CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus

Abstract

Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (106 protoplasts/mL) to produce 60–80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains. Key points • A protocol of high-quality protoplasts for CGES has been developed in M. pilosus. • The GRF of CGES was about five times that of ATMT in M. pilosus. • The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.

Synthetic biology for Monascus: From strain breeding to industrial production

“…Although CRISPR gene editing technology has been used less frequently in Monascus, gene editing studies in Monascus is expected to progress rapidly given its efficient editing ability in other Aspergillus and the optimization of the Aspergillus transformation system. [104] These emerging technologies have been applied in some filamentous…”

“…RT-qPCR test confirmed the successful constructions of site-specific gene editing strains, the gene replacement frequencies were 27.4‰ for mpclr4, 37.5‰ for mpdot1, and 33.3‰ for mplig4. [104] Ganoderma lucidum PEG-mediated protoplast transformation Gene disruption of ura3 by codon-optimized Cas9 and in vitro transcribed gRNAs.…”