Ribosomally synthesized lanthionine-containing peptides (lanthipeptides) have emerged as a promising source of antimicrobials against multidrug resistance pathogens. An effective way to discover and engineer lanthipeptides is through heterologous expression of their biosynthetic gene clusters (BGCs) in a host of choice. Here we report a plug-and-play pathway refactoring strategy for rapid evaluation of lanthipeptide BGCs in Bacillus subtilis based on the T7 expression system. As a proof of concept, we used this strategy to not only observe the successful production of a known lanthipeptide haloduracin β but also discover two new human-microbiota-derived lanthipeptides that previously failed to be produced in Escherichia coli. The resulting B. subtilis plug-and-play T7 expression system should enable the genome mining of new lanthipeptides in a high-throughput manner.