Recombinant Production and Characterization of Six Ene‐reductases from Penicillium steckii

Fungi, known for their adaptability, are valuable sources of enzymes, making them promising for biocatalyst discovery. This study explored Penicillium steckii, primarily recognized for secondary metabolite production, as a source of ene-reductases (ERs), which reduce α,β-unsaturated compounds. Eleven ER-encoding genes were identified, and plasmids for Escherichia coli expression were generated. Six ERs (PsOYE1-6) were successfully produced and purified as soluble FMN-containing proteins. Sequence analysis classified them into Class II (PsOYE1, PsOYE4, PsOYE6), Class III (PsOYE2, PsOYE3), and Class V (PsOYE5) OYEs. All were active on p-benzoquinone and maleimide, with varying activity on other substrates. Their pH optima ranged from 6 to 7, and they exhibited moderate thermostability (35-50 °C). PsOYE2 was crystallized, and its 2.3 Å structure revealed a stable dimer with a unique active site. PsOYE3, PsOYE4, and PsOYE5 were tested for R-carvone conversion and stereoselectivity, all favouring one diastereomer. These fungal ERs expand the enzymatic toolbox for biocatalysis, emphasizing the need for tailored strategies based on specific applications.