The functional expression in yeast of two unusual acidic peroxygenases from
Candolleomyces aberdarensis
by adopting evolved secretion mutations
Fungal unspecific peroxygenases (UPOs) are emergent biocatalysts that perform highly selective C-H oxyfunctionalizations of organic compounds, yet their heterologous production at high levels is required for their practical use in synthetic chemistry. Here, we achieved functional expression in yeast of two new unusual acidic peroxygenases from Candolleomyces ( Psathyrella ) aberdarensis ( Pab UPO) and their production at large scale in bioreactor. Our strategy was based on adopting secretion mutations from Agrocybe aegerita UPO mutant −PaDa-I variant− designed by directed evolution for functional expression in yeast, which belongs to the same phylogenetic family as Pab UPOs –long-type UPOs− and that shares 65% sequence identity. After replacing the native signal peptides by the evolved leader sequence from PaDa-I, we constructed and screened site-directed recombination mutant libraries yielding two recombinant Pab UPOs with expression levels of 5.4 and 14.1 mg/L in S. cerevisiae . These variants were subsequently transferred to P. pastoris for overproduction in fed-batch bioreactor, boosting expression levels up to 290 mg/L with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/L, with veratryl alcohol as substrate). With a broad pH activity profile, ranging from 2.0 to 9.0, these highly secreted, active and stable peroxygenases are promising tools for future engineering endeavors, as well as for their direct application in different industrial and environmental settings. IMPORTANCE In this work, we incorporated several secretion mutations from an evolved fungal peroxygenase to enhance the production of active and stable forms of two unusual acidic peroxygenases. The tandem-yeast expression system based on S. cerevisiae for directed evolution and P. pastoris for overproduction in a ∼300 mg/L scale, is a versatile tool to generate UPO variants. By employing this approach, we foresee that acidic UPO variants will be more readily engineered in the near future and adapted to practical enzyme cascade reactions that can be performed over a broad pH range to oxyfunctionalize a variety of organic compounds.