Skip to main content
SpringerLink
Log in

Metabolic Engineering of Microalgae for Biofuel Production

  • Protocol
  • First Online:
Book cover Biofuels from Algae

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1980))

Abstract

Microalgae are considered as promising cell factories for the production of various types of biofuels, including bioethanol, biodiesel, and biohydrogen by using carbon dioxide and sunlight. In spite of unique advantages of these microorganisms, the commercialization of microalgal biofuels has been hindered by poor economic features. Metabolic engineering is among the most promising strategies put forth to overcome this challenge. In this chapter, metabolic pathways involved in lipid and hydrogen production by microalgae are reviewed and discussed. Moreover, metabolic and genetic engineering approaches investigated for improving the rate of lipid (as a feedstock for biodiesel production) and biohydrogen synthesis are presented. Finally, genetic engineering tools and approaches employed for engineering microalgal metabolic pathways are elaborated. A thorough step-by-step protocol for reconstructing the metabolic pathway of various microorganisms including microalgae is also presented.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Gimpel JA, Henríquez V, Mayfield SP (2015) In metabolic engineering of eukaryotic microalgae: potential and challenges come with great diversity. Front Microbiol 6:1376

    PubMed  PubMed Central  Google Scholar 

  2. Stephens E et al (2010) An economic and technical evaluation of microalgal biofuels. Nat Biotechnol 28(2):126

    CAS  PubMed  Google Scholar 

  3. Peng K et al (2018) The bioeconomy of microalgal biofuels. In: Energy from microalgae. Springer, New York, pp 157–169

    Google Scholar 

  4. Cheng JJ, Timilsina GR (2011) Status and barriers of advanced biofuel technologies: a review. Renew Energy 36(12):3541–3549

    CAS  Google Scholar 

  5. Singh J, Gu S (2010) Commercialization potential of microalgae for biofuels production. Renew Sustain Energy Rev 14(9):2596–2610

    CAS  Google Scholar 

  6. Banerjee C, Dubey KK, Shukla P (2016) Metabolic engineering of microalgal based biofuel production: prospects and challenges. Front Microbiol 7:432

    PubMed  PubMed Central  Google Scholar 

  7. Brown LM, Zeiler KG (1993) Aquatic biomass and carbon dioxide trapping. Energ Conver Manage 34(9-11):1005–1013

    CAS  Google Scholar 

  8. Chisti Y (2008) Biodiesel from microalgae beats bioethanol. Trends Biotechnol 26(3):126–131

    CAS  PubMed  Google Scholar 

  9. Schenk PM et al (2008) Second generation biofuels: high-efficiency microalgae for biodiesel production. Bioenergy Res 1(1):20–43

    Google Scholar 

  10. Greenwell H et al (2010) Placing microalgae on the biofuels priority list: a review of the technological challenges. J R Soc Interface 7:703–726

    PubMed Central  Google Scholar 

  11. Pienkos PT, Darzins A (2009) The promise and challenges of microalgal-derived biofuels. Biofuels Bioprod Biorefin 3(4):431–440

    CAS  Google Scholar 

  12. De Bhowmick G, Koduru L, Sen R (2015) Metabolic pathway engineering towards enhancing microalgal lipid biosynthesis for biofuel application—a review. Renew Sustain Energy Rev 50:1239–1253

    Google Scholar 

  13. Ho S-H et al (2014) Perspectives on engineering strategies for improving biofuel production from microalgae—a critical review. Biotechnol Adv 32(8):1448–1459

    CAS  PubMed  Google Scholar 

  14. Zhang F, Rodriguez S, Keasling JD (2011) Metabolic engineering of microbial pathways for advanced biofuels production. Curr Opin Biotechnol 22(6):775–783

    CAS  PubMed  Google Scholar 

  15. Liu D, Evans T, Zhang F (2015) Applications and advances of metabolite biosensors for metabolic engineering. Metab Eng 31:35–43

    PubMed  Google Scholar 

  16. Majidian P, Tabatabaei M, Zeinolabedini M, Naghshbandi MP, Chisti Y (2018) Metabolic engineering of microorganisms for biofuel production. Renew Sustain Energy Rev 82:3863–3885

    CAS  Google Scholar 

  17. Jagadevan S et al (2018) Recent developments in synthetic biology and metabolic engineering in microalgae towards biofuel production. Biotechnol Biofuels 11(1):185

    PubMed  PubMed Central  Google Scholar 

  18. Costa JAV, De Morais MG (2011) The role of biochemical engineering in the production of biofuels from microalgae. Bioresour Technol 102(1):2–9

    CAS  PubMed  Google Scholar 

  19. Zorrilla López U et al (2013) Engineering metabolic pathways in plants by multigene transformation. Int J Dev Biol 57(6–8):565–576

    PubMed  Google Scholar 

  20. Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25(3):294–306

    CAS  PubMed  Google Scholar 

  21. Gaurav N et al (2017) Utilization of bioresources for sustainable biofuels: a review. Renew Sustain Energy Rev 73:205–214

    CAS  Google Scholar 

  22. Bellou S et al (2014) Microalgal lipids biochemistry and biotechnological perspectives. Biotechnol Adv 32(8):1476–1493

    CAS  PubMed  Google Scholar 

  23. Yu W-L et al (2011) Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae. Microb Cell Fact 10(1):91

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Radakovits R et al (2010) Genetic engineering of algae for enhanced biofuel production. Eukaryot Cell 9(4):486–501

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Post-Beittenmiller D, Jaworski J, Ohlrogge J (1991) In vivo pools of free and acylated acyl carrier proteins in spinach. Evidence for sites of regulation of fatty acid biosynthesis. J Biol Chem 266(3):1858–1865

    CAS  PubMed  Google Scholar 

  26. Post-Beittenmiller D, Roughan G, Ohlrogge JB (1992) Regulation of plant fatty acid biosynthesis: analysis of acyl-coenzyme A and acyl-acyl carrier protein substrate pools in spinach and pea chloroplasts. Plant Physiol 100(2):923–930

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Chang W-C, Zheng H-Q, Chen C-NN (2016) Comparative transcriptome analysis reveals a potential photosynthate partitioning mechanism between lipid and starch biosynthetic pathways in green microalgae. Algal Res 16:54–62

    Google Scholar 

  28. Liang M-H, Jiang J-G (2013) Advancing oleaginous microorganisms to produce lipid via metabolic engineering technology. Prog Lipid Res 52(4):395–408

    CAS  PubMed  Google Scholar 

  29. Dunahay TG et al (1996) Manipulation of microalgal lipid production using genetic engineering. Appl Biochem Biotechnol 57(1):223

    Google Scholar 

  30. Sheehan J et al (1998) A look back at the US Department of Energy’s aquatic species program: biodiesel from algae. National Renewable Energy Laboratory, Golden, p 328

    Google Scholar 

  31. Gomma AE et al (2015) Improvement in oil production by increasing malonyl-CoA and glycerol-3-phosphate pools in scenedesmus quadricauda. Indian J Microbiol 55(4):447–455

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Zhang X, Agrawal A, San KY (2012) Improving fatty acid production in Escherichia coli through the overexpression of malonyl coA-Acyl carrier protein transacylase. Biotechnol Prog 28(1):60–65

    PubMed  Google Scholar 

  33. Fan Y et al (2018) Characterization of 3-ketoacyl-coA synthase in a nervonic acid producing oleaginous microalgae Mychonastes afer. Algal Res 31:225–231

    Google Scholar 

  34. Dehesh K, Tai H, Edwards P, Byrne J, Jaworski JG (2001) Overexpression of 3-ketoacyl-acyl-carrier protein synthase IIIs in plants reduces the rate of lipid synthesis. Plant Physiol 125(2):1103–1114

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Sun X-M et al (2018) Enhancement of lipid accumulation in microalgae by metabolic engineering. Biochim Biophys Acta Mol Cell Biol Lipids

    Google Scholar 

  36. Klok A et al (2014) Edible oils from microalgae: insights in TAG accumulation. Trends Biotechnol 32(10):521–528

    CAS  PubMed  Google Scholar 

  37. Shan D et al (2010) GPAT3 and GPAT4 are regulated by insulin-stimulated phosphorylation and play distinct roles in adipogenesis. J Lipid Res 51(7):1971–1981. https://doi.org/10.1194/jlr.M006304

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Oelkers P et al (2002) The DGA1 gene determines a second triglyceride synthetic pathway in yeast. J Biol Chem 277(11):8877–8881

    CAS  PubMed  Google Scholar 

  39. Cases S et al (1998) Identification of a gene encoding an acyl CoA: diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. Proc Natl Acad Sci 95(22):13018–13023

    CAS  PubMed  Google Scholar 

  40. Li J et al (2014) Choreography of transcriptomes and lipidomes of nannochloropsis reveals the mechanisms of oil synthesis in microalgae. Plant Cell 26(4):1645–1665

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Niu Y-F et al (2013) Improvement of neutral lipid and polyunsaturated fatty acid biosynthesis by overexpressing a type 2 diacylglycerol acyltransferase in marine diatom Phaeodactylum tricornutum. Mar Drugs 11(11):4558–4569

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Chen CY et al (2016) Expression of type 2 diacylglycerol acyltransferse gene DGTT1 from Chlamydomonas reinhardtii enhances lipid production in Scenedesmus obliquus. Biotechnol J 11(3):336–344

    CAS  PubMed  Google Scholar 

  43. Hung C-H et al (2013) Functional study of diacylglycerol acyltransferase type 2 family in Chlamydomonas reinhardtii. FEBS Lett 587(15):2364–2370

    CAS  PubMed  Google Scholar 

  44. La Russa M et al (2012) Functional analysis of three type-2 DGAT homologue genes for triacylglycerol production in the green microalga Chlamydomonas reinhardtii. J Biotechnol 162(1):13–20

    PubMed  Google Scholar 

  45. Boyle NR, Morgan JA (2009) Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii. BMC Syst Biol 3(1):4

    PubMed  PubMed Central  Google Scholar 

  46. Ma Y-H et al (2014) Antisense knockdown of pyruvate dehydrogenase kinase promotes the neutral lipid accumulation in the diatom Phaeodactylum tricornutum. Microb Cell Fact 13(1):100

    PubMed  PubMed Central  Google Scholar 

  47. Xue J et al (2015) Genetic improvement of the microalga Phaeodactylum tricornutum for boosting neutral lipid accumulation. Metab Eng 27:1–9

    PubMed  Google Scholar 

  48. Guan Y et al (2004) Two-stage photo-biological production of hydrogen by marine green alga Platymonas subcordiformis. Biochem Eng J 19(1):69–73

    CAS  Google Scholar 

  49. Melis A, Melnicki MR (2006) Integrated biological hydrogen production. Int J Hydrogen Energy 31(11):1563–1573

    CAS  Google Scholar 

  50. Eroglu E, Melis A (2016) Microalgal hydrogen production research. Int J Hydrogen Energy 41(30):12772–12798

    CAS  Google Scholar 

  51. Gimpel JA et al (2013) Advances in microalgae engineering and synthetic biology applications for biofuel production. Curr Opin Chem Biol 17(3):489–495

    CAS  PubMed  Google Scholar 

  52. Shuba ES, Kifle D (2018) Microalgae to biofuels: ‘Promising’ alternative and renewable energy, review. Renew Sustain Energy Rev 81:743–755

    CAS  Google Scholar 

  53. Beer LL et al (2009) Engineering algae for biohydrogen and biofuel production. Curr Opin Biotechnol 20(3):264–271

    CAS  PubMed  Google Scholar 

  54. Khetkorn W et al (2017) Microalgal hydrogen production—a review. Bioresour Technol 243:1194–1206

    CAS  PubMed  Google Scholar 

  55. Surzycki R et al (2007) Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas. Proc Natl Acad Sci 104(44):17548–17553

    CAS  PubMed  Google Scholar 

  56. Dubini A, Ghirardi ML (2015) Engineering photosynthetic organisms for the production of biohydrogen. Photosynth Res 123(3):241–253

    CAS  PubMed  Google Scholar 

  57. Esquível MG et al (2011) Efficient H2 production via Chlamydomonas reinhardtii. Trends Biotechnol 29(12):595–600

    PubMed  Google Scholar 

  58. Lee H-S, Vermaas WF, Rittmann BE (2010) Biological hydrogen production: prospects and challenges. Trends Biotechnol 28(5):262–271

    CAS  PubMed  Google Scholar 

  59. 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

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Heydarizadeh P et al (2013) Plastids of marine phytoplankton produce bioactive pigments and lipids. Mar Drugs 11(9):3425–3471

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Terashima M, Specht M, Hippler M (2011) The chloroplast proteome: a survey from the Chlamydomonas reinhardtii perspective with a focus on distinctive features. Curr Genet 57(3):151–168

    CAS  PubMed  Google Scholar 

  62. Coragliotti AT et al (2011) Molecular factors affecting the accumulation of recombinant proteins in the Chlamydomonas reinhardtii chloroplast. Mol Biotechnol 48(1):60–75

    CAS  PubMed  Google Scholar 

  63. Rasala BA, Mayfield SP (2015) Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses. Photosynth Res 123(3):227–239

    CAS  PubMed  Google Scholar 

  64. Cerutti H et al (2011) RNA-mediated silencing in algae: biological roles and tools for the analysis of gene function. Eukaryot Cell 10(9):1164–1172. https://doi.org/10.1128/EC.05106-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Molnar A et al (2009) Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J 58(1):165–174

    CAS  PubMed  Google Scholar 

  66. Johanningmeier U, Fischer D (2010) Perspective for the use of genetic transformants in order to enhance the synthesis of the desired metabolites: engineering chloroplasts of microalgae for the production of bioactive compounds. In: Bio-farms for nutraceuticals. Springer, New York, pp 144–151

    Google Scholar 

  67. Purton S et al (2013) Genetic engineering of algal chloroplasts: progress and prospects. Russ J Plant Physiol 60(4):491–499

    CAS  Google Scholar 

  68. Anand V et al (2017) Proteomic approaches in microalgae: perspectives and applications. 3 Biotech 7(3):197

    PubMed  PubMed Central  Google Scholar 

  69. Banerjee C, Singh PK, Shukla P (2016) Microalgal bioengineering for sustainable energy development: recent transgenesis and metabolic engineering strategies. Biotechnol J 11(3):303–314

    CAS  PubMed  Google Scholar 

  70. Kasai Y et al (2015) Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea. Biotechnol Biofuels 8(1):94

    PubMed  PubMed Central  Google Scholar 

  71. Shin S-E et al (2016) CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii. Sci Rep 6:27810

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Sizova I et al (2013) Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases. Plant J 73(5):873–882

    CAS  PubMed  Google Scholar 

  73. Andrianantoandro E et al (2006) Synthetic biology: new engineering rules for an emerging discipline. Mol Syst Biol 2(1)

    Google Scholar 

  74. Bashir KMI et al (2016) Microalgae engineering toolbox: selectable and screenable markers. Biotechnol Bioprocess Eng 21(2):224–235

    CAS  Google Scholar 

  75. Jang Y-S et al (2012) Engineering of microorganisms for the production of biofuels and perspectives based on systems metabolic engineering approaches. Biotechnol Adv 30(5):989–1000

    CAS  PubMed  Google Scholar 

  76. Thiele I, Palsson BØ (2010) A protocol for generating a high-quality genome-scale metabolic reconstruction. Nat Protoc 5(1):93

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Chen JW et al (2017) Identification of a malonyl CoA-acyl carrier protein transacylase and its regulatory role in fatty acid biosynthesis in oleaginous microalga Nannochloropsis oceanica. Biotechnol Appl Biochem 64(5):620–626

    CAS  PubMed  Google Scholar 

  78. Li Z et al (2018) Overexpression of malonyl-CoA: ACP transacylase in Schizochytrium sp. to improve polyunsaturated fatty acid production. J Agric Food Chem 66(21):5382–5391

    CAS  PubMed  Google Scholar 

  79. Balamurugan S et al (2017) Occurrence of plastidial triacylglycerol synthesis and the potential regulatory role of AGPAT in the model diatom Phaeodactylum tricornutum. Biotechnol Biofuels 10(1):97

    PubMed  PubMed Central  Google Scholar 

  80. Yamaoka Y et al (2016) Identification of a Chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content. Plant Biotechnol J 14(11):2158–2167

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Zou L-G et al (2018) High-efficiency promoter-driven coordinated regulation of multiple metabolic nodes elevates lipid accumulation in the model microalga Phaeodactylum tricornutum. Microb Cell Fact 17(1):54

    PubMed  PubMed Central  Google Scholar 

  82. Niu Y-F et al (2016) Molecular characterization of a glycerol-3-phosphate acyltransferase reveals key features essential for triacylglycerol production in Phaeodactylum tricornutum. Biotechnol Biofuels 9(1):60

    PubMed  PubMed Central  Google Scholar 

  83. Iskandarov U et al (2016) Cloning and characterization of a GPAT-like gene from the microalga Lobosphaera incisa (Trebouxiophyceae): overexpression in Chlamydomonas reinhardtii enhances TAG production. J Appl Phycol 28(2):907–919

    CAS  Google Scholar 

  84. Wei H et al (2017) A type-I diacylglycerol acyltransferase modulates triacylglycerol biosynthesis and fatty acid composition in the oleaginous microalga, Nannochloropsis oceanica. Biotechnol Biofuels 10(1):174

    PubMed  PubMed Central  Google Scholar 

  85. Iwai M et al (2014) Enhancement of extraplastidic oil synthesis in C hlamydomonas reinhardtii using a type-2 diacylglycerol acyltransferase with a phosphorus starvation–inducible promoter. Plant Biotechnol J 12(6):808–819

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Zienkiewicz K et al (2017) Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts. Biotechnol Biofuels 10(1):8

    PubMed  PubMed Central  Google Scholar 

  87. Li D-W et al (2016) A type 2 diacylglycerol acyltransferase accelerates the triacylglycerol biosynthesis in heterokont oleaginous microalga Nannochloropsis oceanica. J Biotechnol 229:65–71

    CAS  PubMed  Google Scholar 

  88. Hsieh H-J, Su C-H, Chien L-J (2012) Accumulation of lipid production in Chlorella minutissima by triacylglycerol biosynthesis-related genes cloned from Saccharomyces cerevisiae and Yarrowia lipolytica. J Microbiol 50(3):526–534

    CAS  PubMed  Google Scholar 

  89. Beacham TA, Ali ST (2016) Growth dependent silencing and resetting of DGA1 transgene in Nannochloropsis salina. Algal Res 14:65–71

    Google Scholar 

  90. Deng X-D et al (2012) The roles of acyl-CoA: diacylglycerol acyltransferase 2 genes in the biosynthesis of triacylglycerols by the green algae Chlamydomonas reinhardtii. Mol Plant 5(4):945–947

    CAS  PubMed  Google Scholar 

  91. Trentacoste EM et al (2013) Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Proc Natl Acad Sci 110(49):19748–19753

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Microbial Biotechnology, School of Biology, College of Science, University of Tehran, Tehran, Iran

    Mohammad Pooya Naghshbandi

  2. Microbial Biotechnology Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education, and Extension Organization (AREEO), Karaj, Iran

    Meisam Tabatabaei

  3. Biofuel Research Team (BRTeam), Karaj, Iran

    Meisam Tabatabaei

  4. Department of Mechanical Engineering of Agricultural Machinery, Faculty of Agricultural Engineering and Technology, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

    Mortaza Aghbashlo

  5. Institute of Industrial Biotechnology, Government College University, Lahore, Pakistan

    Muhammad Nauman Aftab

  6. Department of Zoology, Lahore College for Women University, Lahore, Pakistan

    Irfana Iqbal

Authors
  1. Mohammad Pooya Naghshbandi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Meisam Tabatabaei
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mortaza Aghbashlo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Muhammad Nauman Aftab
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Irfana Iqbal
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Meisam Tabatabaei or Mortaza Aghbashlo .

Editor information

Editors and Affiliations

  1. Finnish Environment Institute, Helsinki, Finland

    Kristian Spilling

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media New York

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Naghshbandi, M.P., Tabatabaei, M., Aghbashlo, M., Aftab, M.N., Iqbal, I. (2019). Metabolic Engineering of Microalgae for Biofuel Production. In: Spilling, K. (eds) Biofuels from Algae. Methods in Molecular Biology, vol 1980. Humana, New York, NY. https://doi.org/10.1007/7651_2018_205

Download citation

  • DOI: https://doi.org/10.1007/7651_2018_205

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9415-1

  • Online ISBN: 978-1-4939-9416-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation