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Background  Butanol is an important biofuel and chemical. The development of butanol-tolerant strains and the identification of functional butanol-tolerant genes is essential for high-yield bio-butanol production due to the toxicity of butanol.    Results   Escherichia coli  BW25113 was subjected for the first time to error-prone PCR-based whole-genome shuffling. The resulting mutants BW1847 and BW1857 were found to tolerate 2% (v/v) butanol and short-chain alcohols, including ethanol, isobutanol, and 1-pentanol. The mutants exhibited good stability under butanol stress, indicating that they are potential host strains for the construction of butanol pathways. BW1847 had better butanol tolerance than BW1857 under 0–0.75% (v/v) butanol stress, but showed a lower tolerance than BW1857 under 1.25–2% (v/v) butanol stress. Genome resequencing and PCR confirmation revealed that BW1847 and BW1857 had nine and seven single nucleotide polymorphisms, respectively, and a common 14-kb deletion. Functional complementation experiments of the SNPs and deleted genes demonstrated that the mutations of  acrB  and  rob  gene and the deletion of  TqsA  increased the tolerance of the two mutants to butanol. Genome-wide site-specific mutated strains DT385 ( acrB  C 1198 T) and DT900 ( rob  AT 686–7 ) also showed significant tolerance to butanol and had higher butanol efflux ability than the control, further demonstrating that their mutations yield an inactive protein that enhances butanol resistance characteristics.    Conclusions  Stable  E. coli  mutants with enhanced short alcohols and high concentrations of butanol tolerance were obtained through a rapid and effective method. The key genes of butanol tolerance in the two mutants were identified by comparative functional genomic analysis.    Electronic supplementary material  The online version of this article (10.1186/s13068-019-1405-z) contains supplementary material, which is available to authorized users.

Identification of functional butanol-tolerant genes from Escherichia coli mutants derived from error-prone PCR-based whole-genome shuffling