Journal
METABOLIC ENGINEERING
Volume 47, Issue -, Pages 60-72Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2018.03.004
Keywords
Isopentenyl pyrophosphate (IPP); IPP toxicity; Isoprenol; ApppI; Mevalonate pathway; Multi-omics
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Funding
- US Department of Energy, Office of Science, Office of Biological and Environmental Research [DE-AC02-05CH11231]
- NIH [S10RR029668, S10RR027303]
- United States Government
- NNF Center for Biosustainability [Synthetic Biology Tools for Yeast] Funding Source: researchfish
- Novo Nordisk Fonden [NNF10CC1016517] Funding Source: researchfish
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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 coll. 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. coil 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. coil, 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.
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