Lack of protective osmolytes limits final cell density and volumetric productivity of ethanologenic Escherichia coli KO11 during xylose fermentation.

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 x 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 x ml(-1) OD(550) unit(-1)), trehalose (9.9 nmol x ml(-1) OD(550) unit(-1)), and betaine (19.8 nmol x 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.

Flux through citrate synthase limits the growth of ethanologenic Escherichia coli KO11 during xylose fermentation.

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.

Glycine betaine: reserve form of choline in Penicillium fellutanum in low-sulfate medium.

In spite of choline's importance in fungal metabolism, its sources in cytoplasm have not been fully established. 13C nuclear magnetic resonance analysis of mycelial extracts from day-5 Penicillium fellutanum cultures showed that, as well as choline-O-sulfate, intracellular glycine betaine is another reserve form of choline, depending on the availability of sulfate in the culture medium. These observations are discussed relative to the multiple roles of choline and its precursors in P. fellutanum.

Utilization of phosphocholine from extracellular complex polysaccharide as a source of cytoplasmic choline derivatives in Penicillium fellutanum.

Penicillium fellutanum produces a phosphorylated, choline-containing extracellular polysaccharide, peptidophosphogalactomannan (pP(x)GM) [where x is the number of phosphodiester residues]). The 13C-methyl-labeled pP(x)GM ([methyl-13C]pP(x)GM) was prepared from the cultures supplemented with L-[methyl-13C]methionine and was used as a probe to monitor the fate of phosphocholine in this polymer. The addition of [methyl-13C]pP(x)GM to growing cultures in low-phosphate medium resulted in the disappearance within 5 days of [methyl-13C]phosphocholine and N,N'-dimethylphosphoethanolamine from the added [methyl-13C]pP(x)GM. Two 13C-methyl-enriched cytoplasmic solutes, choline-O-sulfate and glycine betaine, were found in mycelial extracts, suggesting that phosphocholine-containing extracellular pP(x)GM of P. fellutanum is a precursor of intracellular choline-O-sulfate and glycine betaine. The mycelia cultured in low-phosphate (2 mM) medium contained glycine betaine and 1.5-fold more choline-O-sulfate than those grown in high-phosphate (20 mM) medium. The high levels of extracellular nonspecific phosphocholine:phosphocholine hydrolase and acid phosphomonoesterase observed in the low-phosphate culture medium are likely related to the release of phosphocholine from pP(x)GM and hydrolysis of phosphocholine, respectively. These results suggest that extracellular pP(x)GM of P. fellutanum provides phosphate needed as the environment becomes depleted of this nutrient. Choline, in excess of that needed immediately, is stored in the cytoplasm in forms that can be reutilized.

1H NMR study of lithium D-lactate.

Polycrystalline D-lactic acid lithium salt [(R)-2-hydroxypropanoic acid lithium salt, lithium D-lactate] has been investigated by pulsed proton magnetic resonance methods between 77 and 300 K at 25 MHz. The main relaxation mechanism is methyl rotation; the motion is characterized by an activation energy Ea = 14.5 +/- 0.5 kJ/mol and time factor tau 0 = (1.5 +/- 0.5) x 10(-13) s. The activation energy is higher than the potential barrier obtained by ESR and ENDOR techniques for methyl rotation in the lactate radical. The methyl rotation is also responsible for a reduction of the dipolar second moment. Below 100 K the reduction of the dipolar second moment is ascribed to quantum-mechanical tunneling; an excitation energy of 6.1 +/- 1 kJ/mol is derived from a contribution to the spin-lattice relaxation times from the tunneling.