Heterologous Expression of Candida antarctica Lipase B in Aspergillus niger Using CRISPR/Cas9-mediated Multi-Gene Editing.

Aspergillus niger, a filamentous fungus, is known as a cell factory due to its ability to produce large amounts of organic acids and industrial enzymes. Lipase B from Candida antarctica (CALB) is one of the most widely used lipases in industrial applications, including oil processing, papermaking, food, pharmaceuticals, and personal care products. In this study, the CRISPR/Cas9 technique was employed to knock out the pyrG and kusA genes in A. niger. The CALB gene was integrated into the high-production protein gene loci, such as glaA and amyA, to construct a multi-copy CALB production engineered strain. Additionally, the pepA, aglU, and bglA genes were deleted, which minimized the background level of secreted proteins in A. niger and increased the production of CALB. After two rounds of gene editing, the A. niger with multi-copy CALB was created, and the engineered A. niger CCTCC 206047.09 with high CALB yield was isolated. After 120 h of liquid fermentation, the lipase activity reached 17.84 U/mL and the protein yield reached 10.21 mg/mL. In summary, an engineered A. niger strain with high lipase activity was successfully isolated by employing a CRISPR/Cas9 system to integrate CALB into high-expression loci, while simultaneously knocking out the host's highly expressed protein genes. These results provide an effective strategy for the high expression of both heterologous and homologous enzymes in A. niger.