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Title: Dirhamnose-lipid production by recombinant nonpathogenic bacterium Pseudomonas chlororaphis

item Ashby, Richard - Rick
item Gunther, Nereus - Jack

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/23/2015
Publication Date: 2/11/2015
Citation: Solaiman, D., Ashby, R.D., Gunther, N.W., Zerkowski, J.A. 2015. Dirhamnose-lipid production by recombinant nonpathogenic bacterium Pseudomonas chlororaphis. Applied Microbiology and Biotechnology. DOI: 10.1007/s00253-015-6433-4.

Interpretive Summary: Rhamnolipids (RLs) are biobased surfactants produced by bacteria. They are “green” chemicals that are friendly to the environment, and are sustainable because they can be produced from non-petroleum-based renewable feedstocks. RL has a wide-range application such as detergent, fungicide, antibiotics, and wound-healing ingredient. The structure of RL is basically composed of two parts: The water-soluble part makes up of one or two molecules of a sugar called rhamnose, and the water-insoluble part consists of one or two fatty-acid molecules. RLs that contain only one rhamnose sugar (called R1L) are expected to have different properties and thus applications than those that contain two rhamnose (R2L). The most popular bacterium used in RL production is an opportunistic human pathogen called Pseudomonas aeruginosa. We have previously succeeded in identifying a non-pathogenic bacterium called Pseudomonas chlororaphis to make R1L. In the present work, we succeeded in genetically engineer this bacterium to enable it to make R2L. The ability to produce R1L and R2L using a non-pathogenic bacterium will greatly expand the application field of this “green” biobased chemical to the food and medical areas.

Technical Abstract: We previously discovered that Pseudomonas chlororaphis NRRL B-30761 produces monorhamnolipids with predominantly 3-hydroxydodecanoyl-3-hydroxydodecanoate (C12-C12) or 3-hydroxydodecenoyl-3-hydroxydodecanoate (C12:1-C12) 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 (rhlAPch, 894 bps), rhamnosyltransferase chain B (rhlBPch, 1272 bps) and N-acyl-homoserine lactone-dependent transcriptional regulatory protein (rhlRPch, 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 60-65% 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, confirming the genetic basis for monorhamnose-lipid (R1L) synthesis in this organism. We genetically constructed a dirhamnose-lipid (R2L) synthesizing P. chlororaphis by cloning and expressing a rhamnosyltransferase C (rhlC) gene of P. aeruginosa using an expression vector (pBS29-P2-gfp) containing a P. 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 zero production of 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 both in the range of 0.1 mM, and their minimum surface tensions were approximately 26 mN/m but with R1L showing a slightly lower value.