Engineering of Unspecific Peroxygenases Using a Superfolder-Green-Fluorescent-Protein-Mediated Secretion System in Escherichia coli .

Unspecific peroxygenases (UPOs), secreted by fungi, demonstrate versatility in catalyzing challenging selective oxyfunctionalizations. However, the number of peroxygenases and corresponding variants with tailored selectivity for a broader substrate scope is still limited due to the lack of efficient engineering strategies. In this study, a new unspecific peroxygenase from Coprinopsis marcescibilis ( Cma UPO) is identified and characterized. To enhance or reverse the enantioselectivity of wildtype (WT) Cma UPO catalyzed asymmetric hydroxylation of ethylbenzene, Cma UPO was engineered using an efficient superfolder-green-fluorescent-protein ( sf GFP)-mediated secretion system in Escherichia coli . Iterative saturation mutagenesis (ISM) was used to target the residual sites lining the substrate tunnel, resulting in two variants: T125A/A129G and T125A/A129V/A247H/T244A/F243G. The two variants greatly improved the enantioselectivities [21% ee ( R ) for WT], generating the ( R )-1-phenylethanol or ( S )-1-phenylethanol as the main product with 99% ee ( R ) and 84% ee ( S ), respectively. The sf GFP-mediated secretion system in E. coli demonstrates applicability for different UPOs ( Aae UPO, Cci UPO, and Pab UPO-I). Therefore, this developed system provides a robust platform for heterologous expression and enzyme engineering of UPOs, indicating great potential for their sustainable and efficient applications in various chemical transformations.