The Peptide Antibiotic Corramycin Adopts a ß-Hairpin-like Structure and Is Inactivated by the Kinase ComG.

The rapid development of antibiotic resistance, especially among difficult-to-treat Gram-negative bacteria, is recognized as a serious and urgent threat to public health. The detection and characterization of novel resistance mechanisms are essential to better predict the spread and evolution of antibiotic resistance. Corramycin is a novel and modified peptidic antibiotic with activity against several Gram-negative pathogens. We demonstrate that the kinase ComG, part of the corramycin biosynthetic gene cluster, phosphorylates and thereby inactivates corramycin, leading to the resistance of the host. Remarkably, we found that the closest structural homologues of ComG are aminoglycoside phosphotransferases; however, ComG shows no activity toward this class of antibiotics. The crystal structure of ComG in complex with corramycin reveals that corramycin adopts a ß-hairpin-like structure and allowed us to define the changes leading to a switch in substrate from sugar to peptide. Bioinformatic analyses suggest a limited occurrence of ComG-like proteins, which along with the absence of cross-resistance to clinically used drugs positions corramycin as an attractive antibiotic for further development.

Darobactins Exhibiting Superior Antibiotic Activity by Cryo-EM Structure Guided Biosynthetic Engineering.

Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated novel darobactins with enhanced antibacterial activity. Here we utilize an optimized purification method and present cryo-EM structures of the Bam complex with darobactin 9 (D9), which served as a blueprint for the biotechnological generation of twenty new darobactins including halogenated analogs. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, without observing toxic effects.

Structure Elucidation, Total Synthesis, Antibacterial In Vivo Efficacy and Biosynthesis Proposal of Myxobacterial Corramycin.

Herein, we describe the myxobacterial natural product Corramycin isolated from Corallococcus coralloides. The linear peptide structure contains an unprecedented (2R,3S)-γ-N-methyl-ß-hydroxy-histidine moiety. Corramycin exhibits anti-Gram-negative activity against Escherichia coli (E. coli) and is taken up via two transporter systems, SbmA and YejABEF. Furthermore, the Corramycin biosynthetic gene cluster (BGC) was identified and a biosynthesis model was proposed involving a 12-modular non-ribosomal peptide synthetase/polyketide synthase. Bioinformatic analysis of the BGC combined with the development of a total synthesis route allowed for the elucidation of the molecule's absolute configuration. Importantly, intravenous administration of 20 mg kg-1 of Corramycin in an E. coli mouse infection model resulted in 100 % survival of animals without toxic side effects. Corramycin is thus a promising starting point to develop a potent antibacterial drug against hospital-acquired infections.

Improved broad-spectrum antibiotics against Gram-negative pathogens via darobactin biosynthetic pathway engineering.

The development of new antibiotics is imperative to fight increasing mortality rates connected to infections caused by multidrug-resistant (MDR) bacteria. In this context, Gram-negative pathogens listed in the WHO priority list are particularly problematic. Darobactin is a ribosomally produced and post-translationally modified bicyclic heptapeptide antibiotic selectively killing Gram-negative bacteria by targeting the outer membrane protein BamA. The native darobactin A producer Photorhabdus khanii HGB1456 shows very limited production under laboratory cultivation conditions. Herein, we present the design and heterologous expression of a synthetically engineered darobactin biosynthetic gene cluster (BGC) in Escherichia coli to reach an average darobactin A production titre of 13.4 mg L-1. Rational design of darA variants, encoding the darobactin precursor peptide with altered core sequences, resulted in the production of 13 new 'non-natural' darobactin derivatives and 4 previously hypothetical natural darobactins. One of the non-natural compounds, darobactin 9, was more potent than darobactin A, and showed significantly improved activity especially against Pseudomonas aeruginosa (0.125 µg mL-1) and Acinetobacter baumannii (1-2 µg mL-1). Importantly, it also displayed superior activity against MDR clinical isolates of E. coli (1-2 µg mL-1) and Klebsiella pneumoniae (1-4 µg mL-1). Independent deletions of genes from the darobactin BGC showed that only darA and darE, encoding a radical forming S-adenosyl-l-methionine-dependent enzyme, are required for darobactin formation. Co-expression of two additional genes associated with the BGCs in hypothetical producer strains identified a proteolytic detoxification mechanism as a potential self-resistance strategy in native producers. Taken together, we describe a versatile heterologous darobactin platform allowing the production of unprecedented active derivatives in good yields, and we provide first experimental evidence for darobactin biosynthesis processes.