Advertisement

pp 1-20 | Cite as

Metabolic Engineering of Microalgae for Biofuel Production

  • Mohammad Pooya Naghshbandi
  • Meisam TabatabaeiEmail author
  • Mortaza AghbashloEmail author
  • Muhammad Nauman Aftab
  • Irfana Iqbal
Protocol
Part of the Methods in Molecular Biology book series

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.

Keywords

Microalgae Biodiesel Biohydrogen Genetic engineering Metabolic engineering 

References

  1. 1.
    Gimpel JA, Henríquez V, Mayfield SP (2015) In metabolic engineering of eukaryotic microalgae: potential and challenges come with great diversity. Front Microbiol 6:1376Google Scholar
  2. 2.
    Stephens E et al (2010) An economic and technical evaluation of microalgal biofuels. Nat Biotechnol 28(2):126Google Scholar
  3. 3.
    Peng K et al (2018) The bioeconomy of microalgal biofuels. In: Energy from microalgae. Springer, New York, pp 157–169Google Scholar
  4. 4.
    Cheng JJ, Timilsina GR (2011) Status and barriers of advanced biofuel technologies: a review. Renew Energy 36(12):3541–3549Google Scholar
  5. 5.
    Singh J, Gu S (2010) Commercialization potential of microalgae for biofuels production. Renew Sustain Energy Rev 14(9):2596–2610Google Scholar
  6. 6.
    Banerjee C, Dubey KK, Shukla P (2016) Metabolic engineering of microalgal based biofuel production: prospects and challenges. Front Microbiol 7:432Google Scholar
  7. 7.
    Brown LM, Zeiler KG (1993) Aquatic biomass and carbon dioxide trapping. Energ Conver Manage 34(9-11):1005–1013Google Scholar
  8. 8.
    Chisti Y (2008) Biodiesel from microalgae beats bioethanol. Trends Biotechnol 26(3):126–131Google Scholar
  9. 9.
    Schenk PM et al (2008) Second generation biofuels: high-efficiency microalgae for biodiesel production. Bioenergy Res 1(1):20–43Google Scholar
  10. 10.
    Greenwell H et al (2010) Placing microalgae on the biofuels priority list: a review of the technological challenges. J R Soc Interface 7:703–726Google Scholar
  11. 11.
    Pienkos PT, Darzins A (2009) The promise and challenges of microalgal-derived biofuels. Biofuels Bioprod Biorefin 3(4):431–440Google Scholar
  12. 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 50:1239–1253Google Scholar
  13. 13.
    Ho S-H et al (2014) Perspectives on engineering strategies for improving biofuel production from microalgae—a critical review. Biotechnol Adv 32(8):1448–1459Google Scholar
  14. 14.
    Zhang F, Rodriguez S, Keasling JD (2011) Metabolic engineering of microbial pathways for advanced biofuels production. Curr Opin Biotechnol 22(6):775–783Google Scholar
  15. 15.
    Liu D, Evans T, Zhang F (2015) Applications and advances of metabolite biosensors for metabolic engineering. Metab Eng 31:35–43Google Scholar
  16. 16.
    Majidian P, Tabatabaei M, Zeinolabedini M, Naghshbandi MP, Chisti Y (2018) Metabolic engineering of microorganisms for biofuel production. Renew Sustain Energy Rev 82:3863–3885Google Scholar
  17. 17.
    Jagadevan S et al (2018) Recent developments in synthetic biology and metabolic engineering in microalgae towards biofuel production. Biotechnol Biofuels 11(1):185Google Scholar
  18. 18.
    Costa JAV, De Morais MG (2011) The role of biochemical engineering in the production of biofuels from microalgae. Bioresour Technol 102(1):2–9Google Scholar
  19. 19.
    Zorrilla López U et al (2013) Engineering metabolic pathways in plants by multigene transformation. Int J Dev Biol 57(6–8):565–576Google Scholar
  20. 20.
    Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25(3):294–306Google Scholar
  21. 21.
    Gaurav N et al (2017) Utilization of bioresources for sustainable biofuels: a review. Renew Sustain Energy Rev 73:205–214Google Scholar
  22. 22.
    Bellou S et al (2014) Microalgal lipids biochemistry and biotechnological perspectives. Biotechnol Adv 32(8):1476–1493Google Scholar
  23. 23.
    Yu W-L et al (2011) Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae. Microb Cell Fact 10(1):91Google Scholar
  24. 24.
    Radakovits R et al (2010) Genetic engineering of algae for enhanced biofuel production. Eukaryot Cell 9(4):486–501Google Scholar
  25. 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):1858–1865Google Scholar
  26. 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):923–930Google Scholar
  27. 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 16:54–62Google Scholar
  28. 28.
    Liang M-H, Jiang J-G (2013) Advancing oleaginous microorganisms to produce lipid via metabolic engineering technology. Prog Lipid Res 52(4):395–408Google Scholar
  29. 29.
    Dunahay TG et al (1996) Manipulation of microalgal lipid production using genetic engineering. Appl Biochem Biotechnol 57(1):223Google Scholar
  30. 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 328Google Scholar
  31. 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):447–455Google Scholar
  32. 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):60–65Google Scholar
  33. 33.
    Fan Y et al (2018) Characterization of 3-ketoacyl-coA synthase in a nervonic acid producing oleaginous microalgae Mychonastes afer. Algal Res 31:225–231Google Scholar
  34. 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):1103–1114Google Scholar
  35. 35.
    Sun X-M et al (2018) Enhancement of lipid accumulation in microalgae by metabolic engineering. Biochim Biophys Acta Mol Cell Biol LipidsGoogle Scholar
  36. 36.
    Klok A et al (2014) Edible oils from microalgae: insights in TAG accumulation. Trends Biotechnol 32(10):521–528Google Scholar
  37. 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.M006304CrossRefGoogle Scholar
  38. 38.
    Oelkers P et al (2002) The DGA1 gene determines a second triglyceride synthetic pathway in yeast. J Biol Chem 277(11):8877–8881Google Scholar
  39. 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):13018–13023Google Scholar
  40. 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):1645–1665Google Scholar
  41. 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):4558–4569Google Scholar
  42. 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):336–344Google Scholar
  43. 43.
    Hung C-H et al (2013) Functional study of diacylglycerol acyltransferase type 2 family in Chlamydomonas reinhardtii. FEBS Lett 587(15):2364–2370Google Scholar
  44. 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):13–20Google Scholar
  45. 45.
    Boyle NR, Morgan JA (2009) Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii. BMC Syst Biol 3(1):4Google Scholar
  46. 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):100Google Scholar
  47. 47.
    Xue J et al (2015) Genetic improvement of the microalga Phaeodactylum tricornutum for boosting neutral lipid accumulation. Metab Eng 27:1–9Google Scholar
  48. 48.
    Guan Y et al (2004) Two-stage photo-biological production of hydrogen by marine green alga Platymonas subcordiformis. Biochem Eng J 19(1):69–73Google Scholar
  49. 49.
    Melis A, Melnicki MR (2006) Integrated biological hydrogen production. Int J Hydrogen Energy 31(11):1563–1573Google Scholar
  50. 50.
    Eroglu E, Melis A (2016) Microalgal hydrogen production research. Int J Hydrogen Energy 41(30):12772–12798Google Scholar
  51. 51.
    Gimpel JA et al (2013) Advances in microalgae engineering and synthetic biology applications for biofuel production. Curr Opin Chem Biol 17(3):489–495Google Scholar
  52. 52.
    Shuba ES, Kifle D (2018) Microalgae to biofuels: ‘Promising’ alternative and renewable energy, review. Renew Sustain Energy Rev 81:743–755Google Scholar
  53. 53.
    Beer LL et al (2009) Engineering algae for biohydrogen and biofuel production. Curr Opin Biotechnol 20(3):264–271Google Scholar
  54. 54.
    Khetkorn W et al (2017) Microalgal hydrogen production—a review. Bioresour Technol 243:1194–1206Google Scholar
  55. 55.
    Surzycki R et al (2007) Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas. Proc Natl Acad Sci 104(44):17548–17553Google Scholar
  56. 56.
    Dubini A, Ghirardi ML (2015) Engineering photosynthetic organisms for the production of biohydrogen. Photosynth Res 123(3):241–253Google Scholar
  57. 57.
    Esquível MG et al (2011) Efficient H2 production via Chlamydomonas reinhardtii. Trends Biotechnol 29(12):595–600Google Scholar
  58. 58.
    Lee H-S, Vermaas WF, Rittmann BE (2010) Biological hydrogen production: prospects and challenges. Trends Biotechnol 28(5):262–271Google Scholar
  59. 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):1373–1383Google Scholar
  60. 60.
    Heydarizadeh P et al (2013) Plastids of marine phytoplankton produce bioactive pigments and lipids. Mar Drugs 11(9):3425–3471Google Scholar
  61. 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):151–168Google Scholar
  62. 62.
    Coragliotti AT et al (2011) Molecular factors affecting the accumulation of recombinant proteins in the Chlamydomonas reinhardtii chloroplast. Mol Biotechnol 48(1):60–75Google Scholar
  63. 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):227–239Google Scholar
  64. 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.05106-11CrossRefGoogle Scholar
  65. 65.
    Molnar A et al (2009) Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J 58(1):165–174Google Scholar
  66. 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 144–151Google Scholar
  67. 67.
    Purton S et al (2013) Genetic engineering of algal chloroplasts: progress and prospects. Russ J Plant Physiol 60(4):491–499Google Scholar
  68. 68.
    Anand V et al (2017) Proteomic approaches in microalgae: perspectives and applications. 3 Biotech 7(3):197Google Scholar
  69. 69.
    Banerjee C, Singh PK, Shukla P (2016) Microalgal bioengineering for sustainable energy development: recent transgenesis and metabolic engineering strategies. Biotechnol J 11(3):303–314Google Scholar
  70. 70.
    Kasai Y et al (2015) Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea. Biotechnol Biofuels 8(1):94Google Scholar
  71. 71.
    Shin S-E et al (2016) CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii. Sci Rep 6:27810Google Scholar
  72. 72.
    Sizova I et al (2013) Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases. Plant J 73(5):873–882Google Scholar
  73. 73.
    Andrianantoandro E et al (2006) Synthetic biology: new engineering rules for an emerging discipline. Mol Syst Biol 2(1)Google Scholar
  74. 74.
    Bashir KMI et al (2016) Microalgae engineering toolbox: selectable and screenable markers. Biotechnol Bioprocess Eng 21(2):224–235Google Scholar
  75. 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):989–1000Google Scholar
  76. 76.
    Thiele I, Palsson BØ (2010) A protocol for generating a high-quality genome-scale metabolic reconstruction. Nat Protoc 5(1):93Google Scholar
  77. 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):620–626Google Scholar
  78. 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):5382–5391Google Scholar
  79. 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):97Google Scholar
  80. 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):2158–2167Google Scholar
  81. 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):54Google Scholar
  82. 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):60Google Scholar
  83. 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):907–919Google Scholar
  84. 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):174Google Scholar
  85. 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):808–819Google Scholar
  86. 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):8Google Scholar
  87. 87.
    Li D-W et al (2016) A type 2 diacylglycerol acyltransferase accelerates the triacylglycerol biosynthesis in heterokont oleaginous microalga Nannochloropsis oceanica. J Biotechnol 229:65–71Google Scholar
  88. 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):526–534Google Scholar
  89. 89.
    Beacham TA, Ali ST (2016) Growth dependent silencing and resetting of DGA1 transgene in Nannochloropsis salina. Algal Res 14:65–71Google Scholar
  90. 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):945–947Google Scholar
  91. 91.
    Trentacoste EM et al (2013) Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Proc Natl Acad Sci 110(49):19748–19753Google Scholar

Copyright information

© Springer Science+Business Media New York 2019

Authors and Affiliations

  • Mohammad Pooya Naghshbandi
    • 1
  • Meisam Tabatabaei
    • 2
    • 3
    Email author
  • Mortaza Aghbashlo
    • 4
    Email author
  • Muhammad Nauman Aftab
    • 5
  • Irfana Iqbal
    • 6
  1. 1.Department of Microbial Biotechnology, School of Biology, College of ScienceUniversity of TehranTehranIran
  2. 2.Microbial Biotechnology DepartmentAgricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education, and Extension Organization (AREEO)KarajIran
  3. 3.Biofuel Research Team (BRTeam)KarajIran
  4. 4.Department of Mechanical Engineering of Agricultural Machinery, Faculty of Agricultural Engineering and Technology, College of Agriculture and Natural ResourcesUniversity of TehranKarajIran
  5. 5.Institute of Industrial BiotechnologyGovernment College UniversityLahorePakistan
  6. 6.Department of ZoologyLahore College for Women UniversityLahorePakistan

Personalised recommendations