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31P nuclear magnetic resonance studies of the fermentation of glucose to ethanol by Zymomonas mobilis.

High resolution 31P nuclear magnetic resonance spectroscopy has been employed to study the fermentation of glucose to ethanol by Zymomonas mobilis, strain ZM4, a bacterium which uses the Entner-Doudoroff pathway. The levels of nucleoside triphosphates, sugar phosphates, UDP-sugars and Pi in intact fermenting cells have been studied with a time resolution of 1 min. It is suggested that a pH gradient is established across the cell membrane during fermentation and that the intracellular pH does not rise above approximately 6.4. 31P resonances from most phosphorus-containing intermediates in the Entner-Doudoroff pathway, as well as adenosine and uridine nucleotides and a number of other intracellular metabolites, have been assigned in perchloric extracts of fermenting cells by means of a number of techniques, including two-dimensional homonuclear J-resolved and two-dimensional homonuclear shift-correlated spectroscopy. Quantification of these metabolites in spectra of extracts of fermenting cells indicates that the rate-limiting steps in the Entner-Doudoroff pathway in Z. mobilis are the conversions of glucose 6-phosphate to 6-phosphogluconate and of 3-phosphoglycerate to 2-phosphoglycerate.