Abstract：
Developing effective bacterial autolytic systems for fast release of intracellular bioproducts could simplify purification procedures and help with the high throughput screening of mutant libraries in protein engineering. Here, we developed a fast and tightly regulated E. coli autolytic system, named the FhuD-lysozyme-SsrA mediated autolytic (FLSA) system, by integrating the secretion signal peptide, T7 lysozyme, and E. coli ClpX/P-SsrA protein degradation machinery. To decrease the cytotoxicity of leaky T7 lysozymes, the SsrA tag was fused to the C-terminus of T7 lysozyme to confer a tight regulation of its production. Using sfGFP as a reporter, we demonstrated that anchoring the Sec-Tat dual pathway signal peptide FhuD to the N-terminus of T7 lysozyme-SsrA could give the highest cell lysing efficiency. The optimization of the FLSA system indicated that weak alkaline conditions (pH 8.0) and 0.5% Triton X-100 could further increase the lysing efficiency by about 24%. The FLSA system was validated by efficient production of sfGFP and human growth hormone 1 (hGH1) in a shake flask, with a cell lytic efficiency of approximately 82% and 80%, respectively. Besides, the FLSA system was applied for large-scale fermentation, in which approximately 90% sGFP was released with a cell density OD600 of 110. Moreover, the FLSA system was also tested for α-amylase mutant library screening in microplates, and the results showed that intracellular α-amylase can be efficiently released out of cells for activity quantitation. In all, the FLSA system can facilitate the release of intracellular recombinant proteins into the cell culture medium, which has the potential to serve as an integrated system for large-scale production of recombinant targets and high throughput enzyme engineering in synthetic biology.
摘要：
开发有效的细菌自溶系统以快速释放细胞内生物产物可以简化纯化程序，并有助于蛋白质工程中突变文库的高通量筛选。这里,我们开发了一个快速和严格调控的大肠杆菌自溶系统，命名为FhuD-溶菌酶-SsrA介导的自溶（FLSA）系统，通过整合分泌信号肽，T7溶菌酶，和大肠杆菌ClpX/P-SsrA蛋白降解机制。为了减少泄漏的T7溶菌酶的细胞毒性，将SsrA标签融合到T7溶菌酶的C末端以赋予其生产的严格调节。使用sfGFP作为报告者，我们证明，将Sec-Tat双途径信号肽FhuD锚定到T7溶菌酶-SsrA的N末端可以提供最高的细胞裂解效率。FLSA系统的优化表明，弱碱性条件（pH8.0）和0.5％TritonX-100可以进一步将裂解效率提高约24％。通过在摇瓶中有效生产sfGFP和人生长激素1（hGH1）来验证FLSA系统，细胞裂解效率约为82%和80%，分别。此外,FLSA系统应用于大规模发酵，其中约90％的sGFP被释放，细胞密度OD600为110。此外，还在微孔板中测试了FLSA系统的α-淀粉酶突变体文库筛选，结果表明，细胞内α-淀粉酶可以有效地释放出细胞以进行活性定量。总之,FLSA系统可以促进细胞内重组蛋白释放到细胞培养基中，具有作为合成生物学中大规模生产重组靶标和高通量酶工程的集成系统的潜力。
