Uncovering the Cryptic Gene Cluster for 3-amino-4-hydroxybenzoate Derived Ahbamycins, by Searching SARP Regulator Encoding Genes in the Genome

Genome mining using standard bioinformatics tools has allowed for the uncovering of hidden biosynthesis gene clusters for specialized metabolites in genomes. In this work, we have used an alternative approach consisting in seeking " Antibiotic Regulatory Proteins" (SARP) encoding genes and analyzing their surrounding DNA region to unearth cryptic gene clusters that cannot be identified using standard bioinformatics tools. This strategy has allowed the unveiling of the new cluster in , which had not been retrieved before using antiSMASH. The cluster is highly preserved in other strains, which suggests a role for their encoding compounds in specific environmental conditions. By combining overexpression of three regulatory genes and generation of different mutants, we were able to activate the cluster, and to identify and chemically characterize the encoded compounds that we have named ahbamycins (AHBs). These constitute a new family of metabolites derived from 3-amino-4-hydroxybenzoate (3,4-AHBA) known for having antibiotic and antitumor activity. Additionally, by overexpressing three genes of the cluster (, and ) for the synthesis and activation of 3,4-AHBA, a new hybrid compound, AHB18, was identified which had been produced from a metabolic crosstalk between the AHB and the argimycin P pathways. The identification of this new BGC opens the possibility to generate new compounds by combinatorial biosynthesis.