Display of the self-sufficient CYP102A1 on the surface of E. coli-derived Outer Membrane Vesicles

Abstract

The cytochrome P450 (CYP) monooxygenase superfamily offers the unique ability to catalyze regio-and stereospecifical oxidation of a non-activated C-H bond. CYPs found applications in the synthesis of pharmaceuticals and drug metabolites as well as in bioremediation. They are typically used in whole-cell bioconversion processes, due to their low stability and the need for a redox partner and cofactor. Unfortunately, substrate uptake and/or product transport limitations are frequently encountered and side reactions occur due to other enzymes in the cellular environment. Here, we present a proof-of-principle of a novel cell-free cytochrome P-450 nanocatalyst based on surface display on bacterial outer membrane vesicles. The self-sufficient CYP 102A1 from Bacillus megaterium was engineered to be translocated on the outer membrane vesicles of Escherichia coli. The resulting vesicles can simply be isolated from the culture supernatant. Moreover, no expensive and elaborate enzyme purification is required. This approach shows great promise as an alternative strategy to recombinantly produce CYP enzymes for a variety of applications, such as in fine chemical production and in the development of biosensors.

Functionalization of OMVs for Biocatalytic Applications

“…Another example of using OMVs as biocatalysts is the display of CYP102A1 protein, a self-sufficient monooxygenase, on E. coli -derived OMVs to convert para -nitrophenoxydodecanoic acid or 12-pNCA substrate to para -nitrophenol, expanding the application of OMVs in the field of biosensors. Immobilization of CYP102A1 with OMVs demonstrates increased the stability and ease of biocatalyst use without the need for expensive enzyme purification [ 59 ]. Apparently, using OMVs as biocatalysts offers a promising platform for the efficient and sustainable production of high-value compounds.…”