Journal of Applied Phycology

, Volume 31, Issue 1, pp 255–267 | Cite as

De novo transcriptomic analysis of the oleaginous alga Botryococcus braunii AC768 (Chlorophyta)

  • Xiaolin Zhang
  • Fang Wen
  • Zhenyu Xu
  • Deying Sun
  • Wee Chew
  • Jianhua LiuEmail author


The oleaginous alga Botryococcus braunii (Chlorophyta) is known to accumulate hydrocarbons up to 80% of cell dry weight. Races A, B, and L of B. braunii species accumulate alkadiene/triene, botryococcene, and lycopadiene, respectively. Transcriptome analyses in race A and race B have identified various transcripts encoding enzymes involved in the biosynthesis of alkadiene/triene and botryococcene. However, the transcriptome of B. braunii race L has not been analyzed. In this study, we report the de novo assembly of the transcriptome of B. braunii AC768 race L. AC768 culture in 2×BB medium reaches a maximum density (cell dry weight) of 5.5 g L−1, of which, 6% is lycopadiene. Transcriptional profiles of AC768 show that ESTs involved in energy metabolisms are most abundantly expressed, suggesting an energy requirement for lipid accumulation. Transcriptomic analysis indicates the presence of a putative lycopaoctaene synthase that synthesizes the precursor of lycopadiene in AC768. Comparative analysis between races reveals that botryococcene synthase and lycopaoctaene synthase are specific to races B and L of B. braunii, respectively. Taken together, our results indicate that hydrocarbon chemicals accumulated in different races of B. braunii are determined by their transcriptional profiles.


Botryococcus Hydrocarbon Lycopadiene Race L Squalene synthase-like proteins Transcriptome 



The authors would like to thank the anonymous reviewer for his/her thoughtful comments and suggestions that have greatly improved this manuscript.

Author contributions

XZ, DS, WC, and JL performed the biological and biochemical studies; FW, ZX, and JL carried out the bioinformatics and statistical studies; JL conceived of the study and participated in its design and coordination; JL drafted the manuscript. All authors read and approved the final manuscript.

Funding information

This work was partly supported by a grant from the National Natural Science Foundation of China (Grant No. 31571392) and grants from the Zhoushan Municipal Science and Technology Bureau, Zhejiang Province, China (Grant No. 2012C31019 and 2014C51020) to JL, and a grant from Joint Council Office, A-STAR, Singapore (Grant No. 1031C002) to JL and WC. FW and ZX are recipients of the M.Sc. scholarship in Ocean College, Zhejiang University, Zhejiang Province, China.

Compliance with ethical standards

This study does not involve human and animal materials.

Competing interests

The authors declare that they have no competing interests.

Supplementary material

10811_2018_1577_MOESM1_ESM.ods (623 kb)
ESM 1 (ODS 622 kb)
10811_2018_1577_MOESM2_ESM.ods (26 kb)
ESM 2 (ODS 26 kb)
10811_2018_1577_MOESM3_ESM.ods (24 kb)
ESM 3 (ODS 23 kb)
10811_2018_1577_MOESM4_ESM.ods (538 kb)
ESM 4 (ODS 537 kb)
10811_2018_1577_MOESM5_ESM.ods (590 kb)
ESM 5 (ODS 590 kb)
10811_2018_1577_MOESM6_ESM.ods (28 kb)
ESM 6 (ODS 28 kb)
10811_2018_1577_MOESM7_ESM.ods (28 kb)
ESM 7 (ODS 28 kb)
10811_2018_1577_MOESM8_ESM.docx (20 kb)
ESM 8 (DOCX 19 kb)
10811_2018_1577_MOESM9_ESM.ods (44 kb)
ESM 9 (ODS 44 kb)
10811_2018_1577_MOESM10_ESM.ods (20 kb)
ESM 10 (ODS 20 kb)


  1. Andrews S (2010) FastQC: a quality control tool for high throughput sequence data.
  2. Baba M, Ioki M, Nakajima N, Shiraiwa Y, Watanabe MM (2012) Transcriptome analysis of an oil-rich race A strain of Botryococcus braunii (BOT-88-2) by de novo assembly of pyrosequencing cDNA reads. Bioresour Technol 109:282–286CrossRefGoogle Scholar
  3. Banerjee A, Sharma R, Chisti Y, Banerjee UC (2002) Botryococcus braunii: a renewable source of hydrocarbons and other chemicals. Crit Rev Biotechnol 22:245–279CrossRefGoogle Scholar
  4. Blanc G, Agarkova I, Grimwood J, Kuo A, Brueggeman A, Dunigan DD, Gurnon J, Ladunga I, Lindquist E, Lucas S, Pangilinan J, Proschold T, Salamov A, Schmutz J, Weeks D, Yamada T, Lomsadze A, Borodovsky M, Claverie JM, Grigoriev IV, Van Etten JL (2012) The genome of the polar eukaryotic microalga Coccomyxa subellipsoidea reveals traits of cold adaptation. Genome Biol 13(5):R39CrossRefGoogle Scholar
  5. Bland JM, Altman DG (1995) Multiple significance tests: the Bonferroni method. BMJ 310(6973):170CrossRefGoogle Scholar
  6. Bold HC (1949) The morphology of Chlamydomonas chlamydogama sp. Bull Torrey Bot Club 76:101–108CrossRefGoogle Scholar
  7. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120CrossRefGoogle Scholar
  8. Breitenbach J, Visser H, Verdoes JC, van Ooyen AJ, Sandmann G (2011) Engineering of geranylgeranyl pyrophosphate synthase levels and physiological conditions for enhanced carotenoid and astaxanthin synthesis in Xanthophyllomyces dendrorhous. Biotechnol Lett 33:755–761CrossRefGoogle Scholar
  9. ChanYong T-P, Largeau C, Casadevall E (1986) Biosynthesis of non-isoprenoid hydrocarbons by the microalga Botryococcus braunii: evidence for an elongation decarboxylation mechanism; activation of decarboxylation. Nouv J Chim 10:701–707Google Scholar
  10. Dennis MW, Kolattukudy PE (1991) Alkane biosynthesis by decarbonylation of aldehyde catalyzed by a microsomal preparation from Botryococcus braunii. Arch Biochem Biophys 287:268–275CrossRefGoogle Scholar
  11. Dennis MW, Kolattukudy PE (1992) A cobalt-porphirin enzyme converts a fatty aldehyde to a hydrocarbon and CO. Proc Natl Acad Sci U S A 89:5306–5310CrossRefGoogle Scholar
  12. Fang L, Sun D, Xu Z, He J, Qi S, Chen X, Chew W, Liu J (2015) Transcriptomic analysis of a moderately growing subisolate Botryococcus braunii 779 (Chlorophyta) in response to nitrogen deprivation. Biotechnol Biofuels 8:130CrossRefGoogle Scholar
  13. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotech 29:644–652CrossRefGoogle Scholar
  14. Ioki M, Baba M, Nakajima N, Shiraiwa Y, Watanabe MM (2012a) Transcriptome analysis of an oil-rich race B strain of Botryococcus braunii (BOT-22) by de novo assembly of pyrosequencing cDNA reads. Bioresour Technol 109:292–296CrossRefGoogle Scholar
  15. Ioki M, Baba M, Nakajima N, Shiraiwa Y, Watanabe MM (2012b) Transcriptome analysis of an oil-rich race B strain of Botryococcus braunii (BOT-70) by de novo assembly of 5′-end sequences of full-length cDNA clones. Bioresour Technol 109:277–281CrossRefGoogle Scholar
  16. Jin J, Dupré C, Yoneda K, Watanabe MM, Legrand J, Grizeau D (2016) Characteristics of extracellular hydrocarbon-rich microalga Botryococcus braunii for biofuels production: recent advances and opportunities. Process Biochem 51:1866–1875CrossRefGoogle Scholar
  17. Kanehisa M, Goto S (2000) KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res 28:27–30CrossRefGoogle Scholar
  18. Kawachi M, Tanoi T, Demura M, Kaya K, Watanabe MM (2012) Relationship between hydrocarbons and molecular phylogeny of Botryococcus braunii. Algal Res 1:114–119CrossRefGoogle Scholar
  19. Kuswik-Rabiega G, Rilling HC (1987) Squalene synthetase. Solubilization and partial purification of squalene synthetase, copurification of presqualene pyrophosphate and squalene synthetase activities. J Biol Chem 262:1505–1509PubMedGoogle Scholar
  20. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, W A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948CrossRefGoogle Scholar
  21. Lohr M, Schwender J, Polle JE (2012) Isoprenoid biosynthesis in eukaryotic phototrophs: a spotlight on algae. Plant Sci 185-186:9–22CrossRefGoogle Scholar
  22. Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard L, Marshall WF, Qu LH, Nelson DR, Sanderfoot AA, Spalding MH, Kapitonov VV, Ren Q, Ferris P, Lindquist E, Shapiro H, Lucas SM, Grimwood J, Schmutz J, Cardol P, Cerutti H, Chanfreau G, Chen CL, Cognat V, Croft MT, Dent R, Dutcher S, Fernandez E, Fukuzawa H, Gonzalez-Ballester D, Gonzalez-Halphen D, Hallmann A, Hanikenne M, Hippler M, Inwood W, Jabbari K, Kalanon M, Kuras R, Lefebvre PA, Lemaire SD, Lobanov AV, Lohr M, Manuell A, Meier I, Mets L, Mittag M, Mittelmeier T, Moroney JV, Moseley J, Napoli C, Nedelcu AM, Niyogi K, Novoselov SV, Paulsen IT, Pazour G, Purton S, Ral JP, Riano-Pachon DM, Riekhof W, Rymarquis L, Schroda M, Stern D, Umen J, Willows R, Wilson N, Zimmer SL, Allmer J, Balk J, Bisova K, Chen CJ, Elias M, Gendler K, Hauser C, Lamb MR, Ledford H, Long JC, Minagawa J, Page MD, Pan J, Pootakham W, Roje S, Rose A, Stahlberg E, Terauchi AM, Yang P, Ball S, Bowler C, Dieckmann CL, Gladyshev VN, Green P, Jorgensen R, Mayfield S, Mueller-Roeber B, Rajamani S, Sayre RT, Brokstein P, Dubchak I, Goodstein D, Hornick L, Huang YW, Jhaveri J, Luo Y, Martinez D, Ngau WCA, Otillar B, Poliakov A, Porter A, Szajkowski L, Werner G, Zhou K, Grigoriev IV, Rokhsar DS, Grossman AR (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245–250CrossRefGoogle Scholar
  23. Metzger P, Casadevall E (1983) Structure de trois nouveaux botryococcènes synthétisés par une souche de Botryococcus braunii cultivée en laboratoire. Tet Lett 24:4013–4016CrossRefGoogle Scholar
  24. Metzger P, Casadevall E (1987) Lycopadiene, a tetraterpenoid hydrocarbon from new strains of the green alga Botryococcus braunii. Tet Lett 28:3931–3934CrossRefGoogle Scholar
  25. Metzger P, Largeau C (2005) Botryococcus braunii: a rich source for hydrocarbons and related ether lipids. Appl Microbiol Biotechnol 66:486–496CrossRefGoogle Scholar
  26. Metzger P, Berkaloff C, Couté A, Casadevall E (1985) Alkadiene- and botryococcene-producing races of wild strains of Botryococcus braunii. Phytochemistry 24:2305–2312CrossRefGoogle Scholar
  27. Metzger P, Allard B, Casadevall E, Berkaloff C, Couté A (1990) Structure and chemistry of a new chemical race of Botryococcus braunii that produces lycopadiene, a tetraterpenoid hydrocarbon. J Phycol 26:258–266CrossRefGoogle Scholar
  28. Misawa N, Truesdale MR, Sandmann G, Fraser PD, Bird C, Schuch W, Bramley PM (1994) Expression of a tomato cDNA coding for phytoene synthase in Escherichia coli, phytoene formation in vivo and in vitro, and functional analysis of the various truncated gene products. J Biochem 116:980–985CrossRefGoogle Scholar
  29. Molnar I, Lopez D, Wisecaver JH, Devarenne TP, Weiss TL, Pellegrini M, Hackett JD (2012) Bio-crude transcriptomics: gene discovery and metabolic network reconstruction for the biosynthesis of the terpenome of the hydrocarbon oil-producing green alga, Botryococcus braunii race B (Showa). BMC Genomics 13:576CrossRefGoogle Scholar
  30. Niehaus TD, Okada S, Devarenne TP, Watt DS, Sviripa V, Chappell J (2011) Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii. Proc Nat Acad Sci U S A 108:12260–12265CrossRefGoogle Scholar
  31. Palenik B, Grimwood J, Aerts A, Rouze P, Salamov A, Putnam N, Dupont C, Jorgensen R, Derelle E, Rombauts S, Zhou K, Otillar R, Merchant SS, Podell S, Gaasterland T, Napoli C, Gendler K, Manuell A, Tai V, Vallon O, Piganeau G, Jancek S, Heijde M, Jabbari K, Bowler C, Lohr M, Robbens S, Werner G, Dubchak I, Pazour GJ, Ren Q, Paulsen I, Delwiche C, Schmutz J, Rokhsar D, van de Peer Y, Moreau H, Grigoriev IV (2007) The tiny eukaryote Ostreococcus provides genomic insights into the paradox of plankton speciation. Proc Nat Acad Sci U S A 104:7705–7710CrossRefGoogle Scholar
  32. Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14:417–419CrossRefGoogle Scholar
  33. Prochnik SE, Umen J, Nedelcu AM, Hallmann A, Miller SM, Nishii I, Ferris P, Kuo A, Mitros T, Fritz-Laylin LK, Hellsten U, Chapman J, Simakov O, Rensing SA, Terry A, Pangilinan J, Kapitonov V, Jurka J, Salamov A, Shapiro H, Schmutz J, Grimwood J, Lindquist E, Lucas S, Grigoriev IV, Schmitt R, Kirk D, Rokhsar DS (2010) Genomic analysis of organismal complexity in the multicellular green alga. Science 329:223–226CrossRefGoogle Scholar
  34. Rivals I, Personnaz L, Taing L, Potier MC (2007) Enrichment or depletion of a GO category within a class of genes: which test? Bioinformatics 23:401–407CrossRefGoogle Scholar
  35. Robinson GW, Tsay YH, Kienzle BK, Smith-Monroy CA, Bishop RW (1993) Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation. Mol Cell Biol 13:2706–2717CrossRefGoogle Scholar
  36. Senousy HH, Beakes GW, Hack E (2004) Phylogenetic placement of Botryococcus braunii (Trebouxiophyceae) and Botryococcus sudeticus isolate UTEX 2629 Chlorophyceae. J Phycol 40:412–423CrossRefGoogle Scholar
  37. Shi Q, Pavey ES, Carter RE (2012) Bonferroni-based correction factor for multiple, correlated endpoints. Pharm Stat 11:300–309CrossRefGoogle Scholar
  38. Sun D, Zhu J, Fang L, Zhang X, Chow Y, Liu J (2013) De novo transcrip- tome profiling uncovers a drastic downregulation of photosynthesis upon nitrogen deprivation in the nonmodel green alga Botryosphaerella sudeticus. BMC Genomics 14:715CrossRefGoogle Scholar
  39. Takahashi I, Ogura K (1981) Farnesyl pyrophosphate synthetase from Bacillus subtilis. J Biochem 89:1581–1587CrossRefGoogle Scholar
  40. Tasić MB, Pinto LFR, Klein BC, Veljković VB, Filho RM (2016) Botryococcus braunii for biodiesel production. Renew Sust Energy Rev 64:260–270CrossRefGoogle Scholar
  41. Thabet I, Guirimand G, Guihur A, Lanoue A, Courdavault V, Papon N, Bouzid S, Giglioli-Guivarch N, Simkin A, Clastre M (2012) Characterization and subcellular localization of geranylgeranyl diphosphate synthase from Catharanthus roseus. Mol Biol Rep 39:3235–3243CrossRefGoogle Scholar
  42. Thapa HR, Naik MT, Okada S, Takada K, Molnár I, Xu Y, Devarenne TP (2016) A squalene synthase-like enzyme initiates production of tetraterpenoid hydrocarbons in Botryococcus braunii race L. Nat Commun 7:11198CrossRefGoogle Scholar
  43. Thapa HR, Tang S, Sacchettini JC, Devarenne TP (2017) Tetraterpene synthase substrate and product specificity in the green microalga Botryococcus braunii race L. ACS Chem Biol 12:2408–2416CrossRefGoogle Scholar
  44. Uchida H, Sumimoto K, Oki T, Nishii I, Mizohata E, Matsunaga S, Okada S (2018) Isolation and characterization of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene from Botryococcus braunii, race B. J Plant Res.
  45. Viguera E, Canceill D, Ehrlich SD (2001) Replication slippage involves DNA polymerase pausing and dissociation. EMBO J 20:2587–2595CrossRefGoogle Scholar
  46. Worden AZ, Lee JH, Mock T, Rouze P, Simmons MP, Aerts AL, Allen AE, Cuvelier ML, Derelle E, Everett MV, Foulon E, Grimwood J, Gundlach H, Henrissat B, Napoli C, McDonald SM, Parker MS, Rombauts S, Salamov A, von Dassow P, Badger JH, Coutinho PM, Demir E, Dubchak I, Gentemann C, Eikrem W, Gready JE, John U, Lanier W, Lindquist EA, Lucas S, Mayer KFX, Moreau H, Not F, Otillar R, Panaud O, Pangilinan J, Paulsen I, Piegu B, Poliakov A, Robbens S, Schmutz J, Toulza E, Wyss T, Zelensky A, Zhou K, Armbrust EV, Bhattacharya D, Goodenough UW, van de Peer Y, Grigoriev IV (2009) Green evolution and dynamic adaptations revealed by genomes of the marine picoeukaryotes Micromonas. Science 324:268–272CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Ocean CollegeZhejiang UniversityZhoushanChina
  2. 2.Genome Institute of SingaporeSingaporeSingapore
  3. 3.Biopolis Shared Facilities, A-STARSingaporeSingapore
  4. 4.Instittute of Chemical and Engineering SciencesSingaporeSingapore
  5. 5.Ocean Research Center of ZhoushanZhejiang UniversityZhoushanChina

Personalised recommendations