高通量构建与筛选信号肽库提高枯草芽孢杆菌外源蛋白的表达分泌

High-throughput screening of signal peptides to improve the expression and secretion of heterologous proteins in Bacillus subtilis

摘要:

【目的】 基于信号肽在分泌表达系统中的重要作用，利用自动化高通量设备，搭建枯草芽孢杆菌(Bacillus subtilis)酶蛋白信号肽库的高通量自动化构建与筛选平台，探索枯草芽孢杆菌中不同信号肽对酶蛋白的表达分泌作用。【方法】 首先以大肠杆菌-芽孢杆菌穿梭载体pHP13以及pMA5为骨架，将编码毒性蛋白的ccdB基因插入大肠杆菌-芽孢杆菌穿梭载体的启动子和目标基因之间，构建针对绿色荧光蛋白(green fluorescent protein, GFP)以及普鲁兰酶(pullulanase, PulA)的信号肽筛选载体。以枯草芽孢杆菌168基因组为模板，扩增173条信号肽序列，基于高通量自动化设备搭建枯草芽孢杆菌外源蛋白表达及活性筛选平台，构建含有不同信号肽的外源蛋白重组菌株，筛选并考察不同信号肽对外源蛋白表达分泌的影响。【结果】 RpmG、AspB、CitH、LytF和YkwD这5个信号肽具有较强的引导GFP胞外蛋白分泌能力，其中RpmG引导GFP胞外分泌能力最强，其重组菌株与对照菌株相比胞外GFP的荧光值提高了236%。针对PulA的信号肽筛选，其中41个信号肽与PulA的适配性很低，2个信号肽RpmG、AspB引导PulA胞外分泌的能力较强。含AspB信号肽的PulA重组菌株分泌率可达到74%，与对照菌株相比分泌率提高了68%；RpmG信号肽引导的PulA酶活与其他信号肽相比最高，总酶活可达116 U/mL，细胞外酶活为52 U/mL。【结论】 本研究建立了枯草芽孢杆菌酶蛋白信号肽库的高通量自动化构建与筛选平台，分别获得可以提高GFP和PulA表达分泌的信号肽。该自动化平台允许大量样品的并行处理，这简化了纯手工的重复性实验工作，并且我们发现高通量实验平台不论在时间和成本方面都优于手工操作。自动化高通量平台的优势将随着样本量的增加而更加显著。综上所述，我们建立的自动化高通量筛选平台不仅可以加速外源蛋白的信号肽筛选优化过程，也为其他高附加值蛋白酶的工业菌种改造提升和迭代提供新的技术支撑。

Abstract:

[Objective] Considering the important role of signal peptides in the secretory expression of heterologous proteins, we devised an automated high-throughput platform for the automatic screening of signal peptides, aiming to explore the effects of different signal peptides in Bacillus subtilis on the expression of heterologous proteins. [Methods] First, using the Escherichia coli-B.subtilis shuttle vectors pHP13 and pMA5 as the skeleton, we amplified the cell division B lethal gene (ccdB) and then ligated it to the middle of the promoter and the target gene to build the signal peptide screening vector. With the genomic DNA of B. subtilis 168 as the template, 173 signal peptides were amplified. An automated platform was established for the expression and screening of heterologous proteins in B. subtilis. Furthermore, the recombinant strains of heterologous proteins containing different signal peptides were constructed, and the effects of different signal peptides on the secretory expression of heterologous proteins were investigated. [Results] Five signal peptides (RpmG, AspB, CitH, LytF, and YkwD) showed strong abilities to induce the export of GFP from B. subtilis. Among them, RpmG had the strongest ability to induce the export of GFP, and the extracellular GFP fluorescence of the recombinant strain increased by 236% compared with that of the control strain. In addition, 41 signal peptides were not compatible with pullulanase (PulA), while the two signal peptides RpmG and AspB showed strong abilities to export PulA. The highest PulA activity of 116 U/mL was detected from the recombinant strain carrying the signal peptide RpmG, and the extracellular enzyme activity was 52 U/mL. The secretion rate of the PulA recombinant strain carrying the signal peptide AspB reached 74%, which was 68% higher than that of the control strain. [Conclusion] We developed an automated platform for high-throughput screening of the heterologous protein signal peptides in B. subtilis and obtained the signal peptides capable of improving the secretory expression of GFP and PulA. This automated platform allowed the parallel processing of a considerable number of samples, which simplified the repetitive manual laboratory work. This platform outperformed manual operation in terms of both time consumption and cost. The advantage of the automated high-throughput platform will be more significant with the increase in sample size. In summary, the established automatic high-throughput screening platform not only accelerates the screening process of signal peptides of heterologous proteins, but also provides new technical support for the modification and iteration of industrial strains of other value-added proteases.