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

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