Method of producing a low molecular weight organic compound in a cell
A method of producing a low molecular weight organic compound (e.g. a plant or bacteria secondary metabolite) in increased yields involving use of a microorganism cell, which comprises a gene involved in the biosynthesis pathway leading to a low molecular weight organic aglycon compound and a glycosyltransferase gene capable of glycosylating the produced aglycon.
1. A microbial cell for higher production of vanillin, said cell comprising at least the following three heterologous enzymatic activities:
i) a 3-dehydroshikimate dehydratase
ii) an aromatic carboxylic acid reductase; and
iii) a 3-O-methyl transferase;
wherein said cell has been transformed with an expression vector comprising a heterologous nucleic acid encoding an UDPG-glycosyltransferase capable of glycosylating vanillin.
2. The cell according to claim 1 , wherein the cell is E. coli.
3. The cell according to claim 1 , wherein the cell is Saccharomyces cerevisiae.
4. The cell according to claim 1 , wherein the 3-dehydroshikimate dehydratase is capable of catalysing conversion of 3-dehydroshikimate to protocatechuic acid.
5. The cell according to claim 1 , wherein the 3-dehydroshikimate dehydratase is 3-dehydroshikimate dehydratase of an organism selected from the group consisting of Neurospora crassa, Aspergillus nidulans, Podospora pauciseta and Acinetobacter calcoaceticus.
6. The cell according to claim 1 , wherein the aromatic carboxylic acid reductase is capable of catalysing conversion of protocatechuic acid to protocatechualdehyde and/or conversion of vanillic acid to vanillin.
7. The cell according to claim 1 , wherein the aromatic carboxylic acid reductase is an ATP-dependent aromatic carboxylic acid reductase.
8. The cell according to claim 1 , wherein the aromatic carboxylic acid reductase is an ATP-dependent aromatic carboxylic acid reductase of an organism selected from the group consisting of actinomycetes and basidiomycetes.
9. The cell according to claim 1 , wherein the aromatic carboxylic acid reductase is an ATP-dependent aromatic carboxylic acid reductase of an organism selected from the group consisting of Nocardia sp., Neurospora crassa, Trametes, Dichomitus, Bjerkandera and Pleurotus.
10. The cell according to claim 1 , wherein the 3-O-methyl transferase is capable of catalysing conversion of protocatechualdehyde to vanillin.
11. The cell according to claim 1 , wherein the 3-O-methyl transferase is 3-O-methyl transferase of an organism selected from the group consisting of strawberry, aspen, almond and Vanilla planifolia.
12. A method of producing vanillin comprising the steps of cultivating the microbial cell according to claim 1 and purifying vanillin from said culture.
13. A method of preparing a microbial cell for higher production of vanillin, said method comprising the steps of:
(i) providing a microbial cell transformed with an expression vector comprising a heterologous nucleic acid encoding UDPG-glycosyltransferase; and
ii) introducing into said cell a heterologous nucleic acid encoding a 3-O-methyl transferase; and
iii) introducing into the cell produced in step ii) a heterologous nucleic acid encoding an aromatic carboxylic acid reductase; and
iv) introducing into the cell produced in step iii) a heterologous nucleic acid encoding a 3-dehydroshikimate dehydratase;
thereby obtaining the microbial cell for higher production of vanillin.