Directed evolution toward improved production of L-ribose from ribitol.
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 error-prone PCR and screened using a dinitrosalicylate reagent were prepared. 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(-1)d(-1) in 50 mL shaken flasks at 34 degrees 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 large-scale production of L-ribose.
Christ
Trevor N
TN
tchrist@zuchem.com
Deweese
Kara A
KA
Woodyer
Ryan D
RD
D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D008354:Mannitol Dehydrogenases / Q000235:genetics; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000235:genetics; D012255:Ribitol / Q000378:metabolism*; D012266:Ribose / Q000096:biosynthesis*
D011994:Recombinant Proteins; D012255:Ribitol; D012266:Ribose; D008354:Mannitol Dehydrogenases