Co-factor regeneration at the cell surface – upgrading the biotechnological potential of whole cell biocatalysts.

Schüürmann J, Lang K, Jose J

Abstract

Whole cell biocatalysts offer significant advantages compared to purified enzymes, more specifically cheap and efficient production combined with obsolete purification. However, there are limitations to theapplicability of whole cells. Cross reactions with endogenous enzymes and membrane impermeability of substrates and products might complicate their use. Autodisplay presents enzymes on the surface ofE.coli cells eliminating mass transfer problems and possible side reactions. The technique replaces the passenger domain of a native autotransporter protein by a peptide or protein of choice. However, a majorchallenge for some enzymes to be used in biotechnological applications, is the regeneration of co-factors, which are too expensive to be added stoichiometrically. Here, we report the development of a cellsurface based NADPH regeneration system. To circumvent laborious protein purification and keep the advantages of surface displayed catalysts we used the Autodisplay technology to present variousdehydrogenases on the cell surface of E.coli. Surface display was confirmed by protease accessibility tests and FACS analysis. NADPH production was measured photometrically at 340 nm. We first concentratedour efforts on an engineered formate dehydrogenase. The enzyme showed similar specific activity compared to the soluble enzyme, but the efficiency of the system was limited by the number of enzymes onthe bacterial surface and high amounts of whole-cell catalysts were needed for effective production of NADPH. Thus, we are currently exploring alternative enzymes with higher specific activities for surfacedisplay and cell associated cofactor regeneration.