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Title: Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli

Abstract

Isopentenyl pyrophosphate (IPP) toxicity presents a challenge in engineered microbial systems since its formation is unavoidable in terpene biosynthesis. In this work, we develop an experimental platform to study IPP toxicity in isoprenol-producing Escherichia coli. We first characterize the physiological response to IPP accumulation, demonstrating that elevated IPP levels are linked to growth inhibition, reduced cell viability, and plasmid instability. We show that IPP toxicity selects for pathway “breakage”, using proteomics to identify a reduction in phosphomevalonate kinase (PMK) as a probable recovery mechanism. Next, using multi-omics data, we demonstrate that endogenous E. coli metabolism is globally impacted by IPP accumulation, which slows nutrient uptake, decreases ATP levels, and perturbs nucleotide metabolism. We also observe the extracellular accumulation of IPP and present preliminary evidence that IPP can be transported by E. coli, findings that might be broadly relevant for the study of isoprenoid biosynthesis. Finally, we discover that IPP accumulation leads to the formation of ApppI, a nucleotide analog of IPP that may contribute to observed toxicity phenotypes. This comprehensive assessment of IPP stress suggests potential strategies for the alleviation of prenyl diphosphate toxicity and highlights possible engineering targets for improved IPP flux and high titer isoprenoid production.

Authors:
 [1];  [1];  [2];  [1];  [1];  [1];  [1];  [1];  [1];  [3];  [4];  [5];  [1]
  1. Lawrence Berkeley National Laboratory
  2. BATTELLE (PACIFIC NW LAB)
  3. University of Eastern Finland
  4. Joint BioEnergy Institute
  5. Lawrence Berkley National Lab
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1512692
Report Number(s):
PNNL-SA-138198
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 47
Country of Publication:
United States
Language:
English

Citation Formats

George, Kevin W., Thompson, Mitchell G., Kim, Joonhoon, Baidoo, Edward, Wang, George, Benites, Veronica, Petzold, Christopher J., Chan, Leanne, Yilmaz, Suzan, Turhanen, Petri, Adams, Paul, Keasling, Jay D., and Lee, Taek Soon. Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli. United States: N. p., 2018. Web. doi:10.1016/j.ymben.2018.03.004.
George, Kevin W., Thompson, Mitchell G., Kim, Joonhoon, Baidoo, Edward, Wang, George, Benites, Veronica, Petzold, Christopher J., Chan, Leanne, Yilmaz, Suzan, Turhanen, Petri, Adams, Paul, Keasling, Jay D., & Lee, Taek Soon. Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli. United States. doi:10.1016/j.ymben.2018.03.004.
George, Kevin W., Thompson, Mitchell G., Kim, Joonhoon, Baidoo, Edward, Wang, George, Benites, Veronica, Petzold, Christopher J., Chan, Leanne, Yilmaz, Suzan, Turhanen, Petri, Adams, Paul, Keasling, Jay D., and Lee, Taek Soon. Tue . "Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli". United States. doi:10.1016/j.ymben.2018.03.004.
@article{osti_1512692,
title = {Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli},
author = {George, Kevin W. and Thompson, Mitchell G. and Kim, Joonhoon and Baidoo, Edward and Wang, George and Benites, Veronica and Petzold, Christopher J. and Chan, Leanne and Yilmaz, Suzan and Turhanen, Petri and Adams, Paul and Keasling, Jay D. and Lee, Taek Soon},
abstractNote = {Isopentenyl pyrophosphate (IPP) toxicity presents a challenge in engineered microbial systems since its formation is unavoidable in terpene biosynthesis. In this work, we develop an experimental platform to study IPP toxicity in isoprenol-producing Escherichia coli. We first characterize the physiological response to IPP accumulation, demonstrating that elevated IPP levels are linked to growth inhibition, reduced cell viability, and plasmid instability. We show that IPP toxicity selects for pathway “breakage”, using proteomics to identify a reduction in phosphomevalonate kinase (PMK) as a probable recovery mechanism. Next, using multi-omics data, we demonstrate that endogenous E. coli metabolism is globally impacted by IPP accumulation, which slows nutrient uptake, decreases ATP levels, and perturbs nucleotide metabolism. We also observe the extracellular accumulation of IPP and present preliminary evidence that IPP can be transported by E. coli, findings that might be broadly relevant for the study of isoprenoid biosynthesis. Finally, we discover that IPP accumulation leads to the formation of ApppI, a nucleotide analog of IPP that may contribute to observed toxicity phenotypes. This comprehensive assessment of IPP stress suggests potential strategies for the alleviation of prenyl diphosphate toxicity and highlights possible engineering targets for improved IPP flux and high titer isoprenoid production.},
doi = {10.1016/j.ymben.2018.03.004},
journal = {Metabolic Engineering},
number = ,
volume = 47,
place = {United States},
year = {2018},
month = {5}
}