Monday, November 5, 2007 - 3:30 PM

Fermentative Production Of L-Sugars From Polyols

Ryan D. Woodyer1, Mike Racine1, David Demirjian1, and Badal C. Saha2. (1) zuChem, 2201 W Campbell Park Dr, Chicago, IL 60612, (2) Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, 1815 N University Street, Peoria, IL 61604

Demand for carbohydrate-based molecules has increased rapidly with their use in biochemical and pharmaceutical research. However, significant challenges exist in creating or obtaining carbohydrate-based molecules of known chemical structure. The recognition of the role of carbohydrates in many cellular functions and diseases as well as their importance in biologically active natural products has resulted in the need for large quantities of starting materials based on rare sugars for medicinal chemistry. For example, the unnatural rare sugar L-ribose is used in many different applications, including several nucleoside-based pharmaceuticals (current and in clinical trials), suggesting the large demand for L-ribose is still growing. Here, we describe a new synthetic platform with potential for the production of several rare sugars, with L-ribose being the model target. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically synthesized for optimal expression in Escherichia coli. This MDH catalyzes the interconversion of several polyols with their rare sugar counterparts including ribitol to L-ribose. Recombinant expression was successfully achieved in active form and one step purification was demonstrated. The synthetic utility of the recombinant E. coli strain as a whole cell catalyst was demonstrated with the production of L-ribose. The conversion of ribitol to L-ribose was optimized using shaken flasks. The final achieved conversions were >70% at a concentration of 40 g/L and >50% at a concentration of 100 g/L. The optimized conditions were then scaled up to a 1 L fermentation that resulted in a 55% conversion of 100 g/L ribitol in 72 hours for a volumetric productivity of 17.3 g/L*day. This system represents a significantly improved method for the large scale production of L-ribose. Application of this system as the first scalable commercial process for the production of L-galactose and L-gulose will be discussed.