Random Mutagenesis And Batch Fermentation Of Clostridium Butyricum And Its Tolerance Mechanism To 1,3-Propanediol

1,3-propanediol(1,3-PD)is an important chemical raw material which has been utilized in a wide range of applications in food,medicine,cosmetics,and other uses.The biosynthesis of 1,3-PD is considered as low-cost and environmentally friendly process.However,the natural 1,3-PD producing strains face various problems such as low product tolerance and toxicity of by-products to the organisms.As one of the natural producers,Clostridium butyricum is probiotics and requires no addition of expensive coenzymes in the production of 1,3-PD.Therefore,this study aimed to solve the problem of C.butyricum low tolerance against the biosynthesis products in the 1,3-PD production.The physical and chemical combined mutagenesis method was conducted on wild strains,revealing a mutant strain with high 1,3-PD-tolerance strain.Furthermore,batch fermentation of 1,3-PD by C.butyricum high-tolerant strain was optimized by one-factor-at-one-time and by employing response surface methodology.Finally,high-throughput multi-omics of wild-type and mutant strains were sequenced and genes and proteins related to 1,3-PD tolerance were analyzed from the perspective of bioinformatics.The function of some proteins was verified preliminary.The main research results are as follows:(1)The media with a series of 1,3-PD concentrations were used to determine the initial tolerance of wild strain C.butyricum XYB11,physical and chemical combined mutagenesis of N-methyl-N'-nitro-N-nitrosoguanidine(NTG)and Atmospheric and room-temperature plasma(ARTP)were performed to obtain mutant strain C.butyricum YP855,which has more tolerance to 1,3-PD(30.8% higher than the original starting strain).(2)The 1,3-PD batch fermentation of mutant C.butyricum YP855 were optimized by one-factor-at-one-time and response surface methodology.The optimal conditions for batch fermentation were found to be as follows: glycerol concentration 81.0 g/L,yeast extract concentration 1.6 g/L,fermentation time 21.7 h,fermentation temperature 36.7°C.Eventually,batch fermentation of 1,3-PD under optimal conditions got a final production of 37.20 g/L,a yield of 0.51 g/g,and a productivity of 1.71 g/(L·h),increased by 29.48%,18% and 29% respectively compared with the wild strain.(3)Complete genome of C.butyricum XYB11 was sequenced,and prediction of gene structure and annotation of functions were performed.Comparative analyses for C.butyricum XYB11 wild and mutant strain were conducted by multi-omics.The transcriptome was sequenced to obtain differentially expressed genes,and the proteome was quantified by iTRAQ to determine the differentially expressed proteins.The functional annotations were performed on differential genes and proteins.Then,bioinformatics method was used to analyze the data of the omics.The enrichment analysis and comparison of GO and KEGG pathways were performed.From functional perspective,the relationship between C.butyricum and its 1,3-PD tolerance was analyzed.Seven key genes that may be correlated to the 1,3-PD tolerance of C.butyricum were preliminary screened for subsequently initial validation of their function.(4)Those screened key genes were cloned from C.butyricum XYB11,and seven recombinant plasmids pET-gene were successfully constructed by seamless cloning technology.Each recombinant plasmid was transformed and the corresponding recombinant protein was successfully expressed in E.coli BL21(DE3).The 1,3-PD tolerance of each engineered strain was tested,and the function of these key proteins was preliminarily verified.Finally,the ABC transporter,histidine kinase,isocitrate dehydrogenase and methyl-binding chemotaxis protein were considered to be related to the 1,3-PD tolerance of C.butyricum.Together with results from multi-omics,the possible molecular mechanisms of high-tolerance were analyzed.