An improved method for culturing Streptomyces sahachiroi: Biosynthetic origin of the enol fragment of azinomycin B
- Additional Document Info
- View All
Azinomycin B is an environmental DNA crosslinking agent produced by the soil microorganism Streptomyces sahachiroi. While the agent displays potent cytotoxic activities against leukemic cell lines and animal mouse models, the lack of a consistent supply of the natural product has hampered detailed biological investigations on the compound, including its mode of action and biosynthesis. We report here a significant methodological improvement in the culturing of the bacterium, which allows reliable and steady production of the natural product in good yields. The key experimental step involves the culturing of the strain on dehydrated plates, followed by the generation of a two-stage starter culture and subsequent fermentation of the strain under nutrient-starved conditions. We illustrate use of this culture system by investigating the formation of the enol fragment of the molecule in isotopic labeling experiments with threonine and several advanced precursors (beta-ketoamino acid 3, beta-hydroxyamino aldehyde 4, and beta-ketoaminoaldehyde 5). The results unequivocally show that threonine is the most advanced precursor accepted by the NRPS (non-ribosomal peptidyl synthetase) machinery for final processing and construction of the enol moiety of the natural product.
author list (cited authors)
Kelly, G. T., Sharma, V., & Watanabe, C.