caa a22 4500 605502129 CHVBK 20210128100604.0 cr unu---uuuuu 210128e20150501xx s 000 0 eng 10.1007/s00253-015-6433-4 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6433-4 Dirhamnose-lipid production by recombinant nonpathogenic bacterium Pseudomonas chlororaphis [Elektronische Daten] [Daniel Solaiman, Richard Ashby, Nereus Gunther IV, Jonathan Zerkowski] 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 , 894bps), rhamnosyltransferase chain B (rhlB Pch , 1272bps), and N-acyl-homoserine lactone-dependent transcriptional regulatory protein (rhlR Pch , 726bps). 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. 0.1mM, and their minimum surface tensions were ca. 26mN/m with no significant difference. Springer-Verlag Berlin Heidelberg (outside the USA), 2015 Biosurfactant nationallicence Microbial glycolipid nationallicence Pseudomonas chlororaphis nationallicence Rhamnolipid nationallicence Rhamnosyltransferases nationallicence Surface tension nationallicence Solaiman Daniel Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 E. Mermaid Lane, 19038, Wyndmoor, PA, USA aut Ashby Richard Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 E. Mermaid Lane, 19038, Wyndmoor, PA, USA aut Gunther IV Nereus Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 E. Mermaid Lane, 19038, Wyndmoor, PA, USA aut Zerkowski Jonathan Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 E. Mermaid Lane, 19038, Wyndmoor, PA, USA aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/10(2015-05-01), 4333-4342 0175-7598 99:10<4333 2015 99 253 https://doi.org/10.1007/s00253-015-6433-4 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6433-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Solaiman Daniel Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 E. Mermaid Lane, 19038, Wyndmoor, PA, USA aut NATIONALLICENCE 700 1- Ashby Richard Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 E. Mermaid Lane, 19038, Wyndmoor, PA, USA aut NATIONALLICENCE 700 1- Gunther IV Nereus Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 E. Mermaid Lane, 19038, Wyndmoor, PA, USA aut NATIONALLICENCE 700 1- Zerkowski Jonathan Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 E. Mermaid Lane, 19038, Wyndmoor, PA, USA aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/10(2015-05-01), 4333-4342 0175-7598 99:10<4333 2015 99 253