (MATa ura3-52 trpl-289 leu2-3_l 12 his3Al MAL2-8C SUC2) that had been modified to increase sesquiterpene flux via integration of two copies of the catalytic region of HMG-CoA reductase 1 under control of convergent P(gall) promoters at the homothallic switching endonuclease (YDL227C) locus on chromosome 4 (see SEQ ID NO: 53 shown below). See: Entian et al, Yeast Genetic Strain and Plasmid Collections. Methods in Microbiology. 2007; (36): 629-666; tHMGl, Donald et al, Effects of overproduction of the catalytic domain of 3- hydroxy-3-methylglutaryl coenzyme A reductase on squalene synthesis in Saccharomyces cerevisiae. Appl Environ Microbiol. 1997 Sep;63(9):3341-4; Qzaydin et al, Carotenoid-based phenotypic screen of the yeast deletion collection reveals new genes with roles in isoprenoid production. Metab Eng. 2013 Jan;15:174-83, each of which is hereby incorporated by reference in its entirety). Competition for fanesyl pyrophosphate was reduced in these cells by replacing the Erg9 (Farnesyl-diphosphate famesyl transferase) promoter with the methionine-repressible Met3 promoter as shown below in SEQ ID NO: 54 and incubating in media containing methionine (see: Ro et al, Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature. 2006 Apr 13;440(7086):940-3; and Asadollahi et al, Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of ERG9 repression on sesquiterpene biosynthesis. Biotechnol Bioeng. 2008 Feb 15;99(3):666-77, each of which is hereby

incorporated by reference in its entirety for this purpose). This strain with downregulated Erg9 and containing two copies of galactose-inducible tHMGl on chromosome 4 was designated tl 19889.

The transformation of the chimeric sesquiterpene vectors into strain tl 19889 was performed employing the chemical transformation techniques demonstrated in Gietz et al., Yeast transformation by the LiAc/SS Carrier DNA/PEG method. Methods Mol Biol. 2006;313:107-20, which is hereby incorporated by reference in its entirety for this purpose.

Sesquiterpene Production and Extraction

Transformant colonies were inoculated into 300 pi of SC-ura medium (Synthetic

Complete with 2% dextrose, no uracil added) in 96 deep well plates. The plates were covered with Excel Scientific AeroSeal membranes (BS-25) and incubated for 48 hours at 30 °C in a shaking incubator. 30 mΐ of the cultures (1:15 dilution) were mixed into 420 mΐ of SC -ura induction medium containing 1.8% galactose and 0.2% raffinose as the carbon sources, yielding a starting optical density at 600 nm (ODeoo) of approximately 0.1-0.2. A 0.88% dodecane overlay (4 mΐ) was added to each well and the plates were covered with AeroSeal membranes and incubated at 30°C in a shaking incubator for four days. 15 mΐ of each culture was removed to measure ODeoo at the end of the four days. 350 mΐ of ethyl acetate (250 mM tridecane internal) was added to directly to each well and mixed (1:1 Extraction). The 96- well plates were then centrifuged and the ethyl acetate extractions were stored at -80 °C in glass vials until analysis by GC-MS.

Sesquiterpene structure identification Ethyl Acetate samples (l.OuL) were injected into the Agilent/Gerstel 7890B GC System, where the GC inlet was set to 250C with a split ratio of 2:1. The capillary column was an Agilent DB-5MS (20mx0.18 mmx0.18pm) with carrier gas (helium) flow set to 1.5ml/min. The GC oven temperature was set to 100°C (hold for 0.10 min) with a ramp of 40°C/min to 155°C, where the ramp was then 15°C/min to 190°C and then finally the ramp was changed to 75°C/min to 280C (5-minute method). For a more comprehensive analysis of targets, the GC oven

temperature was set to 100°C (hold for 2.0 min) with a 10°C/min ramp to 250°C (hold for 2.0 min) was utilized (20-minute method). The MS source and quadruple for both methods were set to 230°C and 180°C on the Agilent 5977B MSD (Etune), respectively. The mass scan range was set to 40-250 mz where spectra and linear retention index calculations were matched against the NIST MS database (2008 version), in addition to available standards and essential oils.

Peaks present in the extracted ion chromatogram (204.2mz parent mass) were identified in one of six ways (see Table 3). The authentic standards utilized in this screen for verification of products were beta-caryophyllene (Sigma- Aldrich catalog # W225207-SAMPLE-K), beta- farnesene (Sigma- Aldrich catalog # 73492- 1ML-F), trans-nerolidol (Sigma- Aldrich catalog # 18143-100MG-F), and alpha-humulene (Sigma-Aldrich catalog # 53675-1ML). Sesquitperene rich essential oils used to aid structure identification were derived from the following plants: Rhodendron, Sweet Basil, Black Pepper, Citronella, Ylang, Balsam copaiba, and Patchouli. AHO( YDL227C)::2xP(gal)-//7/V/G7 integration on chromosome 4.

AGGGTTCGCAAGTCCTGTTTCTATGCCTTTCTCTTAGTAATTCACGAAATAAACCT ATGGTTTACGAAATGATCCACGAAAATCATGTTATTATTTACATCAACATATCGCG AAAATTCATGTCATGTCCACATTAACATCATTGCAGAGCAACAATTCATTTTCATAG AGAAATTTGCTACTATCACCCACTAGTACTACCATTGGTACCTACTACTTTGAATTG TACTACCGCTGGGCGTTATTAGGTGTGAAACCACGAAAAGTTCACCATAACTTCGA ATAAAGTCGCGGAAAAAAGTAAACAGCTATTGCTACTCAAATGAGGTTTGCAGAAG CTTGTTGAAGCATGATGAAGCGTTCTAAACGCACTATTCATCATTAAATATTTAAA GCTCATAAAATTGTATTCAATTCCTATTCTAAATGGCTTTTATTTCTATTACAACTA TTAGCTCGATGCACGAGCGCAACGCTCACAACGCTCGTCCAACGCCGGCGGACCTACG GATTAGAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTC G TCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAAT AA AGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCC CACA AACCTT CAAA T GAA C GAA T CAAA TTAA CAACCA TA GGA T GA TAA TGC GA TTA GTTTTTTA GCCTT AA CTGCA TAA C CA CTTTAACTAA TA CTTT CAA CA TTTT C GGTTT GTA TTACTTCTTA TT CAAA TGT AATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAA AACTA

TAATGGCTGCAGACCAATTGGTGAAGACTGAAGTCACCAAGAAGTCTTTTACTGCT CCTGTACAAAAGGCTTCTACACCAGTTTTAACCAATAAAACAGTCATTTCTGGATC GAAAGTCAAAAGTTTATCATCTGCGCAATCGAGCTCATCAGGACCTTCATCATCTA GTGAGGAAGATGATTCCCGCGATATTGAAAGCTTGGATAAGAAAATACGTCCTTTA GAAGAATTAGAAGCATTATTAAGTAGTGGAAATACAAAACAATTGAAGAACAAAGA GGTCGCTGCCTTGGTTATTCACGGTAAGTTACCTTTGTACGCTTTGGAGAAAAAAT TAGGTGATACTACGAGAGCGGTTGCGGTACGTAGGAAGGCTCTTTCAATTTTGGC AGAAGCTCCTGTATTAGCATCTGATCGTTTACCATATAAAAATTATGACTACGACC GCGTATTTGGCGCTTGTTGTGAAAATGTTATAGGTTACATGCCTTTGCCCGTTGGT GTTATAGGCCCCTTGGTTATCGATGGTACATCTTATCATATACCAATGGCAACTAC AGAGGGTTGTTTGGTAGCTTCTGCCATGCGTGGCTGTAAGGCAATCAATGCTGGC GGTGGTGCAACAACTGTTTTAACTAAGGATGGTATGACAAGAGGCCCAGTAGTCC GTTTCCCAACTTTGAAAAGATCTGGTGCCTGTAAGATATGGTTAGACTCAGAAGAG GGACAAAACGCAATTAAAAAAGCTTTTAACTCTACATCAAGATTTGCACGTCTGCA ACATATTCAAACTTGTCTAGCAGGAGATTTACTCTTCATGAGATTTAGAACAACTA CTGGTGACGCAATGGGTATGAATATGATTTCTAAGGGTGTCGAATACTCATTAAAG CAAATGGTAGAAGAGTATGGCTGGGAAGATATGGAGGTTGTCTCCGTTTCTGGTA ACTACTGTACCGACAAAAAACCAGCTGCCATCAACTGGATCGAAGGTCGTGGTAA GAGTGTCGTCGCAGAAGCTACTATTCCTGGTGATGTTGTCAGAAAAGTGTTAAAAA GTGATGTTTCCGCATTGGTTGAGTTGAACATTGCTAAGAATTTGGTTGGATCTGCA ATGGCTGGGTCTGTTGGTGGATTTAACGCACATGCAGCTAATTTAGTGACAGCTGT TTTCTTGGCATTAGGACAAGATCCTGCACAAAATGTCGAAAGTTCCAACTGTATAA CATTGATGAAAGAAGTGGACGGTGATTTGAGAATTTCCGTATCCATGCCATCCATC GAAGTAGGTACCATCGGTGGTGGTACTGTTCTAGAACCACAAGGTGCCATGTTGG ACTTATTAGGTGTAAGAGGCCCACATGCTACCGCTCCTGGTACCAACGCACGTCAA TTAGCAAGAATAGTTGCCTGTGCCGTCTTGGCAGGTGAATTATCCTTATGTGCTGC CCTAGCAGCCGGCCATTTGGTTCAAAGTTATATGACCCACAACAGGAAACCTGCTG AACCAACAAAACCTAACAATTTGGACGCCACTGATATAAATCGTTTGAAAGATGGG TCCGTCACCTGCATTAAATCCTAAGCTAGCTAAGA TCCGCTCTAA CCGAAAAGGAAGG AGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAG AA CGTTA TTTA TA TTTCAAA TTTTTCTTTTTTTTCTGTA CA GA CGCGTGTA CGCA TGTAA CA TT A TA CTGAAAA CCTTGCTTGA GAAGGTTTTGGGA CGCTCGAA GA TCCA GCTCGGCCGT ACG A A A ATC GTT ATT GT CTTG A AGGT G A A ATTT CT ACT CTT ATT A AT GGT G A AC GTT A AGCT G ATGCTATGATGGAAGCTGATTGGTCTTAACTTGCTTGTCATCTTGCTAATGGTCATATGG CTCGTGTTATTACTTAAGTTATTTGTACTCGTTTTGAACGTAATGCTAATGATCATCTTA T GGAATAATAGTGAACGGCCG agctggatcttcgagcgtcccaaaaccttctcaagcaaggttttcagtataatgtacatg c gtacacgcgtctgtacagaaaaaaaagaaaaatttgaaatataaataacgttcttaatac taacataactataaaaaaataaatagggacct agacttcaggttgtctaactccttccttttcggttagagcggatcfl AGCT AGCttaggatttaatgcaggtgacggacccatctttcaaa cgatttatatcagtggcgtccaaattgttaggttttgttggttcagcaggtttcctgttg tgggtcatataactttgaaccaaatggccggctg ctagggcagcacataaggataattcacctgccaagacggcacaggcaactattcttgcta attgacgtgcgttggtaccaggagcggta gcatgtgggcctcttacacctaataagtccaacatggcaccttgtggttctagaacagta ccaccaccgatggtacctacttcgatggatg gcatggatacggaaattctcaaatcaccgtccacttctttcatcaatgttatacagttgg aactttcgacattttgtgcaggatcttgtcctaa tgccaagaaaacagctgtcactaaattagctgcatgtgcgttaaatccaccaacagaccc agccattgcagatccaaccaaattcttagc aatgttcaactcaaccaatgcggaaacatcactttttaacacttttctgacaacatcacc aggaatagtagcttctgcgacgacactcttac cacgaccttcgatccagttgatggcagctggttttttgtcggtacagtagttaccagaaa cggagacaacctccatatcttcccagccatac tcttctaccatttgctttaatgagtattcgacacccttagaaatcatattcatacccatt gcgtcaccagtagttgttctaaatctcatgaaga gtaaatctcctgctagacaagtttgaatatgttgcagacgtgcaaatcttgatgtagagt taaaagcttttttaattgcgttttgtccctcttct gagtctaaccatatcttacaggcaccagatcttttcaaagttgggaaacggactactggg cctcttgtcataccatccttagttaaaacagt tgttgcaccaccgccagcattgattgccttacagccacgcatggcagaagctaccaaaca accctctgtagttgccattggtatatgataa gatgtaccatcgataaccaaggggcctataacaccaacgggcaaaggcatgtaacctata acattttcacaacaagcgccaaatacgc ggtcgtagtcataatttttatatggtaaacgatcagatgctaatacaggagcttctgcca aaattgaaagagccttcctacgtaccgcaac cgctctcgtagtatcacctaattttttctccaaagcgtacaaaggtaacttaccgtgaat aaccaaggcagcgacctctttgttcttcaattg ttttgtatttccactacttaataatgcttctaattcttctaaaggacgtattttcttatc caagctttcaatatcgcgggaatcatcttcctcact agatgatgaaggtcctgatgagctcgattgcgcagatgataaacttttgactttcgatcc agaaatgactgttttattggttaaaactggtg tagaagccttttgtacaggagcagtaaaagacttcttggtgacttcagtcttcaccaatt ggtctgcagccatTAT asttttttctccttsac gttaaagtatagaggtatattaacaattttttgttgatacttttattacatttgaataag aagtaatacaaaccgaaaatgttgaaagtattagttaa agtggttatgcagtttttgcatttatatatctgttaatagatcaaaaatcatcgcttcgc tgattaattaccccagaaataaggctaaaaaactaat cgcattatcatcctatggttgttaatttgattcgttcatttgaaggtttgtggggccagg ttactgccaatttttcctcttcataaccataaaagctagt attgtagaatctttattgttcggagcagtgcggcgcgaggcacatctgcgtttcaggaac gcgaccggtgaagacgaggacgcacggagga gagtcttccttcggagggctgtcacccgctcggcggcttctaatccgt/ GGLCCGCCGGCGLLGG/ CG/ GCGLLGLG

AGCGTTGCGCTCGTGCATCaatgtgtatattagtttaaaaagttgtatgtaataaaa gtaaaatttaatattttggatgaaa aaaaccatttttagactttttcttaactagaatgctggagtagaaatacgccatctcaag atacaaaaagcgttaccggcactgatttgttt caaccagtatatagattattattgggtcttgatcaactttcctcagacatatcagtaaca gttatcaagctaaatatttacgcgaaagaaaa acaaatattttaattgtgatacttgtgaattttattttattaaggatacaaagttaagag aaaacaaaatttatatacaatataagtaatatt catatatatgtgatgaatgcagtcttaacgagaagacatggccttggtgacaactctctt caaaccaacttcagcctttctcaattcatcag cagatgggtcttcgatttgcaaagcagcca (SEQ ID NO: 53)

Upper case, bold: HO upstream homology sequence (SEQ ID NO: 56)

Upper case, italicized and underlined: P(gall) (SEQ ID NO: 57)

Upper case, underlined and bold: tHMGl (SEQ ID NO: 58)

Upper case, bold and italicized : CYC 1 terminator (SEQ ID NO: 59)

Lower case, bold and italicized : CYC 1 terminator, reverse complement (SEQ ID NO: 60)

Uower case, underlined and bold: tHMGl , reverse complement (SEQ ID NO: 61)

Lower case, italicized and underlined: P(gall), reverse complement (SEQ ID NO: 62)

Uower case, bold: HO downstream homology sequence (SEQ ID NO: 63)

P(met3) integration upstream of Erg9 with flanking genes included.

ATGTCCGGTAAATGGAGACTAGTGCTGACTGGGATAGGCAATCCAGAGCCTCAGT

ACGCTGGCACCCGTCACAATGTAGGGCTATATATGCTGGAGCTGCTACGAAAGCG

GCTTGGTCTGCAGGGGAGAACCTATTCCCCTGTGCCTAATACGGGCGGCAAAGTG

CATTATATAGAAGACGAACATTGTACGATACTAAGATCGGATGGCCAGTACATGAA

TCTAAGTGGAGAACAGGTGTGCAAGGTCTGGGCCCGGTACGCCAAGTACCAAGCC

CGACACGTTGTTATTCATGACGAGTTAAGTGTGGCGTGTGGAAAAGTGCAGCTCA

GAGCCCCCAGCACCAGTATTAGAGGTCATAATGGGCTGCGAAGTCTACTGAAATG

CTCCGGAGGCCGTGTACCCTTTGCCAAATTGGCTATTGGAATCGGCAGAGAACCT

GGGTCCCGCTCTAGAGACCCTGCGAGCGTCTCCCGCTGGGTTCTGGGAGCTCTAA

CTCCGCAGGAACTACAAACCTTGCTTACACAGAGTGAACCTGCTGCCTGGCGTGCT

CTGACTCAGTACATTTCATAGGTTTAACTTGATACTACTAGATTTTTTCTCTTCATT TAT A A A ATTTTT GGTT AT A ATT G A AGCTTT AG A AGT AT G A A A A A ATCCTTTTTTTTC ATT CTTT GCAACCAAAATAAGAAGCTTCTTTTATTCATTGAAATGATGAATATAAACCTAACAAAA GAAAAAGACTCGAATATCAAACATTAAAAAAAAATAAAAGAGGTTATCTGTTTTCCCAT TTAGTTGGAGTTTGCATTTTCTAATAGATAGAACTCTCAATTAATGTGGATTTAGTTTCT CTGTTCGTTTTTTTTTGTTTTGTTCTCACTGTATTTACATTTCTATTTAGTATTTAGTTA TT

CATATAATCTTAACTTCTCGAGGAGCTCGATCTTGAAACTGAGTAAGATGCTCAGAA TA

CCCGTCAAGATAAGAGTATAATGTAGAGTAATATACCAAGTATTCAGCATATTCTCC TC

TT CTTTTGT AT A A AT C AC GG A AGGG AT G ATTT AT A AG A A A A AT G A AT ACT ATT AC ACTT

CATTTACCACCCTCTGATCTAGATTTTCCAACGATATGTACGTAGTGGTATAAGGTG AGG

GGGTCCACAGATATAACATCGTTTAATTTAGTACTAACAGAGACTTTTGTCACAACT AC

ATATAAGTGTACAAATATAGTACAGATATGACACACTTGTAGCGCCAACGCGCATCC TA

CGGATTGCTGACAGAAAAAAAGGTCACGTGACCAGAAAAGTCACGTGTAATTTTGTA A

CTCACCGCATTCTAGCGGTCCCTGTCGTGCACACTGCACTCAACACCATAAACCTTA GC

AACCTCCAAAGGAAATCACCGTATAACAAAGCCACAGTTTTACAACTTAGTCTCTTA TG

AAGTGTCTCTCTCTGTCGTAACAGTTGTGATATCGGAAGAAGAGAAAAGACGAAGAG C AGAAGCGGAAAACGTATACACGTCACATATCACACACACACAatgggaaagctattacaa ttggcat tgcatccggtcgagatgaaggcagctttgaagctgaagttttgcagaacaccgctattct ccatctatgatcagtccacgtctccatatctc ttgcactgtttcgaactgttgaacttgacctccagatcgtttgctgctgtgatcagagag ctgcatccagaattgagaaactgtgttactctc ttttatttgattttaagggctttggataccatcgaagacgatatgtccatcgaacacgat ttgaaaattgacttgttgcgtcacttccacgag aaattgttgttaactaaatggagtttcgacggaaatgcccccgatgtgaaggacagagcc gttttgacagatttcgaatcgattcttattga attccacaaattgaaaccagaatatcaagaagtcatcaaggagatcaccgagaaaatggg taatggtatggccgactacatcttagatg aaaattacaacttgaatgggttgcaaaccgtccacgactacgacgtgtactgtcactacg tagctggtttggtcggtgatggtttgacccgt ttgattgtcattgccaagtttgccaacgaatctttgtattctaatgagcaattgtatgaa agcatgggtcttttcctacaaaaaaccaacatc atcagagattacaatgaagatttggtcgatggtagatccttctggcccaaggaaatctgg tcacaatacgctcctcagttgaaggacttca tgaaacctgaaaacgaacaactggggttggactgtataaaccacctcgtcttaaacgcat tgagtcatgttatcgatgtgttgacttatttg gccggtatccacgagcaatccactttccaattttgtgccattccccaagttatggccatt gcaaccttggctttggtattcaacaaccgtgaa gtgctacatggcaatgtaaagattcgtaagggtactacctgctatttaattttgaaatca aggactttgcgtggctgtgtcgagatttttgac tattacttacgtgatatcaaatctaaattggctgtgcaagatccaaatttcttaaaattg aacattcaaatctccaagatcgaacagtttatg gaagaaatgtaccaggataaattacctcctaacgtgaagccaaatgaaactccaattttc ttgaaagttaaagaaagatccagatacga tgatgaattggttccaacccaacaagaagaagagtacaagttcaatatggttttatctat catcttgtccgttcttcttgggttttattatatat acactttacacagagcgtga (SEQ ID NO: 54)

Uppercase, bold and underlined: Upstream sequence PTH1 (YHR18S 64)

Uppercase and underlined: P(met3) (SEQ ID NO: 65)

Lowercase, bold and underlined: Erg9 (YHR190W) (SEQ ID NO: 66)

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All references, including patent documents, disclosed herein are incorporated by reference in their entirety, particularly for the disclosure referenced herein.