Discovery of Tricyclic Aromatic Polyketides Reveals Hidden Chain-Length Flexibility in Type II Polyketide Synthases

Type II polyketide synthases (PKSs) collectively generate polyketide intermediates of varying chain lengths, which undergo cyclization and further tailoring to produce structurally diverse aromatic polyketides. The length of the polyketide chain is a critical factor shaping the core scaffold of the final product. However, individual type II PKSs typically produce intermediates with a fixed chain length, thereby limiting the structural diversity accessible from a single biosynthetic system. In this study, we report the discovery of two pairs of novel tricyclic aromatic polyketides, varsomycin C/C' and oxtamycin A/A', along with two known analogues. These compounds are derived from the var and oxt gene clusters in Streptomyces varsoviensis/varR1, which primarily produce decaketide-derived tetracycline natural products, varsomycin A-B and oxytetracycline. Bioinformatic analysis combined with metabolite profiling of gene-disrupted mutants indicated that varsomycin C and C' are co-produced by enzymes encoded in the var cluster, with contributions from oxtJ and oxtF in the oxt cluster, resulting in nonaketide-derived tricyclic scaffolds. Oxtamycin A and A', along with the two analogues, are predicted to be biosynthesized by the oxt cluster. These results suggest that the minimal PKSs from both clusters possess intrinsic flexibility in controlling polyketide chain length, enabling the production of both decaketide and nonaketide intermediates, which represents a rare example of dual chain-length programming in type II PKSs. This flexibility reveals new natural sources of nonaketide biosynthetic enzymes and enriches the chemical diversity of tricyclic aromatic polyketides. Our findings deepen the understanding of type II PKS chain-length regulation and provide a foundation for future engineering of PKSs to produce customized bioactive aromatic polyketides.