College of Food Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
Hubei International Scientific and Technological Cooperation Base of Traditional Fermented Foods, Huazhong Agricultural University, Wuhan, 430070, China.
Appl Microbiol Biotechnol. 2024 Jan 19;108(1):154. doi: 10.1007/s00253-023-12865-x.
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 (10 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.
长毛红曲菌长期以来一直被用于生产富含莫纳可林 K（MK）的降脂药物。基因组挖掘表明，这种真菌中仍有许多有潜力的基因值得探索。因此，高效的遗传操作工具将极大地加速这一进程。在本研究中，我们首先开发了用于 CRISPR/Cas9 系统受体的原生质体制备方案。随后，将载体和供体 DNA 共转化到受体（10 个原生质体/mL）中，每个测试产生 60-80 个转化体。选择三个与 DNA 损伤反应（DDR）相关的基因（mpclr4、mpdot1 和 mplig4），比较农杆菌介导转化（ATMT）和 CRISPR/Cas9 基因编辑系统（CGES）在长毛红曲菌 MS-1 中的基因替换频率（GRF）。结果表明，CGES 的 GRF 大约是 ATMT 的五倍，这表明 CGES 作为长毛红曲菌 MS-1 的靶向基因编辑工具优于 ATMT。mpclr4 的失活通过非同源末端连接（NHEJ）促进 DDR，并提高了对 DNA 损伤剂的耐受性。mpdot1 的失活阻断了 DDR，导致对 DNA 损伤剂的耐受性降低。mplig4 的失活主要阻断了 NHEJ 途径，导致对 DNA 损伤剂的耐受性明显降低。液体发酵表明，与野生型相比，Δmpclr4 菌株生产 MK 的能力提高了 52.6%。本研究为更有效地探索红曲菌基因功能提供了思路。关键点：• 已在长毛红曲菌中开发出用于 CGES 的高质量原生质体制备方案。• CGES 在长毛红曲菌中的 GRF 约为 ATMT 的五倍。• 与野生型相比，Δmpclr4 的 MK 产量提高了 52.6%。
