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.

Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element.

The RiPP precursor recognition element (RRE) is a conserved domain found in many prokaryotic ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic gene clusters (BGCs). RREs bind with high specificity and affinity to a recognition sequence within the N-terminal leader region of RiPP precursor peptides. Lasso peptide biosynthesis involves an RRE-dependent leader peptidase, which is discretely encoded or fused to the RRE as a di-domain protein. Here we leveraged thousands of predicted BGCs to define the RRE:leader peptidase interaction through evolutionary covariance analysis. Each interacting domain contributes a three-stranded ß-sheet to form a hydrophobic ß-sandwich-like interface. The bioinformatics-guided predictions were experimentally confirmed using proteins from discrete and fused lasso peptide BGC architectures. Support for the domain-domain interface derived from chemical shift perturbation, paramagnetic relaxation enhancement experiments, and rapid variant activity screening using cell-free biosynthesis. Further validation of selected variants was performed with purified proteins. We developed a p-nitroanilide-based leader peptidase assay to illuminate the role of RRE domains. Our data show that RRE domains play a dual function. RRE domains deliver the precursor peptide to the leader peptidase, and the rate is saturable as expected for a substrate. RRE domains also partially compose the elusive S2 proteolytic pocket that binds the penultimate threonine of lasso leader peptides. Because the RRE domain is required to form the active site, leader peptidase activity is greatly diminished when the RRE domain is supplied at substoichiometric levels. Full proteolytic activation requires RRE engagement with the recognition sequence-containing portion of the leader peptide. Together, our observations define a new mechanism for protease activity regulation.

Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme-Substrate Tolerance.

Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like, threaded conformation. Owing to a locked three-dimensional structure, lasso peptides can be unusually stable toward heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. All known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent class of RiPPs; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address longstanding challenges associated with lasso peptide production. We report the successful use of CFB for the formation of an array of sequence-diverse lasso peptides that include known examples as well as a new predicted lasso peptide from Thermobifida halotolerans. We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants to evaluate the substrate tolerance of the biosynthetic pathway. By evaluating more than 1000 randomly chosen variants, we show that the lasso-forming cyclase from the fusilassin pathway is capable of producing millions of sequence-diverse lasso peptides via CFB. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.

Pseudomonas Virulence Factor Pathway Synthesizes Autoinducers That Regulate the Secretome of a Pathogen.

Cell-to-cell communication via chemical signals is an essential mechanism that pathogenic bacteria use to coordinate group behaviors and promote virulence. The Pseudomonas virulence factor (pvf) gene cluster is distributed in more than 500 strains of proteobacteria including both plant and human pathogens. The pvf cluster has been implicated in the production of signaling molecules important for virulence; however, the regulatory impact of these signaling molecules on virulence had not been elucidated. Using the insect pathogen Pseudomonas entomophila L48 as a model, we demonstrated that pvf-encoded biosynthetic enzymes produce PVF autoinducers that regulate the expression of pvf genes and a gene encoding the toxin monalysin via quorum sensing. In addition, PVF autoinducers regulate the expression of nearly 200 secreted and membrane proteins, including toxins, motility proteins, and components of the type VI secretion system, which play key roles in bacterial virulence, colonization, and competition with other microbes. Deletion of pvf also altered the secondary metabolome. Six major compounds upregulated by PVF autoinducers were isolated and structurally characterized, including three insecticidal 3-indolyl oxazoles, the labradorins, and three antimicrobial pyrrolizidine alkaloids, the pyreudiones. The signaling properties of PVF autoinducers and their wide-ranging regulatory effects indicate multifaceted roles of PVF in controlling cell physiology and promoting virulence. The broad genome distribution of pvf suggests that PVF-mediated signaling is relevant to many bacteria of agricultural and biomedical significance.

Specificity of Nonribosomal Peptide Synthetases in the Biosynthesis of the Pseudomonas virulence factor.

The Pseudomonas virulence factor (pvf) biosynthetic operon has been implicated in bacterial virulence and signaling. We identified 308 bacterial strains containing pvf homologues that likely produce signaling molecules with distinct structures and biological activities. Several homologues of the nonribosomal peptide synthetase (NRPS), PvfC, were biochemically characterized and shown to activate l-Val or l-Leu. The amino acid selectivity of PvfC and its homologues likely direct pvf signaling activity. We explored the natural diversity of the active site residues present in 92% of the adenylation domains of PvfC homologues and identified key residues for substrate selection and catalysis. Sequence similarity network (SSN) analysis revealed grouping of PvfC homologues that harbor the same active site residues and activate the same amino acids. Our work identified PvfC as a gatekeeper for the structure and bioactivity of the pvf-produced signaling molecules. The combination of active site residue identification and SSN analysis can improve the prediction of aliphatic amino acid substrates for NRPS adenylation domains.

Discovery of (Dihydro)pyrazine N-Oxides via Genome Mining in Pseudomonas.

Overexpression of the Pseudomonas virulence factor ( pvf) biosynthetic operon led to the identification of a family of pyrazine N-oxides (PNOs), including a novel dihydropyrazine N,N'-dioxide (dPNO) metabolite. The nonribosomal peptide synthetase responsible for production of (d)PNOs was characterized, and a biosynthetic pathway for (d)PNOs was proposed. This work highlights the unique chemistry catalyzed by pvf-encoded enzymes and sets the stage for bioactivity studies of the metabolites produced by the virulence pathway.