Construction of lipoxygenase‐like gene of pseudomonas aeruginosa PA01 into pET‐28A

Abstract

Naturally occurring renewable fatty acids in vegetable oil and their derivatives are industrial useful compounds. Castor oil contains greater than 85% of its fatty acid component as ricinoleic acid which is an industrial useful compound. Soapstock is a plentiful and inexpensive byproduct of edible oil refining. About 100 million pounds of soapstock are produced in the United States annually. The acidulated soapstock contains about 15% of free fatty acids primarily as oleic and linoleic acids. We are interested in promoting the cash value of soapstock by converting fatty acids in soapstock to value-added hydroxyfatty acid derivatives. We previously reported that Pseudomonas aeruginosa PA01 (PA01) is capable of transforming oleic acid to 10-hydroxy-8(E)-octadecenoic (HOD) and 7, 10-dihydroxy-8(E)- octadecenoic (DOD). The transformation of oleic to HOD and then DOD has been proposed to include lipoxygenase and hydroxylase. Our interest in lipoxygenase-like enzyme stems partly from the fact that HOD is a ricinoleic acid-like compound with potential industrial applications. To search for a putative lipoxygenase gene in PA01, we have previously cloned a lipoxygenase-like gene (accession no. AE004547) by polymerase chain reaction and expressed it in GST expression system. However, the expressed protein was found in insoluble inclusion body. In the present study, we report sucloning of the putative lipoxygenase gene into pET28a. This expression system will result in expression of a His-tagged protein. The protein will be purified and characterized. The research is supported by USDA- CSREES2004-35504-14712 and the University Research Council at Western Illinois University.