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

Xueting He,Junyan Zhang,Zhiquan Wang,Jianjun Qiao,Hao Song,Minhua Zhang,Tingli Xue,Jiefang Hong,Yuanyuan Ma,Lanfeng Hui

doi:10.1186/s13068-019-1405-z

Abstract

BackgroundButanol 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.ResultsEscherichia 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 C1198T) and DT900 (rob AT686–7) 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.ConclusionsStable 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.

Highlights

Butanol is an important biofuel and chemical
Heat shock protein (HSP) genes respond to butanol stress; as such, their upregulation or overexpression could lead to an increase in tolerance [13]
The mutant strains BW1847 and BW1857 were obtained from the second round of shuffling, reaching a maximum O D600 of 0.69 and 0.50, respectively, which was 5.0- and 3.6-fold higher than that of BW25113, respectively

Summary

Introduction

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. Butanol-tolerant E. coli strains are obtained using random engineering strategies, such as global transcription machinery engineering (GTME) and evolution engineering, among others [14,15,16,17]. These mutants developed by random mutation and selection generally show a greater tolerance than those with functional changes in one gene, and could tolerate 1–2.0% (v/v) butanol [16, 18]. When membrane-related functional genes are overexpressed in butanol-tolerant E. coli through evolution, the final engineered strain can grow under 2% butanol stress [18], indicating that a combinational strategy could effectively improve tolerance. The genes of many mutants have not yet been functionally identified, which limits our understanding of the butanol tolerance mechanism as well as the potential to obtain butanol-tolerant characteristics

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Results

Discussion

Conclusion