Improved method for the production of natural vanillin

A process for producing vanillin, comprising the steps of

• i. Providing an aqueous medium comprising a solvent, one or more eugenol oxidase, one or more alkene cleavage oxygenase and eugenol,
• ii. Incubating the aqueous medium and
• iii. Optionally isolating the vanillin, from the reaction mixture,
• wherein the one or more eugenol oxidase is capable of catalysing the reaction from eugenol to coniferyl alcohol in an aqueous medium comprising water, eugenol oxidase and eugenol, and
• wherein the one or more alkene cleavage oxygenase is capable of catalysing the reaction from coniferyl alcohol to vanillin in an aqueous medium comprising water, alkene cleavage oxygenase and coniferyl alcohol,
• wherein after incubation at least 20% of the eugenol have been converted to vanillin.

A recombinant construct comprising a nucleic acid molecule encoding a eugenol oxidase and a nucleic acid molecule encoding a alkene cleavage oxygenase,

• wherein the eugenol oxidase is comprising a sequence encoding an amino acid molecule selected from the group consisting of
• a. The amino acid molecule of SEQ ID NO:
• 2 and
• b. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO:
• 2 or a functional fragment thereof,
• c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO:
• 1 or a functional fragment thereof, and
• d. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO:
• 1 or a functional fragment thereof, and
• wherein the amino acid molecule as defined in b, c, d is catalysing the reaction from eugenol to coniferyl alcohol in an aqueous medium, and
• wherein the alkene cleavage oxygenase is comprising a sequence encoding an amino acid molecule selected from the group consisting of
• e. The amino acid molecule of SEQ ID NO:
• 30, 24, 26, 38, 40, 64, 66, 74, 76, 82, 98, 120, 128, 130, 132, 134 or 135, and
• f. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO:
• 30, 24, 26, 38, 40, 64, 66, 74, 76, 82, 98, 120, 128, 130, 132, 134 or 135, or a functional fragment thereof, and
• g. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO:
• 29, 23, 25, 37, 39, 63, 65, 73, 75, 81, 97, 119, 127, 129, 131, or 133, or a functional fragment thereof, and
• h. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO:
• 29, 23, 25, 37, 39, 63, 65, 73, 75, 81, 97, 119, 127, 129, 131, or 133, or a functional fragment thereof, and
• wherein the amino acid molecule as defined in f, g, h is catalysing the reaction from coniferyl alcohol to vanillin in an aqueous medium.

A composition comprising water, one or more eugenol oxidase and one or more alkene cleavage oxygenase, eugenol,

• wherein the eugenol oxidase is selected from the group consisting of
• a. The amino acid molecule of SEQ ID NO:
• 2 and
• b. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO:
• 2 or a functional fragment thereof, and
• c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO:
• 1 or a functional fragment thereof, and
• d. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO:
• 1 or a functional fragment thereof, and
• wherein the amino acid molecule as defined in b, c, d is catalysing the reaction from eugenol to coniferyl alcohol in an aqueous medium and
• wherein the alkene cleavage oxygenase is selected from the group consisting of
• e. The amino acid molecule of SEQ ID NO:
• 30, 24, 26, 38, 40, 64, 66, 74, 76, 82, 98, 120, 128, 130, 132, 134 or 135, and
• f. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO:
• 30, 24, 26, 38, 40, 64, 66, 74, 76, 82, 98, 120, 128, 130, 132, 134 or 135, or a functional fragment thereof, and
• g. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO:
• 29, 23, 25, 37, 39, 63, 65, 73, 75, 81, 97, 119, 127, 129, 131, or 133, or a functional fragment thereof, and
• h. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO:
• 29, 23, 25, 37, 39, 63, 65, 73, 75, 81, 97, 119, 127, 129, 131, or 133, or a functional fragment thereof, and
• wherein the amino acid molecule as defined in f, g, h is catalysing the reaction from coniferyl alcohol to vanillin in an aqueous medium.

A recombinant microorganism comprising an introduced, increased or enhanced activity and/or expression of one or more eugenol oxidase and one or more alkene cleavage oxygenase,

• wherein the one or more eugenol oxidase is capable of catalysing the reaction from eugenol to coniferyl alcohol, and
• wherein the one or more alkene cleavage oxygenase is capable of catalysing the reaction from coniferyl alcohol to vanillin, and
• wherein the eugenol oxidase is comprising a sequence selected from the group consisting of
• a. The amino acid molecule of SEQ ID NO:
• 2 and
• b. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO:
• 2 or a functional fragment thereof, and
• c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO:
• 1 or a functional fragment thereof, and
• d. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO:
• 1 or a functional fragment thereof, and
• wherein the amino acid molecule as defined in b., c., d is catalysing the reaction from eugenol to coniferyl alcohol and
• wherein the alkene cleavage oxygenase is comprising a sequence selected from the group consisting of
• e. The amino acid molecule of SEQ ID NO:
• 30, 24, 26, 38, 40, 64, 66, 74, 76, 82, 98, 120, 128, 130, 132, 134 or 135, and
• f. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO:
• 30, 24, 26, 38, 40, 64, 66, 74, 76, 82, 98, 120, 128, 130, 132, 134 or 135, or a functional fragment thereof, and
• g. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO:
• 29, 23, 25, 37, 39, 63, 65, 73, 75, 81, 97, 119, 127, 129, 131, or 133, or a functional fragment thereof, and
• h. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO:
• 29, 23, 25, 37, 39, 63, 65, 73, 75, 81, 97, 119, 127, 129, 131, or 133, or a functional fragment thereof, and
• wherein the amino acid molecule as defined in f, g, h is catalysing the reaction from coniferyl alcohol to vanillin.