Characterization of the Biosynthetic Gene Cluster and Shunt Products Yields Insights into the Biosynthesis of Balmoralmycin

Angucyclines are a family of structurally diverse, aromatic polyketides with some members that exhibit potent bioactivity. Angucyclines have also attracted considerable attention due to the intriguing biosynthetic origins that underlie their structural complexity and diversity. Balmoralmycin (compound 1) represents a unique group of angucyclines that contain an angular benz[a]anthracene tetracyclic system, a characteristic C-glycosidic bond-linked deoxy-sugar (D-olivose), and an unsaturated fatty acid chain. In this study, we identified a Streptomyces strain that produces balmoralmycin and seven biosynthetically related coproducts (compounds 228). Four of the coproducts (compounds 528) are novel compounds that feature a highly oxygenated or fragmented lactone ring, and three of them (compounds 325) exhibited cytotoxicity against the human pancreatic cancer cell line MIA PaCa-2 with IC₅₀ values ranging from 0.9 to 1.2 mg/mL. Genome sequencing and CRISPR/dCas9-assisted gene knockdown led to the identification of the;43 kb balmoralmycin biosynthetic gene cluster (bal BGC). The bal BGC encodes a type II polyketide synthase (PKS) system for assembling the angucycline aglycone, six enzymes for generating the deoxysugar D-olivose, and a hybrid type II/III PKS system for synthesizing the 2,4-decadienoic acid chain. Based on the genetic and chemical information, we propose a mechanism for the biosynthesis of balmoralmycin and the shunt products. The chemical and genetic studies yielded insights into the biosynthetic origin of the structural diversity of angucyclines. IMPORTANCE Angucyclines are structurally diverse aromatic polyketides that have attracted considerable attention due to their potent bioactivity and intriguing biosynthetic origin. Balmoralmycin is a representative of a small family of angucyclines with unique structural features and an unknown biosynthetic origin. We report a newly isolated Streptomyces strain that produces balmoralmycin in a high fermentation titer as well as several structurally related shunt products. Based on the chemical and genetic information, a biosynthetic pathway that involves a type II polyketide synthase (PKS) system, cyclases/aromatases, oxidoreductases, and other ancillary enzymes was established. The elucidation of the balmoralmycin pathway enriches our understanding of how structural diversity is generated in angucyclines and opens the door for the production of balmoralmycin derivatives via pathway engineering.