The article introduces a unified biocatalysis (UniBioCat) system based on cell-free gene expression for rapid biosynthesis and engineering of ribosomally synthesized and post-translationally modified peptides (RiPPs). The system is demonstrated by reconstituting a full biosynthetic pathway for the de novo biosynthesis of salivaricin B, a lanthipeptide RiPP. By deleting several protease/peptidase genes from the source strain, the performance of UniBioCat is enhanced, enabling the synthesis and screening of salivaricin B variants with enhanced antimicrobial activity. The system is further shown to be generalizable by synthesizing and evaluating the bioactivity of ten uncharacterized lanthipeptides. UniBioCat offers a fast, streamlined, and promising approach to study RiPP natural products, accelerating their discovery, characterization, and synthesis.The article introduces a unified biocatalysis (UniBioCat) system based on cell-free gene expression for rapid biosynthesis and engineering of ribosomally synthesized and post-translationally modified peptides (RiPPs). The system is demonstrated by reconstituting a full biosynthetic pathway for the de novo biosynthesis of salivaricin B, a lanthipeptide RiPP. By deleting several protease/peptidase genes from the source strain, the performance of UniBioCat is enhanced, enabling the synthesis and screening of salivaricin B variants with enhanced antimicrobial activity. The system is further shown to be generalizable by synthesizing and evaluating the bioactivity of ten uncharacterized lanthipeptides. UniBioCat offers a fast, streamlined, and promising approach to study RiPP natural products, accelerating their discovery, characterization, and synthesis.