A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases

Abstract Fungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing haem-thiolate active site and rely hydrogen peroxide the oxygen source. However, their heterologous production in fast-growing organism suitable for high throughput screening has only succeeded once—enabled by intensive directed evolution campaign. We developed applied modular Golden Gate-based secretion system, allowing first of four UPOs yeast, one-step purification application enantioselective conversion preparative scale. The Gate setup was designed to be universally applicable consists three module types: i) signal peptides secretion, ii) UPO genes, iii) protein tags split-GFP detection. episomal system is use Saccharomyces cerevisiae transferred chromosomally integrated expression cassettes Pichia pastoris . Shake flask productions yielded up 24 mg/L secreted enzyme, which employed scale phenethylamine derivative reaching 98.6 % ee Our results demonstrate rapid, yeast workflow yielding biotransformations.