High-Yield Lasso Peptide Production in a Burkholderia Bacterial Host by Plasmid Copy Number Engineering.

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 Burkholderia 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 E. coli, 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 Mycetohabitans sp. B13, leading to the isolation of mycetolassin-15 and mycetolassin-18 in combined titers of 11 mg/L. These results validate Burkholderia sp. FERM BP-3421 as a production platform for lasso peptide discovery.

Biotechnology approaches for natural product discovery, engineering, and production based on Burkholderia bacteria.

Bacterial natural products (NPs) retain high value in discovery efforts for applications in medicine and agriculture. Burkholderia ß-Proteobacteria are a promising source of NPs. In this review, we summarize the recently developed genetic manipulation techniques used to access silent/cryptic biosynthetic gene clusters from Burkholderia native producers. We also discuss the development of Burkholderia bacteria as heterologous hosts and the application of Burkholderia in industrial-scale production of NPs. Genetic engineering and fermentation media optimization have enabled the industrial-scale production of at least two Burkholderia NPs. The biotechnology approaches discussed here will continue to facilitate the discovery and development of NPs from Burkholderia.