CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Fusarium fujikuroi and Its Application in Strain Engineering for Gibberellic Acid Production
CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Fusarium fujikuroi and Its Application in Strain Engineering for Gibberellic Acid Production
复制标题
基于CRISPR/Cas9的丝状真菌藤黑镰刀菌基因组编辑及其在赤霉酸生产菌株工程中的应用
DOI：
10.1021/acssynbio.8b00478
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发表时间：
2019-02-01
影响因子：
4.7
通讯作者：
Ji, Xiao-Jun
中科院分区：
文献类型：
--
作者：
Shi, Tian-Qiong;Gao, Jian;Ji, Xiao-Jun
丝状真菌藤井镰刀菌以其产生天然植物生长激素--一系列赤霉酸(GAS)而闻名。GA_1、GA_3、GA_4、GA_7等具有生物活性，已广泛应用于农业生产。然而，传统基因工具的低效率限制了进一步的研究，使这种真菌更有效，并能够产生定制的气体。在这里，我们建立了一个高效的基于CRISPR/Cas9的Fujikuroi基因组编辑工具。首先，我们比较了三种不同的核定位信号(NLS)，并从组蛋白H2B中选择了一个有效的NLS(HTBNLS)来使Cas9蛋白能够进入真菌的细胞核。然后，探索了不同的sgRNA体外表达策略和不同的基于启动子的体内表达策略。Fujikuroi中鉴定的U6小核RNA和SS rRNA启动子的编辑效率最高。SS rRNA启动子驱动的基因组编辑效率高达79.2%。此外，还对多基因编辑进行了探索，并取得了良好的效果。最后，我们使用开发的基因组编辑工具对负责在丝状真菌F.fujikuroi中积累一系列气体的代谢途径进行了工程设计，并成功地将其GA产物从GA3改变为定制的GA4和GA7混合物。由于这些混合物在农业上的使用效率更高，特别是在水果生长方面，所开发的菌株将极大地提高工业GA的生产。
The filamentous fungus Fusarium fujikuroi is well-known for its production of natural plant growth hormones: a series of gibberellic acids (GAs). Some GAs, including GA1, GA3, GA4, and GA7, are biologically active and have been widely applied in agriculture. However, the low efficiency of traditional genetic tools limits the further research toward making this fungus more efficient and able to produce tailor-made GAs. Here, we established an efficient CRISPR/Cas9-based genome editing tool for F. fujikuroi. First, we compared three different nuclear localization signals (NLS) and selected an efficient NLS from histone H2B (HTBNLS) to enable the import of the Cas9 protein into the fungal nucleus. Then, different sgRNA expression strategies, both in vitro and different promoter-based in vivo strategies, were explored. The promoters of the U6 small nuclear RNA and SS rRNA, which were identified in F. fujikuroi, had the highest editing efficiency. The SS rRNA-promoter-driven genome editing efficiency reached up to 79.2%. What's more, multigene editing was also explored and showed good results. Finally, we used the developed genome editing tool to engineer the metabolic pathways responsible for the accumulation of a series GAs in the filamentous fungus F. fujikuroi, and successfully changed its GA product profile, from GA3 to tailor-made GA4 and GA7 mixtures. Since these mixtures are more efficient for agricultural use, especially for fruit growth, the developed strains will greatly improve industrial GA production.