title: Enoate reductases from non conventional yeasts: bioconversion, cloning, and functional expression in Saccharomyces cerevisiae.
abstract: Old yellow enzymes (OYEs, EC 1.6.99.1) are flavin-dependent oxidoreductases that catalyze the stereoselective trans-hydrogenation of the double bond, representing a promising alternative to metal-based catalysis. Bioconversion of ketoisophorone (KIP) by 28 non-conventional yeasts belonging to 16 different species was investigated. Growing cells of most of the strains reduced KIP via OYE and showed high stereoselectivity, producing R-levodione as major product. Competition by carbonyl reductase (CR) activity was observed in several strains. The best performing yeasts belong to Candida castellii, Kazachstania spencerorum and Kluyveromyces marxianus exhibited yields of levodione ≥77% up to 95% e.e., and. Candida freyschussii, the sole strain lacking the OYE gene, reduced KIP only to unsaturated alcohols via CR. Nine unedited OYE genes were cloned, sequenced, and heterologously expressed in Saccharomyces cerevisiae BY4741ΔOye2, a mutant that showed negligible OYE and CR activities. Compared with the corresponding wild-type yeasts, growing cells of the recombinant strains bioconverted KIP with improved yields of OYE products, minor competition by CR activity, and lower enantioselectivity. In particular, resting cells of recombinant S. cerevisae presented the best performance in KIP bioconversion. Based on the results herein reported, selected strains of non-conventional yeasts and novel OYE genes can be profitably used as innovative biocatalysts in asymmetric reductions.
authors.affiliation: Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, Modena, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Raimondi
authors.forename: Diego
authors.initials: D
authors.lastname: Romano
authors.forename: Alberto
authors.initials: A
authors.lastname: Amaretti
authors.forename: Francesco
authors.initials: F
authors.lastname: Molinari
authors.forename: Maddalena
authors.initials: M
authors.lastname: Rossi
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003512:Cyclohexanones / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009252:NADPH Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016415:Sequence Alignment; D013237:Stereoisomerism; D015003:Yeasts / Q000201:enzymology* / Q000235:genetics
chemical_list: D003512:Cyclohexanones; D005656:Fungal Proteins; D011994:Recombinant Proteins; C494596:levodione; C476259:ketoisophorone; D009252:NADPH Dehydrogenase