A new synthetic platform with potential for the production of several rare sugars, with L-ribose as the model target, is described. 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 enzyme catalyzes the interconversion of several polyols and their L-sugar counterparts, including the conversion of ribitol to L-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of L-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 M ZnCl 2 and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter ؊1 day ؊1 . This system represents a significantly improved method for the large-scale production of L-ribose.Optically pure carbohydrates are important intermediates for the preparation of pharmaceutical, food, and agrochemical products (2,4,15,22). In particular, these carbohydrates are increasingly important in biochemical research and in development of new pharmaceutical therapies since carbohydrates are involved in cellular recognition, signaling, extra-and intracellular targeting, and even the development of disease states (1,2,7,22,27). Access to consistent, optically pure, and inexpensive carbohydrate starting materials is critical to the continuation of this research.The unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (33-38, 44) acts on the 1 position of D-mannitol, producing D-mannose (Fig. 1), in contrast to the more common 2-mannitol dehydrogenase, which interconverts D-mannitol and D-fructose (37). This novel regioselectivity combined with stringent stereoselectivity at the 2 position allows the MDH enzyme from A. graveolens to catalyze several interesting conversions, including the conversion of ribitol to L-ribose (Fig. 1), the conversion of D-sorbitol to L-gulose, and the conversion of galactitol to L-galactose (33). Sugars with the L configuration are often available only in limited amounts or at a high cost. To address these availability and economic concerns, utilization of MDH from A. graveolens is proposed.For this study, L-ribose was chosen as a model target since it is the potential starting material for many L-nucleoside-based pharmaceutical compounds, including Clevudine, Tyzeka, Valtorcitabine, Elvucitabine, and Epivir (12,14,40). The interest in L-nucleosides has increased, as noted in Table 1, which shows several L-nucleoside-based pharmaceutical compounds recently app...