Systematic Screening of Optimal Signal Peptides for Secretory Production of Heterologous Proteins in Bacillus subtilis
Systematic Screening of Optimal Signal Peptides for Secretory Production of Heterologous Proteins in Bacillus subtilis
复制标题
系统筛选枯草芽孢杆菌分泌产生异源蛋白的最佳信号肽。
DOI：
10.1021/acs.jafc.8b04183
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发表时间：
2018-12-19
影响因子：
6.1
通讯作者：
Zhang, Dawei
中科院分区：
文献类型：
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作者：
Fu, Gang;Liu, Jinlan;Zhang, Dawei
枯草芽孢杆菌广泛用于大规模工业化生产异源蛋白。由于其高的内在分泌能力，它可以通过一般的Sec型分泌途径有效地将蛋白质分泌到培养上清液中。本研究以α-淀粉酶AmyS为报告基因，构建了包含173个来自B的Sec型信号肽（SP）的文库。枯草杆菌使用快速，序列独立的方法。将所得的在表达载体中含有不同信号肽的文库DNA用于转化B。枯草芽孢杆菌直接在高效率，和15个SP产生显着增加的产量的AmyS被确定使用淀粉碘为基础的高通量测定。此外，最佳性能的信号肽的序列之间的相关性和它们的分泌效率进行了分析，这揭示了这些SP的几个共同的性质。最后，对信号肽产生菌进行高密度发酵，66 h时淀粉酶产量达到最大值5086 U/mL，产酶能力达77.1 U/mL·h。
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.