Efficient Production of L-Ribose with a Recombinant Escherichia coli Biocatalyst

• Published in: Applied and Environmental Microbiology Vol. 74; no. 10; pp. 2967 - 2975
• Main Authors: Woodyer, Ryan D, Wymer, Nathan J, Racine, F. Michael, Khan, Shama N, Saha, Badal C
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.05.2008; American Society for Microbiology (ASM)
• Subjects: alcohol oxidoreductases; Apium graveolens; Apium graveolens - enzymology; Apium graveolens - genetics; biocatalysts; Biological and medical sciences; biosynthesis; Catalysts; Cells; Chlorides - pharmacology; Cloning, Molecular; Coenzymes - pharmacology; E coli; enzyme activity; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Fermentation; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes; genetically engineered microorganisms; Glycerol - metabolism; L-ribose; mannitol-1-dehydrogenase; Microbiology; Physiology and Biotechnology; Polymers - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Ribitol - metabolism; ribose; Ribose - metabolism; Sugar Alcohol Dehydrogenases - genetics; Sugar Alcohol Dehydrogenases - metabolism; synthetic genes; transgenes; zinc; Zinc Compounds - pharmacology; Bacteria; Escherichia coli; Genetically modified microorganism; Production; Enterobacteriaceae; Biocatalyst
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.02768-07

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 μM ZnCl₂ 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.