Abstract
The genetic manipulation of basidiomycete mushrooms is notoriously difficult and immature, and there is a lack of research reports on clustered regularly interspaced short palindromic repeat (CRISPR) based gene editing of functional genes in mushrooms. In this work, Ganoderma lucidum, a famous traditional medicinal basidiomycete mushroom, which produces a type of unique triterpenoid-anti-tumor ganoderic acids (GAs), was used, and a CRISPR/CRISPR-associated protein-9 nuclease (Cas9) editing system for functional genes of GA biosynthesis was constructed in the mushroom. As proof of concept, the effect of different gRNA constructs with endogenous u6 promoter and self-cleaving ribozyme HDV on ura3 disruption efficiency was investigated at first. The established system was applied to edit a cytochrome P450 monooxygenase (CYP450) gene cyp5150l8, which is responsible for a three-step biotransformation of lanosterol at C-26 to ganoderic acid 3-hydroxy-lanosta-8, 24-dien-26 oic acid. As a result, precisely edited cyp5150l8 disruptants were obtained after sequencing confirmation. The fermentation products of the wild type (WT) and cyp5150l8 disruptant were analyzed, and a significant decrease in the titer of four identified GAs was found in the mutant compared to WT. Another CYP gene involved in the biosynthesis of squalene-type triterpenoid 2, 3; 22, 23-squalene dioxide, cyp505d13, was also disrupted using the established CRISPR-Cas9 based gene editing platform of G. lucidum. The work will be helpful to strain molecular breeding and biotechnological applications of G. lucidum and other basidiomycete mushrooms.
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Acknowledgments
We thank Ms. Han Li for her assistance in earlier stage of fermentation experiments and Mr. Wen-Fang Wang for his discussion on HPLC data and GA biosynthetic pathway.
Funding
This work was supported by National Key Research and Development Program of China (No. 2018YFA0901904), National Natural Science Foundation of China (nos. 31770037 and 31971344), and Shanghai Municipal Natural Science Foundation (nos. 17ZR1448900 and 18ZR1420300).
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JJZ, PAW, and HX are the co-inventors of a functional gene editing technology in Lingzhi, which is covered under a Chinese patent (Application No. 201811522445.0) with a priority date Dec. 13, 2018.
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Wang, PA., Xiao, H. & Zhong, JJ. CRISPR-Cas9 assisted functional gene editing in the mushroom Ganoderma lucidum. Appl Microbiol Biotechnol 104, 1661–1671 (2020). https://doi.org/10.1007/s00253-019-10298-z
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DOI: https://doi.org/10.1007/s00253-019-10298-z