In 2004, the fungal heme-thiolate enzyme subfamily of unspecific peroxygenases (UPOs) was first described in the basidiomycete Agrocybe aegerita. As UPOs naturally catalyze a broad range of oxidative transformations by using hydrogen peroxide as electron acceptor and thus possess a great application potential, they have been extensively studied in recent years. However, despite their versatility to catalyze challenging selective oxyfunctionalizations, the availability of UPOs for potential biotechnological applications is restricted. Particularly limiting are the identification of novel natural biocatalysts, their production, and the description of their properties. It is hence of great interest to further characterize the enzyme subfamily as well as to identify promising new candidates. Therefore, this review provides an overview of the state of the art in identification, expression, and screening approaches of fungal UPOs, challenges associated with current protein production and screening strategies, as well as potential solutions and opportunities.

Biocatalysis is constantly providing novel options for the synthesis of active pharmaceutical ingredients (APIs). In addition to drug development and manufacturing, biocatalysis also plays a role in drug discovery and can support many active ingredient syntheses at an early stage to build up entire scaffolds in a targeted and preparative manner. Recent progress in recruiting new enzymes by genome mining and screening or adapting their substrate, as well as product scope, by protein engineering has made biocatalysts a competitive tool applied in academic and industrial spheres. This is especially true for the advances in the field of nonribosomal peptide synthesis and enzyme cascades that are expanding the capabilities for the discovery and synthesis of new bioactive compounds via biotransformation. Here we highlight some of the most recent developments to add to the portfolio of biocatalysis with special relevance for the synthesis and late-stage functionalization of APIs, in order to bypass pure chemical processes.

The active vitamin D metabolites 25‐OH−D and 1α,25‐(OH)2−D play an essential role in controlling several cellular processes in the human body and are potentially effective in the treatment of several diseases, such as autoimmune diseases, cardiovascular diseases and cancer. The microbial synthesis of vitamin D2 (VD2) and vitamin D3 (VD3) metabolites has emerged as a suitable alternative to established complex chemical syntheses. In this study, a novel strain, Kutzneria albida, with the ability to form 25‐OH−D2 and 25‐OH−D3 was identified. To further improve the conversion of the poorly soluble substrates, several solubilizers were tested. 100‐fold higher product concentrations of 25‐OH−D3 and tenfold higher concentrations of 25‐OH−D2 after addition of 5 % (w/v) 2‐hydroxypropyl β‐cyclodextrin (2‐HPβCD) were reached. Besides the single‐hydroxylation products, the human double‐hydroxylation products 1,25‐(OH)2−D2 and 1,25‐(OH)2−D3 and various other potential single‐ and double‐hydroxylation products were detected. Thus, K. albida represents a promising strain for the biotechnological production of VD2 and VD3 metabolites.

Biocatalytic oxidation reactions are in high demand. Among the applied enzymes, the heme-thiolate enzyme subfamily of unspecific peroxygenases (UPOs), which relies on hydrogen peroxide as cosubstrate and oxidant, has generated great interest. Almost two decades after their first description, databases provide thousands of putative UPO sequences, but only a few enzymes have been characterized. To address this gap, efficient screening methods for the identification of novel UPOs are necessary. Here, a new screening strategy is presented based on solid cultivation of wild-type fungi. The identification of a promising candidate strain highlights the applicability of this approach.