Influence of C₄‐Dcu transporters on hydrogenase and formate dehydrogenase activities in stationary phase‐grown fermenting Escherichia coli

Abstract

During mixed‐acid fermentation, Escherichia coli transports succinate mainly via transporters of the Dcu family. Here, we analyze the influence of Dcu transporters on hydrogenase (Hyd) and fermentative formate dehydrogenase (FDH‐H) activities and how this is affected by external pH and carbon source. Using selected dcu mutations, it was shown that Dcu carriers mainly affect Hyd and FDH‐H activities during glycerol but not glucose fermentation at acidic pH. During glycerol fermentation at pH 5.5, inactivation of either one or all Dcu carriers increased total Hyd activity by 60% compared with wild type. Under the same growth conditions, a dcuACBD mutant had a twofold higher FDH‐H activity. When glucose was fermented in dcuD single mutant at pH 5.5, the FDH‐H activity was also increased twofold compared with wild type. Interestingly, in dcuD or dcuACBD mutants at pH 7.5, Hyd activity was lowered by 20%. Taken together, it can be concluded that during glucose fermentation at pH 7.5, lack of DcuD affects Hyd enzyme activity, but at pH 5.5, it has a stronger effect on FDH‐H activity. During glycerol fermentation, lack of Dcu carriers increased Hyd and FDH‐H activities as revealed at pH 5.5. The results suggest that impairing Dcu transport function increases intracellular formate levels and thus affects H2 cycling and proton‐motive force generation.

A Synthetic Pathway for the Production of Benzylsuccinate in Escherichia coli

“…To achieve the conversion of benzoyl-CoA to benzylsuccinate, we intended to use the reverse-running β-oxidation pathway of benzylsuccinate to benzoyl-CoA, which participates in anaerobic toluene degradation. Because we intended to couple the reaction with anaerobically grown E. coli cultures producing succinate as a fermentation product [ 36 , 37 , 38 ], we decided to use the bbs operon of the ferric-iron-reducing bacterium G. metallireducens (genes Gmet_1521–1531 ) [ 39 ] as the source for the enzymes. Compared to denitrifying toluene-degrading bacteria, the bbs operons of G. metallireducens and other strictly anaerobic toluene degraders contain additional genes for the subunits of a special electron-transfer flavoprotein (ETF; Gmet_1525 and 1526 ) which serves as an electron acceptor for benzylsuccinyl-CoA dehydrogenase [ 25 ] and an ETF:menaquinone oxidoreductase ( Gmet_1527 ) [ 40 ], which may result in a better performance of the pathway under anaerobic conditions.…”

A Synthetic Pathway for the Production of Benzylsuccinate in <em>Escherichia coli</em>

“…To achieve the conversion of benzoyl-CoA to benzylsuccinate, we intended to use the reverse-running β-oxidation pathway of benzylsuccinate to benzoyl-CoA, which participates in anaerobic toluene degradation. Because we intended to couple the reaction with anaerobically grown E. coli cultures producing succinate as fermentation product [36][37][38], we decided to use the bbs operon of the ferric iron reducing bacterium G. metallireducens (genes Gmet_1521 -1531) [39] as the source for the enzymes. Compared to denitrifying toluene-degrading bacteria, the bbs operons of G. metallireducens and other strictly anaerobic toluene degraders contain additional genes for the subunits of a special electron-transfer flavoprotein (ETF; Gmet_1525 and 1526) which serves as electron acceptor for benzylsuccinyl-CoA dehydrogenase [40] and an ETF:menaquinone oxidoreductase (Gmet_1527) [41], which may result a better performance of the pathway under anaerobic conditions.…”