Discovery and biocatalytic exploration of fungal ene-reductases
Old Yellow Enzymes (OYEs) catalyze the double bond reduction of α,β–unsaturated compounds, particularly those possessing an electron-withdrawing group. This is of interest to synthetic chemistry applications that target synthesis of enantiomerically pure compounds. OYEs surpass chemical catalysts due to their high enantioselectivity/stereoselectivity and operability under mild conditions. The first OYE was described in 1932, hence its name. Many other OYE-type reductases have since been discovered, yet numerous ene-reductases remain undiscovered across diverse sources. Recent classification categorizes these enzymes into seven classes based on sequence and structural features. This study concentrated on OYEs from fungi, specifically Penicillium citrinum CBMAI 1186 and Penicillium steckii (both mesophilic species), along with Aspergillus thermomutatus, Chaetomium thermophilium, Lachancea thermotolerans, Ogatae polymorpha and Thermothielavioides terrestris (all mesothermophilic species). The study involving P. citrinum CBMAI 1186 involved whole-cell reactions to reduce α,β,γ,δ–unsaturated compounds. For P. steckii, eleven OYE-encoding genes were selected, yielding six expressed and purified enzymes. Characterization of these enzymes included reaction rates, thermostability, pH optimum, and substrate acceptance. To broaden the toolbox of OYEs with robust variants, five genes from thermophilic fungi were selected for an exploratory biocatalytic study that resulted in the production and characterization of five thermostable OYEs. Next, four newly discovered OYEs were selected for performing whole-cell conversions using Escherichia coli. When using a biphasic system, efficient conversions could be achieved. In summary, this work focused on the acquisition, examination, and application of fungal OYEs, and to contribute to new insights that complement the existing understanding of this enzyme group, while enriching the available toolbox of OYEs for biocatalysis, thereby expanding the prospects for future applications.