Regulatory Part Engineering for High-Yield Protein Synthesis in an All-Streptomyces-Based Cell-Free Expression System

Streptomyces-based cell-free expression systems have been developed to meet the demand for synthetic biology applications. However, protein yields from the previous Streptomyces systems are relatively low, and there is a serious limitation of available genetic tools such as plasmids for gene (co)expression. Here, we sought to expand the plasmid toolkit with a focus on the enhancement of protein production. By screening native promoters and ribosome binding sites, we were able to construct a panel of plasmids with different abilities for protein synthesis, which covered a nearly 3-fold range of protein yields. Using the most efficient plasmid, the protein yield reached up to a maximum value of 515.7 ± 25.3 μg/mL. With the plasmid toolkit, we anticipate that our Streptomyces cell-free system will offer great opportunities for cell-free synthetic biology applications such as in vitro biosynthesis of valuable natural products when cell-based systems remain difficult or not amenable.