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

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.

The Family Keeps on Growing: Four Novel Fungal OYEs Characterized

“…In both cases, genome analysis found 6 [ 21 ] and 10 [ 29 ] putative OYEs, respectively, for which the modeling-predicted structural differences suggested specific substrate-enzyme matching. Afterward, the expression profiles of the genes encoding the putative isoenzymes were analyzed for both species and clearly showed differential activation in response to environmental stimuli (plant infection or oxidative stress for A. rabiei ) [ 2 , 21 ] or exposure to different substrates (for M. circinelloides ) [ 30 ]. Thus, the low activities registered for our two An OYEs could be ascribed to the fact that standard substrates generally used during screenings of OYEs may not mimic the natural substrates (still unknown) of these enzymes, leading to a limited activity spectrum and low specific activities being recorded.…”

Bio‐detoxification of Jatropha curcas L. cake by a soil‐borne Mucor circinelloides strain using a zebrafish survival model and solid‐state fermentation

“…The multiple abilities of M. circinelloides to transform and decompose environmental toxins may be accredited to a variety of enzymes it secretes, e.g., the old yellow enzymes, which are flavin‐dependent oxidoreductases, can mediate C = C reduction (Romagnolo et al . 2017); alcohol dehydrogenase 1, which contributes to the Crabtree effect, connects fermentative and oxidative metabolism in the M. circinelloides (Rangel‐Porras et al . 2019); and HMG‐CoA reductase, plays an important role in degrading terpenoid compounds (Nagy et al .…”

Discovery, Characterization, Engineering, and Applications of Ene-Reductases for Industrial Biocatalysis

“…This suggested the presence of at least eight ERs within M. circinelloides that showed high reduction of the industrially important α-methyl cinnamaldehyde (>99% conversion after 20 h; Scheme 1B). 32…”

“…31 A further whole-cell biotransformation study of M. circinelloides was performed with cyclohexen-2-one, α-methylcinnamaldehyde, and methyl cinnamate as model substrates. 32 In silico analysis identified 10 potential OYEs from M. circinelloides, and the expression of the individual genes was monitored parallel to product formation. This suggested the presence of at least eight ERs within M. circinelloides that showed high reduction of industrially important α-methylcinnamaldehyde (>99% conversion after 20 h) (Scheme 1B).…”