Directed evolution of unspecific peroxygenase in organic solvents

Abstract

Fungal unspecific peroxygenases (UPOs) are efficient biocatalysts that insert oxygen atoms into nonactivated C-H bonds with high selectivity. Many oxyfunctionalization reactions catalyzed by UPOs are favored in organic solvents, a milieu in which their enzymatic activity is drastically reduced. Using as departure point the UPO secretion mutant from Agrocybe aegerita (PaDa-I variant), in the current study we have improved its activity in organic solvents by directed evolution. Mutant libraries constructed by random mutagenesis and in vivo DNA shuffling were screened in the presence of increasing concentrations of organic solvents that differed both in regard to their chemical nature and polarity. In addition, a palette of neutral mutations generated by genetic drift that improved activity in organic solvents was evaluated by site directed recombination in vivo. The final UPO variant of this evolutionary campaign carried nine mutations that enhanced its activity in the presence of 30% acetonitrile (vol/vol) up to 23-fold over PaDa-I parental type, and it was also active and stable in aqueous acetone, methanol and dimethyl sulfoxide mixtures. These mutations, which are located at the surface of the protein and in the heme channel, seemingly helped to protect UPO from harmful effects of cosolvents by modifying interactions with surrounding residues and influencing critical loops.

Assessing Peroxygenase-Mediated Oxidations in the Presence of High Concentrations of Water-Miscible Co-Solvents

“…This feature makes the use of this peroxygenase already remarkable as other heme-containing enzymes such as the peroxygenase HspUPO or the frequently investigated cytochrome P450 BM3 get already inactivated in presence of low concentrations of water-miscible co-solvents of typically less than 20 vol% (Seng Wong et al, 2004;Rotilio et al, 2021). Further improvements may be even expected using evolved variants of rAaeUPO with even higher solvent tolerance (Martin-Diaz et al, 2021).…”

Peroxide-Mediated Oxygenation of Organic Compounds by Fungal Peroxygenases

“…The latter leads to the formation of phenoxy radicals and subsequently to undesired coupling products and quinones. This shortcoming can be circumvented by applying radical scavengers such as ascorbic acid, which reduce the radicals formed [ 49 , 50 ], but engineered UPOs offer a more elegant way to prevent these side activities [ 51 ]; furthermore, these molecular techniques can be applied to improve other UPO shortcomings, such as stability in organic solvents [ 52 ].…”

Broadening the Biocatalytic Toolbox—Screening and Expression of New Unspecific Peroxygenases

“…Nonetheless, the results presented here increase the toolbox of UPOs available for genetic engineering and heterologous expression—a general requirement for the industrial application of this enzyme type. The described expression of new UPOs, the (evolutionary) optimization of the enzymes [ 6 , 9 , 24 , 29 , 30 , 31 ], as well as process-engineering approaches [ 32 , 33 , 34 , 35 , 36 , 37 ] will certainly make UPOs a key player in technical oxy-functionalization in the future.…”