Discovery and Biosynthetic Studies of a Highly Reduced Cinnamoyl Lipid, Tripmycin A, from an Endophytic Streptomyces sp.

A Tripterygium wilfordii endophyte, Streptomyces sp. CB04723, was shown to produce an unusually highly reduced cytotoxic cinnamoyl lipid, tripmycin A (1). Structure-activity relationship studies revealed that both the cinnamyl moiety and the saturated fatty acid side chain are indispensable to the over 400-fold cytotoxicity improvement of 1 against the triple-negative breast cancer cell line MDA-MB-231 compared to 5-(2-methylphenyl)-4-pentenoic acid (2). Bioinformatical analysis, gene inactivation, and overexpression revealed that Hxs15 most likely acted as an enoyl reductase and was involved with the side chain reduction of 1, which provides a new insight into the biosynthesis of cinnamoyl lipids.

Lipopeptide surfactin ameliorates the cell uptake of platensimycin and enhances its therapeutic effect on treatment of MRSA skin infection.

OBJECTIVES: The rapid development of drug-resistant bacteria, especially MRSA, poses severe threats to global public health. Adoption of antibiotic adjuvants has proved to be one of the efficient ways to solve such a crisis. Platensimycin and surfactin were comprehensively studied to combat prevalent MRSA skin infection. METHODS: MICs of platensimycin, surfactin or their combinations were determined by resazurin assay, while the corresponding MBCs were determined by chequerboard assay. Growth inhibition curves and biofilm inhibition were determined by OD measurements. Membrane permeability analysis was conducted by propidium iodide staining, and morphological characterizations were performed by scanning electron microscopy. Finally, the therapeutic effects on MRSA skin infections were evaluated in scald-model mice. RESULTS: The in vitro assays indicated that surfactin could significantly improve the antibacterial performance of platensimycin against MRSA, especially the bactericidal activity. Subsequent mechanistic studies revealed that surfactin not only interfered with the biofilm formation of MRSA, but also disturbed their cell membranes to enhance membrane permeability, and therefore synergistically ameliorated MRSA cellular uptake of platensimycin. Further in vivo assessment validated the synergistic effect of surfactin on platensimycin and the resultant enhancement of therapeutical efficacy in MRSA skin-infected mice. CONCLUSIONS: The combination of effective and biosafe surfactin and platensimycin could be a promising and efficient treatment for MRSA skin infection, which could provide a feasible solution to combat the major global health threats caused by MRSA.

Genome mining of Streptomyces sp. CB00271 as a natural high-producer of ß-rubromycin and the resulting discovery of ß-rubromycin acid.

ß-rubromycin (ß-RUB) (1) is an efficient inhibitor of human telomerase possessing a unique spiroketal moiety as a potential pharmacophore and regarded as a promising anticancer drug lead. But the development of (ß-RUB) (1) has long been hampered by its low titer and very poor water solubility. By adopting a genome mining strategy, an FAD-dependent monooxygenase RubN involving with the formation of the spiro system was applied as the probe and Streptomyces sp. CB00271 was screened out from our strain collection as an alternative natural high producer of ß-RUB (1). After a series of fermentation optimizations, CB00271 could produce 124.8 ± 3.4 mg/L ß-RUB (1), which was the highest titer up to now. Moreover, the enhanced production of ß-RUB (1) in fermentation broth also led to the discovery of a new congener ß-RUB acid (7), which was structurally elucidated as the acid form of ß-RUB (1). Comparing to ß-RUB (1), the substituted carboxyl group endowed ß-RUB acid (7) much better solubility in serum and resulted in its higher activity towards tumor cells. Our work set up a solid base for the pilot-scale production of ß-RUB (1) and its congeners to facilitate their future development as promising anticancer drug leads, and also provide an alternative and practical strategy for the exploitation of other important microbial natural products.

Deoxidized gulose moiety attenuates the pulmonary toxicity of 6'-deoxy-bleomycin Z without effect on its antitumor activity.

Bleomycins (BLMs) are broad-spectrum antitumor drugs, but the dose-dependent lung toxicity has restricted their therapeutic applications. Many efforts have contributed to develop novel BLM analogues, but mainly focused on single functional domain owing to the structural complexity of BLM. Benefit from the engineered production of two novel analogues 6'-deoxy-BLM Z (6'-DO-BLM Z) and BLM Z, they together with clinical BLM-sulfate comprised a good model with varied sugar or C-terminal domain in any two of them, allowing us to study their structure-activity relationships pairwise. Our investigations suggested the biological activities of BLM or its analogues are mainly depended on the C-terminal amine, while the changed C-terminal amine endowed BLM Z with much higher pulmonary toxicity comparing to BLM-sulfate, whereas the deoxidized gulose unit with same C-terminal amine evidently attenuated the pulmonary toxicity of 6'-DO-BLM Z without effect on antitumor activity. Further mechanistic studies revealed that the alleviation of pulmonary toxicity in 6'-DO-BLM Z by a slight change in the sugar moiety could attribute to the decrease of ROS production and thereby reduce the subsequent caspase-1 activity and resulting inflammatory response. Therefore, the synergistic modifications on C-terminal amine and sugar moiety provide new insights to efficiently develop potential BLM candidate with good clinical performance.

Surfactin Ameliorated the Internalization and Inhibitory Performances of Bleomycin Family Compounds in Tumor Cells.

The cationic glycopeptide bleomycin (BLM) is a broad-spectrum chemotherapy drug clinically applied to treat various malignant tumors. The poor cell membrane permeability of BLM, which is prone to high dose usage and may consequently induce dose-dependent lung toxicity, is a sticking point to limit clinical applications of BLM. As a commercial biosurfactant, the anionic lipopeptide surfactin (SF) is well known for its potent ability to disturb membranes and widely applied in cosmetic area as a permeabilization synergist. In this work, our in vitro investigations showed that SF could ameliorate the cell internalization of BLM, and the combined usage of SF notably improved the antitumor activity of BLM or its analogues while having no obvious effects on normal cells. Subsequent in vivo assessments on the subcutaneous treatment of A375 melanoma in mice demonstrated that SF could also enhance the therapeutic effects of BLM family compounds in subeffective doses, with no obvious toxicities on lungs and skin. Also, our preliminary results suggested the formation of complex micelles at the nanoscale by the self-assembly of BLM and SF, which may contribute to the ameliorated internalization and the antitumor effect of BLM. Therefore, SF could be applied as a potential synergist for BLM to reduce its treatment dose while maintaining the therapeutic effect on treatment of skin carcinoma, which provides us an alternative way to minimize the side effects of clinical BLM and facilitate the development of new BLM-type drugs.

The discovery and development of microbial bleomycin analogues.

The bleomycins (BLMs) belong to a subfamily of glycopeptide antibiotics and are clinically applied in combination chemotherapy regimens to treat various malignancies. But the therapeutic applications of BLMs are restricted by the accompanied dose-dependent lung toxicity and potential incidence of lung fibrosis. Many efforts have been devoted to develop novel BLM analogues, for seeking of drug leads with improved antitumor activity and/or reduced lung toxicity. The progresses in the biosynthetic studies of BLMs have greatly expedited the process to achieve such goals. This review highlights the discovery and development of microbial BLM analogues in the past two decades, especially those derived from engineered biosynthesis. Moreover, the summarized structure-activity relationship, which is specifically focusing on the sugar moiety, shall shed new insights into the prospective development of BLM analogues.

Strain improvement by combined UV mutagenesis and ribosome engineering and subsequent fermentation optimization for enhanced 6'-deoxy-bleomycin Z production.

The bleomycins (BLMs) are important clinical drugs extensively used in combination chemotherapy for the treatment of various cancers. Dose-dependent lung toxicity and the development of drug resistance have restricted their wide applications. 6'-Deoxy-BLM Z, a recently engineered BLM analogue with improved antitumor activity, has the potential to be developed into the next-generation BLM anticancer drug. However, its low titer in the recombinant strain Streptomyces flavoviridis SB9026 has hampered current efforts, which require sufficient compound, to pursue preclinical studies and subsequent clinical development. Here, we report the strain improvement by combined UV mutagenesis and ribosome engineering, as well as the fermentation optimization, for enhanced 6'-deoxy-BLM production. A high producer, named S. flavoviridis G-4F12, was successfully isolated, producing 6'-deoxy-BLM at above 70 mg/L under the optimized fermentation conditions, representing a sevenfold increase in comparison with that of the original producer. These findings demonstrated the effectiveness of combined empirical breeding methods in strain improvement and set the stage for sustainable production of 6'-deoxy-BLM via pilot-scale microbial fermentation.