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

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.