Production of dirhamnose-lipid in recombinant nonpathogenic bacterium Pseudomonas chlororaphis

Patent metadata

Field	Value
Publication number	US-10190144-B2
Application number	US-201414892793-A
Country	US
Kind code	B2
Filing date	Jun 3, 2014
Priority date	Jun 6, 2013
Publication date	Jan 29, 2019
Grant date	Jan 29, 2019

Abstract

Pseudomonas chlororaphis NRRL B-30761 produces monorhamnolipids with predominantly 3-hydroxydodecenoyl-3-hydroxydecanoate (C 12:1 -C 10 ) or 3-hydroxydodecanoyl-3-hydroxydecanoate (C 12 -C 10 ) as the lipid moiety under static growth conditions. The cloning and sequencing of three genes and proteins involved in the biosynthesis of monorhamnose-lipid (R 1 L) is described. Expression of two of these genes, i.e., rhlA and rhlB, together in P. chlororaphis NRRL B-30761 increases R 1 L production by at least 10-fold. Also the generation of a recombinant P. chlororaphis NRRL B-30761 capable of synthesizing dirhamnose-lipid (R 2 L) is described. Characterization of R 1 L and R 2 L produced by the recombinant P. chlororaphis NRRL B-30761 is also described.

First claim

We, the inventors, claim:

1. A recombinant Pseudomonas chlororaphis that is derived from P. chlororaphis NRRL B-30761, said recombinant P. chlororaphis further comprises an expression vector, wherein said expression vector comprises a promoter operably linked to a heterologous polynucleotide encoding a heterologous Pseudomonas rhamnosyltransferase C, wherein rhlA and rhlB genes in said recombinant P. chlororaphis are operably linked to endogenous promoters.

2. The recombinant Pseudomonas chlororaphis of claim 1 wherein said heterologous Pseudomonas rhamnosyltransferase C comprises the amino acid sequence of SEQ ID NO: 26.

3. The recombinant Pseudomonas chlororaphis of claim 2 wherein said heterologous Pseudomonas rhamnosyltransferase C comprises the nucleotide sequence of SEQ ID NO: 25.

4. A method of producing dirhamnose-lipid comprising
culturing said recombinant P. chlororaphis of claim 1 in medium capable of supporting rhamnose-lipid biosynthesis.

5. The method of claim 4 wherein said heterologous Pseudomonas rhamnosyltransferase C comprises the amino acid sequence of SEQ ID NO: 26.

6. The method of claim 5 wherein said heterologous Pseudomonas rhamnosyltransferase C comprises the nucleotide sequence of SEQ ID NO: 25.

7. A method for producing dirhamnose-lipid comprising growing said recombinant Pseudomonas chlororaphis of claim 1 in medium capable of supporting dirhamnose-lipid biosynthesis.

8. The method of claim 7 , wherein said heterologous rhamnosyltransferase C comprises the amino acid sequence of SEQ ID NO: 26.

9. The method of claim 8 , wherein said heterologous rhamnosyltransferase C comprises the nucleotide sequence of SEQ ID NO: 25.

Assignees

• Us Agriculture

Inventors

• Solaiman Daniel
• Ashby Richard D
• Zerkowski Jonathan A
• Gunther Nereus W

Classifications

• C12P7/6409

  Fatty acids · CPC title

• C12Y204/01

  Hexosyltransferases (2.4.1) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• C12P19/44 Primary

  Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)} · CPC title