构建链霉菌无细胞平台挖掘套索肽类天然产物

Developing a Streptomyces-based cell-free platform for discovery of lasso peptides

摘要:

【目的】套索肽作为一类核糖体翻译后修饰肽(RiPPs)广泛分布于放线菌中，以其独特的修饰结构和多样的生理活性受到了广泛的关注。为了更好地研究未知的套索肽，期望开发基于链霉菌的无细胞转录翻译平台(下称“无细胞平台”)实现无细胞合成套索肽或其前体肽。【方法】首先尝试以不同的链霉菌构建无细胞合成平台，并以绿色荧光蛋白为报告蛋白对平台产率进行优化；在构建合适稳定的表达体系后，将包含有套索肽生物合成基因的质粒引入体系中以探索套索肽的无细胞合成。【结果】在对基于模式菌株Streptomyces lividans TK24的无细胞体系进行制备工艺、体系组分、反应条件等多个参数进行优化后，该体系最高能达到90µg/mL的荧光蛋白表达量；基于该体系成功表达了目标套索肽的前体肽，并通过融合SUMO标签增加前体肽在该体系中的稳定性。【结论】本研究成功构建了一类链霉菌无细胞平台，为丰富来源的基因表达提供了可能性。尽管该体系在对表达套索肽未知蛋白的适用性上仍有待进一步提升，但无细胞平台在天然产物的探索中将起到越来越重要的作用。

Abstract:

[Objective]As a type of ribosomally synthesized and post-translationally modified peptides (RiPPs) rich in Actinomycetes, lasso peptides have received increasing attention for their distinct modified structures and diverse biological activities. Therefore, we endeavored to develop a Streptomyces-based cell-free protein transcription/translation platform suitable for cell-free synthesis of lasso peptides or their precursors. [Methods] We first developed the cell-free platform with different Streptomyces strains and then optimized the preparation method, system components, and reaction conditions to improve the platform efficiency according to the expression of enhanced green fluorescent protein (eGFP). The core genes of the targeted lasso peptides were then introduced to the optimized platform for expression. [Results]We increased the titer of eGFP in a cell-free platform based on Streptomyces lividans TK24 to 90 µg/mL under the optimized conditions. With this cell-free platform, we expressed the precursors of lasso peptides and further fused SUMO-tag to the targeted precursors to increase their stability. [Conclusion] In this study, we constructed a Streptomyces-based cell-free platform for the expression of unexplored genes. Although the applicability of the platform for expressing unknown lasso peptides still calls for further improvement, cell-free platforms will facilitate the research on natural products.