Engineering Escherichia coli for efficient conversion of glucose to pyruvate

Abstract

Escherichia coli TC44, a derivative of W3110, was engineered for the production of pyruvate from glucose by combining mutations to minimize ATP yield, cell growth, and CO2 production (⌬atpFH ⌬adhE ⌬sucA) with mutations that eliminate acetate production [poxB::FRT (FLP recognition target) ⌬ackA] and fermentation products (⌬focA-pflB ⌬frdBC ⌬ldhA ⌬adhE). In mineral salts medium containing glucose as the sole carbon source, strain TC44(⌬focA-pflB ⌬frdBC ⌬ldhA ⌬atpFH ⌬adhE ⌬sucA poxB::FRT ⌬ackA) converted glucose to pyruvate with a yield of 0.75 g of pyruvate per g of glucose (77.9% of theoretical yield; 1.2 g of pyruvate liters ؊1 ⅐h ؊1 ). A maximum of 749 mM pyruvate was produced with excess glucose. Glycolytic flux was >50% faster for TC44 producing pyruvate than for the wild-type W3110 during fully aerobic metabolism. The tolerance of E. coli to such drastic changes in metabolic flow and energy production implies considerable elasticity in permitted pool sizes for key metabolic intermediates such as pyruvate and acetyl-CoA. In strain TC44, pyruvate yield, pyruvate titer, and the rate of pyruvate production in mineral salts medium were equivalent or better than previously reported for other biocatalyts (yeast and bacteria) requiring complex vitamin feeding strategies and complex nutrients. TC44 offers the potential to improve the economics of pyruvate production by reducing the costs of materials, product purification, and waste disposal.metabolic engineering ͉ glycolytic flux ͉ fermentation ͉ ATPase

Homolactate Fermentation by Metabolically Engineered Escherichia coli Strains

“…The second strategy examined for prevention of succinate accumulation was a knockout of frdABCD (3,33). In E. coli, two distinct enzymes catalyze succinate oxidation and fumarate reduction (4): succinate-ubiquinone oxidoreductase functions aerobically as part of the TCA cycle, and menaquinol-fumarate oxidoreductase (QFR) is mainly used for anaerobic respiration and succinate production.…”

OptStrain: A computational framework for redesign of microbial production systems

“…This goal parallels the continuing quest in biotechnology to construct microbial strains capable of accomplishing an ever-expanding array of desired biotransformations. These biotransformations are aimed at products that range from simple precursor chemicals (Nakamura and Whited 2003;Causey et al 2004) or complex molecules such as carotenoids (Misawa et al 1991), to electrons in biofuel cells (Liu et al 2004) or batteries (Bond et al 2002;Bond and Lovley 2003), to even microbes capable of precipitating heavy metal complexes in bioremediation applications (Finneran et al 2002;Lovley 2003;Methe et al 2003). Recent developments in molecular biology and recombinant DNA technology have ushered in a new era in the ability to shape the gene content and expression levels for microbial production strains in a direct and targeted fashion (Stephanopoulos 2002).…”

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

“…Much research has reported that E. coli could be genetically modified to produce various amino acids and organic acids by plasmid-mediated homologous recombination (Zhou et al, 2003;Causey et al, 2004;Imaizumi et al, 2005;Zhang et al, 2007). At present, as far as we are aware, gene inactivation of the C. glutamicum genome is mainly carried out by plasmidmediated homologous recombination.…”