CRISPR-Cas9 assisted in-situ complementation of functional genes in the basidiomycete Ganoderma lucidum

Abstract

Basidiomycetes can produce various valuable secondary metabolites with bioactivities. However, the low titer of metabolites has greatly limited their application. Currently, shortage of mutant complementation techniques, especially in-situ complementation, impeded the elucidation of their metabolites biosynthesis pathways. In this work, Ganoderma lucidum, a traditional Chinese medicinal basidiomycete, which can produce various bioactive ganoderic acids (triterpenoids), was used; and a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) mediated in-situ complementation system for two functional genes was established. For a disruptant of gl26097, which encodes a Cys2-His2 (C2H2)-type zinc finger transcription factor involved in calmodulin-calcineurin signaling, one in-situ complemented strain of 8 transformants was obtained,concluded by sequencing confirmation. For the function of gl26097, both the cell growth and content of four identified individual ganoderic acids (GAs) were restored in the complemented strain compared to the gl26097 disruptant. For a disruptant of cyp5150l8, which is responsible for transforming lanosterol to a GA, one in-situ complemented strain from 8 transformants was obtained as confirmed by sequencing. The titer of four identified GAs increased significantly in the complemented strain compared to the cyp5150l8 disruptant. To the best of our knowledge, this is the first research report on applying CRISPR-Cas9 to complement mutants in-situ in basidiomycetes. The CRISPR-Cas9 assisted gene complementation technology will be helpful to study gene functions and GA biosynthesis pathway in G. lucidum, which may be also applied to other basidiomycetes.