A simple and efficient method for lyophilization of recombinant E. coli JM109 (DE3) whole-cells harboring active Rieske non-heme iron dioxygenases

Abstract

Rieske non-heme iron dioxygenases are a class of intriguing enzymes covering a broad reaction and substrate spectrum and have been studied extensively in the last decades. In nature, these biocatalysts are essential for the production of cis -dihydroxylated metabolites, as a first step during the degradation of aromatic compounds in microorganisms. The enzymes are able to produce relevant amounts of compounds in short reaction times, but the effort for constant cultivation of recombinant cells and production of cell mass for biotransformations is high. To overcome the steady production process, our task was to find a way to make the biocatalysts durable and storable. In this way, laboratories lacking equipment for microbiology, e.g. chemistry laboratories, can be supplied with the enzymes to open up new possibilities in the production of molecules. We present a quick and efficient method that uses lyophilization to freeze-dry recombinant whole-cells that harbor the enzyme of interest. By washing the cells with a cryoprotectant before lyophilization, we could conserve the enzyme activity to the level of freshly harvested cells. Moreover, this simple to apply method enables subsequent steps like storage of the cell powder for transportation and on demand use in biotransformations. The method was established with the cumene dioxygenase (CDO) of Pseudomonas fluorescens IP01 and its variant CDO M232A expressed in E. coli JM109 (DE3) cells, employing R -limonene and naphthalene, respectively, as substrates in biotransformations. The method could be successfully applied in the analytical and semi-preparative reaction scale. Preservation of biocatalysts in recombinant whole-cells. Ready-to-use enzymatic reaction. Semi-preparative biotransformation with lyophilized whole-cells.

Enhanced Semi‐Preparative Biotransformation of Cumene Dioxygenase: From Analytical Scale to Product Isolation

“…In the literature, semi-preparative biotransformations of ROs have been carried out in orbital shakers in flasks, baffled flasks or on magnetic stirrers in round bottom flasks [11,13,[18][19][20][21]. However, to the best of our knowledge no comparison of different semi-preparative biotransformation setups for ROs has been performed so far.…”

A Career in Catalysis: Bernhard Hauer

“…Also, undesired side reactions, such as product degradation by dehydrogenases, occur in whole cell systems. This was countered by the knockout of the responsible dehydrogenases and optimization of E. coli strains. ,− In a recent study, the glycerol dehydrogenase GldA in E. coli BW25113 catalyzing the degradation of cis -1,2-dihydrocatechol ( 44 ) was identified. By using the knockout strain E. coli BW25113 GldA from the KEIO collection, the degradation of cis -1,2-dihydrocatechol ( 44 ) produced from the TDO-catalyzed dihydroxylation from benzene ( 43 ) was dramatically reduced.…”