Glucaric acid production by a recombinant E.coli strain: investigation of myo inositol oxygenase soluble expression

Glucaric acid has a wide range of potential applications including polyamide synthesis, phosphate replacement in detergents, and bio-based polyurethane production. The 6 carbon diacid 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 (myo-inositol-1-phosphate synthase), MIOX (myo-inositol oxygenase), and Udh (uronate dehydrogenase) genes resulting in production of D-glucaric acid from glucose.

We have developed a semi-defined production medium with a “glucose auto-feed system”, consisting of soluble starch and amyloglucosidase, to mimic in shake flasks and well plates the glucose fed-batch processes typically used at the industrial scale. A maximum optical density (OD600) of 27 and a glucaric acid titer of 3.6 gm/L were achieved in shake flasks using this small scale process.

Previous studies have indicated that MIOX activity is the lowest of the 3 enzymes expressed.  Using Western blot analysis, we have compared soluble MIOX expression using the semi-defined medium and (slow) “glucose auto-feed” culture conditions to conditions of excess glucose, higher IPTG concentrations and higher temperatures.  MIOX is expressed as a full-length (33 kDa) soluble protein when cells are grown using “glucose auto-feed” at a reduced temperature and IPTG concentration.  Alternatively, when cells are grown under conditions of excess glucose at higher temperatures and IPTG concentrations, an insoluble and truncated (25 kDa) MIOX is produced, perhaps explaining low MIOX activity levels.