The Omics Study Of Bacillus Subtilis 7-3-3 And Construction Of Efficient Ramie-degumming Strains

Alkaline pectinase usually refers to polygalacturonate lyase(PGL).It has wide application in ind-ustrial production,such as textile processing,paper industry,degumming of fibers,treatment of wastewater and so oa B.subtilis 7-3-3 is a strain screened by our laboratory and could produce more PGL.In this thesis,the genome of the strain,B.subtilis 7-3-3,was sequenced.The proteome of the B.subtilis 7-3-3,engineering strain B-pN-pelA,and B-pN-pelA+pelC were analyzed,and genetic engineering strains were constructed based on the differences in proteome of the three strains.The results were as follows:CAZyme,protease,and degumming related enzymes were annotated and gathered based on genome data analysis.It was found that there were not only p pelB,pelC gene,but also yesW(rhamnogalacturonanendolyase)in PL family,all the genes were related to degumming.In addition,some genes were also found which were related to rhamnogalacturonan I degrading and exhibited in the form of gene cluster.The results may provide hetofiil reference for the construction of efficient degumming strains.The fermentation broths of B.subtilis 7-3-3 at different fermentation time were analyzed by MS,and found the fermentation broth at 48 h contained the most number of proteins according to proteome analysis.Based on the highest content of secreted protein,promoters were screened and applied for over-express ion of gene pelA and gene pelC.Three methods,siuch as enzyme cut-connection,homologous recombinatbn and Inserting the SD Sequence of peIA were respectively used to construct over-expression strains.It was found that,using the enzyme cut-connection method,only csn gene's promoter could over-express the gene pelA.The PGL activity of pelA-overexpressed strain B-pN-CHP-A by HP1 promoter using homotogous recombination reached the level of PGL activity of strain B-pN-pelA.Using the mothed of Inserting the SD Sequence of pelA,gene pelA could be successfiully overexpressed by csn promoter,and the PGL activity of engineering strain B-pN-Ccsn-2A was 1.3 times higher than that of strain B-pN-pelA.For pelC gene overexpression,the three methods can't make significant improvement in PGL activity,assurrd that promoter replacement had little effect on the expression of pelC gene,or polygalacturonate was not the optimum substrate for PelC.The fermentation broth of B.subtilis 7-3-3,B-pN-pelA,and B-pN-pelA-pelC at 55 h was also analyzed by MS.Differences in total extracellular proteins and the predicted secreted protein was analyzed,and the unique protein and the differential expression proteins of the different strains were summarized.Then,the coding genes and pelA gene were co-expressed in B.subtilis 7-3-3.It was found that co-expression gene aprE,yesWand gmuG improved PGL activity.Meanwhile,yes W and gmuG had synergistic effect on deguuming of fibers.There was not obvious difference in the pectirase activity and degumming effect by co-expressing aprE gene with pelA gene,although enzyme system of fermentation broth was changed.There was almost no effect on PGL activity by co-expressing yciB and pelAy but co-egression of spo VR and showed a negative effect on PGL activity.