Efficient production of L-ribose with a recombinant Escherichia coli biocatalyst.
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 microM 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.
Woodyer
Ryan D
RD
zuChem, Inc., 2225 W. Harrison, Suite F, Chicago, Illinois 60612, USA. rwoodyer@zuchem.com
Wymer
Nathan J
NJ
Racine
F Michael
FM
Khan
Shama N
SN
Saha
Badal C
BC
D000077102:Apium / Q000201:enzymology / Q000235:genetics; D002712:Chlorides / Q000494:pharmacology; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005990:Glycerol / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012255:Ribitol / Q000378:metabolism; D012266:Ribose / Q000378:metabolism*; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D003067:Coenzymes; D011108:Polymers; D011994:Recombinant Proteins; D017967:Zinc Compounds; C024617:polyol; D012255:Ribitol; D012266:Ribose; C016837:zinc chloride; D013401:Sugar Alcohol Dehydrogenases; C077257:mannitol-mannose 1-oxidoreductase; D005990:Glycerol