Enoate reductases from non conventional yeasts: bioconversion, cloning, and functional expression in Saccharomyces cerevisiae

Old yellow enzymes (OYEs, EC 1.6.99.1) are flavin-dependent oxidoreductases that catalyze the stereoselectivetrans-hydrogenation of the double bond, representing a promising alternative to metal-basedcatalysis. Bioconversion of ketoisophorone (KIP) by 28 non-conventional yeasts belonging to 16 differentspecies was investigated. Growing cells of most of the strains reduced KIP via OYE and showed highstereoselectivity, producing R-levodione as major product. Competition by carbonyl reductase (CR) activitywas observed in several strains. The best performing yeasts belong to Candida castellii, Kazachstaniaspencerorum and Kluyveromyces marxianus exhibited yields of levodione ≥77% up to 95% e.e., and. Candidafreyschussii, the sole strain lacking the OYE gene, reduced KIP only to unsaturated alcohols via CR. Nineunedited OYE genes were cloned, sequenced, and heterologously expressed in Saccharomyces cerevisiaeBY4741Oye2, a mutant that showed negligible OYE and CR activities. Compared with the correspondingwild-type yeasts, growing cells of the recombinant strains bioconverted KIP with improved yields ofOYE products, minor competition by CR activity, and lower enantioselectivity. In particular, resting cellsof recombinant S. cerevisae presented the best performance in KIP bioconversion. Based on the resultsherein reported, selected strains of non-conventional yeasts and novel OYE genes can be profitably usedas innovative biocatalysts in asymmetric reductions.