Complementary DNA cloning and functional analysis of lycopene β-cyclase in the brown alga Undaria pinnatifida

  • Akira InoueEmail author
  • Toshiyuki Iwayama
  • Takao Ojima
Original Article Chemistry and Biochemistry


To date, there is no functional evidence of the enzymes catalyzing carotenogenesis in brown algae. In this study, we have investigated the activity of brown algal lycopene β-cyclase that converts lycopene to β-carotene. A homology search for a lycopene β-cyclase candidate was performed after de novo transcriptome analysis of Undaria pinnatifida. The complementary DNA of the candidate, encoding 625 residues, termed Undaria pinnatifida lycopene β-cyclase-like protein (UpLCYB), was cloned. UpLCYB has a longer N-terminal region consisting of 76 residues than the 11 lycopene β-cyclase candidates of other brown algae. The N-terminal region consisting of 89 residues of the UpLCYB was predicted to be the chloroplast transition signal peptide. To assay the activity of UpLCYB, lycopene-producing Escherichia coli was prepared by introducing three genes encoding geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase from Flavobacterium sp. strain UMI-01. Recombinant UpLCYB (residues 90–625) was then expressed in this strain. The cells showed a yellow color, in contrast with cells without the UpLCYB gene, which exhibited a red color. Analysis of the extracted carotenoids from UpLCYB-expressing cells indicated that β-carotene was the main carotenoid, but only lycopene was detected in cells that did not express UpLCYB. These results suggest that UpLCYB plays a catalytic role in the conversion of lycopene to β-carotene.


Carotenoid β-carotene Flavobacterium Carotenogenesis De novo transcriptome analysis N-terminus Signal peptide 



This work was supported by Japan Society for the Promotion of Science KAKENHI grant no. 16H04977. We would like to express our gratitude to Dr. Tomohiro Hirose (Open Facility Division, Global Facility Center, Creative Research Institution, Hokkaido University) for performing the APCI-MS on an Exactive Mass Spectrometer and providing insight and expertise that greatly assisted our research.

Author contributions

A. I. conceived the experiment and wrote the manuscript. A. I. and T. I. conducted the experiments. A. I. and T. O. prepared the brown algal cDNA.


  1. Botella-Pavía P, Besumbes Ó, Phillips MA et al (2004) Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors. Plant J 40:188–199. CrossRefPubMedGoogle Scholar
  2. Cao T-J, Huang X-Q, Qu Y-Y et al (2017) Cloning and functional characterization of a lycopene β-cyclase from macrophytic red alga Bangia fuscopurpurea. Mar Drugs 15:116. CrossRefGoogle Scholar
  3. Cock JM, Sterck L, Rouzé P et al (2010) The Ectocarpus genome and the independent evolution of multicellularity in brown algae. Nature 465:617–621. CrossRefPubMedGoogle Scholar
  4. Cunningham FX Jr, Sun Z, Chamovitz D et al (1994) Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp. strain PCC7942. Plant Cell 6:1107. CrossRefPubMedGoogle Scholar
  5. Cunningham FX, Pogson B, Sun Z et al (1996) Functional analysis of the β and ε lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation. Plant Cell 8:1613–1626. PubMedGoogle Scholar
  6. Cunningham FX, Lee H, Gantt E (2007) Carotenoid biosynthesis in the primitive red alga Cyanidioschyzon merolae. Eukaryot Cell 6:533–545. CrossRefPubMedGoogle Scholar
  7. Dambek M, Eilers U, Breitenbach J et al (2012) Biosynthesis of fucoxanthin and diadinoxanthin and function of initial pathway genes in Phaeodactylum tricornutum. J Exp Bot 63:5607–5612. CrossRefPubMedGoogle Scholar
  8. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016. CrossRefPubMedGoogle Scholar
  9. Epstein G, Smale DA (2017) Undaria pinnatifida: a case study to highlight challenges in marine invasion ecology and management. Ecol Evol 7:8624–8642. CrossRefPubMedGoogle Scholar
  10. Grabherr MG, Haas BJ, Yassour M et al (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652. CrossRefPubMedGoogle Scholar
  11. Haas BJ, Papanicolaou A, Yassour M et al (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8:1494–1512. CrossRefPubMedGoogle Scholar
  12. Inoue A, Takadono K, Nishiyama R et al (2014) Characterization of an alginate lyase, FlAlyA, from Flavobacterium sp. strain UMI-01 and its expression in Escherichia coli. Mar Drugs 12:4693–4712. CrossRefPubMedGoogle Scholar
  13. Inoue A, Nishiyama R, Mochizuki S, Ojima T (2015) Identification of a 4-deoxy-l-erythro-5-hexoseulose uronic acid reductase, FlRed, in an alginolytic bacterium Flavobacterium sp. strain UMI-01. Mar Drugs 13:493–508. CrossRefPubMedGoogle Scholar
  14. Inoue A (2018) Characterization of PL-7 family alginate lyases from marine organisms and their applications. Methods Enzymol 605:499–524. CrossRefPubMedGoogle Scholar
  15. Konotchick T, Dupont CL, Valas RE et al (2013) Transcriptomic analysis of metabolic function in the giant kelp, Macrocystis pyrifera, across depth and season. New Phytol 198:398–407. CrossRefPubMedGoogle Scholar
  16. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. CrossRefPubMedGoogle Scholar
  17. Larkin MA, Blackshields G, Brown NP et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948. CrossRefPubMedGoogle Scholar
  18. Maeda H (2015) Nutraceutical effects of fucoxanthin for obesity and diabetes therapy: a review. J Oleo Sci 64:125–132. CrossRefPubMedGoogle Scholar
  19. Mata-Gómez L, Montañez J, Méndez-Zavala A, Aguilar C (2014) Biotechnological production of carotenoids by yeasts: an overview. Microb Cell Fact 13:12. CrossRefPubMedGoogle Scholar
  20. Mikami K, Hosokawa M (2013) Biosynthetic pathway and health benefits of fucoxanthin, an algae-specific xanthophyll in brown seaweeds. Int J Mol Sci 14:13763–13781. CrossRefPubMedGoogle Scholar
  21. Misawa N, Nakagawa M, Kobayashi K et al (1990) Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J Bacteriol 172:6704–6712. CrossRefPubMedGoogle Scholar
  22. Misawa N, Shimada H (1998) Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts. J Biotechnol 59:169–181. CrossRefGoogle Scholar
  23. Miyashita K, Nishikawa S, Beppu F et al (2011) The allenic carotenoid fucoxanthin, a novel marine nutraceutical from brown seaweeds. J Sci Food Agric 91:1166–1174. CrossRefPubMedGoogle Scholar
  24. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6. CrossRefPubMedGoogle Scholar
  25. Nishitsuji K, Arimoto A, Iwai K et al (2016) A draft genome of the brown alga, Cladosiphon okamuranus, S-strain: a platform for future studies of “mozuku” biology. DNA Res 23:561–570. CrossRefPubMedGoogle Scholar
  26. Peng J, Yuan J-P, Wu C-F, Wang J-H (2011) Fucoxanthin, a marine carotenoid present in brown seaweeds and diatoms: metabolism and bioactivities relevant to human health. Mar Drugs 9:1806–1828. CrossRefPubMedGoogle Scholar
  27. Ramos A, Coesel S, Marques A et al (2008) Isolation and characterization of a stress-inducible Dunaliella salina Lcy-β gene encoding a functional lycopene β-cyclase. Appl Microbiol Biotechnol 79:819–828. CrossRefPubMedGoogle Scholar
  28. Sandmann G (2002) Molecular evolution of carotenoid biosynthesis from bacteria to plants. Physiol Plant 116:431–440. CrossRefGoogle Scholar
  29. Shan TF, Pang SJ, Li J, Li X (2014) De novo transcriptome analysis of the gametophyte of Undaria pinnatifida (Phaeophyceae). J Appl Phycol 27:1011–1019. CrossRefGoogle Scholar
  30. Steinbrenner J, Linden H (2003) Light induction of carotenoid biosynthesis genes in the green alga Haematococcus pluvialis: regulation by photosynthetic redox control. Plant Mol Biol 52:343–356. CrossRefPubMedGoogle Scholar
  31. Stickforth P, Steiger S, Hess WR, Sandmann G (2003) A novel type of lycopene ε-cyclase in the marine cyanobacterium Prochlorococcus marinus MED4. Arch Microbiol 179:409–415. CrossRefPubMedGoogle Scholar
  32. Takaichi S (2011) Carotenoids in algae: distributions, biosyntheses and functions. Mar Drugs 9:1101–1118. CrossRefPubMedGoogle Scholar
  33. Wang G, Sun J, Liu G et al (2014) Comparative analysis on transcriptome sequencings of six Sargassum species in China. Acta Oceanol Sin 33:37–44. CrossRefGoogle Scholar
  34. Ye N, Zhang X, Miao M et al (2015) Saccharina genomes provide novel insight into kelp biology. Nat Commun 6:6986. CrossRefPubMedGoogle Scholar

Copyright information

© Japanese Society of Fisheries Science 2019

Authors and Affiliations

  1. 1.Laboratory of Marine Biotechnology and Microbiology, Division of Marine Life Science, Graduate School of Fisheries SciencesHokkaido UniversityHakodateJapan

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