4.8 Article

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

Journal

CHEMICAL SCIENCE
Volume 12, Issue 35, Pages 11882-11893

Publisher

ROYAL SOC CHEMISTRY
DOI: 10.1039/d1sc02725e

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The development of new antibiotics is crucial in combating infections caused by multidrug-resistant bacteria, with Gram-negative pathogens posing particular challenges. Through synthetic engineering of darobactin biosynthetic gene cluster in Escherichia coli, high yields of darobactin A and its active derivatives have been successfully produced, showing improved efficacy against various bacteria. This study also sheds light on the potential self-resistance mechanism in native darobactin producers.
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 mu g mL(-1)) and Acinetobacter baumannii (1-2 mu g mL(-1)). Importantly, it also displayed superior activity against MDR clinical isolates of E. coli (1-2 mu g mL(-1)) and Klebsiella pneumoniae (1-4 mu g 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.

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