Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea.
Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), Daejeon 34113, Korea.
Int J Mol Sci. 2020 Nov 17;21(22):8668. doi: 10.3390/ijms21228668.
Lysozyme is widely used as a model protein in studies of structure-function relationships. Recently, lysozyme has gained attention for use in accelerating the degradation of secondary sludge, which mainly consists of bacteria. However, a high-throughput screening system for lysozyme engineering has not been reported. Here, we present a lysozyme screening system using a genetically encoded biosensor. We first cloned bacteriophage T4 lysozyme (T4L) into a plasmid under control of the promoter. The plasmid was expressed in with no toxic effects on growth. Next, we observed that increased soluble T4L expression decreased the fluorescence produced by the genetic enzyme screening system. To investigate T4L evolution based on this finding, we generated a T4L random mutation library, which was screened using the genetic enzyme screening system. Finally, we identified two T4L variants showing 1.4-fold enhanced lytic activity compared to native T4L. To our knowledge, this is the first report describing the use of a genetically encoded biosensor to investigate bacteriophage T4L evolution. Our approach can be used to investigate the evolution of other lysozymes, which will expand the applications of lysozyme.
溶菌酶被广泛用作研究结构-功能关系的模型蛋白。最近，溶菌酶因其在加速主要由细菌组成的剩余污泥降解方面的应用而受到关注。然而，目前还没有报道用于溶菌酶工程的高通量筛选系统。在这里，我们使用遗传编码的生物传感器展示了一种溶菌酶筛选系统。我们首先将噬菌体 T4 溶菌酶 (T4L) 克隆到受 启动子控制的质粒中。该质粒在 中表达，对生长没有毒性作用。接下来，我们观察到可溶性 T4L 表达的增加降低了遗传酶筛选系统产生的荧光。为了基于这一发现研究 T4L 的进化，我们生成了一个 T4L 随机突变文库，并用遗传酶筛选系统进行筛选。最后，我们鉴定出两种 T4L 变体，与天然 T4L 相比，其溶菌活性提高了 1.4 倍。据我们所知，这是首次报道使用遗传编码的生物传感器来研究噬菌体 T4L 的进化。我们的方法可用于研究其他溶菌酶的进化，这将扩大溶菌酶的应用。
