Overexpression of phosphoenolpyruvate carboxykinase (PCK) was reported to cause the harboring of higher intracellular ATP concentration in Escherichia coli, accompanied with a slower growth rate. For systematic determination of the relationship between the artificial increase of ATP and growth retardation, PCKWT enzyme was directly evolved in vitro and further overexpressed. The evolved PCK67 showed a 60 % greater catalytic efficiency than that of PCKWT. Consequently, the PCK67-overexpressing E. coli showed the highest ATP concentration at the log phase of 1.45 μmol/gcell, with the slowest growth rate of 0.66 h−1, while the PCKWT-overexpressing cells displayed 1.00 μmol/gcell ATP concentration with the growth rate of 0.84 h−1 and the control had 0.28 μmol/gcell with 1.03 h−1. To find a plausible reason, PCK-overexpressing cells in a steady state during chemostat growth were applied to monitor intracellular reactive oxygen species (ROS). Higher amount of intracellular ROS were observed as the ATP levels increased. To confirm the hypothesis of slower growth rate without perturbation of the carbon flux by PCK-overexpression, phototrophic Gloeobacter rhodopsin (GR) was expressed. The GR-expressing strain under illumination harbored 81 % more ATP concentration along with 82 % higher ROS, with a 54 % slower maximum growth rate than the control, while both the GR-expressing strain under dark and dicarboxylate transporter (a control membrane protein)-expressing strain showed a lower ATP and increased ROS, and slower growth rate. Regardless of carbon flux changes, the artificial ATP increase was related to the ROS increase and it was reciprocally correlated to the maximum growth rate. To verify that the accumulated intracellular ROS were responsible for the growth retardation, glutathione was added to the medium to reduce the ROS. As a result, the growth retardation was restored by the addition of 0.1 mM glutathione. Anaerobic culture even enabled the artificial ATP-increased E. coli to grow faster than control. Collectively, it was concluded that artificial ATP increases inhibit the growth of E. coli due to the overproduction of ROS.
|Number of pages||10|
|Journal||Journal of Industrial Microbiology and Biotechnology|
|Publication status||Published - 2015 Jun 1|
Bibliographical noteFunding Information:
This study was financially supported by the Korean Ministry of Science, ICT and Future Planning (Intelligent Synthetic Biology Center program 2012M3A6A8054887 and Basic Research Program 2013R1A1A2009023). P. Kim was supported by a fellowship from the Catholic University of Korea (2014 fund).
© 2015, Society for Industrial Microbiology and Biotechnology.
All Science Journal Classification (ASJC) codes
- Applied Microbiology and Biotechnology