Substrate interactions guide cyclase engineering and lasso peptide diversification.

Lasso peptides are a diverse class of naturally occurring, highly stable molecules kinetically trapped in a distinctive [1]rotaxane conformation. How the ATP-dependent lasso cyclase constrains a relatively unstructured substrate peptide into a low entropy product has remained a mystery owing to poor enzyme stability and activity in vitro. In this study, we combined substrate tolerance data with structural predictions, bioinformatic analysis, molecular dynamics simulations and mutational scanning to construct a model for the three-dimensional orientation of the substrate peptide in the lasso cyclase active site.

Piperacillin triggers virulence factor biosynthesis via the oxidative stress response in .

Natural products have been an important source of therapeutic agents and chemical tools. The recent realization that many natural product biosynthetic genes are silent or sparingly expressed during standard laboratory growth has prompted efforts to investigate their regulation and develop methods to induce their expression. Because it is difficult to intuit signals that induce a given biosynthetic locus, we recently implemented a forward chemical-genetic approach to identify such inducers.

Identification of the Hypertension Drug Guanfacine as an Antivirulence Agent in Pseudomonas aeruginosa.

An alternative solution to the cyclical development of new antibiotics is the concept of disarming pathogens without affecting their growth, thereby eliminating the selective pressures that lead to resistant phenotypes. Here, we have employed our previously developed HiTES methodology to identify one such compound against the ESKAPE pathogen Pseudomonas aeruginosa. Rather than induce silent biosynthetic gene clusters, we used HiTES to suppress actively expressed virulence genes.

Recent advances in activating silent biosynthetic gene clusters in bacteria.

The explosion of microbial genome sequences has shown that bacteria harbor an immense, largely untapped potential for the biosynthesis of diverse natural products, which have traditionally served as an important source of pharmaceutical compounds. Most of the biosynthetic genes that can be detected bioinformatically are not, or only weakly, expressed under standard laboratory growth conditions. Herein we review three recent approaches that have been developed for inducing these so-called silent biosynthetic gene cluster: insertion of constitutively active promoters using CRISPR-Cas9, high-throughput elicitor screening for identification of small molecule inducers, and reporter-guided mutant selection for creation of overproducing strains.

Antibiotic dialogues: induction of silent biosynthetic gene clusters by exogenous small molecules.

Natural products have traditionally served as a dominant source of therapeutic agents. They are produced by dedicated biosynthetic gene clusters that assemble complex, bioactive molecules from simple precursors. Recent genome sequencing efforts coupled with advances in bioinformatics indicate that the majority of biosynthetic gene clusters are not expressed under normal laboratory conditions.

Mapping the Trimethoprim-Induced Secondary Metabolome of Burkholderia thailandensis.

While bacterial genomes typically contain numerous secondary metabolite biosynthetic gene clusters, only a small fraction of these are expressed at any given time. The remaining majority is inactive or silent, and methods that awaken them would greatly expand our repertoire of bioactive molecules. We recently devised a new approach for identifying inducers of silent gene clusters and proposed that the clinical antibiotic trimethoprim acted as a global activator of secondary metabolism in Burkholderia thailandensis.