| Bacillus subtilis is an attractive host for the production of heterologous secretory proteins for several reasons:it is non pathogenic and capable of secreting functional extracellular proteins directly to the culture medium, a great deal of vital information concerning genetic manipulation and large scale fermentation has now been acquired. Its application is restricted by secreting a variety of proteases and the unstable of recombinant expression plasmids. In the near past years, with the development of molecular biology and genetic engineering, there are many important progresses for Bacillus subtilis as the host in the genetic engineering were achieved. There is a good prospect for the application of B. subtilis expression system. Meanwhile, different genetic manipulation systems have been developed and these methods promote the development in B. subtilis research. This work mainly focused on establishing a simple and efficient B. subtilis genome manipulation system and applied it to improving the host strain to enhance the expressing level of heterologous protein. Some works were also performed to study the transformation of wild Bacillus strains and display methyl parathion hydrolase (MPH) on the surface of B. subtilis spore; these works pave a way for the search of novel expression systems and the development of new bio-remedation form.The existing B. subtilis genetic manipulation systems are depending on the plasmids construction and the recombination system of B. subtilis. In this work, we developed a fast and accurate method which based on PCR and mutant Cre/lox system to engineer B. subtilis genome. Instead of the classical vector construction dependent mutation and counter-selectable marker dependent selection of antibiotic-resistant marker losing cell, efficient PCR and Cre/lox site recombination system were used to realize mutation and marker elimination. The whole process could be finished in about four days. The mutation process could also be accomplished in two days by using a strain containing a cre IPTG-inducible expression cassette in the chromosome as recipient or using the lox sites flanked cassette containing both the cre (IPTG)-inducible expression cassette and resistance marker, which can be applied to genome-scale mutations. This method was used successfully to inactivate a specific gene, to delete long fragment, to realize the in-frame deletion of a target gene, and to carry out multi-manipulations in a same background. Furthermore, it can also be applied to large genome rearrangement.Three antibiotics resistance genes were left in the chromosome of B. subtilis WB800 in its construction process, which affects further modification of this strain using these markers. Using the method above, we deleted these antibiotics resistance genes, getting a clean protease deficient expression host WB8003.The mutation delivery system above can also be modified to be a new fast method which applies to introducing foreign genes into the chromosome of B. subtilis without any antibiotics resistance marker left. Using this method, we integrated the mpd gene express cassette at the amyE site of WB8003, yielding genetically engineered strain BSM1.DNA sequencing of the B. subtilis chromosomal rrnE region revealed a 13 kb region with a lower G+C content and lower gene density between the groESL operon and the gut operon; this region was called prophage 3, which encodes both the genes for BsuM modification (ydiO and ydiP) and BsuM restriction (ydiR, ydiS, and ydjA). BsuM restriction has been shown to reduce the transformation efficiency of B. subtilis with recombinant plasmids. Using the system above, we deleted both BsuM restriction and BsuM modification systems and study the effect on plasmids transformation. The result showed that the transformation efficiency of the plasmid containing XhoI site was remarkably reduced and the the transformation efficiency of the plasmid to 168 strain was not affected by the modification of E. coli methylation system.Picha pastoris expression system is widely used for heterologous protein expression at present and sometimes expression host need to been modified to enhance expression level. But efficiency of the existing mutation methods is too low. As in B. subtilis, based on PCR and mutant Cre/lox system, we established a fast gene knockout method in Picha pastoris, which were successfully used to delete the his4 and pep4 gene.The Bacillus spore offers unique resistance properties and can survive extremes of temperature, desiccation, and exposure to solvents and other noxious chemicals. These unique attributes would make the spore an attractive vehicle for delivery of heterologous antigens or, indeed, any bioactive molecule to extreme environments such as the gastrointestinal tract. In this work, we displayed the MPH on the surface of B. subtilis spore, but low MPH activity was detected. Among the spore coat proteins, cotB, cotC and cotG can be used for the target sites, so several protein can been displayed on on the surface of B. subtilis spore at the same time to transfer pesticide degrading path way to the spore surface.In the previous work in our lab, it was found that when used B. subtilis 168 and wild Bacillus strains as donors and recipients, mixed them on filter membrane and incubated, pUB110 derived recombinant plasmids could transfer from donors to recipients, but the mechanism was not clear. In this work, we proved that wild Bacillus could take plasmid through developing natural competence. Furthermore, the form and source of the plasmid remarkably affect its transformation efficiency to wild Bacillus. This offers referrence to the plasmid introduction into wild Bacillus and establishing new efficient expession system. |
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