Subcellular localization of aphidicolin biosynthetic enzymes heterologously expressed in Aspergillus oryzae

Akihiko Ban, Mizuki Tanaka, Ryuya Fujii, Atsushi Minami, Hideaki Oikawa, Takahiro Shintani, Katsuya Gomi

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)139-147
Number of pages9
JournalBioscience, Biotechnology and Biochemistry
Volume82
Issue number1
DOIs
Publication statusPublished - 2018

Keywords

  • Aphidicolin
  • Aspergillus oryzae
  • Protease protection assay
  • Secondary metabolite biosynthetic enzyme
  • Subcellular localization

ASJC Scopus subject areas

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

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