Catabolic regulation analysis of Escherichia coli and its crp, mlc, mgsA, pgi and ptsG mutants
Authors: Yao Ruilian , Hirose Yuki , Sarkar Dayanidhi , Nakahigashi Kenji , Ye Qin , Shimizu Kazuyuki 
Published: August 2011

Background

Most bacteria can use various compounds as carbon sources. These carbon sources can be either co-metabolized or sequentially metabolized, where the latter phenomenon typically occurs as catabolite repression. From the practical application point of view of utilizing lignocellulose for the production of biofuels etc., it is strongly desirable to ferment all sugars obtained by hydrolysis from lignocellulosic materials, where simultaneous consumption of sugars would benefit the formation of bioproducts. However, most organisms consume glucose prior to consumption of other carbon sources, and exhibit diauxic growth. It has been shown by fermentation experiments that simultaneous consumption of sugars can be attained byptsG, mgsAmutants etc., but its mechanism has not been well understood. It is strongly desirable to understand the mechanism of metabolic regulation for catabolite regulation to improve the performance of fermentation.

Results

In order to make clear the catabolic regulation mechanism, several continuous cultures were conducted at different dilution rates of 0.2, 0.4, 0.6 and 0.7 h-1using wild typeEscherichia coli. The result indicates that the transcript levels of global regulators such ascrp, cra, mlcandrpoSdecreased, while those offadR, iclR, soxR/Sincreased as the dilution rate increased. These affected the metabolic pathway genes, which in turn affected fermentation result where the specific glucose uptake rate, the specific acetate formation rate, and the specific CO2evolution rate (CER) were increased as the dilution rate was increased. This was confirmed by the13C-flux analysis. In order to make clear the catabolite regulation, the effect ofcrpgene knockout (Δcrp) and crp enhancement (crp+) as well asmlc, mgsA, pgiandptsGgene knockout on the metabolism was then investigated by the continuous culture at the dilution rate of 0.2 h-1and by some batch cultures. In the case of Δcrp(and also Δmlc) mutant, TCA cycle and glyoxylate were repressed, which caused acetate accumulation. In the case ofcrp+mutant, glycolysis, TCA cycle, and gluconeogenesis were activated, and simultaneous consumption of multiple carbon sources can be attained, but the glucose consumption rate became less due to repression ofptsGandptsHby the activation of Mlc. Simultaneous consumption of multiple carbon sources could be attained bymgsA, pgi, andptsGmutants due to increase incrpas well ascyaA, while glucose consumption rate became lower.

Conclusions

The transcriptional catabolite regulation mechanism was made clear for the wild typeE. coli, and itscrp, mlc, ptsG, pgi, and mgsAgene knockout mutants. The results indicate that catabolite repression can be relaxed andcrpas well ascyaAcan be increased bycrp+, mgsA, pgi, andptsGmutants, and thus simultaneous consumption of multiple carbon sources including glucose can be made, whereas the glucose uptake rate became lower as compared to wild type due to inactivation ofptsGin all the mutants considered.