Discovery of thiazostatin D/E using UPLC-HR-MS2-based metabolomics and σ-factor engineering of sp. SE50/110.

As the natural producer of acarbose, sp. SE50/110 has high industrial relevance. Like most Actinobacteria, the strain carries several more putative biosynthetic gene clusters (BGCs) to produce further natural products, which are to be discovered. Applying a metabolomics-guided approach, we tentatively identified five further compounds that are produced by the strain: watasemycin, thiazostatin, isopyochelin, pulicatin, and aerugine. A comparison of the genomic context allowed the identification of the putative BGC, which is highly similar to the watasemycin biosynthetic gene cluster of . In addition to the identified molecules, a thiazostatin-like compound was found. Isolation and structure elucidation with 1D and 2D NMR and HRMS were applied. The fraction containing 369.0929 [M + H] comprised two highly similar compounds identified as thiazostatin D and thiazostatin E. The compounds possessed the same phenol-thiazole-thiazole molecular scaffold as the previously reported thiazostatin and watasemycin and have anti-proliferative activity against the breast adenocarcinoma cell line MCF7 and human melanoma cell line A2058, while no activity again the non-malignant immortalized fibroblast cell line MRC-5 was observed. We further showed that the manipulation of global transcriptional regulators, with () and anti-anti-σ factor coding as an example, enabled the production manipulation of the 2-hydroxyphenylthiazoline family molecules. While the manipulation of enabled the shift in the peak intensities between the five products of this pathway, manipulation prevented their production. The production of a highly polar compound with 462.1643 [M + H] and calculated elemental composition CHNO was activated under the expression and is exclusively produced by the engineered strain.