Glucose consumption in carbohydrate mixtures by phosphotransferase-system mutants of Escherichia coli.

Escherichia coli lacking the glucose phosphotransferase system (PTS), mannose PTS and glucokinase are supposedly unable to grow on glucose as the sole carbon source (Curtis SJ, Epstein W. J Bacteriol 1975;122:1189-1199). We report that W ptsG manZ glk (ALS1406) grows slowly on glucose in media containing glucose with a second carbon source: ALS1406 metabolizes glucose after that other carbon source, including arabinose, fructose, glycerol, succinate or xylose, is exhausted. Galactose is an exception to this rule, as ALS1406 simultaneously consumes both galactose and glucose. The ability of ALS1406 to metabolize glucose in a xylose-glucose mixture was unchanged by an additional knockout in any single gene involved in carbohydrate transport and utilization, including agp (periplasmic glucose-1-phosphatase), galP (galactose permease), xylA (xylose isomerase), alsK (allose kinase), crr (glucose PTS enzyme IIA), galK (galactose kinase), mak (mannokinase), malE (maltose transporter), malX (maltose PTS enzyme IIBC), mglB (methyl-galactose transporter subunit), nagE (N-acetyl glucosamine PTS enzyme IICBA), nanK (N-acetyl mannosamine kinase) or pgm (phosphoglucose mutase). Glucose metabolism was only blocked by the deletion of two metabolic genes, pgi (phosphoglucose isomerase) and zwf (glucose-6-phosphate 1-dehydrogenase), which prevents the entry of glucose-6-phosphate into the pentose phosphate and Embden-Meyerhof-Parnas pathways. Carbon-limited steady-state studies demonstrated that xylose must be sub-saturating for glucose to be metabolized, while nitrogen-limited studies showed that xylose is partly converted to glucose when xylose is in excess. Under transient conditions, ALS1406 converts almost 25 % (mass) xylose into glucose as a result of reversible transketolase and transaldolase and the re-entry of carbon into the pentose phosphate pathway via glucose-6-phosphate 1-dehydrogenase.

Adaptation of Escherichia coli to elevated sodium concentrations increases cation tolerance and enables greater lactic acid production.

Adaptive evolution was employed to generate sodium (Na(+))-tolerant mutants of Escherichia coli MG1655. Four mutants with elevated sodium tolerance, designated ALS1184, ALS1185, ALS1186, and ALS1187, were independently isolated after 73 days of serial transfer in medium containing progressively greater Na(+) concentrations. The isolates also showed increased tolerance of K(+), although this cation was not used for selective pressure. None of the adapted mutants showed increased tolerance to the nonionic osmolyte sucrose. Several physiological parameters of E. coli MG1655 and ALS1187, the isolate with the greatest Na(+) tolerance, were calculated and compared using glucose-limited chemostats. Genome sequencing showed that the ALS1187 isolate contained mutations in five genes, emrR, hfq, kil, rpsG, and sspA, all of which could potentially affect the ability of E. coli to tolerate Na(+). Two of these genes, hfq and sspA, are known to be involved in global regulatory processes that help cells endure a variety of cellular stresses. Pyruvate formate lyase knockouts were constructed in strains MG1655 and ALS1187 to determine whether increased Na(+) tolerance afforded increased anaerobic generation of lactate. In fed-batch fermentations, E. coli ALS1187 pflB generated 76.2 g/liter lactate compared to MG1655 pflB, which generated only 56.3 g/liter lactate.

A substrate-selective co-fermentation strategy with Escherichia coli produces lactate by simultaneously consuming xylose and glucose.

We describe a new approach for the simultaneous conversion of xylose and glucose sugar mixtures which potentially could be used for lignocellulosic biomass hydrolysate. In this study we used this approach to demonstrate the production of lactic acid. This process uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. In addition to knockouts in pflB encoding for pyruvate formate lyase, the xylose-selective (glucose deficient) strain E. coli ALS1073 has deletions of the glk, ptsG, and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1074 has a xylA deletion. By combining these two strains in a single process the xylose and glucose in a mixed sugar solution are simultaneously converted to lactate. Furthermore, the biomass concentrations of each strain can readily be adjusted in order to optimize the overall product formation. This approach to the utilization of mixed sugars eliminates the problem of diauxic growth, and provides great operational flexibility.

High glycolytic flux improves pyruvate production by a metabolically engineered Escherichia coli strain.

We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h(-1). Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g . h), pyruvate formation rate (1.11 g/g h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h(-1) achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter h and yield of 0.68 g/g.

The Promiscuous sumA Missense Suppressor from Salmonella enterica Has an Intriguing Mechanism of Action.

While most missense suppressors have very narrow specificities and only suppress the allele against which they were isolated, the sumA missense suppressor from Salmonella enterica serovar Typhimurium is a promiscuous or broad-acting missense suppressor that suppresses numerous missense mutants. The sumA missense suppressor was identified as a glyV tRNA Gly3(GAU/C) missense suppressor that can recognize GAU or GAC aspartic acid codons and insert a glycine amino acid instead of aspartic acid. In addition to rescuing missense mutants caused by glycine to aspartic acid changes as expected, sumA could also rescue a number of other missense mutants as well by changing a neighboring (contacting) aspartic acid to glycine, which compensated for the other amino acid change. Thus the ability of sumA to rescue numerous missense mutants was due in part to the large number of glycine codons in genes that can be mutated to an aspartic acid codon and in part to the general tolerability and/or preference for glycine amino acids in proteins. Because the glyV tRNA Gly3(GAU/C) missense suppressor has also been extensively characterized in Escherichia coli as the mutA mutator, we demonstrated that all gain-of-function mutants isolated in a glyV tRNA Gly3(GAU/C) missense suppressor are transferable to a wild-type background and thus the increased mutation rates, which occur in glyV tRNA Gly3(GAU/C) missense suppressors, are not due to the suppression of these mutants.

Effect of overexpressing nhaA and nhaR on sodium tolerance and lactate production in Escherichia coli.

BACKGROUND: Like other bacteria, Escherichia coli must carefully regulate the intracellular concentration of sodium ion (Na+). During the bacterial production of any organic acid, cations like Na+ invariably accumulate during a process which must maintain a near neutral pH. In this study, the E. coli nhaA gene encoding the Na+/H+ antiporter membrane protein and the nhaR gene encoding the NhaA regulatory protein were overexpressed in wild-type E. coli MG1655 and in MG1655 pflB (ALS1317) which lacks pyruvate formate lyase activity and thus accumulates lactate under anaerobic conditions. RESULTS: Expression of either the nhaA or nhaR gene on the high copy inducible expression vector pTrc99A caused a significant reduction in the growth rate of MG1655. No change in growth rate was observed for MG1655 or ALS1317 for Na+ concentrations of 0.75-0.90 M when the medium copy pBR322 plasmid was used to overexpress the two genes. In a fed-batch process to produce the model acid lactate with NaOH addition for pH control, lactate accumulation ceased in MG1655, MG1655/pBR322, MG1655/pBR322-nhaR and MG1655/pBR322-nhaA when the concentration reached 55-58 g/L. In an identical process lactate accumulation in MG1655/pBR322-nhaAR did not terminate until the concentration reached over 70 g/L. CONCLUSIONS: Although overexpression the genes did not improve growth rate at high Na+ concentrations, the overexpression of nhaA and nhaR together led to a 25% increase in lactate production. Thus, the observed (absence of) impact that these genetic modifications had on growth rate is a poor indicator of their effect on acid accumulation. The overexpression of nhaAR did not cause faster lactate production, but permitted the culture to continue accumulating lactate at 10% greater Na+ concentration.