Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals by the appropriate microbes. Due to the differences in the optimum conditions for the activity of the fungal cellulases that are required for depolymerization of cellulose to fermentable sugars and the growth and fermentation characteristics of the current industrial microbes, simultaneous saccharification and fermentation (SSF) of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity, leading to a higher-than-required cost of cellulase in SSF. We have isolated bacterial strains that grew and fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to L(؉)-lactic acid at 50°C and pH 5.0, conditions that are also optimal for fungal cellulase activity. Xylose was metabolized by these new isolates through the pentose-phosphate pathway. As expected for the metabolism of xylose by the pentose-phosphate pathway, [13 C]lactate accounted for more than 90% of the total 13 C-labeled products from [ 13 C]xylose. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans, although the B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. These new B. coagulans isolates have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource, for the production of fuels and chemicals.

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...

The PduO enzyme of Salmonella enterica is an ATP:cob(I)alamin adenosyltransferase that catalyzes the final step in the conversion of vitamin B 12 to coenzyme B 12 . The primary physiological role of this enzyme is to support coenzyme B 12 -dependent 1,2-propanediol degradation, and bioinformatic analysis has indicated that it has two domains. Here the PduO adenosyltransferase was produced in Escherichia coli, solubilized from inclusion bodies, purified to apparent homogeneity, and partially characterized biochemically. The K m values of PduO for ATP and cob(I)alamin were 19.8 and 4.5 M, respectively, and the enzyme V max was 243 nmol min ؊1 mg of protein ؊1 . Further investigations showed that PduO was active with ATP and partially active with deoxy-ATP, but lacked measurable activity with other nucleotides. 31 P nuclear magnetic resonance established that triphosphate was a product of the PduO reaction, and kinetic studies indicated a ternary complex mechanism. A series of truncated versions of the PduO protein were produced in Escherichia coli, partially purified, and used to show that adenosyltransferase activity is associated with the N-terminal domain. The N-terminal domain was purified to near homogeneity and shown to have biochemical properties and kinetic constants similar to those of the full-length enzyme. This indicated that the C-terminal domain was not directly involved in catalysis or substrate binding and may have another role.

Limited cell growth and the resulting low volumetric productivity of ethanologenic Escherichia coli KO11 in mineral salts medium containing xylose 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 pathway. The results of nuclear magnetic resonance investigations of intracellular osmolytes under different growth conditions coupled with those of studies using genetically modified strains have confirmed and extended this hypothesis. During anaerobic growth in mineral salts medium containing 9% xylose (600 mM) and 1% corn steep liquor, proline was the only abundant osmolyte (71.9 nmol ml ؊1 optical density at 550 nm [OD 550 ] unit ؊1 ), and growth was limited. Under aerobic conditions in the same medium, twice the cell mass was produced, and cells contained a mixture of osmolytes: glutamate (17.0 nmol ml ؊1 OD 550 unit ؊1 ), trehalose (9.9 nmol ml ؊1 OD 550 unit ؊1 ), and betaine (19.8 nmol ml ؊1 OD 550 unit ؊1 ). Two independent genetic modifications of E. coli KO11 (functional expression of Bacillus subtilis citZ encoding NADH-insensitive citrate synthase; deletion of ackA encoding acetate kinase) and the addition of a metabolite, such as glutamate (11 mM) or acetate (24 mM), as a supplement each increased the intracellular glutamate pool during fermentation, doubled cell growth, and increased volumetric productivity. This apparent requirement for a larger glutamate pool for increased growth and volumetric productivity was completely eliminated by the addition of a protective osmolyte (2 mM betaine or 0.25 mM dimethylsulfoniopropionate), consistent with adaptation to osmotic stress rather than relief of a specific biosynthetic requirement.

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The ethanologenic bacteria Escherichia coli strains KO11 and LYO1, and Klebsiella oxytoca strain P2, were investigated for their ability to metabolize furfural. Using high performance liquid chromatography and 13C-nuclear magnetic resonance spectroscopy, furfural was found to be completely biotransformed into furfuryl alcohol by each of the three strains with tryptone and yeast extract as sole carbon sources. This reduction appears to be constitutive with NAD(P)H acting as electron donor. Glucose was shown to be an effective source of reducing power. Succinate inhibited furfural reduction, indicating that flavins are unlikely participants in this process. Furfural at concentrations >10 mM decreased the rate of ethanol formation but did not affect the final yield. Insight into the biochemical nature of this furfural reduction process may help efforts to mitigate furfural toxicity during ethanol production by ethanologenic bacteria.