Old Yellow Enzyme homologues in Mucor circinelloides: expression profile and biotransformation

Alice Romagnolo,Anna Poli,Luisa Lanfranco,Giovanna Cristina Varese,Bianca Serito,Federica Spina,Elisabetta Brenna,Michele Crotti,Daniela Monti,Sara Risso

doi:10.1038/s41598-017-12545-7

Abstract

The reduction of C=C double bond, a key reaction in organic synthesis, is mostly achieved by traditional chemical methods. Therefore, the search for enzymes capable of performing this reaction is rapidly increasing. Old Yellow Enzymes (OYEs) are flavin-dependent oxidoreductases, initially isolated from Saccharomyces pastorianus. In this study, the presence and activation of putative OYE enzymes was investigated in the filamentous fungus Mucor circinelloides, which was previously found to mediate C=C reduction. Following an in silico approach, using S. pastorianus OYE1 amminoacidic sequence as template, ten putative genes were identified in the genome of M. circinelloides. A phylogenetic analysis revealed a high homology of McOYE1-9 with OYE1-like proteins while McOYE10 showed similarity with thermophilic-like OYEs. The activation of mcoyes was evaluated during the transformation of three different model substrates. Cyclohexenone, α-methylcinnamaldehyde and methyl cinnamate were completely reduced in few hours and the induction of gene expression, assessed by qRT-PCR, was generally fast, suggesting a substrate-dependent activation. Eight genes were activated in the tested conditions suggesting that they may encode for active OYEs. Their expression over time correlated with C=C double bond reduction.

Highlights

The reduction of C=C double bonds is a key reaction in organic chemistry but it is usually carried out by metal catalysts with a strong impact on the technical and economic feasibility of the process[1,2]
Nine McOYEs showed a similarity with S. pastorianus OYE1 of about 40% while McOYE10 showed a lower similarity (25.33%; Table 1)
Hydrogenation of C=C double bonds is an important reaction in several manufacturing processes for the production of bulk and fine chemicals; researchers and industries are moving towards more sustainable approaches as biocatalysis and in recent years, several research groups have focused on the identification of Old Yellow Enzymes (OYEs) homologues to be exploited in different processes[1]

Summary

Introduction

The reduction of C=C double bonds is a key reaction in organic chemistry but it is usually carried out by metal catalysts with a strong impact on the technical and economic feasibility of the process[1,2]. The biological reduction of activated C=C double bonds may be carried out by flavin-dependent oxidoreductases, namely ene reductases (ERs), belonging to the Old Yellow Enzyme (OYE) family (EC 1.6.99.1)[5]. They catalyze the asymmetric hydrogenation of C=C double bond conjugated with electron withdrawing groups (EWGs) in the presence of NAD(P)H as cofactor[2]. Most of the literature evidences focused on Ascomycetes and Basidiomycetes[9,12] but the presence of OYE homologue within Zygomycota phylum has never been assessed

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Conclusion