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

Abstract

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.

Quinone Reduction by the Na+-Translocating NADH Dehydrogenase Promotes Extracellular Superoxide Production inVibrio cholerae

“…Superoxide and H 2 O 2 formation activities from three to four experiments are presented. The protein content of V. cholerae cell suspensions was estimated from the optical density, assuming that 1 unit of absorbance at 550 nm corresponds to 0.33 g total dry weight liter Ϫ1 (42) and that 55% of the total dry weight represents protein (32). Inhibition studies.…”

High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered Escherichia coli Strain

“…Another explanation for the observed termination of cell growth and pyruvate formation is the high osmotic stress caused by the K ϩ ion required to maintain the pH. Organic osmolytes, such as betaine, often accumulate naturally to protect cells from high osmotic stress (44), and the production of ethanol (38) and lactate (50) has been improved by the addition of betaine to the medium. Our fed-batch results similarly demonstrated that 5 mM betaine prolonged cell growth and thereby increased the final pyruvate concentration by at least 25%.…”

“…Because the accumulation of pyruvate in our current study would simultaneously require the accumulation of K ϩ (or another counterion) to maintain the pH, we conducted an experiment to determine whether the addition of the known osmoprotectant betaine (38,50) would improve pyruvate accumulation. Without betaine present in the medium under acetate-limited conditions, strain ALS929 was able to accumulate over 56 g/liter pyruvate in about 28 h (Fig.…”

Enhanced Trehalose Production Improves Growth of Escherichia coli under Osmotic Stress

“…Although glutamate and proline provide transient relief from osmotic stress, allosteric control of proline biosynthesis in E. coli and the negative charge of glutamate limit their effectiveness. Recent studies have shown that the addition of betaine and dimethylsulfoniopropionate stimulated the growth of and ethanol production by recombinant E. coli (41). Betaine also improved the growth of Bacillus subtilis at elevated temperatures (20).…”