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

Genome mining using standard bioinformatics tools has allowed for the uncovering of hidden biosynthesis gene clusters for specialized metabolites in Streptomyces genomes. In this work, we have used an alternative approach consisting in seeking Streptomyces 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 ahb cluster in Streptomyces argillaceus, which had not been retrieved before using antiSMASH. The ahb cluster is highly preserved in other Streptomyces 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 ahb 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 (ahbH, ahbI, and ahbL2) 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.

Automated summary

Genome mining using bioinformatics tools allowed for uncovering hidden biosynthesis gene clusters, while an alternative approach involving the analysis of Streptomyces Antibiotic Regulatory Proteins (SARP) encoding genes was used to identify cryptic gene clusters. This strategy led to the discovery of the new ahb cluster in Streptomyces argillaceus and the identification of ahbamycins as a new family of metabolites with antibiotic and antitumor activity. Further investigation revealed a hybrid compound, AHB18, produced through metabolic crosstalk between the ahb and argimycin P pathways. This finding opens up possibilities for generating new compounds through combinatorial biosynthesis.