ABSTRACT Lasso peptides are bacterial metabolites characterized by a unique knotted configuration that confers thermal stability, proteolytic resistance, and potent target affinity in biological applications. Known lasso peptides have a diversity of biological functions, including as antibiotics. In terms of modification for drug development, they are recognized for their ability to be engineered via epitope grafting and genetically encoded libraries. However, the lack of a viable platform to produce lasso peptides has hindered the discovery and development of lasso-based drug leads. Chemical synthesis of lasso peptides is impeded by the lasso fold, positioning microbial production as a favorable alternative. However, heterologous expression of lasso peptide gene clusters (most commonly using E. coli as the bacterial host) results in low yield (<15 mg/L). In an experiment to test the expression of the model lasso peptide capistruin in an alternative Burkholderia bacterial host, a stochastic overproducer clone was identified. Preliminary investigation into the underlying molecular mechanisms of capistruin overproduction led to the recapitulation of high yield capistruin production (up to 130%) by way of plasmid copy number modulation. Production of capistruin with this alternative Burkholderia host corresponds to a yield 800-fold greater than that reported for production using E. coli. Our production platform was validated when Burkholderia was employed to express an orphan gene cluster from the endosymbiont Mycetohabitans sp. B13 (Burkholderiaceae), leading to the discovery of two lasso peptides. The objectives of this proposal are to further establish an effective production platform for novel lasso peptides while simultaneously deepening our understanding of the molecular determinants of compound overproduction by the Burkholderia host.