Identification of Enzymes from Pseudogluconobacter saccharoketogenes Producing d-Glucaric Acid from d-Glucose

In 2004, the US Department of Energy listed d-glucaric acid as one of the top 12 bio-based chemicals and a potential biopolymer building block. In this study, we show that Pseudogluconobacter saccharoketogenes strains can produce d-glucaric acid from d-glucose, although in low yield because of the generation of the byproduct 2-keto-d-gluconic acid in large quantities. To improve d-glucaric acid yield, we generated Rh47-3, a P. saccharoketogenes IFO14464 mutant, which produced d-glucaric acid from d-gluconic acid and d-glucose with 81 and 53 mol% yields, respectively. Furthermore, the key enzymes involved in d-glucaric acid production, alcohol dehydrogenase (Ps-ADH), aldehyde dehydrogenase (Ps-ALDH), and gluconate 2-dehydrogenase (Ps-GADH), were purified and their roles in d-glucaric acid synthesis were evaluated. Ps-ADH and Ps-ALDH catalyzed d-glucaric acid production, which was mediated by d-gluconic acid and d-glucuronic acid pathways. In contrast, Ps-GADH inhibited d-glucaric acid production by promoting the formation of 2-keto-d-gluconic acid from d-glucose.