Integrated Metabolomic and Genomic Insights into Amino Acid Incorporation Within the Hybrid Polyketide-Alkaloid Antibiotic TLN-05220

Actinobacteria are a rich source of bioactive compounds and unique biosynthetic chemistry. <i>Micromonospora echinospora</i> subsp. <i>challisensis</i> NRRL 12255 produces the aromatic polyketide TLN-05220, which exhibits nanomolar activity against antibiotic-resistant human pathogens, including vancomycin-resistant <i>Enterococcus faecalis</i> and methicillin-resistant <i>Staphylococcus aureus</i>. The pentangular polyphenol core of TLN-05220 is decorated with a piperazinone moiety; yet, the enzymes responsible for the construction of this uncommon modification from amino acid precursors are unknown. Synthetic piperazinone-containing molecules have diverse antimicrobial, antiviral, anticancer, and anti-inflammatory bioactivity profiles, and determining biosynthetic routes for the assembly of this heterocycle may enhance drug discovery and medicinal chemistry efforts. We identified a putative TLN-05220 biosynthetic gene cluster (BGC) in the commercially available strain <i>M. echinospora</i> ATCC 15837 that contains both type-I and type-II polyketide synthases, two predicted asparagine synthetase-like enzymes, and two genes (<i>tln</i>1 and <i>tln</i>5) that putatively encode pyridoxal 5'-phosphate (PLP)-dependent amino acid synthases. Stable isotopic feeding studies coupled with liquid chromatography-mass spectrometry (LC-MS) identified l-alanine, l-serine, and glycine as metabolic precursors of TLN-05220. Subsequent in vitro enzymology established that Tln1 is a PLP-dependent alanine racemase, while Tln5 performs a stereoselective β-substitution reaction of <i>O</i>-phospho-l-serine with a preferential d-alanine nucleophile. Alanine racemization and pseudodipeptide l-serine-Cβ-<i>N</i>-d-alanine (d,l-PDP) incorporation into TLN-05220 were further supported using deuterated intermediates and mass spectrometry techniques. Establishing the enzymes that catalyze amino acid installation within TLN-05220 inspires further biosynthetic discovery and engineering while enabling the biocatalytic syntheses of novel amino acid-containing polyketide antibiotics.