Metabolic Engineering of Escherichia coli W3110 for Redox Neutral and Oxidized Products

Thomas B. Causey, Shengde Zhou, and L. O. Ingram

Florida Center for Renewable Chemicals and Fuels, University of Florida, Gainesville, FL 32611

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 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 4 ATPs per glucose, this strain grows well in mineral salts medium and has no auxotrophic requirement. Glycolytic flux in TC36 (0.5 µmol min-1 mg-1 protein) was 1.5-2.0 fold 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 strain TC36 may prove useful for the commercial production of a variety of commodity chemicals.

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