Abstract
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.
Highlights
Old Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes
Recombinant strains bearing heterologous OYE genes were used as whole-cells biocatalysts to reduce three molecules with electron-poor double bond activated by different electron-withdrawing groups (Figure 1), i.e. ketoisophorone (2,6,6-trimethylcyclohex-2-ene-1,4-dione, 1a), α-methyl-trans-cinnamaldehyde ((E)-2-methyl-3-phenyl2-propenal, 2a), and trans-β-methyl-β-nitrostyrene ((E)1-phenyl-2-nitropropene, 3a)
Even though high efficiency of 1a transformation was observed, and the reaction proceeded with preference for the R-levodione (1b), the enantioselectivity generally decreased compared to that obtained with the corresponding wild type yeasts [16]
Summary
Old Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes. Due to the increasing demand of optically pure building blocks, considerable efforts are addressed at identifying, characterizing, improving, and developing Old Yellow Enzymes (OYE) biocatalysts, i.e. flavin-dependent enoate reductases (EC 1.6.99.1), that catalyze the chemoand stereoselective hydrogenation of electron-poor alkenes [3,4]. Their biotechnological potential lies on the ability to generate up to two stereocenters by the stereoselective anti-hydrogenation of the activated C = C double bond, which is highly demanded in asymmetric synthesis, since conventional chemical methods mostly give synhydrogenation [5,6]. OYE genes from unconventional yeasts were cloned and expressed in Saccharomyces cerevisiae which was used as a whole-cell biocatalyst, demonstrating the important role of unconventional yeasts as sources of novel biocatalysts and their potential for the expansion of known genetic biodiversity [16]
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