BIOSYNTHESIS OF CANNFLAVIN A AND B

Field

The present invention relates to polypeptides. More specifically, the present invention is, in embodiments, concerned with polypeptides encoding enzymes involved in the biosynthesis of Cannflavin A and/or Cannflavin B and related compositions and methods.

Background

Flavonoids are compounds produced by many plants that influence the color of flowers, among other things. Flavonoids are defined by a 15-carbon backbone that includes two phenyl groups. Like terpenoids, they are“secondary metabolites,” advantageous to the plant (attracting a pollinator, inhibiting growth of a mold, etc.) but not“primary” components like the proteins, lipids, and carbohydrates needed for life itself.

In the 1980s, Dr. Marilyn Barrett identified two prenylated flavonoids in cannabis which were previously unknown. She named them“Cannflavin” A and B. Cannflavins are now being studied for anti inflammatory activity. To date, Cannflavins have been obtained primarily through their extraction from cannabis plant material and their synthetic pathways in cannabis were unknown.

Manassi et al. (Org Lett. 2008 Jun 5; 10(11 ):2267-70) describe the use of orthogonal protecting groups and the development of a modified Robinson flavone synthesis that avoids harsh acidic conditions to develop a regioselective synthesis of 6- and 8-prenylflavones from the same prenylated disilylated phloracetophenone, targeting Cannflavin B, the COX-inhibiting principle of marijuana, and its unnatural isomer Isocannflavin B as model compounds.

U.S. Patent No. 6,403, 126 describes a method of extracting cannabinoids, cannflavins, and/or essential oils from hemp and/or of producing a whole hemp extract lacking A ⁹ -THC. The industrial hemp is harvested and the chaff is threshed from the seeds. The chaff is then ground and the ground chaff is extracted with an organic solvent. The extract is then loaded onto a chromatographic column selected to fractionate specific cannabinoids, cannflavins, and essential oils.

U.S. Patent Application Publication No. 2015/0297653 describes methods of obtaining an extract of Cannabis plant material as well as subsequent processing of the extract to provide a concentrate of Cannabis. Also described are pharmaceutical dosage forms (e.g. , oral thin films and transdermal patches) that include the concentrate (or extract) of Cannabis, as well as methods of medical treatment that include administering the pharmaceutical dosage forms.

U.S. Patent No. 9,687,469 describes a pharmaceutical composition for the prevention and treatment of cancer with specific flavonoid-based compounds selected from among the groups of Flavone, Flavanone and Flavanol, a method for the prevention and treatment of cancer and inflammation using the specific flavonoid-based pharmaceutical compositions, a method for isolating the flavonoid- based pharmaceutical compositions from raw plant material, and a method for synthesizing said specific flavonoid-based pharmaceutical compositions.

U.S. Patent No. 9,730,911 describes the extraction of pharmaceutically active components from plant materials, and more particularly to the preparation of a botanical drug substance (BDS) for incorporation in to a medicament. It also relates to a BDS, for use in pharmaceutical formulations. In particular it relates to BDS comprising cannabinoids obtained by extraction from cannabis. European Patent No. 2 044 935 relates to a composition comprising at least one non psychotropic cannabinoid and/or at least one phenolic or flavonoid compound and/or Denbinobin and their uses for the prevention and treatment of gastrointestinal inflammatory diseases and for the prevention and treatment of gastrointestinal cancers. It also relates to a phytoextract obtained from the plant Cannabis sativa, more particularly from the selected variety CARMA.

U.S. Patent Application Publication Nos. 2014/0057251 and 2016/0177404 describe the generation of a draft de novo reference sequence for the Cannabis (C.) sativa and C. indica genomes as well as four full length contiguous sequences with homology to THCA and CBDA synthases and 10 partially homologous contigs with truncated ORFs. Described is an (one or more) isolated sequence (e g., nucleic acid sequence, DNA, RNA, genomic sequence, polypeptide) of a Cannabis genome and uses thereof.

International Patent Application Publication No. WO 2008/034648 describes a process for the production of a fine chemical in an organism such as a microorganism, a non-human animal or plant, or in a part thereof. The invention furthermore relates to nucleic acid molecules, antisense, RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, co-suppression molecule, or ribozyme molecules, or polypeptides, nucleic acid constructs, vectors, antibodies, host cells, plant tissue, propagation material, harvested material, plants, microorganisms as well as agricultural compositions and to their use.

U.S. Patent Application Publication No. 2017/0114373 describes methods for producing vanilloid compounds in a recombinant host, and in particular for converting a protocatechuic aldehyde into a substantially pure vanilloid. It further relates to novel yeast strains that are suitable for producing such vanilloid compounds.

There is a need for alternative therapies to overcome or mitigate at least some of the deficiencies of the prior art, and/or to provide a useful alternative.

Description of the Drawings

The present invention will be further understood from the following description with reference to the Figures, in which:

Figure 1. Enzymatic characterization of CsOMT6. (A) Chemical structures of the three flavones (Apigenin, Luteolin and Chrysoeriol) and two flavonols (Quercetin and Kaempferol) that typically accumulate in C. sativa and that were tested as potential substrates for CsOMT6. (B) Relative enzyme activity and substrate preference. Recombinant CsOMT6 was provided with the selected flavonoids above, along with [ ¹⁴ C]-SAM as a methyl donor, and O-methyltransferase enzyme activity was monitored. Data are the means ± SD from three independent experiments and are presented as relative activity compared to that observed with the preferred substrate, quercetin.

Figure 2. Phylogenetic analysis of aromatic prenyltransferases from C. sativa. The eight putative CsPTs identified in the C. sativa genome were aligned with the amino acid sequences of various plant aromatic prenyltransferases that have been previously identified using Clustal W and a Neighbor-joining phylogenetic tree (1000 replicates) to illustrate their evolutionary relationships was then constructed using the MEGA 6.0 software package. Bootstrap values (> 60%) are indicated at the nodes of each branch and the branch lengths are proportional to the number of amino acid substitutions per sequence. Note that the eight CsPTs (bolded) distinctly fall into three groups, which are involved in either tocopherol (II), plastoquinone (V), or terpenophenolic (VI) biosynthesis. Figure 3. Enzymatic synthesis of 6-prenylflavone standards using recombinant GuA6DT from Glycyrrhiza uralensis. Yeast microsomes containing GuA6DT were supplied apigenin, chrysoeriol, or luteolin as flavone substrates, along with DMAPP as a prenyl donor. The reaction products were resolved by HPLC and representative chromatograms are shown that illustrate the separation of the 6- prenylflavones from their corresponding substrate. The Q-TOF mass spectra (inset) of each enzymatic product indicates their exact mass, which is consistent with the addition of a single prenyl group (68 amu) to the parent flavone backbone.

Figure 4. Evidence for Cannflavin A and B biosynthesis by CsPT3. (A) Representative HPLC chromatograms are shown for authentic 6-dimethylallyl chrysoeriol/Cannflavin B (top) and for the separation of the extracted enzymatic reaction products from assays with recombinant CsPT3, chrysoeriol, and DMAPP (bottom). Note that in the enzyme assays, chrysoeriol is converted to a product that shares the same HPLC retention time and CID-Q-TOF mass spectra (inset) with 6-dimethylallyl chrysoeriol/Cannflavin B. (B) A representative HPLC chromatogram illustrating the enzymatic reaction products that were extracted from assays conducted with CsPT3, chrysoeriol, and GPP. Note the conversion of chrysoeriol to a product with greater hydrophobicity than 6-dimethylallyl

chrysoeriol/Cannflavin B and which exhibits a Q-TOF mass spectra (inset) consistent with geranylated chrysoeriol ([M+H] ⁺ 437).

Figure 5. Key HMBC correlations of Cannflavin A (above) and Cannflavin B (below). Arrows indicate magnetization transfer from a proton to a carbon and bold arrows indicate correlations that were observed from both the 5-OH hydroxyl proton and the 1" proton on the geranyl or prenyl group to the same carbon resonance.

Figure 6. Phylogenetic analysis of type 1 o-methyltransferases from C. sativa. A neighbour joining phylogenetic tree (1000 replicates) of type 1 o-methyltransferases from diverse plants was constructed using the MEGA 6.0 software package. Included in this analysis are twenty four unique type 1 o-methyltransferases (CsOMTs) that were identified in the C. sativa genome. Branch lengths indicate the number of amino acid substitutions per sequence and bootstrap values (>60%) are indicated next to each branch. Note that the CsOMTs (bolded) are uniformly distributed amongst four main groups of plant type 1 proteins.

Figure 7. Enzymatic characterization of CsOMT21. (A) Three flavones (Apigenin, Luteolin and Chrysoeriol) and two flavonols (Quercetin and Kaempferol) that typically accumulate in C. sativa were tested as potential substrates. (B) Relative enzyme activity and substrate preference. Recombinant CsOMT21 was purified by Ni ²⁺ -affinity chromatography (inset). Proteins were resolved by SDS-PAGE and stained with Coomassie Blue. Lanes 1 and 2 contain crude E. coli extracts containing CsOMT21 and the purified protein, respectively; Molecular weights (kDa) are indicated. The selected flavones and flavonols (as numbered above) were provided to recombinant CsOMT21 in enzyme assays along with [ ¹⁴ C]-SAM as a methyl donor. Data are the means ± SD from three independent experiments and are presented as relative activity compared to that observed with the preferred substrate, luteolin. (C) Evidence for the conversion of luteolin to chrysoeriol by CsOMT21. Enzyme assays with CsOMT21 together with luteolin and unlabelled SAM as co-substrates were extracted and analyzed by HPLC. Note the identical retention time and CID-Q-TOF mass spectral fragmentation pattern of the enzymatic product from these assays (bottom panel) with that of an authentic chrysoeriol standard ( top panel). (D) Kinetic analysis of CsOMT21. Recombinant CsOMT21 was assayed under standard assay conditions at the indicated concentrations of luteolin. Kinetic parameters were determined by non-linear regression analysis using the Michaelis-Menten kinetics model of the SigmaPlot 12.3 software.

Figure 8. Proposed biosynthetic pathway toward Cannflavin A and B synthesis. Solid arrows indicate core steps of the flavonoid biosynthetic pathway for which enzymes have been identified in a variety of plants. Dashed arrows represent proposed enzymatic reactions in C. sativa, based on the evidence presented in this study.

Summary

In accordance with an aspect, there is provided a polypeptide comprising the sequence of SEQ ID NO:3 (CsPT3), a variant thereof having at least 80% sequence identity to SEQ ID NO:3, or a fragment of the polypeptide or the variant thereof.

In an aspect, the variant has at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3.

In an aspect, the polypeptide, variant, or fragment comprises up to 100, 150, 200, 250, 300, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 amino acids.

In an aspect, the polypeptide comprises the sequence of SEQ ID NO:3.

In an aspect, the polypeptide consists of the sequence of SEQ ID NO:3.

In an aspect, the polypeptide encodes an enzyme.

In an aspect, the enzyme is a flavone prenyltransferase.

In an aspect, the enzyme prenylates chrysoeriol using GPP to produce Cannflavin A.

In an aspect, the enzyme prenylates chrysoeriol using DMAPP to produce Cannflavin B.

In accordance with an aspect, there is provided a polypeptide comprising the sequence of SEQ ID NO:59 (CsOMT21 ), a variant thereof having at least 80% sequence identity to SEQ ID NO:59, or a fragment of the polypeptide or the variant thereof.

In an aspect, the variant has at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:59.

In an aspect, the polypeptide, variant, or fragment comprises up to 100, 150, 200, 250, 300, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 amino acids.

In an aspect, the polypeptide comprises the sequence of SEQ ID NO: 59.

In an aspect, the polypeptide consists of the sequence of SEQ ID NO: 59.

In an aspect, the polypeptide encodes an enzyme.

In an aspect, the enzyme is an O-methyltransferase.

In an aspect, the enzyme methylates luteolin to produce chrysoeriol.

In an aspect, the polypeptide is for use in synthesizing Cannflavin A and/or Cannflavin B.

In an aspect, the polypeptide is recombinant.

In accordance with an aspect, there is provided a nucleic acid encoding at least one polypeptide described herein.

In an aspect, the nucleic acid is cDNA.

In accordance with an aspect, there is provided a vector comprising at least one nucleic acid described herein. In accordance with an aspect, there is provided a host cell comprising at least one vector described herein.

In accordance with an aspect, there is provided a host cell expressing at least one polypeptide described herein.

In accordance with an aspect, there is provided a host cell expressing at least two different polypeptides described herein.

In an aspect, the host cell is an algal cell, a plant cell (e.g. , Nicotians spp.), a bacterial cell (e.g. ,

E. coli or Agrobacterium tumefaciens ), or a yeast cell (e.g., S. cerevisiae), optionally wherein the plant cell is not a cannabis plant cell.

In an aspect, the host cell is grown in the presence of and/or expresses luteolin, chrysoeriol,

GPP, and/or DMAPP, or functional fragments or variants thereof.

In accordance with an aspect, there is provided an expression system comprising at least one polypeptide described herein; the nucleic acid described herein, the vector described herein, or the host cell described herein.

In accordance with an aspect, the expression system further comprises luteolin, chrysoeriol,

GPP, and/or DMAPP, or functional variants or fragments thereof.

In accordance with an aspect, there is provided substantially pure Cannflavin A and/or B, for example, at least about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9% pure.

In accordance with an aspect, there is provided THC-free Cannflavin A and/or B, for example, comprising less than about 5%, about 4%, about 3%, about 2%, about 1 %, about 0.5%, about 0.1 %, or about 0.01 % THC by weight.

In an aspect, the Cannflavin A and/or Cannflavin B is produced by a method using at least one polypeptide described herein; the nucleic acid described herein, the vector described herein, or the host cell described herein.

In accordance with an aspect, there is provided a cosmetic composition comprising the

Cannflavin A and/or Cannflavin B described herein and at least one cosmetically acceptable carrier.

In accordance with an aspect, there is provided a pharmaceutical composition comprising the Cannflavin A and/or Cannflavin B described herein and at least one pharmaceutically acceptable carrier.

In accordance with an aspect, there is provided a natural health product comprising the

Cannflavin A and/or Cannflavin B described herein, such as a supplement, beverage, or food.

In accordance with an aspect, there is provided a use of the Cannflavin A and/or Cannflavin B described herein in a cosmetic, pharmaceutical, or natural health product.

In accordance with an aspect, there is provided a recombinant method of producing Cannflavin A or Cannflavin B, the method comprising expressing at least one polypeptide described herein in a cell, wherein the cell expresses luteolin, chrysoeriol, GPP, and/or DMAPP, or functional fragments or variants thereof.

In accordance with an aspect, there is provided a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1-38, a variant thereof having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1 -38, or a fragment of the polypeptide or the variant thereof. In an aspect, the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 1 , 3, 4, 7, and 8.

In an aspect, the variant has at least 85% 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

In an aspect, the polypeptide, variant, or fragment comprises up to 100, 150, 200, 250, 300, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 amino acids.

In an aspect, the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 1-38.

In an aspect, the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 1 , 3, 4, 7, and 8.

In an aspect, the polypeptide consists of a sequence selected from the group consisting of SEQ ID NO: 1-38.

In an aspect, the polypeptide consists of a sequence selected from the group consisting of SEQ ID NO: 1 , 3, 4, 7, and 8.

In an aspect, the polypeptide encodes an enzyme.

In an aspect, the enzyme is a flavone prenyltransferase.

In accordance with an aspect, there is provided a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NO:39-87, a variant thereof having at least 80% sequence identity to a sequence of selected from the group consisting of SEQ ID NO:39-87, or a fragment of the polypeptide or the variant thereof.

In an aspect, the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:44, 50, and 59.

In an aspect, the variant has at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

In an aspect, the polypeptide, variant, or fragment comprises up to 100, 150, 200, 250, 300, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 amino acids.

In an aspect, the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 1-38.

In an aspect, the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:44, 50, and 59.

In an aspect, the polypeptide consists of a sequence selected from the group consisting of SEQ ID NO: 1-38.

In an aspect, the polypeptide consists of a sequence selected from the group consisting of SEQ ID NO:44, 50, and 59.

In an aspect, the polypeptide encodes an enzyme.

In an aspect, the enzyme is an O-methyltransferase.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description.

Detailed Description

Described herein is the biosynthetic pathway responsible for Cannflavin A and B synthesis in cannabis plants. Novel enzymes are described, namely CsOMT21 , which methylates luteolin to produce chrysoeriol, and CsPT3, which prenylates chrysoeriol using GPP or DMAPP to produce Cannflavin A or Cannflavin B, respectively. Expression systems, such as plants, algae, bacteria, or yeast, can be engineered to express these enzymes and thereby produce substantially pure Cannflavin A and/or Cannflavin B as desired. Cannflavin A and Cannflavin B are known to inhibit PGE ₂ and may find use in anti-inflammatory compositions and methods.

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. See, e.g.

Singleton et al. , Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989), each of which are incorporated herein by reference. For the purposes of the present invention, the following terms are defined below.

As used herein, the phrase“exogenous polynucleotide” or“exogenous polypeptide” refers to a heterologous nucleic acid or polypeptide sequence that may not be naturally expressed within the organism in question (e.g., a nucleic acid or polypeptide sequence from a different species) or which overexpression in the species is desired. The exogenous polynucleotide or polypeptide may be introduced into the species, such as a plant, in a stable or transient manner, so as to produce a ribonucleic acid (RNA) molecule and/or a polypeptide molecule. It should be noted that the exogenous polynucleotide may comprise a nucleic acid sequence which is identical or partially homologous to an endogenous nucleic acid sequence of the species.

The term“endogenous” as used herein refers to any polynucleotide or polypeptide which is present and/or naturally expressed within a particular species or cell thereof.

“Variants” of the sequences described herein are biologically active sequences that have a peptide sequence that differs from the sequence of a native or wild-type sequence, by virtue of an insertion, deletion, modification and/or substitution of one or more amino acids within the native sequence. Such variants generally have less than 100% sequence identity with a native sequence. Ordinarily, however, a biologically active variant will have an amino acid sequence with at least about 70% sequence identity with the sequence of a corresponding naturally occurring sequence, typically at least about 75%, more typically at least about 80%, even more typically at least about 85%, even more typically at least about 90%, and even more typically of at least about 95%, 96%, 97%, 98%, or 99% sequence identity. The variants nucleotide fragments of any length that retain a biological activity of the corresponding native sequence. Variants also include sequences wherein one or more amino acids are added at either end of, or within, a native sequence. Variants also include sequences where a number of amino acids are deleted and optionally substituted by one or more different amino acids.

"Percent sequence identity" is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the sequence of interest after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. None of 5', 3', or internal extensions, deletions or insertions into the candidate sequence shall be construed as affecting sequence identity or homology. Methods and computer programs for the alignment are well known in the art, such as "BLAST".

"Active" or "activity" for the purposes herein refers to a biological activity of a native or naturally- occurring molecule, wherein "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring molecule. Thus, "biologically active" or "biological activity" when used in conjunction with the polypeptides described herein refers to a polypeptide that exhibits or shares an effector function of the native polypeptide. For example, the polypeptides described herein have the biological activity of converting luteolin to chrysoeriol or of converting chrysoeriol to Cannflavin A or Cannflavin B. "Biologically active" or "biological activity" when used in conjunction with variant sequences means that the variant sequences exhibit or share an effector function of the parent sequence. The biological activity of the variant sequence may be increased, decreased, or at the same level as compared with the parent sequence.

"Isolated" refers to a molecule that has been purified from its source or has been prepared by recombinant or synthetic methods and purified. Purified polypeptides are substantially free of contaminating components, such as THC, cannabinoids, and/or terpenes, for example.

"Substantially free" herein means less than about 5%, typically less than about 2%, more typically less than about 1 %, even more typically less than about 0.5%, most typically less than about 0.1 % contamination, such as with other polypeptides or non-peptide molecules. An "essentially pure" polypeptide composition means a composition comprising at least about 90% by weight of the polypeptide, based on total weight of the composition, typically at least about 95% by weight, more typically at least about 90% by weight, even more typically at least about 95% by weight, and even more typically at least about 99% by weight, based on total weight of the composition.

As used herein, "treatment" or“therapy” is an approach for obtaining beneficial or desired clinical results. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" and“therapy” can also mean prolonging survival as compared to expected survival if not receiving treatment or therapy. Thus, "treatment" or“therapy” is an intervention performed with the intention of altering the pathology of a disorder. Specifically, the treatment or therapy may directly prevent, slow down or otherwise decrease the pathology of a disease or disorder such as inflammation, or may render the inflammation more susceptible to treatment or therapy by other therapeutic agents.

The terms "therapeutically effective amount", "effective amount" or "sufficient amount" mean a quantity sufficient, when administered to a subject, including a mammal, for example a human, to achieve a desired result, for example an amount effective to treat inflammation. Effective amounts of the Cannflavin A and B molecules described herein may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage or treatment regimes may be adjusted to provide the optimum therapeutic response, as is understood by a skilled person. Likewise, an“effective amount” of the polypeptides described herein refers to an amount sufficient to function as desired, such as to convert luteolin to chrysoeriol or to convert chrysoeriol to Cannflavin A or B or to treat inflammation.

Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

The term“pharmaceutically acceptable” means that the compound or combination of compounds is compatible with the remaining ingredients of a formulation for pharmaceutical use, and that it is generally safe for administering to humans according to established governmental standards, including those promulgated by the United States Food and Drug Administration.

"Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed. Often the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmacologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine;

monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, and dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

By“recombinant host” or“genetically modified host” is meant a host cell whose genetic material has been modified, either by suppression or inactivation of genes, and/or by addition of exogenous genetic material.

The expressions“at least a gene” or“at least a nucleic acid” encompass“one geneVone nucleic acid”, and“one or more genesVone or more nucleic acids”. It also encompasses a gene that is present in the genome of a recombinant host in multiple copies.

In understanding the scope of the present application, the articles“a”,“an”,“the”, and“said” are intended to mean that there are one or more of the elements. Additionally, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

It will be understood that any embodiments described as“comprising” certain components may also“consist of” or“consist essentially of,” wherein“consisting of” has a closed-ended or restrictive meaning and“consisting essentially of” means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase“consisting essentially of” encompasses any known pharmaceutically acceptable additive, excipient, diluent, carrier, and the like. Typically, a composition consisting essentially of a set of components will comprise less than 5% by weight, typically less than 3% by weight, more typically less than 1 % by weight of non-specified components. It will be understood that any component defined herein as being included may be explicitly excluded from the claimed invention by way of proviso or negative limitation. For example, in embodiments, THC, cannabinoids, and/or terpenes are explicitly excluded from the compositions and methods described herein.

In addition, all ranges given herein include the end of the ranges and also any intermediate range points, whether explicitly stated or not.

Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

Polypeptides

Described herein are polypeptides encoding enzymes that are typically prenyltransferases or O- methyltransferases. These polypeptides are typically derived from Cannabis sativa and find use in converting cannabis compounds into compounds with medicinal activity and/or precursors to such compounds. Analogous polypeptides are found in a number of other species as noted below.

For example, described herein are polypeptides comprising one or more of the following sequences, variants thereof, or fragment of the polypeptides or variants. In the following sequences, amino acid sequences and GenBank accession numbers of aromatic prenyltransferases and O- methyltransferases from Cannabis sativa and their plant relatives are shown. Species abbreviations: Ah,

>CsPTl [PK28436] SEQ ID NO : 1

MGLSSVCTFSFQTNYHTLLNPHNNNPKTSLLCYRHPKTPIKYSYNNFPSKHCSTKSFHLQ NKCSESLSIANNSIRAATTNQTEPPE SDNHSVATKILNFGKACWKLQRPYTIIAFTSCACGLFGKELLHNTNLISWSLMFKAFFFL VAVLCIASFTTTINQIYDLHIDRINK PDLPLASGEISVNTAWIMSIIVALFGLIITIKMKGGPLYIFGYCFGIFGGIVYSVPPFRW KQNPSTAFLLNFLAHIITNFTFYYAS RAALGLPFELRPSFTFLLAFMKSMGSALALIKDASDVEGDTKFGI STLASKYGSRNLTLFCSGIVLLSYVAAILAGI IWPQAFNSN VMLLSHAILAF LILQTRDFALTNYDPEAGRRFYEFMWKLYYAEYLVYVFI (395 )

>Cs PT2 [PK02092] SEQ ID NO : 2

MPMDSLLLGSFTTPS ILTKGGNNWRRCNLRNVDFSGSYGAYRVSKFSGWNVKETHCCSGFQPCSLKHCTKSTETS VFHYRKNNRFL LNAAAEQPLESEPKNVLNSAKDALDAFYRFSRPHTVIGTALSIVSVSLLAVEKLSDFSPL FFAGMLEAVVAALLMNIYIVGLNQLY DIDIDKVNKPYLPLASGEYSVQTGVMIVASFAI LSFGLGWIVGSWPLFWALFISFILGTAYSIDVPLLRWKRFALVAAMCI LAVRA VIVQLAFFLHMQTHVFKRPASFSRPLI FATAEMSFFSVVIALFKDIPDIDGDRIFGIRSFTVRLGQKRVFWICIALLEIAYTVALL VGASSGFLWSKVVTVLGHTILASILWTNAKSVDLSSKAAITSFYMFIWKVFYAEYLLIPL VR (406)

>Cs PT3 [PK17697] SEQ ID NO : 3

MVFSSVCSFPSSLGTNFKLVPRSNFKASSSHYHEINNFINNKPIKFSYFSSRLYCSAKPI VHRENKFTKSFSLSHLQRKSS IKAHG EIEADGSNGTSEFNVMKSGNAIWRFVRPYAAKGVLFNSAAMFAKELVGNLNLFSWPLMFK ILSFTLVILCIFVSTSGINQIYDLDI DRLNKPNLPVASGEISVELAWLLTIVCTISGLTLTIITNSGPFFPFLYSASIFFGFLYSA PPFRWKKNPFTACFCNVMLYVGTSVG VYYACKASLGLPANWSPAFCLLFWFISLLSIPISIAKDLSDIEGDRKFGIITFSTKFGAK PIAYICHGLMLLNYVSVMAAAIIWPQ FFNSSVILLSHAEMAIWVLYQAWILEKSNYATETCQKYYIFLWIIFSLEHAFYLEM (400)

>CsPT4 [ PK15523 ] SEQ ID NO : 4

MGLSLVCTFSFQTNYHTLLNPHNKNPKNSLLSYQHPKTPI IKSSYDNFPSKYCLTKNFHLLGLNSHNRI SSQSRS IRAGSDQIEGS PHHESDNSIATKILNFGHTCWKLQRPYVVKGMI SIACGLFGRELFNNRHLFSWGLMWKAFFALVPILSFNFFAAIMNQIYDVDIDR INKPDLPLVSGEMSIETAWILSIIVALTGLIVTIKLKSAPLFVFIYIFGIFAGFAYSVPP IRWKQYPFTNFLITISSHVGLAFTSY SATTSALGLPFVWRPAFSFIIAETTTVMGMTIAFAKDISDIEGDAKYGVSTVATKLGARN MTFVVSGVLLLNYLVSISIGIIWPQVF KSNIMILSHAILAFCLIFQTRELALANYASAPSRQFFEFIWLLYYAEYFVYVFI (398)

>Cs PT5 [PK11068] SEQ ID NO : 5

MELSLSLGGPTIFPRYRASYTSTKLTTHFSNFPSKFSTKNFHQTLSFYGPTRGSKSLLNT HQWRNSIRACAEAGAAGSNPVLNKVS DFRDACWRFLRPHTIRGTTLGSIALVARALIENPNLIKWSLLLKAFSGLLALICGNGYIV GINQIYDIGIDKVNKPYLPIAAGDLS VQSAWYLVI LFAVAGLLTVGFNFGPFI TSLYCLGLVLGT IYSVPPFRMKRFPVAAFLI IATVRGFLLNFGVYYATRAALGLTFEWS SAVAFITTFVTLFALVIAITKDLPDVEGDRKFQISTFATKLGVRNIAYLGSGLLLLNYIG AIAAAIYMPQAFKRNLMLPIHTILAL SLVFQAWVLEQANYTKEAIAGFYRFIWNLFYVEYI IFPFI (384 )

>Cs FT 6 [PK13891] SEQ ID NO : 6

MDSLFLSSFSTPSVLSKGGNNWRRCNLKNVEFENTGSYVAYNVSRLRVWKVREKPCSAVF QPSSLKHCAKGSETFVFYQRPNERFL VKAAGGQPLESEPKNDMNSAKDALDAFYRFSRPHTVIGTALSIVSVSLLAIEKLSDFSPL FFVGMLEAIVAALLMNIYIVGLNQLY DIDIDKVNKPYLPLASGEYSIQTGVMIVASFSILSFGVGWLVGSWPLE ALFISFVLGTAYSINVPLLRWKRFALVAAMCI LAVRA VIVQLAFFLHIQTHVFKRPAVFSRPLIFATAETTSFFSVVIALFKDIPDIDGDRIYGIRS FTVRLGQKRVFWICISLLEIAYTVALL VGASSGFLWSKVVTVLGHTILASILWTNAKSVDLSSKAAITSFYMFIWKLFYAEYLLIPL VR (406)

>Cs PT7 [PK29226] SEQ ID NO : 7

MELSSICNFSFQTNYHTLLNPHNKNPKSSLLSHQHPKTPIITSSYNNFPSNYCSNKNFHL QNRCSKSLLIAKNSIRTDTANQTEPP ESNTKYSVVTKILSFGHTCWKLQRPYTFIGVISCACGLFGRELFHNTNLLSWSLMLKAFS SLMVILSVNLCTNIINQITDLDIDRI NKPDLPLASGEMSIETAWIMSIIVALTGLILTIKLNCGPLFISLYCVSILVGALYSVPPF RWKQNPNTAFSSYEMGLVIVNFTCYY ASRAAFGLPFEMSPPFTFILAFVKSMGSALFLCKDVSDIEGDSKHGISTLATRYGAKNIT FLCSGIVLLTYVSAI LAAI IWPQAFK SNVMLLSHATLAFWLIFQTREFALTNYNPEAGRKFYEETTWKLHYAEYLVYVFI (397 )

>CsPT8 [PK07278] SEQ ID NO : 8

MMFSSVCSSLSPLGTNITVPRSNYFKSTSSSSHYYKINNKPITFSSFSSTQDYSAKGTLV YTENKFTKSFLQRKTSIRANGEIEAD GSDGTTEFNVMKSGNTIWRFARPYAAKGVLFNYAALFAKELVGNLNLCSWSLILKIPSFV LVILCVTICVNGINQIYDLDIDRLNK PDLPLASGEMSIEMAWVLTIFCAISGLILTITMNSGPLFPFLYCGSIFVAGFLYSAPPFR FKNNHFTALLCNYVMFVSTTLQIYCA YKAGLGLPLNWSPAFCLLVWFLSLIAVTICIGKDLSDIEGDRKFGVTTFPTEYGAKPIAL ICHGLILLDYVGLMAAAIIWPQLFNS KLI LLSHAFMAVWVVYQAWILEKSNYTTEACQKYYMYLWTIYSVEHILYLFM (396)

>H1PT1 [AB543053 ] SEQ ID NO : 9

MELSSVSSFSLGTNPFISIPHNNNNLKVSSYCCKSKSRVINSTNSKHCSPNNNNNTSNKT THLLGLYGQSRCLLKPLSFISCNDQR GNS IRASAQIEDRPPESGNLSALTNVKDFVSVCWEYVRPYTAKGVI ICSSCLFGRELLENPNLFSRPLI FRALLGMLAILGSCFYT AGINQIFDMDIDRINKPDLPLVSGRISVESAWLLTLSPAIIGFILILKLNSGPLLTSLYC LAILSGTIYSVPPFRWKKNPI TAFLC ILMIHAGLNFSVYYASRAALGLAFAWSPSFSFITAFITFMTLTLASSKDLSDINGDRKFG VETFATKLGAKNITLLGTGLLLLNYV AAI STAI IWPKAFKSNIMLLSHAILAFSLIFQARELDRTNYTPEACKSFYEFIWILFSAEYVVYLFI (411)

>H1PT2 [KM222442] SEQ ID NO:10

MELSSACNLSLKPNYYYYPTSLFPSNNSYNNLKASSYYQTQRPIKCCSYSPSKYCSTKKL QTTHLLGLYAKHKCLKPFSIGHLPRP NSLTAWSHQSEFPSTIVTKGSNFGHASWKFVRPIPFVAVSIICTSLFGAELLKNPNLFSW QLMFDAFQGLVVILLYHIYINGLNQI YDLESDRINKPDLPLAAEEMSVKSAWFLTIFSAVASLLLMIKLKCGLFLTCMYCCYLVIG AMYSVPPFRWKMNTFTSTLWNFSEIG IGINFLINYASRATLGLPFQWRPPFTFIIGFVSTLSIILSILKDVPDVEGDKKVGMSTLP VIFGARTIVLVGSGFFLLNYVAAIGV AIMWPQAFKGYIMIPAHAIFASALIFKTWLLDKANYAKEASDSYYHFLWFLMIAEYILYP FIST (408)

>AtVTE2-l [AAM10489] SEQ ID NO:ll

MESLLSSSSLVSAAGGFCWKKQNLKLHSLSEIRVLRCDSSKVVAKPKFRNNLVRPDGQGS SLLLYPKHKSRFRVNATAGQPEAFDS NSKQKSFRDSLDAFYRFSRPHTVIGTVLSILSVSFLAVEKVSDISPLLFTGILEAVVAAL MMNIYIVGLNQLSDVEIDKVNKPYLP LASGEYSVNTGIAIVASFSIMSFWLGWIVGSWPLFWALFVSETTLGTAYSINLPLLRWKR FALVAAMCILAVRAI IVQIAFYLHIQT HVFGRPILFTRPLIFATAEMSFFSVVIALFKDIPDIEGDKIFGIRSFSVTLGQKRVFWTC VTLLQMAYAVAILVGATSPFIWSKVI SVVGHVILATTLWARAKSVDLSSKTEI TSCYMFIWKLFYAEYLLLPFLK (393)

>GmVTE2-l [ABB70126] SEQ ID NO:12

MDSMLLRSFPNINNASSLATTGSYLPNASWHNRKIQKEYNFLRFRWPSLNHHYKSIEGGC TCKKCNIKFVVKATSEKSFESEPQAF DPKSILDSVKNSLDAFYRFSRPHTVIGTALSIISVSLLAVEKISDISPLFFTGVLEAVVA ALFMNIYIVGLNQLSDVEIDKINKPY LPLASGEYSFETGVT IVASFS ILSFWLGWVVGSWPLFWALFVSFVLGTAYS INVPLLRWKRFAVLAAMCILAVRAVIVQLAFFLHI QTHVYKRPPVFSRSLIFATAFMSFFSVVIALFKDIPDIEGDKVFGIQSFSVRLGQKPVFW TCVILLEIAYGVALLVGAASPCLWSK IVTGLGHAVLASILWFHAKSVDLKSKASITSFYMFIWKLFYAEYLLIPFVR (395)

>TaVTE2-l [ABB70123] SEQ ID NO:13

MDSLRLRPSSLRSAPGAAAARRRDHILPSFCSIQRNGKGRVTLSIQASKGPTINHCKKFL DWKYSNHRISHQSINTSAKAGQSLQP ETEAHDRASFWKPISSSLDAFYRFSRPHTIIGTALSIVSVSLLAVESLSDISPLFLTGLL EAVVAALEITNIYIVGLNQLFDIEIDK VNKPTLPLASGEYSPATGVAIVSVFAAMSFGLGWVVGSPPLFWALFISFVLGTAYSVNLP YFRWKRSAVVAALCI LAVRAVIVQLA FFLHIQTFVFRRPAVFSKPLIFATAETTTFFSVVIALFKDIPDIEGDRIFGIQSFSVRLG QSKVFWTCVGLLEVAYGVAILMGVTSS SLWSKSLTVVGHAILASILWSSARSIDLTSKAAITSFYMLIWRLFYAEYLLIPLVR (400)

>ZmVTE2-l [ABB70122] SEQ ID NO:14

MDALRLRPSLLSVRPGAARPRDHFLPPCCSIQRNGEGRICFSSQRTQGPTLHHHQKFFEW KSSYCRISHRSLNTSVNASGQQLQSE

PETHDSTTIWRAISSSLDAFYRFSRPHTVIGTALSIVSVSLLAVQSLSDISPLFLTG LLEAVVAALEMNIYIVGLNQLFDIEIDKV NKPTLPLASGEYTPATGVAIVSVFAAMSFGLGWAVGSQPLFWALFISFVLGTAYSINLPY LRWKRFAVVAALCILAVRAVIVQLAF FLHIQTFVFRRPAVFSRPLLFATGETTTFFSVVIALFKDI PDIEGDRIFGIRSFSVRLGQKKVFWICVGLLEMAYSVAILMGATSSC LWSKTATIAGHSILAAILWSCARSVDLTSKAAI TSFYMFIWKLFYAEYLLI PLVR (399)

>ApVTE2-l [ABB70124] SEQ ID N0:15

MLSMDSLLTKPVVIPLPSPVCSLPILRGSSAPGQYSCRNYNPIRIQRCLVNYEHVKPRFT TCSRSQKLGHVKATSEHSLESGSEGY TPRSIWEAVLASLNVLYKFSRPHTIIGTAMGIMSVSLLVVESLSDISPLFFVGLLEAVVA ALFMNVYIVGLNQLFDIEIDKVNKPD LPLASGEYSPRAGTAIVIASAIMSFGIGWLVGSWPLF ALFISFVLGTAYSINLPFLRWKRSAVVAAICILAVRAVIVQLAFFLHI QSFVFKRPASFTRPLIFATAFMSFFSVVIALFKDIPDIDGDKIFGIHSFSVRLGQERVFW ICIYLLEMAYTVVMVVGATSSCLWSK CLTVIGHAI LGSLLWNRARSHGPMTKTTITSFYMFVWKLFYAEYLLIPFVR (395 )

>CpVTE2-l [ABB70125] SEQ ID N0:16

MRMESLLLNSFSPSPAGGKICRADTYKKAYFATARCNTLNSLNKNTGEYHLSRTRQRFTF HQNGHRTYLVKAVSGQSLESEPESYP NNRWDYVKSAADAFYRFSRPHTIIGTALSIVSVSLLAVEKLPELNSMFFTGLLEVILAAL EMNIYIVGLNQLSDIDIDKVNKPYLP LASGEFSVGTGVTIVTSFLIMSE LGWVVGSWPLE ALFISFVLGTAYSIDMPMLRWKRSAVVAALCILAVRAVIVQIAFFLHMQM HVYGRAAALSRPVIFATGFMSFFSIVIALFKDIPDIEGDKIFGIRSFTVRLGQERVFWIC ISLLEMAYAVAILVGSTSPYLWSKVI TVSGHVVLASILWGRAKSIDFKSKAALTSFYMFIWKLFYAEYLLI PLVR (393)

>AtVTE2-2 [ABB70127] SEQ ID N0:17

MELSISQSPRVRFSSLAPRFLAASHHHRPSVHLAGKFISLPRDVRFTSLSTSRMRSKFVS TNYRKISIRACSQVGAAESDDPVLDR IARFQNACWRFLRPHTIRGTALGSTALVTRALIENTHLIKWSLVLKALSGLLALICGNGY IVGINQIYDIGIDKVNKPYLPIAAGD LSVQSAWLLVIFFAIAGLLVVGFNFGPFITSLYSLGLFLGTIYSVPPLRMKRFPVAAFLI IATVRGFLLNFGVYHATRAALGLPFQ WSAPVAFITSFVTLFALVIAITKDLPDVEGDRKFQISTLATKLGVRNIAFLGSGLLLVNY VSAISLAFYMPQVFRGSLMIPAHVIL ASGLIFQTWVLEKANYTKEAISGYYRFIWNLFYAEYLLFPFL (386)

>GmVTE2-2 [ABB70128] SEQ ID N0:18

MELSLSPTSHRVPSTIPTLNSAKLSSTKATKSQQPLFLGFSKHFNSIGLHHHSYRCCSNA VPERPQRPSSIRACTGVGASGSDRPL AERLLDLKDACWRFLRPHTIRGTALGSFALVARALIENTNLIKWSLFFKAFCGLFALICG NGYIVGINQIYDISIDKVNKPYLPIA AGDLSVQSAWFLVIFFAAAGLSIAGLNFGPFIFSLYTLGLFLGTIYSVPPLRMKRFPVAA FLI IATVRGFLLNFGVYYATRASLGL AFEWSSPVVFITTFVTFFALVIAITKDLPDVEGDRKYQISTFATKLGVRNIAFLGSGILL VNYIVSVLAAIYMPQAFRRWLLIPAH TIFAISLIYQARILEQANYTKDAISGFYRFIWNLFYAEYAIFPFI (389)

>OsVTE2-2 [XP_015646905] SEQ ID N0:19

MASLASPPLPCRAAATASRSGRPAPRLLGPPPPPASPLLSSASARFPRAPCNAARWSRRD AVRVCSQAGAAGPAPLSKTLSDLKDS CWRFLRPHTIRGTALGSIALVARALIENPQLINWWLVFKAFYGLVALICGNGYIVGINQI YDIRIDKVNKPYLPIAAGDLSVQTAW LLVVLFAAAGFSIVVTNFGPFITSLYCLGLFLGTIYSVPPFRLKRYPVAAFLIIATVRGF LLNFGVYYATRAALGLTFQWSSPVAF ITCFVTLFALVIAITKDLPDVEGDRKYQISTLATKLGVRNIAFLGSGLLIANYVAAIAVA FLMPQAFRRTVMVPVIIAALAVGI IFQ TWVLEQAKYTKDAISQYYRFIWNLFYAEYIFFPLI (379)

VOsHGGT [AAP43913] SEQ ID NO:20

MQASSAAAAAACSAIKPAAHQHTVQVQEDKRGSEFRARFGTRKLSWGGKLSVENSALHQC QSLTRSIRRQKRQHSPVLQVRCYAIA GDQHESIATEFEEICKEVPQKLGAFYRFCRPHTIFGTIIGITSVSLLPMRSLDDFTMKAL WGFLEALSSSLCMNIYVVGLNQLYDI QIDKVNKPSLPLASGEFSVATGAVLVLTSLIMS IAIGIRSKSAPLLCALFI SFFLGSAYSVDAPLLRWKRNAFLAASCILFVRAVL VQLAFFAHMQQHVLKRPLAPTKSVVFATLEMCCFSSVIALFKDIPDIDGDRHFGVESLSV RLGPERVYWLCINILLTAYGAAILAG ASSTNLCQMI ITVFGHGLLAFALWQRAQHCDVENKAWITSFYMFIIVKLFYAEYFLIPFVQ ( 404 )

VHvHGGT [AAP43911] SEQ ID NO: 21

MQAVTAAAAAGQLLTDTRRGPRCRARLGTTRLSWTGRFAVEAFAGQCQSSATTVMHKFSA ISQAARPRRNTKRQCSDDYPALQAGC SEVN1NDQNGSNANRLEEIRGDVLKKLRSFYEFCRPHTIFGTIIGITSVSLLPMKSIDDF TVTVLRGYLEALTAALCMNIYVVGLNQ LYDIQIDKINKPGLPLASGEFSVATGVFLVLAFLIMSFSIGIRSGSAPLMCALIVSFLLG SAYSIEAPFLRIAIKRHALLAASCILFV RAILVQLAFFAHMQQHVLKRPLAATKSLVFATLETTCCFSAVIALFKDIPDVDGDRDFGI QSLSVRLGPQRVYQLCISILLTAYGAA TLVGASSTNLFQKI I TVSGHGLLALTLWQRAQHFEVENQARVTSFYMFIINKLFYAEYFLIPFVQ (408)

VTaHGGT [AAP43912] SEQ ID NO:22

MQATTAAAAAQLLTDTRRGPRCSRARLGATRLSWPGRFAVEAFAGRCQSSATTVTHRFSA ISQATSPRRKARRQCSDDQSALQAGC SKVNRDQHGYDVNWFEEISQEVSKKLRAFYQFCRPHTIFGTIIGITSVSLLPMKSIDDFT ATVLKGYLEALAAALCMNIYVVGLNQ LYDIQIDKINKPGLPLAAGEFSVATGVFLVVTFLIMSFSIGIHSGSVPLMYALVVSFLLG SAYSIEAPLLRIAIKRHALLAASCILFV RAILVQLAFFAHMQQHVLKRPLAATKSLVFATLEMCCFSAVIALFKDIPDVDGDRDFGIQ SLSVRLGPQRVYQLCISILLTAYLAA TVVGASSTHLLQKI I TVSGHGLLALTLWQRARHLEVENQARVTSFYMFIWKLFYAEYFLIPFVQ (408)

>SfN8DT-l [BAG12671] SEQ ID NO:23

MGSMLLASFPGASSI TTGGSCLRSKQYAKNYDASSYVTTSlAfYKKRKIQKEHCAAIFSKHNLKQHYKVNEGGST SNTSKECEKKYVV NAISEQSFEYEPQTRDPESIWDSVNDALDIFYKFCRPYAMFTIVLGATFKSLVAVEKLSD LSLAFFIGWLQVVVAVICIHIFGVGL NQLCDIEIDKINKPDLPLASGKLSFRNVVI ITASSLILGLGFAWIVDSWPLFWTVFI SCMVASAYNVDLPLLRINKKYPVLTAINFI ADVAVTRSLGFFLHMQTCVFKRPTTFPRPLIFCTAIVSIYAIVIALFKDIPDMEGDEKFG IQSLSLRLGPKRVFWICVSLLEMTYG VTILVGATSPILWSKIITVLGHAVLASVLINYIIAKSVDLTSNVVLHSFYMFIINKLHTA EYFLIPLFR (410) >GuA6DT [ KJ123716 ] SEQ ID NO:24

MAKNSLNPI SFFGQKERHSPSFGGNIWQSQNCTKNYYASSYAPKASWHKKNIQKEYFFLRFKQSSSNHL YKDIEGGSTYRECNRKY VVKAAPGPSFESESPAFDSKNILESVKNFINVFFKLISPYAMIAAALSITSASLLAVEKL SDISPQFFIGLLQGLIPNLEMGVYMA GLNQLCDIE IDKINKPHLPLASGEISFTTGVI I IASSFIVSLWLGSIVGSWPSLWALISFCVIWTGYSVNVPLLRWKRHPALAAMC IIATWGFIFPIGYFLHIQTFVFKRSAVFSRPVVFSTIEMSFFSLVIALFKDIPDIEGDQA FGVQSFSASLGQKRVFWICVSLLETA YGVALLMGATSSCLWSKI ITVLGHAILALVLFYRAKSINLKSKAS IASFEMFIWKLLYAEYFLVPLVR (412)

>LaPTl [AER35706 ] SEQ ID NO:25

MSAMLASCFTIPSSIKAGGNRPRSKQCGKTYYASSNVPTLWHKTEKIQKEHCAMMSSNSL QHRCKVIEDGFKYQQWKRKCTINAIS EQSFEPESQAQYKKSMKDSVKDGLVAFYEFTRPYSAIPI ILEATCMSLLAVEKSSDLSLIFFKGWVQTVVATLLMI IVNCGLNQLC DLEIDKINKPHLPLTSGALSIKAAIAIVAASAFLGLWFSWSSGSWPLEWNVLYNNVLAVF YSVDLPLLRWKKSSFLTAVYILTNIG VVIPIGSFLHMQTHVFKRAATLPRSMLLSTTVLSIFCIVISMIKDIPDMEGDEKFGIKSF ALSLGQKRVFSICISLLQMSYGVGIL VGATSPYLWSKIFTVVGHATLALVLQYRAKSVDPKSKDSVQSFYMFIWKKLFIAECLLLP LFRS (408)

>SfiLDT [BAK52290] SEQ ID NO : 26

MGFVLPASFPRSSSI TTGSYGTTLWHKSEKIQKEYCVMLSSSHNLKHRHKVIHRGSSCQECERKYVVNATSGQLF EYEPQATDIKS NWDSIKDALNVFYSFMRPYSAIAAAMGATSVSLLAVEKLSDLSLPFFIGWLQAVVFSFIV NIFNCGLNELCDVELDKINKPNLPLV SGELSFRTGVLIVASSLIMSFGLTLIVGSWPLFWSQFASSLLAAAYSINLPLLRWKKYPI LAATSILTNVAVAVPLGYFLHMQTHV FKRPATFPRPLNFCIAILSLFFVVIALFKDIPDIEGDKKFGVQSLAVRLGQKRVFWICIS LLEMAYGVTILVGATSPFLWSKISTG LGHAVLASIVWNRAKSVDLKNKDSYKSFYMFIWKLICAEYCLIPLFR (391)

>SfG6DT [BAK52291] SEQ ID NO:27

MGFVLPASFYGASSIKTGGSCWRSKQYAKNHYASSYLTTLCHKTGENKKEYREMMSSQPN LRHHYRIMEGGSTCQENEKKY IVKAT SKQTFEYEPHAQHSKSIWDSIKNAFDAFYRFSRPYAAIEAALGATSISFLAVEKLSDLSV VFFIGLLQVVVASFEMNIFHCGFNQL CDIEIDKINKPYLPLASGELSFRNSVLIVASSLMLCFGLAWIEGSWPLFWGFFVCAMLTA AYS INLPLLRWKKSSMLAAINIFVNA GVLRPLGYFLHMQTCVFKRPTTFPRPLIFCMAILSLFFVVIALFKDIPDTEGDKKFGIRS LSAQLGQKQVFWICISLLQMAYGITI LAGVTSPFLWSKISMVLGHAILASILGYQVKSVDLKNNDALQSFYLFIWKLLTVEYCLIP LFR (407 )

>GmG4DT [BAH22520] SEQ ID NO: 28

MDWGLAISSHPKPYSVTTGGNLWRSKHTTKNIYFASSWI SKASRHKRETQIEHNVLRFQQPSLDHHYKCIRGGSTYQECNRKFVVK AISKQPLGFEAHASNPKNILDSVKNVLSAFYWFSYPYTMIGITLCAFSSSLLAVEKLSDI SLSFLIGVLQGVLPQLFIEIYLCGVN QLYDLEIDKINKPHLPMASGQFSFKTGVIISAAFLALSFGFTWITGSWPLICNLVVIASS WTAYSIDVPLLRWKRYPFVAAMCMIS TWALALPISYFHHMQTVVLKRPIGFPRSLGFLVAEMTFYSLGLALSKDIPDVEGDKEHGI DSFAVRLGQKRAFWICVSFFEMAFGV GILAGASCSHFWTKIFTGMGNAVLASILWYQAKSVDLSDKASTGSFYMFIWKLLYAGFFL MALIR (409)

>AhR4DT-l [AQM74172] SEQ ID NO : 29

MAFGHLVLI PRSTSS IATTAASCWKSKKFADNYYANSYGRRALWQSDRNLTKDHSIKTSLQHNISKLHYNPIERG SRCNKIEKTYL TNASSSAQSHESEPEVHESPKALESIKKGLVMFLQFCRLYAFLGMIPAGLSSSLLAVDNF SEI SPLLFLKGVLQY IVTFFFTSQFV MGVNQLSDVEIDKINKPDLPLASGEYSFTSGVI LVTSFLLAGFGVAWMLGSQPLIWSVVVTAALMGAYSVNFPLLRWKRSI ILTSL SNAIAMLASFHIGPFLHMKTFVLKKAATFPRSMILGCVVIGLFYT I ITLTKDLGDVEGDKAAGLKTLPIRLGVKPVFWLCVSLIQM AYGIAITMGALSPVLWSKIVTVVAHAFMVFYVWNHALNSVDLSSKDSLHSFHLEMFKLVT VEGILIQFVR (414 )

>AhR3'DT-l [AQM74173] SEQ ID NO:30

MAFGVVAASFSRAPSIVTTRGCYVTRASLPNKSLKFSKEYNLKTSLQHNWKHNSRSIFER GSTITTCDKYDEKKYLMNVTQSHEAE PHSQSILKSIIDALDAFRKFSRFYAFIAMVVGSLSTSLLAVDNLTELYPAFFNGFLQCMA AYFEMHLYIVGINQLADLEIDKINKP YLPLASGNYSFRTAVITVTSFLFTGFGIAWI IGSKPLLWTIFASFVLMTAYSVNLPLLRWKKSTILTVMGNTLSMVISFNLGPFYH MKTHVLKKAATFPRSLLFAVVVMSMYYIVIALTKDIPDIEGDKEAGLQTLAIRLGPKTVF WSSVALLEMAYGAAIIIGASSPFLWS KISVVLSHAILALFVWYRSTLVDLSNKDSLQAFYMLIFKLFSVENILMLFVR (396)

>AhR3'DT-2 [AQM74174] SEQ ID NO:31

MPFGLSASFLKSRSFHHHGTRRALWNNNGKLSKEYCIKMQHNYWKNHCTNLKGGSMMSDD KFEKKYLVNATSKNSHDEPKKSQPIL EFIKDGMDTFRQFSRLYAFFSFISSGLSSSLLAVDNLSNISPKMFLIGFLQFLIPNCIMF QYIVGVNQLADIEIDKINKPYLPLAS GKYSLRNAI I IVASSLLMGFGSAWVLGSRPMFWCLVISTMLMTAYSVNLPLLRWKRSTILATLSLASSMT IGQHIAPFLHMKTVLK KALNYPRSLVFTVVVVSLFYTVISLAKDIPDIEGDKAAGHKTLAIHLGPRRVFWFCISLL QMTYGIAIIMGALSPILWSKIFTVVT HFIMSI ILWYRANSVDLSNNDSLQSFYMAIFVFLSVENFLVLFVR (389)

>AhR3'DT-4 [AQM74176] SEQ ID NO:32

MASTSRLLLHASLPPPTTSISKTNSGSHAVIRS IWHNNGKYPKEKTCIETPLLLQHNQKHHYTCDQIKRKHFVKATHAQSKNEPEP QADSAKPIWNSIKDVMHTIQKFSVFYALIGLLSGILSSSLLAVEKLSDLSPTFFISMLQE MAAYSSMQLYTTGVNQLADIEIDKIN KPYRPLASSKISFGGGLAIVAASLEMSFGLALMIGSKPLLWGLILIFILMTAYSVNLPFL RWKKSTILTLLSGVPTILTAYNLAPY LHMKTFVLKKPFIFTRSLAFTTVVMTFFYVVISLFKDIPDIEGDKKEGLQTLSIRLGPKR VFWLCISLLEMTYGIAIIMGLTSPFL WSKIFTVMAHAINAWILWFRANSVDLKSKEDFQSFYMFI FKLLYLENVLVLFVR (398)

>C1PT1 [BAP27988] SEQ ID NO:33

MLQMHSNSSFSPKCYYPLQHAGCVKTLQLPLTKVHGGLNRSESKNYAIKCTQSDSFYSTN KIRNNENSSSRNCKPFNKYRVAVTLQ QQDCASNNEDDINSTSFRDVLLKKLHALYVFTRPFAMIGTIVGITSIAILPLQSFADLTP KYFMEFLKALLSAVLMNNYVGTVNQV ADVEIDKVNKPGLPLASGDLSVGTGLAITLILSLTSLAIALSLQSPPLIFGLIVWFLLGT AYSVDLPFLRWKTNPFLAGMCMVIVF GLVYQFSFFIHFQKYVLGRPVVITRPLIFAAAI ISTISAVMSLLKDIPDEDGDKQFGYQSISSKLGKENVLRLCVYALFFAYGVAV IVGASSSFQLGKLVS I IGHSTLAFLLWLRAQTVDLSNNASTFSFYLFVWKLFYGEYLLIHFLR (407 ) >PcPT [BA031627] SEQ ID NO:34

MSQTLMHSRFSSGFLHHQPEKGFLTLQTQRRHAKTLKGEKEFPSRVVSCHKNVDSSKNFS SSCEKSKTQEKILAQTLGATSDGEAI VQPNNDFEVTWQNTLRRKWDAFSIFSRPYSAICTIIGISSVSLLPLTSVADFSPAYFVGL LQALIPFLCANIYTSAINQLVDVDID KINKPYLPLVSGEFSMGEGRAIVSALTFTCFAMAIMSHSVPLFVGVLVYFLIGTAYSVEH PLLRWKTKPAMAAFSMAGLMGLTIQP TVFYHIQNVLGKPMVFSRSVAFATMFFSIFAACLGAIKDIPDVEGDREFGNLTFSVRYGQ EKVFSFCLNVLLLAYGSAVVVGASSS SLLCKTVSVIGHTVLASLLVLRAKSTNPKDPESTQSFYMFLFKLLYAEYVLIHEMR (400)

>PsPTl [AJW31563] SEQ ID NO:35

MAQTIMHSRLSSGFLHLQRDKGFRTLPTQRRHAKVVNGDQEFAFRVVSCDKNLDSTKNFS GSCEKPIRTHTNKLLQTISATSDREA IIQPKDDYEAPWQNTLRRKWDAFCTFGRPYSAICTIIGISSVSLLPLTSVKDFSAPYFVG LLQALIPFLCANIYTSGINQLVDVDI DPINKPYLPLVSGEFSLGEGRAIVSALAEMCLAVGILSHSTPLFVGVLVYFLIGTAYSVE LPLLRWKTKPAMAAFSMAGLMGLTIQ PAVFYHIQNALGKPMVFSKTVAFATIFFSVFAAVLGAIKDVPDVEGDTAFGNRTFSVRYG QEKVFSVCLNILLLAYGFAVVVGASS SFLLCKIVSVMGHTTLASLLLLRAKSTNPKDPESTQSFYMFLFKLLYAEYVLIHEMR (401 )

>Ps PT2 [AJW31564] SEQ ID NO : 36

MTQTLMHSRFSSGFLHLQRRERSGFLTSFPTRRRHATILNKDKELTVRVVSCDKILDSTN NSSRSCEKPINRTNTSTLLQTLGASS EGEVIIQPKAEYEETWDSIEWRKWDAFVTFGRPYSVLGSIIGISSVSLLPLTSVGDFSLA VFVGFVQALIPFVCANIYASGINQVV DYE IDKINKPYLPLVSGDFSMGEGKAVVSATGFLCLAMT IMFGSLPLFLGVLGYFLYATAYSVELPFLRWKTKPFMAAFSMAGLMG LTIQPSVYYHIQNVLGRPMVLTKPVVFATSFISVFSAVLAMIKDLPDVEGDRALGNLTFS VRYGQEKVFNICVGIMLAAYASAVVT GSFSSLLICKLVSVIGHTALAFLLMLRAKSIDVNDPESTQSFYMFAFQLLYAEYVLIHFI R (405)

>MaIDT [AJD80982] SEQ ID NO:37

MELSISHSSLRLPAI IPQRCKASSHEKRLFSIKPTTKNIKSSNFPSNCSTANKLILPLGLYGERKLSKSLLYGQH RRNSTT IRASA EAHESANNSDGTFAKFSSFGSALYKFLRIYALSHTIVSTVSLFARVLVENPHLFKWSLVL KAFPGLIAMTLANAYYIGINQIYDAD IDRVNKPYLPIPAGELSLKHAWILVISFAVGALSILRLMNADWITTSIFCFGLFLAHFYS APPLRFKQSPIATSIVNPLNAGIVHN LGLIYATRASLGLPFVWNPSTLFIVNFITPFFLAITNLKDLTDMEGDSKHNIRTLPTIYG PRKITFFFVGMLLTHYVAAALAGILL PKVFNPYVMAPAHAI LGLLLFLKTRELDKANYTVEASETFYKFIWKILLLEFVIFPFI (402)

>Ct IDT [AJD80983] SEQ ID NO:38

MAFSISHSSLRLSPIAPHQRFIRASSHDNNSVLSIKNKIKSPNFPSKSSTDKLIFPVGLY GENQFSKSLSYGKDRRNNIRASFEAQ PADSKTTLAKVSRFGSTCYKFLRPYAMSHTVASAIGLFARVLVDNPQFLKWSVVLKAFPG LIAMILATAYYIGINQIYDADIDRVN KPYLPIPSGELSLKQAWFLVISDLLAGLLILRLMNADLITTSLYCAGLFLGTFYSAPPLR FKESSFQTSIVNPLMAGILHNIGVLY ASTVSLGLPFNLSSPPVVFIVIFITLYFIVITNLKDLTDIEGDIKHNIRTLPAIFGPRKT TFFFAGILLATYVGSMAAGICMPQAF RPYVMVPAHAILGALTFFKVRKLDKANYSMEESADFYQFLWKILCLEFVIFPFI (398)

YCsOMTl [ PK03555 ] SEQ ID NO : 39

MEADGEDAVLRGQVE IWKYMLSFADSMALKCAVELQLADI IHSHTSPITLSQIASAI PGATSPDLSCLARIMRLLVRRRI FTQHQK PKSDGEEEEALYGPTHSSRWLLTKTNDHDQLTLAPMILMENDPRLMAPWHCFSRCVKEGG VAFKKAHNGQSIWEFGAENPE INKLF NDAMECTAKVVMKAI LSHYSDGGFSDIKSMVDVGGGTGGSISEIVRSYPHIKGINFDLPHVIATAPPYSGVSHVG GDMFRSVPTAD AIFMKWILHDWSDEDCVKILKNCRKAIAEESGKVIIVESVLEEESNNNNNNNEVFGDTAL MLDLVMVAHTTGGKERTQKQWKTILE QGGFPRYNFIKIKALPSI IEAYPN (368)

>CsOMT2 [PK03696] SEQ ID NO : 40

SELLFQAQTHLYNHTLSYISSMCLKCAIELGIPDIINNHGQPHIPLPQLVSSLRLPPTKT DILRRLMRPLVHFGYFTTTKVVINSQ NKEEEEEEVDAYGLTSSSKLLFVNNNGNNKIPSMSTIVCLQLDQAEMTPWHSLGNWLRKD EATTLFESAHEMSFWEYTSKNTKFGH LFNEAMADDSKLMLKLVIEDVKPVFEGLTSLVDVGGGTGEVCKILTQVFPHLKCSVLELS HVVANLPNAQNLKFIEGDMFQAIPPA NAVLLKWILHNWSDEECVTILKKCREAIASNEGGKVVI IDVVINSKKDEHEVNEAKI SFDLMMMVLFNGRERSQKEWENLFFKAGF TRYKITPIFGLESLIEVFP (363)

>CsOMT3 [ RK04621] SEQ ID NO : 41

MASAVKGAILAIDNEASFQDQAEIWKYMLNFGDSMALKCAVELRLPDILHSADGPMTLSE IAAAIPNAPSPQASHLFRVLRLLVRR KIFTSEEDKVSGETLYGPTKLSSWLLHEPAPTSDSDASIMTLAPMLIMENHPWHVDPWHK LSEFIREGGLAPFEKEHGYMFDFAAK NPLYNKLLNNAMACTARITIRTLLSHCGDDLFNGVGSVVDVGGGTGRFISE IVKSYPHIKGINFDRSHVISTAPAYPGVTHIGGDM FQEVPSADAVVTKWVLHDWGDELCVKLLKNCRKAIPEKGGKVI IVDIVVEADGEGLFDDTGVVFDVLMLAHNTGGKERTEKEWKSL LDQSGFPRYKITKIPALQSVIEAYPN (370)

>CsOMT4 [PK05994] SEQ ID NO:42

MMSSINDNTITTTQQLSLGYVNLYKHMLSYASSMALKSAVELGIPDIIFNKGKAQTISLH ELVSALQIPPSRTNFLRRVMRVLVHS GIFTNEKGYNDDKEEEEVYGLTPSSKLLLTNGNNSEVPSVGPFVLSVLEPVTVTAFHLIG NWLKNEDSPATPFHLANDDGLGLYEY WGKNTDGFGDRLNEGMESDSGTLKFVLKNFKSTFEGITTLVDVGGNTGTMCKMLIEAFPH LKCSVFDLPYVVEANSHNNTENLKFI EGDMFQTIPEADAILFKLVLCGCSDDESIKILKNCREAISRNGKGKVLIIENNVINSEKD ELLELEAKLYFDMLLLASVTGRERSK KDWENIFFQAGFTHYEITPMFGLEALIEVFP ( 375 )

>CsOMT5 [PK07724] SEQ ID NO:43

MDALSRDQAEIFEHMFSFVDSMALKCAVELRIADIIHSQDCPISLTQIASKIITNSHNSP MISSSPDNNTMLYLNRIMTSLVRKKI FTAQYDHDQNNNQTVLYYGVTSKSRWLLRDAKPSLAPLI LMENHPIQMAPWHYFSHI IKDQEGSATAYEKAHGCGIFELASVNGEL NKI FNDGMACLGEMVMGAILPAYDVFGCMGSLVDVGGGIGGDLAE IVKSHPHIKGINFDLPHVTATAPESNGVTHVAGNMFESVPS ADAIFIKWVLHDWCDEECVKI LRNCRKAIPEKNGKVI IVEIVLKDSSQNKENDDVFDETRMIFDMVMMAHTCRGKERTEFEWKKLL EEAGFPRYKITKIPAIPSIIEAYPF (369) >CsOMT6 [PK08183] SEQ ID NO : 44

MAPTQISEELEASLFAMQLAGTSSILPMVLKTALELDLLEIIAMAGPNAFLSPSDIAAQL PTNNPNASMMLDRMLRLLASYNVLTY LLRDKVTSDGKVLVERLYGLAPLSKFLTKNEDGASIAPLCLMVQDKVEMESWYHLKDAIL DGGIAFDKAHGMPAFKYNQIDKRFNK IFNKGMFDHSSITMKKILETYKGFEGLNSMVDVGGGSGAVLSMIVTKYPSIKGINFDLHH VIEDAPPFPGVAHVGGDMFVSVPKGD AIFIKWICHDWSDEDCLKLLKNCYDAVPHHGKVIVAEFI LSYARDSSLAAKCTAHSDMIMLVDHGGKERTQKEFEELAKAAGFKGF KVVCNAFNTYIMEFLKTN (362)

>CsOMT7 [PK08793] SEQ ID NO: 45

MAVETHKDELIWIPKEDEEERARVDIYKYIFGFVEMAVVKCAIELGIADAIESHGRPMSL LELSSALGCAAPALHRIMRFLTNRKL FKE IRINENVQDSEQPSLYAQTALSRLILRSGEKSMATFVLMESSPPMLAPWHGLSARVKTEV DDSSAPSPFEVANGKDVWSYAAA NPGHSQLINEAMACNARVTVAAILDGCLDVFDGIGTIVDVGGGNGTALRMLVRACPWIRG INFDLPHVVSVALKSEGVEHVGGDMF KFVPKADAAFLMSVLHDWEDDECIQILKKCREAIPGDKGKVIMVECVIEENNNNVEEKHE ELELKDVGLFLDMVMIAHTNKGKERT LEEWAYVLAQAGFNRYNIRAINAIYSI IEAFPN (377 )

>CsOMT8 [PK10317] SEQ ID NO : 46

MAKMQEGGDHHDELTCRAKEEDEQEEERARIDIYKYVFGFVEMAVVKCAIELGIADT IECHGRPMSLKELSSALGCTPHNIHRIMR FLVNRRIFKEIKNDIVNDEGAGTLYVQTSLSRLLIKSGERSMASFVLMESSKPMLAPWHC LSSRLKAEVIDNSLTPFEEANGQDLW SYTAANPEHSQLLNEAMACNARVTVAAILDSCLEVFDGVGSIVDVGGGNGTAMQLLVKGC PWIKEGILFDLPHVVSVALKSDRVVH VGGDMFDSVPKADAAFLMWVLHDWEDKDCIQILKNCREAISEKGKVI IVESVIENNKEQNNGMKKDELEFKDVGLFLDMVMMAHTN KGKERTLDQWVYVLHQAGFTRYNVRSIKGAISSLIEAFPI (384 )

>CsOMT9 [PK10819] SEQ ID NO:47

MEKLKSFSYLNNNIDLAMNEENNTKLLGAQAHIWNQIFNFINSMCLKCAVELGIPDI INNYGKPMTISQLTLALSINKNKFHCLYR LMRLLTHSGFFALEKVEIEGEKEEEGYVITEASKLLLKDNPMSVTPFLLLVLNPILTKSF DVLDTWFQNDSPTPFDTANGRTFWDY GSHEPKLVQLFNDGMASDARLVTSVVIEKCKGVFEGVERLVDVGGGTGTVAKSIATAFPQ IECSVLDLPHVVADLEDENNLKFIGG DMFVEIPTADVVLLKWILHDWNDEESVKILKNCREAVYKSKKKSGKLI I IDMNIKNDNNENSFETQLFFDMLMMALVSGRERNEKE WSKLFKDAGFSRYKI TPILGLRFVIEVYP (373)

KCsOMTlO [PK11500] SEQ ID NO:48

MDGIQEGDHHDELTLRLNEKEEEERARIDIYKYVFGFVEMAVVKCAIELGIADTIESHGR PISLLDLSSALSCNPHNLHRIMRFLV NRRIFKEIKNDTVNDKGCLYVQTSLSRLLIKSGERSMASFVLMESSNPMLAPWHGLSARV KAEATDALTPFEAANGVDVWSYAAAN PDHSQLINEAMACNARVTVAAILNGCLDVFDGVGSIVDVGGGNGTTLQLLVKGCPWINQG INFDLPHVVSVALKSDGVVHVGGDMF DSVPKADAAFLMWVLHDWGDQECIQILKKCKEAIPEKGKVI IVESVIENNKLEENVMKKELELKDVGLFLDMTMMAHTNKGKERSL DEWVYVLHQAGFTRYNVRSIDGAVSSVIEAFPA (377 )

>CsOMTll [PK12774] SEQ ID NO:49

MGSISENTTTLELSQGYVNVYKHMLSYASSMALKCAIQLHIPDIIFTKGKDQTITLPELA SALQIPPSRISCLRRVMRVLVHSGIF SNKNQHDDDKTEEEEVYGLTSSSKILLTGGNNNGVPSVGGYVLAVLEPVTVTAFHLIGSW LKKESPRTPFHLANDEGLSLYEYWGK NIDGFGDRLDEGMESDSGVLKFVLKDLKSSFEDITTLVDVGGNTGSMCMMLIEEFPHLKC TVFDLPYAIEANSHNSTSNLKFVEGD MFQSTFPEADAFLLKSVLSGCSDEECVKILKNCGEAISRNGGGKVMVIDNNVINTKNDEA AELEAKLYFDMLEMTALTGRERTKKD YENIFYQAGFTRIKI TRMFGLKSLIEGFL (373)

>CsOMT12 [PK13022] SEQ ID NO:50

MTTPTQMSEELEANYLFAMKLASATVLPMVLKTALELGLLEI IVMAGPGAFLSPSNIVAQLPTKNPNAPVMLDRMLRLLASYNVLT YSIRDGERLYGMTPLSKFLTKNEGGLS IAPLCHMDQDKVI IDCWYHMKDAVLDGGI PFNKAHGMPIFEYTQRDQRLNKIVNRAMST LST I IMKNI LETYNGFKGLNS IVDVGGGTGATLSMI IAKYPSIKGINFDLHHVIQHAPPLPGVEHVSGDMFVSVPKGDAIFMKRIC HDWSDEECLKLLKNCYDALDDDGKVIVEELIVPAAPDSSPSTKNSFHYDILMMVNLNGKE RTQKEYEQLAMEAGFKAFKIHCIAFN SYIMEFLAKGPKVFWSVRVPPLL (367)

>CsOMT13 [PK15692] SEQ ID NO:51

MESSQLRGQELICQLIFSYYNTMALKCAVELRIADIIHSHGKPITISQIASDIQSNSNSK SPINIDNLFRIMRILVRKGVFIEHHD DDHGDSTISLYGLCDSSRCLLWDFDSSLVPFILLNTHPLMMASSHNFGKSVIGDKGNPFE NDQDVWSFASNNPIFNKLFNDAMISG SHMVLRHVLSTYKDSFNCIKGTMVDVGGGVGQVISEIVKSHRHIKGINFDLPHVIATAPT YDGVTHVGGDMFES IPSADVVFLKWI IHGWNDDACVKILKNCRKAIGEKKNGKI 11VDMVLDPNSNEIFQETRLAMDLVMLANSNNGKERTELEWKKLLNEAGFSRYKITKN QNI LDI IEAFPF (356)

>CsOMT14 [PK17162] SEQ ID NO:52

MGSINENTITTTQELSQGYVNLYKHMLSYASSMALKSAVELGIPDIIFTKGKAQTISLHE LVSALQIPPSRTNFLRRVMRVLVHSG IFTNEKGYNDDKEEEEVYGLTPSSKLLLTGGKNNGVPSVGPYVLAVLEPVTVTAFGS IGNWLKKESPTTPFHLANDEGLSLYEYWG KNTDGFGDRLNEGMESDSGVLKFVLKDFKSVFEGITTLVDVGGNTGLMCKMLLEAFPHLQ CSVYDLAYAVDANSHNNTQNLKFIEG DMFQTVPQADAILFKCVLSGCSDEECTKILKNCRDAISRNGGGKVLI IDNNVINSKTEDHLAMETLLYFDMLMMTALTGRERTKKD WEKIFFEAGFSSCKVTPMFGVKTLIEVFP (373)

>CsOMT15 [ PK19674.1] SEQ ID NO:53

MGSELEGTTEVVVDLKRKQEEESFCYAAQLLNTNVLTKSLQTTIELGIFDI IAKAGEGGKLSAREIVAQLPTNNPDAPMMVDRILR MLASYSVLVCSVVADDQRAYSLNNVSKCFVTNEDGVSLGPLMLLLEDKVFSDSWSQLKGA ILEGGIPFNRFHGMNAFEYPALDSRF NKVFNRAMQSMTTMLAKQTIESYKGFENLKQLVDVGGGLGVTLKE ITSTYPHIKGINFDLPHVVQHAPSYPGVEHVGGDMFESVPS GDAIEMKWI LHDWSDEQCVKVLKNCYKAIPENGKVIVMEGLLPMLPEASYGDNIMSKTDVLMMTQNPGG KERSKQEFQALASGAGF NGIRFECCVSGFWIMEFFK (363) >CsOMTl 6 [ PK19674.2] SEQ ID NO:54

MAPPSEELANTPQIVNDERKQEEENFAYAAQLVNSSVLSMSLQSAIELGVFDI IAKAGDAAKLSAQDIVAQMPTTNPDAPRMLDRI LRMLASHSVLACSLENEDLRVYCLNDVSKLFVTNEDGVSLGPLMLLQDKVFLDSWSQLKG AILEGGIPFNRVHGMHAFEYPSLDQK FNQVFNKAMYNQTTLVLKKILEVYKGFENLEKVVDVGGGLGGTLNQITSKYPHIKGINFD LPHVVEHAPSYPGVEHVGGDMFESVP TGAIEMKWI LHDWSDEHCLKLLKNCYKAIPDNGNVIVMEAILPT IPETNSADRCTSQTDVLMMTQNPGGKERSKQEFQALASGAGF NGIRFECCVSGFWIMEFFK (363)

>CsOMT17 [ PK19674.3] SEQ ID NO:55

MMGSDQLEVIVDLKRPKQEESFCYALQLLSTNI LIKSLQATVELGIFDI IAKAGEGSKLSAAE IVAQLPTNNPDAVMMVDRILRML AGHSVLTCSVVADNPRVYSHNTVSKCFVTDEDGVSLGSLISLLDDKVYSDSWSQLKGAIL EGGIPFNRLHGMNSFEYTALDSRFNK VFNRAMQSMTTMIAKQTIESYKGFENLKQLVDVGGGLGVTLKEITSTYPHIKGINFDLPH VVQHAPSYPGVEHVGGDMFESVPSGD AIFMKWILHDWSDEQCVKVLKNCYKAI PENGKVIVMEGLLPMLPEASYGDNIMSKTDVLMMTQNPGGKERSKQEFQALASGAGFNG IRFECCVSGFWIMEFFK (361)

>CsOMT18 [ PK19674.4] SEQ ID NO:56

MAPPSEELANTPQIVNDERKQEEENFAYAAQLVNSSVLSMSLQSAIELGVFDI IAKAGDAAKLSAQDIVAQMPTTNPDAPRMLDRI LRMLASHSVLACSLENEDLRVYCLNDVSKLFVTNEDGVSLGPLMSLLQDKVFLDSWSQLK GAILEGGIPFNRVHGMHAFEYPSLDQ KFNQVFNKAMYNQTTLVLKKILEVYKGFENLEKVVDVGGGLGGTLNQITSKYPHIKGINF DLPHVVEHAPSYPGVEHVGGDMFESV PTGDAIEMKWILHDWSDEHCLKLLKNCYKAIPDNGNVIVMEAILPTIPETNSADRCTSQT DVLMMTQNPGGKERSKKEFLALATGA GFSGIRFECFVCNEMIMEFYK (365)

>CsOMTl 9 [PK19715] SEQ ID NO:57

MEKSRNSSSHVDLVVNEDNNTKLLRAQAHIWNHICKFINSMSLKCAIELGIPDIVNNHGK PMTISQLTLALPINKNKSHCLYRLMR LLTHSGFFALEKTEIKGEEEEEGYVITEASKLLLKDNPMSVTPLLLVLLDPTLTKPYDVL STWFRNDDSTPFVTTNGMAIWDYYSH EPKLAQSFNEAMASDARLVTSVLIEKCKGVFEGVDSLVDVGGGTGTVAKS IATTFPQIQCSVLDLPHVVAGLQGEKNLNFIAGDMF VEVPTAQVVLLKWILHDWSDENSVKILKKCKEAITKSGKKIGKVVVIDMI IENEKGE IDDESYETQLEMDMTMTLVSGRERNEKEL SKLFKDAGFSHYKITPILGLRSLIEIYP (372 )

>CsOMT20 [PK23308] SEQ ID NO:58

MEKLKSFRHLNNNIDLVLNEENSIELLRAQGHIWNQIFNFINSMSLKCAIQLGIPDIINN YGKPMTISQLKLALPINQKKSSCVYR LMRILTHSNFFALQKVEGREGEEEEEGYVITDASKLLLKDNPMSVTPFLLAMLDPVITKP WDFLSNWFQNDDPTPFDTANGMTEWD YGSHQPNLARFFNDAMASDARLVTSVVIEKCRWVFEGVESLVDVGGGTGTVATTIATSFP QIQCSVLDLPHVVADLQGANNLVNFI GGDMFVEVPPAEVVLLKWILHDAINDEESVKILKKCKEAI TKNNKKGGKVI 11DMKVENEKDEDDESYETQLFFDMLMMALVTGKER NEKEWAKLFKDAGFSDYKITPILGLRSLIEVYP (377 )

>CsOMT21 [PK24150] SEQ ID NO : 59

MGSTGIETQMTPTQI SDEEANLFAMQLASASVLPMVLKAALELDLLEI IAKAGPGAFLSPSDIAQQLPTQNPDAPVMLDRMLRLLA SYNVVTYSLRERETAEEEGKVERLYGLAPVSKYLTKNEDGVSIAPLCLMNQDKVLMESWY HLKDAVLDGGIPFNKAYGMTAFEYHG TDQRFNKIFNRGMSDHSTITMKKILETYKGFEGLNSIVDVGGGTGAVVNMIVSKYPTIKG INFDLPHVIEDAPPLTGVEHVGGDMF VSVPKGDAI EIIKWICHDWSDEHCLKFLKNCIIAALPEHGKVIVAECILPVAPDSSLATKSTVHIDVIM LAHNPGGKERTEKEFEALA KGAGFKGFKVHCNAFNTHIMEFLKTI (370)

>CsOMT22 [PK27112] SEQ ID NO:60

MNLIMGEGELVSCRELVEAQELIYNCSLSHIKPMSLKCAIELGIPDIIHNHGQPITLSKL ISSLPIHPSKAHCIHRLMRILVHFGF FTTQLLLPQQQEETYSLTLASKLFLKDCPIKATPFFLVQLNPLLLKPWHFLSTWLQKGED DDDHPSTPFEMANGINEWDGVGNDPM VKYMFTEAMATDSYLMSKVIVEEGKEVFEGLSSLVDVGGGTGIMANAIVEAFTNIKCTVL DLPYIVADLKGTHNLNFVEGDMFKKI PSANAVLLKWTLHDWNDEEVVVILKKCREAIWSKDKGGKVIVIDMVIDDDEEPKSSVETQ LCFDMLMMVNLTGKERNEKEWENLFL AAGFSHYKINPIVGFRSLIEVFP (367)

>CsOMT23 [PK27154] SEQ ID NO:61

MEKLKSFRHLNNNIDLVLNEENSIELLGAQGHIWNQIFNFINSMSLKCAIQLGIPDIVNN YRKPMTISQLVLALPINQKKSPCVYR LMRILIHSGFFALQKVEGGGEGEEEEGYVITDASKLLLKDNPMSVTPFLLSMLDPVMTKP WDFLSNWFQNDDPTPFDTANGMTEWD YGSHQPNLARFFNDAMASDARLVTSVVIEKCKWVFEGVESLVDVGGGTGTVATSIATNFP QIQCTVLDLPHVVADLQGGNNLNFVG GDMFVEVPTAEVVLLKWILHDWNDEESVKILKKCKEAIMKSKKKGGKVIIIDMKVENEKD EDDESYETQLFFDMLMMTLLTGKERN EKEWAKLFKDAGFSDYKITPI LGLRSVIEVYP (376)

>CsOMT24 [PK29262] SEQ ID NO:62

MQKGQKGCQINQIPMSIERNNVEEDESFFYAVELRSSVVLPMSLYATIELGVFEILAKAG DGAKLSSSDIASHLPTENPDAPMMLD RILTLLASHSVLDCVVVGEGSSMRKLYSLSPVSKHFLPKEDGVSSHALMKLGLDKVSLES WFELKNAVLEGGTSFKRAHGMNVFEY GKSDSRFGEVFNSAMYNQAKIVTKKI IESYKGFENNIKTLVDVGGGFGVTVSLIVSKYPQIKAINFDLPHVIKNAPTYPGVEHVGG DMFEKIPNGDAIFMKWILHD1NNDEDCVKILKKCYEAIPSNGKVIVVDMVVPIMAETTHK AKSI FQLDLVMLSQNPGGKERNQHEFQ AIANAAGFSTINFACSIENVKVIEFIK (371)

>OsOMT9 SEQ ID NO: 63

MGSTAADMAAAADEEACMYALQLASSS ILPMTLKNAIELGLLETLQSAAVAGGGGKAALLTPAEVADKLPSKANPAAADMVDRMLR LLASYNVVRCEMEEGADGKLSRRYAAAPVCKWLTPNEDGVSMAALALMNQDKVLMESINY YLKDAVLDGGIPFNKAYGMTAFEYHGT DARFNRVFNEGMKNHSVI ITKKLLDLYTGFDAASTVVDVGGGVGATVAAVVSRHPHIRGINYDLPHVISEAPPFPGVE HVGGDMFA SVPRGGDAI LMKWILHDWSDEHCARLLKNCYDALPEHGKVVVVECVLPESSDATAREQGVFHVDMIMLA HNPGGKERYEREFRELA RAAGFTGFKATYIYANAWAIEFTK (368) >SlaGOMT SEQ ID NO: 64

MENPKELLNAQAHIWNHIFAYHRSAALKCAVELGIPDTIEKHGNPMTLQDLANSLAI TPTKTLSLYRLLRLLVHSNFFSMTKLVDG EEAYANNINSQLLLKDHPCTLAPFTLGMLDPAMTEPPHYLSKWFQNQDESVFHVIHGRSF WEHAGLTPGFNQLFNRAMGSDASFVS IALVANKDFAKMVEGIGSLVDVAGGDGTVAKI IARAYPWLKCTVFDLPQVVDGLQGNGSNLEYVAGDMFKEIPSADVVMLKWILHD WSDEHCVRI LERCKEAIPSNGKI 11 IDMVVDPQAQNNNHFQAQLLSDMEMMALNVGGIERTEDQWKKLFLQAGFNHYNIFPILGI R SVIEVRCL (352)

VRhOOMTl SEQ ID NO: 65

MERLNSFRHLNQKWSNGEHSNELLHAQAHIWNHIFSFINSMSLKSAIQLGI PDI INKHGYPMTLSELTSALPIHPTKSHSVYRLMR ILVHSGFFAKKKLSKTDEEGYTLTDASQLLLKDHPLSLTPYLTAMLDPVLTNPWNYLSTW FQNDDPTPFDTAHGMTFWDYGNHQPS IAHLFNDAMASDARLVTSVI INDCKGVFEGLESLVDVGGGTGTLAKAIADAFPHIECTVLDLPHVVADLQGSKNLKYTGG DMFEAV PPADTVLLKWILHDWSDEECIKILERSRVAITGKEKKGKVI I IDMMMENQKGDEESIETQLFFDMLMMALVGGKERNEKEWAKLFT DAGFSDYKI TPISGLRSLIEVYP (367)

>RhOOMT2 SEQ ID NO: 66

MERLNSFKHLNQKWSNGEHSNELLHAQAHIWNHIFSFINSMSLKSAIQLGI PDI INKHGPMTLSELTSALPIHPTKSHSVYRLMRI LVHSGFFAKKKLSKTDEEGYTLTDASQLLLKDHPLSLTPFLTAMLDPVLTTPWNYLSTWF QNEDPTPFDTAHGMTFWDYGNHQPSI AHLFNDAMASDARLVTSVI IDDCKGVFEGLESLVDVGGGTGTVAKAIADAFPHIECTVLDLPHVVADLQGSKNLKYTGG DMFEAVP PADTVLLKWILHDWNDEECIKILKRSRVAITSKDKKGKVIIIDMMMENQKGDEESIETQL FFDMLMMALVRGQERNEKEWAKLFTD AGFSDYKITPILGLRSLIEVYP (366)

VNtCTOMT SEQ ID NO: 67

MESSTKSQIPTQSEEERNCTYAMQLLSSSVLPFVLHSTIQLEVFEILAKSNDTKLSASQI VSQIPNCKNPDAATMLDRMLYVLASY SLFTCSIVEDEENNGGQKRVYGLSQVGKFFVRDEDGASMGPLLALLQDKVFINSWFELKD AVLEGGVPFDRVHGVVHAFEYPKSDP KFNDVFNKAMINHTTVVMKKI LENYKGFENLKTLVDVGGGLGVNLKMITSKYPTIKGTNFDLPHVVQHAPSYPGVEHVGGD MFESV PEGDAIEMKWILHDWSDSHNLKLLKNCYKALPDNGKVIVVEAILPVKPDIDTAVVGVSQC DLIMMAQNPGGKERSEEEFRALATEA GFKGVNLICCVCNFWVMEFCK (365)

VNtCAOMT SEQ ID NO: 68

MESSTKSQIPTQSEEERNCTYAMQLLSSSVLPFVLHSTIQLEVFEILAKSNDTKLSASQI VSQIPNCTKPEAPTMLNRMLYVLASY SLFTCSIVEDEKNNGGQKRVYGLSQVGKFFVKNENGASMGPLLALLQNKVFINSWFELKD AVLEGGVPFDRVHGVHAFEYPKSDPK FNDVFNKAMINHTTVVMKKILENYKGFENLKTLVDVGGGLGVNLKMITSKYPTIKGTNFD LPHVVQHAPSYPGVEHVGGDMFESVP EGDAIEMKWILHDWSDSHNLKLLKNCYKALPDNGKVIVVEAILPVKPDIDTAVVGVSQCD LIMMAQNPGGKERSEEEFRALATEAG FKGVNLICCVCNFWVMEFCK (364)

VRgANMT SEQ ID NO: 69

MGSLSESHTQYKHGVEVEEDEEESYSRAMQLSMAIVLPMATQSAIQLGVFE I IAKAPGGRLSASEIATI LQAQNPKAPVMLDRMLR LLVSHRVLDCSVSGPAGERLYGLTSVSKYFVPDQDGASLGNEMALPLDKVFMESWMGVKG AVMEGGIPFNRVHGMHIFEYASSNSK FSDTYHRAMFNHSTIALKRILEHYKGFENVTKLVDVGGGLGVTLSMIASKYPHIQAINFD LPHVVQDAASYPGVEHVGGNMFESVP EGDAILMKWILHCWDDEQCLRILKNCYKATPENGKVIVMNSVVPETPEVSSSARETSLLD VLLMTRDGGGRERTQKEFTELAIGAG FKGINFACCVCNLHIMEFFK (364)

>S1CT0MT SEQ ID NO : 70

MGSTANIQLATQSEDEERNCTYAMQLLSSSVLPFVLHST IQLDVFDILAKDKAATKLSALEIVSHMPNCKNPDAATMLDRMLYVLA SYSLLDCSVVEEGNGVTERRYGLSRVGKFFVRDEDGASMGPLLALLQDKVFINSWFELKD AVLEGGVPFDRVHGVHAFEYPKLDPK FNDVFNQAMINHTTVVMKRILENYKGFENLKTLVDVGGGLGVNLKMITSKYPTIKGTNFD LPHVVQHAPSYPGVDHVGGDMFESVP QGDAIEMKWILHDWSDGHCLKLLKNCHKALPDNGKVIVVEANLPVKPDTDTTVVGVSQCD LIMMAQNPGGKERSEQEFRALASEAG FKGVNLICCVCNFWVMEFYK (364)

VHIOMTI SEQ ID NO : 71

MESLRGQEQIWQLMFSFVDSMALKCAIELRIADIIHSHGKPITLSQIASGIRSNSNSSIS PNIPYLSRIMRFLVRKNIFTEHQEDN DEVISLYGLSDSSRWLLRDFKSSLAPMVLMQTHPLSMAVWHFLEDYVPNSSNTFEKAHGC NIWEFSSANPDFNKI FNNAMASIVPI YMGAVLSSYKDGLGCIKGTVVDVGGGTGGSISELMKYYPNIKGINFDLPHVIATAPALDG VTHISGDIFESIPSADAVLMKGVLHC FSDEKCVKVLRNCRKAITDKKNGKIIILEIVLDPTSNQIFDETRMVYDLLIPXFSGGKER TELEWKRLLNEAGFTSIKITKIPIIP AIIEAFLV (352)

VH10MT2 SEQ ID NO: 72

MELARNDQTEAALRGEANVWKSINGIADEMVMKCALELRIPDIVHSHSAPI TLAQIASSVPDSPSLNLSYLSRIMRLLVRRKIFSQ HKSLDGEEVLYGPTHSSRLLLSKTTLPDQVTLAPFVAEMTHPYLSAPWSCLARCVKEGGN GFEMVHGGRQLWDLSPGNPEFNKVFN DGMASTARI TTMAILSEYRDVFCGICSLVDVGGEFGGSI SAIVKSHPHIKGINYDLPHVVATAPTYTGLVSHVGGNMFEWI PTAVA VEMKWILHDWADEDCVKILKNCRRAMPEKGGKI I IVDIVLEPEGNGLFDDAAVMLDIALMALTRGKERTEKEWKRVLEEGGFPRYQ ILKIPALTSVIEAYPQ (360)

VH10MT3 SEQ ID NO: 73

MEKLKSFRHLNNNIDLILNEENSTEILGAQAHIWNQIFNFINSMSLKCAIQLGIPDI INNHGKPMTISQLTLALPINRKKSPCVYR LMRILIHSGFFALQKAEVGEEGGGEEEGYVITDASKLLLKDNPMSVTPFLLAMLDPVMTK PWDFLSNWFQNGDPTPFDTANGMAFW DYGSHEPKLARFFNDAMASDARLVTSVVIEKCKGVFEGVESLVDVGGGTGTVASSIAAAF PHIQCTVFDLPHVVADLQGGNNLNFV GGDMFVDVPATEVVLLKWILHDWNDEESVKILKKCKEAI SKSNKKGGKVI I IDMKVENEKDEDDESYETQLFFDMLMMALVTGRER NEKEWAKLFKDAGFSNYKITPILGLRSLIEVYP (377 ) >GeOMT SEQ ID NO : 74

MAFSTNGSEEIELYHAQIHLYKHVYNFVSSMALKSAMELGIADVIHNHGKPITLPELASA LKLHPSKVGILYRFLRLLTHNGFFAK TTVPSQNGKDGEEEEETAYALTPPSKLLVKGKPTCLASIVRGALHPSSLDMWPSSEKWFK EDKELTLFESATGESEWDFLNKDSES GTLSMFQEAMAADSQMFKLALKECRHVFEGLESLVDVGGGTGGVTKLIHEEFPHLKCTVF DQPQVVGNLSGNENLKFVGGDMFKSI PPADAVLLKWVLHDWNDELSLKILKNSKEAISGKGKEGKVIIIDISIDEASGDRELTELQ LDYDLVMLTMFNGKEREKKEWEKLIS DAGFSSYKI TPICGFKSLIEVFP (367)

ASbCOMT SEQ ID NO: 75

MGSTAEDVAAVADEEACMYAMQLASSSILPMTLKNALELGLLEVLQKDAGKALAAEEVVA RLPVAPTNPAAADMVDPMLRLLASYD VVKCQMEDKDGKYERRYSAAPVGKWLTPNEDGVSMAALALMNQDKVLMESWYYLKDAVLD GGIPFNKAYGMTAFEYHGTDPRFNRV FNEGMKNHSVIITKKLLEFYTGFDESVSTLVDVGGGIGATLHAITSHHSHIRGVNFDLPH VISEAPPFPGVQHVGGDMFKSVPAGD AILMKWILHDWSDAHCATLLKNCYDALPEKGGKVIVVECVLPVTTDAVPKAQGVFHVDMI MLAHNPGGRERYEREFRDLAKAAGFS GFKATYIYANAWAIEFIK (362)

AZ COMT SEQ ID NO : 76

MGSTAGDVAAVVDEEACMYAMQLASSSILPMTLKNAIELGLLEVLQKEAGGGKAALAPEE VVARMPAAPSDPAAAAAMVDRMLRLL ASYDVVRCQMEDRDGRYERRYSAAPVCKWLTPNEDGVSMAALALMNQDKVLMESWYYLKD AVLDGGIPFNKAYGMTAFEYHGTDAR FNRVFNEGMKNHSVIITKKLLDFYTGFEGVSTLVDVGGGVGATLHAITSRHPHISGVNFD LPHVISEAPPFPGVRHVGGDMFASVP AGDAILMKWILHDWSDAHCATLLKNCYDALPENGKVIVVECVLPVNTEATPKAQGVFHVD MIMLAHNPGGKERYEREFRELAKGAG FSGFKATYIYANAWAIEFIK (364)

>VvOMT SEQ ID NO: 77

MDLANGVISAELLHAQAHVWNHIFNFIKSMSLKCAIQLGIPDIIHNHGKPMTLPELVAKL PVHPKPSQCVYRLMRILVHSGFLAAQ RVQQGKEEEGYVLTDASRLLLMDDSLSIRPLVLAMLDPILTKPWHYLSAWFQNDDPTPFH TAHERSETAIDYAGHEPQLNNSFNEAMA SDARLLTSVLLKEGQGVFAGLNSLVDVGGGTGKVAKAIANAFPHLNCTVLDLPHVVAGLQ GSKNLNYFAGDMFEAIPPADAILLKW ILHDWSDEECVKILKRCREAIPSKENGGKVIIIDMIMMKNQGDYKSTETQLFFDMTMMIF APGRERDENEWEKLFLDAGFSHYKIT PILGLRSLIEVYP (357)

AObCVOMT SEQ ID NO : 78

MALQNMDISLSTEQLLQAQAHVWNHMYAFANSMSLKCAIQLGIPDILHKHDHPMTLSQLL KAIPINKEKSQSFQRLMRALVNSNFF IEENSNNQEVCYWLTPASRLLLKGAPLTVAPLVQVVLDPTFTNPWHYMSEWFKHENHATQ FEAANGCTFWEKLANKPSMGRFFDEA MSCDSRLVAHVLTKDYKHVIDGIRTLVDVGGGNGTMAKAIVEAVPTMKCTVLDLPHVVAG LESTDKLSYIGGDMFQSIPSADAILL KFIIHDWDDEEGLKILKPCKDAVGIGGKVIIIDVVVGVNHDVDEVLEDQLHFDMAMMSYF NAKERTMNEWEKLISAAGFTSYKLTP AFGVRSLIEAYP (356)

AObEOMT SEQ ID NO : 79

MALQKVDISLSTEQLLQAQVHVWNHMYAFANSMSLKCAIQLGIPDILHKHGRPMTLSQLL QSIPINKEKTQCFQRLMRALVNSNFF IEENNSNNQEVCYWLTPASCLLLKEAPLTVTPLVQVVLDPTFTNPWHHMSEWFTHEKHAT QFEAANGCTEWEKLANEPSKGRFFDE AMSCDSRLIAHVFTKDYKHVIEGIRTLVDVGGGNGTMAKAIVEAMPTIKCTVIDLPHVVA GLESTDNLNYIGGDMFQSIPSADAIL LKSIIHDWDDVEGLKILKKCKDAVVMGGKVIIIDVVVGVNHDIDEVLEDQLHFDMAMMCY FNAKERTMSEWEKLIYDAGFKSYKLT PAFGVRSLIEAYP (357)

AShMOMTl [ADZ76433 ] SEQ ID NO: 80

MALSMDNIVISNEEEICMMKAMHLPCGLYLNMVLKAAIELDLFEIIAKSTTQKLSSYEIA SQIPTKNPNASSLVLERILRFLASQS LLTCNITKNDDGNVHTTYNLTPLSQSLISDKDGTSIAPFLLLATDPVGVHACFHLKDAIL EGEIPFNKAHGVHAFEYHGKDSPMNG LFNKAMQNLTCIEMKRIVECYNGFQGVKEIIDVGGGLGISLASIISKYPNIKGINFDLPH VIKDAPTYEGIEHVGGDMWDSIPQGE LIILKAVLHSLDDEDCVKILKNCWRALPNDGKVVVIEQIQPKYPETNLLSKRSFSFDISM MIMFHGGKERTKQQFEDLAKQAGFTY IKVVARAYYSWLIELYKY (362)

>ShMOMT2 [ADZ76434 ] SEQ ID NO: 81

MASNNNCAYELIEAEAQSWDYILSYLRPSCIKCAIQLGIPDILHKNADPIMSLSDLIAAL PNLNPSKTTFIPILMRVLVDFGLFNY HQQQGDGYSLTTVGRLLVENHHFGNRSFFLFAQHPVVLNTAASVGDWLKDDLRTAFETAD GKSHWDYCGADPEFNGVFNDAMAGDS RLMSNLLISDCCAGVFEGLTSLVDIGGGTGAVAMAIAGAFPSLKCIVLDLPHVIADRKGS GNLEFVAGSMFDKIPHANAILLKWIL HNWDDEDCVKLLKKCKESISSRENGGKVIIIDMIMEDNYNNKQLVQSQHLMDLIMRITYA SKERTEKEWEKLFLEAGFSGYKIITS LGLRSLIEIYP (355)

>AtOMT1 [AAB96879] SEQ ID NO: 82

MGSTAETQLTPVQVTDDEAALFAMQLASASVLPMALKSALELDLLEIMAKNGSPMSPTEI ASKLPTKNPEAPVMLDRILRLLTSYS VLTCSNRKLSGDGVERIYGLGPVCKYLTKNEDGVSIAALCLMNQDKVLMESWYHLKDAIL DGGIPFNKAYGMSAFEYHGTDPRFNK VFNNGMSNHSTITMKKILETYKGFEGLTSLVDVGGGIGATLKMIVSKYPNLKGINFNLPH VIEDAPSHPGIEHVGGDMFVSVPKGD AIFMKWICHDWSDEHCVKFLKNCYESLPEDGKVILAECILPETPDSSLSTKQVVHVDCIM LAHNPGGKERTEKEFEALAKASGFKG IKVVCDAFGVNLIELLKKL (363)

>CrFOMT 6 SEQ ID NO: 83

MDLQTAEFREAQAKIWSQAFSFANCAALKCAVKLGIADAIDNHDKKALTLSELTEELSIK PSKSPFLQRLMRQLVNAGFFTEAKQL RDDNKDGRTTTAYALTPVSRLLLKNEQWNLRGIVLTMLDPAELKAWSVLNDWFKNDDPTA FQTAHEKNYWDYTAENTQHCQIFEDA MANDSVLVSKLLVTEYKFLFEGLTSLIDLGGSTGTIAKALAKSFPNLKCTVFDLPHVVAN LESTKNLEFVGGDMFEKLPPSNAILL KWILHDWNDEDCVKILKNCKKAIQEKGNGGKVIIIDTVVYSQKNEKELVDLQISMDMAMV INFAAKERTEEEWEHLIREAGFSGHK IFPMYDFRSIIEVYP (359) >CdFOMT5 SEQ ID NO: 84

MGS IVDGERDQSFAYASQLVMGTVLPMAIQAVYELGIFE ILDKVGPGAKLCASDIAAQLLTKNKDAPMMLDRILRLLASYSVVECS LDASGARRLYSLNSVSKYYVPNKDGVLLGPLLQMNQDKVLLESWSQLKDAI LEGGIPFNRAHGVHIFEYAGLDPKFNKRFNTAMYN YTSLVLSNI LESYKGFDNIKQLVDVGGSLGVTLQAITTKYPYIKGINFDQPHVIDHAPPHPRIEHVGGD MFQSVPKGDAI IMKSVL HDWDDEHCLKLLKNCYKSIPEDGKVIVVESMLPEVPNTS IESKSNSHLDVLMMIQSPGGKERTRHEEMTLATGAGFGGISCELAIG SLWVMEFYK (353)

>MpOMT3 [AAR09601] SEQ ID NO: 85

MEASFENGRKRSSSSSSEEESAFSFAMELAAGSVLPMVIKSAIDLNLLELIKRGGEEGAS AYELAAQINAENPKAAAEMIDRILQL LAAHSVLTCRVETPPSRRRRYSLAAVCKFLTRDEDGASLAPLSLLVQDRVFMEPWYHLKD VIVEGGVAFERAYGVHAFEYHAKDPK FNKIFNQAMHNQSIIEMKRILEIYKGFEGVKSLVDVGGGTGASSKMIVSKYPLIKAINFD LPHVIQDASPHPEVEHVGGDMFVSVP KADAIFLKWICHDWSDEHCRKLLKNCYDAILGNGKVI IAESTLPEDPNSGPDTIHAIRGDVIMLTVNPGGKERTEKEFRTLALQAG FKRLVKVCAAFHTCIMECHK (364)

>CaFOMT [AAA80579] SEQ ID NO : 86

MLFAMQLASASVLPMVLKSAIELDLLE I IASQDTCMSPTEIASHLPTTNPHAPTMIDRILRLLSSYSIVTCSVRSVDDQRVYSPAP

VCKYLTKNQDGVSIAALCVAAQDKVLMECWYHMKDAVLDGGIPFNKAYGMPIFDYFA KDLGSNKVFNKGMSDFSSMIIKKILETYK

GFQGLTSLVDVGGGTGATLTKILSKYPTIRGINFDLPHVIQDAPEYPGIEHVGGDMF VSVPKGDAIEMKWICHDWNEEQCLKLLKN

CYDALPNNGKVIVAEYILPVVPDSSLASKLSVTADVMIVTQNSGGKERTEKEFEALA KAAGFQGFQVFCNAFTIYIIEFSKNISN

(343)

>TaFOMT [ABB03907] SEQ ID NO:87

MGS IAAGADEDACMYALQLVSSSILPMTLKNAIELGLLETLMAAGGKFLTPAEVAAKLPSAAN PEAPDMVDRMLRLLASYNVVSCR TEDGKDGRLSRRYGAAPVCKYLTPNEDGVSMSALALMNQDKVLMESWYYLKDAVLDGGIP FNKAYGMSAFEYHGTDPRFNRVFNEG MKNHS111TKKLLESYKGFEGLGTLVDVGGGVGATVAAI TAHYPT IKGINFDLPHVI SEAPPFPGVTHVGGDMFQKVPSGDAILMK WILHDWSDEHCATLLKNCYDALPAHGKVVLVECILPVNPEATPKAQGVFHVDMIMLAHNP GGRERYEREFEALAKGAGFAAMKTTY IYANAWAIEFTK (356)

The variants of the polypeptides described herein may have any degree of sequence identity to the native polypeptides, provided they retain some degree of native activity, for example

prenyltransferase or methyltransferase activity. For example, the variants typically have at least about 70%, about 71 %, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity. Typically the variants have at least about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the corresponding native polypeptide.

Likewise, the fragments of the polypeptides or variants described herein may have any length, provided they retain some degree of native activity, for example prenyltransferase or methyltransferase activity. For example, the fragments may be missing about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 , about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 250, or about 300 amino acid residues as compared to polypeptide in question. Thus, the fragments of the polypeptides or variants described herein may have about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, or about 370 or more amino acid residues. The polypeptides, variants, and fragments described herein may also be fused to other polypeptides and could therefore comprise additional amino acid residues, such as for example about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 , about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1500, or about 2000 or more additional amino acids.

The polypeptides described herein may comprise, consist of, or consist essentially of the sequences of any one or more of SEQ ID NO: 1-87, such as SEQ ID NO: 1 , 3, 4, 7, 8, 44, 50, or 59, and typically encode an enzyme. Typically, the enzyme is a flavone prenyltransferase, such as CsPT3, which converts chrysoeriol to Cannflavin A or Cannflavin B, in the presence of GPP or DMAPP, respectively. In other embodiments, the enzyme is an O-methyltransferase, such as CsOMT21 , which converts luteolin to chrysoeriol.

The polypeptides described herein are typically expressed in a host cell or organism, such as a bacterium, an archaeon, a yeast, a protozoon, an alga, a fungus, or a plant, including single cells and cell cultures of any thereof for enzymatically acting on a molecule present in the host cell or organism or its cell culture medium. The polypeptides described herein may instead be used in a cell-free system for acting on a molecule present in the system.

A species and strain selected for use in expressing the polypeptides described herein is typically first analyzed to determine whether the polypeptide genes are endogenous to the strain. Genes for which an endogenous counterpart is not present in the strain are assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function(s).

In embodiments, the hosts described herein endogenously express and/or are engineered to express at least one nucleic acid coding for an O-methyltransferase polypeptide that is suitable for methylating luteolin or variants thereof to produce chrysoeriol or variants thereof, such as a nucleic acid coding for said polypeptide from Cannabis sativa, typically CsOMT21 or variants or fragments thereof.

In additional or alternate embodiments, the hosts described herein endogenously express and/or are engineered to express at least one nucleic acid coding for a flavone prenyltransferase polypeptide that is suitable for prenylating chrysoeriol or variants thereof using geranyl diphosphate (GPP) or dimethylallyl diphosphate (DMAPP) (or variants of either thereof) to produce Cannflavin A or B, respectively, or variants thereof, such as a nucleic acid coding for said polypeptide from Cannabis sativa, typically CsPT3 or variants or fragments thereof.

Host cells described herein can be any cell capable of producing at least one protein described herein, and include bacterial, fungal (including yeast), parasite, arthropod, animal, algal, and plant cells. The cells may be prokaryotic or eukaryotic. Typical host cells are yeast, algal and plant cells. In a typical embodiment, the plant cell is a seed cell, in particular, a cell in a cotyledon or endosperm of a seed. In one embodiment, the cell is an animal cell. The animal cell may be of any type of animal such as, for example, a non-human animal cell, a non-human vertebrate cell, a non-human mammalian cell, or cells of aquatic animals such as fish or Crustacea, invertebrates, insects, etc. Non limiting examples of arthropod cells include insect cells such as Spodoptera frugiperda (Sf) cells, for example, Sf9, Sf21 , Trichoplusia ni cells, and Drosophila S2 cells. An example of a bacterial cell useful as a host cell of the present invention is Synechococcus spp. (also known as Synechocystis spp.), for example Synechococcus elongatus. Examples of algal cells useful as host cells of the present invention include, for example,

Chlamydomonas sp. (for example, Chlamydomonas reinhardtii), Dunaliella sp. , Haematococcus sp. Chlorella sp., Thraustochytrium sp., Schizochytrium sp., and Volvox sp.

Host cells for expression of the instant nucleic acids may include microbial hosts that grow on a variety of feedstocks, including simple or complex carbohydrates, organic acids and alcohols and/or hydrocarbons over a wide range of temperature and pH values. The host cells may be of an organism suitable for a fermentation process, such as, for example, Yarrowia lipolytica or other yeasts.

Further exemplary prokaryotic and eukaryotic host cell species are described in more detail below. However, it will be appreciated that other species not specifically described may be suitable.

For example, a recombinant host can be an Ascomycete. A recombinant host can be a cyanobacterium selected from the group consisting of Synechocystis, Synechococcus, Anabaena, Cyanothece, Thermosynechococcus, Rhodopseudomonas. A recombinant host can be of a genus selected from the group consisting of Aspergillus, Candida, Pichia, Saccharomyces and Rhodotorula.

A recombinant host can be a photosynthetic microorganism. For example, the organism can be of a genus selected from the group consisting of Chlamydomonas, Dunaliella, Chlorella, Botryococcus, Nannochloropsis, Physcomitrella and Ceratodon.

A recombinant host can be a prokaryote such as Escherichia coli, Rhodobacter sphaeroides, or Rhodobacter capsulatus. It will be appreciated that certain microorganisms can be used to screen and test genes of interest in a high throughput manner, while other microorganisms with desired productivity or growth characteristics can be used for large-scale production of Cannflavin A and/or B compounds and/or their precursors.

A recombinant host can be of the genus Saccharomyces, which includes, for example,

Zygosaccharomyces fermentatii, Zygosaccharomyces bisporus, Debaromyces occidentalis, Torulaspora delbrueckii, Kluyvezromyces lactis, Pichia pastoris, Saccharomyces cerevisae and Schizosaccharomyces pombe.

A recombinant host can be a yeast such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, or a bacteria such as Escherichia coli.

According to one exemplary embodiment, Saccharomyces cerevisiae strains can be isogenic haploids derived from BY4741 , which are obtainable from EUROSCARF (haploid a-mater BU00 or a- mater BY10). For reference, the yeast strain BY4741 is derived from a strain collection that contains knock outs of auxotrophic (-ura3, -Ieu2, his3) marker genes. Thus, the yeast strain can be a yeast strain that contains knock outs of auxotrophic (-ura3, -Ieu2, his3) marker genes.

Thus, it will be understood that the polypeptides described herein can be expressed in a variety of expression host cells e.g., bacteria, yeasts, mammalian cells, insect cells, plant cells, algal cells such as Chlamydomonas, or in cell-free expression systems. In some embodiments the nucleic acid can be subcloned from the cloning vector into a recombinant expression vector that is appropriate for the expression of fusion polypeptide in bacteria, mammalian, insect, yeast, or plant cells or a cell-free expression system such as a rabbit reticulocyte expression system. Some vectors are designed to transfer coding nucleic acid for expression in mammalian cells, insect cells and year in one single recombination reaction. For example, some of the GATEWAY® (Invitrogen) destination vectors are designed for the construction of baculovirus, adenovirus, adeno-associated virus (AAV), retrovirus, and lentiviruses, which upon infecting their respective host cells, permit heterologous expression of fusion polypeptides in the appropriate host cells. Transferring a gene into a destination vector is accomplished in just two steps according to manufacturer's instructions. There are GATEWAY® expression vectors for protein expression in insect cells, mammalian cells, and yeast. Following transformation and selection in E. coli, the expression vector is ready to be used for expression in the appropriate host.

Examples of other expression vectors and host cells are the strong CMV promoter-based pcDNA3.1 (Invitrogen) and pCINEO vectors (Promega) for expression in mammalian cell lines such as CHO, COS, HEK-293, Jurkat, and MCF-7; replication incompetent adenoviral vector vectors pADENO- X™, pAd5F35, pLP-ADENO™-X-CMV (CLONTECH®), pAd/CMV/V5-DEST, pAd-DEST vector

(Invitrogen) for adenovirus-mediated gene transfer and expression in mammalian cells; pLNCX2, pLXSN, and pLAPSN retrovirus vectors for use with the RETRO-X™ system from Clontech for retroviral-mediated gene transfer and expression in mammalian cells; pLenti4/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells; adenovirus-associated virus expression vectors such as pAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene) for adeno-associated virus-mediated gene transfer and expression in mammalian cells; BACpak6 baculovirus (Clontech) and pFASTBAC™ HT (Invitrogen) for the expression in S. frugiperda 9 (Sf9), Sf11 , Tn-368 and BTI-TN-5B4-1 insect cell lines; pMT/BiP/V5-His (Invitrogen) for the expression in Drosophila schneider S2 cells; Pichia expression vectors pPICZa, pPICZ, pFLDa and pFLD (Invitrogen) for expression in P. pastoris and vectors pMETa and pMET for expression in P.

methanolica, pYES2/GS and pYD1 (Invitrogen) vectors for expression in yeast S. cerevisiae.

Recent advances in the large scale expression heterologous proteins in Chlamydomonas reinhardtii are described in Griesbeck., 34 Mol. Biotechnol. 213 (2006); Fuhrmann, 94 Methods Mol Med. 191 (2006), which is incorporated herein by reference in its entirety. Foreign heterologous coding sequences are inserted into the genome of the nucleus, chloroplast and mitochondria by homologous recombination. The chloroplast expression vector p64 carrying the most versatile chloroplast selectable marker aminoglycoside adenyl transferase (aadA), which confer resistance to spectinomycin or streptomycin, can be used to express foreign protein in the chloroplast. The biolistic gene gun method can be used to introduce the vector in the algae. Upon its entry into chloroplasts, the foreign DNA is released from the gene gun particles and integrates into the chloroplast genome through homologous recombination.

In some embodiments, any of the polypeptides described herein is produced by expression from a recombinant baculovirus vector. In another embodiment, any of the polypeptides described herein is expressed by an insect cell. In yet another embodiment, any of the polypeptides described herein is isolated from an insect cell. There are several benefits of protein expression with baculovirus in insect cells, including high expression levels, ease of scale-up, production of proteins with posttranslational modifications, and simplified cell growth. Insect cells do not require C0 ₂ for growth and can be readily adapted to high-density suspension culture for large-scale expression. Many of the post-translational modification pathways present in mammalian systems are also utilized in insect cells, allowing the production of recombinant protein that is antigenically, immunogenically, and functionally similar to the native mammalian protein. Baculoviruses are DNA viruses in the family Baculoviridae. These viruses are known to have a narrow host-range that is limited primarily to Lepidopteran species of insects (butterflies and moths). The baculovirus Autographs californica Nuclear Polyhedrosis Virus (AcNPV), which has become the prototype baculovirus, replicates efficiently in susceptible cultured insect cells. AcNPV has a double-stranded closed circular DNA genome of about 130,000 base-pairs and is well characterized with regard to host range, molecular biology, and genetics. The Baculovirus Expression Vector System (BEVS) is a safe and rapid method for the abundant production of recombinant proteins in insect cells and insects. Baculovirus expression systems are powerful and versatile systems for high-level, recombinant protein expression in insect cells. Expression levels up to 500 mg/ml have been reported using the baculovirus expression system, making it an ideal system for high-level expression. Recombinant baculoviruses that express foreign genes are constructed by way of homologous recombination between baculovirus DNA and chimeric plasmids containing the gene sequence of interest. Recombinant viruses can be detected by virtue of their distinct plaque morphology and plaque-purified to homogeneity.

Recombinant polypeptides described herein can be produced in insect cells including, but not limited to, cells derived from the Lepidopteran species S. frugiperda. Other insect cells that can be infected by baculovirus, such as those from the species Bombyx mori, Galleria mellanoma, Trichplusia ni, or Lamanthria dispar, can also be used as a suitable substrate to produce recombinant proteins described herein. Baculovirus expression of recombinant proteins is well known in the art. See U.S. Pat. No. 4,745,051 ; U.S. Pat. No. 4,879,236; U.S. Pat. No. 5,179,007; U.S. Pat. No. 5,516,657; U.S. Pat. No. 5,571 ,709; U.S. Pat. No. 5,759,809, each of which is incorporated by reference herein in its entirety. It will be understood by those skilled in the art that the expression system is not limited to a baculovirus expression system. The recombinant proteins described herein can also be expressed in other expression systems such as Entomopox viruses (the poxviruses of insects), cytoplasmic polyhedrosis viruses (CPV), and transformation of insect cells with the recombinant gene or genes constitutive expression. A good number of baculovirus transfer vectors and the corresponding appropriately modified host cells are commercially available, for example, pAcGP67, pAcSECG2TA, pVL1392, pVL1393, pAcGHLT, and pAcAB4 from BD Biosciences; pBAC-3, pBAC-6, pBACgus-6, and pBACsurf-1 from NOVAGEN®, and pPolh-FLAG and pPolh-MAT from SIGMA ALDRICH®.

In one embodiment, described herein are expression vectors comprising the coding DNA sequence for the polypeptides described herein for the expression and purification of the recombinant polypeptide produced from a protein expression system using host cells selected from, e.g., bacteria, mammalian, insect, yeast, or plant cells. The expression vector should have the 5' upstream and 3' downstream regulatory elements such as promoter sequences, ribosome recognition and TATA box, and 3' UTR AAUAAA transcription termination sequence for efficient gene transcription and translation in its respective host cell. The expression vector is, preferably, a vector having the transcription promoter selected from a group consisting of CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, b-actin promoter, SV40 (simian virus 40) promoter and muscle creatine kinase promoter, and the transcription terminator selected from a group consisting of SV40 poly(A) and BGH terminator; more preferably, an expression vector having the early promoter/enhancer sequence of cytomegalovirus and the adenovirus tripartite leader/intron sequence and containing the replication origin and poly(A) sequence of SV40. The expression vector can have additional coding regions, such as those encoding, for example, 6x-histidine, V5, thioredoxin, glutathione-S-transferase, c-Myc, VSV-G, HSV, FLAG, maltose binding peptide, metal-binding peptide, HA and“secretion” signals (Honeybee melittin, a-factor, PHO, Bip), which can be incorporated into the expressed fusion polypeptide. In addition, there can be enzyme digestion sites incorporated after these coding regions to facilitate their enzymatic removal if they are not needed. These additional nucleic acids are useful for the detection of fusion polypeptide expression, for protein purification by affinity chromatography, enhanced solubility of the recombinant protein in the host cytoplasm, and/or for secreting the expressed fusion polypeptide out into the culture media or the spheroplast of the yeast cells. The expression of the fusion polypeptide can be constitutive in the host cells or it can be induced, e.g., with copper sulfate, sugars such as galactose, methanol, methylamine, thiamine, tetracycline, infection with baculovirus, and (isopropyl-beta-D-thiogalactopyranoside) IPTG, a stable synthetic analog of lactose.

In another embodiment, the expression vector comprising a polynucleotide described herein is a viral vector, such as adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus vectors, among others. Recombinant viruses provide a versatile system for gene expression studies and therapeutic applications.

When more than one polypeptide is expressed in a host cell, the polypeptides can be translated from independent nucleic acids, or as part of the same nucleic acid, for instance in the form of a polycistronic nucleic acid and/or for encoding a polyprotein.

Thus, in embodiments, the host cells are suitable for use in the methods described herein. For example, the host cells described herein are suitable for producing a Cannflavin, such as Cannflavin A and/or B.

In embodiments, the host cells described herein are suitable for producing a substantially pure Cannflavin, and to a host cell suitable for converting a chrysoeriol or variants thereof into a substantially pure Cannflavin, such as Cannflavin A and/or B, or variants thereof.

In additional or alternate embodiments, the host cells described herein are suitable for producing a substantially pure chrysoeriol, and to a host cell suitable for converting a luteolin or variants thereof into a substantially pure chrysoeriol or variants thereof.

Thus, in embodiments, the host cells are suitable for producing a substantially pure Cannflavin and express at least a nucleic acid coding for a prenyltransferase polypeptide, such as a flavone prenyltransferase, such as CsPT3 (or fragments or variants thereof), which is suitable for prenylating chrysoeriol to produce Cannflavin A and/or Cannflavin B. In embodiments, the host cells additionally express a nucleic coding for an O-methyltransferase polypeptide, such as CsOMT21 (or fragments or variants thereof) that is suitable for methylating luteolin to produce chrysoeriol.

In embodiments, the host cells are suitable for converting a chrysoeriol into a substantially pure Cannflavin, such as Cannflavin A and/or B and/or are suitable for converting a luteolin into a substantially pure chrysoeriol.

It will be understood that an O-methyltransferase may be optional when a Cannflavin precursor such as chrysoeriol is produced and/or available to the host cell, the host cell may also comprise a gene encoding an O-methyltransferase. Likewise, genes encoding one or more enzymes involved in the upstream production of luteolin, apigenin, naringenin, and phenylalanine, such as F3’H, FNS, CHI, CHS, 4CL, C4H, or PAL, may also be expressed in the host cells and/or sources of these precursor molecules may be provided and/or available to the host cell, exogenously or endogenously. Examples of appropriate mediums for a selection of recombinant hosts are provided in

International Patent Application Publication No. WO 2013/022881 , incorporated herein by reference.

Cannflavin A and/or Cannflavin B Compositions

Also described herein are compositions comprising a Cannflavin obtainable or obtained by one of the methods as disclosed above, and to the use of said composition as a medicinal agent, such as an anti-inflammatory, for pharmacological purposes and/or cosmetic purposes.

The composition comprises a Cannflavin, such as Cannflavin A and/or B, obtainable or obtained by one of the methods as disclosed above, from said host or from the culture supernatant thereof, which includes clarified culture supernatant. According to some embodiments the composition is a culture supernatant or a clarified culture supernatant.

Provided herein are compositions comprising substantially pure Cannflavin A and/or B, which are, for example, at least about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9% pure. Likewise, provided herein are compositions comprising Cannflavin A and/or B, which are substantially free of THC, cannabinoids, and/or terpenes. Such THC-, cannabinoid-, and/or terpene-free compositions comprise, for example, less than about 5%, about 4%, about 3%, about 2%, about 1 %, about 0.5%, about 0.1 %, or about 0.01 % THC, cannabinoid, and/or terpene by weight.

The Cannflavin A and/or B compositions may be formulated for use by a subject, such as a mammal, including a human. Compositions comprising the Cannflavin A and/or B molecules described herein may comprise about 0.00001 % to about 99% by weight of the active and any range there-in- between, such as from about 0.00001 %, about 0.0001 %, about 0.001 %, about 0.01 %, about 0.1 %, about 0.5%, about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.7%, or about 99.9%, to about 0.0001 %, about 0.001 %, about 0.01 %, about 0.1 %, about 0.5%, about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.7%, about 99.9%, about 99.99%. For example, typical doses may comprise from about 0.1 pg to about 100 pg of the molecules described herein per 300 mg dose, such as about 0.5 pg, about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 25 pg, about 50 pg, or about 75 pg per 300 mg dose, such as from about 0.1 pg to about 10 pg, or from about 1 pg to about 5 pg, or from about 1 pg to about 2 pg per 300 mg dose (and all related increments and percentages by weight).

The Cannflavin A and/or B molecules described herein may be used in any suitable amount, but are typically provided in doses comprising from about 1 to about 10000 ng/kg, such as from about 1 to about 1000, about 1 to about 500, about 10 to about 250, or about 50 to about 100 ng/kg, such as about 1 , about 10, about 25, about 50, about 75, about 100, about 150, about 200, about 250, about 300, or about 500 ng/kg. Typically, Cannflavins are present at from about 0.001 % to about 0.1 %, such as from about 0.005% to about 0.05%, such as from about 0.01 % to about 0.02%, such as about 0.014% in dried cannabis herb. Similar amounts, higher amounts, or lower amounts could be used for administration.

The Cannflavin A and/or B molecules may be administered over a period of hours, days, weeks, or months, depending on several factors, including the severity and type of the inflammation or other condition being treated, whether a recurrence is considered likely, or to prevent the inflammation or other condition, etc. The administration may be constant, e.g., constant infusion over a period of hours, days, weeks, months, etc. Alternatively, the administration may be intermittent, e.g., the molecules may be administered once a day over a period of days, once an hour over a period of hours, or any other such schedule as deemed suitable.

The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically or cosmetically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in "Handbook of Pharmaceutical Additives" (compiled by Michael and Irene Ash, Gower Publishing Limited, Aldershot, England (1995)). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and may be contained in buffered solutions with a suitable pH and/or be iso-osmotic with physiological fluids. In this regard, reference can be made to U.S. Patent No. 5,843,456 (the entirety of which is incorporated herein by reference).

Pharmaceutically acceptable carriers are well known to those skilled in the art and include, for example, sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, cannabis oil, and water. Furthermore the composition may comprise one or more stabilizers such as, for example, carbohydrates including sorbitol, mannitol, starch, sucrose, dextrin and glucose, proteins such as albumin or casein, and buffers like alkaline phosphates.

The Cannflavin A and/or B molecules described herein can, in embodiments, be administered for example, by parenteral, intravenous, subcutaneous, intradermal, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, intrarectal, intravaginal, aerosol, oral, topical, or transdermal administration. Typically, the compositions of the invention are administered orally or topically directly to the site of inflammation or in a cosmetic oil, lotion, cream, or gel to a desired body location, such as the face.

It is understood by one of skill in the art that the Cannflavin A and/or B molecules described herein can be used in conjunction with known therapies for prevention and/or treatment of inflammation in subjects and/or with compositions for preventing the signs of aging or other cosmetic compositions. Similarly, the Cannflavin A and/or B molecules described herein can be combined with one or more other cannabis-derived products, such as cannabinoids, terpenes, or THC. The Cannflavin A and/or B molecules described herein may, in embodiments, be administered in combination, concurrently or sequentially, with conventional treatments for inflammation, including non-steroidal anti-inflammatory drugs, for example. The Cannflavin A and/or B molecules described herein may be formulated together with such conventional treatments when appropriate.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient.

Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

Examples

Example 1 : Biosynthesis of Cannflavin A and B from Cannabis sativa L.

ABSTRACT

In addition to the psychoactive constituents that are typically associated with Cannabis sativa L., there exist a number of other‘secondary metabolites’ in this plant that are believed to contribute to its medicinal versatility. This study focused on two such compounds, known as Cannflavin A and B, which are prenylated flavonoids that specifically accumulate in C. sativa and are known to exhibit potent anti inflammatory activity in various animal cell models. Using a combination of phylogenomic and biochemical approaches, an aromatic prenyltransferase from C. sativa (CsPT3) was identified that catalyzes the regiospecific addition of either geranyl diphosphate (GPP) or dimethylallyl diphosphate (DMAPP) to position 6 of the methylated flavone, chrysoeriol, to produce Cannflavin A and B, respectively. Further evidence is presented for an o-methyltransferase (CsOMT21 ) encoded within the C. sativa genome that specifically converts the widespread plant flavone known as luteolin to chrysoeriol, both of which accumulate in C. sativa. These results therefore imply the following reaction sequence for Cannflavin A and B biosynthesis: Luteolin > Chrysoeriol > Cannflavin A and B. Taken together, the identification of these two unique enzymes represent a novel branch point from the general flavonoid pathway in C. sativa and also offer a tractable route towards metabolic engineering strategies that are designed to produce these two medicinally relevant Cannabis compounds.

INTRODUCTION

Since antiquity, Cannabis sativa L. has been cultivated for a variety of industrial, medicinal and recreational uses. In recent years, a worldwide socio-political movement aimed towards the legalization of C. sativa has spurred a resurgence of interest in this versatile plant and has afforded researchers the opportunity to explore its metabolic diversity (McPartland & Russo 2001 ; van Bakel et al. 2011 ; Sawler et al. 2015; Booth et al. 2017; Sexton et al. 2018). Apart from the psychoactive A ⁹ -tetrahydrocannabinolic acid (THC) and the pharmacologically related cannabinoids (CBDs) that typically accumulate in a variety of C. sativa cultivars, there exists a plethora of specialized metabolites in this plant species that are believed to contribute to its medicinal properties (ElSohly & Slade 2005; Radwan et al. 2008; Flores- Sanchez & Verpoorte 2008; McPartland & Russo 2014; Booth 2017). One such class of compounds are the prenylated flavonoids, known as Cannflavin A and B (Barrett et al. 1985).

The interest surrounding Cannflavin A and B within the Cannabis community stems from three seminal studies: First, Fairbairn and Pickens (1981 ) demonstrated that THC and CBD-free extracts from C. sativa could reduce the cataleptic effects of THC in mice and that this effect could be reversed by prostaglandin E ₂ (PGE ₂ ) administration. Barrett and colleagues purportedly identified the causal agent in these extracts as Cannflavin A and B and verified that these prenylated flavonoids could inhibit the production of PGE ₂ in human rheumatoid synovial cells and provide anti-inflammatory benefits that were approximately thirty times more effective than aspirin (Barrett et al. 1985, 1986). It was later

demonstrated that the underlying basis for their potent anti-inflammatory properties was that Cannflavin A and B act to inhibit the in vivo production of two pro-inflammatory mediators, prostaglandin E ₂ (PGE ₂ ) and the leukotrienes (LTs) (Werz et al. 2014). Surprisingly, however, since these striking reports of two non psychoactive constituents from C. sativa that have medicinal potential, little attention has been focused on how these unique flavonoids are actually synthesized within C. sativa (Andre et al. 2016; Pollastro 2018).

Cannflavin A and B belong to the class of plant flavonoids known as flavones, which occur in several plant lineages (Winkel-Shirley 2001 ; Ross & Kasum 2002; Andersen & Markham 2005; Jiang et al. 2016). Flavones perform a myriad of in planta functions that range from regulators of auxin transport to mediators in plant-pathogen interactions (Johnson et al. 2007; Zhang et al. 2009; Ferreyra et al. 2012). In addition, the dietary consumption of various plant flavones is well established to offer neuroprotective, antioxidant, and anticancer properties in several animal cell models (Pietta 2000; Nabavi et al. 2015; Madunic et al. 2018). While the flavone biosynthetic pathway has been extensively studied in several plants, almost nothing is known about this process in Cannabis (Flores-Sanchez & Verpoorte 2008;

Andre et al. 2016). It is possible that the core flavone pathway is similarly embedded within C. sativa, given that this plant also accumulates three widespread flavones (apigenin, luteolin and chrysoeriol) as well as their glycosylated derivatives (Turner et al. 1980; Radwan et al. 2008).

Cannflavin A and B appear to be Cannabis specific (Vanhoenacker et al. 2002; Ross et al. 2005; Werz et al. 2014) and their unique bioactivity appears to be linked to two key modifications of their parent flavone backbone: First is their distinct prenylation pattern in which a prenyl side-chain, in the form of geranyl (C10) or dimethylallyl diphosphate (C5), are affixed to the 6 position of the flavone A-ring, respectively (Barrett et al. 1985; Choi et al. 2004). These prenyl moieties impart lipophilicity to the parent flavone, which are believed to enhance uptake and bioaccumulation into cells and promote their interaction with membrane-bound enzymes and receptors that are involved in numerous cell-signalling pathways (Milligan et al. 1999; Botta et al. 2005; Watjen et al. 2007; Werz et al. 2014; Vochyanova et al. 2017). Second, both Cannflavin A and B are modified at the 3' position of the flavone B-ring with a methoxy group, which also increases lipophilicity and may therefore enhance their cellular retention and access to various cellular targets (Ibrahim 2005; Walle 2007; Berim and Gang 2016). In which order these two unique modifications (prenylation and methoxylation) of the parent flavone occur en route to Cannflavin A and B biosynthesis, however, is not known.

We describe herein that the biosynthesis of Cannflavin A and B occurs via a specific branch point from the conserved C. sativa flavone biosynthetic pathway in which a central flavone must be methoxylated at the 3' position of the flavone B-ring and prenylated at the 6 position of the A-ring. Using a combination of phylogenomic and biochemical approaches, we report the identification and

characterization of two enzymes that catalyze these penultimate and final steps of Cannflavin A and B synthesis in C. sativa.

MATERIALS AND METHODS

Chemicals and reagents

Authentic flavonoid standards for apigenin and luteolin were purchased from Indofine Chemical Company, kaempherol and quercetin were purchased from Sigma-Aldrich and chrysoeriol was from Toronto Research Chemicals. The frans-prenyl diphosphates, isopentenyl diphosphate,

dimethyllalyldiphosphate, and geranyl diphosphate, were obtained from Echelon Biosciences.

Radiolableled S-[Methyl- ¹⁴ C] adenosyl-L-Methionine (58.0 mCi mmol ^'1 ) was from Perkin-Elmer. Synthetic drop-out media lacking histidine for culturing yeast was obtained from US Biological. All primers were synthesized by Sigma-Aldrich and are listed in Table 1. All other chemicals were obtained from Sigma- Aldrich, BioBasic, or Fisher Scientific.

Table 1. Synthetic oligonucleotides used in this study. Primer overhangs are underlined, and restriction sites are italicized. A description of what each primer was used for is provided.

Phylogenetic analysis

The DNA sequences encoding for putative type 1 o-methyltransferases ( CsOMTs ) and aromatic prenyltransferases ( CsPTs ) were first identified in the Cannabis sativa genome by searching the Transcriptome Shotgun Assembly (TSA) database that is accessible through NCBI

(https://blast.ncbi.nlm.nih.gov/Blast.cqi) for transcripts that exhibited homology to the Oryza sativa o- methyltransferase, OsOMT9, and the Giycyrrhiza uralensis prenyltransferase, Gu6ADT, respectively. These sequences were then matched against the Cannabis whole genome contig database, via BLASTn searches, to retrieve the full-length open reading frames for the C. sativa OMT and APT gene families (Figure 2). The in-silico assembled amino acid sequences from these gene sequences were then used to construct phylogenetic relationships using the MEGA software package (version 6.0) by the neighbor joining method with bootstrap analysis of 1000 replicates.

Cloning and recombinant protein expression of CsOMTs in E. coli

The Cannabis sativa L. O-methyltransferase open reading frames were synthesized by

Genscript. These cDNAs for CsOMT6, 12 and 21 were amplified by PCR using the KOD Hot Start DNA polymerase (Novagen) and then ligated between the Nde\/Ase\ and Hind III sites of the pET28b vector system (Novagen) which introduces an N-terminal 6x His tag to each coding sequence. These constructs were then introduced into E. coli BL21-CodonPlus (DE3)-RIPL cells. Bacterial cells expressing recombinant CsOMT6, 12 and 21 were cultured in LB media at 37°C to an OD ₆ oo of 0.6. Isopropyl- -D- thiogalactoside (IPTG) was then added to a final concentration of 1 mM and the cells were incubated at 16°C for an additional 18 h. The bacterial cells were collected by centrifugation, re-suspended in Buffer A (20 mM Tris-HCI, pH 8.0, 500 mM KCI), and then disrupted by sonication. Crude protein extracts were centrifuged at 12,000 x g for 10 min at 4°C to remove unbroken cells and debris and then applied to a 1 mL HisTrap HP column (GE Healthcare) equilibrated in Buffer A. Proteins bound to the Ni ²⁺ affinity matrix were washed with five column volumes of Buffer A containing 20 mM imidazole, eluted with one column volume of Buffer A containing 400 mM imidazole, and then immediately desalted on PD-10 columns (GE Healthcare) equilibrated with 50 mM Tris-HCI, pH 7.5, 5 mM MgCI ₂ , and 10% (v/v) glycerol. Protein concentration was determined by the method of Bradford (1976) using BSA as a standard.

Cloning and recombinant protein expression of CsPTs in yeast

The Cannabis sativa L. and Glycyrrhiza uralensis prenyltransferase open reading frames were synthesized by Genscript and included CsPT 1 , 3, 4, 7, 8 and GuA6DT (Li et al. 2014). These cDNAs were applied by PCR and ligated between the BamHI and Xho\ sites of pESC-HIS (Agilent). The sequence-verified constructs were introduced into the Saccharomyces cerevisiae YPH499 yeast strain (ura3-52 Iys2-801 ^amber ade2-101° ^chre trp1-A63 his3-A200 Ieu2-A1 ) using the method outlined by Gietz and Schiestl (2007) and transformants were selected on synthetic drop-out media lacking histidine, supplemented with 0.67% yeast nitrogen base and 2% glucose. For recombinant protein expression, the yeast transformants were cultured as above at 28°C to an OD ₆ oo of 1.0. Yeast cells were then pelleted by centrifugation (5,000 x rpm, 10 min), washed twice with sterile water, and re-suspended in the same media as above containing 2% galactose instead of glucose. Cells were incubated for an additional 18 h at 28°C to induce protein expression.

O-methyltransferase enzyme assays

Assays for determining o-methyltransferase enzyme activity were performed using ~2 pg of purified recombinant protein incubated in a final reaction volume of 100 pL containing 1 mM substrate and 6.9 pM S-[Methyl- ¹⁴ C] adenosyl-L-methionine in 50 mM Tris-HCI, pH 7.5, 5 mM MgCI ₂ , and 10% (v/v) glycerol for 30 min at 37°C. The enzymatic products were extracted with four volumes of ethyl acetate and quantified using a scintillation counter (Model LS6500, Beckman). For reaction product identification, assays were scaled up to a final volume of 500 pL containing ~50 pg of recombinant protein, 2mM substrate and 2mM S-adenosyl-L-methionine in 50 mM Tris-HCI, pH 7.5, 5 mM MgCI ₂ , and 10% (v/v) glycerol for 60 min at 37°C. Enzymatic products were extracted as above, evaporated to dryness under N ₂ gas, and resuspended in 100 pL of methanol. Samples were applied to a Spherisorb ODS2 reverse- phase column (250 mm x 4.6 mm, 5 pm; Supelco) and resolved by HPLC using a non-linear potassium phosphate buffer (50 mM, pH 3.0) and acetonitrile gradient at a flow rate of 1 mL min ^'1 . The amount of acetonitrile in the mobile phase under the starting conditions was 10% and then increased to 30% during the first 10 min. After being maintained at 30% for an additional 10 min, the acetonitrile concentration was then increased by 5% increments every five min during the next 30 min such that the final concentration was 60% at the 50 min mark. The eluted products were detected by absorption at 270 nm and quantified relative to authentic standards. Mass spectral analysis of the enzymatic products was performed as described below.

Microsome extraction and prenyltransferase enzyme assays

Yeast cells expressing the various prenyltransferases were isolated as described above. The cell pellets were re-suspended in 100 mM Tris-HCI, pH 9.0 and disrupted with one-half volume of acid- washed glass beads (425-600 pM, Sigma-Aldrich) for a total of four min (30 s vortex; 30 s on ice).

Following lysis, cell debris and glass beads were removed by centrifugation (5,000 x rpm, 20 min, 4°C) and microsomes were pelleted from the supernatant by ultracentrifugation (46,000 x rpm, 90 min, 4°C). The resulting supernatant was removed and the pelleted microsomes were then re-suspended in 100 mM Tris-HCI, pH 9.0 and protein concentration was determined by the method of Bradford (1976) using BSA as a standard. Prenyltransferase enzyme assays were conducted with -200 pg of microsomal protein in a final reaction volume of 200 mI_ containing 200 mM of prenyl acceptor substrate and 400 mM DMAPP or GPP in 100 mM Tris-HCI, pH 9.0 and 10 mM MgCI ₂ . Assays were allowed to proceed for 60 min at 37°C and then terminated with the addition of 10 pL of 20% formic acid. Prenylated reaction products were extracted with two volumes of ethyl acetate, evaporated to dryness under N ₂ gas, and then re-suspended in 100 pL of methanol. The samples were applied to a Spherisorb ODS2 reverse-phase column (250 mm x 4.6 mm, 5 pm; Supelco) and eluted with a 20 min linear gradient from 45% to 95% methanol in water, pH 2.7 containing 0.1 % formic acid (v/v). The mobile phase was maintained at 100% methanol for an additional 10 min. Products were detected by absorption at 340 nm and quantified relative to authentic standards.

Synthesis of 6-dimethylallyl flavone standards

A flavonoid prenyltransferase from Glycyrrhiza uralensis (GuA6DT) that catalyzes the regiospecific addition of DMAPP onto position 6 of the A-ring on a variety of flavones (Li et al. 2014) was synthesized and produced in yeast microsomes, as described above. According to the in vitro prenyltransferase assay conditions outlined above, yeast microsomes expressing GuA6DT were supplied with apigenin, chrysoeriol, or luteolin as flavone substrates, along with DMAPP as a prenyl donor. The enzymatic reaction products from these assays were resolved by HPLC and compounds corresponding to 6-dimethylallyl apigenin, 6-dimethylallyl chrysoeriol, and 6-dimethylallyl luteolin were collected off-line at retention times of 19.58, 19.93, and 18.49 min, respectively.

Mass spectrometry analysis of enzymatic reaction products

The prenylated flavones that were produced by GuA6DT or CsPT3, in vitro, were purified by HPLC as described above. Samples were evaporated under nitrogen and then re-suspended in methanol prior to liquid chromatography mass spectrometry analysis performed on an Agilent 1200 HPLC liquid chromatograph interfaced with an Agilent UHD 6530 Q-TOF mass spectrometer. A C18 cartridge column (Agilent Rapid Resolution 2.1 x 30 mm, 3.5pm) at 30 °C was used with the following solvents 1 : 1 water and acetonitrile both with 0.1 % formic acid. The first 2 and last 5 minutes of the isocratic flow were sent to waste and not the spectrometer. The flow rate was maintained at 0.4 mL/min. The mass spectrometer electrospray capillary voltage was maintained at 4.0 kV and the drying gas temperature at 250° C with a flow rate of 8 L/min. Nebulizer pressure was 30 psi and the fragmentor was set to 160 V. Nitrogen was used as both nebulizing, drying gas, and collision-induced dissociation gas. The mass-to-charge ratio was scanned across the m/z range of 100-3000 m/z in 4 GHz extended dynamic range positive-ion MS mode. The instrument was externally calibrated with the ESI TuneMix (Agilent). The sample injection volume was 10 pi. Chromatograms were analyzed within Agilent Qualitative Analysis software B 08.0 finding compounds by the Molecular Feature algorithm and generating possible compound formulas including elements C, H, O, and N. Fragmentation patterns of the various parent (molecular) ions were obtained using collision energies of 5, 10 and 20 V, with 20 V being optimal.

NMR characterization of enzymatic reaction products

The enzymatic reaction products from assays with CsPT3 were resolved by HPLC as described above. Compounds suspected to be Cannflavin A and B eluted at 23.35 min. and 20.03 min, respectively, and were subsequently collected. Approximately 0.5mg of each compound was evaporated to dryness under N ₂ gas, resuspended in acetone-d6, and analyzed using ¹ H and ¹³ C NMR. NMR spectra were collected on a Bruker AVANCE II I 600 MHz spectrometer equipped with a 5 mm TCI cryoprobe. The sample temperature was regulated at 298 ± 1 K. Peak assignments for Cannflavin A and B were determined using standard 2D pulse sequences (COSY: cosygpqf, TOCSY: dipsi2gpphzs, HSQC:

hsqcetgpsisp2.2, HMBC: hmbcgpl2ndqf). The HMBC was collected with 768 increments in the indirect dimension; all other experiments were collected with 256 indirect increments. The TOCSY mixing time was set to 80 msec, and the HMBC coupling constant was set to 10 Hz.

Cannflavin A. ¹ H NMR (Acetone-d6, 600 MHz): d _H 6.68 (1 H, d, J = 2.0 Hz, H-3), 6.62 (1 H, d, J =

2.1 Hz, H-8), 7.60 (1 H, s, H-2’), 7.00 (1 H, d, J = 8.3 Hz, H-5’), 7.57 (1 H, d, J = 8.3 Hz, H-6’), 3.36 (2H, d, J

= 7.1 Hz, H ₂ -1”), 5.29 (1 H, dt, J = 1.1 Hz, 7.2 Hz, H-2”), 1.96 (2H, t, J = 7.1 Hz, H ₂ -4”), 2.05 (2H, m, H ₂ - 5”), 5.07 (1 H, t, J = 7.1 Hz, H-6”), 1.59 (3H, s, H ₃ -8”), 1.54 (3H, s, H ₃ -9”), 1.79 (3H, s, H ₃ -10”), 3.98 (3H, s, -OCH ₃ ), 13.3 (1 H, bs, 5-OH); ¹³ C NMR (Acetone-d6, 150 MHz): 5 _C 164.4 (C-2), 104.2 (C-3), 182.8 (C-4),

159.8 (C-5), 112 (C-6), 162.1 (C-7), 93.9 (C-8), 156.3 (C-9), 104.9 (C-10), 123.4 (C-1 '), 110 (C-2'), 148.5 (C-3'), 151 (C-4'), 115.8 (C-5'), 121 (C-6'), 21.8 (C-1"), 122.9 (C-2"), 135 (C-3"), 40.1 (C-4"), 27.6 (C-5"),

124.8 (C-6"), 131.3 (C-7"), 25.3 (C-8"), 17.3 (C-9"), 16 (C-10"), 56.2 (0-CH3).

Cannflavin B: ¹ H NMR (Acetone-d6, 600 MHz): d _H 6.69 (1 H, s, H-3), 6.62 (1 H, s, H-8), 7.61 (1 H, d, J = 2.1 Hz, H-2'), 7.00 (1 H, d, J = 8.3 Hz, H-5'), 7.59 (1 H, dd, J = 8.3 Hz, 2.1 Hz, H-6'), 3.36 (2H, d, J =

7.2 Hz, H-1 "), 5.28 (1 H, m, H-2"), 1.78 (3H, s, H-4"), 1.65 (3H, d, J = 0.9 Hz, H-5"), 3.99 (3H, s, 0-CH3), 13.3 (0.5H, bs, 5-OH) ; ¹³ C NMR (Acetone-d6, 150 MHz): 5 _C 164.7 (C-2), 104.5 (C-3), 183.2 (C-4), 160.2 (C-5), 112.3 (C-6), 162.3 (C-7), 94.1 (C-8), 156.6 (C-9), 105.3 (C-10), 123.8 (C-Y), 110.5 (C-2'), 148.8 (C- 3'), 151.3 (C-4'), 116.3 (C-5'), 121.3 (C-6'), 22.0 (C-1 "), 123.2 (C-2"), 131.7 (C-3"), 17.9 (C-4"), 25.9 (C- 5"), 56.6 (0-CH3).

RESULTS AND DISCUSSION

Phylogenetic analysis of C. sativa prenyltransferases

To synthesize Cannflavin A and B, a prenyl moiety must be added to position 6 of a flavone that typically accumulates in C. sativa. Therefore, we first searched for gene sequences that were putatively annotated as flavonoid or related aromatic prenyltransferases in the Transcriptome Shotgun Assembly (TSA) database for C. sativa, which is accessible through NCBI. A previously described flavone prenyltransferase from Giycyrrhiza uralensis (GuA6DT; GenBank AIT 11912.1 ) was used as a query in these searches (Li et al. 2014). GuA6DT prenylates apigenin which is a widespread plant flavone that also accumulates in C. sativa (McPartland and Russo 2001 ). This search uncovered eight full-length cDNA sequences from C. sativa that exhibited 22-53% identity at the amino acid level to GuA6DT and were putatively annotated as C. sativa prenyltransferases ( CsPT1-8 ;). One of the prenyltransferases that were identified in this search ( CsPT1 ) matched a previously reported enzyme from C. sativa that is known to be involved in the prenylation of olivetolic acid to cannabigerolic acid in the cannabinoid biosynthesis pathway (Page and Boubakir 2014). We next performed a phylogenetic analysis that included CsPT1 and these newly identified prenyltransferases from C. sativa along with all known plant prenyltransferases that have been previously shown to accommodate aromatic substrates (Figure 2;). This analysis

demonstrated that plant aromatic prenyltransferases fall into six distinct groups, which are conveniently defined by the specific branch of aromatic metabolism in which they participate. The eight CsPTs occupy three of these six groups: CsPT2 and CsPT6 reside in a unique clade of prenyltransferases (Group 2) which have been shown to participate in the tocopherol biosynthetic pathway (Collakova and Dellapenna 2001 ; Savidge et al. 2002; Tian et al. 2007). CsPT5 appears to be orthologous to homogentisate solanesyltransferases (Group V) that function in plastoquinone biosynthesis (Venkatesh et al. 2006; Tian et al. 2007). The five remaining CsPTs ( CsPT1 , 3, 4, 7, and 8) formed a third and distantly related group (Group VI) that includes two prenyltransferases from Humulus lupulus (hops), which are involved in the aromatic prenylation reactions required for terpenophenolic biosynthesis (Nagel et al. 2008; Tsurumaru et al. 2010; Li et al. 2015). Surprisingly, this analysis revealed that none of the CsPTs were closely related to any of the flavonoid or coumarin prenyltransferases (Groups I and IV, respectively) that have been previously identified in various plant species (Sasaki et al. 2008; Akashi et al. 2009; Sasaki et al. 2011 ; Shen et al. 2012; Wang et al. 2014; Munakata et al. 2016; Yoneyama et al. 2016; Yang et al. 2018).

Biochemical characterization of recombinant CsPTs identifies a regiospecific chrysoeriol- 6-prenyltransferase

In pursuit of identifying a flavone prenyltransferase(s) that is involved in Cannflavin A and B biosynthesis, we focused on the sixth group of plant PTs that was identified from our phylogenetic analysis, which included CsPT 1, 3, 4, 7, and 8. These particular CsPTs drew our attention for two reasons: First, they represent the only prenyltransferases in our search of the C. sativa genome that are evolutionarily distinct from those CsPTs that appear to function in tocopherol and plastoquinone biosynthesis, which are central plant compounds (Sattler et al. 2004; Kriese et al. 2004). Second, this group of CsPTs are closely related to two enzymes from hops (a Cannabaceae family relative), which are believed to prenylate naringenin chalcone, a widespread intermediate in the general flavonoid pathway (Nagel et al. 2008; Li et al. 2015).

We therefore introduced each of these CsPTs into a well-established yeast expression system that is typically used to characterize this class of enzymes (Sasaki et al. 2011 ; Shen et al. 2012; Li et al. 2014; Wang et al. 2014). As a positive control for prenyltransferase enzyme activity, we also introduced the open-reading frame of GuA6DT from Glycyrrhiza uralensis into this host yeast strain, which was previously shown to prenylate not only apigenin, but a variety of flavones at position 6 of the A ring using dimethyl diphosphate (DMAPP) as a substrate (Li et al. 2014). As expected, in assays with microsomes expressing GuA6DT together with apigenin and DMAPP as a prenyl donor, we observed a single reaction product whose mass-to-charge ratio ( m/z 339) was consistent with 6-dimethylallyl apigenin (Figure 3). As previously demonstrated, luteolin and chrysoeriol were also converted by GuA6DT to their corresponding 6-dimethylallyl flavones using DMAPP as a substrate (Li et al. 2014), and these monoprenylated flavones were subsequently purified by HPLC to serve as prenylflavone standards in subsequent exploratory assays with recombinant CsPTs (Figure 3). Accordingly, the microsomal fractions from each yeast strain expressing the open-frames of CsPT 1, 3, 4, 7, and 8 were recovered and tested for prenyltransferase activity with three flavone substrates that are present in C. sativa. apigenin, chrysoeriol, and luteolin (McPartland & Russo 2001 ; Brenneisen 2007; Radwan et al. 2008). As potential prenyl donors, DMAPP, IPP and GPP were also included as co-substrates in each of these enzyme assays. The reaction products from these assays were extracted with ethyl acetate, analyzed by reverse-phase HPLC, and compared to the authentic prenylated flavones that were isolated from in vitro assays with GuA6DT (see above). No detectable prenylated flavone products were observed in assays with microsomes obtained from yeast cells harbouring the empty vector or with CsPT 1 , 4, 7, or 8 under the above conditions.

However, this analysis revealed that microsomes containing CsPT3 readily converted apigenin and chrysoeriol, but not luteolin, to their corresponding prenylflavones using DMAPP as a prenyl donor, and kinetic analysis clearly showed that chrysoeriol was the preferred flavone substrate (Table 2). IPP was not accommodated as a prenyl donor in assays with microsomes containing CsPT3. The enzymatic product that was observed in assays with CsPT3, chrysoeriol and DMAPP exhibited the same HPLC retention time and fragmentation pattern, as determined by tandem mass spectrometry, to that of 6- dimethylallyl chrysoeriol, also known as Cannflavin B (Figure 4A). Interestingly, CsPT3 also

accommodated GPP as a prenyl donor in assays with apigenin and chrysoeriol, and kinetic analysis again revealed that chrysoeriol was the preferred substrate (Table 2). The reaction product that was observed with assays including CsPT3, chrysoeriol and GPP exhibited less polarity than 6-dimethylallyl chrysoeriol/Cannflavin B (as indicated by HPLC-UV analysis) and exhibited a mass-to-charge ratio ( mlz 437) that was consistent with the addition of a geranyl moiety onto the chrysoeriol backbone, and was therefore assumed to be Cannflavin A (Figure 4B).

Table 2. Kinetic parameters of CsPT3 with various C. sativa flavone substrates. Measurements were made at 37°C in 100 mM Tris-HCI, pH 9.0 and 10 mM MgCI ₂ . Reactions were initiated by adding either DMAPP or GPP as the prenyl donor substrate to each reaction. Data are the means of three independent determinations ± SE. Kinetic constants were not determined (N. D. ) for assays with luteolin as a flavone substrate due to low conversion rates.

DMAPP GPP

Vmax Vmax

Substrate Km (p M) Km (p M)

(pmol min ^·1 mg ^·1 ) (pmol min ^·1 mg ^·1 )

Apigenin 141.7 ± 15.6 1.2 ± 0.0566 49.4 ± 1 1.4 1.3 ± 0.0993

Chrysoeriol 37.9 ± 5.59 1.3 ± 0.0585 35.6 ± 10.7 1.3 ± 0.122

Luteolin N.D. N. D. N. D. N.D.

The structures of the two enzymatic products that were obtained from assays with CsPT3, chrysoeriol, and either DMAPP or GPP as prenyl donors, were further analyzed by ¹ H and ¹³ C NMR. The ¹ H NMR spectra of Cannflavin A exhibited three peaks of area 3H in the region between 1.5 - 2.0 ppm and 2 peaks of area 1 H in the region between 5.0- 5.5 ppm, consistent with three methyl groups and two vinylic protons, respectively, suggesting the presence of a geranyl group. The ¹ H NMR spectra of Cannflavin B exhibited only two peaks of area 3H in the region between 1.5 - 2.0 ppm and a single peak of area 1 H in the region between 5.0- 5.5 ppm, consistent with the two methyl groups and one vinylic proton of a single prenyl group. COSY, TOCSY, and HMBC spectra also demonstrated peak patterns that were consistent with a geranyl group for Cannflavin A and a prenyl group for Cannflavin B (Crombie & Crombie 1982; Barrett et al. 1986; Choi et al. 2004). Initial N MR assignments for both Cannflavin A and B were performed using the COSY, TOCSY, and HSQC spectra to assign all proton resonances as well as those of proton-bearing carbons and an HMBC experiment was then used to complete the carbon assignments (Figure 5). In both Cannflavin A and Cannflavin B, HMBC correlations were observed from both the 5-OH hydroxyl proton and the 1 " proton on the geranyl or prenyl group to the same carbon resonance (shown as bold arrows in Figure 5). The 5-OH hydroxy proton is too far away from the carbon at position 8 for an HMBC correlation to be observed and we therefore reasoned that the

geranylation/prenylation must be at the 6 position. The complete ¹ H- and ¹³ C-NMR assignments are shown in Table 3. Taken together, these results suggest that CsPT3 appears to be the sole member of the CsPT family that prenylates chrysoeriol, using either GPP or DMAPP, in the final steps of Cannflavin A and B biosynthesis, respectively. Table 3. NMR assignments for Cannflavin A and Cannflavin B.

Cannflavin A Cannflavin B

Position aH ac 3H ac

2 164.4 164.7

3 6.68 104.2 6.69 104.5

4 182.8 183.2

5 159.8 160.2

6 112.0 112.3

7 162.1 162.3

8 6.62 93.9 6.62 94.1

9 156.3 156.6

10 104.9 105.3

1 123.4 123.8

2' 7.60 110.0 7.61 110.5

3' 148.5 148.8

4' 151.0 151.3

5' 7.00 115.8 7.00 116.3

6' 7.57 121.0 7.59 121.3

1 " 3.36 21.8 3.36 22.0

2" 5.29 122.9 5.28 123.2

3" 135.0 131.7

4" 1.96 40.1 1.78 17.9

5" 2.05 27.6 1.65 25.9

6" 5.07 124.8

7" 131.3

8" 1.59 25.3

9" 1.54 17.3

10" 1.79 16.0

0-CH3 3.98 56.2 3.99 56.6

5-0 H 13.30 13.30

Phylogenetic analysis of C. sativa o-methyltransferases involved in the methylation of luteolin to chryseoriol

The observation that CsPT3 preferentially prenylates chrysoeriol, in vitro, and that prenylated luteolin is apparently absent in extracts from C. sativa implies, a priori, that methylation of luteolin to chrysoeriol must occur first in the Cannflavin A and B pathway. We reasoned that the alleged enzyme that methylates luteolin at the 3'-hydroxyl position of the flavone B-ring to yield chrysoeriol would likely fall into the class of S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs), which are widely distributed throughout the plant kingdom (Ibrahim et al. 1998; Ibrahim 2005; Kim et al. 2010). We focused our initial searches of the TSA database for C. sativa on type 1 OMTs, which specifically methylate hydroxyl moieties of phenylpropanoid-based compounds (Noel et al. 2003). Using a previously characterized flavonoid-O-methyltransferase from Oryza sativa (OsROMT9) that methylates the 3'- hydroxyl group on a variety of flavonoids as a query (Kim et al. 2006), this search uncovered 40 unique nucleotide sequences corresponding to partial and/or full-length transcripts that were loosely annotated as‘caffeic acid-o-methyltransferases’. We next compared these transcript sequences via BLASTn searches against the Cannabis whole genome contig database to confirm their corresponding full-length open reading frames. This analysis revealed twenty four unique protein sequences which were subsequently annotated as C. sativa O-methyltransferases (CsOMT1-24).

A phylogenetic analysis of the CsOMT family was then performed to establish their evolutionary relatedness to various plant OMTs that have been previously identified to act on aromatic substrates. This analysis revealed that type 1 CsOMTs are distributed into four general groups (Figure 6). It should be noted however, as Schroder et al. (2002) and Lam et al. (2007) previously pointed out, that assigning substrate preference based on sequence similarity alone for this class of plant enzymes is precarious. For example, the first group of type 1 plant OMTs depicted in our phylogenetic analysis includes enzymes that utilize a broad array of aromatic substrates - from simple phenolic compounds, such as chavicol, guaiacol and orcinol (Gang et al. 2002; Scalliet et al. 2006; Akhtar et al. 2013), to more complex heterocyclic aromatics, such as homoeriodictoyl, myricetin, and resveratrol (Schroder et al. 2004;

Schmidin et al. 2008; Schmidt et al. 2011 ). We found nine CsOMT family members present within this group. The second group of type 1 OMTs appear specific to the Cannabaceae family and include seven CsOMTs along with two OMTs from Humulus lupulus that are involved in the synthesis xanthohumol (Nagel et al. 2008). The third and fourth groups represent two closely related sister clades of type 1 plant OMTs and contain the remaining members of the CsOMT family. Strikingly, all plant OMTs that are known to methylate the 3'-hydroxyl position of various flavonoids are confined to group three and include representatives from Arabidopsis, peppermint, rice, wheat, and American golden saxifrage (Gauthier et al. 1996; Muzac et al. 2000; Willitis et al. 2004; Kim et al. 2006; Zhou et al. 2006). We found three CsOMTs (CsOMT6, 12 and 21 ) that fell into this group.

Identification and biochemical characterization of a luteolin o-methyltransferase

While recognizing that phylogenetic-driven predictions of plant OMT function has its caveats, we were nevertheless intrigued by the presence of CsOMT6, 12 and 21 amongst a group of evolutionary conserved OMTs that exhibit regioselective methylation activity for the 3'-position on a variety of flavonoids. We therefore chose to survey the enzymatic activities of these three CsOMTs to elucidate if their encoded proteins could methylate luteolin at the 3'-hydroxyl position of the flavone B-ring to yield chrysoeriol. In accordance with our hypothesis, this reaction would represent the penultimate step in Cannflavin A and B biosynthesis.

We first introduced the open-reading frames of CsOMT6, 12 and 21 into E. coli cells as N- terminal fusion proteins with a His ₆ tag and subsequently assayed desalted protein extracts from cells expressing each protein for O-methyltransferase enzyme activity. We chose to test three flavones (apigenin, luteolin, and chrysoeriol) and two flavonols (Quercetin and Kaempferol) that typically accumulate in C. sativa as potential substrates for each CsOMT, along with the universal methyl donor ¹⁴ C-labeled S-adenosyl methionine as a co-substrate (Figure 7A). After extracting and quantifying the amount of radiolabel within the enzymatic products that were obtained from these initial assays, it was determined that only CsOMT6 and CsOMT21 exhibited appreciable OMT activity with the flavonoid substrates that were provided. Recombinant CsOMT6 exhibited strict substrate specificity towards quercetin (Figure 1 ), while CsOMT21 methylated luteolin primarily (Figure 7B), yet also accommodated quercetin as a substrate, albeit with less efficiency (57% activity compared to luteolin). Notably, neither apigenin, nor chrysoeriol, nor kaempferol, which lack a free 3'-hydroxyl group on their B-ring, were used as substrates by either enzyme. This observation therefore lends further support to the view (Lam et al. 2007) that CsOMT6, CsOMT21 and the other type 1 OMTs that are present in group three (defined by our phylogenetic analysis) encompass an evolutionarily conserved group of enzymes with regiospecificity for 3'-hydroxyl groups on a variety of flavonoid compounds. It also implies that CsOMT21 is a 3'-0- methyltransferase and likely catalyzes the penultimate step in Cannflavin A and B biosynthesis by converting luteolin to chrysoeriol. To test this possibility, recombinant CsOMT21 was purified via Ni ²⁺ affinity chromatography (Fig. 5B) and assayed with chrysoeriol and unlabelled S-adenosyl methionine as co-substrates. Indeed, the identity of the reaction product was confirmed to be chrysoeriol, based on its identical HPLC retention time and mass spectral fragmentation pattern with the authentic standard (Figure 7C). Recombinant CsOMT21 exhibited Michaelian kinetics with kinetic constants that were similar to those that have been previously reported for this class of enzymes (Figure 7D). While our analysis cannot exclude the possibility that the other twenty one CsOMT family member(s) may convert luteolin to chrysoeriol, these results do provide evidence for a branch point from the general flavonoid pathway that is present in C. sativa, whereby luteolin can be first methylated to chrysoeriol en route towards Cannflavin A and B synthesis.

CONCLUDING REMARKS

Guided by previously published metabolomic data from C. sativa, a targeted phylogenomics approach combined with in-vitro biochemical assays was employed in this study to explore the biosynthetic pathway towards Cannflavin A and B (Figure 8). We provide evidence that a unique branch point from the general plant flavonoid pathway has evolved in C. sativa in which the widespread plant flavone, luteolin, is converted into Cannflavin A and B via regiospecific methylation and prenylation reactions. The identification of these two enzymatic steps opens up new opportunities for the metabolic engineering of Cannflavin A and B biosynthesis and also underscores the value of phylogenomics driven gene discovery, an approach that is largely underutilized in the Cannabis research space.

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The above disclosure generally describes the present invention. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

All publications, patents and patent applications cited above are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Although preferred embodiments of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.