Evolutionary engineering of E. coli MG1655 for tolerance against isoprenol

Abstract

Background Isoprenol is the basis for industrial flavor and vitamin synthesis and also a promising biofuel. Biotechnological production of isoprenol with E. coli is currently limited by the high toxicity of the final product. Adaptive laboratory evolution (ALE) is a promising method to address complex biological problems such as toxicity. Results Here we applied this method successfully to evolve E. coli towards higher tolerance against isoprenol, increasing growth at the half-maximal inhibitory concentration by 47%. Whole-genome re-sequencing of strains isolated from three replicate evolutions at seven time-points identified four major target genes for isoprenol tolerance: fabF, marC, yghB, and rob. We could show that knock-out of marC and expression of mutated Rob H(48) → frameshift increased tolerance against isoprenol and butanol. RNA-sequencing showed that the deletion identified upstream of yghB correlated with a strong overexpression of the gene. The knock-out of yghB demonstrated that it was essential for isoprenol tolerance. The mutated Rob protein and yghB deletion also lead to increased vanillin tolerance. Conclusion Through ALE, novel targets for strain optimization in isoprenol production and also the production of other fuels, such as butanol, could be obtained. Their effectiveness could be shown through re-engineering. This paves the way for further optimization of E. coli for biofuel production.

Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase

“…Low concentrations of isoprenol or prenol do not affect microbial growth, but the addition of 50 mM and 100 mM of each alcohol individually inhibits cellular growth. A recent study also found the half‐maximal inhibitory concentration of isoprenol to be 53 mM [23] . When the two compounds were added in combination, the growth rate was reduced at 37.5 mM each and the addition of both alcohols at 100 mM each inhibits cell growth almost completely.…”

“…A recent study also found the half‐maximal inhibitory concentration of isoprenol to be 53 mM. [23] When the two compounds were added in combination, the growth rate was reduced at 37.5 mM each and the addition of both alcohols at 100 mM each inhibits cell growth almost completely.…”

Long-term ecological and evolutionary dynamics in the gut microbiomes of carbapenemase-producing Enterobacteriaceae colonized subjects

“…Leveraging the availability of multiple timepoints across subjects, we identified SNVs whose populations frequencies varied notably over time (>30%). These were then overlapped across subjects to identify SNVs that have this property recurrently ( Table 3, Supplementary File 6 ), identifying a range of polysaccharide utilization ( lacZ, lacI, treA ), pyruvate metabolism ( pflB, pykF ) and protein synthesis ( dnaK , 30S and 50S ribosomal subunits) genes that have been implicated in adaptive evolution under nutrient limitation 43 , antibiotic 44 and environmental stress 45,46 conditions. In particular, several genes were common to the lists for E. coli and K. pneumoniae ( srmB, pnp, nlpI, pheT ), suggesting that similar selection constraints might be acting on strains for both species.…”

Improving isoprenol productionviasystematic CRISPRi screening in engineeredEscherichia coli

“…Current strategies to alleviate isoprenol toxicity have mainly relied on in situ product removal with organic solvents, 23 over-expression of exporter or methionine biosynthesis regulator proteins, 45 or adaptive laboratory evolution (ALE) of the host strain. 46 The genetic bases of E. coli towards alcohol tolerance like butanol and isobutanol have been more extensively elucidated with genome evolution and genomic library screening. [47][48][49] In this study, considering the structural similarity between isoprenol and butanol or isobutanol, we applied the CRISPRi to target the narrowed scope of major alcohol tolerance associated genes that might also confer isoprenol tolerance.…”