Expression and Characterization ofCYP52Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

Abstract

ABSTRACTThree cytochrome P450 monooxygenaseCYP52gene family members were isolated from the sophorolipid-producing yeastStarmerella bombicola(formerCandida bombicola), namely,CYP52E3,CYP52M1, andCYP52N1, and their open reading frames were cloned into the pYES2 vector for expression inSaccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C16to C20fatty acids preferentially. It converted oleic acid (C18:1) more efficiently than stearic acid (C18:0) and linoleic acid (C18:2) and much more effectively than α-linolenic acid (C18:3). No products were detected when C10to C12fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C14to C20saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism inS. bombicolabut downstream of the initial oxidation steps.