Reengineering Escherichia coli for Succinate Production in Mineral Salts Medium

Abstract

The fermentative metabolism of glucose was redirected to succinate as the primary product without mutating any genes encoding the native mixed-acid fermentation pathway or redox reactions. Two changes in peripheral pathways were together found to increase succinate yield fivefold: (i) increased expression of phosphoenolpyruvate carboxykinase and (ii) inactivation of the glucose phosphoenolpyruvate-dependent phosphotransferase system. These two changes increased net ATP production, increased the pool of phosphoenolpyruvate available for carboxylation, and increased succinate production. Modest further improvements in succinate yield were made by inactivating the pflB gene, encoding pyruvate formate lyase, resulting in an Escherichia coli pathway that is functionally similar to the native pathway in Actinobacillus succinogenes and other succinateproducing rumen bacteria.

Increased Furfural Tolerance Due to Overexpression of NADH-Dependent Oxidoreductase FucO in Escherichia coli Strains Engineered for the Production of Ethanol and Lactate

“…Ampicillin (50 mg liter Ϫ1 ), kanamycin (50 mg liter Ϫ1 ), or chloramphenicol (40 mg liter Ϫ1 ) was added as appropriate. Red recombinase technology (Gene Bridges GmbH, Dresden, Germany) was used to facilitate chromosomal integration as previously described (17,19,41,42). All constructions were verified by DNA sequencing.…”

Improved Utilization of Crude Glycerol By-Product from Biodiesel Production

“…The highest concentration of 5.21 g/L of succinic acid at the 0.018/h dilution rate was achieved. Recently, several studies have been reported regarding the succinate production from glycerol by metabolically engineered strains of Escherichia coli (Blankschien et al;X. Zhang et al, 2009X.…”

Activating Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Combination for Improvement of Succinate Production

“…Although several rumen bacteria, such as Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, and Mannheimia succiniciproducens, can produce large amounts of succinate (2,(6)(7)(8), these microorganisms usually require a complex medium and exhibit relatively low yields. On the other hand, Escherichia coli has been widely engineered for succinate production with high titers and yields (3)(4)(5)(9)(10)(11)(12). Through inactivation of the pflB (encoding pyruvate-formate lyase), ldhA (encoding lactate dehydrogenase), and ptsG (encoding enzyme IICB Glc of the phosphoenolpyruvate: carbohydrate phosphotransferase system [PTS]) genes and overexpression of the pyruvate carboxylase gene, strain AFP111/ pTrc99A-pyc was obtained; it produced 99.2 g/liter succinate with a yield of 1.1 g/g glucose by use of a dual-phase fermentation process (3).…”