TY - JOUR
T1 - Subcellular localization of aphidicolin biosynthetic enzymes heterologously expressed in Aspergillus oryzae
AU - Ban, Akihiko
AU - Tanaka, Mizuki
AU - Fujii, Ryuya
AU - Minami, Atsushi
AU - Oikawa, Hideaki
AU - Shintani, Takahiro
AU - Gomi, Katsuya
N1 - Funding Information:
This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas [JSPS KAKENHI grant number 22108007], from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Publisher Copyright:
© 2017 Japan Society for Bioscience, Biotechnology, and Agrochemistry.
PY - 2018
Y1 - 2018
N2 - The secondary metabolite aphidicolin has previously been produced by Aspergillus oryzae after the heterologous expression of four biosynthetic enzymes isolated from Phoma betae. In this study, we examined the subcellular localization of aphidicolin biosynthetic enzymes in A. oryzae. Fusion of green fluorescent protein to each enzyme showed that geranylgeranyl diphosphate synthase and terpene cyclase are localized to the cytoplasm and the two monooxygenases (PbP450-1 and PbP450-2) are localized to the endoplasmic reticulum (ER). Protease protection assays revealed that the catalytic domain of both PbP450s was cytoplasmic. Deletion of transmembrane domains from both PbP450s resulted in the loss of ER localization. Particularly, a PbP450-1 mutant lacking the transmembrane domain was localized to dot-like structures, but did not colocalize with any known organelle markers. Aphidicolin biosynthesis was nearly abrogated by deletion of the transmembrane domain from PbP450-1. These results suggest that ER localization of PbP450-1 is important for aphidicolin biosynthesis.
AB - The secondary metabolite aphidicolin has previously been produced by Aspergillus oryzae after the heterologous expression of four biosynthetic enzymes isolated from Phoma betae. In this study, we examined the subcellular localization of aphidicolin biosynthetic enzymes in A. oryzae. Fusion of green fluorescent protein to each enzyme showed that geranylgeranyl diphosphate synthase and terpene cyclase are localized to the cytoplasm and the two monooxygenases (PbP450-1 and PbP450-2) are localized to the endoplasmic reticulum (ER). Protease protection assays revealed that the catalytic domain of both PbP450s was cytoplasmic. Deletion of transmembrane domains from both PbP450s resulted in the loss of ER localization. Particularly, a PbP450-1 mutant lacking the transmembrane domain was localized to dot-like structures, but did not colocalize with any known organelle markers. Aphidicolin biosynthesis was nearly abrogated by deletion of the transmembrane domain from PbP450-1. These results suggest that ER localization of PbP450-1 is important for aphidicolin biosynthesis.
KW - Aphidicolin
KW - Aspergillus oryzae
KW - Protease protection assay
KW - Secondary metabolite biosynthetic enzyme
KW - Subcellular localization
UR - http://www.scopus.com/inward/record.url?scp=85040843021&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85040843021&partnerID=8YFLogxK
U2 - 10.1080/09168451.2017.1399789
DO - 10.1080/09168451.2017.1399789
M3 - Article
C2 - 29191129
AN - SCOPUS:85040843021
VL - 82
SP - 139
EP - 147
JO - Bioscience, Biotechnology and Biochemistry
JF - Bioscience, Biotechnology and Biochemistry
SN - 0916-8451
IS - 1
ER -