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

1. An isolated or purified sophorolipid-producing cell transformed with a nucleic acid encoding an E 5 polypeptide;

wherein the E 5 polypeptide (a) 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 E 5 polypeptide has the ability to transfer a sophorolipid out of the sophorolipid-producing cell into the surrounding medium;

wherein said cell may optionally contain a nucleic acid encoding at least one E 1 , E 2 , E 3 or E 4 polypeptide or wherein said cell may optionally have a disruption in an endogenous gene encoding an E 3 and/or E 4 polypeptide; wherein:

E 1 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 E 1 polypeptide catalyzes the conversion of Z-9-octadecenoic acid into 17-hydroxy-Z-9-octadecenoic acid;

E 2 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 E 2 polypeptide catalyzes the conversion of UDP-glucose and 17-hydroxy-Z-9-octadecenoic acid into 17-(β-D-glucopyranosyloxy)-Z-9-octadecenoic acid;

E 3 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 E 3 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;

E 4 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 E 4 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.

2. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell has been transformed with at least one extrachromosomally replicating vector carrying said nucleic acid(s).

3. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said nucleic acid(s) are operably linked to a promoter, a regulation region, a ribosome binding site, an expression cassette or an enhancer that increases the expression of said polypeptide.

4. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said transformed sophorolipid-producing cell expresses more of the polypeptide of SEQ ID NO: 10 than the identical non-transformed cell.

5. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said transformed sophorolipid-producing cell produces a greater yield of sophorolipids than the identical non-transformed cell.

6. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell has been transformed with a nucleic acid encoding the polypeptide of SEQ ID NO: 10.

7. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell has been transformed with a nucleic acid encoding an E 5 polypeptide that 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 E 5 polypeptide has the ability to transfer a sophorolipid out of the sophorolipid-producing cell into the surrounding medium.

8. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell is a yeast or fungal cell.

9. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell is selected from the group consisting of Candida bombicola, Candida bogoriensis, Candida batistae, Candida apicola and Wickerhamiella domericqiae.

10. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell has been further modified to block or partially block β-oxidation in said cell.

11. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell has been further transformed with a nucleic acid encoding at least one E 1 polypeptide.

12. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell has been further transformed with a nucleic acid encoding at least one E 2 polypeptide.

13. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell has been further transformed with a nucleic acid encoding at least one E 3 polypeptide.

14. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell has been further transformed with a nucleic acid encoding at least one E 4 polypeptide.

15. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell has been transformed with nucleic acid(s) encoding a combination of polypeptides selected from the group consisting of E 1 E 5 , E 2 E 5 , E 3 E 5 , E 4 E 5 , E 1 E 2 E 5 , E 1 E 3 E 5 , E 1 E 4 E 5 , E 2 E 4 E 5 , E 3 E 4 E 5 , E 2 E 3 E 4 E 5 , E 1 E 3 E 4 E 5 , E 1 E 2 E 4 E 5 , E 1 E 2 E 3 E 5 , and E 1 E 2 E 3 E 4 E 5 polypeptides.

16. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell has been modified to disrupt at least one endogenous gene encoding an E 3 polypeptide.

17. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell has been modified to disrupt at least one endogenous gene encoding an E 4 polypeptide.

18. The isolated or purified sophorolipid-producing cell of claim 1 , wherein said sophorolipid-producing cell has been further modified to disrupt at least one endogenous gene encoding an E 3 polypeptide and to disrupt at least one endogenous gene encoding an E 4 polypeptide.

19. A process for producing a sophorolipid comprising:

culturing the cell of claim 1 on a medium containing a carbon source under conditions suitable for producing a sophorolipid from the carbon source and, optionally,

isolating or recovering the sophorolipid;

wherein said cell may optionally contain a nucleic acid encoding at least one E 1 , E 2 , E 3 or E 4 polypeptide or wherein said cell may optionally have a disruption in an endogenous gene encoding an E 3 and/or E 4 polypeptide; wherein:

E 1 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 E 1 polypeptide catalyzes the conversion of Z-9-octadecenoic acid into 17-hydroxy-Z-9-octadecenoic acid;

E 2 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 E 2 polypeptide catalyzes the conversion of UDP-glucose and 17-hydroxy-Z-9-octadecenoic acid into 17-β-D-glucopyranosyloxy)-Z-9-octadecenoic acid;

E 3 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 E 3 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;

E 4 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 E 4 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.

20. The process of claim 19 , wherein said sophorolipid-producing cell has been transformed with a nucleic acid encoding the polypeptide of SEQ ID NO: 10.

21. The process of claim 19 , wherein said sophorolipid-producing cell has been transformed with a nucleic acid encoding an E 5 polypeptide that 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 E 5 polypeptide has the ability to transfer a sophorolipid out of the sophorolipid-producing cell into the surrounding medium.

22. The process of claim 19 , wherein said sophorolipid-producing cell has been transformed with nucleic acid(s) encoding a combination of polypeptides selected from the group consisting of E 1 E 5 , E 2 E 5 , E 3 E 5 , E 4 E 5 , E 1 E 2 E 5 , E 1 E 3 E 5 , E 1 E 4 E 5 , E 2 E 4 E 5 , E 3 E 4 E 5 , E 2 E 3 E 4 E 5 , E 1 E 3 E 4 E 5 , E 1 E 2 E 4 E 5 , E 1 E 2 E 3 E 5 , and E 1 E 2 E 3 E 4 E 5 polypeptides.

23. The process of claim 19 , wherein said sophorolipid-producing cell:

(a) has been modified to disrupt endogenous gene(s) encoding E 3 polypeptide(s); and

(b) has been further transformed with a nucleic acid(s) encoding E 1 , E 2 , and/or E 4 polypeptides.

24. The process of claim 19 , wherein said sophorolipid-producing cell:

(a) has been modified to disrupt endogenous gene(s) encoding E 4 polypeptide(s); and

(b) has been further transformed with a nucleic acid(s) encoding E 1 , E 2 , and/or E3 polypeptides.

25. The process of claim 19 , wherein said sophorolipid-producing cell:

(a) has been further modified to disrupt endogenous gene(s) encoding E 3 and E 4 polypeptide(s); and

(b) has been further transformed with a nucleic acid(s) encoding E 1 and/or E 2 .