Cells, nucleic acids, enzymes and use thereof, and methods for the production of sophorolipids

An isolated or purified sophorolipid-producing cell transformed with a nucleic acid encoding:

• (a) the polypeptide of SEQ ID NO:
• 8;
• or
• (b) a polypeptide which catalyzes the conversion of UDP-glucose and 17-hydroxy-Z-9-octadecenoic acid into 17-(β-D-glucopyranosyloxy)-Z-9-octadecenoic acid, wherein said polypeptide comprises a variant of the amino acid sequence of SEQ ID NO:
• 8 which is identical to SEQ ID NO:
• 8 except that at least one residue up to 5% of the amino acid residues of SEQ ID NO:
• 8 have been modified by deletion, substitution, and/or insertion;
• wherein said cell may optionally contain a nucleic acid encoding at least one E1, E3, E4 or E5 polypeptide, or wherein said cell may optionally have a disruption in an endogenous gene encoding an E3 and/or E4 polypeptide;
• wherein
• E1 comprises (a) an amino acid sequence selected from the group consisting of SEQ ID NO:
• 7, 53, 55, 57, 59, 61 and 63;
• or (b) comprises a variant of the amino acid sequence of SEQ ID NO:
• 7, 53, 55, 57, 59, 61 or 63 which is identical to the amino acid sequence of SEQ ID NO:
• 7, 53, 55, 57, 59, 61 or 63 except that at least one residue up to 5% of the amino acid residues of SEQ ID NO:
• 7, 53, 55, 57, 59, 61 or 63 have been modified by deletion, substitution, and/or insertion, wherein the E1 polypeptide has the ability to catalyze the conversion of Z-9-octadecenoic acid into 17-hydroxy-Z-9-octadecenoic acid;
• E3 comprises (a) the amino acid sequence of SEQ ID NO:
• 11;
• or (b) comprises a variant of the amino acid sequence of SEQ ID NO:
• 11 which is identical to the amino acid sequence of SEQ ID NO:
• 11 except that at least one residue up to 5% of the amino acid residues of SEQ ID NO:
• 11 have been modified by deletion, substitution, and/or insertion, wherein the E3 polypeptide has the ability to catalyze the conversion of 17-(β-D-glucopyranosyloxy)-Z-9-octadecenoic acid and UDP-glucose into 17-L-[(2′-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-Z-9-octadecenoic acid;
• E4 comprises the amino acid sequence of SEQ ID NO:
• 9;
• or (b) comprises a variant of the amino acid sequence of SEQ ID NO:
• 9 which is identical to the amino acid sequence of SEQ ID NO:
• 9 except that at least one residue up to 5% of the amino acid residues of SEQ ID NO:
• 9 have been modified by deletion, substitution, and/or insertion, wherein the E4 polypeptide has the ability to catalyze the conversion of:
• (i) 17-L-[(2′-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-Z-9-octadecenoic acid 1′,4″-lactone and acetyl-coenzyme A into 17-L-[(2′-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-Z-9-octadecenoic acid 1′,4″-lactone monoacetate;
• (ii) 17-L-[(2′-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-Z-9-octadecenoic acid 1′,4″-lactone monoacetate and acetyl-coenzyme A into 17-L-[(2′-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-Z-9-octadecenoic acid 1′,4″-lactone diacetate;
• or
• (iii) 17-L-[(2′-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-Z-9-octadecenoic acid 1′,4″-lactone and acetyl-coenzyme A into 17-L-[(2′-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-Z-9-octadecenoic acid 1′,4″-lactone diacetate;
• and
• E5 comprises the amino acid sequence of SEQ ID NO:
• 10;
• or (b) comprises a variant of the amino acid sequence of SEQ ID NO:
• 10 which is identical to the amino acid sequence of SEQ ID NO:
• 10 except that at least one residue up to 5% of the amino acid residues of SEQ ID NO:
• 10 have been modified by deletion, substitution, and/or insertion, wherein the E5 polypeptide has the ability to transfer a sophorolipid out of the sophorolipid-producing cell into the surrounding medium.