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Dirhamnose-lipid production by recombinant nonpathogenic bacterium Pseudomonas chlororaphis

  • Applied genetics and molecular biotechnology
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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.

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Acknowledgments

The authors thank Bun-Hong Lai and Nicole Crocker for providing technical assistance. Nucleic acid sequence determination was performed by David S. Needleman, PhD. and Sue Lawlor of Eastern Regional Research Center Core Technologies’ Integrated Biomolecular Resources group. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.

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  1. Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 E. Mermaid Lane, Wyndmoor, PA, 19038, USA

    Daniel K. Y. Solaiman, Richard D. Ashby, Nereus W. Gunther IV & Jonathan A. Zerkowski

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  1. Daniel K. Y. Solaiman
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  2. Richard D. Ashby
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  3. Nereus W. Gunther IV
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Solaiman, D.K.Y., Ashby, R.D., Gunther, N.W. et al. Dirhamnose-lipid production by recombinant nonpathogenic bacterium Pseudomonas chlororaphis . Appl Microbiol Biotechnol 99, 4333–4342 (2015). https://doi.org/10.1007/s00253-015-6433-4

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