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Cloning, sequencing, and characterization of lipase genes from a polyhydroxyalkanoate (PHA)-synthesizing Pseudomonas resinovorans

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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 polymerase chain reaction-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 bp 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 performed, and its effect on 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.

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Acknowledgments

This work was partially supported via a fellowship to JHL under the OECD Co-operative Research Programme on Biological Resource Management for Sustainable Agricultural Systems. The authors thank Nicole Crocker and Susan Lawlor for technical assistance. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture (USDA). USDA is an equal opportunity provider and employer.

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Authors and Affiliations

  1. Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA, 19038, USA

    Richard D. Ashby, David S. Needleman & Daniel K. Y. Solaiman

  2. Department of Food and Nutrition, Daegu University, Jillyang, Gyeongsan, Gyeongbuk, 712-714, South Korea

    Jeung Hee Lee

  3. Department of Food Science and Technology, Chungnam National University, Daejeon, 305-764, South Korea

    Ki-Teak Lee

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  1. Jeung Hee Lee
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  2. Richard D. Ashby
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  3. David S. Needleman
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  4. Ki-Teak Lee
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  5. Daniel K. Y. Solaiman
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Correspondence to Daniel K. Y. Solaiman.

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Lee, J.H., Ashby, R.D., Needleman, D.S. et al. Cloning, sequencing, and characterization of lipase genes from a polyhydroxyalkanoate (PHA)-synthesizing Pseudomonas resinovorans . Appl Microbiol Biotechnol 96, 993–1005 (2012). https://doi.org/10.1007/s00253-012-4133-x

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