Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae.
In this study, we investigated the effects of proteinase gene disruption on heterologous protein production by Aspergillus oryzae. The human lysozyme (HLY) was selected for recombinant production as a model for the heterologous protein. A tandem HLY construct fused with alpha-amylase (AmyB) was expressed by A. oryzae in which the Kex2 cleavage site was inserted at the upstream of HLY. HLY was successfully processed from AmyB and produced in the medium. We performed a systematic disruption analysis of five proteinase genes (pepA, pepE, alpA, tppA, and palB) in the HLY-producing strain with the adeA selectable marker. Comparative analysis indicated that disruption of the tppA gene encoding a tripeptidyl peptidase resulted in the highest increase (36%) in the HLY production. We further deleted the tppA gene in the pepE or palB disruptant with another selectable marker, argB. Consequently, a double disruption of the tppA and pepE genes led to a 63% increase in the HLY production compared to the control strain. This is the first study to report that the double disruption of the tppA and pepE genes improved the production level of a heterologous protein by filamentous fungi.
Jin
Feng Jie
FJ
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Tokyo 113-8657, Japan.
Watanabe
Taisuke
T
Juvvadi
Praveen Rao
PR
Maruyama
Jun-ichi
J
Arioka
Manabu
M
Kitamoto
Katsuhiko
K
D000626:Aminopeptidases; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D009113:Muramidase / Q000096:biosynthesis*; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D011994:Recombinant Proteins; D009113:Muramidase; D010450:Endopeptidases; D010447:Peptide Hydrolases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases