| The large-scale conversion of lignocellulose to fuel ethanol would greatly reduce the U.S. dependence on imported oil. To facilitate this need, Escherichia coli has been genetically engineered for the homofermentative production of ethanol from all constituent sugars of lignocellulose. However, high levels of complex nutrients are required for rapid fermentation of xylose, the second most abundant sugar in lignocellulose. With low levels of complex nutrients, the rate of xylose fermentation was limited by the growth of the biocatalyst. In a mineral salts medium containing 1% corn steep liquor as a nutrient source (90 g liter-1 xylose), growth was limited by an imbalance in the partitioning of carbon between ethanol production and biosynthetic pathways. Citrate synthase was shown to catalyze the specific growth-limiting reaction. The allosteric controls of citrate synthase regulate carbon flow through the oxidizing arm of the TCA pathway, ultimately producing 2-ketoglutarate and glutamate. Functionally expressing citrate synthase II(citZ) from Bacillus subtilis stimulated growth due to its different allosteric and kinetic properties. Acetyl-CoA served as an antagonist to the NADH-mediated allosteric inhibition of the E. coli citrate synthase. Supplementing the medium with pyruvate, acetate, acetaldehyde, 2-ketoglutarate or glutamate increased growth and ethanol production by activating, relieving or bypassing the allosteric regulation of the E. coli citrate synthase. Conservation of acetyl-CoA by mutating acetate kinase (Delta ackA) also increased growth and ethanol production, presumably by increasing the availability of acetyl-CoA (activating citrate synthase). In addition to biosynthetic needs, large intracellular pools of glutamate (>20 mM) function as a protective osmolyte. During growth in the high osmotic environment of the corn steep liquor medium containing 0.6 M xylose, intracellular glutamate was low (<10 mM) and cells grew poorly, consistent with a glutamate deficiency. The addition of glutamate to the medium and all approaches that stimulated citrate synthase increased the high intracellular pool of glutamate during growth in this medium. Supplementing with other protective osmolytes, such as betaine and dimethylsulfoniopropionate, restored growth without affecting the intracellular pool of glutamate and appear to act directly as alternative osmolytes. These results indicate that the poor growth and ethanol production in 1% corn steep liquor medium (0.6 M xylose), the apparent requirement for high levels of nutrients without a specific auxotrophic requirement and the beneficial effects of increased intracellular glutamate all result from the requirement for high levels of protective osmolytes. Under these conditions, the growth of the biocatalyst (E. coli) and ethanol production are limited by insufficient levels of intracellular osmoprotectants rather than the synthesis of glutamate, per se. |
