|Woodyer, Ryan - ZUCHEM|
|Wymer, Nathan - ZUCHEM|
|Racine, F - ZUCHEM|
|Khan, Shama - ZUCHEM|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 8, 2008
Publication Date: April 1, 2008
Citation: Woodyer, R.D., Wymer, N.J., Racine, F.M., Khan, S.N., Saha, B.C. 2008. Efficient production of L-ribose with a recombinant Escherichia coli biocatalyst. Applied and Environmental Microbiology. 74(10):2967-2975. Interpretive Summary: L-ribose is a rare sugar. It is used as a starting material for many pharmaceutical compounds with antiviral, antimalarial, and anticancer activities. The demand for this rare sugar is increasing. An efficient production method for L-ribose from ribitol was developed using recombinant bacterial cells. The recombinant bacterial strain was constructed by expressing the gene encoding an enzyme from garden celery. This system represents a significantly improved method for large scale production of L-ribose and can be applied for production of other important rare sugars.
Technical Abstract: Carbohydrates have important roles in many cellular functions, diseases, and important natural products; however, the significant challenges in creating or obtaining carbohydrate-based molecules of known chemical structure have hampered their use in biochemical and pharmaceutical research. As a result, there is a large demand for starting materials based on rare sugars. 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 already large demand for L-ribose will continue to grow. 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 constructed 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**-1d**-1. This system represents a significantly improved method for the large scale production of L-ribose and will be applied to production of other L-sugars in the future.