Construction of single, double, or triple mutants within kojic acid synthesis genes kojA, kojR, and kojT by the CRISPR/Cas9 tool in Aspergillus oryzae.

Kojic acid is an industrially important secondary metabolite produced by Aspergillus oryzae. The construction of genetic materials for kojic acid related genes is important for understanding the mechanism of kojic acid synthesis in A. oryzae. However, multigene simultaneous knockout mutants for kojic acid synthesis genes remain limited because A. oryzae is multinuclear and good selectable markers are scarce. Here, we firstly successfully obtained single mutants of kojA, kojR, and kojT by our previously constructed CRISPR/Cas9 system in A. oryzae, which demonstrated the feasibility of the targeting sgRNAs for kojA, kojR, and kojT. Then, the AMA1-based genome-editing system for multiplex gene editing was developed in A. oryzae. In the multiplex gene-editing system, two guide RNA expression cassettes were ligated in tandem and driven by two U6 promoters in the AMA1-based autonomously replicating plasmid with the Cas9-expression cassette. Moreover, the multiplex gene-editing technique was applied to target the kojic acid synthesis genes kojA, kojR, and kojT, and the double and triple mutants within kojA, kojR, and kojT were obtained successfully. Additionally, the selectable marker pyrG was knocked out in the single and triple mutants of kojA, kojR, and kojT to obtain the auxotrophic strains, which can facilitate to introduce a target gene into the single and triple mutants of kojA, kojR, and kojT for investigating their relationship. The multiplex gene-editing system and release of these materials provide a foundation for further kojic acid research and utilization.