Discovery of C-Glycosylpyranonaphthoquinones in Streptomyces sp. MBT76 by a Combined NMR-Based Metabolomics and Bioinformatics Workflow

Abstract

Mining of microbial genomes has revealed that actinomycetes harbor far more biosynthetic potential for bioactive natural products than anticipated. Activation of (cryptic) biosynthetic gene clusters and identification of the corresponding metabolites has become a focal point for drug discovery. Here, we applied NMR-based metabolomics combined with bioinformatics to identify novel C-glycosylpyranonaphthoquinones in Streptomyces sp. MBT76 and to elucidate the biosynthetic pathway. Following activation of the cryptic qin gene cluster for a type II polyketide synthase (PKS) by constitutive expression of its pathway-specific activator, bioinformatics coupled to NMR profiling facilitated the chromatographic isolation and structural elucidation of qinimycins A–C (1–3). The intriguing structural features of the qinimycins, including 8-C-glycosylation, 5,14-epoxidation, and 13-hydroxylation, distinguished these molecules from the model pyranonaphthoquinones actinorhodin, medermycin, and granaticin. Another novelty lies in the unusual fusion of a deoxyaminosugar to the pyranonaphthoquinone backbone during biosynthesis of the antibiotics BE-54238 A and B (4, 5). Qinimycins showed weak antimicrobial activity against Gram-positive bacteria. Our work shows the utility of combining bioinformatics, targeted activation of cryptic gene clusters, and NMR-based metabolic profiling as an effective pipeline for the discovery of microbial natural products with distinctive skeletons.

Aromatic Polyketide GTRI‐02 is a Previously Unidentified Product of the act Gene Cluster in Streptomyces coelicolor A3(2)

“…Metabolic profiling in tandem with chromatographic isolation confirmed the production of 1 in the resultant ex‐conjugant MBT76‐1, but not in the parent (wild‐type) strain. It is noteworthy that the titer of GTRI‐02 was higher than that of the glycosylated pyranonaphthoquinones (qinimycins), whereas the production of nonglycosylated pyranonaphthoquinones and anthraquinones was below the detection limit of HPLC‐DAD (Figure ) . This suggests that under the given growth conditions, GTRI‐02 is a major product of the qin gene cluster.…”

“…Further support for the notion that 1 originates from an act ‐like type II PKS was obtained from our investigations into Streptomyces sp. MBT76 . The qin BGC of Streptomyces sp.…”

“…A) Type II PKS gene cluster ( qin ) was activated in Streptomyces sp. MBT76 through constitutive expression of the pathway‐specific activator gene qin ‐ORF11 . Where relevant, homologous enzymes encoded by the act cluster are given in brackets.…”

“… Proposed biosynthetic pathway for GTRI‐02. GTRI‐02 is a previously undetected side product of the act and qin BGCs. So far, these BGCs have mainly been known for the production of the pyranonaphthoquinones kalafungin and actinorhodin.…”

“…MBT76‐1, which overexpresses the qin biosynthetic gene cluster, was described in ref. . Streptomyces strains were cultured as described without PEG 6000 but in the presence of glycerol (1 %, w/v ) and mannitol (0.5 %, w/v ) as the carbon sources, further supplemented with Bacto peptone (0.8 %, w/v ).…”