Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

Abstract

Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces 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 C 16 to C 20 fatty acids preferentially. It converted oleic acid (C 18:1 ) more efficiently than stearic acid (C 18:0 ) and linoleic acid (C 18:2 ) and much more effectively than ␣-linolenic acid (C 18:3 ). No products were detected when C 10 to C 12 fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their -and -1 positions. CYP52N1 oxidized C 14 to C 20 saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed -hydroxylation of fatty acids. Minor -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 in S. bombicola but downstream of the initial oxidation steps.

Versatile biocatalysis of fungal cytochrome P450 monooxygenases

“…Interestingly, fungal CYPs are involved in the initial and rate limiting step of hydroxylation of n -alkanes and fatty acids [ 42 , 43 ]. In Starmerella bombicola , members of the CYP52 family ( CYP52M1 , CYP52E3 , and CYP52N1 ) performed the ω- and ω-1 hydroxylation of various fatty acids ( 3 ) [ 44 ]. CYP52M1 is involved in biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in the alkane metabolism [ 44 ].…”

“…In Starmerella bombicola , members of the CYP52 family ( CYP52M1 , CYP52E3 , and CYP52N1 ) performed the ω- and ω-1 hydroxylation of various fatty acids ( 3 ) [ 44 ]. CYP52M1 is involved in biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in the alkane metabolism [ 44 ]. Members of the CYP52 family have also been identified in the entomopathogenic fungi Metarhizium robertsii ( MrCYP52 ) and Beauveria bassiana ( CYP52X1 ) and are primarily involved in alkane and insect epicuticle degradation [ 45 , 46 ].…”

Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding

“…The CYP2E, CYP4B, CYP52, CYP86, and CYP153 families are known to hydroxylate n-alkanes or fatty acids in both eukaryotes and prokaryotes [6,7,8,9,10]. Among them, CYP2E, CYP4B, CYP52, and CYP86 are eukaryotic CYPs; these enzymes are membrane-bound proteins, limiting their structural characterization and making it difficult to use them in engineered pathways.…”

Candida pseudoglaebosaandKodamaea ohmeriare capable of degrading alkanes in the presence of heavy metals

“…About 10 genera of hydrocarbonoclastic Saccharomycetales yeasts have been described from 2011 to date, including the following species: Brettanomyces, Candida, Lypomyces, Meyerozyma, Pichia, Pseudozyma, Saccharomycopsis, Starmerella, Yamadazyma, and Yarrowia [30][31][32][33][34][35][36]. To our knowledge, this is the first report of a hydrocarbonoclastic phenotype for a K. ohmeri or C. pseudoglaebosa strain.…”