Construction of Quintuple Protease and Double Amylase Gene Deletant for Heterologous Protein Production in <i>Aspergillus oryzae</i> KBN616

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.

Synthetic Biology Tools for Engineering Aspergillus oryzae

“…To solve this problem, Noriyuki et al performed RNA interference (RNAi) on the taa gene of A. oryzae KBN616 by knocking out five proteases (alp, npII, pepE, npI, and pepA). The results showed that the protease activity of A. oryzae itself was significantly reduced, and the amylase activity completely disappeared [84].…”

Heterologous protein production in filamentous fungi

“…Deletion of protease-encoding genes is one of the methods to establish protease-deficient strains. In A. oryzae , several protease-encoding genes were disrupted to improve the production of CHY and HLY (Jin et al 2007 ; Maruyama and Kitamoto 2008 ; Yoon et al 2011 ; Kitamoto et al 2015 ). In T. reesei , the production levels of human interferon alpha-2b were increased upon deletion of the subtilisin protease gene slp7 and the metalloprotease gene amp2 , with yields of 2.1 g/l and 2.4 g/l, respectively (Landowski et al 2016 ).…”