Liu Huan, Wang Gang, Li Wei, Liu Xingzhong, Li Erwei, Yin Wen-Bing
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, PR China.
School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, PR China.
Microbiology (Reading). 2018 May;164(5):769-778. doi: 10.1099/mic.0.000649.
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 Δ 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 and which play critical roles in non-homologous end-joining recombination were disrupted. The resulting mutant showed remarkable transformation rates of 89 %, while no change was found in the deletion mutant compared with the WT strain. This suggests that play a key role in the non-homologous recombination in this strain. Using this system, the biosynthetic gene cluster of trichothecene () harzianum B was identified by deletion of the in . Comparative genome analysis revealed that the trichothecene biosynthetic gene cluster in shared similar organizations with and , 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.
是一种产生丰富次生代谢产物的真菌寄生物种。然而，目前尚无用于进一步研究的遗传转化方法。在此，我们基于原生质体转化通过尿苷/尿嘧啶生物合成基因的互补开发了一种无标记转化系统。通过使用5'-氟乳清酸作为选择试剂的阳性筛选方案获得了Δ的尿苷/尿嘧啶营养缺陷型突变体。为了提高同源整合率，破坏了在非同源末端连接重组中起关键作用的和的直系同源物。所得突变体显示出89%的显著转化率，而与野生型菌株相比，缺失突变体未发现变化。这表明在该菌株的非同源重组中起关键作用。利用该系统，通过缺失中的鉴定了哈茨木霉B的生物合成基因簇。比较基因组分析表明，中的单端孢霉烯生物合成基因簇与和具有相似的组织，尽管它们编码的产物在结构上有所不同。综上所述，高效的遗传系统为研究生物合成多样性和从真菌中挖掘新型天然产物提供了便利工具。
