Combining Rational Design and Computational Tools in Multi-Parameter Enzyme Engineering to Increase the Fitness of a CYP152 Peroxygenase for α-Hydroxylation of Fatty Acids

Abstract

Enzymes of the CYP152 family have the potential to upgrade fatty acids to α-hydroxy acids by regio-and stereoselective hydroxylation using hydrogen peroxide as the oxidant and forming water as the sole by-product. To achieve such transformations with relevant productivity, a high tolerance towards hydrogen peroxide is required. By designing a minimal library targeting oxidation-prone residues with high solvent exposure and introducing mutations to improve expression/stability identified by a computational strategy (PROSS), the hydrogen peroxide tolerance of the CYP152 peroxygenase POSPα was improved up to four-fold. Of the 12 generated enzyme variants, V3-P04 demonstrated the most pronounced improvements across the investigated parameters, exceeding the parent in terms of hydrogen peroxide tolerance, expression yields, and specific activity for the α-hydroxylation of octanoic acid. The superior performance of variant V3-P04 was further underlined in preparative-scale experiments for the functionalisation of heptanoic acid (50 mM), octanoic acid (150 mM), nonanoic acid (100 mM), and 9-decenoic acid (75 mM) where it reached turnover numbers unmatched by the wildtype enzyme of up to 48,333.