Research Of Surfactin Synthase-Related Genes

Members of the Bacillus genus are often considered microbial factories for the production of a vast array of biologically active molecules that have potentially inhibitory for phytopathogen growth. One of the most commonly used and well-studies organisms, the rhizobacterium B. sublilis, has an average of4-5%of its genome devoted to antibiotic synthesis and has the potential to produce more than two dozen structurally diverse antimicrobial compounds.Among these antimicrobial compounds, the cyclic lipopeptide surfactin has well-recognized potential uses in biotechnology and biopharmaceutical applications because of their surfactant properties and biological activities. And it attracts attention and fancy of the applied microbiologists and biotechnologists worldwide, mainly due to its versatile bioactive properties and potential industrial implications. Lots of efforts have been done on genetics and chemistry to obtain novel peptide antibiotics.Surfactin is synthesized by non ribosomal peptide synthetases (NRPSs). The order of biosynthetic modules in the synthetases and the number and type of catalytic domains within each module determine the order of structural and functional elements in the resulting natural product. Interest developed in rationally redesigning the NRPS template to synthesize new peptide products.The enzyme Sfp is significance to the synthesis. Once the Sfp absences, the synthesis is unable to continue. So it’s essential to take sfp gene into consideration, when analysis the function of NRPS.The thesis presents the investigations on the directed-mutation on srfAC-A (coding for the A domain of the third module of surfactin synthetases) cloned from Bacillus sublilis fmbj, and the reconstructing of B. sublilis168on the sfp gene. The aim of the study is to provide a theoretical support on the research of A domain. The main results of this study are as follows:1. The srfAC-A gene was cloned from B. sublilis fmbj, heterologous expressed in Escherichia coli and the activity of the recombinant enzyme was detected.The srfAC-A gene amplified by PCR from B. subtilis fmbj according to the srfAC gene sequence of B. subtilis168was named FsrfAC-A. The recombinant expression vector, pET-23a-FsrfAC-A was constructed. The FSrfAC-A domain was expressed in E. coli BL21(DE3) and purified by Ni-NTA agarose column. It was confirmed that FsrfAC-A domain has activities towards Leu and Ile, and with a higher activity to Ile.2. Three different mutations were introduced to FsrfAC-A respectively, and recombinant expression vectors were constructed and expressed. Otherwise, recombinant integrative plasmids using in Bacillus subtilis were constructed based on mutated genes.Three mutated FsrfAC-As acquired using overlap extension PCR were ligated to pET23a and pET32a. The SDS-PAGE results suggested that they were expressed but insoluble. Otherwise, the mutated FsrfAC-As were ligated to pE5916, acquired recombinant intergrative plasmids. The competence formation of B. subtilis fmbj failed, thus the experiment behind could not proceed.3. The sfp gene from B. subtilis Nja-9was inserted to B. subtilis168by homologous recombination, resulting that the surfactin producing positive.By analyzing the sfp gene with the sequence up-and down-stream from the strains belonging to Bacillus amyloliquefaciens and Bacillus subtilis, amplified the sfp ORF from Nja-9. The P43promoter and a terminator were ligated to sfp ORF, building up the sfp gene expression frame. The recombinant integrative vector PST-1730was constructed and transformed to B. subtilis168, accomplishing the integration at the amyE site of B. sublilis168, obtained the surfactin-producing strain B. subtilis121.