Mining Of Bacterial Butanol-Tolerant Components And Study On Solvent Tolerance Of Escherichia Coli

Solvent tolerance of microorganisms is essential for the efficient production of large amounts of biofuels and plays an important role in non-aqueous industrial biocatalysis.Therefore,it is extremely valuable to obtain host strains with desirable solvent tolerance for industrial applications.Pseudomonas putida is one of the most important industrial microorganisms,and has been widely applied in environmental remediation,production of bioactive natural products and secondary metabolites,etc.Escherichia coli is a promising platform microorganism with a clear genetic background and is often used as a host strain in molecular engineering.However,the poor organic solvent tolerance of E.coli has limited its industrial applications.In this thesis,high n-butanol tolerant E.coli strains and was contructed based on sRNA and molecular chaperone engineering,solvent tolerant components were mined by atmospheric and room temperature plasma?ARTP?random mutagenesis.sRNA regulatory genes?hfq,fur,sgrs and rpoS?and RNA chaperone genes?hfq,fur,rpoH,stpA,nusB,cspC,cspD,proQ and ffh?were overexpressed in E.coli JM109 to improve its n-butanol tolerance.The tolerance of JM109/pQE80L-rpoS strain was increased by 41%in the presence of 8 g·L^-1 n-butanol compared to the control.Then,the rpoS gene was co-expressed with two chaperone genes?secB and groS?,and the n-butanol tolerance of JM109/pQE80L-groS-secB-rpoS was enhanced by 3-fold in the presence of 9.6 g·L^-1 n-butanol compared with the control.Besides,the co-expression orders of three genes could affect the n-butanol tolerance.Additionally,10,000 mutants were screened by constructing a random mutagenesis library of rpoS gene.Finally,mutants C11 and D5 with 1.5-fold and 1.8-fold increased tolerance were obtained.Pseudomonas putida 901 was mutagenized based on ARTP,and adapted in a series of n-butanol concentrations.A mutant with 2-fold enhanced n-butanol tolerance under the treatment of 8 g·L^-1 n-butanol was obtained,and its maximum tolerance was 12 g·L^-1 n-butanol.The mutant and control strains were then subjected to whole genome resequencing and bioinformatics analysis via the Illumina HiSeq platform.About 19 M reads was obtained with an average read of 150 nt.Compared with the reference genome,the fragment pairing rate was92.03%,and the coverage rate was 90.34%.A total of 40704 single nucleotide variations were detected,with isozygoty of 39319 and heterozygote of 40704.There were 2153 inserts and deletions,with homozygous mutation sites of 1871 and heterozygous mutation sites of 282.All the mutations were functionally annotated according to GO/KEGG database.In summary,the n-butanol tolerance of Escherichia coli was improved based on sRNA and chaperone engineering.This study could provide a theoretical guidance for the identification of solvent tolerant components and engineering of microbial host strains for desirable phenotypes.