ArticleApplied microbiology and biotechnology2023
An efficient method for targeted cloning of large DNA fragments from Streptomyces.
Article in Applied microbiology and biotechnology, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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3 citing papers in PubMed.
- Review
- A highly efficient heterologous expression platform to facilitate the production of microbial natural products in Streptomyces.Microbial cell factories · 2025Article
- Identification of a novel butenolide signal system to regulate high production of tylosin in Streptomyces fradiae.Applied microbiology and biotechnology · 2025Article
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6 authors.
Funding
Abstract
Cloning of large DNA fragments from microorganisms becomes increasingly important but remains seriously challenging due to the complexity and diversity of genetic background. In particular, the methods with high precision and efficiency are in great need for obtaining intact biosynthetic gene clusters (BGCs) of microbial natural products. Here, we report a new strategy for targeted cloning of large DNA fragments (TCLD) from different bacteria. Using this method, precise cloning of desired E. coli chromosomal fragments up to 201 kb was achieved with 53% positive rate. Moreover, its application in cloning of large BGCs with high G + C content and multiple repetitive sequences was also demonstrated, including the 98 kb tylosin BGC (tyl), 128 kb daptomycin BGC (dpt), and 127 kb salinomycin BGC (sal). Subsequently, heterologous expression of the cloned tyl BGC in Streptomyces coelicolor M1146 led to the production of tylosins in the resulting recombinant strains. And also, its introduction into Streptomyces fradiae ATCC 19609, a native producer of tylosin, effectively increased tylosin yield to 230%. Hence, TCLD is a powerful tool for cloning large BGCs and would facilitate the discovery of bioactive substances from microbial resources. KEY POINTS: • TCLD is an efficient method for cloning large DNA fragments. • Repeat sequence-mediated intra-molecular cyclization improves the cloning efficiency. • TCLD combined with scarless editing allows unlimited modifications on BGCs.
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