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Production of Biofuel-Related Isoprenoids Derived from Botryococcus braunii Algae

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Hydrocarbon and Lipid Microbiology Protocols

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Abstract

The colony algae Botryococcus braunii produces large amounts of C30+ triterpene hydrocarbons. Recent discovery of the associated biosynthetic genes has facilitated the metabolic engineering of these triterpene hydrocarbons in alternative hosts – where squalene has served as an analytical standard and a closely associated model hydrocarbon biosynthetic pathway. An extraction and analysis method is provided for both the native and heterologous systems. In the case of the native algae, the hydrocarbons are tightly associated with a complex wall matrix. In addition to quantification of extracted triterpenes by GC-FID, secondary ion mass spectrometry (SIMS) has also provided an assessment from “in vivo” samples at room temperature. For heterologous expression of the triterpene pathway in alternative hosts, the hydrocarbons are found both intracellularly and extracellularly. The highly hydrophobic nature of these triterpenes provides for relatively straightforward recovery by extraction into an organic phase. The methylation of the Botryococcus braunii race B hydrocarbons (which enhances its fuel precursor value) is readily resolved by GC-FID methods for routine analysis.

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References

  1. Wolf FR, Nonomura AM, Bassham JA (1985) Growth and branched hydrocarbon in a strain of Botryococcus braunii (Chlorophyta). J Phycol 21:388–396

    Article  CAS  Google Scholar 

  2. Khatri W, Hendrix R, Niehaus T, Chappell J, Curtis WR (2014) Hydrocarbon production in high density Botryococcus braunii race B continuous culture. Biotechnol Bioeng 111:493–503

    Article  CAS  PubMed  Google Scholar 

  3. Hillen LW, Pollard G, Wake LV, White N (1982) Hydrocracking of the oils of Botryococcus braunii to transport fuels. Biotechnol Bioeng 24:193–205

    Article  CAS  PubMed  Google Scholar 

  4. Glikson M, Lindsay K, Saxby J (1989) Botryococcus—A planktonic green alga, the source of petroleum through the ages: transmission electron microscopical studies of oil shales and petroleum source rocks. Org Geochem 14:595–608

    Article  CAS  Google Scholar 

  5. Niehaus TD, Okada S, Devarenne TP, Watt DS, Sviripa V, Chappell J (2011) Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii. Proc Natl Acad Sci U S A 108:12260–12265

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kirby J, Romanini DW, Paradise EM, Keasling JD (2008) Engineering triterpene production in Saccharomyces cerevisiae-beta-amyrin synthase from Artemisia annua. FEBS J 275:1852–1859

    Article  CAS  PubMed  Google Scholar 

  7. Wu S, Jiang Z, Kempinski C, Eric Nybo S, Husodo S, Williams R, Chappell J (2012) Engineering triterpene metabolism in tobacco. Planta 236:867–877

    Article  CAS  PubMed  Google Scholar 

  8. Niehaus TD, Kinison S, Okada S, Yeo Y, Bell SA, Cui P, Devarenne TP, Chappell J (2012) Functional identification of triterpene methyltransferases from Botryococcus braunii race B. J Biol Chem 287:8163–8173

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Khan NE, Nybo SE, Chappell J, Curtis WR (2015) Triterpene hydrocarbon production engineered into a metabolically versatile host – Rhodobacter capsulatus. Biotechnol Bioeng 112:1523–1532

    Article  CAS  PubMed  Google Scholar 

  10. Khan N (2015) Development of biological platform for the autotrophic production of biofuels. The Pennsylvania State University, State College

    Google Scholar 

  11. Zuroff TR, Barri Xiques S, Curtis WR (2013) Consortia-mediated bioprocessing of cellulose to ethanol with a symbiotic Clostridium phytofermentans/yeast co-culture. Biotechnol Biofuels 6:59

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Nybo SE, Khan N, Woolston BM, Curtis WR (2015) Metabolic engineering in chemolithoautotrophic hosts for the production of fuels and chemicals. Metab Eng 30:105–120

    Article  CAS  PubMed  Google Scholar 

  13. Eroglu E, Okada S, Melis A (2011) Hydrocarbon productivities in different Botryococcus strains: comparative methods in product quantification. J Appl Phycol 23:763–775

    Article  CAS  PubMed  Google Scholar 

  14. Kawachi M, Tanoi T, Demura M, Kaya K, Watanabe MM (2012) Relationship between hydrocarbons and molecular phylogeny of Botryococcus braunii. Algal Res 1:114–119

    Article  Google Scholar 

  15. Largeau C, Casadevall E, Berkaloff C, Index-botryococcus KW (1980) Sites of accumulation and composition of hydrocarbons in Botryococcus braunii. Phytochemistry 19:1043–1051

    Article  CAS  Google Scholar 

  16. Grung M, Metzger P, Liaaen-Jensen S (1989) Primary and secondary carotenoids in two races of the green alga Botryococcus braunii. Biochem Syst Ecol 17:263–269

    Article  CAS  Google Scholar 

  17. Scherholz ML (2012) Achieving pH control through stoichiometrically balanced media in algae photobioreactors. The Pennsylvania State University, State College

    Google Scholar 

  18. Maury J, Asadollahi MA, Formenti LR, Schalk M, Nielsen J (2013) Metabolic engineering of isoprenoid production: reconstruction of multistep heterologous pathways in tractable hosts. In: Bach TJ, Rohmer M (eds) Isoprenoid synthesis in plants and microorganisms: new concepts and experimental approaches. Springer, New York, pp 73–89

    Google Scholar 

  19. Yang J, Xian M, Su S, Zhao G, Nie Q, Jiang X, Zheng Y, Liu W (2012) Enhancing production of bio-isoprene using hybrid MVA pathway and isoprene synthase in E. coli. PLoS One 7, e33509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Chandran SS, Kealey JT, Reeves CD (2011) Microbial production of isoprenoids. Process Biochem 46:1703–1710

    Article  CAS  Google Scholar 

  21. Okada S, Devarenne T, Murakami M, Abe H, Chappell J (2004) Characterization of botryococcene synthase enzyme activity, a squalene synthase-like activity from the green microalga Botryococcus braunii, Race B. Arch Biochem Biophys 422:110–118

    Article  CAS  PubMed  Google Scholar 

  22. Choi SP, Bahn SH, Sim SJ (2013) Improvement of hydrocarbon recovery by spouting solvent into culture of Botryococcus braunii. Bioprocess Biosyst Eng 36:1977–1985

    Article  CAS  PubMed  Google Scholar 

  23. Yoo J (2013) Establishment and maintenance of axenic Botryococcus braunii race B algae culture. The Pennsylvania State University, State College

    Google Scholar 

  24. Wolf FR, Nemethy EK, Blanding JH, Bassham JA (1985) Biosynthesis of unusual acyclic isoprenoids in the alga Botryococcus braunii. Phytochemistry 24:733–737

    Article  CAS  Google Scholar 

  25. Weiss TL, Chun HJ, Okada S, Vitha S, Holzenburg A, Laane J, Devarenne TP (2010) Raman spectroscopy analysis of botryococcene hydrocarbons from the green microalga Botryococcus braunii. J Biol Chem 285:32458–32466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Metzger P, Casadevall E, Pouet M, Pouet Y (1985) Structures of some botryococcenes: branched hydrocarbons from the b-race of the green alga Botryococcus braunii. Phytochemistry 24:2995–3002

    Article  CAS  Google Scholar 

  27. Jackson L (2014) New analytical approaches to understand biological systems with Secondary Ion Mass Spectrometry (SIMS). The Pennsylvania State University, State College

    Google Scholar 

  28. Demura M, Ioki M, Kawachi M, Nakajima N, Watanabe MM (2014) Desiccation tolerance of Botryococcus braunii (Trebouxiophyceae, Chlorophyta) and extreme temperature tolerance of dehydrated cells. J Appl Phycol 26:49–53

    Article  CAS  PubMed  Google Scholar 

  29. Weiss TL, Roth R, Goodson C, Vitha S, Black I, Azadi P, Rusch J, Holzenburg A, Devarenne TP, Goodenough U (2012) Colony organization in the green alga Botryococcus braunii (Race B) is specified by a complex extracellular matrix. Eukaryot Cell 11:1424–1440

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ind AC, Porter SL, Brown MT, Byles ED, de Beyer JA, Godfrey SA, Armitage JP (2009) Inducible-expression plasmid for Rhodobacter sphaeroides and Paracoccus denitrificans. Appl Environ Microbiol 75:6613–6615

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Eckardt NA (2005) Photorespiration revisited. Plant Cell 17(August):2139–2141

    Article  CAS  PubMed Central  Google Scholar 

  32. Wang J, Curtis WR (2015) Proton stoichiometric imbalance during algae photosynthetic growth on various nitrogen sources: towards metabolic pH control. J Appl Phycol, pp 1–10. doi: 10.1007/s10811-015-0551-3

  33. Daugulis AJ (1994) Integrated fermentation and recovery processes. Curr Opin Biotechnol 5:192–195

    Article  CAS  PubMed  Google Scholar 

  34. Corry JP, Reed WL, Curtis WR (1993) Enhanced recovery of solavetivone from Agrobacterium transformed root cultures of Hyoscyamus muticus using integrated product extraction. Biotechnol Bioeng 42:503–508

    Article  CAS  PubMed  Google Scholar 

  35. Metzger P, Berkaloff C, Casadevall E, Coute A (1985) Alkadiene-and botryococcene-producing races of wild strains of Botryococcus braunii. Phytochemistry 24:2305–2312

    Article  CAS  Google Scholar 

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Acknowledgments

We acknowledge the generous efforts of Dr. Joe Chappell throughout our development of algae and heterologous production of triterpenes, including “in-house/hands-on” training on initial extraction and GC analytical methods. Amalie Tuerk and Justin Yoo are acknowledged for their extensive efforts in the culturing of Botryococcus braunii strains and various growth and hydrocarbon production studies. This collaborative work with W. R. C. was supported by US Department of Energy. Grant Number: ARPA-e Electrofuels, DE-AR0000092, and the National Science Foundation Collaborative Grant No. CBET-0828648 titled “Development of a Sustainable Production Platform for Renewable Petroleum Based Oils in Algae.” Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

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Authors and Affiliations

  1. Department of Chemical Engineering, The Pennsylvania State University, 226 Fenske Laboratory, University Park, State College, PA, 16802-4400, USA

    William A. Muzika & Nymul E. Khan

  2. Department of Chemistry, The Pennsylvania State University, 209 Chemistry Building, University Park, State College, PA, 16802, USA

    Lauren M. Jackson & Nicholas Winograd

  3. Department of Chemical Engineering, The Pennsylvania State University, 226B Fenske Laboratory, University Park, State College, PA, 16802-4400, USA

    Wayne R. Curtis

Authors
  1. William A. Muzika
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  2. Nymul E. Khan
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  3. Lauren M. Jackson
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  4. Nicholas Winograd
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  5. Wayne R. Curtis
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Corresponding author

Correspondence to Wayne R. Curtis .

Editor information

Editors and Affiliations

  1. School of Biological Sciences, University of Essex Dept. Biological Sciences, Colchester, Essex, United Kingdom

    Terry J. McGenity

  2. Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

    Kenneth N. Timmis

  3. Department of Biology, University of the Balearic Islands and Mediterranean Institute for Advanced Studies (IMEDEA, UIB-CSIC), Palma de Mallorca, Spain

    Balbina Nogales

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© 2015 Springer-Verlag Berlin Heidelberg

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Muzika, W.A., Khan, N.E., Jackson, L.M., Winograd, N., Curtis, W.R. (2015). Production of Biofuel-Related Isoprenoids Derived from Botryococcus braunii Algae. In: McGenity, T., Timmis, K., Nogales, B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_187

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  • DOI: https://doi.org/10.1007/8623_2015_187

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  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-662-53113-6

  • Online ISBN: 978-3-662-53115-0

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