Enzymatic synthesis of kavalactones and flavokavains

Abstract

Disclosed are methods, compositions, proteins, nucleic acids, cells, vectors, compounds, reagents, and systems for the preparation of kavalactones, flavokavains, and kavalactone and flavokavain biosynthetic intermediates using enzymes expressed in heterologous host cells, such as microorganisms or plants, or using in vitro enzymatic reactions. This invention also provides for the expression of the enzymes by recombinant cell lines and vectors. Furthermore, the enzymes can be components of constructs such as fusion proteins. The kavalactones produced can be utilized to treat anxiety disorder, insomnia, and other psychological and neurological disorders. The flavokavains produced can be utilized to treat various cancers including colon, bladder, and breast cancers.

Claims

1. A method for producing a compound of Formula (V), the method comprising: reducing a compound of Formula (IV), or a salt thereof, with a reducing agent using an enzyme having an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of PmRDCT10 polypeptide of SEQ ID NO: 8 to produce a compound of Formula (V), or a salt thereof: ##STR00050## wherein: is a single bond or a double bond; each of R.sub.1, R.sub.2, R.sub.3, R.sub.6, and R.sub.7 independently is hydrogen, substituted or unsubstituted, cyclic or acyclic aliphatic, or —ORx, or R.sub.1 and R.sub.2 are combined to form a ring, or R.sub.2 and R.sub.3 are combined to form a ring, wherein Rx is hydrogen or substituted or unsubstituted, cyclic or acyclic aliphatic; each of R.sub.4 and R.sub.5 independently is hydrogen or substituted or unsubstituted, cyclic or acyclic aliphatic; and R.sub.8 is hydrogen or substituted or unsubstituted, cyclic or acyclic aliphatic.

2. The method of claim 1, wherein is a single bond.

3. The method of claim 1, wherein is a double bond.

4. The method of claim 1, wherein each of R.sub.1, R.sub.2, and R.sub.3 is independently selected from the group consisting of hydrogen, —OH, and —OCH.sub.3.

5. The method of claim 1, wherein R.sub.4 and R.sub.5 are hydrogen.

6. The method of claim 1, wherein R.sub.1, R.sub.2, and R.sub.3 are hydrogen.

7. The method of claim 6, wherein each of R.sub.6 and R.sub.7 is independently selected from the group consisting of hydrogen, OH, and —OCH.sub.3.

8. The method of claim 7, wherein each of R.sub.6 and R.sub.7 is hydrogen.

9. The method of claim 8, wherein R.sub.8 is —CH.sub.3 or hydrogen.

10. The method of claim 1, wherein the reducing agent is NADPH or NADH.

11. The method of claim 1, wherein the enzyme is a purified enzyme or a partially purified enzyme, and the method is performed in vitro.

12. The method of claim 1, wherein the enzyme is in a cell, and the enzyme is heterologous to the cell containing the enzyme, and the method is performed in the cell.

13. The method of claim 1, wherein the method further comprises, prior to the step of reducing a compound of Formula (IV), or a salt thereof, with a reducing agent, the steps of: condensing a compound of Formula (I), or a salt thereof, with coenzyme A (CoA) using an enzyme having an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of Pm4CL1 polypeptide of SEQ ID NO: 1 (Pm4CL1) to produce a compound of Formula (II), or a salt thereof: ##STR00051## reacting a compound of Formula (II), or a salt thereof, with malonyl-CoA using an enzyme having an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of PmSPS1 polypeptide of SEQ ID NO: 2 or an enzyme that is at least 90% sequence identical to the amino acid sequence of PmSPS2 polypeptide of SEQ ID NO: 3 to produce a compound of Formula (III), or a salt thereof: ##STR00052##  and reacting the compound of Formula (III), or a salt thereof, with an enzyme having an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of PmOMT1 polypeptide of SEQ ID NO: 6 to produce a compound of Formula (IV), or a salt thereof, in which R.sub.8 is hydrogen.

14. The method of claim 13, wherein the method is performed in a cell comprising an enzyme having an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of Pm4CL1 polypeptide of SEQ ID NO: 1; an enzyme having an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of PmSPS1 polypeptide of SEQ ID NO: 2 (PmSPS1) or an enzyme having an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of PmSPS2 polypeptide of SEQ ID NO: 3; an enzyme that is at least 90% sequence identical to the amino acid sequence of PmOMT1 polypeptide of SEQ ID NO: 6; and an enzyme that is at least 90% sequence identical to the amino acid sequence of PmRDCT10 polypeptide of SEQ ID NO: 8; wherein the enzymes are heterologous to the cell.

15. The method of claim 14, wherein the cell is a yeast cell or a bacterial cell.

16. The method of claim 15, wherein the method produces a compound of Formula (V), or a salt thereof, in which is a single bond or double bond; each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7, is hydrogen; and R.sub.8 is hydrogen or —CH.sub.3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings, which constitute a part of this specification, illustrate several exemplary embodiments of the invention and together with the description, serve to explain certain principles of the invention. The embodiments disclosed in the drawings are exemplary and do not limit the scope of this disclosure.

(2) FIG. 1 shows the chemical structures of twenty known kavalactones.

(3) FIG. 2 shows the chemical structures of three known flavokavains.

(4) FIG. 3 shows the biosynthetic pathway of kavalactones and flavokavains.

(5) FIG. 4 shows the in vitro enzymatic production of bisnoryangonin (kavalactone intermediate with 6-styryl-4-hydroxy-2-pyrone backbone) and naringenin chalcone (flavokavain intermediate with chalcone backbone) using purified recombinant PmSPS1, PmSPS2, and PmCHS enzymes from p-coumaric acid. CTAL (p-coumaroyltriacetic acid lactone is a known in vitro derailment byproduct of chalcone synthase (CHS), which is not produced in vivo.

(6) FIG. 5 shows the enzymatic production of compounds with a 6-styryl-4-hydroxy-2-pyrone backbone from carboxylic acid compounds with single bond or double bond at the 7,8-position: p-coumaric acid, phloretic acid, and hydrocinnamic acid.

(7) FIG. 6 shows the in vitro enzymatic production starting from phloretic acid and hydrocinnamic acid of compounds with a 6-styryl-4-hydroxy-2-pyrone backbone and single bond at the 7,8-position.

(8) FIG. 7 shows the positions of hydroxyl groups on the 6-styryl-4-hydroxy-2-pyrone backbone that can be methylated by PmOMT4 and PmOMT1.

(9) FIG. 8 shows liquid chromatography-mass spectrometry (LC-MS) results (m/z at retention time) of final products of enzymatic processes using different substrates and a combination of PmCL1, PmSPS1, and two methyltransferases PmOMT4 and PmOMT1.

(10) FIG. 9 shows the results of an in vitro enzyme assay demonstrating the activity of PmRDCT10 to reduce the C.sub.5-C.sub.6 double bond in kavalactones. While the combination of Pm4CL1, PmSPS1, and PmOMT4 is sufficient to produce the kavalactone desmethoxyyangonin from cinnamic acid, PmRDCT10 is required to produce kavain, which carries a single bond at the C.sub.5-C.sub.6 position. The identity of desmethoxyyangonin and kavain was confirmed with pure standards, including their retention times and utilizing tandem mass spectrometry (MS/MS) as shown in the bottom panel.

(11) FIG. 10 shows the pathway to produce methylenedioxy bridge-containing kavalactones such as 5,6-dehydromethysticin starting from caffeic acid.

(12) FIG. 11 shows the LC-MS traces of mass 273.075 m/z corresponding to [C.sub.15H.sub.12C.sub.5+H].sup.+ ion of 5,6-dehydromethysticin in Agrobacterium-infiltrated N. benthamiana leaves. Each leaf was infiltrated with a mixture of agrobacterial strains carrying plasmids with the indicated enzymes.

(13) FIG. 12 shows the production of bisnoryangonin in vivo in E. coli. The E. coli BW27784 strain carrying expression plasmids with the indicated enzymes was incubated for 24 hours in the presence of 1 mM p-coumaric acid.

(14) FIG. 13 shows the production of bisnoryangonin and naringenin chalcone in vivo in the baker's yeast S. cerevisiae. The yeast strain BY4743 carrying expression plasmids with the indicated enzymes was incubated for 2 days in the presence of 2 mM p-coumaric acid.

(15) FIG. 14 shows the production of the kavalactone yangonin in vivo in the plant Nicotiana benthamiana through Agrobacterium-mediated infiltration. This assay utilized the native Nicotiana 4CL enzyme.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

(16) The present disclosure provides methods, compositions, proteins, nucleic acids, cells, vectors, compounds, reagents, and systems for the production of kavalactones, flavokavains, and intermediates thereto. Described herein are the biosynthetic pathways and enzymes useful for the conversion of cinnamic acid derivatives and phenylpropanoic acid derivatives to kavalactones and flavokavains in a series of in vivo and/or in vitro enzymatic reactions. The enzymatic synthesis of kavalactones utilizes 4-coumarate-CoA ligase Pm4CL1 (or an enzyme that is at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (or an enzyme that is at least 80% identical to SEQ ID NO: 2) or styrylpyrone synthase PmSPS2 (or an enzyme that is at least 80% identical to SEQ ID NO: 3), methyltransferase PmOMT4 (or an enzyme that is at least 80% identical to SEQ ID NO: 5), and any number of methyltranferases (e.g., PmOMT1 (or an enzyme at least 80% identical to SEQ ID NO: 6)), cytochrome P450 enzymes (e.g., PmMDB1 (or an enzyme at least 80% identical to SEQ ID NO: 7)), and/or NADPH-dependent reductases (e.g., PmRDCT10 (or an enzyme at least 80% identical to SEQ ED NO: 8)). The enzymatic synthesis of flavokavains utilizes at least one 4-coumarate-CoA ligase Pm4CL1 (or an enzyme that is at least 80% identical to SEQ ID NO: 1), chalcone synthase PmCHS (or an enzyme that is at least 80% identical to SEQ ID NO: 4), methyltranferase PmOMT4 (or an enzyme that is at least 80% identical to SEQ ID NO: 5, and any number of methyltranferases (e.g., PmOMT1 (or an enzyme at least 80% identical to SEQ ID NO: 6)) and cytochrome P450 enzyme PmMDB1 (or an enzyme at least 80% identical to SEQ ID NO: 7). Any of the methods to produce compounds described herein can optionally utilize chemical means or a combination of reactions utilizing enzymes described herein and chemical means.

(17) Any of the methods described herein may include culturing cells or cultivating plants expressing enzymes described herein and isolating one or more compounds described herein from such cells or plants. Methods described herein can include harvesting tissue (e.g., leaves, roots) of a plant expressing enzymes described herein and processing the harvested tissue to isolate one or more compounds described herein therefrom. Compounds may be isolated using solvent extraction, chromatography, and/or other separation methods known in the art.

(18) Any of the enzymatic individual steps may be combined, omitted, or done through other means and still be within the scope of the invention.

(19) Enzymes and cDNA

(20) Sequence identity is the amount of characters which match exactly between two different sequences. Hereby, gaps are not counted and the measurement is relational to the shorter of the two sequences. This has the effect that sequence identity is not transitive, i.e. if sequence A=B and B=C then A is not necessarily equal C (in terms of the identity distance measure): A: AAGGCTT; B: AAGGC; C: AAGGCAT. Here identity(A,B)=100% (5 identical nucleotides/min(length(A),length(B))). Identity(B,C)=100%, but identity(A,C)=85% ((6 identical nucleotides/7)). So 100% identity does not mean two sequences are the same. Sequence identity can be applied to polypeptides and polynucleotide. For example, the phrase an enzyme “that is at least Y % identical to SEQ ID NO: X”, can be understood to apply the description above for comparing amino acid sequences to determine that an enzyme is at least 80% identical to a enzyme with a SEQ ID NO described herein.

(21) In certain embodiments, the enzyme (polypeptide) or DNA (polynucleotide) is a variant of a natural or artificial enzyme or DNA. A “variant” of a particular polypeptide or polynucleotide has one or more additions, substitutions, and/or deletions with respect to the polypeptide or polynucleotide, which may be referred to as the “original polypeptide” or “original polynucleotide”, respectively. An addition may be an insertion or may be at either terminus. A variant may be shorter or longer than the original polypeptide or polynucleotide. The term “variant” encompasses “fragments”. A “fragment” is a continuous portion of a polypeptide or polynucleotide that is shorter than the original polypeptide or polynucleotide. In some embodiments a variant comprises or consists of a fragment. In some embodiments a fragment or variant is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more as long as the original polypeptide or polynucleotide. A fragment may be an N-terminal, C-terminal, or internal fragment. In some embodiments a variant polypeptide comprises or consists of at least one domain of an original polypeptide. In some embodiments a variant polypeptide or polynucleotide comprises or consists of a polypeptide or polynucleotide that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical in sequence to the original polypeptide or polynucleotide over at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the original polypeptide or polynucleotide. In some embodiments the sequence of a variant polypeptide comprises or consists of a sequence that has N amino acid differences with respect to an original sequence, wherein N is any integer up to 1%, 2%, 5%, or 10% of the number of amino acids in the original polypeptide, where an “amino acid difference” refers to a substitution, insertion, or deletion of an amino acid. In some embodiments a substitution is a conservative substitution. Conservative substitutions may be made, e.g., on the basis of similarity in side chain size, polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved. In some embodiments, conservative substitutions may be made according to Table A, wherein amino acids in the same block in the second column and in the same line in the third column may be substituted for one another other in a conservative substitution. Certain conservative substitutions are substituting an amino acid in one row of the third column corresponding to a block in the second column with an amino acid from another row of the third column within the same block in the second column.

(22) TABLE-US-00001 TABLE A Aliphatic Non-polar G A P I L V Polar - uncharged C S T M N Q Polar - charged D E K R Aromatic H F W Y

(23) In some embodiments, proline (P), cysteine (C), or both are each considered to be in an individual group. Within a particular group, certain substitutions may be of particular interest in certain embodiments, e.g., replacements of leucine by isoleucine (or vice versa), serine by threonine (or vice versa), or alanine by glycine (or vice versa).

(24) In some embodiments a variant is a biologically active variant, i.e., the variant at least in part retains at least one activity of the original polypeptide or polynucleotide. In some embodiments a variant at least in part retains more than one or substantially all known biologically significant activities of the original polypeptide or polynucleotide. An activity may be, e.g., a catalytic activity, binding activity, ability to perform or participate in a biological structure or process, etc. In some embodiments an activity of a variant may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, of the activity of the original polypeptide or polynucleotide, up to approximately 100%, approximately 125%, or approximately 150% of the activity of the original polypeptide or polynucleotide, in various embodiments. In some embodiments a variant, e.g., a biologically active variant, comprises or consists of a polypeptide at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to an original polypeptide over at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 100% of the original polypeptide. In some embodiments an alteration, e.g., a substitution or deletion, e.g., in a functional variant, does not alter or delete an amino acid or nucleotide that is known or predicted to be important for an activity, e.g., a known or predicted catalytic residue or residue involved in binding a substrate or cofactor. Variants may be tested in one or more suitable assays to assess activity.

(25) Each amino acid in the enzyme sequence can be encoded by multiple DNA codons. Therefore, there are many possible cDNA sequences that will translated to the same amino acid sequences described herein and produce the same enzyme. Examples of cDNA sequences that encode the enzymes described herein are shown in Example 6.

(26) In certain embodiments, an enzyme of the present disclosure is at least 80%, 85%, 90%, 95%, or 100% identical to an enzyme with a SEQ ID NO described herein. In certain embodiments, an enzyme of the present disclosure is greater than 95%, 96%, 97%, 98%, or 99% identical to an enzyme with a SEQ ID NO described herein.

(27) The enzymes described herein (e.g., 4-coumarate-CoA ligase Pm4CL1 or an enzyme that is at least 80% identical to SEQ ID NO: 1, styrylpyrone synthase PmSPS1 or an enzyme that is at least 80% identical to SEQ ID NO: 2, styrylpyrone synthase PmSPS2 or an enzyme that is at least 80% identical to SEQ ID NO: 3, chalcone synthase PmCHS or an enzyme that is at least 80% identical to SEQ ID NO: 4, methyltransferase PmOMT4 or an enzyme that is at least 80% identical to SEQ ID NO: 5, methyltransferase PmOMT1 or an enzyme that is at least 80% identical to SEQ ID NO: 6, cytochrome P450 enzyme PmMDB1 or an enzyme that is at least 80% identical to SEQ ID NO: 7, and NADPH-dependent reductase PmRDCT10 or an enzyme that is at least 80% identical to SEQ ID NO: 8) are isolated or derived from wildtype, mutant or recombinant Piper methysticum. In certain embodiments, the enzymes are produced using recombinant technology. In certain embodiments, an acid-thiol ligase (EC 6.2.1.A) is used to form carbon-sulfur bonds, wherein A is an integer between 1 and 100, inclusive. The acid-thiol ligase is isolated or derived from wildtype, mutant, or recombinant Piper methysticum. The acid-thiol ligase can be used as an unpurified enzyme, partially purified enzyme, or purified enzyme. In certain embodiments, the amino acid sequence of the enzyme is at least 80% identical to SEQ ID NO: 1, designated 4-coumarate-CoA ligase PmCL1, wherein SEQ ID NO: 1 is MKMVVDTIATDRCVYRSKLPDIEIKNDMSLHNYCFQNIGAYRDNPCLINGSTGEVYTYG EVETTARRVAAGLHRMGVQQREVIMILLPNSPEFVFAFLGASFRGAMSTTANPFYTPQEI AKQVKASGAKLIVTMSAYVDKVRDLAEERGVKVVCVDAPPPGCSHFSELSGADESELP EVDIDPDDVVALPYSSGTTGLPKGVMLTHRSQVTSVAQQVDGENPNLYFRPDDVLLCV LPLFHIYSLNSVLFCGLRVGAAILIMQKFEITALMELVQKYKVTIAPIVPPIVLAIAKSPLV DKYDLSSIRTVMSGAAPMGKELEDAVRAKLPNAKLGQGYGMTEAGPVLSMCLAFAKE PFEIKSGSCGTVVRNAQLKIVDPETGAYLPRNQPGEICIRGSQIMKGYLNDAAATQRTID KEGWLHTGDIGYVDDDEELFIVDRLKEIIKYKGFQVAPAELEAILITHPNIADAAVVPMK DEAAGEVPVAFVVTSNGSVISEDEIKQFISKQVVFYKRINRVFFVDSIPKAPSGKILRKDL RGRLAAGIPK. In certain embodiments, the acid-thiol ligase is 4-coumarate-CoA ligase (EC 6.2.1.12).

(28) In certain embodiments, a transferase or synthase (EC 2.3.1.B) is used to form styrylpyrones from coenzyme A esters of cinnamic acids, wherein B is an integer between 1 and 300, inclusive. In certain embodiments, a transferase or synthase (EC 2.3.1.B) is used to form styrylpyrones from coenzyme A esters of cinnamic acids, wherein B is an integer between 1 and 300, inclusive. The transferase or synthase is isolated or derived from wildtype, mutant, or recombinant Piper methysticum. The transferase or synthase can be used as an unpurified enzyme, partially purified enzyme, or purified enzyme. In certain embodiments, the synthase belongs to an enzyme family of type III polyketide synthases. In certain embodiments, the amino acid sequence of the enzyme is at least 80% identical to SEQ ID NO: 2, designated styrylpyrone synthase PmSPS1, wherein SEQ ID NO: 2 is MSKTVEDRAAQRAKGPATVLAIGTATPANV VYQTDYPDYYFRVTKSEHMTKLKNKFQRMCDRSTIKKRYMVLTEELLEKNLSLCTYME PSLDARQDILVPEVPKLGKEAADEAIAEWGRPKSEITHLIFCTTCGVDMPGADYQLTKLL GLRSSVRRTMLYQQGCFGGGTVLRLAKDLAENNAGARVLVVCSEITTAVNFRGPSDTH LDLLVGLALFGDGAAAVIVGADPDPTLERPLFQIVSGAQTILPDSEGAINGHLREVGLTIR LLKDVPGLVSMNIEKCLMEAFAPMGIHDWNSIFWIAHPGGPTILDQVEAKLGLKEEKLK STRAVLREYGNMSSACVLFILDEVRKRSMEEGKTTTGEGFDWGVLFGFGPGFTVETVVL HSMPIPKADEGR. In certain embodiments, the amino acid sequence of the enzyme is at least 80% identical to SEQ ID NO: 3, designated styrylpyrone synthase PmSPS2, wherein SEQ ID NO: 3 is MSKMVEEHWAAQRARGPATVLAIGTANPPNVLYQADYPDFYFRVTKSEHMT QLKEKFKRICDKSAIRKRHLHLTEELLEKNPNICAHMAPSLDARQDIAVVEVPKLAKEA ATKAIKEWGRPKSDITHLIFCTTCGVDMPGADYQLTTLLGLRPTVRRTMLYQQGCFAGG TVLRHAKDFAENNRGARVLAVCSEFTVMNFSGPSEAHLDSMVGMALFGDGASAVIVG ADPDFAIERPLFQLVSTTQTIVPDSDGAIKCHLKEVGLTLHLVKNVPDLISNNMDKILEEA FAPLGIRDWNSIFWTAHPGGAAILDQLEAKLGLNKEKLKTTRTVLREYGNMSSACVCFV LDEMRRSSLEEGKTTSGEGLEWGILLGFGPGLTVETVVLRSVPISTAN. In certain embodiments, the amino acid sequence of the enzyme is at least 80% identical to SEQ ID NO: 4, designated chalcone synthase PmCHS, wherein SEQ ID NO: 4 is MSKTVEEIWAAQRARGPA TVLAIGTAAPANVVYQADYPDYYFRITKSEHMTELKEKFRRMCDKSMITKRHMHLSEE LLKNNPDICAYMAPSLDARQDMVVVEVPKLGKEAAAKAIKEWGRPKSAITHLIFCTTSG VDMPGADFQLTKLLGLCPSVRRTMLYQQGCFAGGTVLRLAKDLAENNAGARVLVVCS EITAVTFRGPSETHLDSMVGQALFGDGASAIIVGADPDPVIERPLFQIVSAAQTILPDSDG AIDGHLREVGLTFHLLKDVPGLISKNIEKSLKEAFAPLGIDDWNSIFWIVHPGGPAILDQV EAKLRLKVEKLKTTRTVLSEYGNMSSACVLFILDEMRRNSMEEGKATTGEGLHWGVLF GFGPGLTVETVVLHSLPIAEAN. In certain embodiments, the synthase is chalcone synthase (EC 2.3.1.74).

(29) The amino acid sequences of the polyketide synthases described herein contain conserved catalytic triads. In certain embodiments, the conserved catalytic triad of PmSPS1 is Cys164, His304, and Asn337. In certain embodiments, the conserved catalytic triad of PmSPS2 is Cys164, His303, and Asn336. In certain embodiments, the conserved catalytic triad of PmCHS is Cys164, His303, and Asn336.

(30) In certain embodiments, an O-methyltransferase (EC 2.1.1.C) is used to methylate hydroxyl groups substituting styrylpyrones, wherein C is an integer between 1 and 200, inclusive. In certain embodiments, an O-methyltransferase (EC 2.1.1.C) is used to methylate hydroxyl groups substituting chalcones, wherein C is an integer between 1 and 200, inclusive. The O-methyltransferase is isolated or derived from wildtype, mutant, or recombinant Piper methysticum. The O-methyltransferase can be used as an unpurified enzyme, partially purified enzyme, or purified enzyme. In certain embodiments, the amino acid sequence of the enzyme is at least 80% identical to SEQ ID NO: 5, designated O-methyltransferase PmOMT4, wherein SEQ ID NO: 5 is MEQAVFKDQSPSRDDIDEELFQSALYLSTAVVTVPAAIMAANDLDVLQ IIAKAGPGAHLSPTEIVSHLPTRNPNAAAALHRILRVLASHSILECSSRCEGEAKYGLRPV CKFFLNDKDGVSLNAMPSFVQSRVFIDSWQYMKDAVLEGVVPFEKAYGMPFYQFQAV NTKFKETFAKAMAAHSTLVVKKMLDTYNGFEGLTELMDVAGGTGSTLNLIVSKYPQIK GTNFDLKHVIEAAPNYPGVKHLSGDMFDSIPSAKNIIMKWILHNWSDEHCVKLLKNCYT SLPEFGKLIVVDSIVGEDVDAGLTTTNVFGCDFTMLTFFPNAKERTREEFQDLAKASGFS TFKPICCAYGVWVMEFHK. In certain embodiments, the amino acid sequence of the enzyme is at least 80% identical to SEQ ID NO: 6, designated O-methyltransferase PmOMT1, wherein SEQ ID NO: 6 is MNDQELHGYSQNAQPQLWNLLLSFINSMSLKCAVELGIPDIIHSHAQ TPINITDLAASIPIPPNKTSQFRRLMRLLVHSNVFSVHKREDGDEGFLLTPMSRILVTSND NNGGNLSPFVSMMVDPSLVSPWHFLGQWLKGNDTQGTPFRMCHGEEMWDWANKYP DFNKKFNMAMVCDSQYLMKIIVKKCATAFEGKRSLIDVGGGTGGAARSIAEAFPDIQEV SVLDLPHVVAGLPNDSRVKFVGGDMFHTIPPADVVLLKAIFHGWNDEECIKILKNCKKA IPSKEEGGKVMILDMVVNSAPGDHMITEDQYFMDLMMITYARGLERDENEWKKLFKD AGFTSYKITHGLGTSSLIELYP. In certain embodiments, the synthase is chalcone synthase (EC 2.3.1.74).

(31) In certain embodiments, a methylenedioxy bridge-forming enzyme is used to form a methylenedioxy moiety from a hydroxyl group and a methoxy group each separately bonded to adjacent carbons of an aromatic ring belonging to a styrylpyrone or chalcone compound. The methylenedioxy bridge-forming enzyme is isolated or derived from wildtype, mutant, or recombinant Piper methysticum. The methylenedioxy bridge-forming enzyme can be used as an unpurified enzyme, partially purified enzyme, or purified enzyme. In certain embodiments, methylenedioxy bridge-forming enzyme belongs to the P450 enzyme family. In certain embodiments, methylenedioxy bridge-forming enzyme belongs to the CYP719 enzyme family. In certain embodiments, the amino acid sequence of the enzyme is at least 80% identical to SEQ ID NO: 7, designated methylenedioxy bridge-forming enzyme PmMDB1, wherein SEQ ID NO: 7 is MEQAQWVDPTLLPAFVGIIFFFLGMFFGRSSLGAGKGAAPRSTSSTEWPDGPPKLPII GNLHQLNKGGELVHHNLAKLAQSYDRAMTIWVGSWGPMIVVSDADLAWEVLVTKSP DFAGRVLSKLSHLFNANYNTVVAYDAGPQWQSLRRGLQHGPLGPAHVSAQARFHEED MKLLVSDMMRAAQKGGSNGVVEPLAYVRRATIRFLSRLCFGEAFNDEAFVEGMDEAV EETIGATGHARILDAFYFTRHLPIIRRSFIDTVNAKKKIESLVRPLLSRPAPPGSYLHFLLST DAPENMIIFRIFEVYLLGVDSTASTTTWALAFLVSNQQAQEKLHNELAQYCASQNNQIIK ADDVGKLSYLLGVVKETMRMKPIAPLAVPHKTLKETMLDGKRVAAGTTVVVNLYAVH YNPKLWPEPEQFRPERFVVGASGGNGGGSSEYMLQSYLPFGGGMRSCAGMEVGKLQV AMVVANLVMAFKWLPEEEGKMPDLAEDMTFVLMMKKPLAAKIVPRA.

(32) In certain embodiments, a dehydrogenase or reductase (EC 1.1.1.D) is used to reduce the C.sub.5-C.sub.6 double bond of kavalactones into a single bond (FIG. 1), wherein D is an integer between 1 and 450, inclusive. The dehydrogenase or reductase is isolated or derived from wildtype, mutant, or recombinant Piper methysticum. The dehydrogenase or reductase can be used as an unpurified enzyme, partially purified enzyme, or purified enzyme. In certain embodiments, the reductase is a NADPH-dependent reductase. In certain embodiments, the amino acid sequence of the enzyme is at least 80% identical to SEQ ID NO: 8, designated NADPH-dependent reductase PmRDCT10, wherein SEQ ID NO: 8 is METERKSRICVTGAGG FVASWVVKLFLSKGYLVHGTVRDLGEEKTAHLRKLEGAYHNLQLFKADLLDYESLLGA ITGCDGVLHVATPVPSSKTAYSGTELVKTAVNGTLNVLRACTEAKVKKVIYVSSTAAVL VNPNLPKDKIPDEDCWTDEEYCRTTPFFLNWYCLAKTAAEKNALEYGDKEGINVISICPS YIFGPMLQPTINSSNLELLRLMKGDDESIENKFLLMVDVRDVAEAILLLYEKQETSGRYIS SPHGMRQSNLVEKLESLQPGYNYHKNFVDIKPSWTMISSEKLKKLGWKPRPLEDTISET VLCFEEHGLLENE.

(33) Protein sequencing encompasses the process of determining the amino acid sequence of all or part of a protein or peptide. The two major methods of protein sequencing are Edman degradation using a protein sequenator and mass spectrometry. Typically, only part of the protein's sequence needs to be determined experimentally in order to identify the protein with reference to databases of protein sequences deduced from the DNA sequences of their genes.

(34) Prior to attempting to find the ordered sequence of a protein, it is often desirable to know the unordered amino acid composition of a protein as this knowledge can be used to facilitate the discovery of errors in the sequencing process or to distinguish between ambiguous results. Knowledge of the frequency of certain amino acids may also be used to choose which protease to use for digestion of the protein. The misincorporation of low levels of non-standard amino acids (e.g. norleucine) into proteins may also be determined. A generalized method often referred to as amino acid analysis for determining amino acid frequency is as follows: 1) hydrolyze a known quantity of protein into its constituent amino acids; and 2) separate and quantify the amino acids.

(35) Hydrolysis is done by heating a sample of the protein in 6 M hydrochloric acid to approximately 100 to 110° C. for 24 hours or longer. Proteins with many bulky hydrophobic groups may require longer heating periods. However, these conditions are so vigorous that some amino acids (e.g., serine, threonine, tyrosine, tryptophan, glutamine, and cysteine) are degraded. To circumvent this problem the following strategy is employed: 1) heat separate samples for different times; 2) analyze the composition of each resulting solution; and 3) extrapolating back to zero hydrolysis time. A variety of reagents are known to prevent or reduce degradation, such as thiol reagents or phenol to protect tryptophan and tyrosine from attack by chlorine, and pre-oxidizing cysteine. In addition, measuring the quantity of ammonia evolved allows for the determination of the extent of amide hydrolysis.

(36) After the hydrolysis step, the amino acids can be separated by ion-exchange chromatography and then derivatized to facilitate their detection. More commonly, the amino acids are derivatized then resolved by reversed phase HPLC.

(37) The first major method for protein sequencing is the Edman degradation, which consists of the following steps: 1) break any disulfide bridges in the protein with a reducing agent like 2-mercaptoethanol. A protecting group such as iodoacetic acid may be necessary to prevent the bonds from re-forming; 2) separate and purify the individual chains of the protein complex, if there are more than one; 3) determine the amino acid composition of each chain; 4) determine the terminal amino acids of each chain; 5) break each chain into fragments under 50 amino acids long; 6) separate and purify the fragments; 7) determine the sequence of each fragment; 8) repeat with a different pattern of cleavage; and 9) construct the sequence of the overall protein.

(38) Peptides longer than about 50-70 amino acids long cannot be sequenced reliably by the Edman degradation. Therefore, long protein chains need to be broken up into small fragments that can then be sequenced individually. Digestion is done either by endopeptidases such as trypsin or pepsin or by chemical reagents such as cyanogen bromide. Different enzymes give different cleavage patterns, and the overlap between fragments can be used to construct an overall sequence.

(39) The seventh step of the Edman degradation begins with adsorbing the peptide to be sequenced onto a solid surface. One common substrate is glass fiber coated with polybrene, a cationic polymer. The Edman reagent, phenylisothiocyanate (PITC), is added to the adsorbed peptide, together with a mildly basic buffer solution of trimethylamine. This reacts with the amine group of the N-terminal amino acid. The terminal amino acid can then be selectively detached by the addition of anhydrous acid. The derivative then isomerizes to give a substituted phenylthiohydantoin, which can be washed off and identified by chromatography, and the cycle can be repeated. The efficiency of each step is about 98%, which allows about 50 amino acids to be reliably determined.

(40) Automated Edman sequencers called protein sequenators are now in widespread use, and are able to sequence peptides up to approximately 50 amino acids long. A sample of the protein or peptide is immobilized in the reaction vessel of the protein sequenator and the Edman degradation is performed. Each cycle releases and derivatizes one amino acid from the protein or peptide's N-terminus and the released amino acid derivative is then identified by HPLC. The sequencing process is done repetitively for the whole polypeptide until the entire measurable sequence is established or for a pre-determined number of cycles.

(41) The second major method for protein sequencing is mass spectrometry, which consists of the following steps:

(42) 1) The protein is isolated, typically by SDS-PAGE or chromatography.

(43) 2) The isolated protein may be chemically modified to stabilize cysteine residues (e.g., S-amidomethylation or S-carboxymethylation).

(44) 3) The protein is digested with a specific protease to generate peptides. Trypsin, which cleaves selectively on the C-terminal side of lysine or arginine residues, is the most commonly used protease. Its advantages include i) the frequency of Lys and Arg residues in proteins, ii) the high specificity of the enzyme, iii) the stability of the enzyme and iv) the suitability of tryptic peptides for mass spectrometry.
4) The peptides may be desalted to remove ionizable contaminants and subjected to MALDI-TOF mass spectrometry. Direct measurement of the masses of the peptides may provide sufficient information to identify the protein, but further fragmentation of the peptides inside the mass spectrometer is often used to gain information about the peptides' sequences. Alternatively, peptides may be desalted and separated by reversed phase HPLC and introduced into a mass spectrometer via an ESI source. LC-ESI-MS may provide more information than MALDI-MS for protein identification but typically requires more instrument time.
5) Depending on the type of mass spectrometer, fragmentation of peptide ions may occur via a variety of mechanisms such as collision-induced dissociation (CID) or post-source decay (PSD). In each case, the pattern of fragment ions of a peptide provides information about its sequence.
6) Information including the measured mass of the putative peptide ions and those of their fragment ions is then matched against calculated mass values from the conceptual (in silico) proteolysis and fragmentation of databases of protein sequences. A successful match will be found if its score exceeds a threshold based on the analysis parameters. Even if the actual protein is not represented in the database, error-tolerant matching allows for the putative identification of a protein based on similarity to homologous proteins. A variety of software packages are available to perform this analysis.
7) Software packages usually generate a report showing the identity (accession code) of each identified protein, its matching score, and provide a measure of the relative strength of the matching where multiple proteins are identified.
8) A diagram of the matched peptides on the sequence of the identified protein is often used to show the sequence coverage (% of the protein detected as peptides). When the protein is thought to be significantly smaller than the matched protein, the diagram may suggest whether the protein is an N- or C-terminal fragment of the matched protein.
Production of CoA Esters of Formula (II)

(45) In one aspect, the present disclosure provides methods for producing a compound of Formula (II) from a compound of Formula (I), or a salt thereof, and coenzyme A (CoA) using an enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1), wherein:

(46) ##STR00009##

(47) In certain embodiments, the condensation reaction of a compound of Formula (I) with coenzyme A to produce a compound of Formula (II) utilizes adenosine triphosphate. In certain embodiments, the condensation reaction of a compound of Formula (I) with coenzyme A to produce a compound of Formula (II) is performed in vitro. In certain embodiments, the in vitro condensation reaction is performed with an unpurified enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1). In certain embodiments, the in vitro condensation reaction is performed with a partially purified enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1). In certain embodiments, the in vitro condensation reaction is performed with an purified enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1). In certain embodiments, the condensation reaction of a compound of Formula (I) with coenzyme A to produce a compound of Formula (II) is performed in vivo.

(48) In certain embodiments, a single bond. In certain embodiments, is a double bond.

(49) In certain embodiments, each of R.sub.1, R.sub.2, and R.sub.3 independently is hydrogen, optionally substituted, cyclic or acyclic aliphatic, or —OR.sub.x, wherein R.sub.x is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.1 is hydrogen. In certain embodiments, R.sub.2 is hydrogen. In certain embodiments, R.sub.3 is hydrogen. In certain embodiments, R.sub.1 is —OH. In certain embodiments, R.sub.2 is —OH. In certain embodiments, R.sub.3 is —OH. In certain embodiments, R.sub.1 is —OCH.sub.3. In certain embodiments, R.sub.2 is —OCH.sub.3. In certain embodiments, R.sub.3 is —OCH.sub.3. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are hydrogen. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are —OH. In certain embodiments, R.sub.1 and R.sub.3 are —OH. In certain embodiments, R.sub.2 and R.sub.3 are —OH. In certain embodiments, R.sub.2 is —OCH.sub.3.

(50) In certain embodiments, each of R.sub.4 and R.sub.5 independently is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, both R.sub.4 and R.sub.5 are hydrogen.

(51) The methods to produce a compound of Formula (II) include condensing coenzyme A (CoA) with a compound of Formula (I) selected from the group consisting of:

(52) ##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014## ##STR00015##

(53) The methods to produce a compound of Formula (II) include culturing cells engineered to express an enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1). In certain embodiments, the enzyme is at least 80%, 85%, 90%, 95%, or 100% identical to Pm4CL1 (SEQ ID NO: 1). In certain embodiments, the enzyme is purified before being used in a reaction with a compound of Formula (I). In certain embodiments, the enzyme is partially purified before being used in a reaction with a compound of Formula (I).

(54) In certain embodiments, the enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) is a component of a fusion protein. A fusion protein may be created by joining two or more gene or gene segments that code for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. A polyfunctional protein is a single protein that has at least two different activities, wherein that functionality is a native biological function or the result of an engineered enzyme fusion. Thus, a fusion protein may include multiple activities such as those described herein for the kavalactone or flavokavain pathway enzymes described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)).

(55) The enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) is heterologous to the host cell. In certain embodiments, the enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) is recombinantly produced. In certain embodiments, the enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) is obtained from a wildtype organism. In certain embodiments, the enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO. 1) is obtained from a mutant organism. In certain embodiments, the enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) is obtained from a genetically-modified organism. In certain embodiments, the organism is a non-human organism. In certain embodiments, the non-human organism is selected from group consisting of bacteria, yeast, and plant. In certain embodiments, the organism is a plant. In certain embodiments, the plant is Piper methysticum.

(56) A nucleic acid encoding the enzyme may be introduced into the cell via a vector (e.g., plasmids, viral vectors, cosmids, and artificial chromosomes). In certain embodiments, the nucleic acid encoding the enzyme is cDNA derived from the gene encoding the enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1). In some embodiments, multiple cDNAs comprising sequences from different genes (e.g., 2, 3, 4, 5, or more genes) described herein (e.g., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identity to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)), are introduced into the same cell individually, or together, or as part of a single nucleic acid.

(57) The host cells expressing the enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) may be prokaryotic cells, such as bacterial cells (e.g., Escherichia coli cells), or eukaryotic cells, such as yeast cells or plant cells. In certain embodiments, the host cell is capable of expressing two or more kavalactone or flavokavain pathway enzymes described herein. In certain embodiments, the host cell is a bacteria cell and is a wildtype, mutant, recombinant, or genetically engineered form of Escherichia coli. In certain embodiments, the host cell is a yeast cell and is a wildtype, mutant, recombinant, or genetically engineered form of Saccharomyces cerevisiae, In certain embodiments, the host cell is a plant cell and is a wildtype, mutant, recombinant, or genetically engineered form of Nicotiana benthamiana.

Production of 6-styryl-4-hydroxyl-2-pyrone Compounds of Formula (III)

(58) Some aspects of the present disclosure provides methods for producing a compound of Formula (III) from a compound of Formula (II), or a salt thereof, and malonyl-CoA using an enzyme that is at least 80% identical to PmSPS1 (SEQ ID NO: 2). Some aspects of the present disclosure provides methods for producing a compound of Formula (III) from a compound of Formula (II), or a salt thereof, and malonyl-CoA using an enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3). The structure of a compound of Formula (II) and a structure of a compound of Formula (III) are as follows:

(59) ##STR00016##

(60) In certain embodiments, the reaction of a compound of Formula (II) with malonyl-CoA to produce a compound of Formula (III) utilizes two or more molar equivalents of malonyl-CoA relative to the compound of Formula (II). In certain embodiments, the reaction of a compound of Formula (II) with malonyl-CoA to produce a compound of Formula (III) is performed in vitro. In certain embodiments, the reaction of a compound of Formula (II) with malonyl-CoA to produce a compound of Formula (III) is performed in vivo.

(61) In certain embodiments, is a single bond. In certain embodiments, is a double bond.

(62) In certain embodiments, each of R.sub.1, R.sub.2, R.sub.3, R.sub.6, and R.sub.7 independently is hydrogen, optionally substituted, cyclic or acyclic aliphatic, or OR.sub.x, wherein R.sub.x is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.1 is hydrogen. In certain embodiments, R.sub.2 is hydrogen. In certain embodiments, R.sub.3 is hydrogen. In certain embodiments, R.sub.1 is —OH. In certain embodiments, R.sub.2 is —OH. In certain embodiments, R.sub.3 is —OH. In certain embodiments, R.sub.1 is —OCH.sub.3. In certain embodiments, R.sub.2 is —OCH.sub.3. In certain embodiments, R.sub.3 is —OCH.sub.3. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are hydrogen. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are —OH. In certain embodiments, R.sub.1 and R.sub.3 are —OH. In certain embodiments, R.sub.2 and R.sub.3 are —OH. In certain embodiments, R.sub.2 is —OCH.sub.3. In certain embodiments, Re is hydrogen. In certain embodiments, R.sub.6 is —OH. In certain embodiments, R.sub.7 is hydrogen. In certain embodiments, Re is —OH and R.sub.7 is hydrogen. In certain embodiments, both Re and R.sub.7 are hydrogen.

(63) In certain embodiments, each of R.sub.4 and R.sub.5 independently is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, both R.sub.4 and R.sub.5 are hydrogen.

(64) The methods to produce a compound of Formula (III) include reacting malonyl-CoA with a compound of Formula (II) selected from the group consisting of:

(65) ##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##

(66) The methods to produce a compound of Formula (III) include culturing cells engineered to express an enzyme that is at least 80% identical to PmSPS1 (SEQ ID NO: 2). The methods to produce a compound of Formula (III) include culturing cells engineered to express an enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3). In certain embodiments, the enzyme is at least 80%, 85%, 90%, 95%, or 100% identical to PmSPS1 (SEQ ID NO: 2). In certain embodiments, the enzyme is at least 80%, 85%, 90%, 95%, or 100% identical to PmSPS2 (SEQ ID NO: 3). In certain embodiments, the enzyme is purified before reacting with a compound of Formula (II). In certain embodiments, the enzyme is partially purified before reacting with a compound of Formula (II).

(67) In certain embodiments, the enzyme that is at least 80% identical to PmSPS1 (SEQ ID NO: 2) is a component in a fusion protein. In certain embodiments, the enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3) is a component in a fusion protein. A fusion protein may be created by joining two or more gene or gene segments that code for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. A polyfunctional protein is a single protein that has at least two different activities, wherein that functionality is a native biological function or the result of an engineered enzyme fusion. Thus, a fusion protein may include multiple activities such as those described herein for the kavalactone or flavokavain pathway enzymes described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)).

(68) The enzyme that is at least 80% identical to PmSPS1 (SEQ ID NO: 2) is heterologous to the host cell. The enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3) is heterologous to the host cell. In certain embodiments, the enzyme that is at least 80% identical to PmSPS1 (SEQ ID NO: 2) is recombinantly produced. In certain embodiments, the enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3) is recombinantly produced. In certain embodiments, the enzyme that is at least 80% identical to PmSPS1 (SEQ ID NO: 2) is obtained from a wildtype organism. In certain embodiments, the enzyme that is at least 80% identical to PmSPS1 (SEQ ID NO: 2) is obtained from a mutant organism. In certain embodiments, the enzyme that is at least 80% identical to PmSPS1 (SEQ ID NO: 2) is obtained from a genetically-modified organism. In certain embodiments, the enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3) is obtained from a wildtype organism. In certain embodiments, the enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3) is obtained from a mutant organism. In certain embodiments, the enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3) is obtained from a genetically-modified organism. In certain embodiments, the organism is a non-human organism. In certain embodiments, the non-human organism is selected from group consisting of bacteria, yeast, and plant. In certain embodiments, the organism is a plant. In certain embodiments, the plant is Piper methysticum.

(69) A nucleic acid encoding the enzyme may be introduced into the cell in a vector (e.g., plasmids, viral vectors, cosmids, and artificial chromosomes). In certain embodiments, the nucleic acid is cDNA derived from the amino acid sequence of the enzyme that is at least 80% identical to PmSPS1 (SEQ ED NO: 2). In certain embodiments, the nucleic acid is cDNA derived from the amino acid sequence of the enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3). In some embodiments multiple cDNAs comprising sequences complementary to different genes (e.g., 2, 3, 4, 5, or more genes) described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)), are introduced into the same cell individually, or together, or as part of a single nucleic acid.

(70) The host cells expressing the enzyme that is at least 80% identical to PmSPS1 (SEQ ID NO: 2) may be prokaryotic cells, such as bacterial cells (e.g., Escherichia coli cells), or eukaryotic cells, such as yeast cells or plant cells. The host cells expressing the enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3) may be prokaryotic cells, such as bacterial cells (e.g., Escherichia coli cells), or eukaryotic cells, such as yeast cells or plant cells. In certain embodiments, the host cell is capable of expressing two or more kavalactone or flavokavain pathway enzymes described herein. In certain embodiments, the host cell is a bacteria cell and is a wildtype, mutant, recombinant, or genetically engineered form of Escherichia coli. In certain embodiments, the host cell is a yeast cell and is a wildtype, mutant, recombinant, or genetically engineered form of Saccharomyces cerevisiae. In certain embodiments, the host cell is a plant cell and is a wildtype, mutant, recombinant, or genetically engineered form of Nicotiana benthamiana.

(71) In certain embodiments, the method for producing a compound of Formula (III) utilizes a compound of Formula (I), or a salt thereof, as the starting material and comprises the steps: condensing a compound of Formula (I), or a salt thereof, with coenzyme A (CoA) using a recombinant enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) to produce a compound of Formula (II); and reacting a compound of Formula (II), or a salt thereof, with malonyl-CoA using an enzyme that is at least 80% identical to PmSPS1 (SEQ ID NO: 2) to produce a compound of Formula (III).

(72) In certain embodiments, the method for producing a compound of Formula (III) utilizes a compound of Formula (I), or a salt thereof, as the starting material and comprises the steps: condensing a compound of Formula (I), or a salt thereof, with coenzyme A (CoA) using a recombinant enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) to produce a compound of Formula (II); and reacting a compound of Formula (II), or a salt thereof, with malonyl-CoA using an enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3) to produce a compound of Formula (III).

Production of Methylated 6-styryl-4-hydroxyl-2-pyrone Compounds of Formula (IV)

(73) Some aspects of the present disclosure provides methods for producing a compound of Formula (IV) from a compound of Formula (III), or a salt thereof, and S-adenosylmethionine using an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). Some aspects of the present disclosure provides methods for producing a compound of Formula (IV) from a compound of Formula (III), or a salt thereof, and S-adenosylmethionine using an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). The structure of a compound of Formula (III) and a structure of a compound of Formula (IV) are as follows:

(74) ##STR00023##

(75) In certain embodiments, the reaction of a compound of Formula (III) with S-adenosylmethionine to produce a compound of Formula (IV) is performed in vitro. In certain, embodiments, the reaction of a compound of Formula (III) with S-adenosylmethionine to produce a compound of Formula (IV) is performed in vivo.

(76) In certain embodiments, is a single bond. In certain embodiments, is a double bond.

(77) In certain embodiments, each of R.sub.1, R.sub.2, R.sub.3, R.sub.1a, R.sub.2a, R.sub.3a, R.sub.6, and R.sub.7 independently is hydrogen, optionally substituted, cyclic or acyclic aliphatic, or —OR.sub.x, wherein R.sub.x is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.1 is hydrogen. In certain embodiments, R.sub.2 is hydrogen. In certain embodiments, R.sub.3 is hydrogen. In certain embodiments, R.sub.1 is —OH. In certain embodiments, R.sub.2 is OH. In certain embodiments, R.sub.3 is —OH. In certain embodiments, R.sup.1 is —OCH.sub.3. In certain embodiments, R.sub.2 is —OCH.sub.3. In certain embodiments, R.sub.3 is —OCH.sub.3. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are hydrogen. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are —OH. In certain embodiments, R.sub.1 and R.sub.3 are —OH. In certain embodiments, R.sub.2 and R.sub.3 are —OH. In certain embodiments, R.sub.2 is —OCH.sub.3. In certain embodiments, R.sub.6 is hydrogen. In certain embodiments, R.sub.6 is —OH. In certain embodiments, R.sub.7 is hydrogen. In certain embodiments, R.sub.6 is —OH and R.sub.7 is hydrogen. In certain embodiments, both R.sub.6 and R.sub.7 are hydrogen. In certain embodiments, wherein R.sub.8 is —CH.sub.3. In certain embodiments, R.sub.1a is hydrogen. In certain embodiments, R.sub.2a is hydrogen. In certain embodiments, R.sub.3a is hydrogen. In certain embodiments, R.sub.1a is —OH. In certain embodiments, R.sub.2a is OH. In certain embodiments, R.sub.3a is —OH. In certain embodiments, R.sub.1a is —OCH.sub.3. In certain embodiments, R.sub.2a is —OCH.sub.3. In certain embodiments, R.sub.3a is —OCH.sub.3. In certain embodiments, R.sub.1a, R.sub.2a, and R.sub.3a are hydrogen. In certain embodiments, R.sub.1a, R.sub.2a, and R.sub.3a are —OCH.sub.3. In certain embodiments, R.sub.1a and R.sub.3a are —OCH.sub.3. In certain embodiments, R.sub.2a and R.sub.3a are —OCH.sub.3. In certain embodiments, R.sub.2a and R.sub.3a are —OCH.sub.3 and R.sub.8 is —CH.sub.3. In certain embodiments, R.sub.2a and R.sub.3a are —OH and R.sub.8 is —H.

(78) In certain embodiments, each of R.sub.4 and R.sub.5 independently is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.4 and R.sub.5 are hydrogen.

(79) A compound of Formula (III) can provide different compounds of Formula (IV) depending on the choice to utilize only an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5), or only an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6), or both enzymes (FIG. 7). In certain embodiments, R.sub.8 is —CH.sub.3 when a compound of Formula (III) is reacted with an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). In certain embodiments, R.sub.8 is hydrogen when a compound of Formula (III) is reacted with an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, R.sub.8 is —CH.sub.3 when a compound of Formula (III) is reacted with both an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) and an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, if R.sub.1 is —OH, then R.sub.1a is —OH when a compound of Formula (III) is reacted with an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). In certain embodiments, if R.sup.1 is —OH, then R.sub.1a is —OCH.sub.3 when a compound of Formula (III) is reacted with an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO. 6). In certain embodiments, if R.sub.1 is —OH, then R.sub.1a is —OCH.sub.3 when a compound of Formula (III) is reacted with both an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) and an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, if R.sub.2 is —OH, then R.sub.2a is —OCH.sub.3 when a compound of Formula (III) is reacted with an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). In certain embodiments, if R.sub.2 is —OH, then R.sub.2a is —OH when a compound of Formula (III) is reacted with an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, if R.sub.2 is —OH, then R.sub.2a is —OCH.sub.3 when a compound of Formula (III) is reacted with both an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) and an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, if R.sub.3 is —OH, then R.sub.3a is —OCH.sub.3 when a compound of Formula (III) is reacted with an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). In certain embodiments, if R.sub.3 is —OH, then R.sub.3a is —OH when a compound of Formula (III) is reacted with an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, if R.sub.3 is —OH then R.sub.3a is —OCH.sub.3 when a compound of Formula (III) is reacted with both an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) and an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6).

(80) The methods to produce a compound of Formula (IV) include reacting malonyl-CoA with a compound of Formula (III) selected from the group consisting of:

(81) ##STR00024## ##STR00025## ##STR00026##
wherein X is

(82) ##STR00027##

(83) The methods to produce a compound of Formula (IV) include culturing cells engineered to express an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). The methods to produce a compound of Formula (IV) include culturing cells engineered to express an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, the enzyme is at least 80%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to PmOMT4 (SEQ ID NO: 5). In certain embodiments, the enzyme is at least 80%, 85%, 90%, 95%, or 100% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, the enzyme is purified before reacting with a compound of Formula (III). In certain embodiments, the enzyme is partially purified before reacting with a compound of Formula (III).

(84) In certain embodiments, the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) is a component in a fusion protein. In certain embodiments, the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) is a component in a fusion protein. A fusion protein may be created by joining two or more gene or gene segments that code for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. A polyfunctional protein is a single protein that has at least two different activities, wherein that functionality is a native biological function or the result of an engineered enzyme fusion. Thus, a fusion protein may include multiple activities such as those described herein for the kavalactone or flavokavain pathway enzymes described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)).

(85) The enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) is heterologous to the host cell. The enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) is heterologous to the host cell. In certain embodiments, the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) is recombinantly produced. In certain embodiments, the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) is recombinantly produced. In certain embodiments, the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) is obtained from a wildtype organism. In certain embodiments, the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) is obtained from a mutant organism. In certain embodiments, the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) is obtained from a genetically-modified organism. In certain embodiments, the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) is obtained from a wildtype organism. In certain embodiments, the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) is obtained from a mutant organism. In certain embodiments, the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) is obtained from a genetically-modified organism. In certain embodiments, the organism is a non-human organism. In certain embodiments, the non-human organism is selected from group consisting of bacteria, yeast, and plant. In certain embodiments, the organism is a plant. In certain embodiments, the plant is Piper methysticum.

(86) A nucleic acid encoding the enzyme may be introduced into the cell in a vector (e.g., plasmids, viral vectors, cosmids, and artificial chromosomes). In certain embodiments, the nucleic acid is cDNA derived from the amino acid sequence of the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). In certain embodiments, the nucleic acid is cDNA derived from the amino acid sequence of the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In some embodiments multiple cDNAs comprising sequences complementary to different genes (e.g., 2, 3, 4, 5, or more genes) described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)), are introduced into the same cell individually, or together, or as part of a single nucleic acid.

(87) The host cells expressing the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) may be prokaryotic cells, such as bacterial cells (e.g., Escherichia coli cells), or eukaryotic cells, such as yeast cells or plant cells. The host cells expressing the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) may be prokaryotic cells, such as bacterial cells (e.g., Escherichia coli cells), or eukaryotic cells, such as yeast cells or plant cells. In certain embodiments, the host cell is capable of expressing two or more kavalactone or flavokavain pathway enzymes described herein. In certain embodiments, the host cell is a bacteria cell and is a wildtype, mutant, recombinant, or genetically engineered form of Escherichia coli. In certain embodiments, the host cell is a yeast cell and is a wildtype, mutant, recombinant, or genetically engineered form of Saccharomyces cerevisiae. In certain embodiments, the host cell is a plant cell and is a wildtype, mutant, recombinant, or genetically engineered form of Nicotiana benthamiana.

(88) In certain embodiments, the method for producing a compound of Formula (IV) utilizes a compound of Formula (I), or a salt thereof, as the starting material and comprises the steps: condensing a compound of Formula (I), or a salt thereof, with coenzyme A (CoA) using an enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) to produce a compound of Formula (II); reacting a compound of Formula (II), or a salt thereof, with malonyl-CoA using an enzyme that is at least 80% identical to PmSPS1 (SEQ ID NO: 2) to produce a compound of Formula (III); and alkylating a compound of Formula (III), or a salt thereof, with S-adenosylmethionine using an enzyme that at least 80% identical to PmOMT4 (SEQ ID NO: 5) to produce a compound of Formula (IV).

(89) In certain embodiments, the method for producing a compound of Formula (IV) utilizes a compound of Formula (I), or a salt thereof, as the starting material and comprises the steps: condensing a compound of Formula (I), or a salt thereof, with coenzyme A (CoA) using an enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) to produce a compound of Formula (II); reacting a compound of Formula (II), or a salt thereof, with malonyl-CoA using an enzyme that is at least 80% identical to PmSPS1 (SEQ ID NO: 2) to produce a compound of Formula (III); and alkylating a compound of Formula (III), or a salt thereof, with S-adenosylmethionine using an enzyme that at least 80% identical to PmOMT1 (SEQ ID NO: 6) to produce a compound of Formula (IV).

(90) In certain embodiments, the method for producing a compound of Formula (IV) utilizes a compound of Formula (I), or a salt thereof, as the starting material and comprises the steps: condensing a compound of Formula (I), or a salt thereof, with coenzyme A (CoA) using an enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) to produce a compound of Formula (II); reacting a compound of Formula (II), or a salt thereof, with malonyl-CoA using an enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3) to produce a compound of Formula (III); and alkylating a compound of Formula (III), or a salt thereof, with S-adenosylmethionine using an enzyme that at least 80% identical to PmOMT4 (SEQ ID NO: 5) to produce a compound of Formula (IV).

(91) In certain embodiments, the method for producing a compound of Formula (IV) utilizes a compound of Formula (I), or a salt thereof, as the starting material and comprises the steps: condensing a compound of Formula (I), or a salt thereof, with coenzyme A (CoA) using an enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) to produce a compound of Formula (II); reacting a compound of Formula (II), or a salt thereof, with malonyl-CoA using an enzyme that is at least 80% identical to PmSPS2 (SEQ ID NO: 3) to produce a compound of Formula (III); and alkylating a compound of Formula (III), or a salt thereof, with S-adenosylmethionine using an enzyme that at least 80% identical to PmOMT1 (SEQ ID NO: 6) to produce a compound of Formula (IV).

Production of 6-styryl-4-hydroxyl-5,6-dihydro-2-pyrone Compounds of Formula (V)

(92) Some aspects of the present disclosure provides methods for producing a compound of Formula (V) from a compound of Formula (IV), or a salt thereof, and a reducing agent (i.e., NADPH or NADH) using an enzyme that is at least 80% identical to PmRDCT10 (SEQ ID NO: 8), wherein:

(93) ##STR00028##

(94) In certain, embodiments, the reduction reaction of a compound of Formula (IV) with NADPH to produce a compound of Formula (V) occurs in vitro. In certain embodiments, the reduction reaction of a compound of Formula (IV) with NADPH to produce a compound of Formula (V) occurs in vivo. In certain, embodiments, the reduction reaction of a compound of Formula (IV) with NADH to produce a compound of Formula (V) occurs in vitro. In certain embodiments, the reduction reaction of a compound of Formula (IV) with NADH to produce a compound of Formula (V) occurs in vivo.

(95) In certain embodiments, is a single bond. In certain embodiments, is a double bond.

(96) In certain embodiments, each of Ra, R.sub.2, R.sub.3, R.sub.6, and R.sub.7 independently is hydrogen, optionally substituted, cyclic or acyclic aliphatic, or OR.sub.x, wherein R.sub.x is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.1 is hydrogen. In certain embodiments, R.sub.2 is hydrogen. In certain embodiments, R.sub.3 is hydrogen. In certain embodiments, R.sub.1 is —OH. In certain embodiments, R.sub.2 is OH. In certain embodiments, R.sub.3 is —OH. In certain embodiments, R.sub.1 is —OCH.sub.3. In certain embodiments, R.sub.2 is —OCH.sub.3. In certain embodiments, R.sub.3 is —OCH.sub.3. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are hydrogen. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are —OH. In certain embodiments, R.sub.1 and R.sub.3 are —OH. In certain embodiments, R.sub.2 and R.sub.3 are —OH. In certain embodiments, R.sub.2 is —OCH.sub.3. In certain embodiments, R.sub.6 is hydrogen. In certain embodiments, R.sub.6 is —OH. In certain embodiments, R.sub.7 is hydrogen. In certain embodiments, R.sub.6 is —OH and R.sub.7 is hydrogen. In certain embodiments, both R.sub.6 and R.sub.7 are hydrogen. In certain embodiments, R.sub.8 is hydrogen. In certain embodiments, wherein R.sub.8 is —CH.sub.3. R.sub.1, R.sub.2, and R.sub.3 are —OCH.sub.3. In certain embodiments, R.sub.1 and R.sub.3 are —OCH.sub.3. In certain embodiments, R.sub.2 and R.sub.3 are —OCH.sub.3. In certain embodiments, R.sub.2 and R.sub.3 are —OCH.sub.3 and R.sub.8 is —CH.sub.3. In certain embodiments, R.sub.2 and R.sub.3 are —OH and R.sub.8 is —H.

(97) In certain embodiments, each of R.sub.4 and R.sub.5 independently is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.4 and R.sub.5 are hydrogen.

(98) The methods to produce a compound of Formula (V) include reacting NADPH or NADH with a compound of Formula (IV) selected from the group consisting of:

(99) ##STR00029## ##STR00030## ##STR00031##
wherein X is

(100) ##STR00032##

(101) The methods to produce a compound of Formula (V) include culturing cells engineered to express an enzyme that is at least 80% identical to PmRDCT10 (SEQ ID NO: 8). In certain embodiments, the enzyme is at least 80%, 85%, 90%, 95%, or 100% identical to PmRDCT10 (SEQ ID NO: 8). In certain embodiments, the enzyme is purified before reacting with a compound of Formula (IV). In certain embodiments, the enzyme is partially purified before reacting with a compound of Formula (IV).

(102) In certain embodiments, the enzyme that is at least 80% identical to PmRDCT10 (SEQ ID NO: 8) is a component in a fusion protein. A fusion protein may be created by joining two or more gene or gene segments that code for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. A polyfunctional protein is a single protein that has at least two different activities, wherein that functionality is a native biological function or the result of an engineered enzyme fusion. Thus, a fusion protein may include multiple activities such as those described herein for the kavalactone or flavokavain pathway enzymes described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)).

(103) The enzyme that is at least 80% identical to PmRDCT10 (SEQ ID NO: 8) is heterologous to the host cell. In certain embodiments, the enzyme that is at least 80% identical to PmRDCT10 (SEQ ID NO: 8) is recombinantly produced. In certain embodiments, the enzyme that is at least 80% identical to PmRDCT10 (SEQ ID NO: 8) is obtained from a wildtype organism. In certain embodiments, the enzyme that is at least 80% identical to PmRDCT10 (SEQ ID NO: 8) is obtained from a mutant organism. In certain embodiments, the enzyme that is at least 80% identical to PmRDCT10 (SEQ ID NO: 8) is obtained from a genetically-modified organism. In certain embodiments, the organism is a non-human organism. In certain embodiments, the non-human organism is selected from group consisting of bacteria, yeast, and plant. In certain embodiments, the organism is a plant. In certain embodiments, the plant is Piper methysticum.

(104) A nucleic acid encoding the enzyme may be introduced into the cell in a vector (e.g., plasmids, viral vectors, cosmids, and artificial chromosomes). In certain embodiments, the nucleic acid is cDNA derived from the amino acid sequence of the enzyme that is at least 80% identical to PmRDCT10 (SEQ ID NO: 8). In some embodiments multiple cDNAs comprising sequences complementary to different genes (e.g., 2, 3, 4, 5, or more genes) described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)), are introduced into the same cell individually, or together, or as part of a single nucleic acid.

(105) The host cells expressing the enzyme that is at least 80% identical to PmRDCT10 (SEQ ID NO: 8) may be prokaryotic cells, such as bacterial cells (e.g., Escherichia coli cells), or eukaryotic cells, such as yeast cells or plant cells. In certain embodiments, the host cell is capable of expressing two or more kavalactone or flavokavain pathway enzymes described herein. In certain embodiments, the host cell is a bacteria cell and is a wildtype, mutant, recombinant, or genetically engineered form of Escherichia coli. In certain embodiments, the host cell is a yeast cell and is a wildtype, mutant, recombinant, or genetically engineered form of Saccharomyces cerevisiae. In certain embodiments, the host cell is a plant cell and is a wildtype, mutant, recombinant, or genetically engineered form of Nicotiana benthamiana.

Production of 6-styryl-4-hydroxyl-2-pyrone Compounds with a Methylenedioxy Bridge of Formula (VI)

(106) Some aspects of the present disclosure provides methods for producing a compound of Formula (VI) from a compound of Formula (IV), or a salt thereof, and a reducing agent (i.e., NADPH or NADH) using an enzyme that is at least 80% identical to PmMDB1 (SEQ ID NO: 7), wherein:

(107) ##STR00033##

(108) In certain, embodiments, the reduction reaction of a compound of Formula (IV) with NADPH to produce a compound of Formula (VI) occurs in vitro. In certain embodiments, the reduction reaction of a compound of Formula (IV) with NADPH to produce a compound of Formula (VI) occurs in vivo. In certain, embodiments, the reduction reaction of a compound of Formula (IV) with NADH to produce a compound of Formula (VI) occurs in vitro. In certain embodiments, the reduction reaction of a compound of Formula (IV) with NADH to produce a compound of Formula (VI) occurs in vivo.

(109) In certain embodiments, is a single bond. In certain embodiments, is a double bond.

(110) In certain embodiments, each of R.sub.1, R.sub.2, R.sub.3, R.sub.6, R.sub.7, R.sub.9, R.sub.10, and R.sub.11 independently is hydrogen, optionally substituted, cyclic or acyclic aliphatic, or OR.sub.x, or R.sub.9 and R.sub.10 are combined to form a ring, or R.sub.10 and R.sub.11 are combined to form a ring, wherein R.sub.x is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.1 is hydrogen. In certain embodiments, R.sub.2 is hydrogen. In certain embodiments, R.sub.3 is hydrogen. In certain embodiments, R.sub.1 is —OH. In certain embodiments, R.sub.2 is —OH. In certain embodiments, R.sub.3 is —OH. In certain embodiments, R.sub.1 is —OCH.sub.3. In certain embodiments, R.sub.2 is —OCH.sub.3. In certain embodiments, R.sub.3 is —OCH.sub.3. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are hydrogen. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are —OH. In certain embodiments, R.sub.1 and R.sub.3 are —OH. In certain embodiments, R.sub.2 and R.sub.3 are —OH. In certain embodiments, R.sub.2 is —OCH.sub.3. In certain embodiments, R.sub.6 is hydrogen. In certain embodiments, R.sub.6 is —OH. In certain embodiments, R.sub.7 is hydrogen. In certain embodiments, R.sub.6 is —OH and R.sub.7 is hydrogen. In certain embodiments, both R.sub.6 and R.sub.7 are hydrogen. In certain embodiments, R.sub.8 is hydrogen. In certain embodiments, wherein R.sub.8 is —CH.sub.3. In certain embodiments, R.sub.9 and R.sub.10 are combined to form a methylenedioxy cyclic moiety —OCH.sub.2O— and R.sub.11 is hydrogen. In certain embodiments, R.sub.9 and R.sub.10 are combined to form a methylenedioxy cyclic moiety —OCH.sub.2O— and R.sub.11 is optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.9 and R.sub.10 are combined to form a methylenedioxy cyclic moiety —OCH.sub.2O— and R.sub.11 is —OH. In certain embodiments, R.sub.9 and R.sub.10 are combined to form a methylenedioxy cyclic moiety —OCH.sub.2O— and R.sub.11 is —OCH.sub.3. In certain embodiments, R.sub.10 and R.sub.11 are combined to form a methylenedioxy cyclic moiety —OCH.sub.2O— and R.sub.9 is hydrogen. In certain embodiments, R.sub.10 and R.sub.11 are combined to form a methylenedioxy cyclic moiety —OCH.sub.2O— and R.sub.9 is optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.10 and R.sub.11 are combined to form a methylenedioxy cyclic moiety —OCH.sub.2O— and R.sub.9 is —OH. In certain embodiments, R.sub.10 and R.sub.11 are combined to form a methylenedioxy cyclic moiety —OCH.sub.2O— and R.sub.9 is —OCH.sub.3. In certain embodiments, R.sub.1 is —OH, R.sub.2 is —OCH.sub.3, and R.sub.9 and R.sub.10 are combined to form a methylenedioxy cyclic moiety —OCH.sub.2O—. In certain embodiments, R.sub.1 is —OCH.sub.3, R.sub.2 is —OH, and R.sub.9 and R.sub.10 are combined to form a methylenedioxy cyclic moiety —OCH.sub.2O—. In certain, embodiments, R.sub.2 is —OH, R.sub.3 is —OCH.sub.3, and R.sub.10 and R.sub.11 are combined to form a methylenedioxy cyclic moiety —OCH.sub.2O—. In certain embodiments, R.sub.2 is —OCH.sub.3, R.sub.3 is —OH, and R.sub.10 and R.sub.11 are combined to form a methylenedioxy cyclic moiety —OCH.sub.2O—.

(111) In certain embodiments, each of R.sub.4 and R.sub.5 independently is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.4 and R.sub.5 are hydrogen.

(112) The methods to produce a compound of Formula (VI) include reacting NADPH or NADH with a compound of Formula (IV) selected from the group consisting of:

(113) ##STR00034## ##STR00035## ##STR00036##
wherein X is

(114) ##STR00037##

(115) The methods to produce a compound of Formula (VI) include culturing cells engineered to express an enzyme that is at least 80% identical to PmMDB1 (SEQ ID NO: 7). In certain embodiments, the enzyme is at least 80%, 85%, 90%, 95%, or 100% identical to PmMDB1 (SEQ ID NO: 7). In certain embodiments, the enzyme is purified before reacting with a compound of Formula (IV). In certain embodiments, the enzyme is partially purified before reacting with a compound of Formula (IV).

(116) In certain embodiments, the enzyme that is at least 80% identical to PmMDB1 (SEQ ID NO: 7) is a component in a fusion protein. A fusion protein may be created by joining two or more gene or gene segments that code for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. A polyfunctional protein is a single protein that has at least two different activities, wherein that functionality is a native biological function or the result of an engineered enzyme fusion. Thus, a fusion protein may include multiple activities such as those described herein for the kavalactone or flavokavain pathway enzymes described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)).

(117) The enzyme that is at least 80% identical to PmMDB1 (SEQ ID NO: 7) is heterologous to the host cell. In certain embodiments, the enzyme that is at least 80% identical to PmMDB1 (SEQ ID NO: 7) is recombinantly produced. In certain embodiments, the enzyme that is at least 80% identical to PmMDB1 (SEQ ID NO: 7) is obtained from a wildtype organism. In certain embodiments, the enzyme that is at least 80% identical to PmMDB1 (SEQ ID NO: 7) is obtained from a mutant organism. In certain embodiments, the enzyme that is at least 80% identical to PmMDB1 (SEQ ID NO: 7) is obtained from a genetically-modified organism. In certain embodiments, the organism is a non-human organism. In certain embodiments, the non-human organism is selected from group consisting of bacteria, yeast, and plant. In certain embodiments, the organism is a plant. In certain embodiments, the plant is Piper methysticum.

(118) A nucleic acid encoding the enzyme may be introduced into the cell in a vector (e.g., plasmids, viral vectors, cosmids, and artificial chromosomes). In certain embodiments, the nucleic acid is cDNA derived from the amino acid sequence of the enzyme that is at least 80% identical to PmMDB1 (SEQ ID NO: 7). In some embodiments multiple cDNAs comprising sequences complementary to different genes (e.g., 2, 3, 4, 5, or more genes) described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)), are introduced into the same cell individually, or together, or as part of a single nucleic acid.

(119) The host cells expressing the enzyme that is at least 80% identical to PmMDB1 (SEQ ID NO: 7) may be prokaryotic cells, such as bacterial cells (e.g., Escherichia coli cells), or eukaryotic cells, such as yeast cells or plant cells. In certain embodiments, the host cell is capable of expressing two or more kavalactone or flavokavain pathway enzymes described herein. In certain embodiments, the host cell is a bacteria cell and is a wildtype, mutant, recombinant, or genetically engineered form of Escherichia coli. In certain embodiments, the host cell is a yeast cell and is a wildtype, mutant, recombinant, or genetically engineered form of Saccharomyces cerevisiae. In certain embodiments, the host cell is a plant cell and is a wildtype, mutant, recombinant, or genetically engineered form of Nicotiana benthamiana.

(120) Production of Chalcone Compounds of Formula (VII)

(121) Some aspects of the present disclosure provides methods for producing a compound of Formula (VII) from a compound of Formula (II), or a salt thereof, and malonyl-CoA using an enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4), wherein:

(122) ##STR00038##

(123) In certain embodiments, the reaction of a compound of Formula (II) with malonyl-CoA to produce a compound of Formula (VII) utilizes three or more molar equivalents of malonyl-CoA relative to the compound of Formula (II). In certain embodiments, the reaction of a compound of Formula (II) with malonyl-CoA to produce a compound of Formula (VII) occurs in vitro. In certain embodiments, the reaction of a compound of Formula (II) with malonyl-CoA to produce a compound of Formula (VII) occurs in vivo.

(124) In certain embodiments, is a single bond. In certain embodiments, is a double bond.

(125) In certain embodiments, each of R.sub.1, R.sub.2, R.sub.3, R.sub.6, and R.sub.7 independently is hydrogen, optionally substituted, cyclic or acyclic aliphatic, or OR.sub.x, wherein R.sub.x is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.1 is hydrogen. In certain embodiments, R.sub.2 is hydrogen. In certain embodiments, R.sub.3 is hydrogen. In certain embodiments, R.sub.1 is —OH. In certain embodiments, R.sub.2 is —OH. In certain embodiments, R.sub.3 is —OH. In certain embodiments, R.sub.1 is —OCH.sub.3. In certain embodiments, R.sub.2 is —OCH.sub.3. In certain embodiments, R.sub.3 is —OCH.sub.3. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are hydrogen. In certain embodiments, R.sub.1 and R.sub.2 are hydrogen and R.sub.3 is —OH. In certain embodiments, R.sub.1 and R.sub.2 are hydrogen and R.sub.3 is —OCH.sub.3. In certain embodiments, R.sub.6 is hydrogen. In certain embodiments, R.sub.7 is hydrogen.

(126) In certain embodiments, each of R.sub.4 and R.sub.5 independently is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.4 and R.sub.5 are hydrogen.

(127) The methods to produce a compound of Formula (VII) include reacting malonyl-CoA with a compound of Formula (II) selected from the group consisting of:

(128) ##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044##

(129) The methods to produce a compound of Formula (VII) include culturing cells engineered to express an enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4). In certain embodiments, the enzyme is at least 80%, 85%, 90%, 95%, or 100% identical to PmCHS (SEQ ID NO: 4). In certain embodiments, the enzyme is purified before reacting with a compound of Formula (II). In certain embodiments, the enzyme is partially purified before reacting with a compound of Formula (II).

(130) In certain embodiments, the enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4) is a component in a fusion protein. A fusion protein may be created by joining two or more gene or gene segments that code for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. A polyfunctional protein is a single protein that has at least two different activities, wherein that functionality is a native biological function or the result of an engineered enzyme fusion. Thus, a fusion protein may include multiple activities such as those described herein for the kavalactone or flavokavain pathway enzymes described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)).

(131) The enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4) is heterologous to the host cell. In certain embodiments, the enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4) is recombinantly produced. In certain embodiments, the enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4) is obtained from a wildtype organism. In certain embodiments, the enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4) is obtained from a mutant organism. In certain embodiments, the enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4) is obtained from a genetically-modified organism. In certain embodiments, the organism is a non-human organism. In certain embodiments, the non-human organism is selected from group consisting of bacteria, yeast, and plant. In certain embodiments, the organism is a plant. In certain embodiments, the plant is Piper methysticum.

(132) A nucleic acid encoding the enzyme may be introduced into the cell in a vector (e.g., plasmids, viral vectors, cosmids, and artificial chromosomes). In certain embodiments, the nucleic acid is cDNA derived from the amino acid sequence of the enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4). In some embodiments multiple cDNAs comprising sequences complementary to different genes (e.g., 2, 3, 4, 5, or more genes) described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)), are introduced into the same cell individually, or together, or as part of a single nucleic acid.

(133) The host cells expressing the enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4) may be prokaryotic cells, such as bacterial cells (e.g., Escherichia coli cells), or eukaryotic cells, such as yeast cells or plant cells. In certain embodiments, the host cell is capable of expressing two or more kavalactone or flavokavain pathway enzymes described herein. In certain embodiments, the host cell is a bacteria cell and is a wildtype, mutant, recombinant, or genetically engineered form of Escherichia coli. In certain embodiments, the host cell is a yeast cell and is a wildtype, mutant, recombinant, or genetically engineered form of Saccharomyces cerevisiae. In certain embodiments, the host cell is a plant cell and is a wildtype, mutant, recombinant, or genetically engineered form of Nicotiana benthamiana.

(134) In certain embodiments, the method for producing a compound of Formula (VII) utilizes a compound of Formula (I), or a salt thereof, as the starting material and comprises the steps: condensing a compound of Formula (I), or a salt thereof, with coenzyme A (CoA) using an enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) to produce a compound of Formula (II); and reacting a compound of Formula (II), or a salt thereof, with malonyl-CoA using an enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4) to produce a compound of Formula (VII).

(135) Production of Methylated Chalcone Compounds of Formula (VIII)

(136) Some aspects of the present disclosure provides methods for producing a compound of Formula (VIII) from a compound of Formula (VII), or a salt thereof, and S-adenosylmethionine using an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). Some aspects of the present disclosure provides methods for producing a compound of Formula (VIII) from a compound of Formula (VII), or a salt thereof, and S-adenosylmethionine using an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). The structure of a compound of Formula (VII) and a structure of a compound of Formula (VIII) are as follows:

(137) ##STR00045##

(138) In certain embodiments, the reaction of a compound of Formula (VII) with S-adenosylmethionine to produce a compound of Formula (VIII) occurs in vitro. In certain, embodiments, the reaction of a compound of Formula (VII) with S-adenosylmethionine to produce a compound of Formula (VIII) occurs in vivo.

(139) In certain embodiments, is a single bond. In certain embodiments, is a double bond.

(140) In certain embodiments, each of R.sub.1, R.sub.2, R.sub.3, R.sub.1a, R.sub.2a, R.sub.3a, R.sub.6, and R.sub.7 independently is hydrogen, optionally substituted, cyclic or acyclic aliphatic, or OR.sub.x, wherein R.sub.x is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.1 is hydrogen. In certain embodiments, R.sub.2 is hydrogen. In certain embodiments, R.sub.3 is hydrogen. In certain embodiments, R.sub.1 is —OH. In certain embodiments, R.sub.2 is OH. In certain embodiments, R.sub.3 is —OH. In certain embodiments, R.sub.1 is —OCH.sub.3. In certain embodiments, R.sub.2 is —OCH.sub.3. In certain embodiments, R.sub.3 is —OCH.sub.3. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are hydrogen. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are —OH. In certain embodiments, R.sub.1 and R.sub.3 are —OH. In certain embodiments, R.sub.2 and R.sub.3 are —OH. In certain embodiments, R.sub.2 is —OCH.sub.3. In certain embodiments, R.sub.6 is hydrogen. In certain embodiments, R.sub.7 is hydrogen. In certain embodiments, both R.sub.6 and R.sub.7 are hydrogen.

(141) In certain embodiments, each of R.sub.4 and R.sub.5 independently is hydrogen or optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.4 and R.sub.5 are hydrogen.

(142) In certain embodiments, each of R.sub.12 and R.sub.13 independently is optionally substituted, cyclic or acyclic aliphatic. In certain embodiments, R.sub.12 and R.sub.13 are —CH.sub.3. In certain embodiments, R.sub.14 is hydrogen. In certain embodiments, R.sub.14 and R.sub.13 are —CH.sub.3. In certain embodiments, R.sub.12 is hydrogen. In certain embodiments, R.sub.12 is —CH.sub.3, R.sub.13 is —CH.sub.3, and R.sub.14 is hydrogen.

(143) In certain embodiments, R.sub.1a, R.sub.2a, and R.sub.3a are hydrogen. In certain embodiments, R.sub.1, R.sub.2, R.sub.1a and R.sub.2a are hydrogen and R.sub.3 is —OH. In these instances, a compound of Formula (VII) can provide different compounds of Formula (VIII) depending on the choice to utilize only an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5), or only an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6), or both enzymes. In certain embodiments, R.sub.13 is —CH.sub.3 when a compound of Formula (VII) is reacted with an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). In certain embodiments, R.sub.13 is hydrogen when a compound of Formula (VII) is reacted with an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, R.sub.13 is —CH.sub.3 when a compound of Formula (VII) is reacted with both an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) and an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, R.sub.3a is —OCH.sub.3 when a compound of Formula (VII) is reacted with an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). In certain embodiments, R.sub.3a is —OH when a compound of Formula (VII) is reacted with an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, R.sub.3a is —OCH.sub.3 when a compound of Formula (VII) is reacted with both an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) and an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, R.sub.12 is —CH.sub.3 and R.sub.14 is hydrogen or R.sub.12 is hydrogen and R.sub.14 is —CH.sub.3 when a compound of Formula (VII) is reacted with an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, R.sub.12 and R.sub.14 are hydrogen when a compound of Formula (VII) is reacted with an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). In certain embodiments, R.sub.12 is —CH.sub.3 and R.sub.14 is hydrogen or R.sub.12 is hydrogen and R.sub.14 is —CH.sub.3 when a compound of Formula (VII) is reacted with an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) and an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6).

(144) The methods to produce a compound of Formula (VIII) include reacting malonyl-CoA with a compound of Formula (VII) selected from the group consisting of:

(145) ##STR00046## ##STR00047## ##STR00048##
wherein X is

(146) ##STR00049##

(147) The methods to produce a compound of Formula (VIII) include culturing cells engineered to express an enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). The methods to produce a compound of Formula (VIII) include culturing cells engineered to express an enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, the enzyme is at least 80%, 85%, 90%, 95%, or 100% identical to PmOMT4 (SEQ ID NO: 5). In certain embodiments, the enzyme is at least 80%, 85%, 90%, 95%, or 100% identical to PmOMT1 (SEQ ID NO: 6). In certain embodiments, the enzyme is purified before reacting with a compound of Formula (VII). In certain embodiments, the enzyme is partially purified before reacting with a compound of Formula (VII).

(148) In certain embodiments, the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) is a component in a fusion protein. In certain embodiments, the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) is a component in a fusion protein. A fusion protein may be created by joining two or more gene or gene segments that code for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. A polyfunctional protein is a single protein that has at least two different activities, wherein that functionality is a native biological function or the result of an engineered enzyme fusion. Thus, a fusion protein may include multiple activities such as those described herein for the kavalactone or flavokavain pathway enzymes described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ID NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)).

(149) The enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) is heterologous to the host cell. The enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) is heterologous to the host cell. In certain embodiments, the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) is recombinantly produced. In certain embodiments, the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) is recombinantly produced. In certain embodiments, the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) is obtained from a wildtype organism. In certain embodiments, the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) is obtained from a mutant organism. In certain embodiments, the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) is obtained from a genetically-modified organism. In certain embodiments, the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) is obtained from a wildtype organism. In certain embodiments, the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) is obtained from a mutant organism. In certain embodiments, the enzyme that is at least 80% identical to PmOMT1 (SEQ ED NO: 6) is obtained from a genetically-modified organism. In certain embodiments, the organism is a non-human organism. In certain embodiments, the non-human organism is selected from group consisting of bacteria, yeast, and plant. In certain embodiments, the organism is a plant. In certain embodiments, the plant is Piper methysticum.

(150) A nucleic acid encoding the enzyme may be introduced into the cell in a vector (e.g., plasmids, viral vectors, cosmids, and artificial chromosomes). In certain embodiments, the nucleic acid is cDNA derived from the amino acid sequence of the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5). In certain embodiments, the nucleic acid is cDNA derived from the amino acid sequence of the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6). In some embodiments multiple cDNAs comprising sequences complementary to different genes (e.g., 2, 3, 4, 5, or more genes) described herein (i.e., 4-coumarate-CoA ligase Pm4CL1 (at least 80% identical to SEQ ID NO: 1), styrylpyrone synthase PmSPS1 (at least 80% identical to SEQ ID NO: 2), PmSPS2 (at least 80% identical to SEQ ID NO: 3), PmCHS (at least 80% identical to SEQ ID NO: 4), methyltransferase PmOMT4 (at least 80% identical to SEQ ID NO: 5), methyltransferase PmOMT1 (at least 80% identical to SEQ ED NO: 6), cytochrome P450 enzyme PmMDB1 (at least 80% identical to SEQ ID NO: 7), and NADPH-dependent reductase PmRDCT10 (at least 80% identical to SEQ ID NO: 8)), are introduced into the same cell individually, or together, or as part of a single nucleic acid.

(151) The host cells expressing the enzyme that is at least 80% identical to PmOMT4 (SEQ ID NO: 5) may be prokaryotic cells, such as bacterial cells (e.g., Escherichia coli cells), or eukaryotic cells, such as yeast cells or plant cells. The host cells expressing the enzyme that is at least 80% identical to PmOMT1 (SEQ ID NO: 6) may be prokaryotic cells, such as bacterial cells (e.g., Escherichia coli cells), or eukaryotic cells, such as yeast cells or plant cells. In certain embodiments, the host cell is capable of expressing two or more kavalactone or flavokavain pathway enzymes described herein. In certain embodiments, the host cell is a bacteria cell and is a wildtype, mutant, recombinant, or genetically engineered form of Escherichia coli. In certain embodiments, the host cell is a yeast cell and is a wildtype, mutant, recombinant, or genetically engineered form of Saccharomyces cerevisiae. In certain embodiments, the host cell is a plant cell and is a wildtype, mutant, recombinant, or genetically engineered form of Nicotiana benthamiana.

(152) In certain embodiments, the method for producing a compound of Formula (VIII) utilizes a compound of Formula (I), or a salt thereof, as the starting material and comprises the steps: condensing a compound of Formula (I), or a salt thereof, with coenzyme A (CoA) using an enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) to produce a compound of Formula (II); reacting a compound of Formula (II), or a salt thereof, with malonyl-CoA using an enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4) to produce a compound of Formula (VII); and methylating a compound of Formula (VII), or a salt thereof, with S-adenosylmethionine using an enzyme that at least 80% identical to PmOMT4 (SEQ ID NO: 5).

(153) In certain embodiments, the method for producing a compound of Formula (VIII) utilizes a compound of Formula (I), or a salt thereof, as the starting material and comprises the steps: condensing a compound of Formula (I), or a salt thereof, with coenzyme A (CoA) using an enzyme that is at least 80% identical to Pm4CL1 (SEQ ID NO: 1) to produce a compound of Formula (II); reacting a compound of Formula (II), or a salt thereof, with malonyl-CoA using an enzyme that is at least 80% identical to PmCHS (SEQ ID NO: 4) to produce a compound of Formula (VII); and methylating a compound of Formula (VII), or a salt thereof, with S-adenosylmethionine using an enzyme that at least 80% identical to PmOMT1 (SEQ ID NO: 6).

(154) In Vitro Reactions

(155) In vitro reactions are utilized in the present disclosure. In certain embodiments, the reactions use water as a solvent. In certain embodiments, the reaction is performed at room temperature. In certain embodiments, the reaction is performed for 10 minutes to 24 hours. In certain embodiments, the reaction is performed for 2 hours.

(156) The components of the reactions may include one of more of the following: buffer; MgCl.sub.2; ATP; CoA; malonyl-CoA; a compound of Formula (I), Formula (II), Formula (III), Formula (IV), or Formula (VII); S-adenosylmethionine; NADPH; and an enzyme described herein. In certain embodiments, the buffer is potassium phosphate of pH=7.6. In certain embodiments, the concentration of the buffer is 50 mM. In certain embodiments, the concentration of the MgCl.sub.2 is 2.5 mM. In certain embodiments, the concentration of the ATP is 3 mM. In certain embodiments, the concentration of CoA is 2 mM. In certain embodiments, the concentration of malonyl CoA is 3 mM. In certain embodiments the concentration of the compound described herein is 0.5 mM. In certain embodiments, the concentration of S-adenosylmethionine is 2 mM. In certain embodiments, the concentration of NADPH is 6 mM. In certain embodiments, the concentration of an enzyme described herein is 10 μg/ml final concentration of each enzyme used in the reaction.

EXAMPLES

(157) In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the methods, compositions, and systems provided herein and are not to be construed in any way as limiting their scope.

Example 1. Activity of Pm4CL1, PmSPS1, PmSPS2, and PmCHS

(158) Using purified recombinant enzymes expressed in Escherichia coli, we have shown the activity of PmSPS1, PmSPS2, and PmCHS in vitro (FIG. 4). This assay used p-coumaric acid as a substrate and utilized the purified 4-coumarate-CoA ligase Pm4CL1 to produce p-coumaroyl-CoA.

(159) The PmSPS1 and PmSPS2 enzymes can also utilize substrates derived from cinnamic acid variants phloretic acid and hydrocinnamic (phenylpropanoic) acid, which include a single bond instead of the double bond present in cinnamic acid (FIG. 5). This results in a 6-styryl-4-hydroxy-2-pyrone backbone with a single bond at C.sub.7-C.sub.8 position, which can be used to produce reduced kavalactones, such as 7,8-dihydrokavain or 7,8-dihydroyangonin. This activity was verified using in-vitro enzyme assays monitored by LC-MS (FIG. 6).

Example 2. Activity of PmOMT4 and PmOMT1

(160) Two methyltransferases, PmOMT4 and PmOMT1, add methyl groups to hydroxyl groups at various positions of the 6-styryl-4-hydroxy-2-pyrone backbone. PmOMT4 is the key methyltransferase that adds a methyl group to the 4-position, as seen in all kavalactones. In addition, PmOMT4 can methylate the C.sub.11 and C.sub.12 positions (if hydroxyl groups are present there), as found, for example, in 11-methoxyyangonin (FIG. 7). On the other hand, PmOMT1 adds a methyl group to the C.sub.10 position, as found, for example, in 10-methoxyyangonin (FIG. 7).

(161) The target hydroxyl sites of PmOMT1 and PmOMT4 were determined by coupled enzyme assays using different starting substrates (variants of cinnamic acid with different hydroxy modifications on the aromatic ring). The enzyme assay utilized Pm4CL1 and PmSPS1 to produce the 6-styryl-4-hydroxy-2-pyrone backbone, and increase in mass after adding the methyltransferases was monitored by LC-MS (FIG. 8).

Example 3. Activity of PmRDCT10

(162) The C.sub.5-C.sub.6 double bond in kavalactones can be reduced into a single bond by an NADPH-dependent reductase PmRDCT10, as demonstrated by another in vitro enzyme assay (FIG. 9). This reaction is essential to produce reduced kavalactones such as kavain or methysticin.

Example 4. Activity of PmMDB1

(163) The methylenedioxybridge bridge found at the C.sub.11-C.sub.12 position in several kavalactones (methysticin, dihydromethysticin, or dehydromethysticin) is formed by a cytochrome P450 enzyme PmMDB1, which belongs to the CYP719 family (FIG. 10).

(164) The activity of PmMDB1 was confirmed by Agrobacterium-mediated heterologous expression in Nicotiana benthamiana (FIG. 11). This assay utilized the native Nicotiana 4CL.

Example 5. Enzyme Amino Acid Sequences

(165) TABLE-US-00002 Pm4CL1 (SEQ ID NO: 1): MKMVVDTIATDRCVYRSKLPDIEIKNDMSLHNYCFQNIGAYRDNPCLING STGEVYTYGEVETTARRVAAGLHRMGVQQREVIMILLPNSPEFVFAFLGA SFRGAMSTTANPFYTPQEIAKQVKASGAKLIVTMSAYVDKVRDLAEERGV KVVCVDAPPPGCSHFSELSGADESELPEVDIDPDDVVALPYSSGTTGLPK GVMLTHRSQVTSVAQQVDGENPNLYFRPDDVLLCVLPLFHIYSLNSVLFC GLRVGAAILIMQKFEITALMELVQKYKVTIAPIVPPIVLAIAKSPLVDKY DLSSIRTVMSGAAPMGKELEDAVRAKLPNAKLGQGYGMTEAGPVLSMCLA FAKEPFEIKSGSCGTVVRNAQLKIVDPETGAYLPRNQPGEICIRGSQIMK GYLNDAAATQRTIDKEGWLHTGDIGYVDDDEELFIVDRLKEIIKYKGFQV APAELEAILITHPNIADAAVVPMKDEAAGEVPVAFVVTSNGSVISEDEIK QFISKQVVFYKRINRVFFVDSIPKAPSGKILRKDLRGRLAAGIPK PmSPS1 (SEQ ID NO: 2): MSKTVEDRAAQRAKGPATVLAIGTATPANVVYQTDYPDYYFRVTKSEHMT KLKNKFQRMCDRSTIKKRYMVLTEELLEKNLSLCTYMEPSLDARQDILVP EVPKLGKEAADEAIAEWGRPKSEITHLIFCTTCGVDMPGADYQLTKLLGL RSSVRRTMLYQQGCFGGGTVLRLAKDLAENNAGARVLVVCSEITTAVNFR GPSDTHLDLLVGLALFGDGAAAVIVGADPDPTLERPLFQIVSGAQTILPD SEGAINGHLREVGLTIRLLKDVPGLVSMNIEKCLMEAFAPMGIHDWNSIF WIAHPGGPTILDQVEAKLGLKEEKLKSTRAVLREYGNMSSACVLFILDEV RKRSMEEGKTTTGEGFDWGVLFGFGPGFTVETVVLHSMPIPKADEGR PmSPS2 (SEQ ID NO: 3): MSKMVEEHWAAQRARGPATVLAIGTANPPNVLYQADYPDFYFRVTKSEHM TQLKEKYKRICDKSAIRKRHLHLTEELLEKNPNICAHMAPSLDARQDIAV VEVPKLAKEAATKAIKEWGRPKSDITHLIFCTTCGVDMPGADYQLTTLLG LRPTVRRTMLYQQGCFAGGTVLRHAKDFAENNRGARVLAVCSEFTVMNFS GPSEAHLDSMVGMALFGDGASAVIVGADPDFAIERPLFQLVSTTQTIVPD SDGAIKCHLKEVGLTLHLVICNVPDLISNNMDKILEEAFAPLGIRDWNSI FWTAHPGGAAILDQLEAKLGLNKEKLKTTRTVLREYGNMSSACVCFVLDE MRRSSLEEGKTTSGEGLEWGILLGFGPGLTVETVVLRSVPISTAN PmCHS(SEQ ID NO: 4): MSKTVEEIWAAQRARGPATVLAIGTAAPANVVYQADYPDYYFRITKSEHM TELKEKFRRMCDKSMITKRHMHLSEELLKNNPDICAYMAPSLDARQDMVV VEVPKLGKEAAAKAIKEWGRPKSAITHLIFCTTSGVDMPGADFQLTKLLG LCPSVRRTMLYQQGCFAGGTVLRLAKDLAENNAGARVLVVCSETTAVTFR GPSETHLDSMVGQALFGDGASAIIVGADPDPVIERPLFQIVSAAQTILPD SDGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLKEAFAPLGIDDWNSIF WIVHPGGPAILDQVEAKLRLKVEKLKTTRTVLSEYGNMSSACVLFILDEM RRNSMEEGKATTGEGLHWGVLFGFGPGLTVETVVLHSLPIAEAN PmOMT4 (SEQ ID NO: 5): MEQAVFKDQSPSRDDIDEELFQSALYLSTAVVTVPAAIMAANDLDVLQII AKAGPGAHLSPTEIVSHLPTRNPNAAAALHRILRVLASHSILECSSRCEG EAKYGLRPVCKFFLNDKDGVSLNAMPSFVQSRVFIDSWQYMKDAVLEGVV PFEKAYGMPFYQFQAVNTKFKETFAKAMAAHSTLVVKICMLDTYNGFEGL TELMDVAGGTGSTLNLIVSKYPQIKGTNFDLICHVIEAAPNYPGVKHLSG DMFDSIPSAKNIIMKWILHNWSDEHCVKLLKNCYTSLPEFGKLIVVDSIV GEDVDAGUTTTNVFGCDFTMLTFFPNAKERTREEFQDLAKASGFSTFKPI CCAYGVWVMEFHK PmOMT1 (SEQ ID NO: 6): MNDQELHGYSQNAQPQLWNLLLSFINSMSLKCAVELGIPDIIHSHAQTPI NITDLAASIPIPPNKTSQFRRLMRLLVHSNVFSVHKREDGDEGFLLTPMS RILVTSNDNNGGNLSPFVSMMVDPSLVSPWHFLGQWLKGNDTQGTPFRMC HGEEMWDWANKYPDFNKICFNMAMVCDSQYLMKIIVKKCATAFEGKRSLI DVGGGTGGAARSIAEAFPDIQEVSVLDLPHVVAGLPNDSRVICFVGGDMF HTIPPADVVLLKAIFHGWNDEECIKILKNCKICAIPSKEEGGKVMILDMV VNSAPGDHMITEDQYFMDLMMITYARGLERDENEWKKLFKDAGFTSYKIT HGLGTSSLIELYP PmMDB1 (SEQ ID NO: 7): MEQAQWVDPTLLPAFVGIIFFFLGMFFGRSSLGAGKGAAPRSTSSTEWPD GPPKLPIIGNLHQLNKGGELVHHNLAKLAQSYDRAMTIWVGSWGPMIVVS DADLAWEVLVTKSPDFAGRVLSKLSHLFNANYNTVVAYDAGPQWQSLRRG LQHGPLGPAHVSAQARFHEEDMICLLVSDMMRAAQKGGSNGVVEPLAYVR RATIRFLSRLCFGEAFNDEAFVEGMDEAVEETIGATGHARILDAFYFTRH LPIIRRSFIDTVNAKKKIESLVRPLLSRPAPPGSYLHFLLSTDAPENMII FRIFEVYLLGVDSTASTTTWALAFLVSNQQAQEKLHNELAQYCASQNNQI IKADDVGKLSYLLGVVKETMRMKPIAPLAVPHKTLICETMLDGKRVAAGT TVVVNLYAVHYNPKLWPEPEQFRPERFVVGASGGNGGGSSEYMLQSYLPF GGGMRSCAGMEVGKLQVAMVVANLVMAFKWLPEEEGKMPDLAEDMTFVLM MKKPLAAKIVPRA PmRDCT10 (SEQ ID NO: 8): METERKSRICVTGAGGFVASWVVKLFLSKGYLVHGTVRDLGEEKTAHLRK LEGAYHNLQLFKADLLDYESLLGAITGCDGVLHVATPVPSSKTAYSGTEL VKTAVNGTLNVLRACTEAKVKKVIYVSSTAAVLVNPNLPKDKIPDEDCWT DEEYCRTTPFFLNWYCIAKTAAEKNALEYGDKEGINVISICPSYIFGPML QPTINSSNLELLRLMKGDDESIENKFLLMVDVRDVAEAILLLYEKQETSG RYISSPHGMRQSNLVEKLESLQPGYNYHICNFVDIKPSWTMISSEKLKKL GWKPRPLEDTISETVLCFEEHGLLENE

Example 6. cDNA Sequences

(166) cDNA Sequence Encoding for Pm4CL1 (SEQ ID NO: 9)

(167) TABLE-US-00003 cDNA Sequence Encoding for Pm4CL1 (SEQ ID NO: 9) ATGAAGATGGTAGTAGACACTATTGCTACTGATCGATGTGTATACCGGTCTAAGCTGCCGGACATTGAGATCAAGAACGACAT GTCGTTGCACAATTATTGTTTCCAGAACATTGGTGCTTACCGGGACAATCCTTGTCTCATCAATGGCAGCACCGGCGAGGTGT ACACGTACGGCGAGGTGGAGACGACGGCGAGGAGGGTGGCCGCCGGGCTGCACCGGATGGGGGTGCAGCAGCGGGAGGTGATC ATGATCCTCCTCCCCAACTCGCCGGAGTTCGTCTTCGCCTTTCCTCGGCGCCTCCTTCCGCGGGGCCATGTCCACCACCGCCA ACCCCTTCTACACGCCGCAGGAGATCGCCAAGCAGGTCAAGGCCTCCGGCGCGAAGCTCATCGTCACCATGTCCGCCTACGTC GACAAGGTCAGGGACCTGGCCGAGGAGCGCGGCGTCAAAGTGGTGTGCGTCGACGCGCCGCCCCCGGGGTGCTCCCACTTCTC CGAGCTGTCCGGCGCCGACGAGTCGGAGCTGCCCGAGGTGGATATTGACCCCGACGACGTGGTGGCGCTGCCATACTCCTCCG GCACCACCGGCCTCCCTAAAGGAGTGATGCTCACACACCGCAGCCAGGTGACGAGCGTTGCCCAGCAAGTCGACGGCGAGAAC CCGAATCTATACTTCCGGCCAGACGACGTCCTGCTCTGCGTTCTTCCCCTCTTCCACATCTACTCCCTCAACTCGGTGCTCTT CTGCGGCCTGCGCGTCGGGGCGGCGATCCTCATCATGCAGAAGTTCGAGATCACGGCGCTGATGGAGCTGGTGCAGAAGTACA AGGTGACCATTGCGCCCATCGTTCCGCCCATCGTTCTTGCCATCGCCAAGAGCCCGCTCGTCGACAAGTACGACTTGTCGTCC ATTCGGACGGTGATGTCCGGCGCCGCCCCGATGGGGAAGGAGCTCGAAGACGCCGTCCGGGCCAAGCTTCCCAACGCCAAGCT CGGCCAGGGCTATGGGATGACGGAGGCAGGGCCAGTGCTGTCCATGTGrnGGCCTTCGCCAAGGAGCCCTTCGAGATCAAGTC TGGTTCTTGCGGCACCGTGGTCAGGAACGCCCAGCTCAAGATCGTCGACCCAGAAACCGGTGCCTACCTGCCCAGAAACCAAC CCGGCGAAATTTGCATCCGAGGCTCCCAAATCATGAAAGGGTATCTTAATGACGCGGCGGCTACGCAGAGGACGATCGACAAG GAAGGGTGGCTGCACACCGGCGACATTGGCTATGTCGACGACGACGAGGAGCTCTTCATTGTCGATAGGTTGAAGGAGATCAT TAAGTACAAGGGCTTCCAAGTCGCCCCTGCCGAGCTCGAAGCCATTCTCATTACTCACCCTAACATTGCTGATGCCGCTGTTG TCCCGATGAAAGATGAGGCAGCAGGGGAAGTGCCAGTGGCATTTGTGGTGACCTCCAATGGATCAGTCATCAGTGAGGATGAG ATCAAGCAGTTCATTAGCAAGCAGGTGGTGTTCTACAAGCGAATCAATCGAGTCTTTTTCGTTGATTCAATTCCTAAAGCACC CTCTGGGAAGATTTTGAGGAAGGATTTGAGGGGAAGATTGGCAGCTGGTATACCCAAGTAG cDNA Sequence Encoding for PmSPS1 (SEQ ID NO: 10) ATGTCGAAGACGGTGGAGGATCGGGCAGCGCAGCGGGCAAAGGGGCCGGCAACAGTGCTGGCCATCGGCACGGCTACGCCGGC CAATGTGGTGTACCAGACCGATTTATCCGGACTACTACTTCAGGGTCACCAAGAGCGAGCATATGACCAAACTCAAGAACAAG TTTCAACGCATGTGCGACAGGTCGACGATAAAGAAGAGGTACATGGTTTTGACAGAGGAGCTGCTAGAGAAGAATCTGAGTTT GTGCACCTACATGGAACCCTCCCTCGACGCCCGCCAAGACATTCTCGTGCCGGAGGTCCCCAAGCTCGGCAAGGAGGCCGCCG ACGAGGCCATCGCCGAATGGGGACGCCCCAAGTCGGAAATCACCCACCTCATCTTTTGCACTACCTGCGGCGTCGACATGCCC GGCGCCGACTACCAGCTCACCAAGCTCCTCGGTCTCCGCTCCTCCGTCCGTCGCACCATGCTCTATCAGCAGGGATGCTTTGG CGGAGGCACCGTTCTCCGCCTCGCCAAGGACCTCGCCGAGAACAACGCTGGTGCCCGCGTCCTCGTCGTCTGCTCCGAGATCA CCACTGCCGTCAACTTCCGAGGGCCTTCCGACACCCACCTCGACTTATTGGTCGGCTTAGCCCTGTTCGGCGACGGTGCGGCC GCGGTCATAGTCGGTGCGGATCCAGATCCTACCCTCGAGCGGCCGCTCTTTCAAATCGTATCTGGAGCACAGACGATTTCTAC CGGACTCGGAGGGGGCCATCAACGGCCATCTCCGGGAGGTGGGGCTAACCATCCGCCTACTCAAGGACGTACCTGGGCTTGTG TCGATGAACATTGAGAAGTGCCTCATGGAGGCGTTTGCACCGATGGGCATCCACGACTGGAACTCCATCTTTTGGATAGCCCA TCCCGGGGGGCCCACCATACTAGACCAAGTGGAGGCCAAGCTGGGTCTAAAGGAGGAGAAGCTCAAGTCGACGAGGGCTGTTC TGAGGGAGTATGGCAACATGTCTAGCGCCTGTGTCTTGTTCATACTGGACGAGGTAAGGAAGAGGAGCATGGAGGAGGGGAAG ACGACAACCGGTGAGGGGTTCGATTGGGGAGTTCTATTCGGCTTTGGGCCTGGCTTCACAGTGGAGACCGTCGTCTTGCACAG CATGCCCATCCCCAAAGCCGATGAAGGCAGATAA cDNA Sequence Encoding for PmSPS2 (SEQ ID NO: 11) ATGTCGAAGATGGTGGAGGAGCATTGGGCAGCGCAGCGGGCGAGGGGACCGGCGACAGTGCTGGCCATCGGCACTGCAAATCC TCCCAATGTGTTGTACCAGGCAGATTATCCCGACTTCTACTTTAGGGTCACCAAGAGTGAGCACATGACCCAGCTAAAGGAGA AGTTTAAACGTATATGTGATAAGTCAGCAATAAGAAAGCGCCACCTCCATCTAACCGAGGAGCTGCTGGAGAAGAACCCTAAC ATATGTGCACACATGGCCCCCTCCCTCGACGCCCGGCAAGACATTGCGGTGGTGGAGGTCCCCAAGCTAGCCAAAGAAGCTGC AACCAAGGCCATCAAGGAGTGGGGGCGACCCAAGTCCGACATCACCCACCTCATCTTCTGCACCACCTGCGGCGTGGACATGC CCGGCGCCGACTACCAACTCACCACGCTCCTCGGCCTCCGCCCCACGGTCCGCCGCACCATGCTCTACCAACAGGGCTGCTTC GCCGGCGGCACAGTCCTTCGCCATGCCAAGGACTTCGCCGAGAACAATAGGGGTGCTCGTGTCCTCGCCGTCTGCTCGGAGTT CACCGTCATGAACTTCAGCGGACCGTCGGAGGCCCACTTAGACAGCATGGTCGGTATGGCGCTGTTCGGTGATGGCGCCTCGG CTGTCATCGTCGGCGCCGATCCTGACTTTGCCATTGAACGACCGCTCTTTCAACTGGTTTCTACAACACAAACTATTGTCCCG GACTCGGACGGAGCCATCAAGTGCCATCTCAAGGAGGTGGGCCTAACCCTGCATCTCGTTAAGAATGTACCAGATCTCATATC AAATAACATGGACAAGATCCTCGAAGAGGCATTTGCACCATTGGGCATCAGAGATTGGAACTCAATCTTTTGGACAGCTCATC CAGGTGGAGCAGCCATACTCGACCAGTTGGAGGCCAAGCTCGGTCTGAACAAGGAGAAGCTCAAGACTACAAGAACAGTTCTG AGGGAGTATGGAAACATGTCCAGCGCCTGTGTTTGTTTCGTCCTGGACGAGATGAGGAGAAGTAGCTTGGAGGAGGGGAAGAC AACGTCCGGGGAAGGGTTGGAATGGGGAATTCTGCTAGGGTTTGGGCCTGGGTTGACAGTGGAGACAGTCGTCTTGCGTAGCG TACCCATCTCGACAGCCAATTAA cDNA Sequence Encoding for PmCHS (SEQ ID NO: 12) ATGTCGAAGACCGTAGAGGAGATTTGGGCGGCGCAGCGGGCGAGGGGACCAGCCACGGTGCTGGCCATCGGCACTGCTGCGCC GGCCAATGTGGTGTACCAGGCCGATTATCCGGACTACTACTTTAGGATCACCAAGAGCGAGCACATGACAGAGCTCAAGGAGA AGTTCCGACGAATGTGTGACAAGTCGATGATAACGAAGCGGCACATGCACTTGTCGGAGGAGCTGTTGAAAAACAACCCTGAC ATCTGTGCCTACATGGCCCCTTCCCTCGACGCCCGCCAAGATATGGTCGTGGTGGAGGTACCCAAGCTCGGCAAGGAGGCGGC CGCCAAGGCCATCAAGGAATGGGGCCGCCCAAAGTCGGCCATCACCCACCTCATCTTCTGCACCACCTCCGGCGTCGACATGC CCGGCGCCGATTTCCAGCTCACCAAGCTACTCGGCCTCTGCCCCTCCGTTCGCCGCACCATGCTCTACCAGCAGGGCTGCTTC GCCGGCGGTACGGTTCTCCGCCTTGCCAAGGACCTCGCCGAGAACAATGCGGGCGCGAGGGTCCTCGTCGTCTGCTCCGAGAT CACCGCCGTCACCTTCCGCGGCCCCTCGGAGACTCACCTCGATAGCATGGTCGGCCAGGCCCTGTTCGGTGATGGTGCCTCTG CCATCATCGTCGGTGCCGACCCCGACCCCGTCATAGAAAGGCCACTCTTTCAAATTGTATCTGCGGCTCAGACCATCCTTCCC GACTCGGATGGGGCAATAGACGGCCATCTCCGAGAAGTGGGTCTAACCTTCCACCTCCTCAAGGACGTACCTGGGCTCATCTC AAAGAACATCGAGAAGAGCCTAAAGGAGGCGTTTGCACCGCTGGGCATCGACGACTGGAACTCGATATTTTGGATTGTTCATC CAGGCGGGCCGGCCATTCTAGACCAGGTGGAGGCGAAGCTGCGTCTGAAAGTGGAGAAGCTGAAGACAACGAGAACAGTTTTG AGTGAGTACGGGAATATGTCGAGCGCTTGCGTGTTGTTCATACTTGACGAGATGAGGAGGAACAGCATGGAAGAAGGGAAGGC GACGACCGGTGAAGGGTTACATTGGGGAGTTTTGTTTGGTTTTGGGCCGGGCTTGACAGTGGAGACGGTCGTCTTGCATAGTT TGCCCATCGCCGAGGCCAACTAA cDNA Sequence Encoding for PmOMT4 (SEQ ID NO: 13) ATGGAGCAAGCTGTGTTCAAAGACCAATCCCCAAGCAGGGATGATATTGATGAAGAGCTCTTTCAATCTGCTCTATATCTTAG CACTGCGGTTGTCACCGTGCCGGCGGCAATCATGGCTGCAAATGACCTTGACGTGCTGCAGATAATTGCCAAAGCTGGCCCAG GTGCTCACCTATCTCCGACAGAGATTGTCAGCCACCTTCCCACCCGTAACCCTAATGCCGCGGCGGCGCTTCACCGGATACTC CGAGTACTAGCCAGCCACTCCATCCTTGAATGCTCGTCGAGATGCGAGGGCGAGGCAAAATATGGATTAAGGCCGGTGTGCAA GTTCTTTCTCAATGATAAGGATGGTGTCTCCTTGAATGCCATGCCATCCTTCGTTCAAAGTAGAGTTTTTATAGATAGCTGGC AATATATGAAAGATGCTGTTCTTGAGGGGGTAGTCCCCTTTGAGAAAGCCTATGGTATGCCTTTTTATCAGTTTCAAGCAGTG AACACCAAATTCAAAGAAACCTTCGCCAAAGCCATGGCTGCTCACTCAACTTTGGTAGTAAAAAAGATGCTTGACACATACAA TGGGTTTGAGGGACTCACTGAGTTGATGGATGTTGCTGGTGGAACCGGTTCCACCCTCAACCTCATTGTCTCCAAATACCCAC AAATCAAAGGCACAAACTTTGATCTCAAACATGTCATTGAGGCCGCACCAAACTACCCTGGGGTGAAGCATTTGAGTGGGGAC ATGTTTGATAGCATTCCAAGTGCAAAGAACATTATTATGAAGTGGATACTACATAATTGGAGCGACGAGCACTGTGTAAAACT CCTCAAGAACTGCTACACTTCCTTACCAGAATTTGGGAAGTTGATTGTGGTTGATTCCATTGTGGGTGAGGATGTTGATGCTG GTTTGACGACAACAAATGTCTTTGGATGCGACTTCACAATGCTAACTTTCTTCCCCAATGCAAAAGAGAGGACCCGTGAAGAA TTCCAAGACCTGGCCAAAGCTAGTGGCTTCTCAACGTTCAAACCGATCTGCTGCGCCTATGGCGTGTGGGTTATGGAATTTCA CAAATAA cDNA Sequence Encoding for PmOMT1 (SEQ ID NO: 14) ATGAATGATCAAGAGTTGCATGGATACTCACAAAATGCTCAACCTCAGCTATGGAACCTCCTGTTGAGCTTCATAAATTCCAT GTCCCTTAAGTGTGCAGTGGAGTTGGGCATCCCCGATATAATACATAGCCATGCCCAAACACCAATCAACATAACCGACCTTG CTGCCTCCATACCCATTCCCCCAAACAAAACAAGCCAATTCCGCCGACTCATGCGCCTCCTGGTTCACTCCAACGTCTTTTCC GTCCATAAACGTGAGGATGGTGATGAGGGGTTCCTCCTAACTCCTATGTCCAGGATCCTTGTCACGTCGAACGACAATAATGG AGGTAACTTGTCACCCTTTGTTTCCATGATGGTTGATCCGTCCCTGGTGTCTCCATGGCACTTCCTTGGTCAATGGCTCAAAG GCAATGACACCCAAGGCACACCATTTCGCATGTGCCATGGTGAAGAAATGTGGGACTGGGCCAACAAGTACCCGGACTTCAAC AAGAAGTTCAACATGGCGATGGTCTGTGACAGCCAGTATTTAATGAAAATTATTGTGAAGAAGTGCGCCACTGCCTTTGAAGG CAAGAGGTCCCTGATTGACGTCGGTGGCGGGACTGGTGGCGCCGCACGGTCTATTGCCGAAGCATTTCCAGACATACAGGAGG TGTCTGTATTGGATCTTCCTCATGTGGTTGCAGGTTTGCCCAATGACTCGAGGGTGAAGTTTGTTGGAGGAGACATGTTCCAC ACCATCCCTCCCGCTGATGTTGTCTTATTGAAGGCGATTTTTCATGGTTGGAATGATGAGGAGTGCATCAAGATATTGAAGAA CTGCAAGAAGGCAATTCCAAGCAAGGAAGAGGGAGGCAAGGTGATGATATTGGACATGGTGGTCAATTCCGCCCCGGGTGACC ATATGATTACAGAAGATCAATATTTTATGGATTTGATGATGATAACCTACGCAAGAGGATTGGAGAGAGACGAGAATGAATGG AAGAAGCTGTTTAAAGATGCAGGTTTCACATCGTACAAGATCACCCACGGGCTTGGAACGAGTTCGCTTATCGAGCTCTACCC TTAG cDNA Sequence Encoding for PmMDB1 (SEQ ID NO: 15) ATGGAGCAAGCTCAATGGGTCGACCCAACTCTGCTCCCTGCATTTGTGGGCATCATCTTCTTCTTCCTTGGCATGTTCTTTGG AAGGAGTTCTTTGGGAGCTGGGAAGGGTGCAGCGCCTAGAAGCACCAGTTCTACCGAGTGGCCAGACGGCCCTCCAAAGCTGC CCATCATCGGCAACCTGCACCAGCTCAACAAAGGCGGGGAGCTGGTCCACCACAACCTCGCCAAGCTCGCCCAGTCCTACGAC CGCGCCATGACCATCTGGGTCGGCAGCTGGGGCCCCATGATCGTCGTCAGCGACGCCGACCTTGCATGGGAGGTCCTCGTCAC CAAGTCGCCGGATTCGCCGGCCGGGTGCTCTCCAAGCTCTCGCACTTGTTCAACGCCAACTACAACACCGTCGTCGCCTACGA CGCCGGGCCGCAATGGCAGTCGCTCCGGCGAGGTCTGCAGCACGGGCCGCTCGGCCCCGCCCATGTTTCTGCGCAGGCTCGTT TCCACGAAGAAGACATGAAGCTCCTGGTGAGCGACATGATGAGAGCAGCACAGAAAGGTGGTAGCAATGGAGTGGTTGAACCT CTGGCCTATGTCCGGCGAGCCACTATCCGATTTCTGTCTCGTCTATGCTTTGGGGAGGCCTTCAACGACGAGGCGTTCGTGGA GGGGATGGACGAAGCAGTGGAGGAGACCATCGGAGCCACTGGCCATGCACGCATCCTCGACGCCTTCTACTTCACTCGCCACC TCCCTATCATCCGCCGCAGCTTCATAGATACCGTCAACGCCAAGAAGAAGATCGAGAGCCTTGTCCGGCCGTTGCTCTCCCGG CCGGCGCCACCGGGGTCTTACCTCCACTTCCTCCTTTCCACCGACGCGCCGGAGAATATGATCATCTTTCGAATATTCGAAGT CTACTTGCTGGGCGTGGACAGCACCGCCTCCACCACCACATGGGCTCTCGCCTTCCTCGTCTCCAACCAACAGGCGCAGGAGA AGCTCCACAATGAGCTCGCCCAGTACTGCGCCAGCCAGAACAATCAGATCATCAAAGCAGACGACGTCGGAAAGCTGTCGTAC CTGCTCGGGGTAGTGAAGGAGACGATGAGGATGAAGCCGATAGCGCCGCTGGCCGTCCCCCACAAGACGCTCAAGGAGACGAT GCTCGACGGAAAGAGGGTGGCGGCGGGAACGACGGTGGTAGTGAACCTCTATGCCGTCCACTACAACCCGAAGCTATGGCCGG AGCCGGAGCAGTTCCGCCCGGAGAGGTTCGTGGTCGGCGCCAGCGGCGGCAATGGTGGGGGGTCTTCCGAGTACATGCTGCAG TCGTACCTGCCCTTTGGAGGGGGGATGAGGTCCTGCGCAGGGATGGAGGTGGGAAAGTTGCAGGTGGCGATGGTCGTGGCCAA CCTTGTGATGGCATTTAAATGGTTGCCGGAGGAGGAGGGGAAGATGCCGGACCTGGCTGAAGACATGACCTTCGTGCTCATGA TGAAGAAGCCATTGGCTGCCAAAATCGTTCCACGTGCATGA cDNA Sequence Encoding for PmRDCT10 (SEQ ID NO: 16) ATGGAGACTGAGAGGAAGTCCAGGATCTGTGTCACCGGGGCAGGAGGCTTTGTAGCCTCTTGGGTCGTCAAGCTTTTCCTCTC CAAAGGTTATCTTGTCCATGGCACTGTCAGAGACCTCGGAGAAGAGAAGACTGCCCATTTGAGGAAGTTGGAGGGTGCGTACC ATAATCTGCAGCTGTTCAAGGCTGACTTGTTGGATTATGAGTCCTTGCTCGGGGCCATTACTGGCTGCGATGGAGTTCTCCAT GTTGCAACTCCTGTTCCTTCGAGTAAAACTGCTTATTCCGGAACTGAGTTGGTCAAGACTGCTGTGAATGGAACTCTGAATGT GCTCAGGGCATGTACAGAGGCAAAAGTGAAAAAGGTCATCTATGTTTCATCTACTGCCGCTGTTTTGGTGAATCCTAATTTAC CCAAGGATAAAATCCCGGACGAAGATTGTTGGACAGACGAAGAGTACTGCAGGACAACTCCGTTCTTCCTGAATTGGTATTGC ATCGCCAAAACAGCAGCCGAAAAGAATGCCTTGGAATATGGAGATAAAGAAGGGATCAACGTTATATCTATTTGCCCTTCATA CATCTTTGGACCTATGCTTCAACCGACAATTAATTCAAGCAACTTGGAATTGTTGAGGCTAATGAAAGGAGATGACGAAAGCA TAGAAAACAAATTTCTGCTGATGGTGGATGTGCGAGATGTTGCTGAAGCAATTTTACTATTATATGAGAAGCAAGAAACATCA GGGAGATACATTTCTTCGCCGCATGGTATGCGACAAAGCAACTTGGTTGAGAAGCTGGAGAGCCTGCAGCCGGGCTACAATTA TCATAAGAACTTTGTGGATATTAAACCTAGTTGGACAATGATCAGCTCAGAAAAGCTCAAGAAACTTGGTTGGAAACCTAGAC CACTTGAGGACACTATTTCTGAAACAGTGCTGTGTTTTGAAGAGCATGGTTTGCTGGAAAATGAATAG

REFERENCES

(168) 1. Y. N. Singh, Kava: an overview. Journal of Ethnopharmacology 37, 13-45 (1992). 2. V. Lebot, J. Levesque, The Origin And Distribution Of Kava (Piper methysticum Forst. F., Piperaceae): A Phytochemical Approach. Allertonia 5, 223-281 (1989). 3. J. Sarris, E. LaPorte, I. Schweitzer, Kava: A Comprehensive Review of Efficacy, Safety, and Psychopharmacology. Australian & New Zealand Journal of Psychiatry 45, 27-35 (2011). 4. K. Shinomiya et al, Effects of kava-kava extract on the sleep-wake cycle in sleep-disturbed rats. Psychopharmacology 180, 564-569 (2005). 5. S. Caimey, P. Maruff, A. R. Clough, The neurobehavioural effects of kava. Australian and New Zealand Journal of Psychiatry 36, 807-662 (2002). 6. H. C. Chua et al., Kavain, the Major Constituent of the Anxiolytic Kava Extract, Potentiates GABA.sub.A Receptors: Functional Characteristics and Molecular Mechanism. PLoS One 11, e0157700 (2016). 7. A. Ligresti, R. Villano, M. Allara, I. Ujvary, V. Di Marzo, Kavalactones and the endocannabinoid system: The plant-derived yangonin is a novel CB1 receptor ligand. Pharmacological Research 66, 163-169 (2012). 8. L. D. Dinh et al., Interaction of various Piper methysticum cultivars with CNS receptors in vitro. Planta Med 67, 306-311 (2001). 9. N. Abu, The flavokawains: uprising medicinal chalcones. Cancer Cell Int 12, (2013).

EQUIVALENTS AND SCOPE

(169) In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

(170) Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

(171) This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

(172) Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.