State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China.
Key Laboratory of Environment Correlative Dietology, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China.
ACS Synth Biol. 2020 Aug 21;9(8):2087-2095. doi: 10.1021/acssynbio.0c00178. Epub 2020 Jul 16.
Mycotoxin contamination causes disease and death in both humans and animals. Monascus Red, produced by , is used as a food colorant. However, its application is limited by contamination of the nephrotoxin citrinin, which is also produced by the fungus. Suppressing citrinin production by genetic engineering is difficult in a polykaryotic fungus such as Hence, we developed a CRISPR/Cas system to delete large genomic fragments in polykaryotic fungi. Protoplast preparation and regeneration were optimized, and a dual-plasmid CRISPR/Cas system was designed to enable the deletion of the 15-kb citrinin biosynthetic gene cluster in industrial strain KL-001. The obtained homokaryotic mutants were stable, and citrinin was unambiguously eliminated. Moreover, the Monascus Red pigment production was increased by 2-5%. Our approach provides a powerful solution to solve this long-standing problem in the food industry and enables engineering of polykaryotic fungi for mycotoxin eliminations.
真菌毒素污染会导致人类和动物患病和死亡。红曲霉菌所产生的红曲素有作为食用色素的用途。然而，其应用受到真菌毒素桔青霉素的污染所限制，桔青霉素也是由这种真菌产生的。在多细胞真菌如红曲霉菌中，通过基因工程抑制桔青霉素的产生是困难的。因此，我们开发了一种 CRISPR/Cas 系统来删除多细胞真菌中的大片段基因组。优化了原生质体制备和再生条件，并设计了一种双质粒 CRISPR/Cas 系统，以实现对工业菌株 KL-001 中 15kb 桔青霉素生物合成基因簇的删除。获得的同核突变体是稳定的，并且桔青霉素被明确消除。此外，红曲红色素的产量增加了 2-5%。我们的方法为解决食品工业中的这一长期存在的问题提供了一个有力的解决方案，并为多细胞真菌的真菌毒素消除工程提供了可能性。
