WO2020077367A1 - Biosynthesis of homoeriodictyol - Google Patents

Abstract

The present invention relates to methods, mutant O' -methyltransferases, and transformed host cells for the production of homoeriodictyol from eriodictyol via bioconversion.

Description

BIOSYNTHESIS OF HOMOERIODICTY OL

RELATED APPLICATIONS

[001] This application claims priority to U.S. Provisional Application No. 62/744,962, filed on October 12, 2018, and U.S. Provisional Application No. 62/914,560, filed on October 14, 2019, the contents of both of which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

[002] The field of the invention relates to methods, mutagenized O-methyltransf erases, and transformed host cells useful in the bioconversion of eriodictyol to homoeriodictyol.

BACKGROUND OF THE INVENTION

[003] The production of secondary metabolites such as flavonoids is part of the chemical defense, ecological adaptation, and signaling mechanisms of plants. Starting from common precursors, plants produce an array of specialized compounds, including flavonoids, using several recurring strategies such as the transfer of acyl, sugar, and/or methyl moieties, often requiring prior hydroxylation steps. Such modifications change not only the physicochemical properties of the individual compounds produced but greatly modify, change or create certain physiological properties of the resulting compounds.

[004] Structurally, flavonoids are classified into flavanones, flavonols, flavones, isoflavones, flavan-3-ols (catechols), and anthocyanins, depending on the carbonyl group on carbon 4, the double bond between carbon 2 and 3, the presence of a hydroxyl group on carbon 3, and the location of the B ring (Forkmann and Heller, 1999) (Fig. 1). During the biosynthesis of flavonoids, several hydroxyl groups can be added to the A ring and/or the B ring, and the most predominant positions where they are added include the 3, 5, 6, 7, 3', 4 ', and 5' positions. The position and the number of hydroxyl groups vary among different flavonoids.

[005] Eriodictyol is a flavonoid that can be extracted from Yerba Santa ( Eriodictyon calif ornicum). It has hydroxyl groups at the 5, 7, 3', and 4' positions:

Homoeriodictyol can be converted from eriodictyol via an O-methylation reaction at the 3' position:

This type of O-methylation reaction is one of the most important modifications of flavonoids and the resulting O-methylated flavonoids often display a range of new and important physiochemical properties.

[006] Both eriodictyol and homoeriodictyol can be extracted from the Yerba Santa ( Eriodictyon californicum) plant. In terms of their use in the food and beverage industry, it is known that these compounds can be used to mask the bitterness of other compounds present in food products. For example, in a sensory study, they were demonstrated to significantly decrease the bitter taste of caffeine without exhibiting intrinsic strong flavors or taste characteristics, indicating they have potentially significant uses in the production of food, drink and medicine. Moreover, they have been shown to possess higher biological activity and have more anticancer activity than many other flavonoids (Chu et al. 2016). Between the two, homoeriodictyol is more powerful in its bitterness masking capability and therefore has enhanced value to industrial users in the food and medical industry.

[007] Typically, flavonoids can only be harvested from particular plant species in small amounts, which hampers their cost-effective use and their broad availability. Moreover, some of these species from which they are derived are endangered in their natural habitats, thus further limiting the availability of some plant metabolites. Meanwhile, directing the regio-specific methylation of complex aromatic compounds is still quite challenging for chemical synthesis. Accordingly, there is a need in the art for novel highly scalable methods to produce

homoeriodictyol from eriodictyol to further enable human use and consumption. SUMMARY OF THU INVENTION

[008] In one aspect, the present invention addresses the problems described above by providing mutant O-methyltransferases that have demonstrated higher efficiency in the

bioconversion of eriodictyol to homoeriodictyol compared to the wild-type O-methyltransferase. More specifically, the present mutant O-methyltransferases only include a single amino acid residue mutation compared to the wild-type O-methyltransferase.

[009] In some embodiments, the present mutant O-methyltransferase is a Populus trichocarpa caffeic acid 3 -O-methyltransferase (PtCOMTl) that includes a single amino acid residue mutation. For example, the mutation can be at amino acid residue position 316 of SEQ ID NO: 1, where such mutation replaces isoleucine with an amino acid selected from the group consisting of alanine, glycine, proline, serine, threonine, and valine. Accordingly, in certain embodiments, the present mutant O-methyltransferase can include a mutation at amino acid residue position 316 of SEQ ID NO: 1, where such mutation replaces isoleucine with alanine. In certain embodiments, the present mutant O-methyltransferase can include a mutation at amino acid residue position 316 of SEQ ID NO: 1, where such mutation replaces isoleucine with glycine. In certain embodiments, the present mutant O-methyltransferase can include a mutation at amino acid residue position 316 of SEQ ID NO: 1, where such mutation replaces isoleucine with proline. In certain embodiments, the present mutant O-methyltransferase can include a mutation at amino acid residue position 316 of SEQ ID NO: 1, where such mutation replaces isoleucine with serine. In certain embodiments, the present mutant O-methyltransferase can include a mutation at amino acid residue position 316 of SEQ ID NO: 1, where such mutation replaces isoleucine with threonine. In certain embodiments, the present mutant O-methyltransferase can include a mutation at amino acid residue position 316 of SEQ ID NO: 1, where such mutation replaces isoleucine with valine.

[0010] In some embodiments, the present mutant O-methyltransferase is an Oryza sativa O-methyltransferase (OsOMT9) that includes a single amino acid residue mutation. For example, the mutation can be at amino acid residue position 321 of SEQ ID NO: 3, where such mutation replaces valine with alanine.

[0011] In some embodiments, the present mutant O-methyltransferase is Arabidopsis thaliana O-methyltransferase (AtOMTl) that includes a single amino acid residue mutation. For example, the mutation can be at amino acid residue position 314 of SEQ ID NO: 5, where such mutation replaces valine with alanine.

[0012] In another aspect, the present invention provides a method of producing homoeriodictyol. The method can include preparing a reaction mixture comprising eriodictyol and a mutant O-methyltransferase as described herein; and incubating the reaction mixture for a sufficient time to product homoeriodictyol. In some embodiments, at least some of said eriodictyol in the reaction mixture can be produced in situ. In some embodiments, the reaction mixture can include naringenin, where at least some of said eriodictyol in the reaction mixture can be converted from naringenin. To enable bioconversion of naringenin to eriodictyol, the reaction mixture can further include a flavonoid 3’ -hydroxylase and a flavin reductase.

[0013] In certain embodiments, the flavonoid 3’ -hydroxylase can be a 4-coumarate 3- hydroxylase from Saccharothrix espanaensis (SAM5) having the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the flavonoid 3’ -hydroxylase can be a putative pyoverdine chromophore biosynthetic protein C from Streptomyces sclerotialus (SsPvcC) having the amino acid sequence of SEQ ID NO: 61. In certain embodiments, the flavonoid 3’ -hydroxylase can include an N-terminal tag having the amino acid sequence of MTTASGTNADVQNGVRP. In certain embodiments, the flavonoid 3’ -hydroxylase can be a mutant of SsPvcC. More specifically, the mutant can include one or more mutations selected from the group consisting of M196Y, G315H, and D214N compared to the amino acid sequence of SEQ ID NO: 61. Each of these mutants can include an N-terminal tag having the amino acid sequence of

MTTASGTNADVQNGVRP. In certain embodiments, the flavonoid 3’ -hydroxylase can include an amino acid sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, and SEQ ID NO: 59.

[0014] In certain embodiments, the flavin reductase can be a Saccharothrix espanaensis flavin reductase, a Pseudomonas fluorescens flavin reductase, or the reductase subunit of a 4- hydroxyphenylacetate 3 -monooxygenase (HpaC) from E. coli. The flavin reductase can be a polypeptide comprising the amino acid sequence of SEQ ID NO: 65, 67, or 69.

[0015] In another aspect, the present invention relates to a method of providing a transformed host cell, the method comprising transforming the host cell with a synthetic or recombinant nucleic acid molecule comprising a first polynucleotide sequence that encodes a mutant O-methyltransferase, said mutant O-methyltransferase being selected from the group consisting of i) an O-methyltransferase comprising a mutation at amino acid residue position 316 of SEQ ID NO: 1, wherein such mutation replaces isoleucine with an amino acid selected from the group consisting of alanine, glycine, proline, serine, threonine, and valine; ii) an O- methyltransferase comprising a mutation at amino acid residue position 321 of SEQ ID NO: 3, wherein such mutation replaces valine with alanine; iii) an O-methyltransferase comprising a mutation at amino acid residue position 314 of SEQ ID NO: 5, wherein such mutation replaces valine with alanine. In some embodiments, said method further comprises transforming the host cell with a second polynucleotide sequence to express a heterologous flavonoid 3’ -hydroxylase and a third polynucleotide sequence to express a heterologous flavin reductase. In certain embodiments, the second polynucleotide sequence is selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 and SEQ ID NO: 62. In certain embodiments, the third polynucleotide sequence is selected from the group consisting of SEQ ID NO: 64, SEQ ID NO: 66, and SEQ ID NO: 68.

[0016] The present invention further encompasses transformed host cells obtained by such methods, as well as methods of screening such transformed host cells to select isolated recombinant host cells that are capable of producing the mutant O-methyltransferase described herein, and optionally, also the flavonoid 3’ -hydroxylase and flavin reductase described herein. In some embodiments, such host cells can be selected from the group of microbial species consisting of Escherichia; Salmonella; Bacillus; Acinetobacter; Streptomyces; Corynebacterium;

Methylosinus; Methylomonas; Rhodococcus; Pseudomonas; Rhodobacter; Synechocystis;

Saccharomyces; Zygosaccharomyces; Kluyveromyces; Candida; Hansenula; Debaryomyces; Mucor; Pichia; Torulopsis; Aspergillus; Arthrobotlys; Brevibacteria; Microbacterium;

Arthrobacter ; Citrobacter; Klebsiella; Pantoea; and Clostridium. In certain embodiments, the host cell is E. coli.

[0017] Yet another aspect of the present invention relates to a method of producing homoeriodictyol via microbial fermentation. The method can include culturing a transformed host cell in a suitable medium including eriodictyol, where the transformed host cell includes a synthetic or recombinant nucleic acid molecule having a first polynucleotide sequence that encodes a mutant O-methyltransferase, and where such culturing provides for the synthesis of said mutant O-methyltransferase, resulting in eriodictyol being converted to homoeriodictyol by the transformed host cell. [0018] In various embodiments, the mutant O-methyltransferase can be selected from the group consisting of i) an O-methyltransferase comprising a mutation at amino acid residue position 316 of SEQ ID NO: 1, wherein such mutation replaces isoleucine with an amino acid selected from the group consisting of alanine, glycine, proline, serine, threonine, and valine; ii) an O-methyltransferase comprising a mutation at amino acid residue position 321 of SEQ ID NO: 3, wherein such mutation replaces valine with alanine; iii) an O-methyltransferase comprising a mutation at amino acid residue position 314 of SEQ ID NO: 5, wherein such mutation replaces valine with alanine.

[0019] In some embodiments, the medium can further include naringenin, and at least some of the eriodictyol in the medium can be converted from naringenin by the transformed host cell. In these embodiments, the transformed host cell can be further transformed to heterologous express a second polynucleotide sequence that encodes a flavonoid 3’ -hydroxylase and a third polynucleotide sequence that encodes a flavin reductase. For example, the second polynucleotide sequence can encode a flavonoid 3’ -hydroxylase having an amino acid sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 and SEQ ID NO: 63. The second polynucleotide sequence can be selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 and SEQ ID NO: 62. The third polynucleotide sequence can encode a flavin reductase having an amino acid sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69. The third polynucleotide sequence is selected from the group consisting of SEQ ID NO: 64, SEQ ID NO: 66, and SEQ ID NO: 68.

[0020] Homoeriodictyol produced according to the present teachings can be used in various food products, beverages, pharmaceutical products, and other oral consumable products, where the homoeriodictyol can reduce or mask any unpleasant, bitter, and/or astringent taste present in such products.

[0021] While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawing and will herein be described in detail. It should be understood, however, that the drawings and detailed description presented herein are not intended to limit the disclosure to the particular embodiment disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims. [0022] Other features and advantages of this invention will become apparent in the following detailed description of preferred embodiments of this invention, taken with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 illustrates the generic structure of flavonoids, which include subclasses such as flavan-3-ols, anthocyanidins, flavonols, flavones, flavanones, and isoflavones as shown.

[0024] FIG. 2 illustrates the bioconversion pathway from eriodictyol to homoeriodictyol via an O-methyltransferase, where S-adenosyl-L-methionine (SAM) serves as a methyl group donor and becomes S-adenosyl-L-homocysteine following the O-methylation reaction.

[0025] FIG. 3 illustrates the bioconversion from naringenin to eriodictyol via a flavonoid 3’ -hydroxylase (F3’H) and a flavin reductase (FR).

[0026] FIG. 4 shows the plasmid map of pUVAP.

[0027] FIG. 5 shows the plasmid map of PtCOMTl -pUVAP.

[0028] FIG. 6 shows the plasmid map of OsOMT9-pUVAP.

[0029] FIG. 7 shows the plasmid map of AtOMTl -pUVAP.

[0030] FIG. 8 shows the sequence alignment of AtOMTl, ptCOMTl, and OsOMT9.

[0031] FIG. 9 compares the amount of homoeriodictyol (HER) converted from eriodictyol (ER) produced by E. coli strains transformed with a Populus trichocarpa caffeic acid 3 -O-methyltransferase (PtCOMTl) gene, an Oryza sativa O-methyltransferase (OsOMT9) gene, and an Arabidopsis thaliana O-methyltransferase (AtOMTl) gene, respectively.

[0032] FIG. 10 compares the amount of homoeriodictyol (HER) converted from eriodictyol (ER) produced by E. coli strains transformed with a Populus trichocarpa caffeic acid 3 -O-methyltransferase (PtCOMTl) gene (WT), vis-a-vis various single amino acid residue mutants, where isoleucine at amino acid residue position 316 was replaced by the amino acid shown.

[0033] FIG. 11 shows that using E. coli strains transformed to produce a mutagenized OsOMT9-V32lA (HER02), a mutagenized PtCOMTl-I3 l6A (HER-03), and a mutagenized AtOMTl-I3 l4A (HER04), respectively, the titers of homoeriodictyol produced were significantly higher than an E. coli strain transformed to produce a non-mutagenized OsOMT9 (HER-01). DESCRIPTION

Flavonoids

[0034] FIG. 1 shows the generic chemical backbone of flavonoids. Depending on the absence or presence of a carbonyl group on carbon 4, a single bond versus a double bond between carbon 2 and 3, the presence or absence of a hydroxyl group on carbon 3, and whether the B ring is attached to carbon 2 or carbon 3, flavonoids can be separated into subclasses such as flavanones, flavonols, flavones, isoflavones, flavan-3-ols (catechols), and anthocyanins.

[0035] Referring to FIG. 2, eriodictyol can be methylated at carbon 3’ to provide homoeriodictyol. The O-methylation reaction can be catalyzed by flavonoid 3’-0- methlytransferase in the presence of S-adenosyl-L-methionoine which functions as a methyl group donor.

[0036] Referring to FIG. 3, eriodictyol can be bioconverted from naringenin in the presence of a flavonoid 3’ -hydroxylase (F3’H). The bioconversion rate can be enhanced if in addition to F3TT, a flavin reductase (FR) also is present.

O-Methyltransferase

[0037] Caffeic acid 3-O-methyltransferase (COMT) is an enzyme involved in lignin biosynthesis by catalyzing the methylation of phenylpropanoid derivates. This enzyme shows substrate promiscuity, and has activity toward a number of compounds in monolignol biosynthesis pathway (Parvathi et al 2001). PtCOMTl, a COMT isolated from Populus trichocarpa has activity toward 5-hydroxy coniferaldehyde, caffeic, 5-hydroxyferulic acids (Li et al 2000). AtOMTl, a COMT from Arabidopsis thaliana. was shown to be able to catalyze the 3’ -O-methylation of some flavonols such as quercetin and myricetin (Muzac 2000).

[0038] Wild-type PtCOMTl did not show high activity in catalyzing the methylation of eriodictyol to homoeriodictyol (FIG. 9). However, according to the current invention, the inventors surprisingly found that a single amino acid residue mutation could lead to an increase in that activity by 1,675%. AtOMTl was active toward eriodictyol to form homoeriodictyol. Yet, the inventors were able to increase its activity by 23% via site-directed mutagenesis of that

corresponding amino acid residue (see alignment shown in FIG. 8). Similarly, the corresponding mutation on OsOMT9 led to a similar increase in the activity toward eriodictyol. With the application of this discovery, a titer of 6.2 g/L in 50-liter fermenter was achieved (FIG. 11). [0039] Accordingly, in some embodiments, the present teachings relate to a mutant O- methyltransferase. Such mutant O-methyltransferase can be a Populus trichocarpa caffeic acid 3- O-methyltransferase (PtCOMTl) that includes a single amino acid residue mutation. In preferred embodiments, the mutation can be at amino acid residue position 316 of SEQ ID NO: 1, where such mutation replaces isoleucine with an amino acid selected from the group consisting of alanine, glycine, proline, serine, threonine, and valine. In some embodiments, the present mutant O-methyltransferase is an Oryza sativa O-methyltransferase (OsOMT9) that includes a single amino acid residue mutation. For example, the mutation can be at amino acid residue position 321 of SEQ ID NO: 3, where such mutation replaces valine with alanine. In some embodiments, the present mutant O-methyltransferase is Arabidopsis thaliana O-methyltransferase (AtOMTl) that includes a single amino acid residue mutation. For example, the mutation can be at amino acid residue position 314 of SEQ ID NO: 5, where such mutation replaces valine with alanine.

Flavonoid 3’-Hydroxylases

[0040] The present teachings also encompass novel flavonoid 3’ -hydroxylases that are capable of converting flavonoids, such as naringenin, to 3’-hydroxylated flavonoids, such as eriodictyol, without further hydroxylating the 3’-hydroxylated flavonoids to multi-hydroxylated products. This allows the optional bioconversion of naringenin to eriodictyol, where eriodictyol so produced can then be used as the substrate for bioconversion into homoeriodictyol.

[0041] In various embodiments, the present flavonoid 3’-hydroxylase can include an N- terminal tag having the amino acid sequence of MTTASGTNADVQNGVRP (SEQ ID NO: 71). Said flavonoid 3’-hydroxylase can be the putative py overdine chromophore biosynthetic protein C from Streptomyces sclerotialus having the amino acid sequence of SEQ ID NO: 61 or a mutant thereof. More specifically, the mutant can include one or more mutations selected from the group consisting of M196Y, G315H, and D214N compared to the amino acid sequence of SEQ ID NO: 61. Each of these mutants can include an N-terminal tag having the amino acid sequence of MTTASGTNADVQNGVRP. In some embodiments, the flavonoid 3’-hydroxylase can include the amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59.

[0042] Other flavonoid 3’-hydroxylases known in the art also can be used together with the mutant O’-methyltransferase described herein for converting naringenin to homoeriodictyol via the intermediate production of eriodictyol. For example, the flavonoid 3’-hydroxylase SAM5, which is a 4-coumarate 3 -hydroxylase from Saccharothrix espanaensis and has the amino acid sequence of SEQ ID NO: 63, can be used.

Flavin Reductase

[0043] Referring to FIG. 3, the bioconversion of flavonoid to 3’-hydroxylated flavonoid can be enhanced when the flavonoid 3’ -hydroxylase is co-expressed with a flavin reductase. In various embodiments according to the present teachings, the flavin reductase can be a

Saccharothrix espanaensis flavin reductase, a Pseudomonas fluorescens flavin reductase, or the reductase subunit of a 4-hydroxyphenylacetate 3 -monooxygenase (HpaC) from E. coli. The flavin reductase can be a polypeptide comprising the amino acid sequence of SEQ ID NO: 65, 67, or 69.

Production Systems

[0044] Expression vectors including polynucleotide sequences that encode the mutant O- methyltransferases described herein can be used to transform host cells for producing

homoeriodictyol according to the present teachings. Other elements for the transcription and translation of the polynucleotide sequences can include a promoter, a coding region for the enzymes, and a transcriptional terminator. In addition, the same vectors or additional vectors including polynucleotide sequences that encode flavonoid 3’ -hydroxylase and flavin reductase described herein can be used to transform the host cells, if it is desirable to have the host cell capable of producing homoeriodictyol from naringenin.

[0045] A person of ordinary skill in the art will be aware of the molecular biology techniques available for the preparation of expression vectors. The polynucleotide used for incorporation into the expression vector of the subject technology, as described above, can be prepared by routine techniques such as polymerase chain reaction (PCR). In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed (e.g.- plasmid, cosmid, Lambda phages). A vector containing foreign DNA is considered recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

[0046] A number of molecular biology techniques have been developed to operably link DNA to vectors via complementary cohesive termini. In one embodiment, complementary homopolymer tracts can be added to the nucleic acid molecule to be inserted into the vector DNA. The vector and nucleic acid molecule are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

[0047] In an alternative embodiment, synthetic linkers containing one or more restriction sites provide are used to operably link the polynucleotide of the subject technology to the expression vector. In an embodiment, the polynucleotide is generated by restriction endonuclease digestion. In an embodiment, the nucleic acid molecule is treated with bacteriophage T4 DNA polymerase or A. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3'-ends with their

polymerizing activities, thereby generating blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the product of the reaction is a polynucleotide carrying polymeric linker sequences at its ends. These polynucleotides are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the polynucleotide.

[0048] Alternatively, a vector having ligation-independent cloning (LIC) sites can be employed. The required PCR amplified polynucleotide can then be cloned into the LIC vector without restriction digest or ligation (Aslanidis and de Jong, NUCL. ACID. RES. 18 6069-74,

(1990), Haun, et al, BIOTECHNIQUES 13, 515-18 (1992), each of which are incorporated herein by reference).

[0049] In an embodiment, in order to isolate and/or modify the polynucleotide of interest for insertion into the chosen plasmid, it is suitable to use PCR. Appropriate primers for use in PCR preparation of the sequence can be designed to isolate the required coding region of the nucleic acid molecule, add restriction endonuclease or LIC sites, place the coding region in the desired reading frame.

[0050] In an embodiment, a polynucleotide for incorporation into an expression vector of the subject technology is prepared using PCR appropriate oligonucleotide primers. The coding region is amplified, whilst the primers themselves become incorporated into the amplified sequence product. In an embodiment, the amplification primers contain restriction endonuclease recognition sites, which allow the amplified sequence product to be cloned into an appropriate vector. [0051] The expression vectors can be introduced into plant or microbial host cells by conventional transformation or transfection techniques. Transformation of appropriate cells with an expression vector of the subject technology is accomplished by methods known in the art and typically depends on both the type of vector and cell. Suitable techniques include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, chemoporation or electroporation.

[0052] Successfully transformed cells, that is, those cells containing the expression vector, can be identified by techniques well known in the art. For example, cells transfected with an expression vector of the subject technology can be cultured to produce polypeptides described herein. Cells can be examined for the presence of the expression vector DNA by techniques well known in the art.

[0053] The host cells can contain a single copy of the expression vector described previously, or alternatively, multiple copies of the expression vector,

[0054] In some embodiments, the transformed cell is an animal cell, an insect cell, a plant cell, an algal cell, a fungal cell, or a yeast cell. In some embodiments, the cell is a plant cell selected from the group consisting of: canola plant cell, a rapeseed plant cell, a palm plant cell, a sunflower plant cell, a cotton plant cell, a corn plant cell, a peanut plant cell, a flax plant cell, a sesame plant cell, a soybean plant cell, and a petunia plant cell.

[0055] In certain embodiments, the host cell can be selected from the group consisting of bacterium, yeast, and a combination thereof, or any cellular system that would allow the genetic transformation with the selected genes and thereafter the biosynthetic production of 3’- hydroxylated flavonoid (e.g., eriodictyol) from flavonoid (e.g., naringenin). In various

embodiments, the host cell can be selected from the group of microbial species consisting of Escherichia ; Salmonella ; Bacillus ; Acinetobactep Streptomyces; Corynebacterium; Methylosinus., Methylomonas ; Rhodococcus; Pseudomonas ; Rhodobacter ; Synechocystis; Saccharomyces;

Zygosaccharomyces; Kluyveromyces; Candida ; Hansenula ; Debaryomyces; Mucop Pichia ;

Torulopsis ; Aspergillus ; Arthrobotlyp Brevibacteria; Microbacterium ; Arthrobactep Citrobacter\ Klebsiella ; Pantoea; and Clostridium. In preferred embodiments, the host cell can be A. coli.

[0056] Microbial host cell expression systems and expression vectors containing regulatory sequences that direct high-level expression of foreign proteins are well-known to those skilled in the art. Any of these could be used to construct vectors for expression of the recombinant polypeptide of the subjection technology in a microbial host cell. These vectors could then be introduced into appropriate microorganisms via transformation to allow for high level expression of the recombinant polypeptide of the subject technology.

[0057] Vectors or cassettes useful for the transformation of suitable microbial host cells are well known in the art. Typically the vector or cassette contains sequences directing

transcription and translation of the relevant polynucleotide, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the polynucleotide which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. It is preferred for both control regions to be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a host.

[0058] Termination control regions may also be derived from various genes native to the microbial hosts. A termination site optionally may be included for the microbial hosts described herein.

Production of homoeriodictvol via eriodictvol and optionally via naringenin

[0059] The present teachings provide methods for producing homoeriodictyol, where such methods include culturing a transformed host cell in a suitable medium that includes eriodictyol. In various embodiments, the transformed host cell comprises a synthetic or recombinant nucleic acid molecule that includes a first polynucleotide sequence that encodes a mutant O-methyltransferase according to the present teachings. The transformed host cell is cultured under conditions that lead to the synthesis of the mutant O-methyltransferase, which results in eriodictyol being converted to homoeriodictyol by the transformed host cell.

[0060] In various embodiments, the transformed host cell can be cultured at a temperature range of about 25°C to about 40°C. The culture medium can include one or more amino acids, and optionally glucose and/or an antibiotic. The transformed host cell can be cultured for a sufficient period of time until stable cell growth is reached. This typically is referred to as the cell growth phase and can be between about 15 to about 18 hours. The flavonoid substrate can be added only after stable cell growth is reached, or about 8 to about 20 hours after the transformed host cell is added to the culture medium. Once the flavonoid substrate is added, the bioconversion phase begins. Such bioconversion phase can take place between about 8 to 20 hours after the transformed host cell is added to the culture medium and can last until about 20 to 60 hours after the transformed host cell is added to the culture medium.

[0061] In some embodiments, the eriodictyol can be produced in situ from naringenin. According to these embodiments, the transformed host cell can be further transformed to heterologous express a second polynucleotide sequence that encodes a flavonoid 3’ -hydroxylase and a third polynucleotide sequence that encodes a flavin reductase as described herein.

[0062] In addition to fermentation, homoeriodictyol can be produced from eriodictyol (and optionally from naringenin) enzymatically, where mutant O-methyltransferases (and optionally, flavonoid 3’ -hydroxylases and flavin reductases) described herein are isolated and purified, then incubated with the desired substrate for a sufficient time to produce

homoeriodictyol.

Use of the Homoeriodictyol

[0063] Homoeriodictyol produced according to the present teachings can be used in various food products, beverages, pharmaceutical products, and other oral consumable products, where the present 3’-hydroxylated flavonoids can reduce or mask any unpleasant, bitter, and/or astringent taste present in such products. Such products can include one or more natural or artificial sweeteners which have a bitter aftertaste. One with skill in the art will recognize that the 3’-hydroxylated flavonoids produced by the method described herein can be further purified and mixed with other dietary supplements, medical compositions, cosmeceuticals, for nutrition, as well as in pharmaceutical products.

[0064] Molecular biology plays a pivotal role in innovating cosmoceuticals. Compound identification now begins with the identification of molecular targets. For example, the importance of free radicals in association with skin aging has led in recent years to an intensive search for active substances which eliminate the harmful effects of free radicals and thus protect the tissue from oxidative damage. Skin aging manifests as age spots, more specifically as melasma, dyschromia, melanomas, and wrinkling, mainly attributed to free radical damage to the tissues that triggers cross linking and glycation of structural proteins, and pro-inflammatory enzyme systems. The use of flavonoids in cosmetics or pharmacy is known per se. Natural antioxidants, such as the eriodictyol of the invention, that quench free radicals are an essential component of anti-ageing formulations. They potentially offer protection against damage to the tissues, and against the detrimental effects of environmental and other agents. Biochemical reactions that accelerate the progression of skin ageing have their roots in inflammatory processes, as inflammation generates micro-scars that develop into blemishes or wrinkles.

[0065] Flavonoids including flavones and flavone glycoside derivatives discussed herein are known to be scavengers of oxygen radicals and inhibitors of skin proteases so that they are actively able to counteract the aging of the skin and scar formation. By virtue of their coloring properties, some flavones, such as quercetin, are also useful as food colorants. At the same time, their ability to trap oxygen radicals also enables them to be used as antioxidants. Some flavonoids are inhibitors of aldose reductase which plays a key role in the formation of diabetes damage (ex: vascular damage). Other flavonoids (such as hesperidin and rutin) are used therapeutically, more particularly as vasodilating capillary-active agents.

[0066] Scientific research has confirmed a wide influence of flavonoid compounds on various levels of the skin. The uppermost layer of the skin, the stratum corneum, is a structure very rich in lipids and other easily oxidizable compounds. In this layer flavonoids can play an efficient role as anti-oxidizing agents and free radical scavengers. Their antioxidant properties enable them to influence deeper, epidermal skin layers, preventing UV radiation damage and inhibiting some enzyme functions. In the dermis, the deepest skin layer, flavonoids influence the permeability and fragility of the micro-vessel system. The valuable features of flavonoids described above makes them valuable for the cosmetic industry.

Definitions:

[0067] “Cellular system” is any cells that provide for the expression of ectopic proteins. It included bacteria, yeast, plant cells and animal cells. It includes both prokaryotic and eukaryotic cells. It also includes the in vitro expression of proteins based on cellular components, such as ribosomes.

[0068] "Coding sequence" is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to a DNA sequence that encodes for a specific amino acid sequence.

[0069] Growing the Cellular System. Growing includes providing an appropriate medium that would allow cells to multiply and divide. It also includes providing resources so that cells or cellular components can translate and make recombinant proteins.

[0070] Protein Expression. Protein production can occur after gene expression. It consists of the stages after DNA has been transcribed to messenger RNA (mRNA). The mRNA is then translated into polypeptide chains, which are ultimately folded into proteins. DNA is present in the cells through transfection - a process of deliberately introducing nucleic acids into cells. The term is often used for non-viral methods in eukaryotic cells. It may also refer to other methods and cell types, although other terms are preferred: "transformation" is more often used to describe non- viral DNA transfer in bacteria, non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus- mediated DNA transfer. Transformation, transduction, and viral infection are included under the definition of transfection for this application.

[0071] Yeast. According to the current invention a yeast as claimed herein are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. Yeasts are unicellular organisms which evolved from multicellular ancestors but with some species useful for the current invention being those that have the ability to develop multicellular characteristics by forming strings of connected budding cells known as pseudo hyphae or false hyphae.

Structural Terms:

[0072] As used herein, the singular forms "a, an" and "the" include plural references unless the content clearly dictates otherwise.

[0073] To the extent that the term "include," "have," or the like is used in the description or the claims, such term is intended to be inclusive in a manner similar to the term "comprise" as "comprise" is interpreted when employed as a transitional word in a claim.

[0074] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

[0075] The term "complementary" is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to describe the relationship between nucleotide bases that are capable to hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the subjection technology also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences [0076] The terms "nucleic acid" and "nucleotide" are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally-occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified or degenerate variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.

[0077] The term "isolated" is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and when used in the context of an isolated nucleic acid or an isolated polypeptide, is used without limitation to refer to a nucleic acid or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid or polypeptide can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell.

[0078] The terms "incubating" and "incubation" as used herein means a process of mixing two or more chemical or biological entities (such as a chemical compound and an enzyme) and allowing them to interact under conditions favorable for producing a eriodictyol composition.

[0079] The term "degenerate variant" refers to a nucleic acid sequence having a residue sequence that differs from a reference nucleic acid sequence by one or more degenerate codon substitutions. Degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues. A nucleic acid sequence and all of its degenerate variants will express the same amino acid or polypeptide.

[0080] The terms "polypeptide," "protein," and "peptide" are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art; the three terms are sometimes used interchangeably, and are used without limitation to refer to a polymer of amino acids, or amino acid analogs, regardless of its size or function. Although "protein" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term "polypeptide" as used herein refers to peptides, polypeptides, and proteins, unless otherwise noted. The terms "protein," "polypeptide," and "peptide" are used interchangeably herein when referring to a polynucleotide product. Thus, exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.

[0081] The terms "polypeptide fragment" and "fragment," when used in reference to a reference polypeptide, are to be given their ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino-terminus or carboxy -terminus of the reference polypeptide, or alternatively both.

[0082] The term "functional fragment" of a polypeptide or protein refers to a peptide fragment that is a portion of the full-length polypeptide or protein, and has substantially the same biological activity, or carries out substantially the same function as the full-length polypeptide or protein (e.g., carrying out the same enzymatic reaction).

[0083] The terms "variant polypeptide," "modified amino acid sequence" or "modified polypeptide," which are used interchangeably, refer to an amino acid sequence that is different from the reference polypeptide by one or more amino acids, e.g., by one or more amino acid substitutions, deletions, and/or additions. In an aspect, a variant is a "functional variant" which retains some or all of the ability of the reference polypeptide.

[0084] The term "functional variant" further includes conservatively substituted variants. The term "conservatively substituted variant" refers to a peptide having an amino acid sequence that differs from a reference peptide by one or more conservative amino acid substitutions, and maintains some or all of the activity of the reference peptide. A "conservative amino acid substitution" is a substitution of an amino acid residue with a functionally similar residue.

Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one charged or polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; the substitution of one basic residue such as lysine or arginine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another; or the substitution of one aromatic residue, such as phenylalanine, tyrosine, or tryptophan for another. Such substitutions are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. The phrase "conservatively substituted variant" also includes peptides wherein a residue is replaced with a chemically-derivatized residue, provided that the resulting peptide maintains some or all of the activity of the reference peptide as described herein.

[0085] The term "variant," in connection with the polypeptides of the subject technology, further includes a functionally active polypeptide having an amino acid sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least

97%, at least 98%, at least 99%, and even 100% identical to the amino acid sequence of a reference polypeptide.

[0086] The term "homologous" in all its grammatical forms and spelling variations refers to the relationship between polynucleotides or polypeptides that possess a "common evolutionary origin," including polynucleotides or polypeptides from super families and homologous polynucleotides or proteins from different species (Reeck et al., CELL 50:667, 1987). Such polynucleotides or polypeptides have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or the presence of specific amino acids or motifs at conserved positions. For example, two homologous polypeptides can have amino acid sequences that are at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least

88%, at least 89%, at least 900 at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% identical.

[0087] "Suitable regulatory sequences" is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0088] "Promoter" is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters, which cause a gene to be expressed in most cell types at most times, are commonly referred to as "constitutive promoters." It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0089] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0090] The term "expression" as used herein, is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the subject technology. "Over-expression" refers to the production of a gene product in transgenic or recombinant organisms that exceeds levels of production in normal or non-transformed organisms.

[0091] "Transformation" is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to the transfer of a polynucleotide into a target cell. The transferred polynucleotide can be incorporated into the genome or chromosomal DNA of a target cell, resulting in genetically stable inheritance, or it can replicate independent of the host chromosomal. Host organisms containing the transformed nucleic acid fragments may be referred to as "transgenic."

[0092] The terms "transformed," "transgenic," and "recombinant," when used herein in connection with host cells, are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to a cell of a host organism, such as a plant or microbial cell, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host cell, or the nucleic acid molecule can be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or subjects are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.

[0093] The terms "recombinant," "heterologous," and "exogenous," when used herein in connection with polynucleotides, are to be given their ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to a polynucleotide (e.g., a DNA sequence or a gene) that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of site-directed mutagenesis or other recombinant techniques. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position or form within the host cell in which the element is not ordinarily found.

[0094] Similarly, the terms "recombinant," "heterologous," and "exogenous," when used herein in connection with a polypeptide or amino acid sequence, means a polypeptide or amino acid sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, recombinant DNA segments can be expressed in a host cell to produce a recombinant polypeptide.

[0095] The terms "plasmid," "vector," and "cassette" are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA

molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. "Transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.

[0096] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are described below.

[0097] The disclosure will be more fully understood upon consideration of the following non-limiting Examples. It should be understood that these examples, while indicating preferred embodiments of the subject technology, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of the subject technology, and without departing from the spirit and scope thereof, can make various changes and modifications of the subject technology to adapt it to various uses and conditions.

EXAMPLES

Materials and Methods

Bacterial Strains. Plasmids and Culture Conditions

[0098] E. coli strains of DH5a and BL21 (DE3) were purchased from Invitrogen. Plasmid pRSFDuet-l and pCDFDuet-l were purchased from Novagen for DNA cloning and recombinant protein expression purposes.

DNA Manipulation

[0099] All DNA manipulations were performed according to standard procedures.

Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. All PCR reactions were performed with New England Biolabs’ Phusion PCR system according to the manufacturer’s guidance.

Construction of PtCOMTE OsOMT9 and AtOMTl Expression Vectors [00100] The PtCOMTl gene, previously identified from Populus trichocarpa , was cloned from the stem tissues of Populus trichocarpa following Bhuiya and Liu’s protocol (2009). The ORF of PtCOMTl was amplified with an introduction of Nde I restriction site at the 5’-end and that of Not I site at the 3’-end. A forward primer of 5’-

GGAATTCCATATGGGTTCGACAGGTGAAACTCAGATG-3’ (SEQ ID NO: 7) and a reverse primer of 5’-AAGGAAAAAAGCGGCCGCTTAGTTCTTGCGGAATTCAATGACATG-3’

(SEQ ID NO: 8) were used for PtCOMTl amplification. After digestion with Nde I and Not I, the PCR fragment was ligated into the restriction sites of Nde I and Not I of expression vector pUVAP (FIG. 4), generating a plasmid of PtCOMTl -pUVAP (FIG. 5). After sequencing confirmation, the plasmid was ready for transformation into strains BL21 (DE3) and W3110 of Escherichia coli.

The pUVAP vector sequence is listed herein as SEQ ID NO: 49. After sequencing confirmation, the plasmid was ready for transformation into strain BL21 (DE3) of Escherichia coli. The nucleotide sequence of PtCOMTl open reading frame is listed as SEQ ID NO: 2, and the corresponding deduced protein sequence is listed as SEQ ID NO: 1.

[00101] OsOMT9 gene was synthesized by the GenScript Company with the

corresponding sequence (SEQ ID NO: 4) which is codon optimized for its expression in

Escherichia coli. The deduced protein sequence is listed herein in SEQ ID NO: 3. The synthesized OsOMT9 with Nde I restriction site at 5’ - end and Not I restriction site at 3’-end was inserted into the restriction site of Nde I and Not I of pUVAP vector as described above, resulting in construction of the vector OsOMT9-pUVAP (FIG. 6).

[00102] AtOMTl with GenBank accession number of AY081565.1 (SEQ ID NO: 6) was cloned from Arabidopsis leaf tissues. The deduced protein sequence is listed as SEQ ID NO: 5.

The total RNA was extracted with Triazole Plus RNA Purification Kit (Invitrogen Inc.) from the leaves of Arabidopsis thaliana (ecotype Columbia-0). The synthesis of Arabidopsis cDNA was carried out with Im Prom-II™ Reverse Transcription System from Promega Inc. following the manufacturer’s manual. AtOMTl were amplified from the synthesized cDNA with New England Biolab’s Phusion PCR Kit with a forward primer of AtOMTl -Nde_F 5’- GGAATTCCATATGGGTTCAACGGCAGAGACACAATTAAC-3’ (SEQ ID NO: 9) and a reverse primer of AtOMTl -Not_R 5’-

AAGGAAAAAAGCGGCCGCTTAGAGCTTCTTGAGTAACTCAATAAGG-3’ (SEQ ID NO: 10). The PCR product was digested with Nde I and Not I and then inserted into the restriction site of Nde I and Not I of our vector pUVAP. The resulting expression vector, AtOMTl-pUVAP (FIG. 7), was confirmed by sequencing service.

Bioconversion of Eriodictvol to Homoeriodictvol in Shaking Flasks

[00103] E. coli BL2l(DE3) strains harboring PtCOMTl-pUVAP, OsOMT9-pUVAP,

AtOMTl-pUVAP, respectively, were grown in 20 mL of LB medium with 50 pg/L streptomycin in 125 mL shaking flasks. The cells were grown to OD₆oo about 0.6 in a shaker at 37°C, and then changed to 30°C with the addition of lactose to a final concentration of 1.5% (w/v) to induce the expression of exogenous genes. After 3 hours of expression induction, eriodictyol (40% w/v) dissolved in DMSO was added to the culture. The culture was kept shaking under the same culturing condition. Samples were taken at 6 hours after substrate feeding for HPLC analysis.

Site-Directed Mutagenesis by PCR

[00104] The site-directed mutagenesis was carried out by PCR to substitute isoleucine residue at position 316 of PtCOMTl with other 19 amino acids, respectively. The valine residue at position 321 in OsOMT9 and at position 314 of AtOMTl was mutated to alanine with the same procedure. Each substitution mutation was achieved with two primers carrying the mutation with the sequences listed in Table 1. Table 1. Primers used for site-direct mutagenesis

[00105] The PCR products were treated with DPN I enzyme at 37C for 2 hours and extracted and purified from agarose gels using Gel Purification kit purchased from MidSci Company (St Louis, USA) and transformed into E. coli 10G competent cells (Lucigen Inc., Madison, WI, USA). The resultant colonies were grown in LB medium for plasmid extraction. The plasmids with right mutations confirmed by DNA sequencing were transformed into BL21 (DE3) cells for overexpression and bioconversion.

Transformation of E. coli W3110 with the Constructs

[00106] The expression constructs, OsOMT9-pUVAP, OsOMT9-V32lA-pUVP,

PtCOMT 1 -1316 A-pUVAP, and AtOMTl-I3 l4A-pUVAP, were introduced into E. coli W3110 competent cells respectively with standard chemical transformation protocol, leading to the development of homoeriodictyol -producing A. coli strains (“HER” strains), referred herein as HER-01 (with OsOMT9), HER-02 (with OsOMT9-V32lA), HER-03 (PtCOMT 1 -1316 A), and HER-04 (AtOMTl-I3 l4A), respectively.

Production of Homoeriodictvol with Homoeriodictvol-Producing Strains in 50-Liter Fermenters

[00107] Seed culture was prepared in 500 ml shake flasks containing 100 ml working volume, using LB media plus 50mg/L Amp. After inoculated with 200ul of glycerol tube, the flasks were cultivated at 37°C and 200rpm for 7hrs. At this time, OD₆oo was 2.45. The seed culture was then inoculated into a 5L fermenter with the culture medium prepared with the following recipe: Na₂HP0₄ 12H₂0 l.83g/L, KH₂P0₄ 0.7g/L, NaCl 0.5 g/L, (NH₄)₂S0₄ l2g/L, YE (FM802)

7 g/L, glucose 10 g/L, 1M MgS0₄ 7H₂0 lml/L, 1M CaCl₂ lml/L, Amp 50mg/L. The fermentation parameters were set as follows: airflow: 0.8M3/h; temperature: 30°C; agitation: 300rpm-550rpm; pH: set at 7.3±0.05; DO: cascade to agitation to maintain DO above 30%; tank pressure: 0.02Mpa- 0.025Mpa.

[00108] At elapsed fermentation time (EFT) 9.2 hr, 50% glucose was fed at a rate of 80 ml/h and pH was adjusted to 7.5, while the other conditions were not changed. At EFT 10.8 hr, 40% eriodictyol (ER) was fed at a rate of 6.4ml/h.

HPLC and LC-MS analysis.

[00109] HPLC analysis of flavonoids was carried out with Dionex Ultimate 3000 system. Intermediates were separated by reverse-phase chromatography on a Dionex Acclaim 120 C18 column (particle size 3 pm; 150 by 2.1 mm) with a gradient of 0.15% (vol/vol) acetic acid (eluant A) and acetonitrile (eluant B) in a range of 10 to 40% (vol/vol) eluant B and at a flow rate of 0.6 ml/min. For quantification, all intermediates were calibrated with external standards. The compounds were identified by their retention times, as well as the corresponding spectra, which were identified with a diode array detector in the system.

Results

PtCOMT 1 showed little activity toward eriodictyol but AtOMTl showed comparable

bioconversion rate compared to OsOMT9

[00110] OsOMT9 was reported to be able to catalyze the conversion of eriodictyol to homoeriodictyol (Kim et al. 2006; Liu et al. 2013). The inventors tested two plant 3-0- methyltransferases involved in lignin biosynthesis in plants, namely, PtCOMTl and AtOMTl .

The results demonstrated PtCOMTl converted little amount of eriodictyol to homoeriodictyol. However, the conversion rate by AtOMTl is comparable to that by OsOMT9 under the experimental conditions described above (FIG. 9).

A single amino acid mutation in PtCOMTl dramatically increased the bioconversion of eriodictyol to homoeriodictyol

[00111] As shown in FIG. 9, PtCOMTl demonstrated very little activity toward eriodictyol. To increase conversion activity, the inventors attempted structural modifications by site-directed mutagenesis, and by screening the mutant library, the inventors identified a mutant that shows increased conversion of eriodictyol to homoeriodictyol. Sequencing the plasmids revealed that the increased conversion activity was attributed to a single amino acid residue replacement 1316V. From there, the inventors proceeded to replace 1316 with the other 18 amino acids by site-directed mutagenesis. The A. coli strains harboring these mutated genes were tested for the bioconversion of eriodictyol to homoeriodictyol. As shown in FIG. 10, the mutation of isoleucine to alanine (I316A), glycine (I316G), proline (I316P), serine (I316S), threonine (I316T), and valine (I316V) dramatically increased the bioconversion of eriodictyol to homoeriodictyol. Among them, the mutation 1316A showed the highest conversion rate.

Transformed strains producing mutagenized O-methyltransferases were able to produce homoeriodictyol at much higher titers

[00112] As shown in FIG. 11, the strain with the wild type OsOMT9 gave a final titer of 4.2 g/L under the conditions described above. By comparison, each of the transformed strains HER-02 (with OsOMT9-V32lA), HER-03 (PtCOMTl -1316 A), and HER-04 (AtOMTl -1314 A) led to significantly higher titers. The titers reached by HER-02, HER-03, and HER-04 were 5.8 g/L, 6.2 g/L and 5.6 g/L, respectively.

Sequences of Interest

SEQ ID NO. l Protein Sequence of PtCOMTl

MGSTGETQMTPTQVSDEEAHLFAMQLASASVLPMILKTAIELDLLEIMAKAGPGAFLSTS EIASHLPTKNPDAPVMLDRILRLLASYSILTCSLKDLPDGKVERLYGLAPVCKFLTKNEDG V S V SPLCLMNQDKVLMESW YYLKD AILDGGIPFNKAY GMTAFEYHGTDPRFNKVFNKG MSDHSTITMKKLLETYKGFEGLTSLVDVGGGTGAVVNTIVSKYPSIKGINFDLPHVIEDAP

SYPGVEHVGGDMFVSVPKADAVFMKWICHDWSDAHCLKFLKNCYDALPENGKVILVEC

ILPVAPDTSLATKGVVHIDVIMLAHNPGGKERTEKEFEGLAKGAGFQGFEVMCCAFNTHV

IEFRKN*

SEQ ID NO.2 DNA Sequence of PtCOMTl

AT GGGTTCGAC AGGT GAAACTC AGAT GACTCC AACTC AGGT ATC AGATGAAGAGGC A

CACCTCTTTGCCATGCAACTAGCCAGTGCTTCAGTTCTACCAATGATCCTCAAAACAG

CCATTGAACTCGACCTTCTTGAAATCATGGCTAAAGCTGGCCCTGGTGCTTTCTTGTCC

ACATCTGAGATAGCTTCTCACCTCCCTACCAAAAACCCTGATGCGCCTGTCATGTTAG

ACCGTATCTTGCGCCTCCTGGCTAGCTACTCCATTCTTACTTGCTCTCTGAAAGATCTT

CCTGATGGGAAAGTTGAGAGACTGTATGGCCTTGCTCCTGTTTGCAAATTCTTGACCA

AGAACGAGGACGGTGTCTCTGTCAGCCCTCTCTGTCTCATGAACCAGGACAAGGTCCT

CATGGAAAGCTGGTATTATTTGAAAGATGCAATTCTTGATGGAGGAATTCCATTTAAC

AAGGCCTATGGGATGACTGCATTTGAATATCATGGCACGGATCCAAGATTCAACAAG

GTGTTCAATAAGGGAATGTCTGACCACTCTACCATTACCATGAAGAAGCTTCTTGAGA

CCTACAAAGGCTTTGAAGGCCTCACATCCTTGGTGGATGTTGGTGGTGGGACTGGAGC

TGTCGTTAACACCATCGTCTCTAAATACCCTTCAATTAAGGGCATTAACTTTGATCTGC

CCCACGTCATTGAGGATGCCCCATCTTATCCCGGTGTGGAGCATGTTGGTGGGGACAT

GTTTGTTAGCGTGCCCAAAGCAGATGCCGTTTTCATGAAGTGGATATGCCATGATTGG

AGCGACGCACACTGCTTAAAATTCTTGAAGAATTGCTATGACGCCTTGCCGGAAAAC

GGCAAGGTGATACTTGTTGAGTGCATTCTTCCCGTGGCTCCTGACACAAGCCTTGCCA

CCAAGGGAGTCGTGCACATTGATGTTATCATGCTGGCGCACAACCCCGGTGGGAAAG

AGAGGACCGAAAAGGAATTTGAGGGCTTAGCAAAGGGAGCTGGCTTTCAAGGTTTTG

AAGTAATGTGCTGTGCATTCAACACACATGTCATTGAATTCCGCAAGAACTAA

SEQ ID NO.3 Protein Sequence of OsOMT9

MGSTAADMAAAADEEACMYALQLASSSILPMTLKNAIELGLLETLQSAAVAGGGGKAA

LLTP AE VADKLP SK ANP AAADMVDRMLRLL AS YNVVRCEMEEGADGKLSRRY AAAP V C

KWLTPNEDGV SMAALALMNQDKVLMESW YYLKD AVLDGGIPFNKAY GMTAFEYHGTD

ARFNRVFNEGMKNHSVIITKKLLDLYTGFDAASTVVDVGGGVGATVAAVVSRHPHIRGI

NYDLPHVISEAPPFPGVEHVGGDMFASVPRGGDAILMKWILHDWSDEHCARLLKNCYDA

LPEHGKVVVVECVLPESSDATAREQGVFHVDMIMLAHNPGGKERYEREFRELARAAGFT

GFKATYIYANAWAIEFTK*

SEQ ID NO.4 DNA Sequence of OsOMT9

ATGGGTAGCACCGCAGCAGATATGGCAGCAGCAGCCGATGAAGAGGCATGTATGTAT

GCACTGCAGCTGGCAAGCAGCAGTATTCTGCCGATGACCCTGAAAAATGCAATTGAA

C T GGGT C T GC T GG A A AC C C T GC AG AGC GC AGC AGTT GC C GGT GGT GGT GGT A A AGC A

GCACTGCTGACACCGGCAGAAGTTGCAGATAAACTGCCGAGCAAAGCAAATCCGGCA

GCAGCGGATATGGTTGATCGTATGCTGCGTCTGCTGGCGAGCTATAATGTTGTTCGTT

GTGAAATGGAAGAGGGTGCAGATGGTAAACTGAGCCGTCGTTATGCAGCAGCACCGG

TTTGTAAATGGCTGACCCCGAATGAAGATGGTGTTAGCATGGCAGCACTGGCACTGA

T GA AT C AGGAT A A AGTTCTGAT GGA A AGC T GGT ACT AT C T GA A AGAT GC AGTTCTGG

ATGGTGGTATCCCGTTTAACAAAGCCTATGGTATGACCGCCTTTGAATATCATGGCAC

CGATGCACGTTTTAATCGCGTTTTTAATGAGGGCATGAAAAACCATAGCGTGATCATC

ACCAAAAAACTGCTGGATCTGTATACCGGTTTTGATGCCGCAAGCACCGTTGTTGATG TTGGTGGTGGCGTTGGTGCAACCGTTGCAGCCGTTGTTAGCCGTCATCCGCATATTCG

TGGTATTAACTATGATCTGCCGCATGTTATTAGCGAAGCACCGCCTTTTCCGGGTGTT

GAACATGTGGGTGGTGATATGTTTGCAAGCGTTCCGCGTGGTGGTGATGCAATTCTGA

TGAAATGGATTCTGCATGATTGGTCCGATGAACATTGTGCACGTCTGCTGAAAAATTG

TTATGATGCCCTGCCGGAACATGGTAAAGTTGTTGTGGTTGAATGTGTTCTGCCGGAA

AGCAGTGATGCAACCGCACGTGAACAGGGTGTGTTTCATGTTGATATGATTATGCTGG

CACATAATCCGGGTGGCAAAGAACGTTATGAACGTGAATTTCGTGAACTGGCACGTG

CAGCAGGTTTTACGGGTTTTAAAGCAACCTATATCTATGCAAATGCCTGGGCCATTGA

GTTCACCAAATAA

SEQ ID NO.5 Protein Sequence of AtOMT

MGSTAETQLTPVQVTDDEAALFAMQLASASVLPMALKSALELDLLEIMAKNGSPMSPTEI

ASKLPTKNPEAPVMLDRILRLLTSYSVLTCSNRKLSGDGVERIYGLGPVCKYLTKNEDGV

SIAALCLMNQDKVLMESWYHLKDAILDGGIPFNKAYGMSAFEYHGTDPRFNKVFNNGM

SNHSTITMKKILETYKGFEGLT SLVD VGGGIGATLKMIV SK YPNLKGINFDLPHVIED AP SH

PGIEHVGGDMFVSVPKGDAIFMKWICHDWSDEHCVKFLKNCYESLPEDGKVILAECILPE

TPD SSLS TKQ VVHVD CIML AHNPGGKERTEKEFE AL AK AS GFKGIK V V CD AF GVNLIELL

KKL*

SEQ ID NO.6 DNA Sequence of AtOMTl

ATGGGTTCAACGGCAGAGACACAATTAACTCCGGTGCAAGTCACCGACGACGAAGCT

GCCCTCTTCGCCATGCAACTAGCCAGTGCTTCCGTTCTTCCGATGGCTTTAAAATCCGC

CTTAGAGCTTGACCTTCTTGAGATTATGGCCAAGAATGGTTCTCCCATGTCTCCTACC

GAGATCGCTTCTAAACTTCCGACCAAAAATCCTGAAGCTCCGGTCATGCTCGACCGTA

TCCTCCGTCTTCTTACGTCTTACTCCGTCTTAACCTGCTCCAACCGTAAACTTTCCGGT

GATGGCGTTGAACGGATTTACGGGCTTGGTCCGGTTTGCAAGTATTTGACCAAGAACG

AAGATGGTGTTTCCATTGCTGCTCTTTGTCTTATGAACCAAGACAAGGTTCTCATGGA

AAGCTGGTACCATTTGAAGGATGCAATTCTTGATGGTGGGATTCCATTCAACAAGGCT

TATGGAATGAGCGCGTTCGAGTACCACGGGACTGACCCTAGATTCAACAAGGTCTTT

AACAATGGAATGTCTAACCATTCCACAATCACCATGAAGAAGATTCTTGAGACCTAT

AAGGGTTTTGAAGGATTGACTTCTTTGGTTGATGTTGGTGGTGGCATTGGTGCTACAC

TCAAAATGATTGTCTCCAAGTACCCTAATCTTAAAGGCATCAACTTTGATCTCCCACA

TGTCATCGAAGATGCTCCTTCTCATCCTGGTATTGAGCATGTTGGAGGAGATATGTTT

GTAAGTGTCCCTAAAGGTGATGCCATATTCATGAAGTGGATATGTCATGACTGGAGTG

ACGAACATTGCGTGAAATTCTTGAAGAACTGCTACGAGTCACTTCCAGAGGATGGAA

AAGTGATATTAGCAGAGTGTATACTTCCAGAGACACCAGACTCAAGCCTCTCAACCA

AACAAGTAGTCCATGTCGATTGCATTATGTTGGCTCACAATCCCGGAGGCAAAGAAC

GAACCGAGAAAGAGTTTGAGGCATTAGCCAAAGCATCAGGCTTCAAGGGCATCAAAG

TTGTCTGCGACGCTTTTGGTGTTAACCTTATTGAGTTACTCAAGAAGCTCTAA

SEQ ID NO.7 DNA Sequence of PtCOMTl forward primer

GGAATTCCATATGGGTTCGACAGGTGAAACTCAGATG

SEQ ID NO.8 DNA Sequence of PtCOMTl reverse primer

AAGGAAAAAAGCGGCCGCTTAGTTCTTGCGGAATTCAATGACATG

SEQ ID NO.9 DNA Sequence of AtOMTl forward primer GGA ATTC CAT AT GGGTT C A AC GGC AGAG AC AC A ATT A AC

SEQ ID NO.10 DNA Sequence of AtOMTl reverse primer

AAGGAA AAAAGCGGCCGCTT AGAGCTTCTTGAGT AACTC AAT AAGG

SEQ ID NO.11 DNA Sequence of PtCOMTl-I316G forward primer

GTCGTGCACGGCGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.12 DNA Sequence of PtCOMTl-I316G reverse primer

TAACATCGCCGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.13 DNA Sequence of PtCOMT 1-1316L forward primer

GTCGTGCACCTGGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.14 DNA Sequence of PtCOMT 1-1316L reverse primer

TAACATCCAGGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.15 DNA Sequence of PtCOMT 1-1316P forward primer

GTCGTGCACCCGGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.16 DNA Sequence of PtCOMT 1-1316P reverse primer

TAACATCCGGGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.17 DNA Sequence of PtCOMT 1-1316F forward primer

GTCGTGCACTTTGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.18 DNA Sequence of PtCOMT 1-1316F reverse primer

TAACATCAAAGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.19 DNA Sequence of PtCOMT 1-1316W forward primer

GTCGTGCACTGGGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.20 DNA Sequence of PtCOMT 1-1316W reverse primer

TAACATCCCAGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.21 DNA Sequence of PtCOMTl-I3 l6Y forward primer

GTCGTGCACTATGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.22 DNA Sequence of PtCOMTl-I3 l6Y reverse primer

TAACATCATAGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.23 DNA Sequence of PtCOMT 1-1316D forward primer

GTCGTGCACGATGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.24 DNA Sequence of PtCOMT 1-1316D reverse primer

TAACATCATCGTGCACGACTCCCTTGGTGGCAAGGCTTG SEQ ID NO.25 DNA Sequence of PtCOMT 1-1316E forward primer GTCGTGCACGAAGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.26 DNA Sequence of PtCOMT 1-1316E reverse primer TAACATCTTCGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.27 DNA Sequence of PtCOMT 1-1316R forward primer GTCGTGCACCGCGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.28 DNA Sequence of PtCOMT 1-1316R reverse primer TAACATCGCGGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.29 DNA Sequence of PtCOMT 1-1316H forward primer GTCGTGCACCATGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.30 DNA Sequence of PtCOMT 1-1316H reverse primer TAACATCatgGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.31 DNA Sequence of PtCOMT 1-1316K forward primer GTCGTGCACAAAGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.32 DNA Sequence of PtCOMT 1-1316K reverse primer TAACATCTTTGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.33 DNA Sequence of PtCOMT 1-1316S forward primer GTCGTGCACAGCGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.34 DNA Sequence of PtCOMT 1-1316S reverse primer TAACATCGCTGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.35 DNA Sequence of PtCOMT 1-1316T forward primer GTCGTGCACACCGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.36 DNA Sequence of PtCOMT 1-1316T reverse primer T A AC ATCGGT GT GC ACGAC TC CC TT GGT GGC A AGGC TTG

SEQ ID NO.37 DNA Sequence of PtCOMT 1-1316C forward primer GTCGTGCACTGCGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.38 DNA Sequence of PtCOMT 1-1316C reverse primer TAACATCGCAGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.39 DNA Sequence of PtCOMT 1-1316M forward primer GTCGTGCACATGGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.40 DNA Sequence of PtCOMT 1-1316M reverse primer TAACATCCATGTGCACGACTCCCTTGGTGGCAAGGCTTG SEQ ID NO.41 DNA Sequence of PtCOMT 1-1316N forward primer

GTCGTGCACAACGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.42 DNA Sequence of PtCOMT 1-1316N reverse primer

TAACATCGTTGTGCACGACTCCCTTGGTGGCAAGGCTTG SEQ ID NO.43 DNA Sequence of PtCOMT 1-1316Q forward primer

GTCGTGCACCAGGATGTTATCATGCTGGCGCACAACCCC

SEQ ID NO.44 DNA Sequence of PtCOMT 1-1316Q reverse primer

TAACATCCTGGTGCACGACTCCCTTGGTGGCAAGGCTTG

SEQ ID NO.45 DNA Sequence of OsOMT9-V32lA forward primer

GT GTTT CAT GCGGAT AT GATT AT GC T GGC AC AT A AT C CGG

SEQ ID NO.46 DNA Sequence of OsOMT9-V32lA reverse primer

AATCATATCCGCATGAAACACACCCTGTTCACGTGCGG

SEQ ID NO.47 DNA Sequence of AtOMTl-V314A forward primer

GTAGTCCATGCCGATTGCATTATGTTGGCTCACAATCCC

SEQ ID NO.48 DNA Sequence of AtOMTl-V3 l4lA reverse primer

T AAT GC AATCGGC AT GGACT ACTTGTTT GGTTGAGAGGC

SEQ ID NO.49 DNA Sequence of pUVAP Expression Vector

GGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCATCGTTTA

GGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGAATTGTGAGC

GGATAACAATTTCAACTATAAGAAGGAGATATACATATGGCGGATCCGAATTCGGCG

CGCCAGATCTCAATTGGATATCGGCCGGCCGACGTCGGTACCGCGGCCGCCACCGCT

GAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGC

TGAAAGGAGGAACTATATCCGGGTAACGAATTCAAGCTTGATATCATTCAGGACGAG

CCTCAGACTCCAGCGTAACTGGACTGCAATCAACTCACTGGCTCACCTTCACGGGTGG

GCCTTTCTTCGGTAGAAAATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAAT

CTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAA

GAGCTACCAACTCTTTTTCCGAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATAC

TGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCT

ACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGT

GTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCT

GAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGA

GATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCG

GACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCC

AGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAG

CATCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAAC

GCAGAAAGGCCCACCCGAAGGTGAGCCAGGTGATTACATTTGGGCCCTCATCAGAGG

TTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGG

TCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTC

CAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCC TGTTTGGTCATTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATT

TCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGC

TTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAG

ATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAA

CTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTC

GCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGC

TCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACAT

GATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAG

AAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTT

ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCAT

TCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATA

ATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGG

GCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGT

GCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAA

C AGG A AGGC A A A AT GC C GC A A A A A AGGG A AT A AGGGC G AC AC GG A A AT GT T G A AT A

CTCATAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTT

CATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAAGTCAAAAGCCTCCGGTCGG

AGGCTTTTGACTTTCTGCTATGGAGGTCAGGTATGATTTAAATGGTCAGTATTGAGCG

ATATCTAGAGAATTCGTC

SEQ ID #50 SsPvcC-Ml 96Y NT

Artificial Sequence; site-directed mutagenesis

ATGACGGGCGCCGAATATCTGGATTCGCTGCGTGATGGCCGTGCCGTCTATATTCACG

GCGAACGCGTCCGCGATGTCACCGCGCATCCGGCCTTCCGTAACAGCGCGCGTAGTCT

GGCGCAGCTGTATGATGTGCTGCATGAACCGGATTCGCGTGGCGTTCTGAGCGTCCCG

ACCGATACCGGTAATGGCGGTTTTACGCACCCGTTTTTCAAAACCGCGCGTAGCGCCG

GTGATCTGGTGGCAGCCCGCGATGCAATTGTGGCCTGGCAGCGTCTTGTTTACGGTTG

GAT GGGT C GT ACGC CGGATT AT A A AGC AGC ATTC TTC GGT AC GC TT GA AGC C A AC GC

CGATTTCTATGGCCCGTTCCGTGATAACGCACTGGCATGGTATCGTCGTGCACAGGAA

CGCGTGTTGTATTTCAACCATGCGATCGTGCATCCGCCGGTCGATCGCGATCGCCCGG

CCGATCGCACGGCGGATGTGTGCGTCCACGTGGAAGAAGAAACGGATGCCGGCCTTG

TTGTTTCGGGTGCGAAAGTTGTCGCGACCAGCAGCGCCCTGACGAATGCCAATCTGAT

TGCACACTACGGCTTACCGCTGCGTGATAAACGCTTTGGCGCCATGTTCACCGTGCCG

ATGGATAGCCCGGGCCTGAAACTGTTCTGTCGTACCAGTTATGAAATGCATGCCGCGG

TCTTAGGCTCGCCGTTTGATTACCCGTTAAGCAGCCGCCTTGATGAAAATGATTCAAT

CATGGTTCTTGATCGTGTTTTAGTGCCGTGGGAAAACGTGTTTATGTATGATGCCGCC

AGCGCGAACGCGTTTGCGACCCGCAGCGGTTTCCTGGAACGTTTTACCTTTCATGGCT

GTACGCGCTTAGCGGTGAAACTGGATTTTATTGCCGGTTGCCTGCTGAAAGCAGTTGA

AGTGACCGGCACCAGCGGCTTCCGTGGTGTGCAGGCCCAGATTGGCGAAGTTTTAAA

CTGGCGCGATATGTTTTGGGGTATGTCAGATGCAATGGCGAAATCGCCGACGGATTG

GCACAATGGTGCGGTCCAGCCGAATCTGAACTATGGCCTGGCCTACCGCACCTTCATG

GGCATTGGTTACCCGCGTATCCGTGAAATCATTCAGCAGACCATTGGCTCGGGTCTGA

TTTATTTAAATAGTCACGCAAGCGATTGGAAAAATCCGGAAGTTCGTCCGTACTTAGA

TCGCTACCTGCGCGGTAGTCGTGGTGTTGAAGCGATCGATCGCGTTAAACTGCTTAAA

CTGCTGTGGGATTGCGTGGGCACGGAATTTGCGGGCCGTCACGAACTTTATGAACGC

AATTACGGTGGCGATCATGAAGGTATTCGCGTGCAGACCCTGTTGAGTTATCAGGCGC GCGGTCAGGCGGATGCGCTTAAAGGCTTTGCCGATCAGTGTATGTCAGAATACGATCT

GGATGGCTGGACCCGCCCGGATTTATTCGGTCCAGGTGATTTACCTAGACCAGCTACT

GGAGCGTAA

Seq ID #51 SsPvcC-Ml96Y AA

Artificial Sequence; site-directed mutagenesis

MTGAEYLD SLRDGRAVYIHGERVRD VT AHP AFRN S ARSL AQL YD VLHEPD SRGVLS VPT DTGNGGFTHPFFKT ARS AGDL VAARD AIV AW QRL VY GWMGRTPD YK AAFF GTLEANAD FYGPFRDNALAWYRRAQERVLYFNHAIVHPPVDRDRPADRTADVCVHVEEETDAGLVV SGAK WAT S S ALTNANLIAHY GLPLRDKRF GAMFT VPMD SPGLKLF CRT S YEMHAAVLG SPFD YPL S SRLDEND SIM VLDRVL VP WEN VFM YD A AS AN AF ATRS GFLERF TFHGC TRL A VKLDFI AGCLLK A VE VT GT S GFRGV Q AQIGE VLNWRDMF W GM SD AM AK SPTD WHN GA VQPNLNY GLAYRTFMGIGYPRIREIIQQTIGSGLIYLN SHASDWKNPEVRP YLDRYLRGSR GVEAIDRVKLLKLLWDCVGTEFAGRHELYERNYGGDHEGIRVQTLLSYQARGQ DALK GFADQCMSEYDLDGWTRPDLFGPGDLPRPATGA

Seq ID #52 SsPvcC-Ml96Y-Tag NT

Artificial Sequence; N-terminal tag and site-directed mutagenesis

ATGACGACCGCTAGTGGTACCAATGCAGATGTCCAGAATGGCGTCCGCCCGATGACG

GGCGCCGAATATCTGGATTCGCTGCGTGATGGCCGTGCCGTCTATATTCACGGCGAAC

GCGTCCGCGATGTCACCGCGCATCCGGCCTTCCGTAACAGCGCGCGTAGTCTGGCGCA

GCTGTATGATGTGCTGCATGAACCGGATTCGCGTGGCGTTCTGAGCGTCCCGACCGAT

ACCGGTAATGGCGGTTTTACGCACCCGTTTTTCAAAACCGCGCGTAGCGCCGGTGATC

TGGTGGCAGCCCGCGATGCAATTGTGGCCTGGCAGCGTCTTGTTTACGGTTGGATGGG

TCGTACGCCGGATTATAAAGCAGCATTCTTCGGTACGCTTGAAGCCAACGCCGATTTC

TATGGCCCGTTCCGTGATAACGCACTGGCATGGTATCGTCGTGCACAGGAACGCGTGT

TGTATTTCAACCATGCGATCGTGCATCCGCCGGTCGATCGCGATCGCCCGGCCGATCG

CACGGCGGATGTGTGCGTCCACGTGGAAGAAGAAACGGATGCCGGCCTTGTTGTTTC

GGGTGCGAAAGTTGTCGCGACCAGCAGCGCCCTGACGAATGCCAATCTGATTGCACA

CTACGGCTTACCGCTGCGTGATAAACGCTTTGGCGCCATGTTCACCGTGCCGATGGAT

AGCCCGGGCCTGAAACTGTTCTGTCGTACCAGTTATGAAATGCATGCCGCGGTCTTAG

GCTCGCCGTTTGATTACCCGTTAAGCAGCCGCCTTGATGAAAATGATTCAATCATGGT

TCTTGATCGTGTTTTAGTGCCGTGGGAAAACGTGTTTATGTATGATGCCGCCAGCGCG

AACGCGTTTGCGACCCGCAGCGGTTTCCTGGAACGTTTTACCTTTCATGGCTGTACGC

GCTTAGCGGTGAAACTGGATTTTATTGCCGGTTGCCTGCTGAAAGCAGTTGAAGTGAC

CGGCACCAGCGGCTTCCGTGGTGTGCAGGCCCAGATTGGCGAAGTTTTAAACTGGCG

CGATATGTTTTGGGGTATGTCAGATGCAATGGCGAAATCGCCGACGGATTGGCACAA

TGGTGCGGTCCAGCCGAATCTGAACTATGGCCTGGCCTACCGCACCTTCATGGGCATT

GGTTACCCGCGTATCCGTGAAATCATTCAGCAGACCATTGGCTCGGGTCTGATTTATT

TAAATAGTCACGCAAGCGATTGGAAAAATCCGGAAGTTCGTCCGTACTTAGATCGCT

ACCTGCGCGGTAGTCGTGGTGTTGAAGCGATCGATCGCGTTAAACTGCTTAAACTGCT

GT GGGATT GCGT GGGC ACGGAATTT GCGGGCCGTC ACGAACTTT AT GAACGC AATT A

CGGTGGCGATCATGAAGGTATTCGCGTGCAGACCCTGTTGAGTTATCAGGCGCGCGG

TCAGGCGGATGCGCTTAAAGGCTTTGCCGATCAGTGTATGTCAGAATACGATCTGGAT GGCTGGACCCGCCCGGATTTATTCGGTCCAGGTGATTTACCTAGACCAGCTACTGGAG

CGTAA

Seq ID #53 SsPvcC-Ml96Y-Tag AA

Artificial Sequence; N-terminal tag and site-directed mutagenesis

MTTASGTNADVQNGVRPMTGAEYLDSLRDGRAVYIHGERVRDVTAHPAFRNSARSLAQ LYDVLHEPDSRGVLSVPTDTGNGGFTHPFFKTARSAGDLVAARDAIVAWQRLVYGWMG RTPD YK AAFF GTLE ANADF Y GPFRDNAL AW YRRAQERVL YFNH AIVHPP VDRDRP ADRT AD VC VHVEEETD AGL VVSGAK WATS S ALTNANLIAHY GLPLRDKRF GAMFT VPMD SPG LKLF CRT S YEMHAAVLGSPFD YPL S SRLDEND SIMVLDRVL VPWENVFM YD A AS ANAF A TRSGFLERFTFHGCTRLAVKLDFIAGCLLKAVEVTGTSGFRGVQAQIGEVLNWRDMFWG MSDAMAKSPTDWHNGAVQPNLNYGLAYRTFMGIGYPRIREIIQQTIGSGLIYLNSHASDW KNPEVRP YLDRYLRGSRGVE AIDRVKLLKLLWDC VGTEF AGRHEL YERNY GGDHEGIRV QTLLSYQARGQADALKGFADQCMSEYDLDGWTRPDLFGPGDLPRPATGA

Seq ID #54 SsPvcC-G3 l 5H+Tag NT

Artificial Sequence; N-terminal tag + Site-Directed Mutagenesis

ATGACGACCGCTAGTGGTACCAATGCAGATGTCCAGAATGGCGTCCGCCCGATGACG

GGCGCCGAATATCTGGATTCGCTGCGTGATGGCCGTGCCGTCTATATTCACGGCGAAC

GCGTCCGCGATGTCACCGCGCATCCGGCCTTCCGTAACAGCGCGCGTAGTCTGGCGCA

GCTGTATGATGTGCTGCATGAACCGGATTCGCGTGGCGTTCTGAGCGTCCCGACCGAT

ACCGGTAATGGCGGTTTTACGCACCCGTTTTTCAAAACCGCGCGTAGCGCCGGTGATC

TGGTGGCAGCCCGCGATGCAATTGTGGCCTGGCAGCGTCTTGTTTACGGTTGGATGGG

TCGTACGCCGGATTATAAAGCAGCATTCTTCGGTACGCTTGAAGCCAACGCCGATTTC

TATGGCCCGTTCCGTGATAACGCACTGGCATGGTATCGTCGTGCACAGGAACGCGTGT

TGTATTTCAACCATGCGATCGTGCATCCGCCGGTCGATCGCGATCGCCCGGCCGATCG

CACGGCGGATGTGTGCGTCCACGTGGAAGAAGAAACGGATGCCGGCCTTGTTGTTTC

GGGTGCGAAAGTTGTCGCGACCAGCAGCGCCCTGACGAATGCCAATCTGATTGCACA

CATGGGCTTACCGCTGCGTGATAAACGCTTTGGCGCCATGTTCACCGTGCCGATGGAT

AGCCCGGGCCTGAAACTGTTCTGTCGTACCAGTTATGAAATGCATGCCGCGGTCTTAG

GCTCGCCGTTTGATTACCCGTTAAGCAGCCGCCTTGATGAAAATGATTCAATCATGGT

TCTTGATCGTGTTTTAGTGCCGTGGGAAAACGTGTTTATGTATGATGCCGCCAGCGCG

AACGCGTTTGCGACCCGCAGCGGTTTCCTGGAACGTTTTACCTTTCATGGCTGTACGC

GCTTAGCGGTGAAACTGGATTTTATTGCCGGTTGCCTGCTGAAAGCAGTTGAAGTGAC

CGGCACCAGCCACTTCCGTGGTGTGCAGGCCCAGATTGGCGAAGTTTTAAACTGGCG

C GAT AT GTTTTGGGGT AT GT C AGAT GCA AT GGCGA A ATCGC C GAC GGATT GGC AC A A

TGGTGCGGTCCAGCCGAATCTGAACTATGGCCTGGCCTACCGCACCTTCATGGGCATT

GGTTACCCGCGTATCCGTGAAATCATTCAGCAGACCATTGGCTCGGGTCTGATTTATT

TAAATAGTCACGCAAGCGATTGGAAAAATCCGGAAGTTCGTCCGTACTTAGATCGCT

ACCTGCGCGGTAGTCGTGGTGTTGAAGCGATCGATCGCGTTAAACTGCTTAAACTGCT

GTGGGATTGCGTGGGCACGGAATTTGCGGGCCGTCACGAACTTTATGAACGCAATTA

CGGTGGCGATCATGAAGGTATTCGCGTGCAGACCCTGTTGAGTTATCAGGCGCGCGG

T C AGGC GGAT GC GC TT A A AGGCTTT GC C GAT C AGT GT AT GT C AGA AT ACGATCTGGAT

GGCTGGACCCGCCCGGATTTATTCGGTCCAGGTGATTTACCTAGACCAGCTACTGGAG

CGTAA Seq ID #55 SsPvcC-G3 l5H+Tag AA

Artificial Sequence; N-terminal tag + Site-Directed Mutagenesis

MTTASGTNADVQNGVRPMTGAEYLDSLRDGRAVYIHGERVRDVTAHPAFRNSARSLAQ LYDVLHEPDSRGVLSVPTDTGNGGFTHPFFKTARSAGDLVAARDAIVAWQRLVYGWMG RTPD YKAAFF GTLE ANADF Y GPFRDNAL AWYRRAQERVL YFNHAIVHPPVDRDRP ADRT ADVCVHVEEETDAGLVVSGAKVVATSSALTNANLIAHMGLPLRDKRFGAMFTVPMDSP GLKLF CRT S YEMHAAVLGSPFD YPL S SRLDEND SIMVLDRVL VPWENVFM YD AAS ANAF ATRSGFLERFTFHGCTRLAVKLDFIAGCLLKAVEVTGTSHFRGVQAQIGEVLNWRDMFW GMSDAMAKSPTDWHNGAVQPNLNY GL AYRTFMGIGYPRIREIIQQTIGSGLIYLN SHASD WKNPEVRPYLDRYLRGSRGVEAIDRVKLLKLLWDCVGTEFAGRHELYERNYGGDHEGIR VQTLLSYQARGQADALKGFADQCMSEYDLDGWTRPDLFGPGDLPRPATGA

Seq ID #56 SsPvcC-D2l4N+Tag NT

Artificial Sequence; N-terminal tag + Site-Directed Mutagenesis

ATGACGACCGCTAGTGGTACCAATGCAGATGTCCAGAATGGCGTCCGCCCGATGACG

GGCGCCGAATATCTGGATTCGCTGCGTGATGGCCGTGCCGTCTATATTCACGGCGAAC

GCGTCCGCGATGTCACCGCGCATCCGGCCTTCCGTAACAGCGCGCGTAGTCTGGCGCA

GCTGTATGATGTGCTGCATGAACCGGATTCGCGTGGCGTTCTGAGCGTCCCGACCGAT

ACCGGTAATGGCGGTTTTACGCACCCGTTTTTCAAAACCGCGCGTAGCGCCGGTGATC

TGGTGGCAGCCCGCGATGCAATTGTGGCCTGGCAGCGTCTTGTTTACGGTTGGATGGG

TCGTACGCCGGATTATAAAGCAGCATTCTTCGGTACGCTTGAAGCCAACGCCGATTTC

TATGGCCCGTTCCGTGATAACGCACTGGCATGGTATCGTCGTGCACAGGAACGCGTGT

TGTATTTCAACCATGCGATCGTGCATCCGCCGGTCGATCGCGATCGCCCGGCCGATCG

CACGGCGGATGTGTGCGTCCACGTGGAAGAAGAAACGGATGCCGGCCTTGTTGTTTC

GGGTGCGAAAGTTGTCGCGACCAGCAGCGCCCTGACGAATGCCAATCTGATTGCACA

CATGGGCTTACCGCTGCGTGATAAACGCTTTGGCGCCATGTTCACCGTGCCGATGAAT

AGCCCGGGCCTGAAACTGTTCTGTCGTACCAGTTATGAAATGCATGCCGCGGTCTTAG

GCTCGCCGTTTGATTACCCGTTAAGCAGCCGCCTTGATGAAAATGATTCAATCATGGT

TCTTGATCGTGTTTTAGTGCCGTGGGAAAACGTGTTTATGTATGATGCCGCCAGCGCG

AACGCGTTTGCGACCCGCAGCGGTTTCCTGGAACGTTTTACCTTTCATGGCTGTACGC

GCTTAGCGGTGAAACTGGATTTTATTGCCGGTTGCCTGCTGAAAGCAGTTGAAGTGAC

CGGCACCAGCCATTTCCGTGGTGTGCAGGCCCAGATTGGCGAAGTTTTAAACTGGCGC

GATATGTTTTGGGGTATGTCAGATGCAATGGCGAAATCGCCGACGGATTGGCACAAT

GGTGCGGTCCAGCCGAATCTGAACTATGGCCTGGCCTACCGCACCTTCATGGGCATTG

GTTACCCGCGTATCCGTGAAATCATTCAGCAGACCATTGGCTCGGGTCTGATTTATTT

AAATAGTCACGCAAGCGATTGGAAAAATCCGGAAGTTCGTCCGTACTTAGATCGCTA

CCTGCGCGGTAGTCGTGGTGTTGAAGCGATCGATCGCGTTAAACTGCTTAAACTGCTG

TGGGATTGCGTGGGCACGGAATTTGCGGGCCGTCACGAACTTTATGAACGCAATTAC

GGTGGCGATCATGAAGGTATTCGCGTGCAGACCCTGTTGAGTTATCAGGCGCGCGGT

CAGGCGGATGCGCTTAAAGGCTTTGCCGATCAGTGTATGTCAGAATACGATCTGGAT

GGCTGGACCCGCCCGGATTTATTCGGTCCAGGTGATTTACCTAGACCAGCTACTGGAG

CGTAA

Seq ID #57 SsPvcC-D2l4N+Tag AA Artificial Sequence; N-terminal tag + Site-Directed Mutagenesis

MTTASGTNADVQNGVRPMTGAEYLDSLRDGRAVYIHGERVRDVTAHPAFRNSARSLAQ L DVLHEPDSRGVLSVPTDTGNGGFTHPFFKTARSAGDLVAARDAIVAWQRLVYGWMG RTPD YKAAFF GTLE ANADF Y GPFRDNAL AW YRRAQERVLYFNH AIVHPP VDRDRP ADRT AD VC VHVEEETD AGL VVSGAKVVATS S ALTNANLIAHMGLPLRDKRFGAMFTVPMN SP GLKLF CRT S YEMHAAVLGSPFD YPL S SRLDEND SIMVLDRVL VPWENVFMYD AAS ANAF ATRSGFLERFTFHGCTRLAVKLDFIAGCLLKAVEVTGTSGFRGVQAQIGEVLNWRDMFW GMSDAMAKSPTDWHNGAVQPNLNY GL AYRTFMGIGYPRIREIIQQTIGSGLIYLN SHASD WKNPEVRP YLDRYLRGSRGVEAIDRVKLLKLLWDC VGTEF AGRHEL YERNY GGDHEGIR VQTLLSYQARGQADALKGFADQCMSEYDLDGWTRPDLFGPGDLPRPATGA

Seq ID #58 SsPvcC+Tag NT

Artificial Sequence; N-terminal tag

ATGACGACCGCTAGTGGTACCAATGCAGATGTCCAGAATGGCGTCCGCCCGATGACG

GGCGCCGAATATCTGGATTCGCTGCGTGATGGCCGTGCCGTCTATATTCACGGCGAAC

GCGTCCGCGATGTCACCGCGCATCCGGCCTTCCGTAACAGCGCGCGTAGTCTGGCGCA

GCTGTATGATGTGCTGCATGAACCGGATTCGCGTGGCGTTCTGAGCGTCCCGACCGAT

ACCGGTAATGGCGGTTTTACGCACCCGTTTTTCAAAACCGCGCGTAGCGCCGGTGATC

TGGTGGCAGCCCGCGATGCAATTGTGGCCTGGCAGCGTCTTGTTTACGGTTGGATGGG

TCGTACGCCGGATTATAAAGCAGCATTCTTCGGTACGCTTGAAGCCAACGCCGATTTC

TATGGCCCGTTCCGTGATAACGCACTGGCATGGTATCGTCGTGCACAGGAACGCGTGT

TGTATTTCAACCATGCGATCGTGCATCCGCCGGTCGATCGCGATCGCCCGGCCGATCG

CACGGCGGATGTGTGCGTCCACGTGGAAGAAGAAACGGATGCCGGCCTTGTTGTTTC

GGGTGCGAAAGTTGTCGCGACCAGCAGCGCCCTGACGAATGCCAATCTGATTGCACA

CATGGGCTTACCGCTGCGTGATAAACGCTTTGGCGCCATGTTCACCGTGCCGATGGAT

AGCCCGGGCCTGAAACTGTTCTGTCGTACCAGTTATGAAATGCATGCCGCGGTCTTAG

GCTCGCCGTTTGATTACCCGTTAAGCAGCCGCCTTGATGAAAATGATTCAATCATGGT

TCTTGATCGTGTTTTAGTGCCGTGGGAAAACGTGTTTATGTATGATGCCGCCAGCGCG

AACGCGTTTGCGACCCGCAGCGGTTTCCTGGAACGTTTTACCTTTCATGGCTGTACGC

GCTTAGCGGTGAAACTGGATTTTATTGCCGGTTGCCTGCTGAAAGCAGTTGAAGTGAC

CGGCACCAGCGGCTTCCGTGGTGTGCAGGCCCAGATTGGCGAAGTTTTAAACTGGCG

CGATATGTTTTGGGGTATGTCAGATGCAATGGCGAAATCGCCGACGGATTGGCACAA

TGGTGCGGTCCAGCCGAATCTGAACTATGGCCTGGCCTACCGCACCTTCATGGGCATT

GGTTACCCGCGTATCCGTGAAATCATTCAGCAGACCATTGGCTCGGGTCTGATTTATT

TAAATAGTCACGCAAGCGATTGGAAAAATCCGGAAGTTCGTCCGTACTTAGATCGCT

ACCTGCGCGGTAGTCGTGGTGTTGAAGCGATCGATCGCGTTAAACTGCTTAAACTGCT

GT GGGATT GCGT GGGC ACGGAATTT GCGGGCCGTC ACGAACTTT AT GAACGC AATT A

CGGTGGCGATCATGAAGGTATTCGCGTGCAGACCCTGTTGAGTTATCAGGCGCGCGG

TCAGGCGGATGCGCTTAAAGGCTTTGCCGATCAGTGTATGTCAGAATACGATCTGGAT

GGCTGGACCCGCCCGGATTTATTCGGTCCAGGTGATTTACCTAGACCAGCTACTGGAG

CGTAA

Seq ID #59 SsPvcC+Tag AA

Artificial Sequence; N-terminal tag MTTASGTNADVQNGVRPMTGAEYLDSLRDGRAVYIHGERVRDVTAHPAFRNSARSLAQ LYDVLHEPDSRGVLSVPTDTGNGGFTHPFFKTARSAGDLVAARDAIVAWQRLVYGWMG RTPD YKAAFF GTLE ANADF Y GPFRDNAL AWYRRAQERVLYFNH AIVHPP VDRDRP ADRT AD VC VHVEEETD AGL VVSGAKVVATS S ALTNANLIAHMGLPLRDKRF GAMFTVPMDSP GLKLF CRT S YEMHAAVLGSPFD YPL S SRLDEND SIM VEDRVL VPWENVFMYD AAS ANAF ATRS GFLERFTFHGC TRL A VKLDFI AGCLLK AVE VT GT S GFRGV Q AQIGE VLNWRDMF W GMSDAMAKSPTDWHNGAVQPNLNY GL AYRTFMGIGYPRIREIIQQTIGSGLIYLN SHASD WKNPEVRP YLDRYLRGSRGVE AIDRVKLLKLLWDC VGTEF AGRHEL YERNY GGDHEGIR VQTLLSYQARGQADALKGFADQCMSEYDLDGWTRPDLFGPGDLPRPATGA

Seq ID #60: SsPvc NT

Organism: Streptomyces sclerotialus

ATGACGGGCGCCGAATATCTGGATTCGCTGCGTGATGGCCGTGCCGTCTATATTCACG

GCGAACGCGTCCGCGATGTCACCGCGCATCCGGCCTTCCGTAACAGCGCGCGTAGTCT

GGCGCAGCTGTATGATGTGCTGCATGAACCGGATTCGCGTGGCGTTCTGAGCGTCCCG

ACCGATACCGGTAATGGCGGTTTTACGCACCCGTTTTTCAAAACCGCGCGTAGCGCCG

GTGATCTGGTGGCAGCCCGCGATGCAATTGTGGCCTGGCAGCGTCTTGTTTACGGTTG

GATGGGTCGTACGCCGGATTATAAAGCAGCATTCTTCGGTACGCTTGAAGCCAACGC

CGATTTCTATGGCCCGTTCCGTGATAACGCACTGGCATGGTATCGTCGTGCACAGGAA

CGCGTGTTGTATTTCAACCATGCGATCGTGCATCCGCCGGTCGATCGCGATCGCCCGG

CCGATCGCACGGCGGATGTGTGCGTCCACGTGGAAGAAGAAACGGATGCCGGCCTTG

TTGTTTCGGGTGCGAAAGTTGTCGCGACCAGCAGCGCCCTGACGAATGCCAATCTGAT

TGCACACATGGGCTTACCGCTGCGTGATAAACGCTTTGGCGCCATGTTCACCGTGCCG

ATGGATAGCCCGGGCCTGAAACTGTTCTGTCGTACCAGTTATGAAATGCATGCCGCGG

TCTTAGGCTCGCCGTTTGATTACCCGTTAAGCAGCCGCCTTGATGAAAATGATTCAAT

CATGGTTCTTGATCGTGTTTTAGTGCCGTGGGAAAACGTGTTTATGTATGATGCCGCC

AGCGCGAACGCGTTTGCGACCCGCAGCGGTTTCCTGGAACGTTTTACCTTTCATGGCT

GTACGCGCTTAGCGGTGAAACTGGATTTTATTGCCGGTTGCCTGCTGAAAGCAGTTGA

AGTGACCGGCACCAGCGGCTTCCGTGGTGTGCAGGCCCAGATTGGCGAAGTTTTAAA

CTGGCGCGATATGTTTTGGGGTATGTCAGATGCAATGGCGAAATCGCCGACGGATTG

GCACAATGGTGCGGTCCAGCCGAATCTGAACTATGGCCTGGCCTACCGCACCTTCATG

GGCATTGGTTACCCGCGTATCCGTGAAATCATTCAGCAGACCATTGGCTCGGGTCTGA

TTTATTTAAATAGTCACGCAAGCGATTGGAAAAATCCGGAAGTTCGTCCGTACTTAGA

TCGCTACCTGCGCGGTAGTCGTGGTGTTGAAGCGATCGATCGCGTTAAACTGCTTAAA

CTGCTGTGGGATTGCGTGGGCACGGAATTTGCGGGCCGTCACGAACTTTATGAACGC

AATTACGGTGGCGATCATGAAGGTATTCGCGTGCAGACCCTGTTGAGTTATCAGGCGC

GCGGTCAGGCGGATGCGCTTAAAGGCTTTGCCGATCAGTGTATGTCAGAATACGATCT

GGATGGCTGGACCCGCCCGGATTTATTCGGTCCAGGTGATTTACCTAGACCAGCTACT

GGAGCGTAA

Seq ID #61 SsPvcC AA

Organism: Streptomyces sclerotialus

MTGAEYLD SLRDGRAVYIHGERVRD VT AHP AFRN S ARSL AQL YD VLHEPD SRGVLS VPT DTGNGGFTHPFFKT ARS AGDL VAARD AIV AW QRL VY GWMGRTPD YKAAFF GTLEANAD FYGPFRDNALAWYRRAQERVLYFNHAIVHPPVDRDRPADRTADVCVHVEEETDAGLVV SGAK WAT S S ALTNANLIAHMGLPLRDKRF GAMFT VPMD SPGLKLF CRT S YEMHAAVLG

SPFDYPLSSRLDENDSIMVLDRVLVPWENVFMYDAASANAFATRSGFLERFTFHGCTRLA

VKLDFIAGCLLKAVEVTGTSGFRGVQAQIGEVLNWRDMFWGMSDAMAKSPTDWHNGA

VQPNLNY GLAYRTFMGIGYPRIREIIQQTIGSGLIYLN SHASDWKNPEVRPYLDRYLRGSR

GVEAIDRVKLLKLLWDCVGTEFAGRHELYERNYGGDHEGIRVQTLLSYQARGQ DALK

GFADQCMSEYDLDGWTRPDLFGPGDLPRPATGA

SEQ ID NO.64: Nucleic Acid Sequence of SAM5

Organism: Saccharothrix espanaensis

ATGACGATTACCTCTCCGGCCCCGGCTGGTCGCCTGAACAATGTGCGTCCGATGACGG

GTGAAGAATACCTGGAATCCCTGCGTGACGGTCGTGAAGTGTATATTTACGGCGAAC

GCGTCGATGACGTGACCACGCATCTGGCGTTCCGCAACAGCGTTCGTTCTATCGCCCG

CCTGTATGATGTCCTGCACGATCCGGCCTCCGAAGGTGTTCTGCGCGTCCCGACCGAT

ACCGGTAATGGTGGTTTTACCCATCCGTTTTTCAAAACGGCGCGTAGCTCTGAAGACC

TGGTGGCGGCCCGTGAAGCCATTGTCGGTTGGCAACGCCTGGTGTATGGCTGGATGG

GTCGTACCCCGGATTACAAGGCAGCGTTTTTCGGTACGCTGGACGCTAACGCGGAATT

TTATGGCCCGTTCGAAGCCAATGCACGTCGCTGGTATCGTGATGCACAGGAACGCGTT

CTGTACTTCAACCATGCTATCGTGCATCCGCCGGTCGATCGTGACCGTCCGGCTGATC

GTACCGCCGACATTTGCGTCCATGTGGAAGAAGAAACGGATTCAGGCCTGATCGTGT

CGGGTGCCAAAGTGGTTGCAACCGGTTCTGCTATGACGAACGCGAATCTGATTGCCC

ACTATGGTCTGCCGGTTCGCGATAAAAAGTTTGGCCTGGTGTTCACCGTTCCGATGAA

CAGTCCGGGTCTGAAACTGATCTGTCGTACCTCCTATGAACTGATGGTGGCCACGCAG

GGCTCACCGTTTGATTACCCGCTGAGTTCCCGCCTGGATGAAAATGACAGCATTATGA

TCTTTGATCGTGTTCTGGTCCCGTGGGAAAACGTTTTCATGTACGACGCAGGCGCGGC

CAATAGCTTTGCTACCGGCTCTGGTTTCCTGGAACGCTTTACCTTTCATGGCTGCACGC

GTCTGGCAGTGAAACTGGATTTTATTGCAGGCTGTGTTATGAAGGCTGTGGAAGTTAC

CGGCACCACGCACTTCCGCGGTGTTCAGGCGCAAGTCGGCGAAGTGCTGAACTGGCG

TGATGTCTTTTGGGGTCTGTCGGACGCTATGGCGAAAAGTCCGAACAGCTGGGTGGG

CGGTAGCGTTCAGCCGAACCTGAATTATGGCCTGGCCTACCGCACCTTTATGGGCGTG

GGTTATCCGCGTATTAAAGAAATTATCCAGCAAACGCTGGGCTCTGGTCTGATCTACC

TGAACTCATCGGCAGCTGATTGGAAGAATCCGGACGTTCGCCCGTATCTGGATCGTTA

CCTGCGCGGCAGTCGTGGTATTCAGGCAATCGATCGTGTCAAACTGCTGAAGCTGCTG

TGGGACGCGGTGGGCACCGAATTTGCCGGTCGTCATGAACTGTATGAACGCAACTAC

GGCGGTGATCACGAAGGCATTCGTGTGCAGACCCTGCAAGCCTATCAGGCAAATGGT

CAAGCGGCGGCACTGAAAGGCTTTGCGGAACAGTGCATGAGCGAATACGACCTGGAT

GGCTGGACCCGCCCGGACCTGATTAACCCGGGCACCTGA

SEQ ID NO. 63 Amino Acid Sequence of SAM5

Organism: Saccharothrix espanaensis

MTITSPAPAGRLNNVRPMTGEEYLESLRDGREVYIYGERVDDVTTHLAFRNSVRSIARLY DVLHDPASEGVLRVPTDTGNGGFTHPFFKTARSSEDLVAAREAIVGWQRLVYGWMGRTP D YK AAFF GTLD ANAEF Y GPFE ANARRW YRD AQERVL YFNHAIVHPP VDRDRP ADRT ADI C VHVEEETD S GLI V S GAK V V AT GS AMTN ANLIAH Y GLP VRDKKF GL VF T VPMN SPGLKLI CRTSYELMVATQGSPFDYPLSSRLDENDSIMIFDRVLVPWENVFMYDAGAANSFATGSGF LERFTFHGCTRL AVKLDFIAGC VMK AVE VT GTTHFRGV Q AQ V GEVLNWRD VF W GLSD A MAKSPN SWVGGS VQPNLNY GLAYRTFMGVGYPRIKEIIQQTLGSGLIYLN S S AADWKNP D VRP YLDRYLRGSRGIQ AIDRVKLLKLLWD AV GTEF AGRHELYERNY GGDHEGIRVQTL Q AY Q AN GQ AAALKGF AEQCMSEYDLDGWTRPDLINPGT

Seq ID #64: SeFR NT

Organism: Saccharothrix espanaensis

ATGATGACCGTTTATGATAGCGCACTGACAATGGAAGAAACCACCCTGCGTGATGCA

ATGAGCCGTTTTGCAACCGGTGTTAGCGTTGTTACCGTTGGTGGTGAACATACACATG

GTATGACCGCAAATGCCTTTACCTGTGTTAGCCTGGATCCGCCTCTGGTTCTGTGTTGT

GTTGCACGTAAAGCAACCATGCATGCAGCAATTGAAGGTGCACGTCGTTTTGCAGTTA

GCGTTATGGGTGGTGATCAAGAACGTACCGCACGTTATTTTGCAGATAAACGTCGTCC

GCGTGGTCGTGCACAGTTTGATGTTGTTGATTGGCAGCCTGGTCCGCATACAGGTGCA

CCGCTGCTGAGCGGTGCGCTGGCATGGCTGGAATGTGAAGTTGCACAGTGGCATGAA

GGTGGCGATCATACCATTTTTCTGGGTCGTGTTCTGGGTTGTCGTCGTGGTCCGGATA

GTCCGGCACTGCTGTTTTATGGTAGCGATTTTCATCAGATCCGCTAA

Seq ID #65: SeFR A A

Organism: Saccharothrix espanaensis

MMTVYDSALTMEETTLRDAMSRFATGVSVVTVGGEHTHGMTANAFTCVSLDPPLVLCC VARK ATMHAAIEGARRF AV S VMGGDQERT ARYF ADKRRPRGRAQFD VVDW QPGPHTG APLLSGALAWLECEVAQWHEGGDHTIFLGRVLGCRRGPDSPALLFYGSDFHQIR

Seq ID #66: PfFR NT

Organism: Pseudomonas fluorescens

ATGAATGCAGCAACCGAAACCAAAGTTCATGATCTGCTGGATGCCGAAGGTCGTGAT

GTTCGTGATGCACGTGAACTGCGTAATGTTCTGGGTCAGTTTGCAACCGGTGTTACCG

TTATTACCACCCGTACCGCAGATGGTCGTAATGTTGGTGTGACCGCAAATAGCTTTAG

CAGCCTGAGCCTGAGTCCGGCACTGGTTCTGTGGTCACTGGCACGTACCGCACCGAGC

CTGAAAGTTTTTTGTAGCGCAAGCCATTTTGCCATTAATGTGCTGGGTGCACATCAGC

TGCATCTGAGCGAACAGTTTGCACGTGCCGCAGCAGATAAATTTGCCGGTGTTGCACA

TAGTTATGGTAAAGCGGGTGCACCGGTTCTGGATGATGTTGTTGCAGTTCTGGTTTGC

CGTAATGTTACCCAGTATGAAGGTGGTGATCATCTGATTTTTATCGGCGAAATTGAGC

AGTATCGTTATAGCGGTGCAGAACCGCTGGTTTTTCATGCAGGTCAGTATCGTGGTCT

GGGT AGC AATCGT GC AGAAAGCGTTCTGAAAC AT GAAT AA

Seq ID #67: PfFR AA

Organism: Pseudomonas fluorescens

MNAATETK VHDLLD AEGRD VRD ARELRNVLGQF ATGVT VITTRT ADGRNVGVT ANSF S S LSLSPALVLWSLARTAPSLKVFCSASHFAINVLGAHQLHLSEQFARAAADKFAGVAHSYG KAGAPVLDDVVAVLVCRNVTQYEGGDHLIFIGEIEQYRYSGAEPLVFHAGQYRGLGSNR AESVLKHE

Seq ID #68: HpaC NT

Organism: Escherichia coli ATGCAATTAGATGAACAACGCCTGCGCTTTCGTGACGCAATGGCCAGCCTGTCGGCA GC GGT A A AT ATT ATC AC C AC CGAGGGC GAC GCC GGAC A AT GC GGGATT AC GGC A AC G GCCGTCTGCTCGGTCACGGATACACCACCATCGCTGATGGTGTGCATTAACGCCAACA GTGCGATGAACCCGGTTTTTCAGGGCAACGGTAAGTTGTGCGTCAACGTCCTCAACCA T GAGC AGGA ACTGATGGC ACGCC ACTTCGCGGGC AT GAC AGGC AT GGC GAT GGAAGA GCGTTTTAGCCTCTCATGCTGGCAAAAAGGTCCGCTGGCGCAGCCGGTGCTAAAAGG TTCGCTGGCCAGTCTTGAAGGTGAGATCCGCGATGTGCAGGCAATTGGCACACATCTG GTGTATCTGGTGGAGATTAAAAACATCATCCTCAGTGCAGAAGGTCACGGACTTATCT ACTTTAAACGCCGTTTCC ATCCGGTGATGCTGGAAATGGAAGCTGCGATTTAA

Seq ID #69: HpaC AA

Organism: Escherichia coli MQLDEQRLRFRDAMASLSAAVNIITTEGDAGQCGITATAVCSVTDTPPSLMVCINANSAM NP VF Q GN GKLC VN VLNHEQELM ARHF AGMT GM AMEERF SL S C W QKGPL AQP VLKGSL ASLEGEIRD V Q AIGTHL VYLVEIKNIIL S AEGHGLIYFKRRFHP VMLEMEAAI

Seq ID #70 NT

Artificial Sequence; N-terminal tag

ATGACGACCGCTAGTGGTACCAATGCAGATGTCCAGAATGGCGTCCGCCCG Seq ID #71 AA

Artificial Sequence; N-terminal tag

MTTASGTNADVQNGVRP

Claims

1. A method of producing homoeriodictyol, the method comprising culturing a transformed host cell in a suitable medium comprising eriodictyol, wherein the transformed host cell comprises a synthetic or recombinant nucleic acid molecule comprising a first polynucleotide sequence that encodes a mutant O-methyltransferase, said mutant O- methyltransferase being selected from the group consisting of i) an O-methyltransferase comprising a mutation at amino acid residue position 316 of SEQ ID NO: 1, wherein such mutation replaces isoleucine with an amino acid selected from the group consisting of alanine, glycine, proline, serine, threonine, and valine; ii) an O-methyltransferase comprising a mutation at amino acid residue position 321 of SEQ ID NO: 3, wherein such mutation replaces valine with alanine; iii) an O-methyltransferase comprising a mutation at amino acid residue position 314 of SEQ ID NO: 5, wherein such mutation replaces valine with alanine; and wherein said culturing provides for the synthesis of said mutant O-methyltransferase, resulting in eriodictyol being converted to homoeriodictyol by the transformed host cell.

2. The method of claim 1, wherein the mutant O-methyltransferase is an O- methyltransferase comprising a mutation at amino acid residue position 316 of SEQ ID NO: 1, wherein such mutation replaces isoleucine with an amino acid selected from the group consisting of alanine, glycine, proline, serine, threonine, and valine.

3. The method of claim 1, wherein the mutant O-methyltransferase is an O- methyltransferase comprising a mutation at amino acid residue position 321 of SEQ ID NO: 3, wherein such mutation replaces valine with alanine.

4. The method of claim 1, wherein the mutant O-methyltransferase is an O- methyltransferase comprising a mutation at amino acid residue position 314 of SEQ ID NO: 5, wherein such mutation replaces valine with alanine.

5. The method of claim 1, wherein said transformed host cell has been further transformed to heterologously express a second polynucleotide sequence that encodes a flavonoid 3’- hydroxylase and a third polynucleotide sequence that encodes a flavin reductase.

6. The method of claim 5, wherein at least some of the eriodictyol in the medium is

produced in situ by the transformed host cell.

7. The method of claim 6, wherein said medium further comprises naringenin, and at least some of the eriodictyol in the medium is converted from naringenin by the transformed host cell.

8. The method of claim 5, wherein the second polynucleotide sequence encodes a flavonoid 3’ -hydroxylase having an amino acid sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 and SEQ ID NO: 63.

9. The method of claim 8, wherein the second polynucleotide sequence is selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 and SEQ ID NO: 62.

10. The method of claim 5, wherein the third polynucleotide sequence encodes a flavin

reductase having an amino acid sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69.

11. The method of claim 10, wherein the third polynucleotide sequence is selected from the group consisting of SEQ ID NO: 64, SEQ ID NO: 66, and SEQ ID NO: 68.

12. The method of any one of claims 1-11, wherein the host cell is selected from the group of microbial species consisting of Escherichia; Salmonella; Bacillus; Acinetobacter;

Streptomyces; Corynebacterium; Methylosinus; Methylomonas; Rhodococcus;

Pseudomonas; Rhodobacter; Synechocystis; Saccharomyces; Zygosaccharomyces;

Kluyveromyces; Candida; Hansenula; Debaryomyces; Mucor; Pichia; Torulopsis;

Aspergillus; Arthrobotlys; Brevibacteria; Microbacterium; Arthrobacter ; Citrobacter; Klebsiella; Pantoea; and Clostridium.

13. A method of producing homoeriodictyol, said method comprising preparing a reaction mixture comprising: i) eriodictyol and ii) a mutant O-methyltransferase; and incubating the reaction mixture for a sufficient time to produce homoeriodictyol, wherein said mutant O-methyltransferase is selected from the group consisting of i) an O- methyltransferase comprising a mutation at amino acid residue position 316 of SEQ ID NO: 1, wherein such mutation replaces isoleucine with an amino acid selected from the group consisting of alanine, glycine, proline, serine, threonine, and valine; ii) an O- methyltransferase comprising a mutation at amino acid residue position 321 of SEQ ID NO: 3, wherein such mutation replaces valine with alanine; iii) an O-methyltransferase comprising a mutation at amino acid residue position 314 of SEQ ID NO: 5, wherein such mutation replaces valine with alanine.

14. The method of claim 13, wherein at least some of said eriodictyol in the reaction mixture is produced in situ.

15. The method of claim 14, wherein said reaction mixture further comprises naringenin.

16. The method of claim 15, wherein said reaction mixture further comprises a flavonoid 3’- hydroxylase.

17. The method of claim 16, wherein said flavonoid 3’ -hydroxylase comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 and SEQ ID NO: 63.

18. The method of claim 16, wherein said reaction mixture further comprises a flavin

reductase.

19. The method of claim 18, wherein said flavin reductase comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69.

20. The method of claim 18, said method comprising preparing a reaction mixture

comprising: i) eriodictyol, ii) a mutant O-methyltransferase, iii) naringenin, iv) a flavonoid 3’ -hydroxylase, and v) a flavin reductase; and incubating the reaction mixture for a sufficient time to produce eriodictyol from naringenin, and following the production of eriodictyol, further incubating the reaction mixture for a sufficient time to produce homoeriodictyol from eriodictyol.

21. An isolated recombinant host cell transformed with a nucleic acid construct comprising a first polynucleotide sequence encoding a mutant O-methyltransferase, said mutant O- methyltransferase being selected from the group consisting of i) an O-methyltransferase comprising a mutation at amino acid residue position 316 of SEQ ID NO: 1, wherein such mutation replaces isoleucine with an amino acid selected from the group consisting of alanine, glycine, proline, serine, threonine, and valine; ii) an O-methyltransferase comprising a mutation at amino acid residue position 321 of SEQ ID NO: 3, wherein such mutation replaces valine with alanine; iii) an O-methyltransferase comprising a mutation at amino acid residue position 314 of SEQ ID NO: 5, wherein such mutation replaces valine with alanine.

22. The host cell of claim 21, wherein said host cell has been further transformed to heterologously express a second polynucleotide sequence that encodes a flavonoid 3’- hydroxylase and a third polynucleotide sequence that encodes a flavin reductase.

23. The host cell of claim 22, wherein the second polynucleotide sequence is selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 and SEQ ID NO: 62.

24. The host cell of claim 22, wherein the third polynucleotide sequence encodes a flavin reductase having an amino acid sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69.

25. The host cell of any one of claims 21-24, wherein the host cell is selected from the group of microbial species consisting of Escherichia; Salmonella; Bacillus; Acinetobacter ; Streptomyces; Corynebacterium; Methylosinus; Methylomonas; Rhodococcus;

Pseudomonas; Rhodobacter; Synechocystis; Saccharomyces; Zygosaccharomyces; Kluyveromyces; Candida; Hansenula; Debaryomyces; Mucor; Pichia; Torulopsis;

Aspergillus; Arthrobotlys; Brevibacteria; Microbacterium; Arthrobacter ; Citrobacter; Klebsiella; Pantoea; and Clostridium.

26. A mutant O-methyltransferase comprising a mutation at amino acid residue position 316 of SEQ ID NO: 1, wherein such mutation replaces isoleucine with an amino acid selected from the group consisting of alanine, glycine, proline, serine, threonine, and valine.

27. The mutant O-methyltransferase of claim 26, wherein alanine replaces isoleucine at amino acid residue position 316 of SEQ ID NO: 1.

28. The mutant O-methyltransferase of claim 26, wherein alanine replaces isoleucine at amino acid residue position 316 of SEQ ID NO: 1.

29. The mutant O-methyltransferase of claim 26, wherein glycine replaces isoleucine at amino acid residue position 316 of SEQ ID NO: 1.

30. The mutant O-methyltransferase of claim 26, wherein proline replaces isoleucine at amino acid residue position 316 of SEQ ID NO: 1.

31. The mutant O-methyltransferase of claim 26, wherein serine replaces isoleucine at amino acid residue position 316 of SEQ ID NO: 1.

32. The mutant O-methyltransferase of claim 26, wherein threonine replaces isoleucine at amino acid residue position 316 of SEQ ID NO: 1.

33. The mutant O-methyltransferase of claim 26, wherein valine replaces isoleucine at amino acid residue position 316 of SEQ ID NO: 1.

34. A mutant O-methyltransferase comprising a mutation at amino acid residue position 321 of SEQ ID NO: 3, wherein such mutation replaces valine with alanine.

35. A mutant O-methyltransferase comprising a mutation at amino acid residue position 314 of SEQ ID NO: 5, wherein such mutation replaces valine with alanine.