title: Dirhamnose-lipid production by recombinant nonpathogenic bacterium Pseudomonas chlororaphis.
abstract: We previously discovered that Pseudomonas chlororaphis NRRL B-30761 produces monorhamnolipids (R1Ls) with predominantly 3-hydroxydodecenoyl-3-hydroxydecanoate (C12:1-C10) or 3-hydroxydodecanoyl-3-hydroxydecanoate (C12-C10) as the lipid moiety under static growth conditions only. We have now cloned, sequenced, and analyzed in silico the gene locus of NRRL B-30761 containing the putative coding sequences of rhamnosyltransferase chain A (rhlA Pch , 894 bps), rhamnosyltransferase chain B (rhlB Pch , 1272 bps), and N-acyl-homoserine lactone-dependent transcriptional regulatory protein (rhlR Pch , 726 bps). The putative gene products RhlAPch (297 amino acid residues or a.a.), RhlBPch (423 a.a.), and RhlRPch (241 a.a.) only have between 60 and 65% a.a. identities to their respective closest matched homologs in P. aeruginosa. Polymerase chain reaction (PCR)-based assay did not detect the presence of rhamnosyltransferase C gene (rhlC) in P. chlororaphis, suggesting a genetic basis for the lack of dirhamnose-lipid (R2L) synthesis in this organism. We thus genetically constructed an R2L-synthesizing P. chlororaphis by expressing a rhamnosyltransferase C (rhlC) gene of P. aeruginosa using an expression vector (pBS29-P2-gfp) containing a Pseudomonas syringae promoter. The R2L/R1L ratio is 2.4 in the rhamnolipid (RL) sample isolated from the genetically engineered (GE) P. chlororaphis [pBS29-P2-rhlC], in contrast to undetectable R2L in the GE P. chlororaphis [pBS29-P2-gfp] control cells based on LC-MS analysis. The critical micelle concentrations of the R2L and R1L samples from GE P. chlororaphis [pBS29-P2-rhlC] and the control [pBS29-P2-gfp] cells were ca. 0.1 mM, and their minimum surface tensions were ca. 26 mN/m with no significant difference.
authors: Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 E. Mermaid Lane, Wyndmoor, PA, 19038, USA, dan.solaiman@ars.usda.gov.
authors: Daniel K Y
authors: DK
authors: Solaiman
authors: Richard D
authors: RD
authors: Ashby
authors: Nereus W
authors: NW
authors: Gunther
authors: Jonathan A
authors: JA
authors: Zerkowski
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006017:Glycolipids / Q000096:biosynthesis*; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D006017:Glycolipids; C051310:rhamnolipid hemolysin, Pseudomonas aeruginosa; D006602:Hexosyltransferases