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Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins

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Abstract

Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double (tppA, and pepE) and quintuple (tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes (alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae.

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Acknowledgments

We thank Toshiko Tanaka for experimental help. 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.

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Authors and Affiliations

  1. Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

    Jaewoo Yoon, Jun-ichi Maruyama & Katsuhiko Kitamoto

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  1. Jaewoo Yoon
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  2. Jun-ichi Maruyama
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  3. Katsuhiko Kitamoto
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Corresponding author

Correspondence to Katsuhiko Kitamoto.

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Fig. S1

a Southern blot analysis of the pepA disruptant. The open boxes (1.4 kb each) are the flanking regions for disruption of the pepA gene. The 0.3 kb upstream flanking region of the pepA ORF (hatched box) was attached at 5′-end of the downstream flanking regions, introducing direct repeats. The genomic DNAs were digested with BamHI and SacI. The strain analyzed in the panels exhibited the expected band pattern for disruption of the pepA gene. “P” and “Δ” represents the parental strain (NSPlD-tApEnBdIVdVaA1) and the gene disruptant (NSlD-tApEnBdIVdVaApA1), respectively. b Southern blot analysis of the pyrG-excised strains. The genomic DNAs were digested with BamHI and SacI. The strain analyzed in the panels exhibited the expected band pattern for excision of the pyrG gene from the pepA locus. “P” and “pyrG-” represents the parental strain (NSlD-tApEnBdIVdVaApA1) and the pyrG-excised (NSPlD-tApEnBdIVdVaApA1), respectively. c Southern blot analysis of the AopepAa disruptant. The open boxes (1.5 and 1.4 kb) are the flanking regions for disruption of the AopepAa gene. The 0.4 kb upstream flanking region of the AopepAa ORF (hatched box) was attached at 5′-end of the downstream flanking regions, introducing direct repeats. The genomic DNAs were digested with ScaI and PvuI. The strain analyzed in the panels exhibited the expected band pattern for disruption of the AopepAa gene. “P” and “Δ” represents the parental strain (NSPlD-tApEnBdIVdVaApA1) and the gene disruptant (NSlD-tApEnBdIVdVaApApAa1), respectively. d Southern blot analysis of the pyrG-excised strains. The genomic DNAs were digested with ScaI and PvuI. The strain analyzed in the panels exhibited the expected band pattern for excision of the pyrG gene from the AopepAa locus. “P” and “pyrG-” represents the parental strain (NSlD-tApEnBdIVdVaApApAa1) and the pyrG-excised (NSPlD-tApEnBdIVdVaApApAa1), respectively. e Southern blot analysis of the AopepAd disruptant. The open boxes (1.5 kb each) are the flanking regions for disruption of the AopepAd gene. The 0.3 kb upstream flanking region of the AopepAd ORF (hatched box) was attached at 5′-end of the downstream flanking regions, introducing direct repeats. The genomic DNAs were digested with EcoT22I and SacI. The strain analyzed in the panels exhibited the expected band pattern for disruption of the AopepAd gene. “P” and “Δ” represents the parental strain (NSPlD-tApEnBdIVdVaApApAa1) and the gene disruptant (NSlD-tApEnBdIVdVaApApAapAd1), respectively. f Southern blot analysis of the pyrG-excised strains. The genomic DNAs were digested with EcoT22I and SacI. The strain analyzed in the panels exhibited the expected band pattern for excision of the pyrG gene from the AopepAd locus. “P” and “pyrG-” represents the parental strain (NSlD-tApEnBdIVdVaApApAapAd1) and the pyrG-excised (NSPlD-tApEnBdIVdVaApApAapAd1), respectively. g Southern blot analysis of the cpI disruptant. The open boxes (1.5 kb each) are the flanking regions for disruption of the cpI gene. The 0.3 kb upstream flanking region of the cpI ORF (hatched box) was attached at 5′-end of the downstream flanking regions, introducing direct repeats. The genomic DNAs were digested with NdeI and ScaI. The strain analyzed in the panels exhibited the expected band pattern for disruption of the cpI gene. “P” and “Δ” represents the parental strain (NSPlD-tApEnBdIVdVaApApAapAd1) and the gene (NSlD-tApEnBdIVdVaApApAapAdcI1), respectively (PDF 222 kb)

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Yoon, J., Maruyama, Ji. & Kitamoto, K. Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins. Appl Microbiol Biotechnol 89, 747–759 (2011). https://doi.org/10.1007/s00253-010-2937-0

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