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.

The functionalization of C À Hb onds with nonprecious metal catalysts is an important researcha rea for the development of efficient and sustainable processes.Herein, we describe the development of iron porphyrin catalyzedreactions of diazoacetonitrile with N-heterocycles yielding important precursors of tryptamines,a long with experimental mechanistic studies and proof-of-concept studies of an enzymatic process with YfeX enzyme.B yu sing readily available FeTPPCl, we achieved the highly efficient CÀHfunctionalization of indole and indazole heterocycles.T hese transformations feature mild reaction conditions,e xcellent yields with broad functional group tolerance,c an be conducted on gram scale,a nd thus provide au nique streamlined access to tryptamines.

Unspecific peroxygenases (UPOs) enable oxyfunctionalisations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo-and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed a protein engineering in S. cerevisiae with the novel MthUPO. Experimental approaches were combined with computational modelling resulting in the screening of more than 5,300 transformants. This protein engineering led to a significant reshaping of the active site as elucidated by molecular dynamics. The k cat /K m was improved by 16.5-fold. Variants were identified with high chemo-and regioselectivities in the oxyfunctionalisation of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with excellent enantioselectivities of 95 % ee. Additionally, the first reported effective exchange of the conserved catalytic Glu residue was observed. File list (2) download file view on ChemRxiv Manuscript_Knorrscheidt et al.pdf (566.94 KiB) download file view on ChemRxiv Supporting_Knorrscheidt et al.pdf (5.26 MiB)

Protein engineering of an unspecific peroxygenase (UPO) was performed with three substrates and six products in parallel by a high throughput GC-MS setup. Modified chemo- and regioselective variants were identified for aliphatic substrates.

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