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Construction of quintuple protease gene disruptant for heterologous protein production in Aspergillus oryzae

Abstract

Aspergillus oryzae has received attention as a host for heterologous protein production. However, A. oryzae has 134 protease genes, which is recognized to be one of the major reasons for the proteolytic degradation of heterologously produced proteins. We previously reported that double disruption of the protease genes (tppA and pepE) improved heterologous protein (human lysozyme) production by A. oryzae. In this study, we performed successive round of five protease genes (tppA, pepE, nptB, dppIV, and dppV) disruption in A. oryzae by pyrG marker recycling with highly efficient gene-targeting background (ΔligD). The multiple disruption of protease genes were confirmed by Southern blot analysis. Furthermore, the quintuple protease gene disruptants showed the maximum production level of bovine chymosin (CHY) that was 34% higher than those of the double protease gene disruptant (ΔtppA ΔpepE). Consequently, we successfully constructed a multiple protease gene disruptant bearing enhanced levels of CHY productivity. We presented the first evidence that the quintuple disruption of the protease genes improved the production level of a heterologous protein by A. oryzae.

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Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of Japan.

Author information

Correspondence to Katsuhiko Kitamoto.

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Supplementary Fig. 1

DNA microarray analysis of A. oryzae protease genes in 5 × DPY (pH 8.0). A dendrogram based on their expression patterns was generated in which the signal intensities were per-gene-normalized. Blue rectangles represent down-regulation and red rectangles represent up-regulation as compared to the median value (yellow). The protease genes with low trust (low signal intensities) are colored dark or black. The ID of proteases with signal peptides was indicated in the parentheses. The genes registered with the name were described. The image was adopted from GeneSpring GX 7.3. (PPT 96.3 KB)

Supplementary Fig. 2

Western blot analysis of the culture supernatant of the CHY-expressing strains. The control (ΔtppA ΔpepE) (Δ2) and the NSlD-tApEnBdIVdV (ΔtppA ΔpepE ΔnptB ΔdppIV ΔdppV) strain 2-3 (Δ5) expressing CHY were cultivated in 20 ml 5 × DPY (pH 5.5) medium at 30°C for 3–6 days. Culture supernatants (4 μl) were subjected to Western blot analysis. Bands of size approximately 35.4 and 40.3 kDa were detected using the anti-CHY antibody. “Chymosin” refers to authentic bovine chymosin (approximately 100 ng; Sigma). (PPT 1.08 MB)

Supplementary Fig. 3

Relative extracellular protease activity of the bovine chymosin (CHY) produced by the multiple protease gene disruptants. The control (ΔtppA ΔpepE) and the NSlD-tApEnBdIVdV (ΔtppA ΔpepE ΔnptB ΔdppIV ΔdppV) strain 2-3 expressing CHY were cultivated in 20 ml 5 × DPY (pH 5.5) medium at 30°C for 4 or 6 days. The total extracellular protease activity assay was conducted according to a modification of the method described by Idiris et al. (2006b). (PPT 12.3 KB)

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Yoon, J., Kimura, S., Maruyama, J. et al. Construction of quintuple protease gene disruptant for heterologous protein production in Aspergillus oryzae . Appl Microbiol Biotechnol 82, 691–701 (2009). https://doi.org/10.1007/s00253-008-1815-5

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Keyword

  • Aspergillus oryzae
  • Quintuple protease gene disruption
  • Heterologous protein production
  • DNA microarray analysis
  • Bovine chymosin