An efficient Agrobacterium tumefaciens-mediated transformation method for Simplicillium subtropicum (Hypocreales: Cordycipitaceae)

Abstract Filamentous fungi are the organisms of choice for most industrial biotechnology. Some species can produce a variety of secondary metabolites and enzymes of commercial interest, and the production of valuable molecules has been enhanced through different molecular tools. Methods for genetic manipulation and transformation have been essential for the optimization of these organisms. The genus Simplicillium has attracted increased attention given several potential biotechnological applications. The Simplicillium genus harbors several entomopathogenic species and some isolates have been explored for bioremediation of heavy metal contaminants. Furthermore, the myriad of secondary metabolites isolated from Simplicillium spp. render these organisms as ideal targets for deep exploration and further biotechnological mining possibilities. However, the lack of molecular tools hampered the exploration of this genus. Thus, an Agrobacterium tumefaciens-mediated transformation method was established for Simplicillium subtropicum, employing the far-red fluorescent protein TURBOFP635/Katushka, as a visual marker, and the selection marker SUR gene, that confers resistance to chlorimuron ethyl. Notably, one round of transformation using the established method yielded almost 400 chlorimuron resistant isolates. Furthermore, these transformants displayed mitotic stability for, at least, five generations. We anticipate that this method can be useful for deep molecular exploration and improvement of strains in the Simplicillium genus.摘要：丝状真菌（Filamentous fungi）是多数工业生物技术领域首选的模式生物。部分菌种可产生多种具备商业价值的次级代谢产物（secondary metabolites）与酶类，通过各类分子工具已实现高价值产物的生产强化。遗传操作与转化方法对这类宿主的优化至关重要。简囊孢属（Simplicillium）因具备多项潜在生物技术应用价值而受到越来越多的关注。该属包含多种昆虫病原物种（entomopathogenic species），部分菌株已被应用于重金属污染物（heavy metal contaminants）的生物修复（bioremediation）。此外，从简囊孢属菌株中分离得到的大量次级代谢产物，使其成为深度探索与进一步生物技术开发挖掘的理想靶点。然而，目前缺乏可用的分子工具限制了该属的研究进展。为此，本研究建立了适用于亚热带简囊孢（Simplicillium subtropicum）的根癌农杆菌介导的转化（Agrobacterium tumefaciens-mediated transformation）方法，以远红色荧光蛋白TURBOFP635/Katushka（far-red fluorescent protein）作为可视化标记，并以赋予氯嘧磺乙酯（chlorimuron ethyl）抗性的SUR基因（SUR gene）作为筛选标记（selection marker）。值得注意的是，采用本方法仅一轮转化即可获得近400株氯嘧磺抗性转化子。进一步验证显示，这些转化子至少可稳定传代5代且保持有丝分裂稳定性（mitotic stability）。我们预计该方法将为简囊孢属的深度分子研究与菌株优化提供有力支撑。