Optimization of CRISPR/Cas9-based multi-gene editing system in Aspergillus oryzae and its application in α-amyrin biosynthesis.

Aspergillus oryzae, with its high protein secretion capacity, post-translational modification capabilities, and safety, is a promising host for producing natural products and recombinant proteins. However, the lack of efficient genetic tools and precise genome modification methods has significantly slowed progress in the metabolic engineering of A. oryzae. Although the CRISPR/Cas9-mediated genome editing system has been applied in A. oryzae, the low efficiency of gene disruption and heterologous gene integration limits its widespread application as a chassis cell for industrial strain development. In this study, the CRISPR/Cas9-mediated genome editing system in A. oryzae RIB40 was optimized to significantly improve its editing efficiency. By evaluating the effect of promoters on sgRNA expression, it was determined that the Ao (Up338)5SrRNA promoter effectively enhances the gene disruption efficiency. Utilizing the Ao (Up338)5SrRNA promoter, a multiplex gene editing system based on the tRNAGly-sgRNA array was developed, achieving dual-gene disruption efficiency of 78.84% and triple-gene disruption efficiency of 41.15%. Additionally, when the ratio of the genome-editing plasmid to the circular donor DNA was 1:3, the site-specific integration efficiency of the exogenous gene reached 61.36%. Based on the developed CRISPR/Cas9 genome editing system, the heterologous α-amyrin synthetic gene (CADDS) was precisely integrated into the wA locus to achieving the biosynthesis of α-amyrin in Aspergillus oryzae. Furthermore, acetyl-CoA supply was improved by knocking out the competing ethanol metabolic pathway, resulting in an engineered strain that produced 0.69 mg/g of α-amyrin.