Glucose Auto-feeding for Glucaric Acid Production by a Recombinant E. coli Strain

With a wide range of applications including polyamide synthesis, phosphate replacement, and bio-based polyurethane production, a 2004 DOE report listed glucaric acid, a 6-carbon diacid, as one of the “Top Chemicals from Biomass”.  Glucaric acid can be produced from glucose and possesses the ability to replace other 6 carbon petroleum derived chemical building blocks if it can be produced at a commodity chemical price point. The Prather lab at MIT has developed an Escherichia colistrain expressing the Ino1, Miox, and Udh genes which produces D-glucaric acid from glucose.

Using statistical experimental design methods, we have developed a semi-defined production medium with a “glucose auto feed system” utilizing soluble starch and amyloglucosidase to control the release of glucose from the reducing end of the starch molecules.  A maximum optical density (OD600) of 27 and a glucaric acid titer of 3.6 gm/L were achieved.  Analysis of variance revealed the significant effects of the salts, yeast extract, peptone, and IPTG concentrations (p-value < 0.05) and the two factor interactions glucose feed/yeast extract concentration and salts/IPTG concentrations.  This production medium approximates a fed-batch fermentation process that would be utilized to produce glucaric acid commercially and can be scaled-down to well plates for high throughput screening of potentially improved cultures.