Computational-Aided Engineering of a Selective Unspecific Peroxygenase toward Enantiodivergent -Ionone Hydroxylation

Unspecific peroxygenases (UPOs) perform oxyfunctionalizations for a wide range of substrates utilizing H2O2 without the need for further reductive equivalents or electron transfer chains. Tailoring these promising enzymes toward industrial application was intensely pursued in the last decade with engineering campaigns addressing the heterologous expression, activity, stability, and improvements in chemo- and regioselectivity. One hitherto missing integral part was the targeted engineering of enantioselectivity for specific substrates with poor starting enantioselectivity. In this work, we present the engineering of the short-type MthUPO toward the enantiodivergent hydroxylation of the terpene model substrate, beta-ionone. Guided by computational modeling, we designed a small smart library and screened it with a GC-MS setup. After two rounds of iterative protein evolution, the activity increased up to 17-fold and reached a regioselectivity of up to 99.6% for the 4-hydroxy-beta-ionone. Enantiodivergent variants were identified with enantiomeric ratios of 96.6:3.4 (R) and 0.3:99.7 (S), respectively.