{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T11:52:48Z","timestamp":1789386768288,"version":"build-2803163510"},"reference-count":55,"publisher":"American Society for Microbiology","issue":"3","license":[{"start":{"date-parts":[[2002,3,1]],"date-time":"2002-03-01T00:00:00Z","timestamp":1014940800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2002,3]]},"abstract":"<jats:title>ABSTRACT<\/jats:title><jats:p>Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strain<jats:italic>Escherichia coli<\/jats:italic>KO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD<jats:sup>+<\/jats:sup>. Expression of low-<jats:italic>K<jats:sub>m<\/jats:sub><\/jats:italic><jats:italic>Zymomonas mobilis pdc<\/jats:italic>(pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. In<jats:italic>E. coli<\/jats:italic>, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity of<jats:italic>E. coli<\/jats:italic>citrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning the<jats:italic>Bacillus subtilis citZ<\/jats:italic>gene encoding an NADH-insensitive citrate synthase. Expression of recombinant<jats:italic>citZ<\/jats:italic>in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.<\/jats:p>","DOI":"10.1128\/aem.68.3.1071-1081.2002","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T10:09:29Z","timestamp":1027764569000},"page":"1071-1081","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":72,"title":["Flux through Citrate Synthase Limits the Growth of Ethanologenic<i>Escherichia coli<\/i>KO11 during Xylose Fermentation"],"prefix":"10.1128","volume":"68","author":[{"given":"S. A.","family":"Underwood","sequence":"first","affiliation":[{"name":"Institute of Food and Agricultural Sciences, Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M. 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