Metagenome-Guided Analogue Synthesis Yields Improved Gram-Negative-Active Albicidin- and Cystobactamid-Type Antibiotics.

Natural products are a major source of new antibiotics. Here we utilize biosynthetic instructions contained within metagenome-derived congener biosynthetic gene clusters (BGCs) to guide the synthesis of improved antibiotic analogues. Albicidin and cystobactamid are the first members of a new class of broad-spectrum ρ-aminobenzoic acid (PABA)-based antibiotics. Our search for PABA-specific adenylation domain sequences in soil metagenomes revealed that BGCs in this family are common in nature. Twelve BGCs that were bio-informatically predicted to encode six new congeners were recovered from soil metagenomic libraries. Synthesis of these six predicted structures led to the identification of potent antibiotics with changes in their spectrum of activity and the ability to circumvent resistance conferred by endopeptidase cleavage enzymes.

Iterative Methylations Resulting in the Biosynthesis of the t-Butyl Group Catalyzed by a B12-Dependent Radical SAM Enzyme in Cystobactamid Biosynthesis.

B12-dependent radical SAM enzymes that can perform methylations on sp3 carbon centers are important for functional diversity and regulation of biological activity in several nonribosomal peptides. Detailed studies on these enzymes are hindered by the complexity of the substrates and low levels of expression of active enzymes. CysS can catalyze iterative methylations of a methoxybenzene moiety during the biosynthesis of the cystobactamids. Here, we describe the overexpression, purification, substrate identification, and mechanism of this enzyme.