Li_2025_Synth.Syst.Biotechnol_10_365

The Aspergillus oryzae expression system has been developed into a chassis for the production of heterologous lipases, attributed to its strong capabilities in protein production and secretion, robust post-translational modifications, and favourable safety profile. However, the system's relatively low expression levels remain a challenge, hindering its ability to meet the increasing demands of large-scale production. Strain C19, screened by high-throughput methods combining droplet microfluidics and flow cytometry, was demonstrated to be a potential chassis cell based on fermentation kinetic analysis and transcriptome sequencing. By leveraging the endogenous alpha-amylase's expression elements and integration sites, a combination of random and site-directed integration strategies was employed to enhance the expression of heterologous lipases in strain C19. As a result, lipase production in shake-flask fermentation reached a titer of 113.6 U/L. The study further demonstrated that the different alpha-amylase gene loci could serve as effective integration sites for the multi-copy expression of heterologous proteins because the lipase activity of the 3-amylase site integrated strain C19#1-ABC was 3.3 times higher than that of C19#1. Furthermore, fermentation results in a 5-L bioreactor indicated that optimization of fermentation processes and facilities had the potential to further increase heterologous protein expression levels. These findings offered valuable insights into the advancement of A. oryzae expression systems and the potential for scaling engineered strains for industrial applications.