Network Topology Evaluation and Transitive Alignments for Molecular Networking.

Untargeted tandem mass spectrometry (MS/MS) is an essential technique in modern analytical chemistry, providing a comprehensive snapshot of chemical entities in complex samples and identifying unknowns through their fragmentation patterns. This high-throughput approach generates large data sets that can be challenging to interpret. Molecular Networks (MNs) have been developed as a computational tool to aid in the organization and visualization of complex chemical space in untargeted mass spectrometry data, thereby supporting comprehensive data analysis and interpretation. MNs group related compounds with potentially similar structures from MS/MS data by calculating all pairwise MS/MS similarities and filtering these connections to produce a MN. Such networks are instrumental in metabolomics for identifying novel metabolites, elucidating metabolic pathways, and even discovering biomarkers for disease. While MS/MS similarity metrics have been explored in the literature, the influence of network topology approaches on MN construction remains unexplored. This manuscript introduces metrics for evaluating MN construction, benchmarks state-of-the-art approaches, and proposes the Transitive Alignments approach to improve MN construction. The Transitive Alignment technique leverages the MN topology to realign MS/MS spectra of related compounds that differ by multiple structural modifications. Combining this Transitive Alignments approach with pseudoclique finding, a method for identifying highly connected groups of nodes in a network, resulted in more complete and higher-quality molecular families. Finally, we also introduce a targeted network construction technique called induced transitive alignments where we demonstrate effectiveness on a real world natural product discovery application. We release this transitive alignment technique as a high-throughput workflow that can be used by the wider research community.

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.