Genetic engineering for rhamnolipids production by nonpathogenic organism

Authors: Solaiman, Daniel; Aneja, Kawalpreet; Gunther, Nereus - Jack; Ashby, Richard - Rick; Zerkowski, Jonathan

Submitted to: Annual Meeting and Expo of the American Oil Chemists' Society
Publication Type: Abstract Only
Publication Acceptance Date: 2/21/2007
Publication Date: 5/13/2007
Citation: Solaiman, D., Aneja, K., Gunther, N.W., Ashby, R.D., Zerkowski, J.A. 2007. Genetic engineering for rhamnolipids production by nonpathogenic organism [abstract]. Annual Meeting and Expo of the American Oil Chemists' Society. p. 26.

Interpretive Summary:

Technical Abstract: Rhamnolipid (RL) produced by certain bacteria is a biosurfactant useful in the cleaning, biocontrol and enhanced oil recovery applications. Its component, rhamnose, is also a valued product used in the synthesis of pharmaceuticals and in food and fragrance industries. Pseudomonas aeruginosa, an opportunistic human pathogen, is currently used for commercial production of RL. There is thus a need to identify and develop RL-producing nonpathogenic organisms to expand the application sphere of this biosurfactant. We have developed and applied for patent protection for an energy-saving RL production bioprocess using a nonpathogenic bacterium (herein referred to as strain X). We have now determined by using a chromosomal DNA walking technique the complete nucleotide sequence of the genes (rhlABPx) responsible for RL synthesis in this bacterial strain. BLASTP analysis showed that the amino acid sequences of the putative gene-products, RhlA and RhlB, of strain X have only 63%-identities with those of the corresponding enzymes of P. aeruginosa. We subsequently cloned the rhlABPx genes into a transposon Tn7-based Pseudomonas integration plasmid vector, pUC18-mini-Tn7T-LAC. The recombinant plasmid was introduced into two non-P. aeruginosa bacteria along with a helper plasmid, pTNS2, by electroporation. We confirmed by PCR the successful integration of rhlABPx genes into the chromosome of the target organisms at the specific site downstream of the highly conserved glucosamine-6-phosphate synthetase gene (glmS). Work is in progress to quantify the production of RL by these recombinant nonpathogenic bacteria. The expected outcome of this study is an RL-producing bioprocess that uses non-pathogenic bacteria as biocatalyst and agro-based coproducts (i.e., glycerol from biodiesel production and soy molasses) as feedstocks. Such a process should further expand the application potentials of RL into the foods and medical areas.