Clemmensen S E, Kromphardt K J K, Frandsen R J N
Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.
Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark; University College Absalon, Kalundborg, Denmark(1).
Fungal Genet Biol. 2022 May;160:103689. doi: 10.1016/j.fgb.2022.103689. Epub 2022 Mar 23.
Filamentous fungi are prolific producers of secondary metabolites (SecMets), including compounds with antibiotic properties, like penicillin, that allows the producing fungus to combat competitors in a shared niche. However, the biological function of the majority of these small complex metabolites for the producing fungi remains unclear (Macheleidt et al., 2016). In an effort to address this lack of knowledge, we have chosen to study the microbial community of moldy apples in the hope of shedding more light on the role of SecMets for the dynamics of the microbial community. Penicillium expansum is one of the prevalent fungal species in this system, and in co-culture experiments with other apple fungal pathogens, we have observed up- and downregulation of several SecMets when compared to monocultures. However, molecular genetic dissection of the observed changes is challenging, and new methodologies for targeted genetic engineering in P. expansum are needed. In the current study, we have established a CRISPR-Cas9 dependent genetic engineering toolbox for the targeted genetic manipulation of P. expansum to allow for single-step construction of marker-free strains. The method and effect of different combinations of a Cas9-sgRNA expressing plasmids and repair template substrates in the NHEJ-proficient WT strain is tested by targeted deletion of melA, encoding a PKS responsible for pigment formation, which upon deletion resulted in white mutants. Co-transformation with a linear double-stranded DNA fragment consisting of two 2 kb homology arms flanking the PKS gene proved to be the most efficient strategy with 100% confirmed deletions by diagnostic PCR. Shorter homology arms (500-1000 bp) resulted in 20-30% deletion efficiency. Furthermore, we demonstrate the application of the CRISPR-Cas9 method for targeted deletion of biosynthetic genes without a visible phenotype, insertion of a visual reporter-encoding gene (mRFP), and overexpression of biosynthetic genes. Combined, these tools will advance in enabling the deciphering of SecMet biosynthetic pathways, provide in situ insight into when and where SecMets are produced, and provide an avenue to study the role of P. expansum SecMets in shaping the microbial community development on moldy apples via marker-free targeted genetic engineering of P. expansum.
丝状真菌是次生代谢产物（SecMets）的丰富生产者，包括具有抗生素特性的化合物，如青霉素，这使得产生真菌能够在共享生态位中对抗竞争者。然而，这些小的复杂代谢产物对产生真菌的大多数生物学功能仍不清楚（Macheleidt等人，2016年）。为了解决这一知识空白，我们选择研究发霉苹果的微生物群落，希望能更清楚地了解SecMets在微生物群落动态中的作用。扩展青霉是该系统中普遍存在的真菌物种之一，在与其他苹果真菌病原体的共培养实验中，与单培养相比，我们观察到几种SecMets的上调和下调。然而，对观察到的变化进行分子遗传剖析具有挑战性，因此需要用于扩展青霉靶向基因工程的新方法。在本研究中，我们建立了一个依赖CRISPR-Cas9的基因工程工具箱，用于扩展青霉的靶向基因操作，以实现无标记菌株的一步构建。通过靶向缺失编码负责色素形成的聚酮合酶的melA，测试了Cas9-sgRNA表达质粒和修复模板底物的不同组合在NHEJ能力强的野生型菌株中的方法和效果，缺失后产生白色突变体。用由PKS基因两侧两个2kb同源臂组成的线性双链DNA片段进行共转化被证明是最有效的策略，通过诊断PCR确认缺失率为100%。较短的同源臂（500-1000bp）导致20-30%的缺失效率。此外，我们展示了CRISPR-Cas9方法在无可见表型的生物合成基因靶向缺失、视觉报告基因（mRFP）的插入以及生物合成基因的过表达中的应用。综合起来，这些工具将有助于破译SecMet生物合成途径，原位了解SecMets的产生时间和地点，并通过扩展青霉的无标记靶向基因工程为研究扩展青霉SecMets在塑造发霉苹果上的微生物群落发育中的作用提供一条途径。
