The knotted configuration of lasso peptides confers thermal stability and proteolytic resistance, addressing two shortcomings of peptide-based drugs. However, low isolation yields hinder the discovery and development of lasso peptides. While testing sp. FERM BP-3421 as a bacterial host to produce the lasso peptide capistruin, an overproducer clone was previously identified. In this study, we show that an increase in the plasmid copy number partially contributed to the overproducer phenotype. Further, we modulated the plasmid copy number to recapitulate titers to an average of 160% relative to the overproducer, which is 1000-fold higher than previously reported with , reaching up to 240 mg/L. To probe the applicability of the developed tools for lasso peptide discovery, we targeted a new lasso peptide biosynthetic gene cluster from endosymbiont sp. B13, leading to the isolation of mycetolassin-15 and mycetolassin-18 in combined titers of 11 mg/L. These results validate sp. FERM BP-3421 as a production platform for lasso peptide discovery.