Growth retardation of Escherichia coli by artificial increase of intracellular ATP

Yoon Ah Na, Joo Young Lee, Weon Jeong Bang, Hyo Jung Lee, Su In Choi, Soon Kyeong Kwon, Kwang Hwan Jung, Jihyun F. Kim, Pil Kim

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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, PCK WT enzyme was directly evolved in vitro and further overexpressed. The evolved PCK67 showed a 60 % greater catalytic efficiency than that of PCK WT . Consequently, the PCK67-overexpressing E. coli showed the highest ATP concentration at the log phase of 1.45 μmol/g cell , with the slowest growth rate of 0.66 h −1 , while the PCK WT -overexpressing cells displayed 1.00 μmol/g cell ATP concentration with the growth rate of 0.84 h −1 and the control had 0.28 μmol/g cell 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.

Original languageEnglish
Pages (from-to)915-924
Number of pages10
JournalJournal of Industrial Microbiology and Biotechnology
Volume42
Issue number6
DOIs
Publication statusPublished - 2015 Jun 1

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Adenosinetriphosphate
Escherichia coli
Adenosine Triphosphate
Phosphoenolpyruvate
Reactive Oxygen Species
Growth
Oxygen
Rhodopsin
Carbon Cycle
Glutathione
Dicarboxylic Acid Transporters
Carbon
Chemostats
Fluxes
Lighting
Membrane Proteins
Enzymes
Proteins
Membranes

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

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. / Growth retardation of Escherichia coli by artificial increase of intracellular ATP. In: Journal of Industrial Microbiology and Biotechnology. 2015 ; Vol. 42, No. 6. pp. 915-924.
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Growth retardation of Escherichia coli by artificial increase of intracellular ATP. / 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.

In: Journal of Industrial Microbiology and Biotechnology, Vol. 42, No. 6, 01.06.2015, p. 915-924.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Growth retardation of Escherichia coli by artificial increase of intracellular ATP

AU - Na, Yoon Ah

AU - Lee, Joo Young

AU - Bang, Weon Jeong

AU - Lee, Hyo Jung

AU - Choi, Su In

AU - Kwon, Soon Kyeong

AU - Jung, Kwang Hwan

AU - Kim, Jihyun F.

AU - Kim, Pil

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AB - 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, PCK WT enzyme was directly evolved in vitro and further overexpressed. The evolved PCK67 showed a 60 % greater catalytic efficiency than that of PCK WT . Consequently, the PCK67-overexpressing E. coli showed the highest ATP concentration at the log phase of 1.45 μmol/g cell , with the slowest growth rate of 0.66 h −1 , while the PCK WT -overexpressing cells displayed 1.00 μmol/g cell ATP concentration with the growth rate of 0.84 h −1 and the control had 0.28 μmol/g cell 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.

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