Chen Yingying, Cai Cunlei, Yang Jiafan, Shi Junjie, Song Yongxiang, Hu Dan, Ma Junying, Ju Jianhua
CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China.
Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), No. 1119, Haibin Road, Nansha District, Guangzhou 511458, China.
J Fungi (Basel). 2022 Jul 8;8(7):715. doi: 10.3390/jof8070715.
Marine-derived fungi are emerging as attractive producers of structurally novel secondary metabolites with diverse bioactivities. However, the lack of efficient genetic tools limits the discovery of novel compounds and the elucidation of biosynthesis mechanisms. Here, we firstly established an effective PEG-mediated chemical transformation system for protoplasts in two marine-derived fungi, sp. SCSIO F190 and sp. SCSIO SX7S7. Next, we developed a simple and versatile CRISPR-Cas9-based gene disruption strategy by transforming a target fungus with a single plasmid. We found that the transformation with a circular plasmid encoding , a single-guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted and insertional gene mutations in both marine-derived fungal strains. In addition, the histone deacetylase gene was mutated using the established CRISPR-Cas9 system, thereby activating novel secondary metabolites that were not produced in the wild-type strain. Taken together, a versatile CRISPR-Cas9-based gene disruption method was established, which will promote the discovery of novel natural products and further biological studies.
海洋来源的真菌正逐渐成为具有多种生物活性的结构新颖的次生代谢产物的有吸引力的生产者。然而，缺乏有效的遗传工具限制了新化合物的发现和生物合成机制的阐明。在此，我们首先为两种海洋来源的真菌，即 sp. SCSIO F190 和 sp. SCSIO SX7S7 的原生质体建立了一种有效的聚乙二醇（PEG）介导的化学转化系统。接下来，我们通过用单个质粒转化目标真菌，开发了一种简单且通用的基于CRISPR-Cas9的基因破坏策略。我们发现，用编码 Cas9、单向导RNA（sgRNA）和选择标记的环状质粒进行转化，在两种海洋来源的真菌菌株中都导致了高频的靶向和插入基因突变。此外，利用已建立的CRISPR-Cas9系统对组蛋白脱乙酰酶基因进行了突变，从而激活了野生型菌株中未产生的新型次生代谢产物。综上所述，建立了一种通用的基于CRISPR-Cas9的基因破坏方法，这将促进新型天然产物的发现和进一步的生物学研究。
