Abstract
The sugar phosphotransferase system (PTS) is an essential energy-saving mechanism, particularly under anaerobic conditions. Since the PTS consumes equimolar phosphoenolpyruvate to phosphorylate each molecule of internalized glucose in the process of pyruvate generation, its absence can adversely affect the mixed acid fermentation profile and cell growth under anaerobic conditions. In this study, we report that the ΔptsG mutant cells of Escherichia coli K-12 strain exhibited inefficient glucose utilization, produced a significant amount of succinate, and exhibited a low growth rate. However, cells adapted soon after and started to grow rapidly in the same batch culture. As a result, the adapted ΔptsG cells showed the same mixed acid fermentation profiles as the wild-type cells, which was attributed to the mutation of the mlc gene, a repressor of the D-mannose PTS, another transporter for D-glucose. Similar adaptations were observed in the cells with ΔptsGΔmanX and the cells with ΔptsI that resulted in the production of a substantial amount of succinate and fast growth rate. The genome sequencing showed the presence of null mutations in the exuR gene, which encodes a modulator of exuT-encoded non-PTS sugar transporter, in adapted ΔptsGΔmanX and ΔptsI strains. Results from the RT-qPCR analysis and genetic test confirmed that the enhanced expression of ExuT, a non-PTS sugar transporter, was responsible for the uptake of D-glucose, increased succinate production, and fast growth of adapted cells. In conclusion, our study showed that the regulatory network of sugar transporters can be modulated by short-term adaptation and that downstream metabolic flux could be significantly determined by the choice of sugar transporters.
Highlights
Succinic acid is a dicarboxylic acid widely used as an ion chelator and a chemical precursor for the synthesis of 1,4-butandiol, tetrahydrofuran, N-methylpyrrolidone, and 2-pyrrolidone (McKinlay et al, 2007)
The comparison of reconstructed metabolic pathways based on the genome information of Mannheimia succiniciproducens and the E. coli along with the pyruvate addition experiments showed that intracellular pyruvate pool is closely related with the amount of succinate in the mixed acid fermentation of E. coli in which the ptsG, pykF, and pykA genes were deleted (Lee et al, 2005, 2006)
We identified a new D-glucose transporter in E. coli, which may drive metabolic flux to succinate during fermentation, and discussed the regulatory network of D-glucose transport system
Summary
Succinic acid is a dicarboxylic acid widely used as an ion chelator and a chemical precursor for the synthesis of 1,4-butandiol, tetrahydrofuran, N-methylpyrrolidone, and 2-pyrrolidone (McKinlay et al, 2007). The first one is to abrogate the by-products forming metabolic pathways This approach was used to delete pyruvate formate lyase (encoded by the pflB) and lactate dehydrogenase (ldhA) genes to generate E. coli NZN111 strain. These cells were unable to grow under anaerobic conditions but recovered later and a succinate-overproducing AFP111 strain was obtained from the culture (Bunch et al, 1997; Donnelly et al, 1998). The comparison of reconstructed metabolic pathways based on the genome information of Mannheimia succiniciproducens and the E. coli along with the pyruvate addition experiments showed that intracellular pyruvate pool is closely related with the amount of succinate in the mixed acid fermentation of E. coli in which the ptsG, pykF, and pykA genes were deleted (Lee et al, 2005, 2006). We identified a new D-glucose transporter in E. coli, which may drive metabolic flux to succinate during fermentation, and discussed the regulatory network of D-glucose transport system
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