Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
  • A23L2/52—Adding ingredients
  • A23L2/56—Flavouring or bittering agents
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/20—Synthetic spices, flavouring agents or condiments
  • A23L27/204—Aromatic compounds
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/20—Synthetic spices, flavouring agents or condiments
  • A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
• • 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
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

• the invention disclosed herein relates generally to the field of recombinant production of vanillin. Particularly, the invention provides methods for recombinant production of vanillin and compositions containing vanillin.
• vanilla is recognized as one of the most popular flavors and aromas around the world. Over 100 varieties of the vanilla plant exist, but the three main species grown for commercial use are Vanilla planifolia, Vanilla pompona, and Vanilla tahitensis. Vanilla plants require humid, tropical, or subtropical climates of countries or regions such as Madagascar, Indonesia, Mexico, French Polynesia, and the West Indies.
• vanilla plants have proven time-consuming and tedious. Flowering occurs approximately two to three years after planting. The flowers must then be pollinated by hand because of physical separation of the stigma and stamen because few natural pollinators of the vanilla plant exist. Pollination must be performed daily over a four month period. Approximately eight months after pollination, seed pods are ready to be harvested. It is crucial that harvesting occurs at the proper time. For example, if harvesting is done too early, the vanilla beans may have a lower content of vanillin (4-hydroxy-3- methoxybenzaldehyde, methylprotocatechuic aldehyde, vanillaldehyde, vanillic aldehyde).
• Vanillin (CAS# 121 -33-5) is most responsible for the flavor and fragrance profiles of vanilla, and vanillin content is also affected by the region in which the plants are grown and the curing process following harvesting. Curing may take several months in order to develop the flavor and aroma of the vanilla bean. During this time, glucovanillin is converted to vanillin by the activity of endogenous ⁇ -glucosidase activity. See Voisine et a/., 1995, J. Agric. Food Chem. 43: 2658- 2661 and Ruiz-Teran et a/., 2001 , J. Agric. Food Chem. 49: 5207-5209.
• vanilla In the vanilla plant, tyrosine is converted to 4-coumaric acid, which is then converted to ferulic acid, and ferulic acid is converted into vanillin. In the mature seed pod, vanillin is in the ⁇ -D-glucoside form, known as glucovanillin. See Negishi et al. J. Agric. Food Chem. 57: 9959- 9961 (2009).
• vanilla contains approximately 250 other compounds, including para-hydroxy benzaldehyde and para-hydroxy benzoic acid. One or more of these compounds can alter or contribute to off-flavors of vanilla. These off-flavors can be more or less problematic depending on the food system or application of choice.
• Potential contaminants include p- hydroxybenzoic acid, coumarin, ferulic acid, 4-vinylguaiacol, isoeugenol, 5-formylvanillin, para- hydroxybenzaldehyde, acetovanillon, dehydro-di-vanillin, 5-carboxyvanillin, ethyl vanillin, orthovanillin, 4-(hydroxymethyl)-2-methoxyphenol, mandelic acid, coniferyl alcohol, coniferyl aldehyde, 2-methoxy-4-vinylphenol, guaiacol, eugenol, and tumeric. Conditions not limited to climate, soil nutrients, and extraction methods also influence vanilla compositions.
• vanilla can vary greatly from batch-to-batch, and droughts, natural disasters, and deforestation have contributed to lower production and a higher cost of vanilla. Therefore, there remains a need for an in vivo expression system that can produce high, reproducible, pure yields of vanillin.
• the invention is directed to biosynthesis of vanillin preparations from genetically modified cells.
• the invention is directed to vanillin preparations from genetically modified cells having significantly improved biosynthesis rates and yields.
• This disclosure relates to the production of vanillin.
• this disclosure relates to the production of vanillin having the chemical structure:
• the disclosure provides a recombinant host, for example, a microorganism, comprising one or more heterologous biosynthetic genes introduced thereto, wherein the expression of one or more biosynthetic genes results in production of vanillin.
• the invention provides generally a vanillin composition comprising from about 1 % to about 99.9% w/w of vanillin, wherein the composition has a reduced level of contaminants relative to a plant-derived vanillin extract or a vanillin composition produced by an in vitro process, by whole cell bioconversion, or by fermentation.
• the vanillin composition disclosed herein has less than 0.1 % of contaminants relative to a plant-derived vanillin extract or a vanillin composition produced by the in vitro process, by whole cell bioconversion, or by fermentation.
• At least one of the contaminants in the the vanillin composition disclosed herein is a compound that contributes to off-flavors.
• the composition contains a reduced amount of one or a plurality of 2-methoxy-4-vinylphenol, 3-bromo-4-hydroxybenzaldehyde, 3-methoxy-4-hydroxybenzyl alcohol, 4-vinylguaiacol, acetovanillon, coniferyl alcohol, coniferyl aldehyde, coumarin, dehydro- di-vanillin, ethyl vanillin, eugenol, ferulic acid, glyoxylic acid, guaiacol, isoeugenol, mandelic acid, O-benzylvanillin, orthovanillin, para-hydroxybenzaldehyde, p-hydroxybenzoic acid, 5- carboxyvanillin, 5-formylvanillin, turmeric, and/or 4-(hydroxymethyl)-2-methoxyphenol.
• the composition contains a reduced amount of one or a plurality of 2-methyloctadecane, 8, 11 , 14-eicosatrienoic acid, a-amyrin, ⁇ -amyrin, ⁇ -amyrin, acetate, ⁇ - pinene, ⁇ -sitosterol, calcium gluconate, calcium phytate, carboxymethyl cellulose, carnauba wax, carophyllene, carophyllene derivatives, cellulose acetate, centauredin, copper gluconate, cuprous iodide, decanoic acid, epi-alpha-cadinol, ethyl cellulose, gibberellin, hydroxypropylmethyl cellulose, lupeol, methylcellulose, octacosane, octadecanol, pentacosane, quercetin, sodium carboxymethyl cellulose, spathulenol, stigmasterol, and/
• the composition contains a reduced amount of one or a plurality of compounds of Table 4.
• the invention further provides a method for producing vanillin, comprising:
• the recombinant host expresses polypeptides comprising a COMT polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:8, an AROM polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:4, a 3DSD polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:24, NO:25, NO:26, NO:27, NO:28, NO:29, a ACAR polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO: 12, a VAO polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:16, NO:17, NO:18, NO: 19, NO:20, a OMT polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:21 , NO:22, NO:23, and/or a PPTase polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ
• the recombinant host is a yeast cell, a plant cell, a mammalian cell, an insect cell, a fungal cell, or a bacterial cell.
• the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species.
• the yeast cell is a Saccharomycete.
• the yeast cell is a cell from the Saccharomyces cerevisiae species.
• vanillin is produced by fermentation.
• the culture medium for said recombinant host does not comprise one or a plurality of 2-methoxy-4-vinylphenol, 3-bromo-4-hydroxybenzaldehyde, 3-methoxy-4- hydroxybenzyl alcohol, 4-vinylguaiacol, acetovanillon, coniferyl alcohol, coniferyl aldehyde, coumarin, dehydro-di-vanillin, ethyl vanillin, eugenol, ferulic acid, glyoxylic acid, guaiacol, isoeugenol, mandelic acid, O-benzylvanillin, orthovanillin, para-hydroxybenzaldehyde, p- hydroxybenzoic acid, 5-carboxyvanillin, 5-formylvanillin, turmeric, and/or 4-(Hydroxym ethyl )-2- methoxyphenol.
• the culture medium for said recombinant host does not comprise one or a plurality of compounds of Table 4.
• the invention further discloses a method for producing vanillin comprising an in vitro production process using one or a plurality of the polypeptides comprising a COMT polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:8, an AROM polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:4, a 3DSD polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:24, NO:25, NO:26, NO:27, NO:28, NO:29, an ACAR polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO: 12, a VAO polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO: 16, NO: 17, NO: 18, NO: 19, NO:20, an OMT polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:21 , NO:22, NO:23, and/or a PPTase polypeptide poly
• the bioconversion comprises enzymatic bioconversion or whole cell bioconversion.
• the cell of the whole cell bioconversion is a yeast cell, a plant cell, a mammalian cell, an insect cell, a fungal cell, or a bacterial cell.
• the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species.
• the yeast cell is a Saccharomycete.
• the yeast cell is a cell from Saccharomyces cerevisiae species.
• the invention further provides an in vitro method for producing vanillin, comprising:
• the in vitro method is an enzymatic in vitro method or whole cell in vitro method.
• the cell of the whole cell in vitro method is a yeast cell, a plant cell, a mammalian cell, an insect cell, a fungal cell, or a bacterial cell.
• the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species.
• the yeast cell is a Saccharomycete.
• the yeast cell is a cell from Saccharomyces cerevisiae species.
• the invention further provides vanillin produced by the methods disclosed herein.
• the invention further provides a food product comprising the composition disclosed herein.
• the food product is a beverage or a beverage concentrate.
• the invention further provides a method for producing vanillin by fermentation in a yeast cell, comprising: (a) fermenting the yeast cell in a culture medium, under conditions wherein, genes encoding a COMT polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:8, an AROM polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:4, a 3DSD polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:24, NO:25, NO:26, NO:27, NO:28, NO:29, an AGAR polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO: 12, a VAO polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO: 16, NO: 17, NO:18, NO:19, NO:20, an OMT polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:21 , NO:22, NO:23
• the yeast cell expresses polypeptides comprising a COMT polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:8, an AROM polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:4, a 3DSD polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:24, NO:25, NO:26, NO:27, NO:28, NO:29, a ACAR polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO: 12, a VAO polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:16, NO: 17, NO: 18, NO:19, NO:20, a OMT polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:21 , NO:22, NO:23, and/or a PPTase polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:21
• the culture medium for said yeast cell does not comprise one or a plurality of 2-methoxy-4-vinylphenol, 3-bromo-4-hydroxybenzaldehyde, 3-methoxy-4- hydroxybenzyl alcohol, 4-vinylguaiacol, acetovanillon, coniferyl alcohol, coniferyl aldehyde, coumarin, dehydro-di-vanillin, ethyl vanillin, eugenol, ferulic acid, glyoxylic acid, guaiacol, isoeugenol, mandelic acid, O-benzylvanillin, orthovanillin, para-hydroxybenzaldehyde, p- hydroxybenzoic acid, 5-carboxyvanillin, 5-formylvanillin, turmeric, and/or 4-(Hydroxym ethyl )-2- methoxyphenol.
• the culture medium for said yeast cell does not comprise one or a plurality of 2-methoxy-4-vin
• the culture medium for said yeast cell does not comprise 2-methoxy-4-vinylphenol.
• the culture medium for said yeast cell does not comprise 3-bromo-4-hydroxybenzaldehyde.
• the culture medium for said yeast cell does not comprise 3-methoxy-4-hydroxybenzyl alcohol.
• the culture medium for said yeast cell does not comprise 4-vinylguaiacol.
• the culture medium for said yeast cell does not comprise acetovanillon.
• the culture medium for said yeast cell does not comprise coniferyl alcohol.
• the culture medium for said yeast cell does not comprise coniferyl aldehyde.
• the culture medium for said yeast cell does not comprise coumarin.
• the culture medium for said yeast cell does not comprise dehydro-di-vanillin.
• the culture medium for said yeast cell does not comprise ethyl vanillin.
• the culture medium for said yeast cell does not comprise eugenol.
• the culture medium for said yeast cell does not comprise ferulic acid.
• the culture medium for said yeast cell does not comprise glyoxylic acid.
• the culture medium for said yeast cell does not comprise guaiacol.
• the culture medium for said yeast cell does not comprise isoeugenol.
• the culture medium for said yeast cell does not comprise mandelic acid.
• the culture medium for said yeast cell does not comprise O-benzylvanillin.
• the culture medium for said yeast cell does not comprise orthovanillin.
• the culture medium for said yeast cell does not comprise para-hydroxybenzaldehyde.
• the culture medium for said yeast cell does not comprise p-hydroxybenzoic acid.
• the culture medium for said yeast cell does not comprise 5-carboxyvanillin.
• the culture medium for said yeast cell does not comprise 5-formylvanillin.
• the culture medium for said yeast cell does not comprise turmeric.
• the culture medium for said yeast cell does not comprise 4-(Hydroxymethyl)-2-methoxyphenol.
• the culture medium for said yeast cell does not comprise one or a plurality of compounds of Table 4.
• the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species.
• the yeast cell is a Saccharomycete.
• the yeast cell is a cell from the Saccharomyces cerevisiae species.
• the invention further provides a vanillin produced by the methods disclosed herein.
• Any of the hosts described herein can be a microorganism (e.g., a Saccharomycete, such as Saccharomyces cerevisiae, or Escherichia coli).
• the culture media does not comprise one or a plurality of 2-methoxy-4-vinylphenol, 3-bromo-4-hydroxybenzaldehyde, 3- methoxy-4-hydroxybenzyl alcohol, 4-vinylguaiacol, acetovanillon, coniferyl alcohol, coniferyl aldehyde, coumarin, dehydro-di-vanillin, ethyl vanillin, eugenol, ferulic acid, glyoxylic acid, guaiacol, isoeugenol, mandelic acid, O-benzylvanillin, orthovanillin, para-hydroxybenzaldehyde, p-hydroxybenzoic acid, 5-carboxyvanillin, 5-formylvanillin, turmeric, 4-(Hydroxym ethyl )-2- methoxyphenol, or one or a plurality of compounds of Table 4 prior to fermentation.
• the culture media does not comprise one or a plurality of 2-methoxy-4-vinylphenol, 3-bromo-4-hydroxybenzaldehyde, 3- methoxy-4-hydroxybenzyl alcohol, 4-vinylguaiacol, acetovanillon, coniferyl alcohol, coniferyl aldehyde, coumarin, dehydro-di-vanillin, ethyl vanillin, eugenol, ferulic acid, glyoxylic acid, guaiacol, isoeugenol, mandelic acid, O-benzylvanillin, orthovanillin, para-hydroxybenzaldehyde, p-hydroxybenzoic acid, 5-carboxyvanillin, 5-formylvanillin, turmeric, 4-(Hydroxym ethyl )-2- methoxyphenol, or one or a plurality of compounds of Table 4 after fermentation.
• Figure 1 is a schematic of de novo biosynthesis of vanillin (4) in an organism expressing 3-dehydroshikimate dehydratase (3DSD), aromatic carboxylic acid reductase (ACAR), O-methyltransferase (OMT), UDP glucuronosyltransferases (UGT), and phophopantheteine transferase (PPTase) polypeptides.
• 3DSD 3-dehydroshikimate dehydratase
• ACAR aromatic carboxylic acid reductase
• O-methyltransferase O-methyltransferase
• UDP glucuronosyltransferases UDP glucuronosyltransferases
• PPTase phophopantheteine transferase
• vanillin catabolites and metabolic side products including dehydroshikimic acid (1 ), protocatechuic acid (2), protocatechuic aldehyde (3), protocatechuic alcohol (6), 4-(hydroxymethyl)-2-methoxyphenol alcohol (7), and vanillin ⁇ -D-glucoside (8) are also indicated.
• Open arrows show primary metabolic reactions in yeast, black arrows show enzyme reactions introduced by metabolic engineering, and diagonally striped arrows show undesired innate yeast metabolic reactions.
• Figure 2 shows initial steps of the shikimate pathway in Saccharomyces cerevisiae (S. cerevisiae).
• Figure 3 shows a pathway for vanillin synthesis in E. coli.
• Figure 4 shows levels of vanillin glucoside, vanillin, 4-(hydroxymethyl)-2- methoxyphenol alcohol glucoside, and 4-(hydroxymethyl)-2-methoxyphenol alcohol in yeast strains expressing Penicillium simplicissium (P. simplicissium; PS) or Rhodococcus jostii (R. jostii; RJ) 4-(hydroxymethyl)-2-methoxyphenol alcohol oxidase (VAO) and grown in media supplemented with 3 mM 4-(hydroxymethyl)-2-methoxyphenol alcohol.
• P. simplicissium PS
• Rhodococcus jostii R. jostii; RJ
• VAO 4-(hydroxymethyl)-2-methoxyphenol alcohol oxidase
• Figure 5 shows levels of vanillic acid, vanillin, and vanillin glucoside in yeast strains expressing Nocardia iowensis (N. iowensis) or N. crassa ACAR and of Escherichia coli (E. coli) or S. pombe phosphopantetheinyl transferase (PPTase) and grown in media supplemented with 3 mM vanillic acid.
• N. iowensis Nocardia iowensis
• N. crassa ACAR Nocardia iowensis
• E. coli Escherichia coli
• S. pombe phosphopantetheinyl transferase PPTase
• Figure 6 shows particular contaminants of vanillin.
• Figure 7A shows a UV trace of a vanillin analytical standard
• Figure 7B shows a UV trace of a ferulic acid analytical standard
• Figure 7C shows a UV trace of an ethyl vanillin analytical standard
• Figure 7D shows a UV trace of a mandelic acid analytical standard
• Figure 7E shows a UV trace of a eugenol analytical standard
• Figure 7F shows a UV trace of an isoeugenol analytical standard
• Figure 7G shows a UV trace of a guaiacol analytical standard.
• Figure 8A shows a UV chromatogram of a vanillin analytical standard
• Figure 8B shows an extracted ion chromatogram (EIC) of the expected mass of vanillin present in a vanillin sample produced in yeast
• Figure 8C shows an EIC of the expected mass of ethyl vanillin present in a vanillin sample produced in yeast
• Figure 8D shows an EIC of the expected mass of ferulic acid present in a vanillin sample produced in yeast
• Figure 8E shows an EIC of the expected mass of mandelic acid present in a vanillin sample produced in yeast
• Figure 8F shows an EIC of the expected mass of eugenol/isoeugenol present in a vanillin sample produced in yeast
• Figure 8G shows an EIC of the expected mass of guaiacol present in a vanillin sample produced in yeast.
• Figures 8C-8G show the absense of absence of ethyl vanillin, ferulic acid, mandelic acid, eugenol/isoeugenol, and guaiacol impurities.
• Figure 9A shows a UV chromatogram of a vanillin analytical standard (top panel), an EIC of the expected mass of ferulic acid present in a vanillin sample produced in yeast (middle panel), and an EIC of the expected mass of a ferulic acid analytical sample (bottom panel).
• Figure 9B shows an EIC of the expected mass of ethyl vanillin present in a vanillin sample produced in yeast (top panel) and an EIC of the expected mass of an ethyl vanillin analytical sample (bottom panel).
• Figure 9C shows a UV chromatogram of a vanillin analytical standard (top panel), an EIC of the expected mass of mandelic acid present in a vanillin sample produced in yeast (middle panel), and an EIC of the expected mass of a mandelic acid analytical sample (bottom panel).
• Figure 9D shows an EIC of the expected mass of eugenol present in a vanillin sample produced in yeast (top panel) and an EIC of the expected mass of a eugenol analytical sample (bottom panel).
• Figure 9E shows a UV chromatogram of a vanillin analytical standard (top panel), an EIC of the expected mass of isoeugenol present in a vanillin sample produced in yeast (middle panel), and an EIC of the expected mass of a isoeugenol analytical sample (bottom panel).
• Figure 9F shows an EIC of the expected mass of guaiacol present in a vanillin sample produced in yeast (top panel) and an EIC of the expected mass of a guaiacol analytical sample (bottom panel).
• Figures 9B-9F show the absense of ferulic acid, ethyl vanillin, mandelic acid, eugenol, isoeugenol, and guaiacol impurities.
• Figure 10A shows a fingerprinting mass spectrum of vanillin
• Figure 10B shows a fingerprinting mass spectrum of ferulic acid
• Figure 10C shows a fingerprinting mass spectrum of ethyl vanillin
• Figure 10D shows a fingerprinting mass spectrum of mandelic acid
• Figure 10E shows a fingerprinting mass spectrum of eugenol
• Figure 10F shows a fingerprinting mass spectrum of isoeugenol
• Figure 10G shows a fingerprinting mass spectrum of guaiacol.
• Figure 1 1 shows amino acid and nucleotide sequences used herein.
• nucleic acid means one or more nucleic acids.
• nucleic acid can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof.
• the terms "microorganism,” “microorganism host,” “microorganism host cell,” “recombinant host,” and “recombinant host cell” can be used interchangeably.
• the term “recombinant host” is intended to refer to a host, the genome of which has been augmented by at least one DNA sequence. Such DNA sequences include but are not limited to genes that are not naturally present, DNA sequences that are not normally transcribed into RNA or translated into a protein (“expressed"), and other genes or DNA sequences which one desires to introduce into the non-recombinant host. It will be appreciated that typically the genome of a recombinant host described herein is augmented through stable introduction of one or more recombinant genes.
• introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene.
• the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis.
• Suitable recombinant hosts include microorganisms.
• recombinant gene refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. "Introduced,” or “augmented” in this context, is known in the art to mean introduced or augmented by the hand of man.
• a recombinant gene can be a DNA sequence from another species, or can be a DNA sequence that originated from or is present in the same species, but has been incorporated into a host by recombinant methods to form a recombinant host.
• a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA.
• Said recombinant genes are particularly encoded by cDNA.
• engineered biosynthetic pathway refers to a biosynthetic pathway that occurs in a recombinant host, as described herein, and does not naturally occur in the host.
• endogenous gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell.
• heterologous sequence and “heterologous coding sequence” are used to describe a sequence derived from a species other than the recombinant host.
• the recombinant host is an S. cerevisiae cell
• a heterologous sequence is derived from an organism other than S. cerevisiae.
• a heterologous coding sequence can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence.
• a coding sequence is a sequence that is native to the host.
• vanillin precursor and “vanillin precursor compound” are used interchangeably to refer to intermediate compounds in the vanillin biosynthetic pathway.
• Vanillin precursors include, but are not limited to, dehydroshikimic acid, protocatechuic acid, protocatechuic aldehyde, and protocatechuic alcohol.
• Vanillin and vanillin precursors can be produced in vivo (i.e., in a recombinant host), in vitro (i.e., enzymatically), or by whole cell bioconversion.
• vanillin and vanillin precursors are produced in vivo through expression of one or more enzymes involved in the vanillin biosynthetic pathway in a recombinant host.
• a vanillin-producing recombinant host expressing one or more of a gene encoding a 3DSD polypeptide, a gene encoding an ACAR polypeptide, a gene encoding an OMT polypeptide, a gene encoding a VAO polypeptide, a gene encoding a PPTase polypeptide, a gene encoding a COMT polypeptide, and a gene encoding an AROM polypeptide can produce vanillin and/or vanillin precursors in vivo.
• vanillin and vanillin precursors produced in vivo are produced by fermentation.
• the vanillin-producing strain was cultivated in an aerobic, glucose-limited, 5-day fed-batch process. This process included a -16 hour growth phase in the base medium which was primarily a minimal-defined medium with 4-8 wt% complex carbon source combined with glucose, followed by -100 hours of feeding with glucose utilized as the sole carbon and energy source.
• the glucose feed was combined with trace metals, vitamins, salts, a nitrogen source.
• the pH was kept near pH 5, the dissolved oxygen maintained above 20%, and the temperature setpoint was 30°C.
• vanillin and/or vanillin precursors are produced through contact of a vanillin precursor with one or more enzymes involved in the vanillin pathway in vitro.
• contacting protocatechuic acid with an OMT polypeptide can result in production of vanillin in vitro.
• a vanillin precursor is produced through contact of an upstream vanillin precursor with one or more enzymes involved in the vanillin pathway in vitro.
• contacting dehydroshikimic acid with a 3DSD polypeptide can result in production of protocatechuic acid in vitro.
• vanillin or a vanillin precursor is produced by whole cell bioconversion.
• a host cell expressing one or more enzymes involved in the vanillin pathway takes up and modifies a vanillin precursor in the cell; following modification in vivo, vanillin remains in the cell and/or is excreted into the culture medium.
• a host cell expressing a gene encoding an OMT polypeptide can take up protocatechuic acid and modify vanillin in the cell; following modification in vivo, vanillin is excreted into the culture medium or remains in the cell.
• the term “and/or” is utilized to describe multiple components in combination or exclusive of one another.
• x, y, and/or z can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x and (y or z),” or “x or y or z.”
• "and/or” is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group.
• vanillin is produced through one or more of the following steps: culturing a recombinant cell, synthesizing vanillin in a cell, and isolating vanillin.
• vanillin is synthesized in a recombinant host. See e.g. Hansen et al., Appl. Environ. Microbiol. 75: 2765-2774 (2009) and PCT/US2012/049842. each of which is incorporated by reference in its entirety.
• the invention involves (a) providing a recombinant host capable of producing vanillin, wherein said recombinant host harbors a heterologous nucleic acid encoding an Arom Multifunctional Enzyme (AROM) polypeptide and/or a Catechol-O-Methyl Transferase (COMT) polypeptide; (b) cultivating said recombinant host for a time sufficient for said recombinant host to produce vanillin; and (c) isolating vanillin from said recombinant host or from the cultivation supernatant, thereby producing vanillin.
• AROM Arom Multifunctional Enzyme
• COMP Catechol-O-Methyl Transferase
• a recombinant host comprises a 3- dehydroshikimate dehydratase (3DSD), an aromatic carboxylic acid reductase (ACAR), and/or an O-methyltransferase (OMT).
• the 3DSD comprises a Podospora pauciseta (P. pauciseta) 3DSD
• the ACAR comprises a Nocardia ACAR
• the OMT comprises a Homo sapiens OMT.
• a recombinant host comprises a phosphopantetheine transferase (PPTase) and/or a gene encoding a 4-(hydroxymethyl)-2- methoxyphenol alcohol oxidase (VAO). See Figures 1-3.
• AROM polypeptide refers to a polypeptide involved in a step of the shikimate pathway and has one or more of the following activities: 3- dehydroquinate synthase activity, 3-dehydroquinate dehydratase activity, shikimate 5- dehydrogenase activity, shikimate kinase activity, and 3-phosphoshikimate 1- carboxyvinyltransferase activity.
• AROM polypeptides include the S. cerevisiae polypeptide having the amino acid sequence set forth in SEQ ID NO:4 (GENBANK Accession No. X06077); a Schizosaccharomyces pombe (S.
• pombe polypeptide of GENBANK Accession No. NP_594681.1 ; a Schizosaccharomyces japonicas (S. japonicas) polypeptide of GENBANK Accession No. XP_002171624; a Neurospora crassa (N. crassa) polypeptide of GENBANK Accession No. XP_956000; and a Yarrowia lipolytica (Y. lipolytica) polypeptide of GENBANK Accession No. XP_505337.
• an AROM polypeptide can at least 80% (e.g., at least 85, 90, 95, 96, 97, 98, 99, or 100%) identical to the sequence set forth in SEQ ID NO:4 and possess at least four of the five enzymatic activities of the S. cerevisiae AROM polypeptide, i.e., 3- dehydroquinate synthase activity, 3-dehydroquinate dehydratase activity, shikimate 5- dehydrogenase activity, shikimate kinase activity, and 3-phosphoshikimate 1- carboxyvinyltransferase activity.
• a mutant AROM polypeptide wherein said mutant has decreased shikimate dehydrogenase activity relative to a corresponding wild-type AROM polypeptide.
• the mutant AROM polypeptide can have one or more mutations in domain 5, a deletion of at least a portion of domain 5, or lack domain 5. See Figure 2.
• the AROM polypeptide is a mutant AROM polypeptide with decreased shikimate dehydrogenase activity.
• the mutant AROM polypeptide redirects metabolic flux from aromatic amino acid production to vanillin precursor production ( Figure 2).
• Decreased shikimate dehydrogenase activity can be inferred from the accumulation of dehydroshikimic acid in a recombinant host expressing a mutant AROM polypeptide.
• the mutant AROM polypeptide described herein can have one or more modifications in domain 5 (e.g., a substitution of one or more amino acids, a deletion of one or more amino acids, insertions of one or more amino acids, or combinations of substitutions, deletions, and insertions).
• the AROM gene lacking domain 5 is the AR01 gene.
• a mutant AROM polypeptide can have a deletion in at least a portion of domain 5 (e.g., a deletion of the entire domain 5, i.e., amino acids 1305 to 1588 of the amino acid sequence in SEQ ID NO:4, or can have one or more amino acid substitutions in domain 5, such that the mutant AROM polypeptide has decreased shikimate dehydrogenase activity.
• An exemplary mutant AROM polypeptide lacking domain 5 is provided in SEQ ID NO:2 (corresponding nucleotide sequence set forth in SEQ ID NO:1 ).
• Amino acid substitutions that are particularly useful can be found at, for example, one or more positions aligning with position 1349, 1366, 1370, 1387, 1392, 1441 , 1458, 1500, 1533, or 1571 of the amino acid sequence set forth in SEQ ID NO:4.
• a modified AROM polypeptide can have a substitution at a position aligning with position 1370 or at position 1392 of the amino acid sequence set forth in SEQ ID NO:4.
• a modified AROM polypeptide can have one or more of the following: an amino acid other than valine (e.g., a glycine) at a position aligning with position 1349 of the amino acid sequence set forth in SEQ ID NO:4; an amino acid other than threonine (e.g., a glycine) at a position aligning with position 1366 of the amino acid sequence set forth in SEQ ID NO:4; an amino acid other than lysine (e.g., leucine) at a position aligning with position 1370 of the amino acid sequence set forth in SEQ ID NO:4; an amino acid other than isoleucine (e.g., histidine) at a position aligning with position 1387 of the amino acid sequence set forth in SEQ ID NO:4; an amino acid other than threonine (e.g., lysine) at a position aligning with position 1392 of the amino acid sequence set forth in SEQ ID NO:4; an amino acid other than valine (e
• Exemplary mutant AROM polypeptides with at least one amino acid substitution in domain 5 include the AROM polypeptides A1533P, P1500K, R1458W, V1349G, T1366G, I1387H, W1571V, T1392K, K1370L and A1441 P of SEQ ID NO:4.
• a modified AROM polypeptide is fused to a polypeptide catalyzing the first committed step of vanillin biosynthesis, 3-dehydroshikimate dehydratase (3DSD).
• a polypeptide having 3DSD activity and that is suitable for use in a fusion polypeptide includes the 3DSD polypeptide from P. pauciseta, Ustilago maydis (U. maydis), R. jostii), Acinetobacter sp., Aspergillus niger (A. niger), or N. crassa. See, GENBANK Accession Nos.
• a modified AROM polypeptide lacking domain 5 can be fused to a polypeptide having 3DSD activity (e.g., a P. pauciseta 3DSD).
• SEQ ID NO:7 sets forth the amino acid sequence of such a protein.
• the COMT polypeptide according to the invention may, in certain embodiments be a caffeoyl-O-methyltransferase.
• the COMT polypeptide is preferably a catechol-O-methyltransferase.
• a COMT polypeptide of the invention is a mutant COMT polypeptide having improved meta hydroxyl methylation of protocatechuic aldehyde, protocatechuic acid and/or protocatechuic alcohol relative to that of the Homo sapiens COMT having the amino acid sequence set forth in SEQ ID NO:8.
• a COMT polypeptide can be any amino acid sequence that is at least 80% (e.g., at least 85, 90, 95, 96, 97, 98, 99, or 100%) identical to the Homo sapiens COMT sequence set forth in SEQ ID NO:8 and possesses the catechol-O-methyltransferase enzymatic activities of the wild-type Homo sapiens COMT polypeptide.
• a mutant COMT polypeptide is provided.
• the invention provides mutant COMT polypeptides that preferentially catalyze methylation at the meta position of protocatechuic acid, protocatechuic aldehyde, and/or protocatechuic alcohol rather than at the para position.
• mutant COMT polypeptide refers to any polypeptide having an amino acid sequence which is at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% identical to the Hs COMT sequence set forth in SEQ ID NO:8 and is capable of catalyzing methylation of the -OH group at the meta position of protocatechuic acid and/or protocatechuic aldehyde, wherein the amino acid sequence of said mutant COMT polypeptide differs from SEQ ID NO:8 by at least one amino acid. It is preferred that the mutant COMT polypeptide differs by at least one amino acid from any sequence of any wild type COMT polypeptide.
• mutant COMT polypeptide refers to a polypeptide having an amino acid sequence, which is at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% identical to either SEQ ID NO:9 or SEQ ID NO: 10 and is capable of catalyzing methylation of the -OH group at the meta position of protocatechuic acid and/or protocatechuic aldehyde, wherein the amino acid sequence of said mutant COMT polypeptide differs from each of SEQ ID NO:9 and SEQ ID NO:10 by at least one amino acid.
• the mutant COMT polypeptides described herein can have one or more mutations (e.g., a substitution of one or more amino acids, a deletion of one or more amino acids, insertions of one or more amino acids, or combinations of substitutions, deletions, and insertions) in, for example, the substrate binding site.
• a mutant COMT polypeptide can have one or more amino acid substitutions in the substrate binding site of human COMT.
• a "mutant COMT polypeptide" of the invention differs from SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:1 1 by one or two amino acid residues, wherein the differences between said mutant and wild-type proteins are in the substrate binding site.
• the wild-type Homo sapiens COMT lacks regioselective O-methylation of protocatechuic aldehyde and protocatechuic acid, indicating that the binding site of Homo sapiens COMT does not bind these substrates in an orientation that allows the desired regioselective methylation.
• the active site of Homo sapiens COMT is composed of the co-enzyme S-adenosyl methionine (SAM), which serves as the methyl donor, and the catechol substrate, which contains the hydroxyl to be methylated coordinated to Mg 2+ and proximal to Lys144.
• SAM co-enzyme S-adenosyl methionine
• the O-methylation proceeds via an SN2 mechanism, where Lys144 serves as a catalytic base that deprotonates the proximal hydroxyl to form the oxy-anion that attacks a methyl group from the sulfonium of SAM.
• Lys144 serves as a catalytic base that deprotonates the proximal hydroxyl to form the oxy-anion that attacks a methyl group from the sulfonium of SAM.
• the invention provides a mutant COMT polypeptide, which is capable of catalyzing methylation of an -OH group of protocatechuic acid, wherein said methylation results in generation of at least 4 times more vanillic acid compared to iso-vanillic acid, preferably at least 5 times more vanillic acid compared to iso-vanillic acid, such as at least 10 times more vanillic acid compared to iso-vanillic acid, for example at least 15 times more vanillic acid compared to iso-vanillic acid, such as at least 20 times more vanillic acid compared to iso-vanillic acid, for example at least 25 times more vanillic acid compared to iso-vanillic acid, such as at least 30 times more vanillic acid compared to iso-vanillic acid; and which has an amino sequence which differs from SEQ ID NO:8 by at least one amino acid.
• a mutant COMT polypeptide is capable of catalyzing methylation of an -OH group of protocatechuic aldehyde, wherein said methylation results in generation of at least 4, 5, 10, 15, 20, 25, or 30 times more vanillin compared to iso-vanillin; and/or is capable of catalyzing methylation of an - OH group of protocatechuic alcohol, wherein said methylation results in generation of at least 4, 5, 10, 15, 20, 25, or 30 times more 4-(hydroxymethyl)-2-methoxyphenol alcohol compared to iso-4-(hydroxymethyl)-2-methoxyphenol alcohol.
• an in vitro assay can be conducted.
• protocatechuic acid is incubated with a mutant COMT polypeptide in the presence of a methyl donor and subsequently the level of generated iso-vanillic acid and vanillic acid is determined.
• Said methyl donor may for example be S- adenosylmethionine.
• this may be determined by generating a recombinant host harboring a heterologous nucleic acid encoding the mutant COMT polypeptide to be tested, wherein said recombinant host furthermore is capable of producing protocatechuic acid. After cultivation of the recombinant host, the level of generated iso-vanillic acid and vanillic acid may be determined. In relation to this method it is preferred that said heterologous nucleic acid encoding the mutant COMT polypeptide to be tested is operably linked to a regulatory region allowing expression in said recombinant host.
• the recombinant host expresses at least one 3DSD and at least one ACAR, which preferably may be one of the 3DSDs and ACARs described herein.
• the method may also include determining the level of generated vanillin and iso-vanillin. Alternatively, this may be determined by generating a recombinant host harboring a heterologous nucleic acid encoding the mutant COMT polypeptide to be tested, and feeding protocatechuic acid to said recombinant host, followed by determining the level of generated iso-vanillic acid and vanillic acid.
• an in vitro assay or a recombinant host cell can be used to determine whether a mutant COMT polypeptide is capable of catalyzing methylation of an -OH group of protocatechuic aldehyde, wherein said methylation results in generation of at least X times more vanillin compared to iso-vanillin.
• protecatechuic aldehyde is used as starting material and the level of vanillin and iso-vanillin is determined.
• an in vitro assay or a recombinant host cell can be used to determine whether a given mutant COMT polypeptide is capable of catalyzing methylation of an -OH group of protocatechuic alcohol, wherein said methylation results in generation of at least X times more 4-(hydroxymethyl)-2-methoxyphenol alcohol compared to iso-4-(hydroxymethyl)-2- methoxyphenol alcohol.
• protecatechuic alcohol is used as starting material and the level of 4-(hydroxymethyl)-2-methoxyphenol alcohol and iso-4-(hydroxymethyl)- 2-methoxyphenol alcohol is determined.
• the level of vanillin may be determined by any suitable method useful for detecting these compounds, wherein said method can distinguish between vanillin. Such methods include for example HPLC. Similarly, the level of iso-vanillic acid, vanillic acid, iso-4-(hydroxymethyl)-2- methoxyphenol alcohol and 4-(hydroxymethyl)-2-methoxyphenol alcohol may be determined using any suitable method useful for detecting these compounds, wherein said method can distinguish between vanillin. Such methods include for example HPLC.
• the invention provides a mutant COMT polypeptide, which (1 ) has an amino acid sequence sharing at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% sequence identity with SEQ ID NO:8 determined over the entire length of SEQ ID NO:8; and (2) has at least one amino acid substitution at a position aligning with positions 198 to 199 of SEQ ID NO:8, which may be any of the amino acid substitutions described herein below; and (3) is capable of catalyzing methylation of an -OH group of protocatechuic acid, wherein said methylation results in generation of at least 4, 5, 10, 15, 20, 25 or 30 times more vanillic acid compared to iso-vanillic acid.
• said mutant COMT polypeptide may also be capable of catalyzing methylation of an -OH group of protocatechuic aldehyde, wherein said methylation results in generation of at least 4, 5, 10, 15, 20, 25 or 30 times more vanillin compared to iso-vanillin; and/or be capable of catalyzing methylation of an -OH group of protocatechuic alcohol, wherein said methylation results in generation of at least 4, 5, 10, 15, 20, 25, or 30 times more 4-(hydroxymethyl)-2-methoxyphenol alcohol compared to iso-4-(hydroxymethyl)-2-methoxyphenol alcohol.
• the mutant COMT polypeptide may in one preferred embodiment have an amino acid substitution at the position aligning with position 198 of SEQ ID NO:8.
• the mutant COMT polypeptide may be a mutant COMT polypeptide with the characteristics outlined above, wherein said substitution is a substitution of the leucine at the position aligning with position 198 of SEQ ID NO:8 with another amino acid having a lower hydropathy index.
• the mutant COMT polypeptide may be a mutant COMT polypeptide with characteristics as outlined above, wherein said substitution is a substitution of the leucine at the position aligning with position 198 of SEQ ID NO:8 with another amino acid having a hydropathy index lower than 2.
• the mutant COMT polypeptide may be a mutant COMT polypeptide with characteristics as outlined above, wherein said substitution is a substitution of the leucine at the position aligning with position 198 of SEQ ID NO:8 with an Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, Trp or Tyr, for example Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, Lys, Met, Pro, Ser, Thr, Trp or Tyr.
• said substitution is a substitution of the leucine at the position aligning with position 198 of SEQ ID NO:8 with tyrosine.
• substitution of the leucine aligning with position 198 of SEQ ID NO:8 with methionine increased regioselectivity of meta>para O-methylation for protocatechuic aldehyde.
• the mutant COMT polypeptide may have an amino acid substitution at the position aligning with position 199 of SEQ ID NO:8. Accordingly, the mutant COMT polypeptide may be a mutant COMT polypeptide with characteristics as outlined above, wherein said substitution is a substitution of the glutamic acid at the position aligning with position 199 of SEQ ID NO:8 with another amino acid, which has either a neutral or positive side-chain charge at pH 7.4.
• the mutant COMT polypeptide may be a mutant COMT polypeptide with characteristics as outlined above, wherein said substitution is a substitution of the glutamic acid at the position aligning with position 199 of SEQ ID NO:8 with Ala, Arg, Asn, Cys, Gin, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val.
• said substitution is a substitution of the glutamic acid at the position aligning with position 199 of SEQ ID NO:8 with an alanine or glutamine.
• Substitution of the glutamic acid aligning with position 199 of SEQ ID NO:8 with alanine or glutamine increased regioselectivity of meta>para O-methylation for protocatechuic aldehyde.
• a mutant COMT polypeptide can have one or more of the following mutations: a substitution of a tryptophan, tyrosine, phenylalanine, glutamic acid, or arginine for the leucine at a position aligning with position 198 of the amino acid sequence set forth in SEQ ID NO:8; a substitution of an arginine, lysine, or alanine for methionine at a position aligning with position 40 of the amino acid sequence set forth in SEQ ID NO:8; a substitution of a tyrosine, lysine, histidine, or arginine for the tryptophan at a position aligning with position 143 of the amino acid sequence set forth in SEQ ID NO:8; a substitution of an isoleucine, arginine, or tyrosine for the proline at a position aligning with position 174 of the amino acid sequence set forth in SEQ ID NO:8; a substitution of an argin
• a mutant COMT polypeptide contains substitution of tryptophan for leucine at a position aligning with position 198. This mutation may increase regioselectivity of meta>para O-methylation for protocatechuic acid. Modeling of the protein binding site of a COMT polypeptide containing a L198W mutation, indicates that a steric clash can occur between the mutated residue and the substrate. This steric clash does not occur in the meta reacting conformation as the carboxylic acid of the substrate is distal to this residue.
• the mutant COMT polypeptide is a polypeptide of SEQ ID NO:8, wherein the amino acid at position 198 has been substituted with an amino acid having a lower hydropathy index than leucine.
• the mutant COMT polypeptide may be a polypeptide of SEQ ID NO:8, wherein the leucine at the position 198 has been substituted with an amino acid having a hydropathy index lower than 2.
• the mutant COMT polypeptide may be a polypeptide of SEQ ID NO:8, wherein the leucine at position 198 has been substituted with an Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, Lys, Met, Pro, Ser, Thr, Trp or Tyr, preferably Met or Tyr.
• the mutant COMT polypeptide may be a polypeptide of SEQ ID NO:8, wherein the amino acid at position 199 has been substituted with another amino acid, which has either a neutral or positive side-chain charge at pH 7.4.
• the mutant COMT polypeptide may be a polypeptide of SEQ ID NO:8 where the glutamic acid at the position 199 has been substituted with Ala, Arg, Asn, Cys, Gin, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Ala or Gin.
• a mutant COMT polypeptide has two or more mutations. For example, 2, 3, 4, 5, 6, or 7 of the residues in the substrate binding site can be mutated.
• a mutant COMT polypeptide can have a substitution of an arginine or lysine for methionine at a position aligning with position 40 of the amino acid sequence of SEQ ID NO:8; a substitution of a tyrosine or histidine for tryptophan at a position aligning with position 143 of the amino acid sequence of SEQ ID NO:8; a substitution of an isoleucine for proline at a position aligning with position 174 of the amino acid sequence of SEQ ID NO:8, and a substitution of an arginine or lysine for tryptophan at position 38.
• a mutant COMT polypeptide also can have a substitution of lysine or arginine for tryptophan at a position aligning with position 143 of the amino acid sequence of SEQ ID NO:8 and a substitution of an arginine or tyrosine for proline at position 174 of SEQ ID NO:8.
• a mutant COMT polypeptide also can have a substitution of a phenylalanine, tyrosine, glutamic acid, tryptophan, or methionine for cysteine at a position aligning with position 173 of the amino acid sequence set forth in SEQ ID NO:8, a substitution of an alanine for methionine at a position aligning with position 40 of the amino acid sequence set forth in SEQ ID NO:8, and a substitution of a serine, glutamic acid, or aspartic acid for the arginine at a position aligning with position 201 of the amino acid sequence set forth in SEQ ID NO:8.
• the mutant COMT polypeptide has a substitution of the leucine at a position aligning with position 198 of SEQ ID NO:8 as well as a substitution of the glutamic acid at a position aligning with position 199 of SEQ ID NO:8. Said substitutions may be any of the substitutions described in this section above, It is also possible that the mutant COMT polypeptide has a substitution of the leucine at a position aligning with position 198 of SEQ I D NO:8 as well as a substitution of the arginine at a position aligning with position 201 of SEQ ID NO:8. Said substitutions may be any of the substitutions described in this section above.
• the invention provides mutant A ROM and mutant COMT polypeptides and nucleic acids encoding such polypeptides and use of the same in the biosynthesis of vanillin.
• the method includes the steps of providing a recombinant host capable of producing vanillin in the presence of a carbon source, wherein said recombinant host harbors a heterologous nucleic acid encoding a mutant COMT polypeptide and/or mutant AROM polypeptide; cultivating said recombinant host in the presence of the carbon source; and purifying vanillin isolating vanillin from said recombinant host or from the cultivation supernatant.
• a 3DSD polypeptide according to the present invention may be any enzyme with 3-dehydroshikimate dehydratase activity.
• the 3DSD polypeptide is an enzyme capable of catalyzing conversion of 3-dehydro-shikimate to protocatechuate and H 2 0.
• a 3DSD polypeptide according to the present invention is preferably an enzyme classified under EC 4.2.1.1 18.
• a suitable polypeptide having 3DSD activity includes the 3DSD polypeptide made by P. pauciseta, U. maydis, R. jostii, Acinetobacter sp., A. niger or N. crassa.
• the recombinant host may include a heterologous nucleic acid encoding the 3DSD polypeptide of Podospora anserina (P. anserina), U. maydis, R. jostii, Acinetobacter sp., A. niger or N. crassa or a functional homologue of any of the aforementioned sharing at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 98% sequence identity therewith.
• suitable wild-type OMT polypeptides are known.
• a suitable wild-type OMT polypeptide includes the OMT made by H. sapiens, A. thaliana, or Fragaria x ananassa (see GENBANK Accession Nos. NM_000754, AY062837; and AF220491 ), as well as OMT polypeptides isolated from a variety of other mammals, plants or microorganisms.
• ACAR polypeptides are known.
• An ACAR polypeptide according to the present invention may be any enzyme having aromatic carboxylic acid reductase activity.
• the ACAR polypeptide is an enzyme capable of catalyzing conversion protocatechuic acid to protocatechuic aldehyde and/or conversion of vanillic acid to vanillin.
• An ACAR polypeptide according to the present invention is preferably an enzyme classified under EC 1.2.1.30.
• a suitable ACAR polypeptide is made by Nocardia sp. See, e.g., GENBANK Accession No. AY495697.
• the recombinant host may include a heterologous nucleic acid encoding the ACAR polypeptide of Nocardia sp. or a functional homologue thereof sharing at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 98% sequence identity therewith.
• Suitable PPTase polypeptides are known.
• a PPTase polypeptide according to the present invention may be any enzyme capable of catalyzing phosphopantetheinylation.
• the PPTase polypeptide is an enzyme capable of catalyzing phosphopantetheinylation of ACAR.
• a suitable PPTase polypeptide is made by E. coli, Corynebacterium glutamicum (C. glutamicum), or Nocardia farcinica (N. farcinica). See GENBANK Accession Nos. NP_601 186, BAA35224, and YP_120266.
• the recombinant host may include a heterologous nucleic acid encoding the PPTase polypeptide of E. coli, C. glutamicum, or N. farcinica or a functional homologue of any of the aforementioned sharing at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 98% sequence identity therewith.
• a 4-(hydroxymethyl)-2-methoxyphenol alcohol oxidase (VAO) enzyme (EC 1.1.3.38) can also be expressed by host cells to oxidize any formed 4-(hydroxymethyl)-2-methoxyphenol alcohol into vanillin.
• VAO enzymes are known in the art and include, but are not limited to enzymes from filamentous fungi such as Fusarium onilifomis (F. onilifomis; GENBANK Accession No. AFJ11909) and P. simplicissium (GENBANK Accession No. P56216; Benen, et al. (1998) J. Biol. Chem.
• an endogenous polypeptide in order to divert metabolic intermediates toward biosynthesis.
• pyruvate decarboxylase (PDC1 ) and/or glutamate dehydrogenase activity can be reduced.
• a nucleic acid that inhibits expression of the polypeptide or gene product may be included in a recombinant construct that is transformed into the strain.
• mutagenesis can be used to generate mutants in genes for which it is desired to inhibit function.
• a functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide.
• a functional homolog and the reference polypeptide can be natural occurring polypeptides, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, can themselves be functional homologs.
• Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides ("domain swapping").
• Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide:polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs.
• the term "functional homolog” is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
• Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of vanillin biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of nonredundant databases using a COMT, AROM, 3DSD, ACAR. VAO, OMT, or PPTase amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a vanillin biosynthesis polypeptide.
• Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in vanillin biosynthesis polypeptides, e.g., conserved functional domains.
• conserveed regions can be identified by locating a region within the primary amino acid sequence of a vanillin biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. The information included at the Pfam database is described in Sonnhammer et al., Nucl.
• conserveed regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate.
• polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions.
• conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity).
• a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
• polypeptides suitable for producing vanillin in a recombinant host include functional homologs of COMT, AROM, 3DSD, ACAR, VAO, OMT, or PPTase.
• Methods to modify the substrate specificity of, for example, COMT, AROM, 3DSD, ACAR, VAO, OMT, or PPTase are known to those skilled in the art, and include without limitation site-directed/rational mutagenesis approaches, random directed evolution approaches and combinations in which random mutagenesis/saturation techniques are performed near the active site of the enzyme. For example see Osmani et al., Phytochemistry 70 (2009) 325-347.
• a candidate sequence typically has a length that is from 80% to 200% of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 1 10, 1 15, 120, 130, 140, 150, 160, 170, 180, 190, or 200% of the length of the reference sequence.
• a functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g., 90, 93, 95, 97, 99, 100, 105, 110, 1 15, or 120% of the length of the reference sequence, or any range between.
• A% identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows.
• a reference sequence e.g., a nucleic acid sequence or an amino acid sequence described herein
• ClustalW version 1.83, default parameters
• ClustalW calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments.
• word size 2; window size: 4; scoring method:%age; number of top diagonals: 4; and gap penalty: 5.
• gap opening penalty 10.0; gap extension penalty: 5.0; and weight transitions: yes.
• the ClustalW output is a sequence alignment that reflects the relationship between sequences.
• ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk/clustalw).
• %-identity of a candidate nucleic acid or amino acid sequence to a reference sequence the sequences are aligned using ClustalW, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the% identity value can be rounded to the nearest tenth. For example, 78.11 , 78.12, 78.13, and 78.14 are rounded down to 78.1 , while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
• COMT, AROM, 3DSD, ACAR, VAO, OMT, or PPTase can include additional amino acids that are not involved in glucosylation or other enzymatic activities carried out by the enzyme, and thus such a polypeptide can be longer than would otherwise be the case.
• a recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired.
• a coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence.
• the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.
• the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid.
• the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism.
• a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct.
• stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found.
• regulatory region refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5 ' and 3 ' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof.
• a regulatory region typically comprises at least a core (basal) promoter.
• a regulatory region also can include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
• a regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence.
• the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter.
• a regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
• regulatory regions The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region can be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
• One or more genes can be combined in a recombinant nucleic acid construct in "modules" useful for a discrete aspect of vanillin production. Combining a plurality of genes in a module, particularly a polycistronic module, facilitates the use of the module in a variety of species.
• nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid.
• codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host (e.g., microorganism).
• these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.
• an endogenous polypeptide in order to divert metabolic intermediates towards vanillin biosynthesis.
• it can be desirable to downregulate synthesis of sterols in a yeast strain in order to further increase vanillin production, e.g., by downregulating squalene epoxidase.
• it can be desirable to inhibit degradative functions of certain endogenous gene products, e.g., glycohydrolases that remove glucose moieties from secondary metabolites or phosphatases as discussed herein.
• expression of membrane transporters involved in transport of vanillin can be inhibited, such that secretion of glycosylated vanillin is inhibited.
• Such regulation can be beneficial in that secretion of vanillin can be inhibited for a desired period of time during culture of the microorganism, thereby increasing the yield of glucoside product(s) at harvest.
• a nucleic acid that inhibits expression of the polypeptide or gene product can be included in a recombinant construct that is transformed into the strain.
• mutagenesis can be used to generate mutants in genes for which it is desired to inhibit function.
• Recombinant hosts can be used to express polypeptides for the production of vanillin, including mammalian, insect, and plant cells.
• a number of prokaryotes and eukaryotes are also suitable for use in constructing the recombinant microorganisms described herein, e.g., gram-negative bacteria, yeast and fungi.
• a species and strain selected for use as a vanillin production strain is first analyzed to determine which production genes are endogenous to the strain and which genes are not present. Genes for which an endogenous counterpart is not present in the strain are assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function(s).
• prokaryotic and eukaryotic species are described in more detail below. However, it will be appreciated that other species can be suitable.
• suitable species can be in a genus such as Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Eremothecium, Escherichia, Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces or Yarrowia.
• Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Cyberlindnera jadinii, Physcomitrella patens, Rhodoturula glutinis 32, Rhodoturula mucilaginosa, Phaffia rhodozyma UBV-AX, Xanthophyllomyces dendrorhous, Fusarium fujikuroi/Gibberella fujikuroi, Candida utilis, Candida glabrata, Candida albicans, C. glutamicum, and Y. lipolytica.
• a microorganism can be an Ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, S. pombe, A. niger, Y. lipolytica, Ashbya gossypii, or S. cerevisiae.
• a microorganism can be a prokaryote such as, for example but not limiting to, E. coli (see e.g., Zhang et ai, J Ind Microbiol Biotechnol. 2013 Jun;40(6):643-51 ), C. glutamicum, Rhodobacter sphaeroides, or Rhodobacter capsulatus. It will be appreciated that certain microorganisms can be used to screen and test genes of interest in a high throughput manner, while other microorganisms with desired productivity or growth characteristics can be used for large-scale production of vanillin.
• S. cerevisiae is a widely used chassis organism in synthetic biology, and can be used as the recombinant microorganism platform. There are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S. cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant microorganisms.
• a vanillin biosynthesis gene cluster can be expressed in yeast using any of a number of known promoters.
• Aspergillus species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production, and can also be used as the recombinant microorganism platform. Nucleotide sequences are available for genomes of A. nidulans, A. fumigatus, A. oryzae, A. clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergillus, as well as transcriptomic studies and proteomics studies. A. niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for the production of food ingredients such as vanillin.
• E. coli another widely used platform organism in synthetic biology, can also be used as the recombinant microorganism platform. Similar to Saccharomyces, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield . Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms.
• the vanillin precursors for producing large amounts of vanillin are already produced by endogenous genes.
• modules containing recombinant genes for vanillin biosynthesis polypeptides can be introduced into species from such genera without the necessity of introducing mevalonate or MEP pathway genes.
• Arxula adeninivorans (Blastobotrys adeninivorans)
• Arxula adeninivorans is a dimorphic yeast (it grows as a budding yeast like the baker's yeast up to a temperature of 42°C, above this threshold it grows in a filamentous form) with unusual biochemical characteristics. It can grow on a wide range of substrates and can assimilate nitrate. It has successfully been applied to the generation of strains that can produce natural plastics or the development of a biosensor for estrogens in environmental samples.
• Y. lipolytica is a dimorphic yeast (see Arxula adeninivorans) that can grow on a wide range of substrates. It has a high potential for industrial applications.
• Candida boidinii is a methylotrophic yeast (it can grow on methanol). Like other methylotrophic species such as Hansenula polymorpha and Pichia pastoris, it provides an excellent platform for the production of heterologous proteins. Yields in a multigram range of a secreted foreign protein have been reported.
• a computational method, I PRO recently predicted mutations that experimentally switched the cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH.
• Hansenula polymorpha is another methylotrophic yeast (see Candida boidinii). It can furthermore grow on a wide range of other substrates; it is thermo-tolerant and can assimilate nitrate (see also Kluyveromyces lactis). It has been applied to the production of hepatitis B vaccines, insulin and interferon alpha-2a for the treatment of hepatitis C, furthermore to a range of technical enzymes. Kluyveromyces lactis
• Kluyveromyces lactis is yeast regularly applied to the production of kefir. It can grow on several sugars, most importantly on lactose which is present in milk and whey. It has successfully been applied among others to the production of chymosin (an enzyme that is usually present in the stomach of calves) for the production of cheese. Production takes place in fermenters on a 40,000 L scale.
• Pichia pastoris is a methylotrophic yeast (see Candida boidinii and Hansenula polymorpha). It provides an efficient platform for the production of foreign proteins. Platform elements are available as a kit and it is worldwide used in academia for the production of proteins. Strains have been engineered that can produce complex human N-glycan (yeast glycans are similar but not identical to those found in humans).
• Physcomitrella mosses when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera is becoming an important type of cell for production of plant secondary metabolites, which can be difficult to produce in other types of cells.
• Carbon sources of use in the instant method include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the vanillin.
• suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, ethanol, glycerol, glucose, cellulose, starch, cellobiose or other glucose containing polymer.
• sucrose e.g., as found in molasses
• fructose xylose
• ethanol glycerol
• glucose e.glycerol
• the carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during the fed-batch phase.
• Recombinant hosts described herein can be used in methods to produce vanillin.
• the method can include growing the recombinant microorganism in a culture medium under conditions in which vanillin biosynthesis genes are expressed.
• the recombinant microorganism can be grown in a fed batch or continuous process.
• the recombinant microorganism is grown in a fermentor at a defined temperature(s) for a desired period of time.
• microorganisms include, but are not limited to S. cerevisiae, A. niger, A. oryzae, E. coli, L. lactis and B. subtilis.
• the constructed and genetically engineered microorganisms provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, continuous perfusion fermentation, and continuous perfusion cell culture.
• Levels of substrates, intermediates and side products e.g., dehydroshikimic acid, protocatechuic acid, protocatechuic aldehyde, vanillic acid, protocatechuic alcohol, 4-(hydroxymethyl)-2-methoxyphenol alcohol, vanillin ⁇ -D-glucoside can be determined by extracting samples from culture medium for analysis according to published methods.
• vanillin can then be recovered from the culture using various techniques known in the art.
• a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out.
• vanillin can be extracted from the plant tissue using various techniques known in the art. For example, a crude lysate of the cultured microorganism or plant tissue can be centrifuged to obtain a supernatant.
• the resulting supernatant can then be applied to a chromatography column, e.g., a C18 column such as Aqua® C18 column from Phenomenex or a SynergiTM Hydro RP 80A column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as acetonitrile or methanol.
• a chromatography column e.g., a C18 column such as Aqua® C18 column from Phenomenex or a SynergiTM Hydro RP 80A column
• washed with water to remove hydrophilic compounds
• a solvent such as acetonitrile or methanol.
• the compound(s) can then be further purified by preparative HPLC. See also WO 2009/140394, which is incorporated by reference in its entirety.
• vanillin can be produced using whole cells that are fed raw materials that contain precursor molecules.
• the raw materials may be fed during cell growth or after cell growth.
• the whole cells may be in suspension or immobilized.
• the whole cells may be in fermentation broth or in a reaction buffer.
• a permeabilizing agent may be required for efficient transfer of substrate into the cells.
• a recombinant microorganism can be grown in a mixed culture to produce vanillin.
• a first microorganism can comprise one or more biosynthesis genes for producing vanillin while a second microorganism comprises one or more vanillin biosynthesis genes.
• a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermentor.
• vanillin can then be recovered from the culture using various technigues known in the art, e.g., isolation and purification by extraction, vacuum distillation and multi-stage re- crystallization from aqueous solutions and ultrafiltration (Boddeker, et al. (1997) J. Membrane Sci. 137:155-8; Borges da Silva, et al. (2009) Chem. Eng. Des. 87:1276-92).
• Two-phase extraction processes employing either sulphydryl compounds, such as dithiothreitol, dithioerythritol, glutathione, or L -cysteine (US Patent No.
• Ultrafiltration and membrane contactor (MC) techniques have also been evaluated to recover vanillin (Zabkova, et al. (2007) J. Membr. Sci. 301 :221 -37; Scuibba, et al. (2009) Desalination 241 :357-64).
• conventional techniques such as percolation or supercritical carbon dioxide extraction and reverse osmosis for concentration could be used.
• the vanillin is isolated and purified to homogeneity (e.g., at least 90%, 92%, 94%, 96%, or 98% pure).
• the vanillin is isolated as an extract from a recombinant host.
• vanillin may be isolated, but not necessarily purified to homogeneity.
• the amount of vanillin produced can be from about 1 mg/l to about 20,000 mg/L or higher.
• the recombinant microorganism can be cultured for from 1 day to 7 days, from 1 day to 5 days, from 3 days to 5 days, about 3 days, about 4 days, or about 5 days .
• a vanillin composition has a reduced level of contaminants relative to a vanilla extract or fermented vanillin sample, wherein at least one of said contaminants can be found in Tables 1-4 and Figure 6.
• Table 1 Potential classes of contaminants in a vanilla extract or vanillin sample.
• Table 2 Potential contaminants in a vanilla extract or vanillin sample.
• Table 3 Potential contaminants in a vanilla extract or vanillin sample.
• dioctyl phthalate cis-18- cis-20- isovaleric acid 4-(2-propenyl0- heptacosene-2,4- nonacosene-2,4- dione dione 2,6- dimethoxyphenol valeraldehyde acetal 4-methyl-2- 2-methyl-2-butenal N-amyl alcohol pentanone
• caproic acid 1 -octen-3-ol valeraldehyde ethyl caproate, 1 H-pyrrole-2- diethyl acetal octanal carboxaldehyde furfuryl alcohol p-cymene D-limonene benzyl alcohol gamma- hexalactone gamma-terpinene heptanoic acid 1 -octanol P-cresol hexanal diethyl acetal Compounds
• the compounds in Tables 2-4 which include contaminating compounds, can, inter alia, contribute to off-flavors.
• Table 2 includes compounds Generally Recognized as Safe (GRAS).
• Table 3 includes compounds presented in the literature as being present in fermentation-derived vanillin compositions and in vanilla extracts.
• Table 4 includes compounds found in vanilla extracts from plants grown in Madagascar, Kenya, and Indonesia. See e.g. Zhang and Mueller, J. Agric. Food Chem. 60: 10433-44 (2012).
• the culture medium of a recombinant host does not comprise one or a plurality of the compounds of Tables 1 -4 prior to fermentation. In some embodiments, the culture medium of a recombinant host does not comprise one or a plurality of the compounds of Tables 1-4 after fermentation.
• Vanillin compositions produced herein can be analyzed using methods known in the art including, but not limited to, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), and infrared spectroscopy (IR).
• LC-MS of analysis of vanillin and vanillin precursors is described in Jager et ai, Journal of Chromatography A. 1 145: 83-8 (2007), which is incorporated by reference in its entirety.
• mass spectrometry provides qualitative and/or quantitative data by measuring the masses and abundances of ions in the gas phase.
• MS can be used to determine properties such as molecular weight, molecular structure, mixture components, sample concentration, and sample purity. This sensitive technique can also be used to measure reaction progress and distinguish between substances with the same retention time.
• a mass spectrometer is composed of (a) an ion source, (b) a mass analyzer, and (c) a detector. Prior to separation in the mass spectrometer, molecules are ionized; two methods used to ionize molecules are electron ionization and chemical ionization. An electric field deflects ions in complicated trajectories while migrating from the ionization chamber to the detector.
• Vanillin obtained by the methods disclosed herein can be used to make food and beverage products, and dietary supplements.
• compositions produced by a recombinant microorganism described herein can be incorporated into food products.
• a vanillin composition produced by a recombinant organism can be incorporated into a food product in an amount ranging from about 1.5 mg vanillin/kg food product to about 2000 mg vanillin/kg food product on a dry weight basis, depending on the type of food product.
• a vanillin composition produced by a recombinant organism can be incorporated into a cold confectionary (e.g., ice cream), hard candy, or chocolate such that the food product has a maximum of about 95 mg/kg, 200 mg/kg, or 970 mg vanillin/kg food on a dry weight basis, respectively.
• a vanillin composition produced by a recombinant microorganism can be incorporated into a baked good (e.g., a biscuit) such that the food product has a maximum of about 200 mg vanillin/kg food on a dry weight basis.
• a vanillin composition produced by a recombinant microorganism can be incorporated into a beverage (e.g., a carbonated beverage) such that the beverage has a maximum of about 100 mg vanillin/kg.
• Vanillin sugar sold in supermarkets contains about 12500 mg vanillin/kg. See e.g., FEMA, Scientific Literature Review of Vanillin and Derivatives (1985).
• Example 1 Construction of an AROM Lacking Domain 5
• the 5'-nearest 3912 bp of the yeast AR01 gene which includes all functional domains except domain 5 (having the shikimate dehydrogenase activity), was isolated by PCR amplification from genomic DNA prepared from S. cerevisiae strain S288C, using proof-reading PCR polymerase. The resulting DNA fragment was sub-cloned into the pTOPO vector and sequenced to confirm the DNA sequence.
• the nucleic acid sequence and corresponding amino acid sequence are presented in SEQ ID NO:1 and SEQ ID NO:2, respectively.
• This fragment was subjected to a restriction digest with Spel and Sail and cloned into the corresponding restriction sites in the high copy number yeast expression vector p426-GPD (a 2 ⁇ -based vector), from which the inserted gene can be expressed by the strong, constitutive yeast GPDI promoter.
• the resulting plasmid was designated pVAN133.
• All mutant AROM polypeptides described in this example are polypeptides of SEQ ID NO:4, wherein one amino acid has been substituted for another amino acid.
• the mutant AROM polypeptides are named as follows: XnnnY, where nnn indicates the position in SEQ ID NO:4 of the amino acid, which is substituted, X is the one letter code for the amino acid in position nnn in SEQ ID NO:4 and Y is the one letter code for the amino acid substituting X.
• A1533P refers to a mutant AROM polypeptide of SEQ ID NO:4, where the alanine at position 1533 is replaced with a proline.
• the full 4764 bp yeast AR01 gene was isolated by PCR amplification from genomic DNA prepared from S. cerevisiae strain S288C, using proof-reading PCR polymerase. The resulting DNA fragment was sub-cloned into the pTOPO vector and sequenced to confirm the DNA sequence. The nucleic acid sequence and corresponding amino acid sequence are presented in SEQ ID NO:3 and SEQ ID NO:4, respectively. This fragment was subjected to a restriction digest with Spel and Sail and cloned into the corresponding restriction sites in the low copy number yeast expression vector p416-TEF (a CEN-ARS-based vector), from which the gene can be expressed from the strong TEF promoter. The resulting plasmid was designated pVAN183.
• Plasmid pVANI83 was used to make 10 different domain 5 mutants of AR01, using the QUICKCHANGE II Site-Directed Mutagenesis Kit (Agilent Technologies). With reference to SEQ ID NO:4, the mutants contained the following amino acid substitutions: A1533P, P1500K, R1458W, V1349G, T1366G, I 1387H, W1571V, T1392K, K1370L and A1441 P.
• the expression plasmids containing the A1533P, P1500K, R1458W, V1349G, T1366G, I 1387H, W1571V, T1392K, K1370L and A1441 P substitutions were designated pVAN368-pVAN377, respectively.
• the 5'-nearest 3951 bp of the yeast AR01 gene which includes all functional domains except domain 5 with the shikimate dehydrogenase activity, was isolated by PCR amplification from genomic DNA prepared from S. cerevisiae strain S288C, using proof-reading PCR polymerase. The resulting DNA fragment was sub-cloned into the pTOPO vector and sequenced to confirm the DNA sequence. In order to fuse this fragment to the 3- dehydroshikimate dehydratase (3DSD) gene from the vanillin pathway, the 3DSD gene from P. pauciseta (Hansen, et al.
• pVAN132 The nucleic acid sequence and corresponding amino acid sequence of this fusion protein are presented in SEQ ID NO:6 and SEQ ID NO:7, respectively.
• VAO genes were isolated and cloned into a yeast expression vector.
• the expression vectors were subsequently transformed into a yeast strain expressing glucosyltransferase.
• the transformed strains were tested for VAO activity by growing the yeast for 48 h in medium supplemented with 3 mM 4-(hydroxymethyl)-2- methoxyphenol alcohol.
• the results of this analysis are presented in Figure 4.
• VAO enzymes from both P. simplicissium and R. jostii exhibited activity in yeast.
• Example 5 ACAR Gene from N. crassa
• the vector was transformed into a yeast strain expressing a PPTase, strains were selected for the presence of the ACAR gene, and the selected yeast was cultured for 72 h in medium supplemented with 3 mM vanillic acid to demonstrate ACAR activity.
• the results of this analysis are presented in Figure 5.
• the N. crassa ACAR enzyme was found to exhibit a higher activity in yeast than the N. iowensis ACAR. Therefore, in some embodiments of the method disclosed herein, a N. crassa ACAR enzyme is used in the production of vanillin.
• N. iownsis or N. crassa ACAR proteins may be used, including but not limited to, those isolated from Nocardia brasiliensis (N. brasiliensis; GENBANK Accession No. EHY26728), N. farcinica (GENBANK Accession No. BAD56861 ), P. anserina (GENBANK Accession No. CAP62295), or Sordaria macropora (S. macropora; GENBANK Accession No. CCC14931 ), which significant sequence identity with the N. iownsis or N. crassa ACAR protein.
• Example 6 Mass spectrometry analysis of vanillin produced by fermentation
• Elution was carried out using a mobile phase of eluent A (0.1 % Formic acid in water) and eluent B (0.1 % Formic acid in Acetonitrile) by increasing the gradient from 1 - 50% B from min 0.0 to 3.0 and increasing the gradient from 50- 100% B in min 3.0 to 4.0.
• Vanillin, potential vanillin contaminants, and analytical standards were detected using SIM (Single Ion Monitoring) in positive mode.
• the compounds are considered present in the sample if they have the same retention time as well as the same monoisotopic mass value.
• the extracted ion chromatograms in Figure 8 do not show presence of ferulic acid, ethyl vanillin, mandelic acid, eugenol, isoeugenol, and guiacol in the vanillin sample produced by fermentation.
• the peak in Figure 8 eluting at 2.45 min represents a fragment of the vanillin ion and does not represent presence of guaiacol, which elutes at 2.85 min.

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