Characterization of Aspergillus oryzae mutant and its application in heterologous lipase expression

The <i>Aspergillus oryzae</i> 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 α-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 α-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 <i>A. oryzae</i> expression systems and the potential for scaling engineered strains for industrial applications.