caa a22 4500 606212108 CHVBK 20210128101041.0 cr unu---uuuuu 210128e20150601xx s 000 0 eng 10.1007/s10295-015-1609-6 doi (NATIONALLICENCE)springer-10.1007/s10295-015-1609-6 Growth retardation of Escherichia coli by artificial increase of intracellular ATP [Elektronische Daten] [Yoon-Ah Na, Joo-Young Lee, Weon-Jeong Bang, Hyo Lee, Su-In Choi, Soon-Kyeong Kwon, Kwang-Hwan Jung, Jihyun Kim, Pil Kim] 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 1.45μmol/gcell, with the slowest growth rate of 0.66h−1, while the PCKWT-overexpressing cells displayed 1.00μmol/gcell ATP concentration with the growth rate of 0.84h−1 and the control had 0.28μmol/gcell with 1.03h−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 0.1mM 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. Society for Industrial Microbiology and Biotechnology, 2015 Phosphoenolpyruvate carboxykinase nationallicence High intracellular ATP concentration nationallicence Growth retardation nationallicence ROS-scavenging nationallicence Na Yoon-Ah Department of Biotechnology, The Catholic University of Korea, 420-743, Bucheon, Gyeonggi, Korea aut Lee Joo-Young Department of Biotechnology, The Catholic University of Korea, 420-743, Bucheon, Gyeonggi, Korea aut Bang Weon-Jeong Department of Biotechnology, The Catholic University of Korea, 420-743, Bucheon, Gyeonggi, Korea aut Lee Hyo Department of Biotechnology, The Catholic University of Korea, 420-743, Bucheon, Gyeonggi, Korea aut Choi Su-In Department of Biotechnology, The Catholic University of Korea, 420-743, Bucheon, Gyeonggi, Korea aut Kwon Soon-Kyeong Department of Systems Biology, Yonsei University, 120-749, Seoul, Korea aut Jung Kwang-Hwan Department of Life Science, Sogang University, 121-742, Seoul, Korea aut Kim Jihyun Department of Systems Biology, Yonsei University, 120-749, Seoul, Korea aut Kim Pil Department of Biotechnology, The Catholic University of Korea, 420-743, Bucheon, Gyeonggi, Korea aut Journal of Industrial Microbiology & Biotechnology Springer Berlin Heidelberg 42/6(2015-06-01), 915-924 1367-5435 42:6<915 2015 42 10295 https://doi.org/10.1007/s10295-015-1609-6 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s10295-015-1609-6 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Na Yoon-Ah Department of Biotechnology, The Catholic University of Korea, 420-743, Bucheon, Gyeonggi, Korea aut NATIONALLICENCE 700 1- Lee Joo-Young Department of Biotechnology, The Catholic University of Korea, 420-743, Bucheon, Gyeonggi, Korea aut NATIONALLICENCE 700 1- Bang Weon-Jeong Department of Biotechnology, The Catholic University of Korea, 420-743, Bucheon, Gyeonggi, Korea aut NATIONALLICENCE 700 1- Lee Hyo Department of Biotechnology, The Catholic University of Korea, 420-743, Bucheon, Gyeonggi, Korea aut NATIONALLICENCE 700 1- Choi Su-In Department of Biotechnology, The Catholic University of Korea, 420-743, Bucheon, Gyeonggi, Korea aut NATIONALLICENCE 700 1- Kwon Soon-Kyeong Department of Systems Biology, Yonsei University, 120-749, Seoul, Korea aut NATIONALLICENCE 700 1- Jung Kwang-Hwan Department of Life Science, Sogang University, 121-742, Seoul, Korea aut NATIONALLICENCE 700 1- Kim Jihyun Department of Systems Biology, Yonsei University, 120-749, Seoul, Korea aut NATIONALLICENCE 700 1- Kim Pil Department of Biotechnology, The Catholic University of Korea, 420-743, Bucheon, Gyeonggi, Korea aut NATIONALLICENCE 773 0- Journal of Industrial Microbiology & Biotechnology Springer Berlin Heidelberg 42/6(2015-06-01), 915-924 1367-5435 42:6<915 2015 42 10295