Na Yoon-Ah, Lee Joo-Young, Bang Weon-Jeong, Lee Hyo Jung, Choi Su-In, Kwon Soon-Kyeong, Jung Kwang-Hwan, Kim Jihyun F, Kim Pil
Department of Biotechnology, The Catholic University of Korea, Bucheon, 420-743, Gyeonggi, Korea.
J Ind Microbiol Biotechnol. 2015 Jun;42(6):915-24. doi: 10.1007/s10295-015-1609-6. Epub 2015 Apr 3.
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.
据报道，磷酸烯醇式丙酮酸羧激酶（PCK）的过表达会导致大肠杆菌细胞内ATP浓度升高，同时生长速率减慢。为了系统地确定ATP人工增加与生长迟缓之间的关系，对野生型PCK酶进行了体外直接进化并进一步过表达。进化后的PCK67催化效率比野生型PCK高60%。因此，过表达PCK67的大肠杆菌在对数期的ATP浓度最高，为1.45 μmol/g细胞，生长速率最慢，为0.66 h⁻¹，而过表达野生型PCK的细胞ATP浓度为1.00 μmol/g细胞，生长速率为0.84 h⁻¹，对照细胞的ATP浓度为0.28 μmol/g细胞，生长速率为1.03 h⁻¹。为了找到一个合理的原因，在恒化器生长过程中处于稳定状态的过表达PCK的细胞被用于监测细胞内活性氧（ROS）。随着ATP水平的升高，观察到细胞内ROS的量增加。为了证实过表达PCK不会干扰碳通量但会导致生长速率减慢这一假设，表达了光合嗜盐菌视紫红质（GR）。光照下表达GR的菌株ATP浓度高出81%，ROS高出82%，最大生长速率比对照慢54%，而黑暗中表达GR的菌株和表达二羧酸转运蛋白（一种对照膜蛋白）的菌株ATP浓度较低，ROS增加，生长速率较慢。无论碳通量如何变化，人工增加ATP都与ROS增加有关，并且与最大生长速率呈负相关。为了验证细胞内积累的ROS是生长迟缓的原因，向培养基中添加了谷胱甘肽以降低ROS。结果，添加0.1 mM谷胱甘肽恢复了生长迟缓。厌氧培养甚至使人工增加ATP的大肠杆菌比对照生长得更快。总的来说，得出的结论是，人工增加ATP会由于ROS的过量产生而抑制大肠杆菌的生长。
