Skip to main content
SpringerLink
Log in

Transgenic Expression of a Bacterial Thermophilic Amylase in the Chlamydomonas reinhardtii Chloroplast to Facilitate Algal Biofuel Production

  • Published:
BioEnergy Research Aims and scope Submit manuscript

Abstract

This study investigated expression of a thermophilic amylase (arAmyBH) from hyperthermophilic bacterium Thermotoga neapolitana into Chlamydomonas reinhardtii chloroplast to facilitate in vivo starch hydrolysis for algal biofuel production. The expression and accumulation of the recombinant protein in the algal chloroplast were verified by Western blot analysis. The recombinant arAmyBH was purified using Ni-NTA purification system. The optimum pH and temperature of arAmyBH were 5.5 and 60 °C, respectively. The enzymatic hydrolysis of the algal starch demonstrated that with supplemental commercial alpha-amylase, the transformant completely converted starch into reducing sugars in vivo at an elevated temperature.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Markou G, Angelidaki I, Georgakakis D (2012) Microalgal carbohydrates: an overview of the factors influencing carbohydrates production, and of main bioconversion technologies for production of biofuels. Appl Microbiol Biotechnol 96(3):631–645. doi:10.1007/s00253-012-4398-0

    Article  CAS  PubMed  Google Scholar 

  2. Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25(3):294–306. doi:10.1016/j.biotechadv.2007.02.001

    Article  CAS  PubMed  Google Scholar 

  3. Williams PJL, Laurens LML (2010) Microalgae as biodiesel & biomass feedstocks: review & analysis of the biochemistry, energetics & economics. Energy Environ Sci 3(5):554–590. doi:10.1039/b924978h

    Article  CAS  Google Scholar 

  4. Scott SA, Davey MP, Dennis JS, Horst I, Howe CJ, Lea-Smith DJ, Smith AG (2010) Biodiesel from algae: challenges and prospects. Curr Opin Biotechnol 21(3):277–286. doi:10.1016/j.copbio.2010.03.005

    Article  CAS  PubMed  Google Scholar 

  5. Singh A, Nigam PS, Murphy JD (2011) Renewable fuels from algae: an answer to debatable land based fuels. Bioresour Technol 102(1):10–16. doi:10.1016/j.biortech.2010.06.032

    Article  CAS  PubMed  Google Scholar 

  6. Wiley PE, Campbell JE, McKuin B (2011) Production of biodiesel and biogas from algae: a review of process train options. Water Environ Res 83(4):326–338. doi:10.2175/106143010x12780288628615

    Article  CAS  PubMed  Google Scholar 

  7. Gong YM, Jiang ML (2011) Biodiesel production with microalgae as feedstock: from strains to biodiesel. Biotechnol Lett 33(7):1269–1284. doi:10.1007/s10529-011-0574-z

    Article  CAS  PubMed  Google Scholar 

  8. Sharma KK, Schuhmann H, Schenk PM (2012) High lipid induction in microalgae for biodiesel production. Energies 5(5):1532–1553. doi:10.3390/en5051532

    Article  CAS  Google Scholar 

  9. Feng GD, Cheng LH, Xu XH, Zhang L, Chen HL (2012) Strategies in genetic engineering of microalgae for high-lipid production. Prog Chem 24(7):1413–1426

    CAS  Google Scholar 

  10. de Boer K, Moheimani NR, Borowitzka MA, Bahri PA (2012) Extraction and conversion pathways for microalgae to biodiesel: a review focused on energy consumption. J Appl Phycol 24(6):1681–1698. doi:10.1007/s10811-012-9835-z

    Article  CAS  Google Scholar 

  11. Liang YN (2013) Producing liquid transportation fuels from heterotrophic microalgae. Appl Energy 104:860–868. doi:10.1016/j.apenergy.2012.10.067

    Article  CAS  Google Scholar 

  12. Blatti JL, Michaud J, Burkart MD (2013) Engineering fatty acid biosynthesis in microalgae for sustainable biodiesel. Curr Opin Chem Biol 17(3):496–505. doi:10.1016/j.cbpa.2013.04.007

    Article  CAS  PubMed  Google Scholar 

  13. Pragya N, Pandey KK, Sahoo PK (2013) A review on harvesting, oil extraction and biofuels production technologies from microalgae. Renew Sustain Energy Rev 24:159–171. doi:10.1016/j.rser.2013.03.034

    Article  CAS  Google Scholar 

  14. Nguyen MT, Choi SP, Lee J, Lee JH, Sim SJ (2009) Hydrothermal acid pretreatment of Chlamydomonas reinhardtii biomass for ethanol production. J Microbiol Biotechnol 19(2):161–166

    Article  CAS  PubMed  Google Scholar 

  15. Hirayama S, Ueda R, Ogushi Y, Hirano A, Samejima Y, Hon-Nami K, Kunito S (1998) Ethanol production from carbon dioxide by fermentative microalgae. In: Inui T, Anpo M, Izui K, Yanagida S, Yamaguchi T (eds) Advances in chemical conversions for mitigating carbon dioxide, vol 114, Studies in surface science and catalysis. Elsevier Science, Amsterdam, pp 657–660

    Chapter  Google Scholar 

  16. Scholz MJ, Riley MR, Cuello JL (2013) Acid hydrolysis and fermentation of microalgal starches to ethanol by the yeast Saccharomyces cerevisiae. Biomass Bioenergy 48:59–65. doi:10.1016/j.biombioe.2012.10.026

    Article  CAS  Google Scholar 

  17. Doan QC, Moheimani NR, Mastrangelo AJ, Lewis DM (2012) Microalgal biomass for bioethanol fermentation: implications for hypersaline systems with an industrial focus. Biomass Bioenergy 46:79–88. doi:10.1016/j.biombioe.2012.08.022

    Article  CAS  Google Scholar 

  18. Cheng YS, Zheng Y, Labavitch JM, VanderGheynst JS (2013) Virus infection of Chlorella variabilis and enzymatic saccharification of algal biomass for bioethanol production. Bioresour Technol 137:326–331. doi:10.1016/j.biortech.2013.03.055

    Article  CAS  PubMed  Google Scholar 

  19. Choi SP, Nguyen MT, Sim SJ (2010) Enzymatic pretreatment of Chlamydomonas reinhardtii biomass for ethanol production. Bioresour Technol 101(14):5330–5336. doi:10.1016/j.biortech.2010.02.026

    Article  CAS  PubMed  Google Scholar 

  20. Yao CH, Ai JN, Cao XP, Xue S (2013) Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions. Appl Microbiol Biotechnol 97(13):6099–6110. doi:10.1007/s00253-013-4983-x

    Article  CAS  PubMed  Google Scholar 

  21. Harun R, Danquah MK (2011) Enzymatic hydrolysis of microalgal biomass for bioethanol production. Chem Eng J 168(3):1079–1084. doi:10.1016/j.cej.2011.01.088

    Article  CAS  Google Scholar 

  22. Pen J, Molendijk L, Quax WJ, Sijmons PC, van Ooyen AJ, van den Elzen PJ, Rietveld K, Hoekema A (1992) Production of active Bacillus licheniformis alpha-amylase in tobacco and its application in starch liquefaction. Biotechnology (N Y) 10(3):292–296

    Article  CAS  Google Scholar 

  23. Xu X, Fang J, Wang W, Guo J, Chen P, Cheng J, Shen Z (2008) Expression of a bacterial alpha-amylase gene in transgenic rice seeds. Transgenic Res 17(4):645–650. doi:10.1007/s11248-007-9144-5

    Article  CAS  PubMed  Google Scholar 

  24. Lanahan MB, Basu SS, Batie CJ, Chen W, Craig J, Kinkema M (2011) Self-processing plants and plant parts. US 7 919 681 B2

  25. Rosales-Mendoza S, Paz-Maldonado LM, Soria-Guerra RE (2012) Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives. Plant Cell Rep 31(3):479–494. doi:10.1007/s00299-011-1186-8

    Article  CAS  PubMed  Google Scholar 

  26. Franklin S, Ngo B, Efuet E, Mayfield SP (2002) Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J 30(6):733–744

    Article  CAS  PubMed  Google Scholar 

  27. Sun M, Qian K, Su N, Chang H, Liu J, Shen G (2003) Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast. Biotechnol Lett 25(13):1087–1092

    Article  CAS  PubMed  Google Scholar 

  28. Mayfield SP, Schultz J (2004) Development of a luciferase reporter gene, luxCt, for Chlamyodmonas reinhardtii chloroplast. Plant J 37:449–458

    Article  CAS  PubMed  Google Scholar 

  29. Yang ZQ, Li YN, Chen F, Li D, Zhang ZF, Liu YX, Zheng DX, Wang Y, Shen GF (2006) Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast. Chin Sci Bull 51(14):1703–1709. doi:10.1007/s11434-006-2041-0

    Article  CAS  Google Scholar 

  30. Specht E, Miyake-Stoner S, Mayfield S (2010) Micro-algae come of age as a platform for recombinant protein production. Biotechnol Lett 32(10):1373–1383. doi:10.1007/s10529-010-0326-5

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Bock R (2007) Plastid biotechnology: prospects for herbicide and insect resistance, metabolic engineering and molecular farming. Curr Opin Biotechnol 18(2):100–106. doi:10.1016/j.copbio.2006.12.001

    Article  CAS  PubMed  Google Scholar 

  32. Franklin SE, Mayfield SP (2005) Recent developments in the production of human therapeutic proteins in eukaryotic algae. Expert Opin Biol Ther 5(2):225–235. doi:10.1517/14712598.5.2.225

    Article  CAS  PubMed  Google Scholar 

  33. Harris EH (1989) The Chlamydomonas sourcebook : a comprehensive guide to biology and laboratory use. Academic, San Diego

    Google Scholar 

  34. Sueoka N (1960) Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardi. Proc Natl Acad Sci U S A 46(1):83–91

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Manuell AL, Beligni MV, Elder JH, Siefker DT, Tran M, Weber A, McDonald TL, Mayfield SP (2007) Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast. Plant Biotechnol J 5(3):402–412. doi:10.1111/j.1467-7652.2007.00249.x

    Article  CAS  PubMed  Google Scholar 

  36. Park KM, Jun SY, Choi KH, Park KH, Park CS, Cha J (2010) Characterization of an exo-acting intracellular alpha-amylase from the hyperthermophilic bacterium Thermotoga neapolitana. Appl Microbiol Biotechnol 86(2):555–566. doi:10.1007/s00253-009-2284-1

    Article  CAS  PubMed  Google Scholar 

  37. Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas. Nucleic Acids Res 19(15):4083–4089

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Barnes D, Franklin S, Schultz J, Henry R, Brown E, Coragliotti A, Mayfield SP (2005) Contribution of 5′- and 3′-untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes. Mol Genet Genomics 274(6):625–636. doi:10.1007/s00438-005-0055-y

    Article  CAS  PubMed  Google Scholar 

  39. GuhaMajumdar M, Dawson-Baglien E, Sears BB (2008) Creation of a chloroplast microsatellite reporter for detection of replication slippage in Chlamydomonas reinhardtii. Eukaryot Cell 7(4):639–646. doi:10.1128/EC.00447-07

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Boynton JE, Gillham NW (1993) Chloroplast transformation in Chlamydomonas. Methods Enzymol 217:510–536

    Article  CAS  PubMed  Google Scholar 

  41. Kindle KL, Richards KL, Stern DB (1991) Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 88(5):1721–1725

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  42. Guhamajumdar M, Sears BB (2005) Chloroplast DNA base substitutions: an experimental assessment. Mol Genet Genomics 273(2):177–183. doi:10.1007/s00438-005-1121-1

    Article  CAS  PubMed  Google Scholar 

  43. Yohn CB, Cohen A, Rosch C, Kuchka MR, Mayfield SP (1998) Translation of the chloroplast psbA mRNA requires the nuclear-encoded poly(A)-binding protein, RB47. J Cell Biol 142(2):435–442

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  45. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685

    Article  CAS  PubMed  Google Scholar 

  46. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428. doi:10.1021/ac60147a030

    Article  CAS  Google Scholar 

  47. Ruan Z, Zanotti M, Wang X, Ducey C, Liu Y (2012) Evaluation of lipid accumulation from lignocellulosic sugars by Mortierella isabellina for biodiesel production. Bioresour Technol 110:198–205. doi:10.1016/j.biortech.2012.01.053

    Article  CAS  PubMed  Google Scholar 

  48. Fargo DC, Zhang M, Gillham NW, Boynton JE (1998) Shine-Dalgarno-like sequences are not required for translation of chloroplast mRNAs in Chlamydomonas reinhardtii chloroplasts or in Escherichia coli. Mol Gen Genet 257(3):271–282

    Article  CAS  PubMed  Google Scholar 

  49. Mayfield SP, Manuell AL, Chen S, Wu J, Tran M, Siefker D, Muto M, Marin-Navarro J (2007) Chlamydomonas reinhardtii chloroplasts as protein factories. Curr Opin Biotechnol 18(2):126–133. doi:10.1016/j.copbio.2007.02.001

    Article  CAS  PubMed  Google Scholar 

  50. Buleon A, Gallant DJ, Bouchet B, Mouille G, D’Hulst C, Kossmann J, Ball S (1997) Starches from A to C. Chlamydomonas reinhardtii as a model microbial system to investigate the biosynthesis of the plant amylopectin crystal. Plant Physiol 115(3):949–957

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  51. Lee LP (1974) Temperature effect on the permeability of plasma membranes of advanced germinal cells of the rat testis. Can J Biochem 52(7):586–593

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank Dr. Ramin Vismeh and Dr. A. Daniel Jones from RTSF Mass Spectrometry at Michigan State University for technical support.

Author information

Authors and Affiliations

  1. Department of Biosystems and Agricultural Engineering, Michigan State University, 203 A.W. Farrall Hall, East Lansing, MI, 48824-1323, USA

    Xiaoqing Wang, Zhenhua Ruan, Danielle Boileau, Yan Liu & Wei Liao

  2. Department of Plant Biology, Michigan State University, East Lansing, MI, 48824, USA

    Barbara B. Sears

Authors
  1. Xiaoqing Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhenhua Ruan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Danielle Boileau
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Barbara B. Sears
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wei Liao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yan Liu.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 59 kb)

ESM 2

(DOCX 59 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, X., Ruan, Z., Boileau, D. et al. Transgenic Expression of a Bacterial Thermophilic Amylase in the Chlamydomonas reinhardtii Chloroplast to Facilitate Algal Biofuel Production. Bioenerg. Res. 8, 527–536 (2015). https://doi.org/10.1007/s12155-014-9538-1

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12155-014-9538-1

Keywords

Search

Navigation