Glucose metabolism in ‘Sphingomonas elodea’: pathway engineering via construction of a glucose-6-phosphate dehydrogenase insertion mutant

摘要

‘Sphingomonas (formerly Pseudomonas) elodea’ produces the industrially important polysaccharide gellan when grown in media containing glucose. Glucose catabolic enzymes and enzymes of central carbon metabolism were assayed in crude extracts of glucose-grown cultures of this bacterium. Based on these analyses it was concluded that glucose is converted to either gluconate or glucose 6-phosphate and that both of these products are converted to 6-phosphogluconate, a precursor for the Entner-Doudoroff (ED) and pentose phosphate pathways. Phosphoglucoisomerase (Pgi) activity was detected, but the lack of phosphofructokinase activity indicated that the Embden-Meyerhof glycolytic pathway is non-functional for glucose degradation. Thus, this bacterium utilizes glucose mainly via the ED and pentose phosphate pathways. Enzyme analyses suggested the involvement of glucose-6-phosphate dehydrogenase (Zwf) in glucose utilization and CO2 production. The zwf gene was cloned from ‘S. elodea’ and partially sequenced, and a null zwf mutant was constructed. This mutant exhibited no Zwf activity in in vitro assays, grew normally on glucose minimal medium and accumulated biomass (cells plus gellan) and produced CO2 at the same rates as the parental strain. Potential explanations for this finding are provided. Clones carrying the pgi gene were isolated fortuitously.