Biocatalytic exploitation of enoate reductase from unconventional yeasts

Introduction: Old Yellow Enzymes (OYE, EC 1.6.99.1) are flavindependent oxidoreductases that catalyze the stereoselective transhydrogenation of electron-poor alkenes (enoate reductase activity), which can result in important chiral synthons toward synthesis of many pharmaceuticals and chemicals. In this study, environmental yeasts have been screened for OYE activity with the purpose to develop new enoate reductase biocatalyst. Methods: A set of 30 environmental yeasts belonging to 7 genera were screened for OYE activity, based on conversion of ketoisophorone (KIP). Ten new genes encoding OYE enzymes were cloned, sequenced, and overexpressed in S. cerevisiae BY4741(Oye2). The recombinant strains were tested for the reduction of KIP, isophorone, alpha-methyl-transcinnamaldehyde, 3-Methyl-2-cyclohexenone and nitro-phenylpropene. Two OYE genes were overexpressed in Escherichia coli. Results: Based on the amount of KIP reduction products, the strains belonging to the species Kazachstania spp., Candida spp., and Kluyveromyces marxianus exhibited the highest OYE activity, and their genes were cloned. The OYE proteins revealed an identity ranging from 76% to 99% with the protein Oye2 of Saccharomyces cerevisiae. Resting-cell of S. cerevisiae BY4741(Oye2) expressing recombinant OYEs converted up to 1 g l−1 of substrate, in less than 24 hours and with e.e. of 98%. OYE overexpression in E. coli was successful and allowed protein purification. Discussion: These study identified new enoate reductase biocatalysts within unconventional yeasts. Recombinant strains with enhanced OYE activity have been obtained and could be exploited for production of purified enzyme. Furthermore, the data herein reported attract interest due to promising applications in asymmetric synthesis.