Production of dirhamnose-lipid in recombinant nonpathogenic bacterium pseudomonas chlororaphis

A method of producing dirhamnose-lipid comprising

• transforming a wild-type Pseudomonas chlororaphis with an expression vector comprising a promoter operably linked to DNA encoding a Pseudomonas rhamnosyltransferase C to generate a recombinant P. chlororaphis, wherein said wild-type P. chlororaphis produces rhamnosyltransferase A and rhamnosyltransferase B;
• isolating recombinant P. chlororaphis that produces said Pseudomonas rhamnosyltransferase C;
• and
• culturing said isolated recombinant P. chlororaphis in medium capable of supporting rhamnose-lipid biosynthesis.

A method for controlling the ratio of the amount of monorhamnose-lipid to the amount of dirhamnose-lipid produced by a recombinant Pseudomonas chlororaphis comprising

• transforming a wild-type Pseudomonas chlororaphis with an expression vector comprising an inducible promoter operably linked to DNA encoding a Pseudomonas rhamnosyltransferase C to generate a recombinant P. chlororaphis, wherein said wild-type P. chlororaphis produces rhamnosyltransferase A and rhamnosyltransferase B;
• isolating recombinant P. chlororaphis that produces said Pseudomonas rhamnosyltransferase C;
• culturing said isolated recombinant P. chlororaphis in medium capable of supporting rhamnose-lipid biosynthesis;
• and
• inducing expression of said inducible promoter for a set period of time so that rhamnosyltransferase C is produced and converts monorhamnose-lipid into dirhamnose-lipid for a desired amount of time to control said ratio of the amount of monorhamnose-lipid to the amount of dirhamnose-lipid.

A method for controlling the ratio of the amount of monorhamnose-lipid to the amount of dirhamnose-lipid produced by a recombinant Pseudomonas chlororaphis comprising

• transforming wild-type Pseudomonas chlororaphis with an expression vector, wherein said expression vector comprises a first inducible promoter operably linked to a first heterologous polynucleotide encoding rhamnosyltransferase A, a second inducible promoter operably linked to a second heterologous polynucleotide encoding rhamnosyltransferase B, and a third inducible promotor operably linked to a heterologous polynucleotide encoding rhamnosyltransferase C to produce a recombinant P. chlororaphis, wherein said third inducible promoter is distinct from said first inducible promoter and said second inducible promoter so that one can control the activity of said third inducible promoter independently of the activity of said first inducible promoter and the activity of said second inducible promoter;
• growing said recombinant P. chlororaphis in medium capable of supporting dirhamnose-lipid biosynthesis, and
• inducing the expression of said third inducible promoter for desired amount of time to control the amount of rhamnosyltransferase C produced, wherein said rhamnosyltransferase C converts said monorhamnose-lipid to said dirhamnose-lipid thereby controlling said ratio of the amount of monorhamnose-lipid to the amount of dirhamnose-lipid produced, wherein said wild-type P. chlororaphis does not produce rhamnosyltransferase A, rhamnosyltransferase B, nor rhamnosyltransferase C.