Abstract
BackgroundBleomycin is a broad-spectrum glycopeptide antitumor antibiotic produced by Streptomyces verticillus. Clinically, the mixture of bleomycin A2 and bleomycin B2 is widely used in combination with other drugs for the treatment of various cancers. As a secondary metabolite, the biosynthesis of bleomycin is precisely controlled by the complex extra-/intracellular regulation mechanisms, it is imperative to investigate the global metabolic and regulatory system involved in bleomycin biosynthesis for increasing bleomycin production.ResultsN-acetylglucosamine (GlcNAc), the vital signaling molecule controlling the onset of development and antibiotic synthesis in Streptomyces, was found to increase the yields of bleomycins significantly in chemically defined medium. To mine the gene information relevant to GlcNAc metabolism, the DNA sequences of dasR-dasA-dasBCD-nagB and nagKA in S. verticillus were determined by chromosome walking. From the results of Real time fluorescence quantitative PCR (RT-qPCR) and electrophoretic mobility shift assays (EMSAs), the repression of the expression of nagB and nagKA by the global regulator DasR was released under induction with GlcNAc. The relief of blmT expression repression by BlmR was the main reason for increased bleomycin production. DasR, however, could not directly affect the expression of the pathway-specific repressor BlmR in the bleomycins gene cluster. With at the beginning of bleomycin synthesis, the supply of the specific precursor GDP-mannose played the key role in bleomycin production. Genetic engineering of the GDP-mannose synthesis pathway indicated that phosphomannose isomerase (ManA) and phosphomannomutase (ManB) were key enzymes for bleomycins synthesis. Here, the blmT, manA and manB co-expression strain OBlmT/ManAB was constructed. Based on GlcNAc regulation and assisted metabolic profiling analysis, the yields of bleomycin A2 and B2 were ultimately increased to 61.79 and 36.9 mg/L, respectively.ConclusionsUnder GlcNAc induction, the elevated production of bleomycins was mainly associated with the alleviation of the inhibition of BlmT, so blmT and specific precursor synthesis pathways were genetically engineered for bleomycins production improvement. Combination with subsequent metabolomics analysis not only effectively increased the bleomycin yield, but also extended the utilization of chitin-derived substrates in microbial-based antibiotic production.
Highlights
Bleomycin is a member of glycopeptide antibiotics that was first isolated from the fermentation broth of S. verticillus by Umezawa et al [1]
Bleomycin fermentation characteristics and morphological changes in S. verticillus mediated by GlcNAc The yields of bleomycins A2 and B2 produced by S. verticillus American type culture collection (ATCC) 15003 in complex fermentation medium reached 22.18 ± 3.29 mg/L and 38.26 ± 2.36 mg/L, respectively, while none were detected in modified ISP4 medium, even with the addition of 1 g/L tryptone and 0.5 g/L yeast extract
To find the key factors affecting the synthesis of bleomycins, 5 g/L of glucose, mannose, fructose, arabinose, lactose, galactose, chitin, GlcNAc and d-Glucosamine (GlcN) were individually added to the fermentation medium as supplementary carbon sources
Summary
Bleomycin is a member of glycopeptide antibiotics that was first isolated from the fermentation broth of S. verticillus by Umezawa et al [1]. Multiple regulatory networks are employed by Streptomyces to make a decision whether to continue vegetative growth or form spores. Once it enters the spores development, a series of genes are transcribed, which activate the synthesis of antibiotics and morphological differentiation [7, 8]. The regulation of the GntR/HutC family repressor DasR has been found to be essential for the development and morphogenesis process, which controls the expression of genes involved in chitin/GlcNAc metabolism, the development-related phosphoglucosyltransferase system (PTS), stress response and most the known antibiotic biosynthesis pathways [10, 12,13,14,15]. The biosynthesis of bleomycin is precisely controlled by the complex extra-/intracellular regulation mechanisms, it is imperative to investigate the global metabolic and regulatory system involved in bleomycin biosynthesis for increasing bleomycin production
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