WO2014106189A2 - Procédés de fabrication de vanilline par l'intermédiaire de fermentation microbienne utilisant de l'acide férulique fourni par une acide caféique 3-o-méthyltransférase modifiée - Google Patents

Procédés de fabrication de vanilline par l'intermédiaire de fermentation microbienne utilisant de l'acide férulique fourni par une acide caféique 3-o-méthyltransférase modifiée Download PDF

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WO2014106189A2
WO2014106189A2 PCT/US2013/078328 US2013078328W WO2014106189A2 WO 2014106189 A2 WO2014106189 A2 WO 2014106189A2 US 2013078328 W US2013078328 W US 2013078328W WO 2014106189 A2 WO2014106189 A2 WO 2014106189A2
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expressing
acid
vanillin
making
methyltransferase
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PCT/US2013/078328
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WO2014106189A3 (fr
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Rui Zhou
Mohammad Wadud BHUIYA
Xianpeng CAI
Xiaodan Yu
Robert G. Eilerman
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Conagen Inc.
Givaudan Sa
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Publication of WO2014106189A3 publication Critical patent/WO2014106189A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01068—Caffeate O-methyltransferase (2.1.1.68)

Definitions

  • 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.
  • Vanillin is a phenolic aldehyde, which is an organic compound with the molecular formula CsHgOs. Its functional groups include aldehyde, ether, and phenol.
  • vanillin instead of natural vanilla extract, is now more often used as a flavoring agent in foods, beverages, and pharmaceuticals.
  • 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.
  • 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.
  • 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.
  • p-Coumaric acid is a hydroxycinnamic acid, an organic compound that is a hydroxy derivative of cinnamic acid.
  • coumaric 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.
  • 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).
  • TAL tyrosine ammonia lyase
  • p-Coumaric acid has antioxidant properties and is believed to reduce the risk of stomach cancer by reducing the formation of carcinogenic nitrosamines.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ROS reactive oxygen species
  • 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.
  • ferulic acid may reduce oxidative stress and formation of thymine dimers in skin.
  • oral supplements of ferulic acid can inhibit melanin production in the process of skin whitening.
  • 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.
  • agricultural by-products such as maize cob, wheat bran and rice bran.
  • 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.
  • 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.
  • 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 CH 3 ) instead of a methoxy group (-O-CH 3 ).
  • 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.
  • 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.
  • One pathway is similar to the ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • An additional aspect of the current disclosure is a ferulic acid with less negative or greater 5 13 C compared with ferulic acid derived from C 3 plants.
  • Another aspect of the current disclosure is a vanillin with less negative or greater 8 13 C compared with vanillin derived from C 3 plants.
  • Figure 1 illustrates the enzymatic pathway from p-coumaric acid to ferulic acid.
  • 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.
  • Figure 3 shows the bioconversion of p-coumaric acid to caffeic acid with E. coli containing exogenous expression of HpaBC.
  • Figure 4 illustrates the relative COMT enzyme activity of the recombinant COMT, and fused COMT and MS.
  • Figure 5 shows the production of ferulic acid in E. coli strains containing plant
  • Figure 6 illustrates the production of ferulic acid in different strains in M9B medium in 24 hours.
  • Figure 7 shows the production of ferulic acid in the culture of E. coli containing AtCOMT and fusion MS-AtCOMT.
  • Figure 8 illustrates HPLC analysis of hydroxycinnamic acids in the cell culture with the exogenous expression of 4HPA3H and MS-AtCOMT.
  • Figure 9 shows bioconversion of p-coumaric acid to caffeic acid and ferulic acid in E. coli containing HpaBC and MS-AtCOMT.
  • Figure 10 illustrates the bioconversion of hydroxycinnamic acids in maize cob extract.
  • Figure 11 shows a model of the tertiary structure of poplar COMT (A); Close- up view of the substrate binding site of COMT (B).
  • Figure 12 shows the screening of COMT mutant library.
  • Figure 13 illustrates catalytic efficiency of COMT wild type and mutants.
  • Figure 14 exhibits the ferulic acid biotransformation profile along with vanillin and by-products accumulation.
  • Figure 15 shows the ferulic acid biotransformation profile along with vanillin accumulation during feeding in the presence of Amycolalopsis sp. strain (Zhp06).
  • Figure 16 shows the ferulic acid biotransformation profile along with vanillin accumulation.
  • 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.
  • the Alanine 162 is mutated to proline or threonine.
  • 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.
  • the residue is any residue within the methyl binding pocket that interacts or affects the interaction of caffeic acid.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • feruloyl-CoA synthetase or the enoyl-CoA hydratase/aldoase is purified as recombinant proteins - either singularly or collectively.
  • 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.
  • 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.
  • Another aspect of the current disclosure is a method of making vanillin, wherein the vanillin has a "less negative" or greater 8 13 C compared with vanillin derived from C 3 plants includes feeding p-coumaric acid derived from a C 4 plant.
  • the C4 plant is maize.
  • 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.
  • 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.
  • 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.
  • a microorganism species selected from the group consisting of Pseiidomonas sp. HR 199, Amycolatopsis sp. ATCC 39116, Amycolatopsis sp. HR167, Sphingomonas paiicimobilis SYK-6, Pse
  • Another aspect of the current disclosure is a ferulic acid with less negative or greater 6 13 C compared with ferulic acid derived from C 3 plants. This is possible when C 4 plants are used as the source of p-coumaric acid.
  • the 8 13 C for the ferulic acid can be less negative or greater than -30%.
  • the 5 13 C for the ferulic acid can be less negative or greater than -020%.
  • the 8 13 C for the ferulic acid is between about -10% and about -20%. In another disclosure, the 5 13 C is between about -12% and about -17%.
  • FIG. 1 Another aspect of the current disclosure is a vanillin with less negative or greater 5 13 C compared with vanillin derived from C 3 plants. This is possible when C 4 plants are used as the source of p-coumaric acid or ferulic acid.
  • the 5 13 C for the vanillin can be less negative or greater than -30%.
  • the 8 13 C for vanillin can be less negative or greater than - 20%.
  • the 8 li C is between about -10% and about -20%.
  • the 6 1J C is between about -12% and about -17%.
  • the subject technology relates to the extraction of hydroxy cinnamic acids from 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).
  • 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.
  • 4HPA3H 4-hydroxyphenylacetate 3-hydrolase
  • COMP caffeic acid 3-0- methyltranferase
  • 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.
  • E. coli or other cellular systems e.g., yeast
  • 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.
  • 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.
  • methionine synthase 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.
  • MS greatly increases the conversion of caffeic acid to ferulic acid.
  • 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.
  • 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.
  • 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.
  • 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).
  • pCA p-coumaric acid
  • CA caffeic acid
  • 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.
  • C3H plant p-coumarate 3-hydrolase
  • C3H plant specific cytochrome p450 dependent monooxygenase
  • 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.
  • the enzymes could be expressed by in vitro translation and the enzymes are then purified to exert their enzymatic effect outside the cell.
  • 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.
  • T4 DNA ligase were purchased from New England Biolabs. All PCR reactions were performed with New England Biolabs' Phusion PCR system. Construction of plasmid.
  • Genomic DNA of E. coli strain BL21 (DE3) was extracted using Bacterial
  • 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.
  • 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.
  • pCA could be converted into CA in both media, but with significant difference in the conversion activity.
  • Example 2 Bioconversion of caffeic acid to ferulic acid in E. coli hosting plant caffeic acid 3-O-methyltransferase (COMT) and methionine synthase (MS).
  • COMP caffeic acid 3-O-methyltransferase
  • MS methionine synthase
  • the second step in the bioconversion of ferulic acid from p-coumaric acid involves the conversion of caffeic acid (CA) to ferulic acid.
  • CA caffeic acid
  • COMP 3-O-methyltransferase
  • the co-expression of methionine synthase increases the conversion to ferulic acid.
  • Bacterial strains, plasmids and culture condition Bacterial strains, plasmids and culture condition.
  • 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).
  • 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-IITM 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.
  • 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.
  • AtCOMT 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.
  • 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).
  • Primers for generating fusion genes were generated with the same strategy with the primers and templates listed in Table 3.
  • 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.
  • 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.
  • 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.
  • 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.
  • plsd-Sumo-MtMS:: AtCOMT and plsd-pETDuet-HpcBC were co- transformed into Hi-Control BL21(DE3) (Lucigen Inc, WI) according to standard procedure.
  • 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).
  • the codon NNK has 32- fold degeneracy and encodes all 20 amino acids without rare codons.
  • the PCR mixture (25 ⁇ ) composed of Phusion HF buffer containing 60ng COMT DNA template, 200 ⁇ dNTPS, 0.5 ⁇ forward primers, 0.5 ⁇ reverse primers, 5% DMSO and 0.3 ⁇ 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 ⁇ of PCR products were digested with 1 ⁇ Dpnl (New England Biolabs) at 37°C for 4 hrs to remove the template plasmid. Aliquot of (2 ⁇ ) digestive products was added to 50 ⁇ 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 ⁇ SOC medium was added and grow the cells at 37°C for 1 hr.
  • the cells were centrifuged at 5000 rpm for min, 450 ⁇ 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 ⁇ g/ml).
  • LB Luria-Bertani
  • DNA sequencing was done to confirm the mutant.
  • 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.
  • the harvested cell cultures were lysed with 60 ⁇ 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 ⁇ of the reaction in each well, 200 ⁇ caffeic acid substrates, and 400 ⁇ Sadenosyl-Z-methionine (SAM) and 10 ⁇ of lysate. The reaction mixture was incubated at 30°C for 5 mins. Applicants extracted the reaction product with 200 ⁇ ethyl acetate and moved the extracts into a new microplate.
  • SAM Sadenosyl-Z-methionine
  • 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.
  • 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.
  • 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
  • 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; MgS0 4 -7H 2 0, 0.8g/L; Na 2 HP0 4 -7H 2 0, 7.5g/L; KH 2 P0 4 , 1.0 g/L; and 0.2 niL/L antifoam.
  • the media were autoclaved at 120°C for 15 and 30 minutes respectively.
  • 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.
  • 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.
  • 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.
  • C 3 and C 4 plants have different 6 13 C signatures, allowing C 4 grasses to be detected in 5 13 C .
  • C 4 plants have a 5 13 C of -16 to -10 %o
  • C 3 plants have a 8 13 C of -33 to -24%o.
  • 5 13 C for C 4 plants would generally be "less negative” or "greater” in value compared with the 5 13 C for C 3 plants.
  • vanillin, ferulic acid and caffeic acid made from the current bioconversion process would have unique isotope distribution if the starting materials are derived from C 4 plants, such as maize cobs. They would have 5 13 C in the -16 to -10 o range compared to vanillin, ferulic acid and caffeic acid made from general industrial process using C 3 plants, which would be in the -33 to -24%o range.
  • 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.
  • the ferulic acid and vanillin made from the current bioconversion process using corn extracts (or other C 4 plants) as initial starting materials would have "less negative” or greater 8 13 C compared with ferulic acid and vanillin made from C 3 plants (e.g., rice plants), which is the general industrial process for making ferulic acid and vanillin (See Table 4).
  • the difference in 8 13 C would serve as a way to identify products made by this bioconversion process.
  • the ability to use C 4 plants as starting material in the current bioconversion process would serve to preserve nature by alleviating less reliance on tropical C 3 plants. Enzyme Amino Acid Sequences.
  • 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.
  • 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.
  • SEQ ID NO: 3 Amino sequence of Arabidopsis COMT
  • SEQ ID NO: 4 Amino sequence of Medicago truncatula COMT
  • SEQ ID NO: 5 Amino sequence of Catharansus roseus COMT MGSANPDNKNSMTKEEEEACLSAMRLASASVLPMVL SAIELDLLELIKKSGPGAYVSPSEL AAQLPTQNPDAPVMLDRILRLLASYSVLNCTLKDLPDGGIERLYSLAPVCKFLTKNEDGVSM AALLLMNQDKVLMESWYHLKDAVLEGGIPFNKAYGMTAFEYHGKDPRFNKVFNQGMSNHSTI IMKKILEIYQGFQGL TVVDVGGGTGATLNMIVS YPSIKGINFDLPHVIEDAPSYPGVDHV GGDMFVSVPKGDAIFMKWICHDWSDAHCLKFLKNCHEALPENGKVILAECLLPEAPDSTLST QNTVHVDVIMLAHNPGGKERTEKEFEALAKGAGFRGFIKVCCAYNSWIMELLK .
  • SEQ ID NO: 7 Amino sequence of poplar COMT2 MGSTGETQMSPAQILDEEANFAMQLISSSVLPMVLKTAIELDLLEIMAKAGPGALLSPSDIA SHLPTKNPDAPVMLDRILRLLASYSILICSLRDLPDGKVERLYGLASVCKFLTKNEDGVSVS PLCLMNQDKVLMESWYHL DAILEGGIPFNKAYGMTAFEYHGTDPRFN VFNKGMSDHS IA MKKILETYKGFEGLASLVDVGGGTGAVLSTIVSKYPSIKGINFDLPHVIADAPAFPGVENVG GDMFVSVPQADAVF KWICHDWSDEHCLRFLKNCYDALPENGKVILVECILPVAPDTSLATK GVMHVDAIMLAHNPGGKERTEKEFEGLARGAGFKGFEVMCCAFNTYVIEFRKQA.
  • 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.
  • SEQ ID NO: 8 Amino sequence of Arabidopsis methionine synthase
  • SEQ ID NO: 9 Amino sequence of E. coli methionine synthase
  • SEQ ID NO: 10 Amino sequence of methionine synthase of Saccharomyces cerevisiae

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Abstract

Cette invention concerne un procédé de fabrication de vanilline comprenant une expression de la 4-hydroxyphénylacétate 3-hydroxylase, de l'acide caféique 3-O-méthyltransférase, de la méthionine synthase, de la féruloyl-CoA synthétase et de l'énoyl-CoA hydratase/aldolase dans un mélange, l'introduction d'acide p-coumarique dans le mélange, et la collecte de vanilline. Elle comprend de plus une enzyme, telle que l'acide caféique 3-O-méthyltransférase, qui facilite la conversion accrue d'acide caféique en acide férulique, l'enzyme ayant été modifiée à un reste qui permet la méthylation accrue de l'acide férulique, et un procédé d'utilisation de l'enzyme dans la fabrication d'acide férulique suivi par la vanilline.
PCT/US2013/078328 2012-12-31 2013-12-30 Procédés de fabrication de vanilline par l'intermédiaire de fermentation microbienne utilisant de l'acide férulique fourni par une acide caféique 3-o-méthyltransférase modifiée WO2014106189A2 (fr)

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CN107523582A (zh) * 2016-06-20 2017-12-29 天津大学 一种产松柏醇的工程菌、构建方法及产松柏醇的用途
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US10704070B2 (en) 2015-03-03 2020-07-07 The Regents Of The University Of California Protecting group chemistry for clean, reductant-free dyeing
CN116622532A (zh) * 2022-11-25 2023-08-22 深圳先进技术研究院 合成阿魏酸的酵母菌株和构建方法及其在制备阿魏酸及胡椒代谢物中的应用
US12098405B2 (en) 2021-11-09 2024-09-24 The Regents Of The University Of California Protecting group chemistry for clean, reductant-free dyeing

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Cited By (14)

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WO2015066722A1 (fr) 2013-11-04 2015-05-07 Bgn Tech Llc Procédés de préparation de vanilline par fermentation microbienne d'acide férulique à partir d'eugénol au moyen d'une déshydrogénase végétale
US11203774B2 (en) 2015-03-03 2021-12-21 The Regents Of The University Of California Protecting group chemistry for clean, reductant-free dyeing
US10704070B2 (en) 2015-03-03 2020-07-07 The Regents Of The University Of California Protecting group chemistry for clean, reductant-free dyeing
CN107523582B (zh) * 2016-06-20 2021-01-15 天津大学 一种产松柏醇的工程菌、构建方法及产松柏醇的用途
CN107523582A (zh) * 2016-06-20 2017-12-29 天津大学 一种产松柏醇的工程菌、构建方法及产松柏醇的用途
KR101933919B1 (ko) 2017-06-01 2018-12-31 아주대학교 산학협력단 페룰릴-CoA 합성효소 및 에놀-CoA 수화효소/알돌라아제 과발현 대장균을 이용하는 멜라닌 생산 방법
CN109837315A (zh) * 2017-11-29 2019-06-04 味之素株式会社 用于生产目标物质的方法
EP3502263A3 (fr) * 2017-11-29 2019-10-16 Ajinomoto Co., Inc. Procédé de production d'une substance objective
US11680279B2 (en) 2017-11-29 2023-06-20 Ajinomoto Co., Inc. Method for producing objective substance
CN109837315B (zh) * 2017-11-29 2024-05-14 味之素株式会社 用于生产目标物质的方法
US12098405B2 (en) 2021-11-09 2024-09-24 The Regents Of The University Of California Protecting group chemistry for clean, reductant-free dyeing
CN116622532A (zh) * 2022-11-25 2023-08-22 深圳先进技术研究院 合成阿魏酸的酵母菌株和构建方法及其在制备阿魏酸及胡椒代谢物中的应用
CN116622532B (zh) * 2022-11-25 2024-05-17 深圳先进技术研究院 合成阿魏酸的酵母菌株和构建方法及其在制备阿魏酸及胡椒代谢物中的应用
WO2024108675A1 (fr) * 2022-11-25 2024-05-30 深圳先进技术研究院 Souche de levure pour la synthèse d'acide férulique, procédé de construction et son utilisation dans la préparation d'acide férulique et de métabolite de poivre

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