Methods of producing rhamnolipids

A method of preparing at least one rhamnolipid comprising:

• a) contacting a recombinant cell with a medium containing a carbon source wherein the recombinant cell has been genetically modified such that, compared to the wild-type of the cell, said recombinant cell has increased activity of all three of enzymes E1, E2 and E3, and wherein:
• i) enzyme E1 comprises the sequence of SEQ ID NO:2 or an enzyme comprising a sequence in which up to 10% of the amino acids of SEQ ID NO:2 have been modified and for which more than 50% of the enzymatic activity of SEQ ID NO:2 in converting 3-hydroxy-decanoyl-ACP via 3-hydroxydecanoyl-3-hydroxydecanoic acid-ACP to hydroxydecanoyl-3-hydroxydecanoic acid is maintained;
• ii) enzyme E2 comprises the sequence of SEQ ID NO:7 or an enzyme comprising a sequence in which up to 10% of the amino acids of SEQ ID NO:7 have been modified and for which more than 50% of the enzymatic activity of SEQ ID NO:7 in converting dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to a-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid is maintained;
• ii) enzyme E3 comprises the sequence of SEQ ID NO:12 or an enzyme comprising a sequence in which up to 10% of the amino acids of SEQ ID NO:12 have been modified and for which more than 50% of the enzymatic activity of SEQ ID NO:12 in converting dTDP rhamnose and a-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid to a-L-rhamnopyranosyl-(1-2)-a-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid is maintained;
• b) culturing the recombinant cell under suitable conditions for preparation of the rhamnolipid from the carbon source by the cell;
• c) optionally isolating rhamnolipids from the cells and/or the medium of step b);
• wherein at least 70% of the total carbon content of the medium in which the recombinant cells are cultured is in the form of C4 molecules having exactly four carbon atoms.