Schüürmann J, Lindhorst F, Jose J

NADPH Cofactor Regeneration on the Cell Surface: A Requirement for Pharmaceutical Applications of Surface Displayed P450 Enzymes

Abstract

One solution to circumvent mass transfer problems and possible cross reactions with intracellular enzymes is the presentation of enzymes on the cell surface. The Autodisplay technique replacesthe passenger domain of a native autotransporter by a peptide or protein of choice, which is then transported to the cell surface. The current project focusses on the display of CYP102A1 fromBacillus megaterium for pharmaceutical applications. Surface exposure of the enzyme was confirmed by protease accessibility tests, flow-cytometry analysis and activity measurements. In aproof-of-principle study we analyzed a small library of substances with the known R47L F87V L188Q mutant displayed on the cell surface of E. coli.However, the project also elucidated a major challenge for the use of the technology in industrial applications. Bringing an enzyme to the cell surface blocks the access to intracellular cofactors,which are too expensive to be added stoichiometrically. Keeping the advantages of a surface displayed catalyst, we also used the Autodisplay technology to present a dehydrogenases on thesurface of E.coli cells for the regeneration of the essential cofactor NADPH. We aim at a combination with the CYP102A1 whole cell biocatalyst for an efficient system for biotechnological andpharmaceutical applications.