Functional Identification And Tolerance Mechanisms Of Butanol-resistant Mutant Genes In Escherichia Coli BW1847

Biobutanol as a clean and renewable biofuel and important chemical has received increasing attention.E.coli has been used to produce butanol through the genetic engineering because of its clear genetic background and simple genetic manipulation.However,the toxicity of butanol to the host strain seriously limited the yield of butanol.Therefore,it is very important to explore the mechanism of butanol tolerance and develop butanol-tolerant strains to increase the yield of biobutanol.The butanol-tolerant mutant strain E.coli BW1847 was previously obtained by our lab.In this study,the molecular identification was performed,and the data revealed that BW1847 had 9 mutanted genes.Functional complementation study showed that the acr B and rob gene were the key genes for butanol tolerance of BW1847.Genome-wide site-specific mutant DTacr B(mutation of C1198T in acr B gene)and DTrob(AT deletion in rob gene)strains were obtained in order to study the butanol-tolerant function of the two genes and eliminate the impact of plasmid and inducer L-arabinose.The cell density of strains DTacr B and Dacr B(acr B deletion)was 1.2–1.9 times of the control strain BW25113 under 0.75%(v/v)butanol stress.The butanol concentration in LB media was increased by 9.8%–18.2%,which indicated that the deletion or mutation of acr B gene led to the enhancement of butanol efflux ability and improvement of butanol tolerance.The cell density of the DTrob and Drob(rob deletion)strains was increased by 60%than that of the control strain,and the extracellular butanol concentration was increased by 11.2%–22.1%,indicating that the mutated rob _AT686-687/-results in inactivation of the Rob,which was able to promote butanol efflux from cells.Transcriptional analysis of DTrob strain grown under butanol stress showed that there were 285 differentially expression genes,184 up-regulated and 101 down-regulated.Inactivated Rob mainly thus changed the expression of genes related to membrane and transport,and chromatin immunoprecipitation sequencing(Ch IP-seq)also indicates that these differentially expressed genes can bind to Rob,which further indicates that Rob can regulate genes associated with cell membrane function and transport activity.The DTacr B or DTrob strains were also able to tolerate 0.3–0.4%(v/v)pentanol and 0.5–0.75 mol/L Na Cl,indicating that the mutation of acr B or rob gene increased the tolerance of n-pentanol and Na Cl.In conclusion,this study confirmed that the key genes for butanol tolerance in BW1847.The deleted or mutated strains of acr B can enhance the butanol efflux ability and improve the butanol tolerance.The non-active transcriptional factor Rob was first found to cause changes in genes expression related to membrane and transport,which led to the promotion of butanol efflux from cells and enhance butanol tolerance.This study lays a theoretical foundation for the construction of stress-resistant strains and clarification of the molecular mechanism of butanol tolerance.