Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: Homoacetate production

Abstract

Microbial processes for commodity chemicals have focused on reduced products and anaerobic conditions where substrate loss to cell mass and CO2 are minimal and product yields are high. To facilitate expansion into more oxidized chemicals, Escherichia coli W3110 was genetically engineered for acetate production by using an approach that combines attributes of fermentative and oxidative metabolism (rapid growth, external electron acceptor) into a single biocatalyst. The resulting strain (TC36) converted 333 mM glucose into 572 mM acetate, a product of equivalent oxidation state, in 18 h. With excess glucose, a maximum of 878 mM acetate was produced. Strain TC36 was constructed by sequentially assembling deletions that inactivated oxidative phosphorylation (⌬atpFH), disrupted the cyclic function of the tricarboxylic acid pathway (⌬sucA), and eliminated native fermentation pathways (⌬focA-pflB ⌬frdBC ⌬ldhA ⌬adhE). These mutations minimized the loss of substrate carbon and the oxygen requirement for redox balance. Although TC36 produces only four ATPs per glucose, this strain grows well in mineral salts medium and has no auxotrophic requirement. Glycolytic flux in TC36 (0.3 mol⅐min ؊1 ⅐mg ؊1 protein) was twice that of the parent. Higher flux was attributed to a deletion of membrane-coupling subunits in (F1F0)H ؉ -ATP synthase that inactivated ATP synthesis while retaining cytoplasmic F1-ATPase activity. The effectiveness of this deletion in stimulating flux provides further evidence for the importance of ATP supply and demand in the regulation of central metabolism. Derivatives of TC36 may prove useful for the commercial production of a variety of commodity chemicals. metabolic engineering ͉ glycolytic flux ͉ acetic acid ͉ fermentation ͉ ATPase

Temperature‐dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli

“…For strains BJH58, ita35 and ita36A additionally 2 g/L glucose and 2.5 mM CaCl 2 were added. For the shake flask cultivations, a first preculture in LB 0 ‐medium was conducted at 37°C, the second preculture in Minimal Medium (MM) (Causey, Zhou, Shanmugam, & Ingram, 2003) at the respective starting temperature (37, 30, or 28°C) of the main culture. All main cultures were performed with MM with 4 g/L glucose.…”

Scalable production of mechanically tunable block polymers from sugar

“…To ensure the continued vitality of the polymer enterprise, it is necessary to invent high-performance polymers that are both sustainable and cost-competitive. In recent years, ingenious advances in synthetic biology have enabled the economical production of fuels (2-7), chemicals (8)(9)(10)(11)(12), and complex natural products (13-16) from renewable sugars. This elegant manipulation of existing organisms to efficiently produce valuable metabolites from inexpensive feedstocks represents a triumph of modern science and engineering and offers society the promise of renewable, environmentally compatible next-generation materials.…”

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

“…The sugar constituents of lignocellulose can be effectively metabolized by Escherichia coli, a commercially important biocatalyst for the production of amino acids and recombinant proteins (1,11). This organism has recently been engineered for the production of a variety of bulk chemicals, such as organic acids (4,8,18,20,36,47,53), diols (17,35,44), and fuel ethanol (22). Ethanologenic E. coli KO11 was previously constructed by the chromosomal integration of Zymomonas mobilis genes encoding a pyruvate decarboxylase with a low K m (pdc) and alcohol dehydrogenase II (adhB) (37).…”