Improvement of heterologous protein production in Aspergillus oryzae by RNA interference with alpha-amylase genes.
Aspergillus oryzae RIB40 has three alpha-amylase genes (amyA, amyB, and amyC), and secretes alpha-amylase abundantly. However, large amounts of endogenous secretory proteins such as alpha-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of alpha-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three alpha-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of alpha-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in alpha-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of alpha-amylase is effective in heterologous protein production in A. oryzae.
Nemoto
Takashi
T
Department of Biotechnology, The University of Tokyo, Tokyo, Japan.
Maruyama
Jun-ichi
J
Kitamoto
Katsuhiko
K
D000818:Animals; D001236:Aspergillus oryzae / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D012085:Chymosin / Q000096:biosynthesis; D055029:Inverted Repeat Sequences; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D034622:RNA Interference; D012331:RNA, Fungal / Q000235:genetics; D000516:alpha-Amylases / Q000172:deficiency* / Q000235:genetics*
D012331:RNA, Fungal; D000516:alpha-Amylases; D012085:Chymosin