Improving D-Glucaric Acid Production in E. Coli Through Directed Evolution and Delayed Induction

D-glucaric acid is a naturally occurring compound that has been investigated for a number of potential uses, including cancer treatment, cholesterol reduction, and biopolymer synthesis.  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 Mus musculus, and uronate dehydrogenase (Udh) from Pseudomonas syringae.  Characterization of the pathway’s constituent enzymes revealed MIOX to be the rate-limiting pathway enzyme.  Achievement of industrially relevant titers, therefore, hinges on improving the specific activity and/or stability of MIOX.  Several synthetic biology and metabolic engineering strategies have been explored to improve MIOX activity and stability, including time-delayed induction of pathway enzymes, directed evolution of MIOX for improved activity, and protein fusions for enhancement of soluble expression.  We will present experimental results from the application of these strategies towards enhancing MIOX activity and glucaric acid productivity.