Insights into\nHeterocycle Biosynthesis in the Cytotoxic\nPolyketide Alkaloid Janustatin A from a Plant-Associated Bacterium

Janustatin A is a potently cytotoxic polyketide alkaloid\nproduced\nat trace amounts by the marine bacterial plant symbiont <i>Gynuella\nsunshinyii</i>. Its biosynthetic terminus features an unusual\npyridine-containing bicyclic system of unclear origin, in which polyketide\nand amino acid extension units appear reversed compared to the order\nof enzymatic modules in the polyketide synthase (PKS)-nonribosomal\npeptide synthetase (NRPS) assembly line. To elucidate unknown steps\nin heterocycle formation, we first established robust genome engineering\ntools in <i>G</i>. <i>sunshinyii</i>. A combination\nof gene deletion, complementation, production improvement, and NMR\nexperiments then demonstrated that two desaturase homologues, JanA\nand JanB, are involved in hydroxylation and pyridine formation by\ndesaturation, respectively. Structure–activity relationship\nstudies showed that these modifications substantially increase the\ncytotoxicity and that the fully functionalized heterocyclic system\nis crucial for sub-nanomolar cytotoxicity. Isolation of the early\npost-PKS intermediate janustatin D with an already reversed heterocycle\ntopology supports a noncanonical rearrangement process occurring on\nthe PKS-NRPS assembly line.