Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

Abstract

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 Escherichia coli 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 ؉ . Expression of low-K m Zymomonas mobilis pdc (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 E. coli, 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 E. coli 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 Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.The engineering of metabolic pathways for production of industrial chemicals from renewable carbohydrates offers an alternative to petroleum-based processes and chemical synthesis (3,12,22). Genetically altering metabolic pathways, however, often causes unanticipated changes which may limit utility. Undesirable changes, such as reduced growth, decreased glycolytic flux, and low volumetric productivity, are generally attributed to a lack of ATP (19, 50), creation of futile cycles (10,11,37), changes in intracellular metabolite pools, or a metabolic imbalance (2,7,9,13,28,51,53). Often, these detrimental effects are masked by abundant complex nutrients in laboratory media and are apparent only in more minimal media (7,10,11,31).Salmonella enterica serovar Typhimurium was engineered for succinate production by increasing expression of pyc encoding pyruvate carboxylase (50). Although succinate production increased, the growth rate declined by 18% and the glycolytic flux decreased by 40%. Similar results were reported for an analogous construction in Escherichia coli (19). Donnelly and cow...

Lack of Protective Osmolytes Limits Final Cell Density and Volumetric Productivity of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

“…1). Supplementing fermentations with potassium glutamate (2 g liter Ϫ1 ) or expressing Bacillus subtilis citZ encoding an NADH-insensitive citrate synthase stimulated growth and increased volumetric ethanol production (45). Similar benefits were also observed from the deletion of acetate kinase, a mutation that increased the availability of acetyl coenzyme A (acetyl-CoA) (46).…”

“…During aerobic growth in CSL medium (9% xylose, 600 mM), E. coli KO11 grew to more than twice the cell density of cultures grown anaerobically in the same medium with or without additional minerals and trace metals ( Fig. 2A) (45). ATP production does not appear to limit anaerobic growth in 9% xylose (45,46), since cell densities achieved with xylose (Ͻ0.5 ATP molecule per glucose molecule) were equivalent to those with glucose (Ͼ2.0 ATP molecules per glucose molecule).…”

“…Seed cultures and fermentations were performed in CSL medium at 35°C (pH 6.5, 120 rpm) as described previously (34,45). Fermentations were inoculated to provide an initial cell density of 33 mg (dry weight) liter Ϫ1 .…”

“…The limited cell growth and low volumetric productivity of ethanol in CSL medium (containing mineral salts, 10 g of corn steep liquor liter Ϫ1 , and 90 g of xylose liter Ϫ1 ) have been attributed to inadequate partitioning of carbon skeletons into the synthesis of glutamate and other products derived from the citrate arm of the anaerobic tricarboxylic acid (TCA) pathway (45,46) (Fig. 1).…”

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

“…Citrate lyase, an enzyme which cleaves citrate into an equimolar mixture of oxaloacetate and acetate (Schneider et al 2000), is induced when citrate or isocitrate are directly added to the culture medium. Concentrations of both oxaloacetate-derived fumarate and acetate were higher in fermentations conducted with added citrate than in fermentations with 2-ketoglutarate and other TCA pathway intermediates, which would reflect the induction of citrate lyase, as reported by Underwood et al (2002) for an ethanologenic E. coli strain when growing on xylose. Induction of this enzyme probably limits citrate beneficial effects on biosynthesis, which is in good agreement with the enhanced growth and ethanol synthesis observed when 2-ketoglutarate was added.…”