Functional Expression of Two Unusual Acidic Peroxygenases from Candolleomyces aberdarensis in Yeasts by Adopting Evolved Secretion Mutations

• Published in: Applied and environmental microbiology Vol. 87; no. 19; p. e0087821
• Main Authors: Gomez de Santos, Patricia, Hoang, Manh Dat, Kiebist, Jan, Kellner, Harald, Ullrich, René, Scheibner, Katrin, Hofrichter, Martin, Liers, Christiane, Alcalde, Miguel
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 10.09.2021
• Subjects: Agaricales - enzymology; Agaricales - genetics; Applied and Industrial Microbiology; Batch culture; Biocatalysts; Bioreactors; Biotechnology; Directed evolution; Evolution; Fermentation; Mixed Function Oxygenases - genetics; Mutants; Mutation; Organic compounds; Peptides; pH effects; Phylogeny; Pichia - genetics; Protein Engineering; Protein sorting signals; Recombination; Saccharomyces cerevisiae - genetics; Secretion; Signal peptides; Substrates; Veratryl alcohol; Yeast; Pichia pastoris; unspecific peroxygenase; heterologous functional expression; Saccharomyces cerevisiae; directed evolution
• ISSN: 0099-2240; 1098-5336; 1098-5336
• DOI: 10.1128/AEM.00878-21

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 on an ∼300-mg/liter scale is a versatile tool to generate UPO variants. 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 of two new unusual acidic peroxygenases from Candolleomyces ( Psathyrella ) aberdarensis ( Pab UPO) in yeasts and their production at a large scale in a bioreactor. Our strategy was based on adopting secretion mutations from an Agrocybe aegerita UPO mutant, the 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 shares 65% sequence identity. After replacing the native signal peptides with 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/liter in Saccharomyces cerevisiae . These variants were subsequently transferred to Pichia pastoris for overproduction in a fed-batch bioreactor, boosting expression levels up to 290 mg/liter, with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/liter, with veratryl alcohol as the substrate). With a broad pH activity profile, ranging from pH 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 on an ∼300-mg/liter 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.