WO2014106189A2

WO2014106189A2 - Methods of making vanillin via microbial fermentation utilizing ferulic acid provided by a modified caffeic acid 3-o-methyltransferase

Info

Publication number: WO2014106189A2
Application number: PCT/US2013/078328
Authority: WO
Inventors: Rui Zhou; Mohammad Wadud BHUIYA; Xianpeng CAI; Xiaodan Yu; Robert G. Eilerman
Original assignee: Conagen Inc.; Givaudan Sa
Priority date: 2012-12-31
Filing date: 2013-12-30
Publication date: 2014-07-03
Also published as: WO2014106189A3

Abstract

This disclosure provides a method of making vanillin including expressing 4- hydroxyphenylacetate 3-hyrdroxylase, caffeic acid 3-O-methyltransferase, methionine synthase, feruloyl-CoA synthetase, and enoyl-CoA hydratase/aldolase in a mixture, feeding p-coumaric acid to the mixture, and collecting vanillin. It further includes an enzyme, such as caffeic acid 3-O-methyltransferase, that facilitates the increased conversion of caffeic acid to ferulic acid, wherein the enzyme has been modified at a residue that allows for increased methylation of ferulic acid, and a method of using the enzyme in the making of ferulic acid followed by vanillin.

Description

METHODS OF MAKING VANILLIN VIA MICROBIAL FERMENTATION

UTILIZING FERULIC ACID PROVIDED BY A MODIFIED CAFFEIC ACID 3-0-

METHYLTRANSFERASE Cross-Reference to Related Applications

[0001] This disclosure is a PCT Patent application entitled Methods of Making

Vanillin via Microbial Fermentation Utilizing Ferulic Acid Provided by a Modified Caffeic Acid 3-O-Methyltransferase. This application claims priority to US Provisional Patent application No.61/747,682 filed on December 31, 2012, which is incorporated by reference herein in its entirety.

Background of the Disclosure

[0002] Field of Disclosure: This disclosure relates generally to methods of making vanillin, especially by providing substrate ferulic acid to a cellular system that facilitates its conversion to vanillin. Ferulic acid is produced via a modified caffeic acid 3-0- methyltransferase among other enzymes. Caffeic acid 3-O-methyltransferase is used in the high-yield microbial fermentation of caffeic acid to ferulic acid, which is a substrate for the production of vanillin. [0003] Background Art: Vanillin is a phenolic aldehyde, which is an organic compound with the molecular formula CsHgOs. Its functional groups include aldehyde, ether, and phenol. It is the primary component of the extract of the vanilla bean. Synthetic vanillin, instead of natural vanilla extract, is now more often used as a flavoring agent in foods, beverages, and pharmaceuticals. [0004] The largest use of vanillin is as a flavoring, usually in sweet foods. The ice cream and chocolate industries together comprise 75% of the market for vanillin as a flavoring, with smaller amounts being used in confections and baked goods. Vanillin is also used in the fragrance industry, in perfumes, and to mask unpleasant odors or tastes in medicines, livestock fodder, and cleaning products. It is also used in the flavor industry, as a very important key note for many different flavors, especially creamy profiles like cream soda. In addition, vanillin has been used as a chemical intermediate in the production of pharmaceuticals and other fine chemicals. In 1970, more than half the world's vanillin production was used in the synthesis of other chemicals, but as of 2004 this use accounts for only 13% of the market for vanillin.

[0005] Vanillin can be derived from ferulic acid, which can be made from p-coumaric acid. p-Coumaric acid can be converted to ferulic acid by a two-step enzymatic process, encompassing hydroxylation and methylation, with caffeic acid as an intermediate metabolite. Two types of enzymes are involved in this pathway, 4-hydroxyphenylacetate 3- hydrolase (4HPA3H), catalyzing 4-hydroxylation of p-coumaric acid, and caffeic acid 3-0- methyltransferase (COMT), catalyzing the methylation of caffeic acid to ferulic acid.

[0006] p-Coumaric acid is a hydroxycinnamic acid, an organic compound that is a hydroxy derivative of cinnamic acid. There are three isomers of coumaric acid: o-coumaric acid, m-coumaric acid, and p-coumaric acid - that differ by the position of the hydroxy substitution of the phenyl group. p-Coumaric acid is the most abundant isomer of the three in nature. p-Coumaric acid exists in two forms: trans-p-coumaric acid and cis-p-coumaric acid. [0007] p-Coumaric acid can be bioconverted from cinnamic acid by the action of the

P450-dependent enzyme 4-cinnamic acid hydroxylase (C4H). It can also be produced from L-tyrosine by the action of tyrosine ammonia lyase (TAL). p-Coumaric acid has antioxidant properties and is believed to reduce the risk of stomach cancer by reducing the formation of carcinogenic nitrosamines.

[0008] Caffeic acid is an organic compound that is classified as hydroxycinnamic acid (although it is more specifically a dihydroxycinnamic acid). This yellow solid consists of both phenolic and acrylic functional groups. It is found in all plants because it is a key intermediate in the bioconversion of lignin, one of the principal sources of biomass.

[0009] Caffeic acid is bioconverted by hydroxylation of coumaroyl ester of quinic ester. This hydroxylation produces the caffeic acid ester of shikimic acid, which converts to chlorogenic acid. As mentioned previously, it is the precursor to ferulic acid as well as coniferyl alcohol, and sinapyl alcohol, all of which are significant building blocks in lignin. The transformation to ferulic acid is catalyzed by the enzyme caffeic acid 3-0- methyltransferase.

[00010] Caffeic acid possesses anti-oxidant, anti-virus, anti-cancer and anti- inflammatory properties. Caffeic acid is one of the pivotal intermediates of plant phenylpropanoid pathway starting from the deamination of phenylalanine which generates cinnamic acid.

[00011] Ferulic acid is a hydroxycinnamic acid, a class of polyphenols having a C6-C3 skeleton. It is an abundant phenolic phytochemical found in components of plant cell wall such as arabinoxylans as covalent side chains. It is related to trans-cinnamic acid. As a component of lignin, ferulic acid is a precursor in the manufacture of other aromatic compounds. [00012] Ferulic acid, like many natural phenols, is an antioxidant in vitro in the sense that it is reactive toward free radicals such as reactive oxygen species (ROS). ROS and free radicals are implicated in DNA damage, cancer, and accelerated cell aging. Animal studies and in vitro studies suggest that ferulic acid may have direct antitumor activity against breast cancer and liver cancer. Ferulic acid may have pro-apoptotic effects in cancer cells, thereby leading to their destruction. Ferulic acid may be effective at preventing cancer induced by exposure to the carcinogenic compounds benzopyrene and 4-nitroquinoline 1 -oxide.

[00013] If added to a topical preparation of ascorbic acid and vitamin E, ferulic acid may reduce oxidative stress and formation of thymine dimers in skin. There is also a small amount of research showing oral supplements of ferulic acid can inhibit melanin production in the process of skin whitening.

[00014] Ferulic acid and the related hydroxycinnamic acids are a class of naturally- derived phenolic antioxidants and were shown to have health benefits for human being. It can be obtained from agricultural by-products, such as maize cob, wheat bran and rice bran. In the agroindustrial by-products, ferulic acid and other hydroxycinnamic acids such as p- coumaric acid and caffeic acid are mainly bound to cell wall arabinoxylans, from which it can be hydrolyzed either chemically or with the use of feruloyl esterases in combination with other cell wall degrading enzymes such as xylanases and cellulases. However, these hydroxycinnamic acids extracted from the cell walls are a mixture and are difficult to be separated efficiently in industry because of their very similar chemical structures and physical properties. The conversion of p-coumaric acid and caffeic acid to ferulic acid with high efficiency will lead to high yield of ferulic acid production and reduced cost in industrial use. [00015] Ferulic acid is also useful as a precursor in the manufacturing of vanillin, a flavoring agent often used in place of natural vanilla extract. It is an important substrate for the production of vanillin, an aromatic flavor compound in the food and cosmetics industries. The most intensively studied process for producing vanillin by biotransformation, which then can be designated "natural," is based on the substrate ferulic acid. The use of naturally- derived ferulic acid from agro-industrial waste for microbial conversion to vanillin could provide a means of manufacturing vanillin of "natural origin". With the bioconverted ferulic acid from maize cob extract, applicants produced vanillin with high yield and low cost.

[00016] As mentioned previously, vanillin is a phenolic aldehyde that is the primary component of the extract of the vanilla bean. Synthetic vanillin, instead of natural vanilla extract, is now more often used as a flavoring agent in foods, beverages, and pharmaceuticals. Both vanillin and ethylvanillin are used by the food industry; ethylvanillin is more expensive, but has a stronger note. It differs from vanillin by having an ethoxy group (-0-CH CH₃) instead of a methoxy group (-O-CH₃).

[00017] Natural "vanilla extract" is a mixture of several hundred different compounds in addition to vanillin. Artificial vanilla flavoring is a solution of pure vanillin, usually of synthetic origin. Because of the scarcity and expense of natural vanilla extract, there has long been interest in the synthetic preparation of its predominant component. The first commercial synthesis of vanillin began with the more readily available natural compound eugenol. Today, artificial vanillin is made either from guaiacol or from lignin, a constituent of wood, which is a byproduct of the pulp industry.

[00018] Lignin-based artificial vanilla flavoring is alleged to have a richer flavor profile than oil-based flavoring; the difference is due to the presence of acetovanillone in the lignin-derived product, an impurity not found in vanillin synthesized from guaiacol.

[00019] The bioconversion of vanillin is achieved by the conversion of tyrosine into 4- coumaric acid then into ferulic acid and finally into vanillin. Vanillin is then converted into its corresponding glucoside. The conversion of ferulic acid into vanillin is achieved by conversion of the carboxylic acid into a thioester with acetyl-CoA. The feruloyl CoA is then hydrated into -hydroxy-3-methoxyphenyl-P-hydroxypropionyl CoA (HMPHP CoA). At this point, two different pathways have been proposed for the conversion of HMPHP CoA into vanillin. One pathway is similar to the β-oxidation of fatty acid, beginning with the oxidation of the hydroxyl group, cleavage to release acetyl-CoA to form a shortened thioester and then cleavage of the thioester into an aldehyde. The other pathway contains one enzyme that would simultaneously oxidize the hydroxyl group along with the release of aceyl-CoA.

[00020] The high-yield bioconversion of vanillin is highly desirable. The present disclosure seeks to provide for such bioconversion of vanillin.

Brief Summary of Disclosure

[00021] The present disclosure is a method for the high-yield bioconversion of vanillin from the synthetic pathway of p-coumaric acid to caffeic acid to ferulic acid utilizing bacteria that expresses a modified caffeic acid 3-O-methyltransferase among other enzymes. [00022] An aspect of the current disclosure is an enzyme that facilitates the increased conversion of caffeic acid to ferulic acid, wherein the enzyme has been modified at a residue that allows for increased methylation of caffeic acid. Another aspect of the current disclosure is a modified caffeic acid 3-O-methyltransferase with enhanced activity for the methylation of caffeic acid to ferulic acid comprising a modification of a residue that would bind ferulic acid in an non-covalent manner or an electrostatic manner.

[00023] An additional aspect of the current disclosure is a modified caffeic acid 3-0- methyltransferase that increases conversion of caffeic acid to ferulic acid derived from a plant species. Another aspect of the current disclosure is a recombinant caffeic acid 3-O- methyltransferase encoded by a mutated equivalent of caffeic acid 3-O-methyltransferase, characterized that it has increased methylation activity of caffeic acid to ferulic acid. Its leucine in its methyl binding pocket is mutated.

[00024] Another aspect of the current disclosure is a bioconversion method of making ferulic acid comprising the following: expressing 4-hydroxyphenylacetate 3-hyrdroxylase in a bacteria; expressing caffeic acid 3-O-methyltransferase in the bacteria; expressing methionine synthase in the bacteria; growing the bacteria in medium; feeding p-coumaric acid to the bacteria; incubating the bacteria; and collecting ferulic acid.

[00025] An additional aspect of the current disclosure is a method of making vanillin comprising the following: expressing 4-hydroxyphenylacetate 3-hyrdroxylase in a mixture; expressing caffeic acid 3-O-methyltransferase in the mixture; expressing methionine synthase in the mixture; expressing feruloyl-CoA synthetase in the mixture; expressing enoyl-CoA hydratase/aldolase in the mixture; feeding p-coumaric acid to the mixture; and collecting vanillin.

[00026] An additional aspect of the current disclosure is a method of making vanillin comprising the following: providing ferulic acid in a mixture; expressing feruloyl-CoA synthetase in the mixture; expressing enoyl-CoA hydratase/aldolase in the mixture; and collecting vanillin.

[00027] An additional aspect of the current disclosure is a ferulic acid with less negative or greater 5¹³C compared with ferulic acid derived from C₃ plants. Another aspect of the current disclosure is a vanillin with less negative or greater 8¹³C compared with vanillin derived from C₃ plants.

Brief Descriptions of the Drawings

[00028] For a better understanding of the present disclosure, reference may be made to the accompanying drawings in which:

[00029] Figure 1 illustrates the enzymatic pathway from p-coumaric acid to ferulic acid.

[00030] Figure 2 illustrates the HPLC analysis of the bioconversion of p-coumaric acid into caffeic acid in E. coli with exogenous expression of HpaBC. upper panel: LB medium; lower panel: M9A medium. [00031] Figure 3 shows the bioconversion of p-coumaric acid to caffeic acid with E. coli containing exogenous expression of HpaBC.

[00032] Figure 4 illustrates the relative COMT enzyme activity of the recombinant COMT, and fused COMT and MS.

[00033] Figure 5 shows the production of ferulic acid in E. coli strains containing plant

COMT in different cultural medium. [00034] Figure 6 illustrates the production of ferulic acid in different strains in M9B medium in 24 hours.

[00035] Figure 7 shows the production of ferulic acid in the culture of E. coli containing AtCOMT and fusion MS-AtCOMT.

[00036] Figure 8 illustrates HPLC analysis of hydroxycinnamic acids in the cell culture with the exogenous expression of 4HPA3H and MS-AtCOMT.

[00037] Figure 9 shows bioconversion of p-coumaric acid to caffeic acid and ferulic acid in E. coli containing HpaBC and MS-AtCOMT.

[00038] Figure 10 illustrates the bioconversion of hydroxycinnamic acids in maize cob extract. [00039] Figure 11 shows a model of the tertiary structure of poplar COMT (A); Close- up view of the substrate binding site of COMT (B).

[00040] Figure 12 shows the screening of COMT mutant library.

[00041] Figure 13 illustrates catalytic efficiency of COMT wild type and mutants.

[00042] Figure 14 exhibits the ferulic acid biotransformation profile along with vanillin and by-products accumulation.

[00043] Figure 15 shows the ferulic acid biotransformation profile along with vanillin accumulation during feeding in the presence of Amycolalopsis sp. strain (Zhp06).

[00044] Figure 16 shows the ferulic acid biotransformation profile along with vanillin accumulation.

[00045] 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. Detailed Descriptions of the Disclosure

[00046] An aspect of the current disclosure is an enzyme that facilitates the increased conversion of caffeic acid to ferulic acid, wherein the enzyme has been modified at a residue that allows for increased methylation of caffeic acid. The enzyme is a caffeic acid 3-0- methyltransferase. The enzyme is mutated at a leucine residue in the methyl binding pocket. The enzyme is derived from an alfalfa, an Arabidopsis, a Medicago truncatula, a Populus tric ocarpa, or a Catharamits roseus. Its Leucine 136 is replacing with a tyrosine. The enzyme is mutated at an additional residue. In another disclosure, the Alanine 162 is mutated to proline or threonine.

[00047] Another aspect of the current disclosure is a modified caffeic acid 3-0- methyltransferase with enhanced activity for the methylation of caffeic acid to ferulic acid comprising a modification of a residue that would bind ferulic acid in a non-covalent manner or an electrostatic manner. The residue is in a methyl binding pocket of caffeic acid 3-0- methyltransferase. The modified residue is selected from residues in the methyl binding pocket and a combination thereof. The modified residue is a leucine, wherein it incorporates a hydrophobic group. The hydrophobic group could be an aromatic hydrocarbon. The hydrophobic group could comprise a hydroxyl group. An aspect of the current disclosure is that the modified residue Leu-136 is replaced by a tyrosine. Another aspect of the current disclosure is that an equivalent residue to Leu-136 is mutated. By equivalence, the residue is any residue within the methyl binding pocket that interacts or affects the interaction of caffeic acid.

[00048] An additional aspect of the current disclosure is a modified caffeic acid 3-0- methyltransferase that increases conversion of caffeic acid to ferulic acid derived from a plant species. The plant species is selected from an alfalfa, an Arabidopsis, a Medicago truncatula, a Popiihts trichocarpa, a Catharansns rosens, and a combination thereof. The modified residue is selected from the group consisting of Leu-136, Phe-172, Phe-176, and Ala-162, and a combination thereof. The modified Leu-136 is modified to a tyrosine.

[00049] Another aspect of the current disclosure is a recombinant caffeic acid 3-O- methyltransferase encoded by a mutated equivalent of caffeic acid 3-O-methyltransferase, characterized that it has increased methylation activity of caffeic acid to ferulic acid. Its leucine in its methyl binding pocket is mutated. The leucine is mutated to a tyrosine.

[00050] Another aspect of the current disclosure is a bioconversion method of making ferulic acid comprising the following: expressing 4-hydroxyphenylacetate 3-hyrdroxylase in a bacteria; expressing caffeic acid 3-O-methyltransferase in the bacteria; expressing methionine synthase in the bacteria; growing the bacteria in medium; feeding p-coumaric acid to the bacteria; incubating the bacteria; and collecting ferulic acid. The p-coumaric acid is derived from maize cob extract. The medium is M9B. Expressing 4-hydroxyphenylacetate 3-hydroxylase is expressing a hpaB and a hpaC based on amino acid SEQ ID No. 1 and 2. Expressing 4-hydroxyphenyacetate 3-hydroxylase is based on amino acid sequence selected from E. coli. Expressing caffeic acid 3-O-methyltransferase is based on amino acid sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7. Expressing caffeic acid 3-O-methyltransferase is based on amino acid sequence selected from the species consisting of alfalfa, Arabidopsis, Medicago, Catharansas, Popiihis and a combination thereof. Expressing methionine synthase is based on amino acid sequence selected from the group consisting of SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10, and a combination thereof. Expressing methionine synthase is based on amino acid selected from the species consisting of Arabidopsis, E. coli, Saccharo yces, and a combination thereof. 4-Hydroxyphenylacetate 3-hydroxylase is expressed via a plasmid. Caffeic acid 3-O-methyltransferase is expressed via a plasmid. Methionine synthase is expressed via a plasmid. A combination of 4-hydroxyphenylacetate 3-hydroxylase, caffeic acid 3-O-methyltransferase, and methionine synthase is linked by a peptide linker.

[00051] Another aspect of the current disclosure is a method of making vanillin comprising the following: expressing 4-hydroxyphenylacetate 3-hyrdroxylase in a mixture; expressing caffeic acid 3-O-methyltransferase in the mixture; expressing methionine synthase in the mixture; expressing feruloyl-CoA synthetase in the mixture; expressing enoyl-CoA hydratase/aldolase in the mixture; feeding p-coumaric acid to the mixture; and collecting vanillin. The method further comprises expressing each step singularly or collectively by in vitro translation. A further disclosure comprises expressing each step singularly or collectively in a cellular system. The cellular system is selected from the group consisting of bacteria, yeast, and a combination thereof. The 4-hydroxyphenylacetate 3-hyrdroxylase, the caffeic acid 3-O-methyltransferase, or the methionine synthase can be purified as recombinant proteins.

[00052] In another disclosure, the method of making vanillin wherein expressing feruloyl-CoA synthetase in the mixture and expressing enoyl-CoA hydratase/aldolase in the mixture further comprises expressing each step singularly or collectively by in vitro translation. In a separate disclosure, the method of making vanillin wherein expressing feruloyl-CoA synthetase in the mixture and expressing enoyl-CoA hydratase/aldolase in the mixture further comprises expressing each step singularly or collectively in a cellular system. The cellular system is selected from the group consisting of bacteria, yeast, and a combination thereof. The feruloyl-CoA synthetase or the enoyl-CoA hydratase/aldoase is purified as recombinant proteins - either singularly or collectively. In a further disclosure, expressing feruloyl-CoA synthetase in the mixture and expressing enoyl-CoA hydratase/aldolase in the mixture further comprises expressing in a species selected from the genus consisting of Pseudomonas, Amycolatopsis, Sphingomonas paucimobilis, Rhodococciis, Streptomyces, and a combination thereof. Expressing feruloyl-CoA synthetase in the mixture; and expressing enoyl-CoA hydratase/aldolase in the mixture further comprises expressing in a microorganism species can be selected from the group consisting of Pseudomonas sp. HR 199, Amycolatopsis sp. ATCC 39116, Amycolatopsis sp. HR167, Sphingomonas paiicimobilis SYK-6, Pseudomonas fliiorescens AN 103, Streptomyces seotonii, Streptomyces sannanensis, Amycolalopsis sp. strain (Zhp06) and a combination thereof.

[00053] Another aspect of the current disclosure is a method of making vanillin further comprising expressing a vanillin synthase. The vanillin synthase is expressed by in vitro translation. The vanillin synthase is expressed in a cellular system. The cellular system is selected from the group consisting of bacteria, yeast, and a combination thereof. The vanillin synthase is purified as a recombinant protein. [00054] Another aspect of the current disclosure is a method of making vanillin, wherein the vanillin has a "less negative" or greater 8¹³C compared with vanillin derived from C₃ plants includes feeding p-coumaric acid derived from a C₄ plant. The C4 plant is maize. [00055] Another aspect of the current disclosure is a method of making vanillin comprising the following: providing ferulic acid in a mixture; expressing feruloyl-CoA synthetase in the mixture; expressing enoyl-CoA hydratase/aldolase in the mixture; and collecting vanillin. In a further aspect of the disclosure, expressing feruloyl-CoA synthetase in the mixture and expressing enoyl-CoA hydratase/aldolase in the mixture further comprises expressing in a species selected from the genus consisting of Pseiidomonas, Amycolatopsis, Sphingomonas paiicimobilis, Rhodococcus, Streptomyces, and a combination thereof. In another disclosure, expressing feruloyl-CoA synthetase in the mixture and expressing enoyl- CoA hydratase/aldolase in the mixture further comprises expressing in a microorganism species selected from the group consisting of Pseiidomonas sp. HR 199, Amycolatopsis sp. ATCC 39116, Amycolatopsis sp. HR167, Sphingomonas paiicimobilis SYK-6, Pseiidomonas fl orescens AN103, Streptomyces seotonii, Streptomyces sannanensis, Amycolalopsis sp. strain (Zhp06) and a combination thereof. [00056] Another aspect of the current disclosure is a ferulic acid with less negative or greater 6¹³C compared with ferulic acid derived from C₃ plants. This is possible when C₄ plants are used as the source of p-coumaric acid. The 8¹³C for the ferulic acid can be less negative or greater than -30%. The 5¹³C for the ferulic acid can be less negative or greater than -020%. The 8¹³C for the ferulic acid is between about -10% and about -20%. In another disclosure, the 5¹³C is between about -12% and about -17%.

[00057] Another aspect of the current disclosure is a vanillin with less negative or greater 5¹³C compared with vanillin derived from C₃ plants. This is possible when C₄ plants are used as the source of p-coumaric acid or ferulic acid. The 5¹³C for the vanillin can be less negative or greater than -30%. The 8¹³C for vanillin can be less negative or greater than - 20%. The 8^liC is between about -10% and about -20%. In another disclosure, the 6^1JC is between about -12% and about -17%.

[00058] In one aspect, the subject technology relates to the extraction of hydroxy cinnamic acids from plant materials. Among plant materials, maize cob was found to contain abundant hydroxycinnamic acids in its cell wall with release of 2.8% (w/w) p- coumaric acid and 1.6% (w/w) ferulic acid after digestion with sodium hydroxide solution. The released hydroxycinnamic acids were isolated with resin absorption technology (e.g., Wuxi Kangzhen).

[00059] The subject technology is based on, in part, the recognition that p-coumaric acid can be converted into ferulic acid by two enzyme-catalyzed reactions, hydroxylation and methylation, with caffeic acid as an intermediate metabolite (Figure 1). The ferulic acid made can be used as substrate for the making of vanillin. These two reactions in making ferulic acid are facilitated by two types of enzymes: 4-hydroxyphenylacetate 3-hydrolase (4HPA3H) that catalyzes 3 -hydroxylation of p-coumaric acid, and caffeic acid 3-0- methyltranferase (COMT) that catalyzes the methylation of caffeic acid. An aspect of the current disclosure is the establishment of bioconversion reactions in E. coli cells: to express these enzymes in E. coli or other cellular systems (e.g., yeast) and to culture the cells in a biofermentator to convert p-coumaric acid to ferulic acid.

[00060] The 4-hydroxyphenylacetate 3-hydroxylase complex is comprised of two proteins hpaB and hpaC, both of which can be derived from E. coli. Their amino acid sequences consist of SEQ ID No: 1 and SEQ ID No: 2. An aspect of the current disclosure is the exogenous expression of hpaB and hpaC in bacteria or other microorganisms or cellular systems to facilitate the conversion of p-coumaric acid to caffeic acid.

[00061] Caffeic acid 3-O-methyltransferase is derived from an organism selected from the group consisting of an Arabidopsis, a Medicago truncatala, a Popultis trichocarpa, and a Catharansiis roseas. Variants of the amino acid sequence consists of SEQ ID No: 3, 4, 5, 6, and 7. An aspect of the current disclosure is the exogenous expression of caffeic acid 3-O- methyltransferase in bacteria or other microorganisms or cellular systems to mediate the conversion of caffeic acid to ferulic acid.

[00062] In another aspect, methionine synthase (MS) is derived from an organism selected from the group consisting of an Arabidopsis, an E. coli, and a yeast. Their amino acid sequences consist of SEQ ID No: 8, SEQ ID NO: 9 and SEQ ID No: 10. The presence of MS greatly increases the conversion of caffeic acid to ferulic acid. Accordingly, an aspect of the current disclosure is the exogenous expression of MS along with COMT to enhance the methylation of caffeic acid converting it to ferulic acid. Applicants note that the presence of MT aids in increasing the production of ferulic acid. Its absence would still allow for the production of ferulic acid. [00063] The aforementioned enzymes could be linked together. The link of the fusion protein described herein may be a peptide linker, e.g., a peptide linker compromising of 2-15 amino acids. Exemplary linkers include those essentially compromising of glycine-serine- glycine. Also provided are nucleic acids encoding the fusion proteins described herein, host cells comprising the fusion proteins herein, and host cells comprising the nucleic acids described herein. An aspect of the current disclosure is the expression of fusion protein comprising the aforementioned enzymes linked by a peptide linker.

Example 1. Bioconversion of p-coumaric acid to caffeic acid in E. coli hosting exogenous expression of 4HPA3H.

[00064] The first step in the bioconversion of ferulic acid from p-coumaric acid involves the conversion of p-coumaric acid (pCA) to caffeic acid (CA). This initial reaction requires the hydroxylation of pCA at C-3 position, which is catalyzed by plant p-coumarate 3-hydrolase (C3H), a plant specific cytochrome p450 dependent monooxygenase. However, because of its instability and membrane bound property, C3H is not suitable for the conversion of pCA to CA in a prokaryotic system for industrial use. Here, applicants took advantage of the broad substrate preference of a native E. coli hydroxylase, 4- hydroxylphenylacetate 3 -hydroxylase (4HPA3H), which catalyzes the hydroxylation of some phenyl compounds at C3 position. This enzyme was used to convert p-coumaric acid to caffeic acid in a bioconversion pathway (Lin and Yan 2012 Microbial Cell Factories 11 : 42- 50). Although an aspect of the disclosure is to provide for the expression via a cellular system, such as microbial expression of the enzyme, and to allow enzymatic action through the cellular, an alternative aspect is to purify the enzymes as recombinant proteins and allow enzymatic action outside the cellular system. In another aspect, the enzymes could be expressed by in vitro translation and the enzymes are then purified to exert their enzymatic effect outside the cell.

Materials and Methods

Bacterial strains, plasmids and culture condition. [00065] E. coli TOP 10 and BL21 (DE3), for cloning and recombinant protein expression, were purchased from Invitrogen. Plasmid pETDuet-1 were purchased from Novagen was used for cloning and recombinant protein expression purposes. DNA manipulation.

[00066] All DNA manipulations were performed according to standard procedures.

Restriction enzymes, T4 DNA ligase were purchased from New England Biolabs. All PCR reactions were performed with New England Biolabs' Phusion PCR system. Construction of plasmid.

[00067] Genomic DNA of E. coli strain BL21 (DE3) was extracted using Bacterial

DNA extraction kit. HpaB and HpaC genes for the complex 4HPA3H were amplified from the E. coli genomic DNA with PCR, with introduction of Nde I site at the 5 '-end and Xho I site at the end of 3 '-end. The primers used were forward primer HpaBC_F (5'- GGGAATTCCATATGAAACCAGAAGATTTCCGCG) and reverse primer HpaBC R (5'- CTCGAGCGGTTAAATCGCAGCTTCCATTTCCAGC). The PCR product digested with Nde I and Xho I was ligated with plasmid pETDuet-1 digested with the same enzymes and transformed into E. coli DH5a. The plasmid, plsd-pETDuet-HpaBC, extracted from the colony with the positive insert and confirmed by sequencing was transformed into BL21 (DE3) for protein expression and bioconversion of pCA.

Bioconversion.

[00068] E. coli BL21(DE3) containing plsd-pETDuet-HpaBC was grown in the specified medium described in the Results with 50 mg/L ampicillin to OD600=0.6 in a shaker at 37°C, and then changed to 30°C with addition of lactose to final concentration of 1.5% (w/v) to induce the expression of HpaBC. pCA dissolved in 0.2 M NaOH solution was added to the culture to 0.5 gram/L after 2 hour induction with lactose. The culture was kept shaking under the same culture condition, and samples were taken at intervals for HPLC analysis. HPLC analysis.

[00069] The HPLC analysis of hyroxylcinnamic acids 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 μιη; 150 by 2.1 mm) with a gradient of 0.15% (vol/vol) acetic acid (eluent A) and acetonitrile (eluent B) in a range of 10 to 40% (vol/vol) eluent 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

[00070] Applicants tested the conversion of p-coumaric acid to caffeic with the E. coll strain in two media, LB and M9A (14g/L KH₂P0₄, 16g/L K₂HP0₄, lg/L Na₃ citrate-2H₂0, 7.5g/L (NH₄)₂S0₄, 0.25g/L MgS0₄-7H₂0, 0.015g/L CaCl₂-2H₂0, 0.5% (w/v) glucose, pH 7.0) in the flasks. As shown in Figure 2, pCA could be converted into CA in both media, but with significant difference in the conversion activity. The conversion is lower in LB medium and some other uncharacterized compounds appeared in the culture, indicating the nonspecific conversion, or the changes of pCA and CA to other unknown intermediates. The accumulation of CA in LB medium was very little (less than 20 mg/L in 24 hours) (Figure 2 upper panel). However, the conversion of pCA to CA in the mineral medium, M9A, was faster, leading to higher yield of caffeic acid production of 2.23g/L within 24 hours (Figure 2 low panel, and Figure 3).

Example 2. Bioconversion of caffeic acid to ferulic acid in E. coli hosting plant caffeic acid 3-O-methyltransferase (COMT) and methionine synthase (MS).

[00071] The second step in the bioconversion of ferulic acid from p-coumaric acid involves the conversion of caffeic acid (CA) to ferulic acid. This reaction catalyzed by caffeic acid 3-O-methyltransferase (COMT) requires the methylation of the intermediate metabolite: caffeic acid. The co-expression of methionine synthase increases the conversion to ferulic acid.

Materials and Methods

Bacterial strains, plasmids and culture condition.

[00072] E. coli 10G and Hi-Control BL21 (DE3), and pETite N-His SUMO Kan Vector, for cloning and recombinant protein expression, were purchased from Lucigen Inc (Madison, WI).

RNA extraction and cDNA synthesis.

[00073] COMT and MS were cloned from various plant species. Plant total RNA was extracted from Arabidopsis thaliana (ecotype Columbia-0), Medicago truncatula (ecotype A 17), Catharanthus roseus and Populus trichocarpa with Trizol Plus RNA Purification Kit (Invitrogen Inc). The synthesis of cDNA was carried out with Im Prom-II™ Reverse Transcription System from Promega Inc. following the manufacturer's manual. The genes were amplified from the synthesized cDNA with New England Biolabs' Phusion PCR Kit with the primers listed in Table 1. COMT genes were amplified from all the plant species listed above, and MS genes were from stem tissues of Arabidopsis thaliana and Medicago truncatula.

[00074] The PCR products were cloned into pETite N-His SUMO Kan Vector (Lucigen Inc) according to the manufacturer's manual. The resultant plasmids with the right insert confirmed by sequencing, namely plsd-Sumo-AtCOMT, plsd-Sumo-CrCOMT, plsd- Sumo-MtCOMT, plsd-Sumo-PtCOMTl and plsd-Sumo-PtCOMT2, were transformed into Hi-Control BL21(DE3) for heterogeneous gene expression.

Table 1. Primers for cloning of COMT into SUMO vector.

Sumo_AtMS_F CGCGAACAGATTGGAGGTGCTTCACACATTGTTGGATACCCACG At MS,

Arabidopsis

Sumo_AtMS_R GTGGCGGCCGCTCTATTACTTGGCACTGGCGAGCTGGGAGCGG thaliana

Sumo_MtMS_F CGCGAACAGATTGGAGGTGCTTCTCACATTGTTGGATACC MtMS, Medicago

Sumo_MtMS_R GTGGCGGCCG CTCTATTACTTG G CA AG CTCATTG CG G ATTTG truncatula

Fusion of COMT and MS with PCR approach.

[00075] COMTs from Arabidopsis and Medicago were fused with MS from

Arabidopsis and Medicago via two-round PCR strategy yielding fusion gene of AtCOMT: :MtMS, MtMS:: AtCOMT, MtMS: :MtCOMT, MtCOMT: :MtMS, AtCOMT: :AtMS, and AtMS:: AtCOMT, respectively. The nomenclature of the fusion gene comes the name of the gene at the 5 '-end followed by the gene at the 3 '-end, with a linker of 5'-GGTTCGGGT-3'. The primers used for generating the fusion genes were listed in Table 1 and Table 2. The details for fusing two genes in this study are described below with AtCOMT: MtMS as an example: Firstly the stop codon of AtCOMT and the start codon of MtMS were removed, and the linker between AtCOMT and MtMS was introduced, which was achieved by two PCR. One PCR was carried out with forward primer of Sumo- AtCOMT F and reverse primer of AtCOMT-MtMS-Mid_R and plasmid of Sumo-AtCOMT as the template; another PCR was performed with a pair of primers, AtCOMT-MtMS-Mid_F and Sumo-MtMS_R, and plasmid Sumo-MtMS as the template. Each PCR product was purified and served as the templates for the second round PCR, using a pair of primer, Sumo- AtCOMT_F and Sumo-MtMS_R. The final PCR product with the right size in agarose gel was purified and cloned into pETite N-His SUMO Kan Vector (Lucigen Inc, WI). The resultant plasmid, plsd-Sumo-AtCOMT: MtMS, confirmed by sequencing, was transformed into Hi-Control E. coli BL21(DE3). All the other fusion genes were generated with the same strategy with the primers and templates listed in Table 3. Table 2. Primers for generating fusion genes.

Table 3. The templates and primers used for generating the fusion genes

Reverse Primer MtMS-MtCO T- ld_R Sumo-MtCOMT_R Sumo-MtCOMT_R

Product Up_MtMS::MtCOMT Dn_MtMS::MtCOMT MtMS::MtCOMT

Up_MtCOMT::MtMS

Template plsd__Sumo-MtCOIv1T plsd_Sumo-MtMS +

Dn_MtCOMT::MtMS

MtCOMT::MtMS

Forward Primer Sumo-MtCOMT_F MtCOMT-MtMS-Mid_F Sumo-MtCOMT_F

Reverse Primer MtCOMT-Mtlv1S-Mid_R Sumo-MtMS_R Sumo-MtMS_R

Product Up_MtCOMT::lv1tMS Dn_MtCOMT::MtMS MtCOMT::lvltMS

Up_MtMS::AtCOMT

Template plsd_Sumo-MtMS plsd_Sumo-AtCOMT +

Dn_MtMS::AtCOMT

MtMS::AtCOMT

Forward Primer Sumo-MtMS_F MtMS-AtCOMt-Mid_F Sumo-MtlvlS_F

Reverse Primer MtMS-AtCOMT-lv1id_R Sumo-AtCOMT_R Sumo-AtCOMT_R

Product Up_MtMS::AtCOMT Dn_MtMS::AtCOMT MtMS::AtCOMT

Up_AtCOMT::MtMS

Template plsd_Sumo-AtCOMT plsd_Sumo-MtMS +

Dn_AtCOMT::MtMS

AtCOMT::MtMS

Forward Primer Sumo-AtCOMT_F AtCOMT-MtMS-Mid_F Sumo-AtCOMT_F

Reverse Primer AtCOMT-MtMS-lv1id_R Sumo-MtMS_R Sumo-MtMS_R

Product Up_AtCOMT::MtMS Dn_AtCOMT::MtMS AtCOMT::MtMS

Up_AtMS::AtCOMT

Template plsd_Sumo-AtMS plsd_Sumo-AtCOMT +

AtMS::AtCOMT

Dn_AtMS::AtCOMT

Forward Primer Sumo-AtMS_F AtMS-AtCOMt-Mid_F Sumo-AtMS_F Reverse Primer AtMS-AtCO T-Mid_R Sumo-AtCOMT_R Sumo-AtCOMT_R

Product Up_AtMS::AtCOMT Dn_AtMS::AtCOMT AtMS::AtCOMT

Up_AtCOMT::At S

Template plsd_Sumo-AtCOMT plsd_Sumo-AtMS +

Dn_AtCOMT::AtMS

AtCOMT::AtMS

Forward Primer Sumo-AtCOMT_F AtCOMT-AtMS-Mid_F Sumo-AtCOMT_F

Reverse Primer AtCOMT-AtMS-Mid_R Sumo-AtMS_R Sumo-AtMS_R

Product Up_AtCOMT::AtMS Dn_AtCOMT::AtMS AtCOMT: :AtMS

Bioconversion.

[00076] Hi-Control E. coli BL21(DE3) containing pETDuet-HpaBC was grown in the specified medium described in the Results with 50 μg L ampicillin to OD600=0.6 in a shaker at 37°C, and then changed to 30°C with addition of lactose to final concentration of 1.5% (w/v) to induce the expression of HpaBC. p-Coumaric acid dissolved in 0.2 M NaOH solution was added to the culture to 0.5 gram /L after 2 hour induction with lactose. The culture was kept shaking under the same culture condition, and samples were taken at interval for HPLC analysis.

Results

Screening of plant COMT with high conversion efficiency of caffeic acid to ferulic acid.

[00077] All the COMTs that applicants cloned were expressed in Hi-Control

BL21(DE3) and the recombinant proteins were purified to homogeneity (Figure 4). The activity measurement with the purified recombinant proteins demonstrates that AtCOMT showed highest activity with caffeic acid as a substrate (Figure 5). [00078] The in vivo bioconversion test result is consistent with the in vitro enzyme activity measurements. The conversion of caffeic acid to ferulic acid by the E. coli strains containing plant COMTs was tested in the flasks. All the strains showed the production of ferulic acid with the feeding of caffeic acid in the cell culture, in which the strain containing AtCOMT showed the highest conversion rate with the yield of 0.079 g L in 24 hours. The strain harboring MtCOMT showed the second highest production of ferulic acid (0.062g L) under the same conversion condition (Figure 5). Based on these results, applicants focused on AtCOMT and MtCOMT in further study. These results also indicate that ferulic acid can be made in an in vitro manner outside the cell using recombinant enzymes or in vitro translated enzymes.

The bioconversion of caffeic acid to ferulic acid with fusion protein of COMT and MS.

[00079] With the PCR amplification strategy, AtCOMT and MtCOMT were fused with MtMS and AtMS, respectively, with a link of Gly-Ser-Gly between COMT and MS. The fusion genes were expressed in Hi-Control BL21(DE3), and the recombinant proteins were purified to homogeneity. COMT activity measurement in vitro showed the fusion proteins had lower activity compared to AtCOMT and MtCOMT alone based on the protein weight. However, a simple mathematic conversion of the activity on the molar basis of the protein showed similar activity between the fusion proteins and COMT genes alone, indicating the fusion of MS did not affect COMT activity.

[00080] The six strains with the fusion genes and the strains with MtCOMT and

AtCOMT were grown in M9B medium for the conversion of caffeic acid to ferulic acid. As shown in Figure 6, all the strains containing the fusion gene have higher capacity of ferulic acid production in comparison with these with single COMT gene, indicating the beneficial role of MS in COMT activity in vivo.

[00081] In order to increase the conversion efficiency of plant COMT, applicants fused MS with Arabidopsis COMT. The introduction of MS is very helpful for the conversion of caffeic acid. The E. coli strain harboring the fusion gene of MtMS::AtCOMT had dramatic increase in the conversion activity. The yield of ferulic acid in the flask reached to 0.98 g/liter. Similar result was obtained in a 2-liter fermentator (Figure 7). Example 3. Bioconversion of pCA to FA in E. coli hosting MtMS::AtCOMT and HpaBC.

[00082] p-Coumaric acid is bioconverted to ferulic acid through the expression of

HpaBC, MS and COMT.

Materials and Methods

Co-expression of MtMS : : AtCOMT and HpcBC in E. coli.

[00083] Plasmids of plsd-Sumo-MtMS:: AtCOMT and plsd-pETDuet-HpcBC were co- transformed into Hi-Control BL21(DE3) (Lucigen Inc, WI) according to standard procedure. The resulting E. coli strain with the co-expression of MtMS:: AtCOMT and HpcBC, which is confirmed with colony PCR and SDS-PAGE, were utilized for conversion of pCA to FA.

Bioconversion of pCA to FA.

[00084] Single colony of the E. coli strain was grown in 3 mL LB medium with 100 ig/mL ampicillin and 30 μg/mL kanamycin overnight at 37°C, and then the seed culture was transferred to 50 mL M9B medium with 100 ^ig/mL ampicillin and 30 μg/mL kanamycin. The culture was kept shaking at 200 rpm at 37°C until OD600 reached to 0.6. Lactose was added to final concentration of 1.5% (w/v) and the culture was transferred to 30°C at the same shaking speed. pCA dissolved in 0.2 M NaOH was added to the culture and sample was taken for HPLC analysis at the specific time point.

Results

[00085] As shown in Figure 8, pCA fed into the culture of E. coli with the co- expression of HpaBC and MtMS::AtQMT was converted into CA and FA in the flask. HPLC analysis indicates the conversion of 1 g L pCA was complete in flask within 24 h (Figure 9). A yield of 0.75g L FA and 0.20 g/L of CA was obtained under our condition within 72 hours. [00086] The main components in Applicants' maize cob extract are hydroxycinnamic acids including pCA and FA, with the ratio around 3: 1. The E. coli harboring MtMS::AtCOMT and E. coli HpaBC could reduce pCA level in this extract and correspondingly increase CA and FA concentrations in the cell culture in the flask under our conversion condition, indicating most p-coumaric acid in the maize cob extract is converted into ferulic acid by the engineered E. coli strain. About 0.82 g/L of ferulic acid and 0.11 g/L of caffeic acid were obtained with 1 gram of the hydroxycinnamic acid extract from maize cob with this strain after 72 hour bioconversion under our culture condition (Figure 10).

Example 4 Saturation mutagenesis of COMT.

[00087] As the conventional mutagenesis did not improve COMT activity, applicants applied site directed saturation mutagenesis. Saturation mutagenesis allow change one amino acid to other alternative 19 amino acid residues. Applicants performed saturation mutagenesis at the site 24, 28-29, 124, 127, 130-131, 133, 136, 162-163, 166, 172, 176, 180, 183, 266, 297, 316, 319-320, 323-324 and 354 of poplar COMT by following the modified QuickChange site-directed mutagenesis strategy (Stratagene, CA) using NNK degenerate primers (N represents the mixture of A, T, G, C, and K for G/T). The codon NNK has 32- fold degeneracy and encodes all 20 amino acids without rare codons. The PCR mixture (25 μΐ) composed of Phusion HF buffer containing 60ng COMT DNA template, 200 μΜ dNTPS, 0.5 μΜ forward primers, 0.5μΜ reverse primers, 5% DMSO and 0.3 μΐ polymerase. The PCR was performed by denaturing at 98°C for 20 sec, annealing at 58°C for 30 sec and followed by elongation at 72C for 3 min 30 sec for 25 cycle. The QuikChange PCR products were examined by agarose gel electrophoresis and then 15 μΐ of PCR products were digested with 1 μΐ Dpnl (New England Biolabs) at 37°C for 4 hrs to remove the template plasmid. Aliquot of (2 μΐ) digestive products was added to 50 μΐ BL21-Gold (DE3) competent cells (Stratagene, CA), keep on ice for 30 min. After that, heat shock was done at 42°C for 20 sec, keep on ice for 2 min and then 500 μΐ SOC medium was added and grow the cells at 37°C for 1 hr. The cells were centrifuged at 5000 rpm for min, 450 μΐ supernatant was discarded and cells were suspended with the rest of the SOC medium and were inoculated on Luria-Bertani (LB) agar plates containing kanamycin (50 μg/ml). Applicants isolated the plasmid and DNA sequencing was done to confirm the mutant. Applicants confirmed the quality of the library by DNA sequencing. The library covers 84% of mutagenesis (16 mutants out of 19). To cover 100% (19 out of 19 mutant), applicants screened 150 mutants for each site. Example 5

Screening of COMT mutant library.

[00088] Highly sensitive HPLC was used for screening protein library. The transformants were inoculated in 96-well plates (NUNC, Roskilde, Denmark) containing 100 μΐ LB broth per well and incubated at 37°C, overnight. The cultures were then mixed with equal amount of 50% glycerol and stored at -80°C as the master plate. An aliquot of 10 μΐ culture was inoculated in 2 ml deep-well plates (USA scientific) with 1 ml TB broth per well and incubated at 37°C until the OD₆₀o reached 1.0. The cultures were then induced by 0.2 mM IPTG and incubated at 16 °C for 20 hours with shaking at 250 rpm. [00089] Cells were harvested by centrifugation at 4500 rpm for 20 mins and stored at -

80°C until use. The harvested cell cultures were lysed with 60 μΐ Bugbuster solution per well (Novagen, Darmstadt, Germany). Applicants used the lysate directly in a 96-well plate for screening enzymatic activity. Screening was performed in a polypropylene microplate (Bio- Rad, Hercules, USA) containing 100 μΐ of the reaction in each well, 200 μΜ caffeic acid substrates, and 400 μΜ Sadenosyl-Z-methionine (SAM) and 10 μΐ of lysate. The reaction mixture was incubated at 30°C for 5 mins. Applicants extracted the reaction product with 200 μΐ ethyl acetate and moved the extracts into a new microplate. The extracted product was analyzed by HPLC using a reverse-phase CI 8 column (4.6 x 150-mm, Dionex). The samples were resolved in 0.15% acetic acid (A) with an increasing concentration gradient of acetonitrile containing 0.15% acetic acid (B) for 2 min, 5%; then to 8 min, 50%; then to 10 min, 5%; and 11 min, 5%, at a flow rate of 0.6 ml/min. UV absorption was monitored at 280-, and 320-mn with a multiple wavelength photodiode-array detector.

[00090] Applicants screened 600 clones as methods described previously and selected 45 clones that showed higher activity for rescreening. Applicants have measured both the product ferulic acid and the remaining substrate caffeic acid. After rescreening, applicants found 5 clones (A8, A9, Al 1, Bl and B2) out of 45 that show higher activity compared with COMT wild type (Figure 12). Applicants have isolated plasmid and performed DNA sequence. These 5 clones represent 3 different mutants (A9: COMT-L136Y; A8, Al l and B 1 : COMT-A162P; and B2:COMT-A162T). [00091] In another disclosure, applicants purified protein by affinity chromatography and analyzed kinetic parameters using purified protein. Applicants found one single mutant L136Y that produced higher production of ferulic acid compared with that of wild type (Figure 13). Specifically, the L136Y mutant exhibited an catalytic efficiency of 14685 M^"'S^" ^l, while wild type COMT had a catalytic efficiency of 3858 JVT'S^"1. TWO other mutations, A162P and A162T, had catalytic efficiencies of 4165 M^"1 S^"1 and 3658 M^"'S^"', respectively.

[00092] Leucine 136 is shown to be located in the methyl-binding pocket of caffeic acid 3-O-methyltransferase (Figure 11). This result is consistent with the mutant reported for alfalfa COMT (Zubieta et al, 2002, Plant cell, 14: 1265-1277). Mutagenesis at different sites showed additive effect on protein evolution of methyltransferase (Bhuiya et al, 2010, Journal of Biological Chemistry, 285: 277-285). Mutagenesis at other sites using COMT-L136Y as a template may further increase the production of ferulic acid - maybe in combination with mutation at A 162 or another amino acid in the methyl binding pocket

Example 6

Transformation of ferulic acid to vanillin in fermentor.

[00093] Microbial conversion of ferulic acid to vanillin was carried out. Applicants used the strain Amycolalopsis sp. strain (Zhp06) as disclosed in Chinese Patent CN102321563 granted on April 3, 2013, and US Patent Application Publication No. US 2013/01 15667, entitled "Amycolatopsis Sp. Strain and Methods of Using the Same for Vanillin Production," which are both incorporated by reference herein in their entirety. The seed medium used is Tryptic Soy Broth (Soybean-Casein Digest Medium, TSB) and fermentor medium components are: Yeast extract, 8 g/L; glucose, 30 g/L; MgS0₄-7H₂0, 0.8g/L; Na₂HP0₄-7H₂0, 7.5g/L; KH₂P0₄, 1.0 g/L; and 0.2 niL/L antifoam. The media were autoclaved at 120°C for 15 and 30 minutes respectively. [00094] The Amycolalopsis sp. strain (Zhp06) colony was inoculated into TSB and shakes at 30°C until the late exponential phase, which was used as seed culture for fermentor. The seed culture was sub-cultured into 2L fermentor at 5%. The fermentor (New Brunswick Scientific Bioflo-115 3.0L ) was controlled at 30°C to maintain DO above 20% with rpm and aeration. pH was controlled at 7 during the growth. Ferulic acid was freshly dissolved in 1 M Sodium hydroxide at 100 g/L and then sterilized by filtration. Ferulic acid stock solution was fed into fermentor at about 10% when FV strain reached early stationary phase, which is usually 10-20 hours after inoculation. Samples were analyzed by HPLC using the Dionex UltiMate® 3000 HPLC system, and 7.53 g/L vanillin obtained from 12 g/L ferulic acid added, with only trace amount of vanillin alcohol and guanicol accumulated (Figure 14). The molar yield of vanillin is 79.8%. Similarly, 7.94 g/L vanillin obtained from 12.5 g/L ferulic acid added, which gave a molar yield of 81.3%.

Example 7

Transformation of ferulic acid to vanillin in fermentor

[00095] Same as above, another Amycolalopsis sp. strain (Zhp06) colony was inoculated into TSB and shaken at 30°C until the late exponential phase, and then sub- cultured into 2L fermentor at 5%. The fermentor (New Brunswick Scientific Biofio~115 3.0L ) was controlled at 30°C to maintain DO above 20% with rpm and aeration. pH was controlled at 7 during the growth. Freshly made ferulic acid stock solution in 1 M Sodium hydroxide was fed into fermentor at about 10% during early stationary phase and concentration of ferulic acid, vanillin and vanillin alcohol was followed by HPLC assay. When ferulic acid depleted from the culture, ferulic acid solution was fed to fermentor to maintain DO less than 20%, in order to continue the biotransformation. 13.4 g/L vanillin obtained from 21.6 g L ferulic acid added. The molar yield of vanillin is 79.2%.

Example 8

Transformation of ferulic acid to vanillin in fermentor

[00096] Same as above, another Amycolalopsis sp. strain (Zhp06) colony was inoculated into TSB and shaken at 30°C until the late exponential phase, and then sub- cultured into 2L fermentor at 5%. The fermentor (New Brunswick Scientific Bioflo-115 3.0L ) was controlled at 30°C to maintain DO above 20% with rpm and aeration. pH was controlled at 7 during the growth. Freshly made ferulic acid stock solution in 1 M Sodium hydroxide was fed into fermentor at about 10% during early stationary phase and concentration of ferulic acid, vanillin and vanillin alcohol was followed by HPLC assay. When ferulic acid depleted from the culture, new batch of ferulic acid solution was fed to fermentor to continue the biotransformation. 1 1.5 g/L vanillin obtained from 19.2 g L ferulic acid added. The molar yield of vanillin is 76.5%.

Example 9

Isotopic analysis of vanillin obtained through fermentation, and the ferulic acid substrate used [00097] Different sources of ferulic acid, along with several batch of bio-transformed vanillin were analyzed for ¹³C/¹²C (5¹³C) analysis by EA-IRMS at Isotech Laboratories, Inc. (Isotech Laboratories, Inc. 1308 Parkland Court | Champaign Illinois 61821) (Table 4). The ratio for ¹³C/¹²C is reported as 5¹³C. The definition is, in per mil:

where the standard is an established reference material. [00098] The standard established for carbon-13 work was the Pee Dee Belemnite or

(PDB) and was based on a Cretaceous marine fossil, Belemnitella americana, which was from the Pee Dee Formation in South Carolina. This material had an anomalously high ¹³C:^!2C ratio (0.01 12372), and was established as 5¹³C value of zero. Use of this standard gives most natural material a negative 8¹³C. The standards are used for verifying the accuracy of mass spectroscopy; as isotope studies became more common, the demand for the standard exhausted the supply. Other standards, including one Icnown as VPDB, for Vienna PDB, have replaced the original.

[00099] C₃ and C₄ plants have different 6¹³C signatures, allowing C₄ grasses to be detected in 5¹³C . C₄ plants have a 5¹³C of -16 to -10 %o, C₃ plants have a 8¹³C of -33 to -24%o. Thus, 5¹³C for C₄ plants would generally be "less negative" or "greater" in value compared with the 5¹³C for C₃ plants.

Table 4 Isotopic analysis results

Lab No. Name Date Batch number

313371 1 FV 10/31/2012 -16.1 120423

313372 2 FV 10/31/2012 -15.9 120903

313373 3 FV 10/31/2012 -34.4 120406 Rice bran originated

313374 4 FV 10/31/2012 -32.8 120216 FA

313375 FA 1 10/31/2012 -13.3 120322

313376 FA 2 10/31/2012 -13.1 120528

313377 FA 3 10/31/2012 -13.5 120905-1 [000100] Thus, vanillin, ferulic acid and caffeic acid made from the current bioconversion process would have unique isotope distribution if the starting materials are derived from C₄ plants, such as maize cobs. They would have 5¹³C in the -16 to -10 o range compared to vanillin, ferulic acid and caffeic acid made from general industrial process using C₃ plants, which would be in the -33 to -24%o range. In Table 4, different batches of vanillin (1 and 2 FV) and ferulic acid (FA 1, 2, and 3) made from maize cob starting material were analyzed for their 5¹³C signatures and compared with the 5¹³C signatures of vanillin (3 and 4 FV) made from rice bran, a C₃ plant). Accordingly, vanillin and other products made from ferulic acid and caffeic acid derived from C₄ plants utilizing this present process can have a less negative or greater 5¹³C compared with the 5¹³C of vanillin and other products made from C₃ plants. This unique signature can serve as a way to identify products made from the bioconversion pathway as opposed to current industrial process. By "less negative" the value is closer to 0 compared other negative values, which would make the "less negative" value greater than other values that are "more negative".

Example 10

Transformation of ferulic acid to vanillin in fermentor.

[000101] In another disclosure, Amycolalopsis sp. strain (Zhp06) (previously named Streptomyces viridospor s) seed medium used is Tryptic Soy Broth (Soybean-Casein Digest Medium, TSB) and fermenter medium components are: Yeast extract, 8 g/L; glucose, 30 g/L; MgS04'7H20, 0.8g/L; Na2HP04^«7H20, 7.5g/L; KH2P04, 1.0 g/L; and 0.2 mL/L antifoam. The medium were autoclaved at 120°C for 15 and 30 minutes, respectively. The method for this conversion is disclosed in Chinese Patent Application CN102321563 and US Patent Application Publication No. US 2013/0115667, which are both incorporated in their entirety in this application.

[000102] Single Amycolalopsis sp. strain (Zhp06) colony was inoculated into TSB and shaken at 30°C until the late exponential phase, and then sub-cultured into 2 L fermentor at 5%. The fermentor (New Brunswick Scientific Bioflo-115 3.0L) was controlled at 30°C to maintain DO above 20% with rpm and aeration. pH was controlled at 7 during the growth. Freshly made FA stock solution in 1 M NaOH was fed into fermentor at about 10% during early stationary phase and concentrations of FA, vanillin and vanillin alcohol were produced as shown by HPLC assay. When FA depleted from the culture, new batch of FA solution was fed to fermentor to continue the biotransformation (Figure 16).

Bioconversion of ferulic acid to vanillin.

[000103] The purified FA was used as a substrate for vanillin production. Our result showed a yield of 13.4 g/L vanillin was obtained from 21.6 g/L FA, corresponding to a molar yield of vanillin at 79.2% (Figure 15). The accumulation of vanillin is more or less linear over the fermentation period. However, applicants observed rapid FA reduction at the first 24 hours, suggesting S. viridosporus converts FA into different intermediates before being converted to vanillin.

[000104] Again, the ferulic acid and vanillin made from the current bioconversion process using corn extracts (or other C₄ plants) as initial starting materials would have "less negative" or greater 8¹³C compared with ferulic acid and vanillin made from C₃ plants (e.g., rice plants), which is the general industrial process for making ferulic acid and vanillin (See Table 4). The difference in 8¹³C would serve as a way to identify products made by this bioconversion process. Moreover, the ability to use C₄ plants as starting material in the current bioconversion process would serve to preserve nature by alleviating less reliance on tropical C₃ plants. Enzyme Amino Acid Sequences.

[000105] In certain disclosures, the 4-hydroxyphenylacetate 3 -hydroxylase complex with two proteins hpaB and hpaC, was derived from E. coli. Their amino acid sequences consist of SEQ ID No: 1 and SEQ ID No: 2.

[000106] SEQ ID No: 1. Amino sequence of hpaB

MKPEDFRASTQRPFTGEEYLKSLQDGREIYIYGERVKDVTTHPAFRNAAASVAQLYDALHKP EMQDSLC NTDTGSGGYTHKFFRVAKSADDLRHERDAIAEWSRLSYG MGRTPDYKAAFGCA LGGTPGFYGQFEQNARNWYTRIQETGLYFNHAIVNPPIDRHLPTDKVKDVYIKLEKETDAGI IVSGAKVVATNSALTHYNMIGFGSAQVMGENPDFALMFVAPMDADGVKLISRASYEMVAGAT . GSPYDYPLSSRFDENDAILVMDNVLIPWENVLLYRDFDRCRRWTMEGGFARMYPLQACVRLA VKLDFITALLKKSLECTGTLEFRGVQADLGEVVAWRNTFWALSDSMCSEATPWVNGAYLPDH AALQTYRVLAPMAYAKIKNIIERNVTSGLIYLPSSARDLNNPQIDQYLAKYVRGSNGMDHVQ RIKILKLMWDAIGSEFGGRHELYEINYSGSQDEIRLQCLRQAQSSGNMDKMMAMVDRCLSEY DQNGWTVPHLHNNDDINMLDKLLK .

[000107] SEQ ID No: 2. Amino sequence of hpaC

MQLDEQRLRFRDAMASLSAAVNIITTEGDAGQCGITATAVCSVTDTPPSLMVCINANSA NP VFQGNGKLCVNVLNHEQELMARHFAGMTGMAMEERFSLSCWQKGPLAQPVLKGSLASLEGEI RDVQAIGTHLVYLVEIKNIILSAEGHGLIYFKRRFHPVMLEMEAA .

[000108] In certain disclosures, caffeic acid 3-o-methyltransferase is derived from an organism selected from the group consisting of an Arabidopsis, a Medicago truncatiila, a Popiihis trichocarpa, and a Catharansiis rose s. Their amino acid sequences consist of SEQ ID No: 3, 4, 5, 6, and 7. [000109] SEQ ID NO: 3. Amino sequence of Arabidopsis COMT

MGSTAETQLTPVQVTDDEAALFAMQLASASVLPMALKSALELDLLEIMAKNGSPMSPTEIAS KLPTKNPEAPVMLDRILRLLTSYSVLTCSNRKLSGDGVERIYGLGPVCKYLTKNEDGVSIAA LCLMNQDKVLMESWYHKDAILDGGIPFNKAYGMSAFEYHGTDPRFN VFNNGMSNHSTITMK KILETYKGFEGLTSLVDVGGGIGATLKMIVSKYPNLKGINFDLPHVIEDAPSHPGIEHVGGD MFVSVPKGDAIFMKWICHDWSDEHCVKFLKNCESLPEDGKVILAECILPETPDSSLSTKQVV HVDCIMLAHNPGGKERTEKEFEALAKASGFKGI VVCDAFVNLIELLKKL.

[000110] SEQ ID NO: 4. Amino sequence of Medicago truncatula COMT

MGSTGETQITPTHISDEEANLFAMQLASASVLPMVLKSALELDLLEIIAKAGPGAQISPIEI ASQLPTTNPEAPVMLDRILRLLACYNILTCSVRTQQDGKVQRLYGLATVAKYLVKNEDGVSI SALNLMNQDKVLMESWYHKDAVLDGGIPFNKAYGMTAFEYHGTDPRFNKVFNKGMSDHSTIT MKKILETYTGFEGLKSLVDVGGGTGAVINTIVSKYPTIKGINFDLPHVIEDAPSYPGVEHVG GDMFVSIPKADAVFMKWICHDWSDEHCLKFLKNCYEALPDNGKVIVAECILPVAPDSSLATK GVVHIDAIMLAHNPGGKERTQKEFEDLAKGAGFQGFKVHCNANTYIMEFLKKV.

[000111] SEQ ID NO: 5. Amino sequence of Catharansus roseus COMT MGSANPDNKNSMTKEEEEACLSAMRLASASVLPMVL SAIELDLLELIKKSGPGAYVSPSEL AAQLPTQNPDAPVMLDRILRLLASYSVLNCTLKDLPDGGIERLYSLAPVCKFLTKNEDGVSM AALLLMNQDKVLMESWYHLKDAVLEGGIPFNKAYGMTAFEYHGKDPRFNKVFNQGMSNHSTI IMKKILEIYQGFQGL TVVDVGGGTGATLNMIVS YPSIKGINFDLPHVIEDAPSYPGVDHV GGDMFVSVPKGDAIFMKWICHDWSDAHCLKFLKNCHEALPENGKVILAECLLPEAPDSTLST QNTVHVDVIMLAHNPGGKERTEKEFEALAKGAGFRGFIKVCCAYNSWIMELLK .

[000112] SEQ ID NO: 6. Amino sequence of poplar COMTl

MGSTGETQMTPTQVSDEEAHLFAMQLASASVLPMILKTAIELDLLEIMAKAGPGAFLSTSEI ASHLPTKNPDAPVMLDRILRLLASYSILTCSLKDLPDGKVERLYGLAPVCKFLTKNEDGVSV SPLCLMNQDKVLMESWYYLKDAILDGGIPFNKAYGMTAFEYHGTDPRFNKVFNKGMSDHSTI TMKKLLETYKGFEGLTSLVDVGGGTGAVVNTIVSKYPSIKGINFDLPHVIEDAPSYPGVEHV GGDMFVSVPKADAVFMKWICHDWSDAHCLKFLKNCYDALPENGKVILVECILPVAPDTSLAT GVVHIDVIMLAHNPGGKERTEKEFEGLA GAGFQGFEVMCCAFNTHVIEFRKN.

[000113] SEQ ID NO: 7. Amino sequence of poplar COMT2 MGSTGETQMSPAQILDEEANFAMQLISSSVLPMVLKTAIELDLLEIMAKAGPGALLSPSDIA SHLPTKNPDAPVMLDRILRLLASYSILICSLRDLPDGKVERLYGLASVCKFLTKNEDGVSVS PLCLMNQDKVLMESWYHL DAILEGGIPFNKAYGMTAFEYHGTDPRFN VFNKGMSDHS IA MKKILETYKGFEGLASLVDVGGGTGAVLSTIVSKYPSIKGINFDLPHVIADAPAFPGVENVG GDMFVSVPQADAVF KWICHDWSDEHCLRFLKNCYDALPENGKVILVECILPVAPDTSLATK GVMHVDAIMLAHNPGGKERTEKEFEGLARGAGFKGFEVMCCAFNTYVIEFRKQA.

[000114] In certain disclosures, methionine synthase is derived from an organism selected from the group consisting of an Arabidopsis, a E. coli, and a yeast. Their amino acid sequences consist of SEQ ID No: 8, SEQ ID NO: 9 and SEQ ID No: 10.

[000115] SEQ ID NO: 8. Amino sequence of Arabidopsis methionine synthase

MASHIVGYPRMGPKRELKFALESFWDGKSTAEDLQ VSADLRSSIW QMSAAGTKFIPSNTF AHYDQVLDTTA LGAVPPRYGYTGGEIGLDVYFS ARGNASVPAMEMTKWFDTNYHYIVPEL GPEVNFSYASHKAVNEYKEAKALGVDTVPVLVGPVSYLLLSKAAKGVDKSFELLSLLPKILP IYKEVITELKAAGATWIQLDEPVLVMDLEGQKLQAFTGAYAELESTLSGLNVLVETYFADIP AEAYKTLTSLKGVTAFGFDLVRGTKTLDLVKAGFPEGKYLFAGVVDGRNIWANDFAASLSTL QALEGIVGKDKLVVSTSCSLLHTAVDLINET LDDEI SWLAFAAQKVVEVNALAKALAGQ DEALFSANAAALASRRSSPRVTNEGVQKAAAALKGSDHRRATNVSARLDAQQKKLNLPILPT TTIGSFPQTVELRRVRREYKAKKVSEEDYVKAIKEEIKKVVDLQEELDIDVLVHGEPERNDM VEYFGEQLSGFAFTANGWVQSYGSRCVKPPVIYGDVSRPKAMTVFWSAMAQSMTSRPMKGML TGPVTILNWSFVRNDQPRHETCYQIALAIKDEVEDLEKGGIGVIQIDEAALREGLPLRKSEH AFYLDWAVHSFRITNCGVQDSTQIHTHMCYSHFNDIIHSIIDMDADVITIENSRSDEKLLSV FREGV YGAGIGPGVYDIHSPRIPSSEEIADRVNK LAVLEQNILWVNPDCGLKTRKYTEVK PALKN VDAAKLIRSQLASAK .

[000116] SEQ ID NO: 9. Amino sequence of E. coli methionine synthase

MTILNHTLGFPRVGLRRELKKAQESYWAGNSTREELLAVGRELRARHWDQQKQAGIDLLPVG DFAWYDHVLTTSLLLGNVPARHQNKDGSVDIDTLFRIGRGRAPTGEPAAAAEMTKWFNTNYH YMVPEFVKGQQFKLTWTQLLDEVDEALALGHKVKPVLLGPVTWLWLGKVKGEQFDRLSLLND ILPVYQQVLAELAKRGIEWVQIDEPALVLELPQAWLDAY PAYDALQGQV LLLTTYFEGVT PNLDTITALPVQGLHVDLVHGKDDVAELHKRLPSDWLLSAGLINGRNVWRADLTEKYAQIKD IVGKRDLWVASSCSLLHSPIDLSVETRLDAEVKSWFAFALQKCHELALLRDALNSGDTAALA EWSAPIQARRHSTRVHNPAVEKRLAAITAQDSQRANVYEVRAEAQRARFKLPAWPTTTIGSF PQTTEIRTLRLDFKKGNLDANNYRTGIAEHIKQAIVEQERLGLDVLVHGEAERNDMVEYFGE HLDGFVFTQNGWVQSYGSRCVKPPIVIGDISRPAPITVEWAKYAQSLTDKPVKGMLTGPVTI LCWSFPREDVSRETIAKQIALALRDEVADLEAAGIGIIQIDEPALRQGLPLRRSDWDAYLQW GVEAFRINAAAKDDTQIHTHMCYCEFNDIMDSIAALDRDVITIETSRSDMELLESFEEFDYP NEIGPGVYDIHSPNVPSVEWIEALLKKAKRIPAERLWVNPDCGLKTRGWPETRAALANMVQA AQNLRRG .

[000117] SEQ ID NO: 10. Amino sequence of methionine synthase of Saccharomyces cerevisiae

MVQSAVLGFPRIGPNRELKKATEGY NGKITVDELFKVGKDLRTQNWKLQKEAGVDIIPSND FSFYDQVLDLSLLFNVIPDRYTKYDLSPIDTLFAMGRGLQRKATETEKAVDVTALEMVKWFD SNYHYVRPTFSKTTQFKLNGQKPVDEFLEAKELGIHTRPVLLGPVSYLFLGKADKDSLDLEP LSLLEQLLPLYTEILSKLASAGATEVQIDEPVLVLDLPANAQAAIKKAYTYFGEQSNLPKIT LATYFGTVVPNLDAIKGLPVAALHVDFVRAPEQFDEVVAAIGNKQTLSVGIVDGRNIWKNDF KKSSAIVNKAIEKLGADRVVVATSSSLLHTPVDLNNETKLDAEIKGFFSFATQKLDEVVVIT KNVSGQDVAAALEANAKSVESRGKSKFIHDAAVKRRVASIDEKMSTRAAPFEQRLPEQQKVF NLPLFPTTTIGSFPQTKDIRINRN FNKGTISAEEYE FINSEIE VIRFQEEIGLDVLVHG EPERNDMVQYFGEQINGYAFTVNGWVQSYGSRYVRPPIIVGDLSRPKAMSVKESVYAQSITS KPVKGMLTGPITCLRWSFPRDDVDQKTQAMQLALALRDEVNDLEAAGIKVIQVDEPALREGL PLREGTERSAYYTWAAEAFRVATSGVANKTQIHSHFCYSDLDPNHIKALDADVVSIEFSKKD DANYIAEFKNYPNHIGLGLFDIHSPRIPSKDEFIAKISTILKSYPAEKFWVNPDCGLKTRGW EETRLSLTHMVEAAKYFREQYK .

[000118] As is evident from the foregoing description, certain aspects of the present disclosure are not limited by the particular details of the examples illustrated herein, and it is therefore contemplated that other modifications and applications, or equivalents thereof, will occur to those skilled in the art. It is accordingly intended that the claims shall cover all such modifications and applications that do not depart from the spirit and scope of the present disclosure.

[000119] Moreover, 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 or those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described above.

[000120] Other aspects, objects and advantages of the present disclosure can be obtained from a study of the drawings, the disclosure and the appended claims.

Claims

The Claims Are the Following:

1. An enzyme that facilitates the increased conversion of caffeic acid to ferulic acid, wherein the enzyme has been modified at a residue that allows for increased methylation of caffeic acid.

2. The enzyme of claim 1, wherein the enzyme is a caffeic acid 3-O-methyltransferase.

3. The enzyme of claim 2, wherein the enzyme is mutated at a leucine in a methyl binding pocket of caffeic acid 3-O-methyltransferase.

4. The enzyme of claim 2, wherein the enzyme is mutated at an alanine in a methyl binding pocket of caffeic acid 3-O-methyltransferase.

5. The enzyme of claim 2 is derived from an alfalfa, an Ar bidopsis, a Medicago tnmcatitla, a Popidns trichocarpa, or a Catharansiis roseus.

6. The enzyme of claim 3, wherein a Leucine 136 of the enzyme in the methyl binding pocket or equivalent residue is replaced with a tyrosine.

7. The enzyme of claim 4, wherein an Alanine 162 of the enzyme in the methyl binding pocket or equivalent residue is replaced with a proline or a threonine.

8. The enzyme of claim 3, wherein the enzyme is mutated at an additional residue.

9. The enzyme of claim 4, wherein the enzyme is mutated at an additional residue.

10. A modified caffeic acid 3-0-methyltransferase with enhanced activity for the methylation of caffeic acid to ferulic acid comprising a modification of a residue that would bind caffeic acid in an non-covalent manner or an electrostatic manner.

11. The modified caffeic acid 3-O-methylransferase of claim 10, wherein the residue is in a methyl binding pocket of caffeic acid 3-O-methyltransferase.

12. The modified caffeic acid 3-O-methyltransferase of claim 10, wherein the modified residue is selected from residues in the methyl binding pocket and a combination thereof.

13. The modified caffeic acid 3-O-methyltransferase of claim 12, wherein the modified residue is a leucine replaced by an amino acid with a hydrophobic group.

14. The modified caffeic acid 3-O-methyltransferase of claim 12, wherein the modified residue is an alanine.

15. The modified caffeic acid 3-O-methyltransferase of claim 13, wherein the hydrophobic group is an aromatic hydrocarbon.

16. The modified caffeic acid 3-O-methyltransferase of claim 15, wherein the hydrophobic group comprises a hydroxyl group.

17. The modified caffeic acid 3-O-methyltransferase of claim 13, wherein the modified residue is Leu-136 replaced by a tyrosine.

18. The modified caffeic acid 3-O-methyltransferase of claim 14, wherein the residue is Ala-162.

19. A modified caffeic acid 3-O-methyltransferase that increases conversion of caffeic acid to ferulic acid, wherein the modified caffeic acid 3-O-methyltransferase is derived from a plant species.

20. The modified caffeic acid 3-O-methyltransferase of claim 19, wherein the plant species is selected from an alfalfa, an Arabidopsis, a Medicago truncatula, a Popiihis tric ocarpa, a Catharansus roseas, and a combination thereof.

21. The modified caffeic acid 3-O-methyltransferase of claim 19, wherein the modified residue is selected from the group consisting of Leu-136, Phe-172, Phe-176, and Ala- 162, and a combination thereof.

22. The modified caffeic acid 3-O-methyltransferase of claim 19, wherein Leu-136 is modified to a tyrosine.

23. A recombinant caffeic acid 3-O-methyltransferase encoded by a mutated equivalent of caffeic acid 3-O-methyltransferase, characterized that it has increased methylation activity of caffeic acid to ferulic acid.

24. The recombinant caffeic acid 3-0-methyltransferase of claim 23, wherein its leucine in its methyl binding pocket is mutated.

25. The recombinant caffeic acid 3-0-methyltransferase of claim 24, wherein the leucine is mutated to a tyrosine.

26. A bioconversion method of making ferulic acid comprising

expressing 4-hydroxyphenylacetate 3-hyrdroxylase in a mixture;

expressing caffeic acid 3-O-methyltransferase in the mixture;

feeding p-coumaric acid to the mixture;

incubating the mixture; and

collecting ferulic acid.

27. The bioconversion method of making ferulic acid of claim 26 further comprising expressing methionine synthase in the mixture.

28. The bioconversion method of making ferulic acid of claim 27, wherein

expressing 4-hydroxyphenylacetate 3-hyrdroxylase in a mixture;

expressing caffeic acid 3-O-methyltransferase in the mixture; and

expressing methionine synthase in the mixture further comprises expressing each step singularly or collectively by in vitro translation.

29. The bioconversion method of making ferulic acid of claim 27, wherein

expressing 4-hydroxyphenylacetate 3-hyrdroxylase in a mixture;

expressing caffeic acid 3-O-methyltransferase in the mixture; and expressing methionine synthase in the mixture further comprises expressing each step singularly or collectively in a cellular system.

30. The bioconversion method of making ferulic acid of claim 29, wherein the cellular system is selected from the group consisting of bacteria, yeast, and a combination thereof.

31. The bioconversion method of making ferulic acid of claim 29, wherein the cellular system is grown in a medium.

32. The bioconversion method of making ferulic acid of claim 27, wherein the 4- hydroxyphenylacetate 3-hyrdroxylase, the caffeic acid 3-O-methyltransferase, or the methionine synthase are purified as recombinant proteins.

33. The bioconversion method of making ferulic acid of claim 26, wherein p-coumaric acid is derived from maize cob extract.

34. The bioconversion method of making ferulic acid of claim 31, wherein the medium is M9B.

35. The bioconversion method of making ferulic acid of claim 26, wherein expressing 4- hydroxyphenylacetate 3 -hydroxylase further comprises expressing a hpaB and a hpaC based on amino acid SEQ ID No. 1 and 2.

36. The bioconversion method of making ferulic acid of claim 26, wherein expressing 4- hydroxyphenyacetate 3-hydroxylase is based on amino acid sequence selected from an E. coli.

37. The bioconversion method of making ferulic acid of claim 26, wherein expressing caffeic acid 3-O-methyltransferase is based on amino acid sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID 5, SEQ ID 6, SEQ ID 7 and a combination thereof.

38. The bioconversion method of making ferulic acid of claim 26, wherein expressing caffeic acid 3-O-methyltransferase is based on amino acid sequence selected from the species consisting of alfalfa, Arabidopsis, Medicago, Catharansits, Popiihis and a combination thereof.

39. The bioconversion method of making ferulic acid of claim 27, wherein expressing methionine synthase is based on amino acid sequence selected from the group consisting of SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10 and a combination thereof.

40. The bioconversion method of making ferulic acid of claim 27, wherein expressing methionine synthase is based on amino acid selected from the species consisting of Arabidopsis, E. coli, Saccharomyces, and a combination thereof.

41. The bioconversion method of making ferulic acid of claim 26, wherein 4- hydroxyphenylacetate 3-hydroxylase is expressed via a plasmid.

42. The bioconversion method of making ferulic acid of claim 26, wherein caffeic acid 3- O-methyltransferase is expressed via a plasmid.

43. The bioconversion method of making ferulic acid of claim 27, wherein methionine synthase is expressed via a plasmid.

44. The bioconversion method of making ferulic acid of claim 27, further comprises expressing a fusion protein, wherein the fusion protein is combined of proteins or protein portions selected from a group consisting of 4-hydroxyphenylacetate 3- hydroxylase, caffeic acid 3-O-methyltransferase, methionine synthase and a combination thereof.

45. The bioconversion method of making ferulic acid of claim 44, wherein the proteins are joined by a peptide linker.

46. The bioconversion method of making ferulic acid of claim 27, wherein a fusion protein comprising caffeic acid 3-O-methyltransferase and methionine synthase is expressed.

47. A method of making vanillin comprising:

expressing 4-hydroxyphenylacetate 3-hyrdroxylase in a mixture;

expressing caffeic acid 3-O-methyltransferase in the mixture;

expressing methionine synthase in the mixture;

expressing feruloyl-CoA synthetase in the mixture; expressing enoyl-CoA hydratase/aldolase in the mixture;

feeding p-coumaric acid to the mixture; and

collecting vanillin.

48. The method of making vanillin of claim 47, wherein

expressing 4-hydroxyphenylacetate 3 -hyrdroxylase in a mixture;

expressing caffeic acid 3-O-methyltransferase in the mixture; and

49. The method of making vanillin of claim 47, wherein

expressing 4-hydroxyphenylacetate 3 -hyrdroxylase in a mixture;

expressing caffeic acid 3-O-methyltransferase in the mixture; and

expressing methionine synthase in the mixture further comprises expressing each step singularly or collectively in a cellular system.

50. The method of making vanillin of claim 49, wherein

the cellular system is selected from the group consisting of bacteria, yeast, and a combination thereof.

51. The method of making vanillin of claim 49, wherein the 4-hydroxyphenylacetate 3- hyrdroxylase, the caffeic acid 3-O-methyltransferase, or the methionine synthase are purified as recombinant proteins.

52. The method of making vanillin of claim 47, wherein

expressing feruloyl-CoA synthetase in the mixture; and

expressing enoyl-CoA hydratase/aldolase in the mixture further comprises expressing each step singularly or collectively by in vitro translation.

53. The method of making vanillin of claim 47, wherein

expressing feruloyl-CoA synthetase in the mixture; and

expressing enoyl-CoA hydratase/aldolase in the mixture further comprises expressing each step singularly or collectively in a cellular system.

54. The method of making vanillin of claim 53, wherein the cellular system is selected from the group consisting of bacteria, yeast, and a combination thereof.

55. The method of making vanillin of claim 53, wherein the feruloyl-CoA synthetase or the enoyl-CoA hydratase/aldoase or both are purified as recombinant proteins.

56. The method of making vanillin of claim 53, wherein

expressing feruloyl-CoA synthetase in the mixture; and

expressing enoyl-CoA hydratase/aldolase in the mixture further comprises expressing in a species selected from the genus consisting of Pseadomonas, Amycolatopsis, Sphingomonas pancimobilis, Rhodococciis, Streptomyces, and a combination thereof.

57. The method of making vanillin of claim 53, wherein

expressing feruloyl-CoA synthetase in the mixture; and expressing enoyl-CoA hydratase/aldolase in the mixture further comprises expressing in a microorganism species selected from the group consisting of Pseiidomonas sp. HR 199, Amycolatopsis sp. ATCC 39116, Amycolatopsis sp. HR167, Sphingomonas paacimobilis SYK-6, Pseiidomonas fliiorescens AN103, Streptomyces seotonii, Streptomyces sannanensis, Amycolalopsis sp. strain (Zhp06) and a combination thereof.

58. The method of making vanillin of claim 47, further comprises expressing a vanillin synthase.

59. The method of making vanillin of claim 58, wherein vanillin synthase is expressed by in vitro translation.

60. The method of making vanillin of claim 59, wherein the vanillin synthase is expressed in a cellular system.

61. The method of making vanillin of claim 60, wherein the cellular system is selected from the group consisting of bacteria, yeast, and a combination thereof.

62. The method of making vanillin of claim 61, wherein the vanillin synthase is purified as a recombinant protein.

63. The method of making vanillin of claim 47, wherein the vanillin has a greater 5¹³C compared with vanillin derived from C₃ plants, and further comprising:

feeding p-coumaric acid derived from a C₄ plant.

64. The method of making vanillin of claim 63, wherein the C₄ plant is maize.

65. The method of making vanillin of claim 47, wherein expressing 4- hydroxyphenylacetate 3 -hydroxylase further comprises expressing a hpaB and a hpaC based on amino acid SEQ ID No. 1 and 2.

66. The method of making vanillin of claim 47, wherein expressing 4- hydroxyphenyacetate 3-hydroxylase is based on amino acid sequence selected from E. coli.

67. The method of making vanillin of claim 47, wherein expressing caffeic acid 3-0- methyltransferase is based on amino acid sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID 5, SEQ ID 6, SEQ ID 7 and a combination thereof.

68. The method of making vanillin of claim 47, wherein expressing caffeic acid 3-0- methyltransferase is based on amino acid sequence selected from the species consisting of alfalfa, Arabidopsis, Medicago, Catharansits, Popuhis and a combination thereof.

69. The method of making vanillin of claim 47, wherein expressing methionine synthase is based on amino acid sequence selected from the group consisting of SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10 and a combination thereof.

70. The method of making vanillin of claim 47, wherein expressing methionine synthase is based on amino acid selected from the species consisting of Arabidopsis, E. coli, Saccharomyces, and a combination thereof.

71. The method of making vanillin of claim 47, wherein 4-hydroxyphenylacetate 3- hydroxylase is expressed via a plasmid.

72. The method of making vanillin of claim 47, wherein caffeic acid 3-0- methyltransferase is expressed via a plasmid.

73. The method of making vanillin of claim 47, wherein methionine synthase is expressed via a plasmid.

74. The method of making vanillin of claim 47, wherein further comprises expressing a fusion that comprises a combination of 4-hydroxyphenylacetate 3 -hydroxylase, caffeic acid 3-O-methyltransferase, methionine synthase or a combination thereof linked by a peptide linker.

75. A method of making vanillin comprising:

providing ferulic acid based on the method of claim 26 in a mixture;

expressing feruloyl-CoA synthetase in the mixture;

expressing enoyl-CoA hydratase/aldolase in the mixture; and

collecting vanillin.

76. The method of making vanillin of claim 75, wherein expressing feruloyl-CoA synthetase in the mixture; and

expressing enoyl-CoA hydratase/aldolase in the mixture further comprises expressing in a species selected from the genus consisting of Pseitdomonas, Amycolatopsis, Sphingomonas paiicimobilis, Rhodococc s; Streptomyces, and a combination thereof.

77. The method of making vanillin of claim 75, wherein

expressing feruloyl-CoA synthetase in the mixture; and

expressing enoyl-CoA hydratase/aldolase in the mixture further comprises expressing in a microorganism species selected from the group consisting of Pseudomonas sp. HR 199, Amycolatopsis sp. ATCC 39116, Amycolatopsis sp. HR167, Sphingomonas paiicimobilis SYK-6, Pseitdomonas fliiorescens AN103, Streptomyces seotonii, Streptomyces sannanensis, Amycolalopsis sp. strain (Zhp06) and a combination thereof.

78. A ferulic acid with greater 8¹³C compared with ferulic acid derived from C₃ plants.

79. The ferulic acid of claim 78, wherein the ferulic is made based on the method of claim 26.

80. The ferulic acid of claim 78, wherein the 8¹³C is greater than -30%.

81. The ferulic acid of claim 78, wherein the 8¹³C is greater than -20%.

82. The ferulic acid of claim 78, wherein the 8¹³C is between about -10% and about -20%.

83. The feriilic acid of claim 82, wherein the δ C is between about -12% and about -17%.

84. A vanillin with greater δ C compared with vanillin derived from C₃ plants.

85. The vanillin of claim 84, wherein the vanillin is made based on the method of claim 47.

86. The vanillin of claim 84, wherein the δ^]¾ is greater than -30%.

87. The vanillin of claim 84, wherein the 6¹³C is greater than -20%.

88. The vanillin of claim 84, wherein the δ C is between about -10% and about ~20%>.

89. The vanillin of claim 88, wherein the δ^]¾ is between about -12% and about -17%.