Construction of Chromosomally Located T7 Expression System for Production of Heterologous Secreted Proteins in Bacillus subtilis
Construction of Chromosomally Located T7 Expression System for Production of Heterologous Secreted Proteins in Bacillus subtilis
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DOI：
10.1021/jf100445a
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发表时间：
2010-05-12
影响因子：
6.1
通讯作者：
Yu, Su-May
中科院分区：
文献类型：
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作者：
Chen, Po Ting;Shaw, Jei-Fu;Yu, Su-May
枯草芽孢杆菌最常用于重组蛋白的分泌。为了避免使用质粒所带来的问题，在B中重建了以高效著称的T7表达系统。枯草杆菌。因此，开发了用于DNA的基因组插入的无标记和无复制子的方法。通过引导DNA的同源重组作用，携带目的基因的自杀载体被整合到细菌的基因组基因座中。通过FLP重组酶介导插入的选择标记和侧翼为FRT位点的复制子的去除。通过使用所述系统，B.构建枯草杆菌菌株PT 5以携带位于wprA（编码细胞壁相关蛋白酶）的spac启动子调节的17基因1的基因组拷贝。类似地，褐色热单孢菌基因的17个启动子驱动的纳豆激酶或内切葡聚糖酶E1也整合到菌株PT 5的mpr（编码胞外蛋白酶）中。因此，整合体PT 5/Mmp-T7 N或PT 5/MT 1-E1导致“干净的”生产菌株被剥夺了六种蛋白酶。诱导24 h后，纳豆激酶和内切葡聚糖酶E1的分泌量分别达到10860 CU/mL和8.4 U/mL。该结果清楚地表明所提出的方法对于在B中高分泌重组蛋白的巨大前景。枯草杆菌。
Bacillus subtilis is most commonly employed for secretion of recombinant proteins. To circumvent the problems caused by using plasmids, the T7 expression system known for its high efficiency was rebuilt in B. subtilis. Accordingly, a markerless and replicon-free method was developed for genomic insertion of DNAs. By the act of homologous recombination via the guide DNA, a suicidal vector carrying the gene of interest was integrated into genomic loci of bacteria. Removal of the inserted selection marker and replicon flanked by FRT sites was mediated by the FLP recombinase. By using the mentioned system, B. subtilis strain PT5 was constructed to harbor a genomic copy of the spac promoter-regulated 17 gene 1 located at wprA (encoding the cell wall-associated protease). Similarly, the 17 promoter-driven nattokinase or endoglucanase E1 of Thermomonospora fusca genes were also integrated into mpr (encoding an extracellular protease) of strain PT5. Consequently, the integrant PT5/Mmp-T7N or PT5/MT1-E1 resulted in a "clean" producer strain deprived of six proteases. After 24 h, the strain receiving induction was able to secret nattokinase and endoglucanase El with the volumetric activity reaching 10860 CU/mL and 8.4 U/mL, respectively. This result clearly indicates the great promise of the proposed approach for high secretion of recombinant proteins in B. subtilis.