A Dual-Plasmid CRISPR/Cas System for Mycotoxin Elimination in Polykaryotic Industrial Fungi
Weiwei Liu, Chunyan An, Xian Shu, Xiangxi Meng, Yongpeng Yao, Jun Zhang, Fusheng Chen, Hua Xiang, Shuyuan Yang, Xue Gao, Shu‐Shan Gao
Abstract
Mycotoxin contamination causes disease and death in both humans and animals. Monascus Red, produced by Monascus purpureus, 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 M. purpureus. 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 M. purpureus 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.