A unified biocatalysis (UniBioCat) system based on cell-free gene expression was developed for the rapid biosynthesis and engineering of ribosomally synthesized and post-translationally modified peptides (RiPPs). The system enables the in vitro synthesis of mature RiPPs by co-expressing precursor peptides and modification enzymes. As a model, the system was used to synthesize salivaricin B, a lanthipeptide RiPP, and generate variants through site-specific mutations. The system was further expanded to synthesize and evaluate ten uncharacterized lanthipeptides. UniBioCat offers a fast, streamlined, and efficient platform for the discovery, characterization, and synthesis of RiPPs. The system was shown to be generalizable, as it can be used to synthesize a wide range of RiPPs, including those with antimicrobial activity. The system also allows for the rapid screening of variants with enhanced bioactivity. The results demonstrate that UniBioCat is a promising approach for the study and engineering of RiPPs, with potential applications in the discovery of bioactive natural products. The system was also shown to be robust, as it can simultaneously express multiple proteins and catalyze post-translational modifications. The system was further optimized by deleting protease/peptidase genes in the source strain, which improved the performance of the system. The system was also shown to be capable of synthesizing and purifying RiPPs without the need for extensive purification steps. The system was demonstrated to be effective in the synthesis of salivaricin B and its variants, as well as other lanthipeptides. The system was also shown to be capable of generating a wide range of RiPP variants through site-specific mutations. The system was further validated by testing the antimicrobial activity of the synthesized variants. The results indicate that UniBioCat is a promising platform for the study and engineering of RiPPs, with potential applications in the discovery of bioactive natural products.A unified biocatalysis (UniBioCat) system based on cell-free gene expression was developed for the rapid biosynthesis and engineering of ribosomally synthesized and post-translationally modified peptides (RiPPs). The system enables the in vitro synthesis of mature RiPPs by co-expressing precursor peptides and modification enzymes. As a model, the system was used to synthesize salivaricin B, a lanthipeptide RiPP, and generate variants through site-specific mutations. The system was further expanded to synthesize and evaluate ten uncharacterized lanthipeptides. UniBioCat offers a fast, streamlined, and efficient platform for the discovery, characterization, and synthesis of RiPPs. The system was shown to be generalizable, as it can be used to synthesize a wide range of RiPPs, including those with antimicrobial activity. The system also allows for the rapid screening of variants with enhanced bioactivity. The results demonstrate that UniBioCat is a promising approach for the study and engineering of RiPPs, with potential applications in the discovery of bioactive natural products. The system was also shown to be robust, as it can simultaneously express multiple proteins and catalyze post-translational modifications. The system was further optimized by deleting protease/peptidase genes in the source strain, which improved the performance of the system. The system was also shown to be capable of synthesizing and purifying RiPPs without the need for extensive purification steps. The system was demonstrated to be effective in the synthesis of salivaricin B and its variants, as well as other lanthipeptides. The system was also shown to be capable of generating a wide range of RiPP variants through site-specific mutations. The system was further validated by testing the antimicrobial activity of the synthesized variants. The results indicate that UniBioCat is a promising platform for the study and engineering of RiPPs, with potential applications in the discovery of bioactive natural products.