Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, AlbaNova University Center, SE 10691 Stockholm, Sweden.
Biochim Biophys Acta Biomembr. 2019 Feb 1;1861(2):486-494. doi: 10.1016/j.bbamem.2018.11.012. Epub 2018 Dec 4.
Display of recombinant enzymes on the cell surface of Gram-negative bacteria is a desirable feature with applications in whole-cell biocatalysis, affinity screening and degradation of environmental pollutants. One common technique for recombinant protein display on the Escherichia coli surface is autotransport. Successful autotransport of an enzyme largely depends on the following: (1) the size, sequence and structure of the displayed protein, (2) the cultivation conditions, and (3) the choice of the autotransporter expression system. Common problems with autotransporter-mediated surface display include low expression levels and truncated fusion proteins, which both limit the cell-specific activity. The present study investigated an autotransporter expression system for improved display of tyrosinase on the surface of E. coli by evaluating different variants of the autotransporter vector including: promoter region, signal peptide, the recombinant passenger, linker regions, and the autotransporter translocation unit itself. The impact of these changes on translocation to the cell surface was monitored by the cell-specific activity as well as antibody-based flow cytometric analysis of full-length and degraded passenger. Applying these strategies, the amount of displayed full-length tyrosinase on the cell surface was increased, resulting in an overall 5-fold increase of activity as compared to the initial autotransport expression system. Surprisingly, heterologous expression using 7 different translocation units all resulted in functional expression and only differed 1.6-fold in activity. This study provides a basis for broadening of the range of proteins that can be surface displayed and the development of new autotransporter-based processes in industrial-scale whole-cell biocatalysis.
在革兰氏阴性菌的细胞表面展示重组酶是一种理想的特性，可应用于全细胞生物催化、亲和筛选和环境污染物的降解。在大肠杆菌表面展示重组蛋白的一种常见技术是自转运。酶的成功自转运在很大程度上取决于以下几点：（1）展示蛋白的大小、序列和结构，（2）培养条件，以及（3）自转运体表达系统的选择。自转运体介导的表面展示常见的问题包括表达水平低和截短的融合蛋白，这两者都限制了细胞特异性活性。本研究通过评估不同变体的自转运载体，包括启动子区域、信号肽、重组载体、连接区域和自转运体转运单元本身，研究了一种自转运表达系统，以提高酪氨酸酶在大肠杆菌表面的展示。这些变化对转运到细胞表面的影响通过细胞特异性活性以及基于抗体的流式细胞术分析全长和降解载体来监测。应用这些策略，细胞表面展示的全长酪氨酸酶的数量增加，与初始自转运表达系统相比，活性总体增加了 5 倍。令人惊讶的是，使用 7 种不同的转运单元进行异源表达都导致了功能表达，仅在活性上相差 1.6 倍。本研究为拓宽可表面展示的蛋白质范围以及在工业规模全细胞生物催化中开发新的自转运体基工艺提供了基础。
