263013 Improvement of D-Glucaric Acid Production From a Synthetic Pathway in Escherichia Coli
Improvement of D-Glucaric Acid Production from a Synthetic Pathway in Escherichia coli
Eric Shiue, Kristala Jones Prather
D-glucaric acid is a naturally occurring compound that has been investigated for a number of potential uses, including cancer treatment, cholesterol reduction, and production of biodegradable polymers. Additionally, D-glucaric acid has been labeled a “top value-added chemical from biomass” by the US Department of Energy. A pathway for the biosynthesis of D-glucaric acid from glucose has been constructed in E. coli, consisting of three heterologously expressed natural enzymes: myo-inositol-1-phosphate synthase (INO1) from Saccharomyces cerevisiae, myo-inositol oxygenase (MIOX) from mouse, and uronate dehydrogenase (Udh) from Pseudomonas syringae. Using this pathway, glucaric acid titers of 1.13 g/L were achieved following optimization of culture and induction conditions.
Characterization of the pathway's constituent enzymes has revealed MIOX to be the rate-limiting enzyme. Achievement of industrially relevant titers of D-glucaric acid therefore hinges on improving the activity of MIOX. Various strategies have been explored to improve MIOX activity, including timed induction of pathway enzymes, directed evolution of MIOX for improved activity, and MIOX-MBP fusions for enhancement of soluble expression. We will present experimental results on the application of these strategies towards enhancing MIOX activity and glucaric acid productivity.
 Moon, TS et al. Appl. and Env. Microbiology 75:589-595