Investigation of the Odilorhabdin Biosynthetic Gene Cluster Using NRPS Engineering.

The recently identified natural product NOSO-95A from entomopathogenic Xenorhabdus bacteria, derived from a biosynthetic gene cluster (BGC) encoding a non-ribosomal peptide synthetase (NRPS), was the first member of the odilorhabdin class of antibiotics. This class exhibits broad-spectrum antibiotic activity and inspired the development of the synthetic derivative NOSO-502, which holds potential as a new clinical drug by breaking antibiotic resistance. While the mode of action of odilorhabdins was broadly investigated, their biosynthesis pathway remained poorly understood. Here we describe the heterologous production of NOSO-95A in Escherichia coli after refactoring the complete BGC. Since the production titer was low, NRPS engineering was applied to uncover the underlying biosynthetic principles. For this, modules of the odilorhabdin NRPS fused to other synthetases were co-expressed with candidate hydroxylases encoded in the BGC allowing the characterization of the biosynthesis of three unusual amino acids and leading to the identification of a prodrug-activation mechanism by deacylation. Our work demonstrates the application of NRPS engineering as a blueprint to mechanistically elucidate large or toxic NRPS and provides the basis to generate novel odilorhabdin analogues with improved properties in the future.

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.

Harnessing Gram-negative bacteria for novel anti-Gram-negative antibiotics.

Natural products have proven themselves as a valuable resource for antibiotics. However, in view of increasing antimicrobial resistance, there is an urgent need for new, structurally diverse agents that have the potential to overcome resistance and treat Gram-negative pathogens in particular. Historically, the search for new antibiotics was strongly focussed on the very successful Actinobacteria. On the other hand, other producer strains have been under-sampled and their potential for the production of bioactive natural products has been underestimated. In this mini-review, we highlight prominent examples of novel anti-Gram negative natural products produced by Gram-negative bacteria that are currently in lead optimisation or preclinical development. Furthermore, we will provide insights into the considerations and strategies behind the discovery of these agents and their putative applications.

New Genetically Engineered Derivatives of Antibacterial Darobactins Underpin Their Potential for Antibiotic Development.

Biosynthetic engineering of bicyclic darobactins, selectively sealing the lateral gate of the outer membrane protein BamA, leads to active analogues, which are up to 128-fold more potent against Gram-negative pathogens compared to native counterparts. Because of their excellent antibacterial activity, darobactins represent one of the most promising new antibiotic classes of the past decades. Here, we present a series of structure-driven biosynthetic modifications of our current frontrunner, darobactin 22 (D22), to investigate modifications at the understudied positions 2, 4, and 5 for their impact on bioactivity. Novel darobactins were found to be highly active against critical pathogens from the WHO priority list. Antibacterial activity data were corroborated by dissociation constants with BamA. The most active derivatives D22 and D69 were subjected to ADMET profiling, showing promising features. We further evaluated D22 and D69 for bioactivity against multidrug-resistant clinical isolates and found them to have strong activity.

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.