image: Remarkable enhancement of bleomycin production through precise amplification of its biosynthetic gene cluster in Streptomyces verticillus. view more
Credit: ©Science China Press
Bleomycins (BLMs), a family of glycopeptides produced by Streptomyces verticillus ATCC15003, have been used clinically for the treatment of numerous malignant tumors. Due to its complicated structure, the production of BLM mainly relies on the microbial fermentation and its production is really low. Amplification of the biosynthetic gene cluster (BGC) makes great contribution to the overproduction of secondary metabolites in microorganisms. Since most of the BGCs for the important commercial and clinic secondary metabolites are larger than 40 kb (e.g. the 70-kb bleomycin BGC), application of the ZouA system in amplifying the larger-scale BGCs needs to be investigated. What is more, the correlation between BGC copies and the production of secondary metabolites has not been reported so far. Thus, a method for precisely regulating the BGCs (especially for large-scale BGCs) copies and enhancing the production of natural products is urgently needed in microorganisms.
“We first use the combined strategy of a ZouA-dependent DNA amplification system and double-reporters-guided recombinant selection in Streptomyces verticillus ATCC15003, and remarkably enhanced the bleomycin production through precise amplification of its biosynthetic gene cluster.” said Mr. Li Hong, the first author for this work. The results of this research demonstrate the ability and effectiveness of the combined approach consisting of the ZouA system and DRGRS in precisely regulating the tandem amplification of the large-scale cluster in Streptomyces.
“In our study, the introduction of double-reporters-guided recombinant selection (DRGRS) in screening the recombinants not only facilitates the screening process but also significantly reduces the false positive rate (27.5%) which could be caused by high concentration of apramycin”, said Li. Application of DRGRS facilitates to the screen of the recombinant strains which contain multiple copies of the target BGCs. The recombinant strains with different copies (1-6 copies) of the blm cluster were obtained from 705 colonies through preliminary screening, second-time screening and final determination. These results indicate that high-throughput DRGRS can be used for acquiring the recombinants with high-copy number of the blm cluster and reduce false positive rate of the recombinants obtained by the ZouA system.“Using the combined strategy of ZouA system and DRGRS, the bleomycin production in the recombinant containing 6 copies of the bleomycin gene cluster was increased 9.59-fold compared to the wild-type strain. Our study indicates that precise regulation of BGC amplification is a valuable and generally applicable tool for improving the production of secondary metabolites in microorganisms”, said Professor Gang Liu, the corresponding author.
These encouraging results confirm utility of the combined strategy of a ZouA-dependent DNA amplification system and double-reporters-guided recombinant selection on the production enhancement of secondary metabolites (such as antibiotics) in Streptomyces.
This work was supported by grants from the National Key Research and Development Program of China (2021YFC2100600), the National Natural Science Foundation of China (30970072 and 31170088), and Biological Resources Programme, Chinese Academy of Sciences (KFJBRP-009).
See the article:
Li, H., Gao, W., Cui, Y, et al. (2021). Remarkable enhancement of bleomycin production through precise amplification of its biosynthetic gene cluster in Streptomyces verticillus. Sci China Life Sci, in press, https://engine.scichina.com/doi/10.1007/s11427-021-1998-8
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Science China Life Sciences