Oxygenase Catalysis Based On Recombinant Microorganisms
Oxidoreductases catalyze a large variety of regio-, stereo-, and chemoselective hydrocarbon oxyfunctionalizations, reactions, which are important in industrial organic synthesis but difficult to achieve by chemical means. The especially versatile enzyme class of oxygenases is capable of specifically introducing oxygen from molecular oxygen into a large range of organic molecules under mild conditions. Thus, oxygenase catalysis is of utmost interest for the chemical and pharmaceutical industies. The development of oxygenase-based bioprocesses suitable for industry faces hurdles that are not experienced with easy-to-use enzymes such as hydrolases, isomerases, or lyases. Oxygenases are often unstable, consist of multiple components, of which some might be membrane-bound, and require costly cofactors such as NAD(P)H. Thus, for synthetic applications on medium to large scale, the most promising approach is the use of oxygenase containing whole microbial cells, which regenerate cofactors and continuously synthesize the oxygenase. Based on Escherichia coli, Pseudomonas and yeast strains, we develop recombinant whole-cell catalysts and processes for oxygenase catalysis. For several different oxygenase classes, which contain heme, non-heme iron, or flavin in the active site, we investigate critical issues such as low enzyme activity and specificity, product degradation, cofactor recycling, reactant toxicity, and substrate and oxygen mass transfer. To overcome them, we use both biochemical process engineering and biocatalyst engineering approaches.