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A high throughput GC-MS approach was developed, permitting the simultaneous analysis of up to three substrates and six products quantitatively from one reaction mixture. This screening approach was applied to site-saturation libraries of the novel unspecific peroxygenase MthUPO. Using this setup enabled substantial insights from a small mutant library. Enzyme variants were identified exhibiting selective alkene epoxidation and substantially shifted regioselectivities to 2- and 1-octanol formations. Computational modelling rationalised the observed selectivity changes.
Publications
Unspecific peroxygenases (UPOs) enable oxyfunctionalizations 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 protein engineering in Saccharomyces cerevisiae using the MthUPO from Myceliophthora thermophila. More than 5300 transformants were screened. This protein engineering led to a significant reshaping of the active site as elucidated by computational modelling. The reshaping was responsible for the increased oxyfunctionalization activity, with improved kcat/Km values of up to 16.5-fold for the model substrate 5-nitro-1,3-benzodioxole. Moreover, variants were identified with high chemo- and regioselectivities in the oxyfunctionalization of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with enantioselectivities of up to 95% ee. The proposed evolutionary protocol and rationalization of the enhanced activities and selectivities acquired by MthUPO variants represent a step forward toward the use and implementation of UPOs in biocatalytic synthetic pathways of industrial interest.