科研之友
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摘要
Filamentous fungi are emerging as attractive producers of natural products with novel structures, diverse bioactivities and unprecedented enzymology. But their genetic systems are poorly developed, especially in some non-model endogenic fungi, which have hampered our genetic manipulation of their natural product development. Calcarisporium arbuscula NRRL 3705 is an endophytic filamentous fungus rich in biosynthetic gene clusters and primarily producing mycotoxin aurovertins. Here we optimized Agrobacterium tumefaciens-mediated transformation (ATMT)-based efficient DNA introduction into C. arbuscula. By complementation of the mono-oxygenate gene aurC in Delta aurC mutant as a model, we showed that a strong but down-regulated promoter aurAp and three strong constitutive promoter gpdAp, tef1p and tubCp could be used for gene overexpression. Meanwhile, red fluorescence protein (RFP) was expressed in this fungus under the control of tubCp, potentially paving the way for enzyme localization determination during natural product biosynthesis. Furthermore, we developed efficient and convenient gene disruption in C. arbuscula based on ATMT, as exemplified by deletion of aurA in Delta aurC mutant. Our efficiency of deletion ran at about 40%. These results suggest that ATMT-based transformation for gene ectopic expression or deletion is an efficient strategy for genetic manipulation of C. arbuscula, and can be readily adapted to other rare filamentous fungi, potentially to promote discovery and development of natural products.
- 出版日期2018-8
- 单位浙江大学
