Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes

Microbial Cell Factories

Research

Francesco Molinari¹  Martina Letizia Contente¹  Diego Romano¹  Erica Ruvutuso²  Ivano Eberini²  Cristina Sensi²  Maddalena Rossi³  Stefano Raimondi³  Alberto Amaretti³ 

[1] Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, Via G. Celoria 2, 20133, Milan, Italy;Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Trentacoste 2, 20134, Milan, Italy;Dipartimento di Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, Via G. Campi 183, 41125, Modena, Italy;

关键词: Lower Unoccupied Molecular Orbital;    Recombinant Strain;    Lower Unoccupied Molecular Orbital;    Asymmetric Reduction;    Carbonyl Reductase;   

DOI  :  10.1186/1475-2859-13-60

 received in 2014-01-08, accepted in 2014-04-15,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundOld Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes. Their ability to generate up to two stereocenters by the trans- hydrogenation of the C = C double bond is highly demanded in asymmetric synthesis. Isolated redox enzymes utilization require the addition of cofactors and systems for their regeneration. Microbial whole-cells may represent a valid alternative combining desired enzymatic activity and efficient cofactor regeneration. Considerable efforts were addressed at developing novel whole-cell OYE biocatalysts, based on recombinant Saccharomyces cerevisiae expressing OYE genes.ResultsRecombinant S. cerevisiae BY4741∆Oye2 strains, lacking endogenous OYE and expressing nine separate OYE genes from non-conventional yeasts, were used as whole-cell biocatalysts to reduce substrates with an electron-poor double bond activated by different electron-withdrawing groups. Ketoisophorone, α-methyl-trans- cinnamaldehyde, and trans- β-methyl-β-nitrostyrene were successfully reduced with high rates and selectivity. A series of four alkyl-substituted cyclohex-2-enones was tested to check the versatility and efficiency of the biocatalysts. Reduction of double bond occurred with high rates and enantioselectivity, except for 3,5,5-trimethyl-2-cyclohexenone. DFT (density functional theory) computational studies were performed to investigate whether the steric hindrance and/or the electronic properties of the substrates were crucial for reactivity. The three-dimensional structure of enoate reductases from Kluyveromyces lodderae and Candida castellii, predicted through comparative modeling, resulted similar to that of S. cerevisiae OYE2 and revealed the key role of Trp116 both in substrate specificity and stereocontrol. All the modeling studies indicate that steric hindrance was a major determinant in the enzyme reactivity.ConclusionsThe OYE biocatalysts, based on recombinant S. cerevisiae expressing OYE genes from non-conventional yeasts, were able to differently reduce the activated double bond of enones, enals and nitro-olefins, exhibiting a wide range of substrate specificity. Moreover whole-cells biocatalysts bypassed the necessity of the cofactor recycling and, tuning reaction parameters, allowed the synthetic exploitation of endogenous carbonyl reductases. Molecular modeling studies highlighted key structural features for further improvement of catalytic properties of OYE enzymes.

【 授权许可】

Unknown   
© Romano et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.