期刊
MICROBIOLOGY-SGM
卷 164, 期 5, 页码 769-778出版社
MICROBIOLOGY SOC
DOI: 10.1099/mic.0.000649
关键词
Trichoderma hypoxylon; PEG-mediated transformation; uridine/uracil auxotrophy; non-homologous end joining (NHEJ)
类别
资金
- National Key Research and Development Program [2016YFD0400105]
- National Natural Science Foundation of China [21778075]
- National Postdoctoral Program for Innovative Talents [BX201600185]
Trichoderma hypoxylon is a fungicolous species which produces rich secondary metabolites. However, no genetic transformation method is available for further studies. Here, we developed a marker-less transformation system based on the complementation of an uridine/uracil biosynthetic gene by protoplast transformation. An uridine/uracil auxotrophic mutant of Delta thpyr4 was obtained by using a positive screening protocol with 5'-fluoroorotic acid as a selective reagent. To improve the homologous integration rates, the orthologues of ku70 and lig4 which play critical roles in non-homologous end-joining recombination were disrupted. The resulting thlig4 mutant showed remarkable transformation rates of 89 %, while no change was found in the thku70 deletion mutant compared with the WT strain. This suggests that thlig4 play a key role in the non-homologous recombination in this strain. Using this system, the biosynthetic gene cluster of trichothecene (tri) harzianum B was identified by deletion of the thtri5 in T. hypoxylon. Comparative genome analysis revealed that the trichothecene biosynthetic gene cluster in T. hypoxylon shared similar organizations with T. arundinaceum and T. brevicompactum, even though their encoded products are different in structures. Taken together, the highly efficient genetic system provides a convenient tool for studying the biosynthetic diversity and mining the novel natural product from the fungi.
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