Tianjin Institute of Industrial Biotechnology , Chinese Academy of Sciences , Tianjin 300308 , P.R. China.
Key Laboratory of Systems Microbial Biotechnology , Chinese Academy of Sciences , Tianjin 300308 , P. R. China.
J Agric Food Chem. 2018 Dec 19;66(50):13141-13151. doi: 10.1021/acs.jafc.8b04183. Epub 2018 Dec 7.
Bacillus subtilis is widely used for large-scale industrial production of heterologous proteins. Because of its high intrinsic secretory capacity, it can efficiently secrete proteins into the culture supernatant via the general Sec-type secretion pathway. In this study, the α-amylase AmyS was used as a reporter to construct a library encompassing 173 Sec-type signal peptides (SPs) from B. subtilis using a fast, sequence-independent method. The resulting library DNA which harbored different signal peptides in the expression vector was used to transform B. subtilis directly at high efficiency, and 15 SPs which produced a significantly increased yield of AmyS were identified using a starch-iodine-based high-throughput assay. Furthermore, the correlation between the sequences of the best-performing signal peptides and their secretion efficiency was analyzed, which revealed several common properties of these SPs. Finally, high-cell-density fermentation of the α-amylase-producing strain with the best-performing signal peptide yielded a maximum of 5086 U/mL amylase at 66 h with a high productivity of 77.1 U/mL·h.
枯草芽孢杆菌被广泛用于大规模的异源蛋白工业生产。由于其具有较高的固有分泌能力，它可以通过一般的 Sec 型分泌途径将蛋白质有效地分泌到培养上清液中。在本研究中，使用α-淀粉酶 AmyS 作为报告蛋白，通过一种快速、序列无关的方法，从枯草芽孢杆菌构建了一个包含 173 个 Sec 型信号肽（SP）的文库。将含有不同信号肽的表达载体中的文库 DNA 直接转化枯草芽孢杆菌，效率很高，然后使用基于淀粉-碘的高通量测定法，鉴定出了 15 种能显著提高 AmyS 产量的信号肽。此外，还分析了表现最好的信号肽的序列与其分泌效率之间的相关性，揭示了这些 SP 的一些共同特性。最后，使用表现最好的信号肽对产α-淀粉酶的菌株进行高密度发酵，在 66 小时时获得了最高 5086 U/mL 的淀粉酶，产率为 77.1 U/mL·h。
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