Protein design and host engineering for whole-cell biocatalysis using pyrroloquinoline quinone (PQQ)-dependent oxidoreductases

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The proposed research aims to establish pyrroloquinoline quinone (PQQ)-dependent oxidoreductases as a novel, versatile tool for the whole-cell biocatalytic synthesis of industrially relevant products. For this, a synthetic pathway for the production of glyoxylic acid, an important building block for the synthesis of many agrochemicals and polymers, via the metabolism of ethylene glycol using engineered PQQ-dependent oxidoreductases will be established. Pseudomonas putida KT2440 represents an ideal platform for such an approach, due to the presence of the necessary PQQ-dependent oxidation system including cofactor biosynthesis, the excellent available genetic tools, and the general robustness towards harsh environmental conditions. The latter is especially valuable for reactions, in which substrates - such as alcohols - or the corresponding products - such as aldehydes or acids - are toxic to the cells. The use of PQQ-dependent oxidoreductases, which are located in the periplasm of the cell and, thus, catalyze the oxidation of the substrate outside of the cytosol, represents an intelligent and designable way to further limit the toxic effects for a living biocatalyst. In addition, the unique cofactor of these enzymes (PQQ) which shuttles the electrons from the irreversible oxidation reaction directly into the respiratory chain, eliminates a specific regeneration cycle that usually is needed for other electron-transferring cofactors, such as NAD(P)H or FADH.For the successful establishment of a novel whole-cell biocatalyst, the fundamental knowledge of interfering enzyme reactions and genetic stability of the host, defined biochemical properties for the enzymatic catalyst of interest, and an understanding of the molecular determinants responsible for substrate selectivity, are required. Thus, this proposal will combine biochemical and physiological studies, genetics and ´Omics´ techniques, and bioinformatics-driven protein engineering, to provide new and exciting insights into the regulation and function of PQQ-dependent oxidation systems in Pseudomonads, and will expand the existing toolbox for bio-oxidations with a novel and highly promising biocatalyst.