Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/22/2012
Publication Date: 6/1/2012
Publication URL: http://handle.nal.usda.gov/10113/60253
Citation: Lee, J.H., Ashby, R.D., Needleman, D.S., Lee, K., Solaiman, D. 2012. Cloning, sequencing and characterization of lipase genes from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans. Applied Microbiology and Biotechnology. DOI: 10.1007/s00253-012-4133-x. Interpretive Summary: Renewable and surplus agricultural products could be used as a feedstock to reduce the costs of fermentative production of biobased chemicals and materials. Over the years we have developed bioprocesses that use fats, oils and related coproducts to produce bioplastics and biosurfactants. One such process is the production of bioplastics – specifically a class of polymers called polyhydroxyalkanoates (PHAs) – by a bacterium called Pseudomonas resinovorans using animal fats (i.e., tallow and lard) and vegetable oils (i.e., soy oil, sunflower oil, and coconut oil) as feedstocks. A key biological molecule, called lipase, is required of the bacterium in order to break down the fats and oils to make them available to the organism for cell growth and bioplastic synthesis. A better understanding of the properties of the lipase in P. resinovorans could thus help us optimize the production process by, for example, selecting the appropriate fats, oils, or recycled grease (i.e., so-called substrate specificity) to feed the bacteria. In this paper, we described the cloning and computer-analyses of the genes coding for a lipase and a helper protein (called foldase) from P. resinovorans. By knocking out the lipase gene, we showed that the microorganism ceased to display lipase enzyme activity, and the re-introduction of the lipase gene via genetic engineering resulted in the re-emergence of the enzyme activity. These results conclusively showed that the cloned lipase gene is responsible for the lipase production of P. resinovorans. The genes isolated in this work will allow us to produce the lipase enzyme in large quantities for detailed characterization. The enzyme could also be valuable for use in the production of lipid-based nutriceuticals and biofuels.
Technical Abstract: Lipase (lip) and lipase-specific foldase (lif) genes of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans NRRL B-2649 were cloned using primers based on consensus sequences, followed by PCR-based genome walking. Sequence analyses showed a putative Lip gene-product (314 amino acids, a.a.) with its catalytic active-site (Ser111, Asp258 and His280) identified. The foldase lif gene that is located 55 bps downstream of lip, codes for a putative Lif (345 a.a.). To verify the biological function of the cloned lip gene for lipase expression in P. resinovorans, we constructed a lip knock-out mutant (lip::Tn5<KAN-2>) by transposon-insertion. Complementation of the lip knock-out P. resinovorans mutant with a lipase expression plasmid (pBS29-P2-lip) was constructed, and lipase expression was investigated. The wild-type P. resinovorans and the lip::Tn5<KAN-2>[pBS29-P2-lip] recombinant (but not the lip::Tn5<KAN-2> mutant), showed fluorescence on rhodamine B plates indicative of lipase activity. The wild-type exhibited extracellular lipase activity when grown on medium containing triacylglycerol substrates (tallow, olive oil and tributyrin) as sole carbon sources, but the lip::Tn5<KAN-2> mutant did not show such activity. Lipase activity of various strains was also confirmed by TLC analysis of the composition of acylglycerols and free fatty acid in the extracts of the spent culture medium. We further found that tributyrin was more effective than olive oil in inducing lipase expression in P. resinovorans.