Improvement of the one-step production of L-ribose from ribitol using a recombinant Escherichia coli is described. The gene encoding the enzyme mannitol-1-dehydrogenase (MDH) from Apium graveolens has previously been codon-optimized, cloned into the constitutive pZuc10 vector, and expressed in E. coli. This MDH catalyzes the NAD-dependent conversion of mannitol to D-mannose and has the ability to convert several polyols to their L-sugar counterparts, including ribitol to L-ribose. Here, three rounds of directed evolution using libraries generated through errorprone PCR and screened using a dinitrosalicylate reagent. Mutants were selected for improved conversion of L-ribose, and the best mutant was isolated by combining two round 2 mutations. Libraries were also selected for thermal stability and screened at increasingly higher temperatures with each round of mutagenesis. An overall 19.2-fold improvement was observed with a final conversion of 46.6 ± 1.7% and a productivity of 3.88 ± 0.14 gL-1d-1 in 50 mL shaken flasks at 34°C. Further characterization of the mutants suggests that increased enzyme thermal stability and expression are responsible for the increase in L-ribose production. The mutant E. coli production strain isolated represents an improved system for largescale production of L-ribose.