Engineering Aspergillus oryzae for enhanced amylase production via multi-copy integration and microfluidic screening
摘要
Aspergillus oryzae is an industrial filamentous fungus possessing high-efficiency protein expression and secretion capacity, widely used in enzyme preparation, food fermentation, and other fields. The industrial production strain A. oryzae F1 was selected for this study. Although already used for amylase production, its yield still fails to meet industrial demand. To improve the production of α-amylase, targeted engineering of the strain was performed using CRISPR/Cas9-mediated gene editing combined with high-throughput screening strategies. To enhance the site-specific integration efficiency of exogenous genes in A. oryzae F1, the gene lig4 encoding DNA ligase was knocked out, which increased the efficiency of homology-directed repair (HDR) from 13.4% to 34.7%. On this basis, α-amylase activity was improved by 15.2% through multi-copy gene integration. Furthermore, an excellent mutant strain was obtained by combining ARTP mutagenesis and droplet microfluidic high-throughput screening. In a 50 L fermentation system, the α-amylase activity of this mutant reached 4,052 U g−1, representing a 37.4% increase compared with the parental strain F1. These results not only provide a feasible technical strategy for constructing high-efficiency amylase-producing strains, but also offer technical references for the engineering of filamentous fungal chassis cells.