Branched C5 alcohols are important biofuels and platform chemicals that can be easily converted to isoprene. The microbial production of branched C5 alcohols is a promising approach enabling the efficient synthesis of these compounds from renewable sources. Considerable effort has been made to produce isoprenol by introducing and engineering the heterologous mevalonate pathway in Escherichia coli, where isoprenol is produced by dephosphorylation of isopentenyl diphosphate (IPP). The toxicity of isoprenol and its immediate precursor IPP reduces production titers and prevents the industrially-relevant production of branched C5 alcohols. Recent studies have shown some success in improving E. coli isoprenol tolerance, primarily through the assessment of E. coli’s transcriptomic response to exogenous isoprenol addition. Although this approach was reasonably successful for isoprenol, a similar method cannot be employed to assess the toxicity of pathway intermediates such as IPP that do not freely cross the cell membrane. Furthermore, the stress response resulting from exogenous addition might have little relevance to the response associated with the in vivo production of a molecule in an engineered host. In this study, we designed a three-strain experimental system to study IPP toxicity in vivo in isoprenol-producing E. coli. By comparing a wild-type strain, a metabolically balanced high titer isoprenol production strain, and an imbalanced, IPP-accumulating strain, this experimental system allowed us to resolve the cellular responses due to the IPP toxicity and isoprenol production. We employed multiple omics (transcriptomics, proteomics, and metabolomics) and computational approaches to investigate novel behaviors associated with IPP accumulation. Metabolomics and proteomics analysis showed slowed metabolism and eventual recovery in the IPP-accumulating strain, and metabolic network modeling provided further mechanistic explanations for these behaviors. Transcriptomics analysis revealed interesting, unique, and shared responses between the balanced and imbalanced strains. This work is the first extensive study of IPP toxicity in isoprenoid overproducing strains, and this omics-based approach would also be applicable to study other systems.

Characterizing in vivo isopentenyl diphosphate (IPP) toxicity in isoprenol-producing E. coli

253rd National Meeting (2017)

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