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

Isopentenyl pyrophosphate (IPP) is the universal C5 precursor for isoprenoids, the largest class of natural products and frequent targets for metabolic engineering. In engineered microbial systems, IPP toxicity presents a challenge since its formation is unavoidable in the production of high-value, long-chain terpenes. In this work, we develop an experimental platform to study IPP toxicity in E. coli engineered to produce isoprenol, an IPP-derived C5 alcohol. We first characterize the physiological response to IPP accumulation, demonstrating that elevated levels of IPP are linked to growth inhibition, altered morphology, and reduced cell viability. 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, we demonstrate that endogenous E. coli metabolism is globally impacted by IPP accumulation, which results in inhibited nutrient uptake, reduced ATP levels, and an apparent “pause” in metabolism. Finally, we suggest that IPP toxicity is mediated by the formation of an isoprenyl-ATP analog (ApppI). The complementary data presented here represent the most comprehensive assessment of IPP stress to date and suggest potential strategies for the alleviation of IPP and prenyl diphosphate toxicity. To investigate the physiological reason for the metabolic “pause” and subsequent recovery, we used RNAseq to identify differentially expressed genes between engineered strains and the wild-type control at time points corresponding with IPP accumulation and reduction in strain 3Amk. We sampled each strain prior to pathway induction (t=0), at the onset of IPP toxicity in strain 3Amk (t=1), and during growth recovery in strain 3Amk (t=12). By comparing the transcriptome of all 3 strains, we hoped to isolate the impact of IPP accumulation (differentially expressed genes in strain 3Amk only) from the impact of heterologous protein expression and metabolic burden (differentially expressed genes in both 3A and 3Amk relative to WT).