Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System.
Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System.
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DOI：
10.1021/acssynbio.2c00658
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发表时间：
2023-04
影响因子：
4.7
通讯作者：
J. L. N. Dinglasan;Tien T. Sword;J. W. Barker;M. Doktycz;Constance B. Bailey
中科院分区：
文献类型：
--
作者：
J. L. N. Dinglasan;Tien T. Sword;J. W. Barker;M. Doktycz;Constance B. Bailey
基于裂解物的无细胞表达（CFE）系统是用于表达难以在体内合成的蛋白质（例如非核糖体肽合成酶（NRPS））的可获得平台。NRPS是合成生物活性肽天然产物的大（>100 kDa）模块化酶复合物。该合成过程类似于裂解物中的转录/翻译（TX/TL），导致NRPS表达和NRPS活性在无细胞环境中之间的潜在资源竞争。此外，CFE条件取决于蛋白质的大小和结构。在这里，一个报告系统，用于快速调查和优化NRPS CFE的反应环境。该策略在E.使用蓝色色素合成酶A（BpsA），一种模型NRPS，携带C-末端四半胱氨酸（TC）标签，其与双砷染料FlAsH形成荧光复合物。比色测定法适用于裂解物反应，以检测无细胞表达的BpsA-TC的蓝色色素产物靛蓝，证实标记的酶具有催化活性。用于反应中终点TC/FlAsH复合物测量的优化方案能够快速比较在不同反应条件下表达的全长BpsA-TC，定义与蛋白质催化活性和大小相关的NRPS表达的独特要求。重要的是，这些蛋白依赖性CFE条件能够实现更高的靛蓝滴度并改善其他单模NRPS的表达。值得注意的是，这些条件不同于用于表达超折叠GFP（sfGFP）的那些条件，sfGFP是用于优化基于裂解物的CFE系统的常见报告物，这表明定制报告物优化特定酶类的表达的必要性。报告系统预计将推进裂解物为基础的CFE系统的复杂酶的合成，使天然产物的发现。
Lysate-based cell-free expression (CFE) systems are accessible platforms for expressing proteins that are difficult to synthesize in vivo, such as nonribosomal peptide synthetases (NRPSs). NRPSs are large (>100 kDa), modular enzyme complexes that synthesize bioactive peptide natural products. This synthetic process is analogous to transcription/translation (TX/TL) in lysates, resulting in potential resource competition between NRPS expression and NRPS activity in cell-free environments. Moreover, CFE conditions depend on the size and structure of the protein. Here, a reporter system for rapidly investigating and optimizing reaction environments for NRPS CFE is described. This strategy is demonstrated in E. coli lysate reactions using blue pigment synthetase A (BpsA), a model NRPS, carrying a C-terminal tetracysteine (TC) tag which forms a fluorescent complex with the biarsenical dye, FlAsH. A colorimetric assay was adapted for lysate reactions to detect the blue pigment product, indigoidine, of cell-free expressed BpsA-TC, confirming that the tagged enzyme is catalytically active. An optimized protocol for end point TC/FlAsH complex measurements in reactions enables quick comparisons of full-length BpsA-TC expressed under different reaction conditions, defining unique requirements for NRPS expression that are related to the protein's catalytic activity and size. Importantly, these protein-dependent CFE conditions enable higher indigoidine titer and improve the expression of other monomodular NRPSs. Notably, these conditions differ from those used for the expression of superfolder GFP (sfGFP), a common reporter for optimizing lysate-based CFE systems, indicating the necessity for tailored reporters to optimize expression for specific enzyme classes. The reporter system is anticipated to advance lysate-based CFE systems for complex enzyme synthesis, enabling natural product discovery.