Monday, August 11, 2008
P106

Metabolic Engineering of Escherichia Coli Strain C to Efficiently Produce Succinate In Mineral Salts Media

Kaemwich Jantama1, Xueli Zhang2, Jonathan C. Moore2, Spyros A Svoronos1, and Lonnie O. Ingram2. (1) Department of Chemical Engineering, University of Florida, Room 237, Chemical Engineering Bldg, Gainesville, FL 32611, (2) Microbiology and Cell Science, University of Florida, Bldg 981, Museum Road, Gainesville, FL 32611

Derivatives of Escherichia coli C were engineered to produce succinate in mineral salts media using simple fermentations (anaerobic stirred batch with pH control) without plasmids or foreign genes. This was done by a combination of genetic engineering and metabolic evolution with over 2000 generations of growth-based selection. The resulting strain, KJ091 (ΔldhA ΔadhE ΔfocA-pflB ΔmgsA ΔpoxB), produced 1.2 mol of succinate per mol glucose in mineral salts medium with acetate, malate, and pyruvate as significant co-products. Deletion of the threonine decarboxylase (tdcD; acetate kinase homologue) and 2-ketobutyrate formate-lyase (tdcE; pyruvate formate-lyase homologue) reduced the acetate level by 50% and increased succinate yield (1.3 mol mol-1 glucose) by almost 10% as compared to KJ091.  Deletion of two genes involved in oxaloacetate metabolism, aspartate aminotransferase (aspC) and the NAD+-linked malic enzyme (sfcA) (KJ122) significantly increased succinate yield (1.4 mol mol-1 glucose), succinate titer (approximately 700 mM), and average volumetric productivity (0.90 g L-1 h-1).  Residual pyruvate and acetate were substantially reduced by further deletion of pta encoding phosphotransacetylase to produce KJ134(ΔldhA ΔadhE ΔfocA-pflB ΔmgsA ΔpoxB ΔtdcDE ΔcitF ΔaspC ΔsfcA Δpta-ackA). Strain KJ134 produced 94% of the maximum theoretical yield of succinate (1.7 mol per mol glucose) during simple, anaerobic, batch fermentations using mineral salts medium. Strains KJ122 and KJ134 may be potentially useful as biocatalysts for the commercial production of succinate.

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