Establishing a High-Yield Bacillus subtilis -Based Cell-Free Protein Synthesis System for In Vitro Prototyping and Natural Product Biosynthesis

作者：Xiangyang Ji, Wan-Qiu Liu, Zhiling Cao, Shuhui Huang, Jian Li · 发表于：ACS Synthetic Biology · 年份：2025 · DOI：10.1021/acssynbio.5c00021 · 被引用次数：12 · 研究领域：RNA and protein synthesis mechanisms、Microbial Metabolic Engineering and Bioproduction、Bacterial Genetics and Biotechnology

Cell-free systems are emerging as powerful platforms for synthetic biology with widespread applications in both fundamental research, such as artificial cell construction, and practical uses like recombinant protein production. Among these, cell-free protein synthesis (CFPS) plays a crucial role in gene expression for various downstream applications. However, the development of CFPS systems based on certain chassis, such as Bacillus subtilis, still remains limited due to their low in vitro productivity. Here, we report the development of a highly productive CFPS system derived from an engineered B. subtilis 164T7P strain, which contains a genomic integration of the T7 RNA polymerase gene. This modification allows the preparation of cell extracts that inherently contain T7 RNA polymerase, enabling T7 promoter-based transcription without the supplementation of purified T7 RNA polymerase in CFPS reactions. Through systematic optimization of cell extract preparation and key reaction parameters, we achieved the synthesis of 286 ± 16.7 μg/mL of sfGFP in batch reactions, with yields increasing to over 1100 μg/mL in a semicontinuous format that can replenish substrates and remove inhibitory byproducts. We further demonstrated the system’s versatility by using it for two synthetic biology applications: prototyping ribosome binding site (RBS) elements and synthesizing pulcherriminic acid─a bioactive cyclodipeptide. The system successfully characterized RBS performance, with in vitro an...