The growing interest in a “biomass-based” economy has led to new efforts to construct and improve microorganisms capable of producing new chemicals. While the current focus is largely on liquid biofuels, a successful “biorefinery” is likely to be a mixed-product facility, with many compounds produced from a single, biomass-derived feed. One such “value-added” compound is glucaric acid, a molecule with potential uses ranging from human therapeutics to polymer synthesis. Glucaric acid is a natural product, found in fruits, vegetables, and mammals, but there is no known microbial pathway. We are working towards the construction of two different routes for the synthesis of glucaric acid from glucose in E. coli. The first is a three-step pathway consisting of a series of enzyme reactions derived from yeast, mammalian, and bacterial sources. While the combination of these three steps into a single organism constitutes a “novel” pathway, each step is a naturally-occurring reaction, demonstrated in the original species. In contrast, the second pathway 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. We will present progress on the construction of these pathways towards the microbial synthesis of glucaric acid.