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

摘要

The invention relates to cells, nucleic acids, and enzymes, the use thereof for producing sophorolipids, and methods for producing sophorolipids.

主权项

1. An isolated or purified sophorolipid-producing cell: (A) transformed with a nucleic acid encoding an E4 polypeptide; or (B) modified to disrupt at least one endogenous gene encoding an E4 polypeptide;
wherein said E4 comprises (a) the amino acid sequence of SEQ ID NO: 9; or (b) 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 ′,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; wherein said cell may optionally contain a nucleic acid encoding at least one E1, E2, E3 or E5 polypeptide or wherein said cell may optionally have a disruption in an endogenous gene encoding an E3 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) a variant of the amino acid sequence of SEQ ID NO: 7, 53, 55, 57, 59, 61 or 63 which is identical to 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 catalyzes the conversion of Z-9-octadecenoic acid into 17-hydroxy-Z-9-octadecenoic acid; E2 comprises (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 11; or (b) a variant of the amino acid sequence of SEQ ID NO: 8 or 11 which is identical to the amino acid sequence of SEQ ID NO: 8 or 11 except that at least one residue up to 5% of the amino acid residues of SEQ ID NO: 8 or 11 have been modified by deletion, substitution, and/or insertion, wherein the E2 polypeptide catalyzes the conversion of UDP-glucose and 17-hydroxy-Z-9-octadecenoic acid into 17-(β-D-glucopyranosyloxy)-Z-9-octadecenoic acid; E3 comprises (a) the amino acid sequence of SEQ ID NO: 11; or (b) 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; and E5 comprises (a) the amino acid sequence of SEQ ID NO: 10; or (b) 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.