High-throughput droplet microfluidics screening and genome sequencing analysis for improved amylase-producing Aspergillus oryzae

Background The exceptional protein secretion capacity, intricate post-translational modification processes, and inherent safety features of A. oryzae make it a promising expression system. However, heterologous protein expression levels of existing A. oryzae species cannot meet the requirement for industrial-scale production. Therefore, establishing an efficient screening technology is significant for the development of the A. oryzae expression system. Results In this work, a high-throughput screening method suitable for A. oryzae has been established by combining the microfluidic system and flow cytometry. Its screening efficiency can reach 350 droplets per minute. The diameter of the microdroplet was enlarged to 290 µm to adapt to the polar growth of A. oryzae hyphae. Through enrichment and screening from approximately 450,000 droplets within 2 weeks, a high-producing strain with α-amylase increased by 6.6 times was successfully obtained. Furthermore, 29 mutated genes were identified by genome resequencing of high-yield strains, with 15 genes subjected to editing and validation. Two genes may individually influence α-amylase expression in A. oryzae by affecting membrane-associated multicellular processes and regulating the transcription of related genes. Conclusions The developed high-throughput screening strategy provides a reference for other filamentous fungi and Streptomyces . Besides, the strains with different excellent characteristics obtained by efficient screening can also provide materials for the analysis of genetic and regulatory mechanisms in the A. oryzae expression system.