| Bacillus subtilis is a non-pathogenic gram-positive bacterium and has been an attractivehost for production of recombinant proteins from both prokaryotic and eukaryotic origins. Thesuitability and popularity of B. subtilis as one of main hosts for recombinant proteinproduction is due to several reasons: generally recognized as safe as a result of its lack ofpathogenicity and the absence of endotoxins; capable of secreting functional proteins directlyinto culture media, easy for genetic manipulation and handling, and capable of large-scalefermentation. Bacillus subtilis has been applied in animal production, which acts as probioticsand feed enzyme source. It is attractive to employed genetic engineering of Bacillus subtilisfor alleviate the feed resource and enhance feed efficiency. However, the genetic manipulationof Bacillus subtilis involved in the vector, regulation element and methods limited itsbiotechnological application. To easy the manipulation, we develop and improve the geneticmanipulation of Bacillus subtilis in this study through constructing the E. coli-B. subtilisshuttle vector, expression system and promoter trapping system. The results were shown asfollows:1. The E. coli-B. subtilis shuttle vector pGJ103was developed in this study by ligation ofColE1replicon (about700bp) and pGDV1replicon (about2500bp). A maltose-inducibleexpression vector in Bacillus subtilis based on pGJ103has been developed and characterized.The vector permitted β-galactosidase expression at a high level. Using this vector, wesuccessfully expressed the other two genes, bioA and vgb. However, the system constructed inthis study was repressed badly by the glucose.2. Here, we present the electroporation as a feasible and efficient method for introducingcircularized and linearized DNA into Bacillus subtilis chromosome. Two integrationexperiments were carried out and demonstrated the feasibility and efficiency ofelectroporation to introduce the target DNA into the B. subtilis chromosome. To furtherdemonstrate the application of electroporation in genetic research, the early sporulation genespo0A of B. subtilis was knocked out (named B. subtilis GJ09) and, consequently, the null ofsporulation and logged growth was observed in this study.3. Site-directed mutagenesis was facilitated to enhance the expression strength of Pglv. The production of β-Gal driven by the mutant promoter in B. subtilis1A747reached14U/mLat24h when induced by5%maltose. To further improve the promoter system, the B. subtilisexpression host was reconstructed, in which B. subtilis well-characterized constitutivepromoter P43replaced the promoter of the glv operon in B. subtilis chromosome through adouble crossover event, yelding B. subtilis BCYL. The β-galactosidase production from theimproved system (21U/mL) increased compared to that from origin system. Meanwhile, therepression caused by glucose was further alleviated.4. To screen and isolate strong promoters, a promoter trap vector pShuttleF was used inthis study. Using the vector, approximately200colonies containing likely promoters fromBacillus licheniformis genomic DNA were obtained. Amongst them, pShuttle-09exhibited thehighest β-Gal activities in both Escherichia coli and B. subtilis (14016Mill U and16417MillU, respectively). A sequencing analysis showed that pShuttle-09contained PluxSand truncatedluxS in-frame fused with the reporter gene as well as another fragment upstream of PluxScontaining a putative promoter. Reconstructing the hybrid promoter from pShuttle-09to PlapSfurther improved the β-Gal production by1.6folds. By using of the PlapS, β-Gal and BioI weresuccessfully expressed in B. subtilis.In conclusion, a facilitated E.coli-B. subtilis shuttle vecoter, inducible expression systemand promoter trap system were developed in this study. By using of these expression elements,the target genes were high-level expressed in B. subtilis. Thus, we provided a potential tooland manipulation approach for B. subtilis genetic engendering. |
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