Gakuubi Martin Muthee, Ching Kuan Chieh, Munusamy Madhaiyan, Wibowo Mario, Lim Chun Teck, Ma Guang-Lei, Liang Zhao-Xun, Kanagasundaram Yoganathan, Ng Siew Bee
Singapore Institute of Food and Biotechnology Innovation (SIFBI), Agency for Science, Technology and Research (ASTAR), Singapore, Singapore.
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
Front Microbiol. 2022 Sep 29;13:1012115. doi: 10.3389/fmicb.2022.1012115. eCollection 2022.
is a genus of ascomycetous fungi within the family . Members of this genus have been isolated as endophytes from a wide range of host plants and also from plant debris within terrestrial and marine habitats, where they are thought to function as saprobes. sp. F6932 was isolated from white mangrove () in Pulau Ubin Island, Singapore. Crude extracts from the fungus exhibited strong antibacterial activity, and bioassay-guided isolation and structure elucidation of bioactive constituents led to the isolation of palmarumycin C and a new analog palmarumycin CP. Whole-genome sequencing analysis resulted in the identification of a putative type 1 iterative PKS (iPKS) predicated to be involved in the biosynthesis of palmarumycins. To verify the involvement of palmarumycin (PAL) gene cluster in the biosynthesis of these compounds, we employed ribonucleoprotein (RNP)mediated CRISPR-Cas9 to induce targeted deletion of the ketosynthase (KS) domain in PAL. Double-strand breaks (DSBs) upstream and downstream of the KS domain was followed by homology-directed repair (HDR) with a hygromycin resistance cassette flanked by a 50 bp of homology on both sides of the DSBs. The resultant deletion mutants displayed completely different phenotypes compared to the wild-type strain, as they had different colony morphology and were no longer able to produce palmarumycins or melanin. This study, therefore, confirms the involvement of PAL in the biosynthesis of palmarumycins, and paves the way for implementing a similar approach in the characterization of other gene clusters of interest in this largely understudied fungal strain.
是该科内的一个子囊菌属。该属成员已从多种宿主植物以及陆地和海洋栖息地的植物残骸中作为内生菌分离出来，在这些地方它们被认为起着腐生菌的作用。 种F6932是从新加坡乌敏岛的白骨壤（ ）中分离出来的。该真菌的粗提物表现出很强的抗菌活性，通过生物测定指导的生物活性成分分离和结构解析，得到了棕榈霉素C和一种新的类似物棕榈霉素CP。全基因组测序分析鉴定出一个推定的1型迭代聚酮合酶（iPKS），推测其参与棕榈霉素的生物合成。为了验证棕榈霉素（PAL）基因簇在这些化合物生物合成中的作用，我们采用核糖核蛋白（RNP）介导的CRISPR-Cas9诱导PAL中酮合酶（KS）结构域的靶向缺失。在KS结构域的上游和下游产生双链断裂（DSB），随后用潮霉素抗性盒进行同源定向修复（HDR），该抗性盒在DSB两侧各有50 bp的同源序列。与野生型菌株相比，所得缺失突变体表现出完全不同的表型，因为它们具有不同的菌落形态，并且不再能够产生棕榈霉素或黑色素。因此，这项研究证实了PAL参与棕榈霉素的生物合成，并为在这种研究较少的真菌菌株中对其他感兴趣的基因簇进行表征时采用类似方法铺平了道路。
