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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];  [2];  [3];  [1];  [1];  [1];  [1];  [1];  [1];  [4];  [5];  [6];  [1]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Division
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Chemical and Biological Processes Development Group
  4. Univ. of Eastern Finland, Kuopio (Finland). School of Pharmacy, Biocenter Kuopio
  5. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division
  6. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering; Univ. of California, Berkeley, CA (United States). Dept. of Chemical and Biomolecular Engineering; Technical Univ. of Denmark, Lyngby (Denmark). Novo Nordisk Foundation Center for Biosustainability
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1427525
Alternate Identifier(s):
OSTI ID: 1530358
Report Number(s):
PNNL-SA-138198
Journal ID: ISSN 1096-7176; PII: S1096717617303749
Grant/Contract Number:  
AC05-76RL01830; AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 47; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Isopentenyl pyrophosphate (IPP); IPP toxicity; isoprenol; ApppI; mevalonate pathway; multi-omics

Citation Formats

George, Kevin W., Thompson, Mitchell, Kim, Joonhoon, Baidoo, Edward E. K., Wang, George, Benites, Veronica Teixeira, Petzold, Christopher J., Chan, Leanne Jade G., Yilmaz, Suzan, Turhanen, Petri, Adams, Paul D., 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, Kim, Joonhoon, Baidoo, Edward E. K., Wang, George, Benites, Veronica Teixeira, Petzold, Christopher J., Chan, Leanne Jade G., Yilmaz, Suzan, Turhanen, Petri, Adams, Paul D., 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, Kim, Joonhoon, Baidoo, Edward E. K., Wang, George, Benites, Veronica Teixeira, Petzold, Christopher J., Chan, Leanne Jade G., Yilmaz, Suzan, Turhanen, Petri, Adams, Paul D., Keasling, Jay D., and Lee, Taek Soon. Fri . "Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli". United States. doi:10.1016/J.YMBEN.2018.03.004. https://www.osti.gov/servlets/purl/1427525.
@article{osti_1427525,
title = {Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli},
author = {George, Kevin W. and Thompson, Mitchell and Kim, Joonhoon and Baidoo, Edward E. K. and Wang, George and Benites, Veronica Teixeira and Petzold, Christopher J. and Chan, Leanne Jade G. and Yilmaz, Suzan and Turhanen, Petri and Adams, Paul D. 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 = {3}
}

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Works referencing / citing this record:

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Towards efficient terpenoid biosynthesis: manipulating IPP and DMAPP supply
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Terpenoid Metabolic Engineering in Photosynthetic Microorganisms
journal, October 2018

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  • Genes, Vol. 9, Issue 11
  • DOI: 10.3390/genes9110520

Modular Engineering of Biomass Degradation Pathways
journal, April 2019

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  • DOI: 10.3390/pr7040230