Department of Medical Microbiology, University Medical Center Groningen, 9700 RB Groningen, The Netherlands.
Appl Environ Microbiol. 2010 Feb;76(3):659-69. doi: 10.1128/AEM.01671-09. Epub 2009 Nov 30.
Bacillus subtilis is a well-established cell factory for efficient secretion of many biotechnologically relevant enzymes that are naturally produced by it or related organisms. However, the use of B. subtilis as a host for production of heterologous secretory proteins can be complicated by problems related to inefficient translocation of the foreign proteins across the plasma membrane or to inefficient release of the exported proteins from the cell surface into the surrounding medium. Therefore, there is a clear need for tools that allow more efficient membrane targeting, translocation, and release during the production of these proteins. In the present study, we investigated the contributions of the pre (pre(lip)) and pro (pro(lip)) sequences of a Staphylococcus hyicus lipase to secretion of a heterologous protein, the alkaline phosphatase PhoA of Escherichia coli, by B. subtilis. The results indicate that the presence of the pro(lip)-peptide, in combination with the lipase signal peptide (pre(lip)), contributes significantly to the efficient secretion of PhoA by B. subtilis and that pre(lip) directs PhoA secretion more efficiently than the authentic signal peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses indicate that, under the conditions tested, no known secretion or membrane-cell wall stress responses were provoked by the production of PhoA with any of the pre- and pro-region sequences used. Our data underscore the view that the pre-pro signals of the S. hyicus lipase are very useful tools for secretion of heterologous proteins in B. subtilis.
枯草芽孢杆菌是一种成熟的细胞工厂，可高效分泌许多生物技术相关的酶，这些酶天然存在于该菌或相关生物体内。然而，将枯草芽孢杆菌作为生产异源分泌蛋白的宿主可能会因以下问题而变得复杂：外源蛋白穿过质膜的易位效率低下，或已输出的蛋白从细胞表面到周围介质中的释放效率低下。因此，显然需要一些工具来提高这些蛋白生产过程中的膜靶向、易位和释放效率。在本研究中，我们研究了猪葡萄球菌脂肪酶的前（pre(lip)) 和前导（pro(lip)) 序列对枯草芽孢杆菌异源蛋白碱性磷酸酶 PhoA 分泌的贡献。结果表明，pro(lip)-肽的存在与脂肪酶信号肽（pre(lip)) 共同作用，显著促进了 PhoA 在枯草芽孢杆菌中的高效分泌，并且 pre(lip) 比 PhoA 的天然信号肽更有效地指导 PhoA 的分泌。对宿主细胞反应的全基因组转录分析表明，在测试的条件下，使用任何前导和前肽序列生产 PhoA 均不会引发已知的分泌或膜-细胞壁应激反应。我们的数据强调了这样一种观点，即猪葡萄球菌脂肪酶的前导肽序列是在枯草芽孢杆菌中分泌异源蛋白的非常有用的工具。
