title: Identification and functional analysis of the geranylgeranyl pyrophosphate synthase gene (crtE) and phytoene synthase gene (crtB) for carotenoid biosynthesis in Euglena gracilis.
abstract: BACKGROUND
  Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains β-carotene and xanthophylls (such as zeaxanthin, diatoxanthin, diadinoxanthin and 9'-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated.
  
  
  RESULTS
  To clarify the carotenoid biosynthetic pathway in E. gracilis, we searched for the putative E. gracilis geranylgeranyl pyrophosphate (GGPP) synthase gene (crtE) and phytoene synthase gene (crtB) by tblastn searches from RNA-seq data and obtained their cDNAs. Complementation experiments in Escherichia coli with carotenoid biosynthetic genes of Pantoea ananatis showed that E. gracilis crtE (EgcrtE) and EgcrtB cDNAs encode GGPP synthase and phytoene synthase, respectively. Phylogenetic analyses indicated that the predicted proteins of EgcrtE and EgcrtB belong to a clade distinct from a group of GGPP synthase and phytoene synthase proteins, respectively, of algae and higher plants. In addition, we investigated the effects of light stress on the expression of crtE and crtB in E. gracilis. Continuous illumination at 460 or 920 μmol m(-2) s(-1) at 25 °C decreased the E. gracilis cell concentration by 28-40 % and 13-91 %, respectively, relative to the control light intensity (55 μmol m(-2) s(-1)). When grown under continuous light at 920 μmol m(-2) s(-1), the algal cells turned reddish-orange and showed a 1.3-fold increase in the crtB expression. In contrast, EgcrtE expression was not significantly affected by the light-stress treatments examined.
  
  
  CONCLUSIONS
  We identified genes encoding CrtE and CrtB in E. gracilis and found that their protein products catalyze the early steps of carotenoid biosynthesis. Further, we found that the response of the carotenoid biosynthetic pathway to light stress in E. gracilis is controlled, at least in part, by the level of crtB transcription. This is the first functional analysis of crtE and crtB in Euglena.
authors.affiliation: Department of Biosciences, School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan. shota.kato.680@gmail.com.|Plant Molecular and Cellular Biology Laboratory, Department of Biosciences, School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan. shota.kato.680@gmail.com.
authors.forename: Shota
authors.initials: S
authors.lastname: Kato
authors.affiliation: Department of Biology, Nippon Medical School, 1-7-1 Kyonan-cho, Musashino, Tokyo, 180-0023, Japan. takaichi@nms.ac.jp.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Takaichi
authors.affiliation: Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan. ishikawa@life.shimane-u.ac.jp.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ishikawa
authors.affiliation: Department of Biosciences, School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan. asahina@nasu.bio.teikyo-u.ac.jp.
authors.forename: Masashi
authors.initials: M
authors.lastname: Asahina
authors.affiliation: Department of Biosciences, School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan. takahasi@nasu.bio.teikyo-u.ac.jp.
authors.forename: Senji
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Department of Biosciences, School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan. shinomura@nasu.bio.teikyo-u.ac.jp.|Plant Molecular and Cellular Biology Laboratory, Department of Biosciences, School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan. shinomura@nasu.bio.teikyo-u.ac.jp.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Shinomura
mesh_terms: D000595:Amino Acid Sequence; D002338:Carotenoids / Q000096:biosynthesis*; D003001:Cloning, Molecular; D005056:Euglena gracilis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D017343:Genes, Plant; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase / Q000235:genetics*; D008027:Light; D008969:Molecular Sequence Data; D016415:Sequence Alignment
chemical_list: D002338:Carotenoids; C100185:(all-E) phytoene; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase