Construction Of Butanol-tolerant E.coli Strain And Its Butanol Synthesis Pathway

Biobutanol has recently attracted considerable attentions as important commodity chemical and alternative for petroleum-based fuels with the intensification of environmental pollution,greenhouse effect and the increasing demand for energy.The toxicity of butanol to the strain results in the lower butanol production.In this regard,reducing the product inhibition by applying a new fermentation process to remove butanol,or by improving the tolerance of the fermentation strain to butanol to increase the butanol yield of E.coli.With the rapid development of synthetic biology,E.coli has been a potential platform strain to construct butanol synthesis pathway.In the previous work,a series of butanol-resistant engineering strains named HBT1-HBT12 were constructed using site-specific mutagenesis or CRISPR-Cas9 genome editing technology.We systematically evaluated the butanol tolerance of HBT1-HBT12 under 0-2%(v/v)butanol stress in our study.BW25113 was almost can not survival under 1.5%(v/v)butanol stress,the growth improvement of HBT6,HBT9 and HBT11 strains are 6.8,6.1 and 6.5 times that of BW25113,respectively.When cultured in LB medium with 0.75%(v/v)butanol and different p H(3-6)for 24 hours,the cell density of HBT9 was 9%-36% higher than that of HBT6,indicating a better physiological performance against acid stress and butanol stress of HBT9 than that of HBT6.Under 1.5%(v/v)butanol stress,the HBT11 strain,knocking out ast E and overexpress feo A genes in HBT9,showed an increase by 6.5% of the growth improvement.The cell density under 0.75%,1.25%,1.5% and 2%(v/v)butanol stress was increased by 200%,73%,70% and 60% compared to the control strain BW25113,respectively.The HBT11 strain can maintain great generative stability of butanol tolerance after subcultured serialy to 50 th generation.In a word,the engineered strain HBT11 shows higher butanol tolerance,acid stress tolerance,and generative stability,which can be used as an excellent chassis strain for butanol production.The toxicity of butanol to the strain limits the high yield of butanol,in order to increase butanol production,we introduced the butanol synthesis pathway into the genome of butanol tolerance strain HBT11 and control strain BW25113 using CRISPRCas9 genome editing technology to explore the butanol production capacity of the recombinant strains: HBT25 and EB7 were constructed by cloning and overexpressing the gene crt,hbd,and adh E2 from C.acetobutylicum,ter from T.denticola,ato B from E.coli,fdh from Candida boidinii,and by knocking out the gene ldh A,frd ABCD,pyk A,yqe G and ack A-pta in HBT11 and control strain BW25113.After 40 hours of preliminary test tube fermentation,the butanol production of HBT25 was 1.4 g/L,which was 180% higher than that of EB7.The optimum p H,temperature,and glucose concentration of HBT25 butanol fermentation were 7,30°C,and 2%,respectively.And anaerobic switch period in the early logarithm can obtain higher butanol production.The butanol production of HBT25 and EB7 increased gradually,and reached the maximum value of 5.3 g/L and 3.9g/L in 72 h by shake-flask fermentation under optimal conditions.The butanol titer of HBT25 was increased by 38% compared with EB7,which indicated that butanol-resistant strains as the chassis strains for butanol fermentation can significantly increase butanol production and yield.In this study,butanol production was carried out for the first time in butanol-resistant chassis strains,and a promising butanol-producing engineering strain was obtained,which laid the foundation for the industrial production of butanol in E.coli.