title: Chloroplast-targeting protein expression in the oleaginous diatom Fistulifera solaris JPCC DA0580 toward metabolic engineering.
abstract: The chloroplast plays critical roles in lipid metabolism of microalgae, thus it is recognized as an attractive target of metabolic engineering to enhance biofuel production. It has been well known that recombinant protein expression in microalgal chloroplasts needs specific signal sequence which governs the transition manner of nuclear-encoded polypeptides within the subcellular compartments. However certain microalgae, including diatoms, have complex membrane systems surrounding the chloroplast, and thus chloroplast-targeting protein expression with the signal sequence has rarely been demonstrated except for a few model non-oleaginous diatoms. In this study, we performed recombinant green fluorescence protein (GFP) expression and transportation into the chloroplast of the oleaginous marine diatom, Fistulifera solaris JPCC DA0580. The signal sequence of ATP synthetase gamma subunit, which was predicted to localize in the chloroplast according to a bioinformatics analysis pipeline, was employed as a key factor of this technique. As a result, specific localization of GFP in the chloroplast was observed. It would be useful to engineer the lipid synthesis pathways existing in the chloroplast. Furthermore, intensive gathering of GFP in the rod-like structure was also detected, which has not been observed in model diatom studies. As comparing with electron microscopic observation, the structure was estimated to be a pyrenoid.
authors.affiliation: Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan; Japan Science and Technology Agency (JST), Core Research for Evolutionary Science and Technology (CREST), 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Sunaga
authors.affiliation: Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Maeda
authors.affiliation: Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yabuuchi
authors.affiliation: Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan; Japan Science and Technology Agency (JST), Core Research for Evolutionary Science and Technology (CREST), 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Muto
authors.affiliation: Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Yoshino
authors.affiliation: Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan; Japan Science and Technology Agency (JST), Core Research for Evolutionary Science and Technology (CREST), 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan. Electronic address: tsuyo@cc.tuat.ac.jp.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Tanaka
mesh_terms: D025181:ATP Synthetase Complexes / Q000235:genetics / Q000378:metabolism; D056804:Biofuels / Q000600:supply & distribution; D002736:Chloroplasts / Q000378:metabolism*; D017377:Diatoms / Q000166:cytology* / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D058086:Microalgae / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport
chemical_list: D056804:Biofuels; D021382:Protein Sorting Signals; D049452:Green Fluorescent Proteins; D025181:ATP Synthetase Complexes
keywords: Chloroplast; Fistulifera solaris JPCC DA0580; Marine oleaginous diatom; Pyrenoid; Signal sequence