(251f) Engineering Microbial Production of Glucaric Acid

Organic acids represent a class of compounds with both commercial interest and significant potential for biological production. Glucaric acid is one such molecule, deemed a ?value-added? compound for integration into biorefineries. We have engineered strains of E. coli capable of producing glucaric acid from glucose in three steps. Two of the genes utilized have been previously cloned and reported in the literature. INO1 enables the production of myo-inositol from glucose, and MIOX oxidizes myo-inositol to glucuronic acid. We have cloned and characterized a third enzyme, uronate dehydrogenase, from multiple sources, that facilitates the conversion of glucuronic acid to glucaric acid. Flux towards both glucuronic and glucaric acids is ultimately limited by MIOX, whose activity is dependent upon its substrate concentration. We have used synthetic scaffolds to co-localize INO1 and MIOX, thereby increasing the local concentration of myo-inositol. We will discuss the effects of these synthetic methods on improving the performance of the biosynthetic pathway. In addition, we will present progress to-date on the construction of an alternative designed pathway towards glucuronic acid that proposes putative transformations based on the selection of enzymes primarily according to generalized enzyme reactions and not specific substrate-enzyme pairs. This latter method follows a framework of synthetic biology for ?retro-biosynthetic design? of novel pathways and requires that new activities consisting of enzymes with altered substrate specificity be created.