Cofactor engineering in cyanobacteria to overcome imbalance between NADPH and NADH: A mini review

  • Jongmoon Park
  • Yunnam Choi
Review Article


Cyanobacteria can produce useful renewable fuels and high-value chemicals using sunlight and atmospheric carbon dioxide by photosynthesis. Genetic manipulation has increased the variety of chemicals that cyanobacteria can produce. However, their uniquely abundant NADPH-pool, in other words insufficient supply of NADH, tends to limit their production yields in case of utilizing NADH-dependent enzyme, which is quite common in heterotrophic microbes. To overcome this cofactor imbalance and enhance cyanobacterial fuel and chemical production, various approaches for cofactor engineering have been employed. In this review, we focus on three approaches: (1) utilization of NADPH-dependent enzymes, (2) increasing NADH production, and (3) changing cofactor specificity of NADH-dependent enzymes from NADH to NADPH.


NADH-dependent enzyme NADPH-dependent enzyme transhydrogenase site-directed mutagenesis enzyme engineering 


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This research was supported by the Marine Biotechnology Program (Marine BioMaterials Research Center) funded by the Ministry of Oceans and Fisheries, Korea, and BK21+ program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology. This work was also conducted under the framework of Research and Development Program of the Korea Institute of Energy Research (KIER) (B4-2474-02).


  1. 1.
    Parmar A, Singh N K, Pandey A, Gnansounou E, Madamwar D. Cyanobacteria and microalgae: A positive prospect for biofuels. Bioresource Technology, 2011, 102(22): 10163–10172CrossRefPubMedGoogle Scholar
  2. 2.
    Machado I M, Atsumi S. Cyanobacterial biofuel production. Journal of Biotechnology, 2012, 162(1): 50–56CrossRefPubMedGoogle Scholar
  3. 3.
    Nozzi N E, Oliver J W, Atsumi S. Cyanobacteria as a platform for biofuel production. Frontiers in Bioengineering and Biotechnology, 2013, 1: 1–6CrossRefGoogle Scholar
  4. 4.
    Deng M D, Coleman J R. Ethanol synthesis by genetic engineering in cyanobacteria. Applied and Environmental Microbiology, 1999, 65(2): 523–528PubMedPubMedCentralGoogle Scholar
  5. 5.
    Dexter J, Fu P. Metabolic engineering of cyanobacteria for ethanol production. Energy & Environmental Science, 2009, 2(8): 857–864CrossRefGoogle Scholar
  6. 6.
    Gao Z, Zhao H, Li Z, Tan X, Lu X. Photosynthetic production of ethanol from carbon dioxide in genetically engineered cyanobacteria. Energy & Environmental Science, 2012, 5(12): 9857–9865CrossRefGoogle Scholar
  7. 7.
    Choi Y N, Park J M. Enhancing biomass and ethanol production by increasing NADPH production in Synechocystis sp. PCC 6803. Bioresource Technology, 2016, 213: 54–57CrossRefPubMedGoogle Scholar
  8. 8.
    Kusakabe T, Tatsuke T, Tsuruno K, Hirokawa Y, Atsumi S, Liao J C, Hanai T. Engineering a synthetic pathway in cyanobacteria for isopropanol production directly from carbon dioxide and light. Metabolic Engineering, 2013, 20: 101–108CrossRefPubMedGoogle Scholar
  9. 9.
    Lan E I, Liao J C. Metabolic engineering of cyanobacteria for 1-butanol production from carbon dioxide. Metabolic Engineering, 2011, 13(4): 353–363CrossRefPubMedGoogle Scholar
  10. 10.
    Lan E I, Liao J C. ATP drives direct photosynthetic production of 1-butanol in cyanobacteria. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(16): 6018–6023CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Atsumi S, Higashide W, Liao J C. Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nature Biotechnology, 2009, 27(12): 1177–1180CrossRefPubMedGoogle Scholar
  12. 12.
    Angermayr S A, Paszota M, Hellingwerf K J. Engineering a cyanobacterial cell factory for production of lactic acid. Applied and Environmental Microbiology, 2012, 78(19): 7098–7106CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Varman A M, Yu Y, You L, Tang Y J. Photoautotrophic production of D-lactic acid in an engineered cyanobacterium. Microbial Cell Factories, 2013, 12(1): 1–8CrossRefGoogle Scholar
  14. 14.
    Zhou J, Zhang H, Meng H, Zhang Y, Li Y. Production of optically pure D-lactate from CO2 by blocking the PHB and acetate pathways and expressing D-lactate dehydrogenase in cyanobacterium Synechocystis sp. PCC 6803. Process Biochemistry, 2014, 49(12): 2071–2077CrossRefGoogle Scholar
  15. 15.
    Angermayr S A, Van der Woude A D, Correddu D, Vreugdenhil A, Verrone V, Hellingwerf K J. Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis sp. PCC6803. Biotechnology for Biofuels, 2014, 7(1): 1–15CrossRefGoogle Scholar
  16. 16.
    Li C, Tao F, Ni J, Wang Y, Yao F, Xu P. Enhancing the light-driven production of D-lactate by engineering cyanobacterium using a combinational strategy. Scientific Reports, 2015, 5: 1–11CrossRefGoogle Scholar
  17. 17.
    Miyake M, Takase K, Narato M, Khatipov E, Schnackenberg J, Shirai M, Kurane R, Asada Y. Polyhydroxybutyrate production from carbon dioxide by cyanobacteria. Applied Biochemistry and Biotechnology, 2000, 84–86(1-9): 991–1002CrossRefPubMedGoogle Scholar
  18. 18.
    Tyo K E, Jin Y S, Espinoza F A, Stephanopoulos G. Identification of gene disruptions for increased poly-3-hydroxybutyrate accumulation in Synechocystis PCC 6803. Biotechnology Progress, 2009, 25(5): 1236–1243CrossRefPubMedGoogle Scholar
  19. 19.
    Zhou J, Zhu T, Cai Z, Li Y. From cyanochemicals to cyanofactories: A review and perspective. Microbial Cell Factories, 2016, 15(1): 1–9CrossRefGoogle Scholar
  20. 20.
    Wang Y, San K Y, Bennett G N. Cofactor engineering for advancing chemical biotechnology. Current Opinion in Biotechnology, 2013, 24(6): 994–999, 99CrossRefPubMedGoogle Scholar
  21. 21.
    Akhtar MK, Jones P R. Cofactor Engineering for enhancing the flux of metabolic pathways. Frontiers in Bioengineering and Biotechnology, 2014, 2: 1–6CrossRefGoogle Scholar
  22. 22.
    Tamoi M, Miyazaki T, Fukamizo T, Shigeoka S. The calvin cycle in cyanobacteria is regulated by CP12 via the NAD(H)/NADP(H) ratio under light/dark conditions. Plant Journal, 2005, 42(4): 504–513CrossRefPubMedGoogle Scholar
  23. 23.
    Cooley J W, Vermaas W F. Succinate dehydrogenase and other respiratory pathways in thylakoid membranes of Synechocystis sp. strain PCC 6803: Capacity comparisons and physiological function. Journal of Bacteriology, 2001, 183(14): 4251–4258CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Dempo Y, Ohta E, Nakayama Y, Bamba T, Fukusaki E. Molarbased targeted metabolic profiling of cyanobacterial strains with potential for biological production. Metabolites, 2014, 4(2): 499–516CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Hirokawa Y, Maki Y, Tatsuke T, Hanai T. Cyanobacterial production of 1,3-propanediol directly from carbon dioxide using a synthetic metabolic pathway. Metabolic Engineering, 2016, 34: 97–103CrossRefPubMedGoogle Scholar
  26. 26.
    Li H, Liao J C. Engineering a cyanobacterium as the catalyst for the photosynthetic conversion of CO2 to 1,2-propanediol. Microbial Cell Factories, 2013, 12(1): 1–9CrossRefGoogle Scholar
  27. 27.
    Oliver J W, Machado I M, Yoneda H, Atsumi S. Cyanobacterial conversion of carbon dioxide to 2,3-butanediol. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(4): 1249–1254CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Savakis P E, Angermayr S A, Hellingwerf K J. Synthesis of 2,3-butanediol by Synechocystis sp. PCC 6803 via heterologous expression of a catabolic pathway from lactic acid-and enterobacteria. Metabolic Engineering, 2013, 20: 121–130CrossRefPubMedGoogle Scholar
  29. 29.
    Niederholtmeyer H, Wolfstadter B T, Savage D F, Silver P A, Way J C. Engineering cyanobacteria to synthesize and export hydrophilic products. Applied and Environmental Microbiology, 2010, 76(11): 3462–3466CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    McNeely K, Xu Y, Bennette N, Bryant D A, Dismukes G C. Redirecting reductant flux into hydrogen production via metabolic engineering of fermentative carbon metabolism in a cyanobacterium. Applied and Environmental Microbiology, 2010, 76(15): 5032–5038CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Kumaraswamy G K, Guerra T, Qian X, Zhang S, Bryant D A, Dismukes G C. Reprogramming the glycolytic pathway for increased hydrogen production in cyanobacteria: Metabolic engineering of NAD+-dependent GAPDH. Energy & Environmental Science, 2013, 6(12): 3722–3731CrossRefGoogle Scholar
  32. 32.
    Jarboe L R, Yqh D. A broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals. Applied Microbiology and Biotechnology, 2011, 89(2): 249–257CrossRefPubMedGoogle Scholar
  33. 33.
    Wee Y J, Kim J N, Ryu H W. Biotechnological production of lactic acid and its recent applications. Food Technology and Biotechnology, 2006, 44(2): 163–172Google Scholar
  34. 34.
    Joseph A, Aikawa S, Sasaki K, Tsuge Y, Matsuda F, Tanaka T, Kondo A. Utilization of lactic acid bacterial genes in Synechocystis sp. PCC 6803 in the production of lactic acid. Bioscience, Biotechnology, and Biochemistry, 2013, 77(5): 966–970CrossRefPubMedGoogle Scholar
  35. 35.
    Polizzi K M, Chaparro-Riggers J F, Vazquez-Figueroa E, Bommarius A S. Structure-guided consensus approach to create a more thermostable penicillin G acylase. Biotechnology Journal, 2006, 1(5): 531–536CrossRefPubMedGoogle Scholar
  36. 36.
    Terao Y, Miyamoto K, Ohta H. Introduction of single mutation changes arylmalonate decarboxylase to racemase. Chemical Communications, 2006, 34(34): 3600–3602CrossRefGoogle Scholar
  37. 37.
    Vázquez-Figueroa E, Chaparro-Riggers J, Bommarius A S. Development of a thermostable glucose dehydrogenase by a structure-guided consensus concept. ChemBioChem, 2007, 8(18): 2295–2301CrossRefPubMedGoogle Scholar
  38. 38.
    Jochens H, Bornscheuer U T. Natural diversity to guide focused directed evolution. ChemBioChem, 2010, 11(13): 1861–1866CrossRefPubMedGoogle Scholar
  39. 39.
    Ema T, Nakano Y, Yoshida D, Kamata S, Sakai T. Redesign of enzyme for improving catalytic activity and enantioselectivity toward poor substrates: Manipulation of the transition state. Organic & Biomolecular Chemistry, 2012, 10(31): 6299–6308CrossRefGoogle Scholar
  40. 40.
    Holmberg N, Ryde U, Bulow L. Redesign of the coenzyme specificity in l-lactate dehydrogenase from bacillus stearothermophilus using site-directed mutagenesis and media engineering. Protein Engineering, Design & Selection, 1999, 12(10): 851–856CrossRefGoogle Scholar
  41. 41.
    Ma C, Zhang L, Dai J, Xiu Z. Relaxing the coenzyme specificity of 1,3-propanediol oxidoreductase from Klebsiella pneumoniae by rational design. Journal of Biotechnology, 2010, 146(4): 173–178CrossRefPubMedGoogle Scholar
  42. 42.
    Richter N, Zienert A, Hummel W. A single-point mutation enables lactate dehydrogenase from Bacillus subtilis to utilize NAD+ and NADP+ as cofactor. Engineering in Life Sciences, 2011, 11(1): 26–36CrossRefGoogle Scholar
  43. 43.
    Meng H, Liu P, Sun H, Cai Z, Zhou J, Lin J, Li Y. Engineering a Dlactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors. Scientific Reports, 2016, 6: 1–8CrossRefGoogle Scholar
  44. 44.
    Steiner K, Schwab H. Recent advances in rational approaches for enzyme engineering. Computational and Structural Biotechnology Journal, 2012, 2(3): 1–12CrossRefGoogle Scholar
  45. 45.
    Li Y, Cirino P C. Recent advances in engineering proteins for biocatalysis. Biotechnology and Bioengineering, 2014, 111(7): 1273–1287CrossRefPubMedGoogle Scholar

Copyright information

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Department of Chemical EngineeringPohang University of Science and TechnologyGyeongbukKorea
  2. 2.School of Environmental Science and EngineeringPohang University of Science and TechnologyGyeongbukKorea
  3. 3.Division of Advanced Nuclear EngineeringPohang University of Science and TechnologyGyeongbukKorea

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