Genome Mining for Diazo-Synthesis-Related Genes in Streptomyces sp. CS057 Unveiled the Cryptic Biosynthetic Gene Cluster crx for the Novel 3,4-AHBA-Derived Compound Crexazone 2

Natural products play a crucial role in drug development, addressing the escalating microbial resistance to antibiotics and the treatment of emerging diseases. Progress in genome sequencing techniques, coupled with the development of bioinformatics tools and the exploration of uncharted habitats, has highlighted the biosynthetic potential of actinomycetes. By in silico screening for diazo-related gene genomes from twelve <i>Streptomyces</i> strains isolated from <i>Attini</i> leaf-cutting ants, the new <i>crx</i> biosynthetic gene cluster (BGC) was identified in <i>Streptomyces</i> sp. CS057. This cluster, highly conserved in several <i>Streptomyces</i> strains, contains genes related to diazo group formation and genes for the biosynthesis of 3,4-AHBA. By overexpressing the LuxR-like regulatory gene <i>crxR1</i>, we were able to activate the <i>crx</i> cluster, which encodes the biosynthesis of three 3,4-AHBA-derived compounds that we named crexazones (CRXs). The chemical structure of crexazones (CRXs) was determined by LC-DAD-HRMS-based dereplication and NMR spectroscopic analyses and was found to correspond to two known compounds, 3-acetamido-4-hydroxybenzoic acid (CRX1) and the phenoxazinone texazone (CRX3), and a novel 3,4-AHBA-containing compound herein designated as CRX2. Experimental proof linking the <i>crx</i> BGC to their encoded compounds was achieved by generating mutants in selected <i>crx</i> genes.