Screening Butanol Tolerant Escherichia Coli By Using Error-prone PCR Whole Genome Shuffling

Butanol is shown to be superior to ethanol as a new type of potential alternative biofuel becaurse it has higher energy density,lower hygroscopicity and it can be manufactured based on the already-established infrastructures for ethanol production.Butanol has been used in a wide range of fields,including food,plastics and pharmaceutical industries.Traditional bio-butanol production from biomass is achieved by anaerobic acetone-butanol-ethanol（ABE）fermentation using Clostridia.However,the butanol production in Clostridia is coupled to its strictly anaerobic fermentation and its complex metabolic mechanisms and butanol toxicity.Recently,Escherichia coli as a host strain,has been used to construct a butanol production pathway.The poor butanol tolerance of bacteria also result in the inhibition of cell growth,which decrease the butanol production.Therefore,economic production of butanol will rely on the improvement of butanol tolerance of the producers.It is,for the first time,to improve the butanol tolerance of E.coli BW25113 by using error-prone PCR whole genome shuffling,and Red recombinant system is used to improve the recombination efficiency.Via three rounds of genome shuffling,two mutant strains,BW1847A2 and BW1857A2,are successfully obtained and are able to tolerate 1.5%（v/v）butanol.The maximum OD₆₀₀ value of BW1847A2 and BW1857A2 strain cultured in 1.5%（v/v）butanol is 144 and 33%higher than that of control strain,respectively.After 50 subculture passages,the butanol tolerance of BW1847A2 and BW1857A2 has no significant difference from that of the original ones,demonstrating their genetic stability.The BW1847A2 and BW1857A2 are able to tolerate 1.5%（v/v）isobutanol,0.6%（v/v）1-pentanol and 5.5–7%（v/v）ethanol.After whole genome resequencing,the BW1847A2 and BW1857A2 strains have 12and 8 mutant genes,respectively.The butanol tolerance analysis of gene deletion mutants indicates that the deletion of RS22900 and RS02385 gene can increase the butanol tolerance of mutants,and the tolerance of RS08395,RS14165 and RS19735genes deletion mutants is decreased.The error-prone PCR whole genome shuffling is also used to improve the butanol tolerance of E.coli K12,and the isolated strain KP1-2 is able to tolerate 3%（v/v）butanol and 13%（v/v）ethanol.The KP1-2 strain is subsequently identified as Staphylococcus aureus by 16S rRNA sequencing.It,for the first time shows that the isolated S.aureus exhibits high resistance to butanol.S.aureus also shows good growth in common LB medium,and it can be easily genetically modified.Therefore,S.aureus can be used as host strains to construct a novel butanol or ethanol biosynthetic pathway.In conclusion,this study for the first time screens the high-butanol tolerant E.coli strain by using error-prone PCR genome shuffling.The isolated mutants BW1847A2and BW1857A2 can be used as the host strains for the construction of butanol biosynthetic pathway.Preliminary gene function analysis of mutation genes provides a theoretical basis for investigation of the gene function invovled in tolerate butanol and the elaborate clarification of the molecular mechanism of butanol tolerance.