Abstract
We constructed a quintuple protease (alp, npII, pepE, npI, pepA) and double amylase (taaG3, taaG1) gene deletant, KO4, for the development of a heterologous gene expression system for an industrial shoyu koji mold, Aspergillus oryzae KBN616. Multiple gene deletion was performed using the Latour system, a simple and effective chromosome modification method developed in Schizosaccharomyces pombe. Gene deletion was confirmed by Southern blot analysis for protease genes and PCR amplification for amylase genes. Deletion of the five protease genes reduced the extracellular protease activity to approximately 1.0% of the level of the parent strain. Double deletion of the amylase genes completely eliminated all detectable α-amylase activity. These results indicate the construction of a host strain applicable to the efficient production and purification of heterologous proteins.
