Genetic modification of lignin biosynthesis for improved biofuel production

  • Hiroshi Hisano
  • Rangaraj Nandakumar
  • Zeng-Yu WangEmail author
Invited Review


The energy in cellulosic biomass largely resides in plant cell walls. Cellulosic biomass is more difficult than starch to break down into sugars because of the presence of lignin and the complex structure of cell walls. Transgenic down-regulation of major lignin genes led to reduced lignin content, increased dry matter degradability, and improved accessibility of cellulases for cellulose degradation. This review provides background information on lignin biosynthesis and focuses on genetic manipulation of lignin genes in important monocot species as well as the dicot potential biofuel crop alfalfa. Reduction of lignin in biofuel crops by genetic engineering is likely one of the most effective ways of reducing costs associated with pretreatment and hydrolysis of cellulosic feedstocks, although some potential fitness issues should also be addressed.


Biomass Biofuel crops Genetic engineering Lignin modification 



This work was supported by the BioEnergy Science Center and the Samuel Roberts Noble Foundation. The BioEnergy Science Center is supported by the Office of Biological and Environmental Research in the DOE Office of Science.


  1. Barriere Y. O.; Argiller O. Brown-midrib genes of maize: A review. Agronomie 13: 865–876; 1993. doi: 10.1051/agro:19931001.CrossRefGoogle Scholar
  2. Barriere Y.; Ralph J.; Mechin V.; Guillaumie S.; Grabber J. H.; Argillier O.; Chabbert B.; Lapierre C. Genetic and molecular basis of grass cell wall biosynthesis and degradability: II. Lessons from brown-midrib mutants. C R Biol 327: 847–860; 2004. doi: 10.1016/j.crvi.2004.05.010.PubMedCrossRefGoogle Scholar
  3. Baucher M.; Bernard-Vailhe M. A.; Chabbert B.; Besle J. M.; Opsomer C.; Van Montagu M.; Botterman J. Downregulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility. Plant Mol Biol 39: 437–447; 1999. doi: 10.1023/A:1006182925584.PubMedCrossRefGoogle Scholar
  4. Baucher M.; Monties B.; Van Montagu M.; Boerjan W. Biosynthesis and genetic engineering of lignin. Crit Rev Plant Sci 17: 125–197; 1998. doi: 10.1016/S0735-2689(98)00360-8.CrossRefGoogle Scholar
  5. Biswas G.; Ransom C.; Sticklen M. Expression of biologically active Acidothermus cellulolyticus endoglucanase in transgenic maize. Plant Sci 171: 617–623; 2006. doi: 10.1016/j.plantsci.2006.06.004.CrossRefGoogle Scholar
  6. Boudet A. M.; Kajita S.; Grima-Pettenati J.; Goffner D. Lignins and lignocellulosics: a better control of synthesis for new and improved uses. Trends Plant Sci 8: 576–581; 2003. doi: 10.1016/j.tplants.2003.10.001.PubMedCrossRefGoogle Scholar
  7. Bouton J. H. Molecular breeding of switchgrass as a bioenergy crop. Curr Opin Gen Develop 17: 553–558; 2007. doi: 10.1016/j.gde.2007.08.012.CrossRefGoogle Scholar
  8. Chapple C.; Ladisch M.; Melian R. Loosening lignin’s grip on biofuel production. Nat Biotechnol 25: 746–747; 2007. doi: 10.1038/nbt0707-746.PubMedCrossRefGoogle Scholar
  9. Capell T.; Christou P. Progress in plant metabolic engineering. Curr Opin Biotechnol 15: 148–154; 2004. doi: 10.1016/j.copbio.2004.01.009.PubMedCrossRefGoogle Scholar
  10. Chen F.; Dixon R. A. Lignin modification improves fermentable sugar yields for biofuel production. Nat Biotechnol 25: 759–761; 2007. doi: 10.1038/nbt1316.PubMedCrossRefGoogle Scholar
  11. Chen F.; Reddy M. S. S.; Temple S.; Jackson L.; Shadle G.; Dixon R. A. Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wall-bound ferulic acid in alfalfa (Medicago sativa L.). Plant J 48: 113–124; 2006. doi: 10.1111/j.1365-313X.2006.02857.x.PubMedCrossRefGoogle Scholar
  12. Chen L.; Auh C.; Chen F.; Cheng X. F.; Aljoe H.; Dixon R. A.; Wang Z. Y. Lignin deposition and associated changes in anatomy, enzyme activity, gene expression and ruminal degradability in stems of tall fescue at different developmental stages. J Agric Food Chem 50: 5558–5565; 2002. doi: 10.1021/jf020516x.PubMedCrossRefGoogle Scholar
  13. Chen L.; Auh C.; Dowling P.; Bell J.; Lehmann D.; Wang Z. Y. Transgenic down-regulation of caffeic acid O-methyltransferase (COMT) led to improved digestibility in tall fescue (Festuca arundinacea). Funct Plant Biol 31: 235–245; 2004. doi: 10.1071/FP03254.CrossRefGoogle Scholar
  14. Chen L.; Auh C. K.; Dowling P.; Bell J.; Chen F.; Hopkins A.; Dixon R. A.; Wang Z. Y. Improved forage digestibility of tall fescue (Festuca arundinacea) by transgenic down-regulation of cinnamyl alcohol dehydrogenase. Plant Biotechnol J 1: 437–449; 2003. doi: 10.1046/j.1467-7652.2003.00040.x.PubMedCrossRefGoogle Scholar
  15. Cherney J. H.; Cherney D. J. R.; Akin D. E.; Axtell J. D. Potential of brown-midrib, low-lignin mutants for improving forage quality. Adv Agron 46: 157–198; 1991. doi: 10.1016/S0065-2113(08)60580-5.CrossRefGoogle Scholar
  16. Conger, B. V.; Songstad, D. D.; McDaniel, J. K.; Bond, J. Genetic transformation of Dactylis glomerata by microprojectile bombardment. Proc XVII Intl Grasslands Congr. 1034–1036; 1993.Google Scholar
  17. Denchev P. D.; Songstad D. D.; McDaniel J. K.; Conger B. V. Transgenic orchardgrass (Dactylis glomerata) plants by direct embryogenesis from microprojectile bombarded leaf cells. Plant Cell Rep 16: 813–819; 1997. doi: 10.1007/s002990050326.CrossRefGoogle Scholar
  18. Dixon R. A.; Bouton J. H.; Narasimhamoorthy B.; Saha M.; Wang Z. Y.; May G. D. Beyond structural genomics for plant science. Adv. Agron 95: 77–161; 2007. doi: 10.1016/S0065-2113(07)95002-6.CrossRefGoogle Scholar
  19. Dixon R. A.; Chen F.; Gua D.; Parvathi K. The biosynthesis of monolignols: a “metabolic grid” or independent pathways to guaiacyl and syringyl units? Phytochemistry 57: 1069–1084; 2001. doi: 10.1016/S0031-9422(01)00092-9.PubMedCrossRefGoogle Scholar
  20. Ericksson M. E.; Israelsson M.; Olsson O.; Moritiz T. Increased gibberellin biosynthesis in transgenic trees promotes growth, biomass production and xylem fiber length. Nat Biotechnol 18: 784–788; 2000. doi: 10.1038/77355.CrossRefGoogle Scholar
  21. Grand C.; Parmentier P.; Boudet A.; Boudet A. M. Comparison of lignins and of enzymes involved in lignification in normal and brown midrib (bm3) mutant corn seedlings. Physiologie Végétale 23: 905–911; 1985.Google Scholar
  22. Gray K. A.; Zhao L.; Emptage M. Bioethanol Curr Opin Chem Biol 10: 141–146; 2006. doi: 10.1016/j.cbpa.2006.02.035.CrossRefGoogle Scholar
  23. Gressel J. Transgenics are imperative for biofuel crops. Plant Sci 174: 246–263; 2008. doi: 10.1016/j.plantsci.2007.11.009.CrossRefGoogle Scholar
  24. Guillaumie S.; Goffner D.; Barbier O.; Martinant J. P.; Pichon M.; Barrière Y. Expression of cell wall related genes in basal and ear internodes of silking brown-midrib-3, caffeic acid O-methyltransferase (COMT) down-regulated and normal maize plants. BMC Plant Biol 8: 71; 2008. doi: 10.1186/1471-2229-8-71.PubMedCrossRefGoogle Scholar
  25. Guillaumie S.; Pichon M.; Martinant J. P.; Bosio M.; Goffner D.; Barriere Y. Differential expression of phenylpropanoid and related genes in brown-midrib bm1, bm2, bm3, and bm4 young near-isogenic maize plants. Planta 226: 235–250; 2007. doi: 10.1007/s00425-006-0468-9.PubMedCrossRefGoogle Scholar
  26. Guo D.; Chen F.; Inoue K.; Blount J. W.; Dixon R. A. Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa: impacts on lignin structure and implications for the biosynthesis of G and S lignin. Plant Cell 13: 73–88; 2001a.PubMedCrossRefGoogle Scholar
  27. Guo D.; Chen F.; Wheeler J.; Winder J.; Selman S.; Peterson M.; Dixon R. A. Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases. Transgenic Res 10: 457–464; 2001b. doi: 10.1023/A:1012278106147.PubMedCrossRefGoogle Scholar
  28. Halpin C.; Holt K.; Chojecki J.; Oliver D.; Chabbert B.; Monties B.; Edwards K.; Barakate A.; Foxon G. A. Brown-midrib maize (bm1)—a mutation affecting the cinnamyl alcohol dehydrogenase gene. Plant J 14: 545–553; 1998. doi: 10.1046/j.1365-313X.1998.00153.x.PubMedCrossRefGoogle Scholar
  29. He X.; Hall M. B.; Gallo-Meagher M.; Smith R. L. Improvement of forage quality by downregulation of maize O-methyltransferase. Crop Sci 43: 2240–2251; 2003.Google Scholar
  30. Iiyama K.; Lam T. B. T. Structural characteristics of cell walls of forage grasses: their nutritional evaluation for ruminants. Asian-Austr J Anim Sci 14: 862–879; 2001.Google Scholar
  31. Ishida Y.; Saito H.; Ohta S.; Hiei Y.; Komari T.; Kumashiro T. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14: 745–750; 1996. doi: 10.1038/nbt0696-745.PubMedCrossRefGoogle Scholar
  32. Keating J. D.; Panganiban C.; Mansfield S. D. Tolerance and adaptation of ethanologenic yeasts to lignocellulosic inhibitory compounds. Biotechnol Bioeng 93: 1196–1206; 2006. doi: 10.1002/bit.20838.PubMedCrossRefGoogle Scholar
  33. Koonin S. E. Getting serious about biofuels. Science 311: 435; 2006. doi: 10.1126/science.1124886.PubMedCrossRefGoogle Scholar
  34. Kuc J.; Nelson O. E. The abnormal lignins produced by the brown midrib mutants of maize. I. The brown midrib mutant. Arch Biochem Biophys 105: 103; 1964. doi: 10.1016/0003-9861(64)90240-1.PubMedCrossRefGoogle Scholar
  35. Li X.; Weng J. K.; Chapple C. Improvement of biomass through lignin modification. Plant J 54: 569–581; 2008. doi: 10.1111/j.1365-313X.2008.03457.x.PubMedCrossRefGoogle Scholar
  36. Li Y.; Kajita S.; Kawai S.; Katayama Y.; Morohoshi N. Down-regulation of an anionic peroxidase in transgenic aspen and its effect on lignin characteristics. J Plant Res 116: 175–182; 2003. doi: 10.1007/s10265-003-0087-5.PubMedCrossRefGoogle Scholar
  37. Lu R.; Martin-Hernandez A. M.; Peart J. R.; Malcuit I.; Baulcombe D. C. Virus-induced gene silencing in plants. Methods 30: 296–303; 2003. doi: 10.1016/S1046-2023(03)00037-9.PubMedCrossRefGoogle Scholar
  38. Marita J. M.; Vermerris W.; Ralph J.; Hatfield R. D. Variations in the cell wall composition of maize brown midrib mutants. J Agric Food Chem 51: 1313–1321; 2003. doi: 10.1021/jf0260592.PubMedCrossRefGoogle Scholar
  39. Miki D.; Itoh R.; Shimamoto K. RNA silencing of single and multiple members in a gene family of rice. Plant Physiol 138: 1903–1913; 2005. doi: 10.1104/pp.105.063933.PubMedCrossRefGoogle Scholar
  40. Oraby H.; Venkatesh B.; Dale B.; Ahamd R.; Ransome C.; Oehmke J.; Sticklen M. B. Enhanced conversion of plant biomass into glucose using transgenic rice-produced endoglucanase for cellulosic ethanol. Trans Res 16: 739–749; 2007. doi: 10.1007/s11248-006-9064-9.CrossRefGoogle Scholar
  41. Pillonel C.; Mulder M. M.; Boon J. J.; Forster B.; Binder A. Involvement of cinnamyl-alcohol dehydrogenase in the control of lignin formation in Sorghum bicolor L. Moench Planta 185: 538–544; 1991.Google Scholar
  42. Piquemal J.; Chamayou S.; Nadaud I.; Beckert M.; Barrière Y.; Mila I.; Lapierre C.; Rigau J.; Puigdomenech P.; Jauneau A.; Digonnet C.; Boudet A. M.; Goffner D.; Pichon M. Downregulation of caffeic acid O-methyltransferase in maize revisited using a transgenic approach. Plant Physiol 130: 1675–1685; 2002. doi: 10.1104/pp.012237.PubMedCrossRefGoogle Scholar
  43. Ragauskas A. J.; Williams C. K.; Davison B. H.; Britovsek G.; Cairney J.; Eckert C. A.; Frederick W. J.; Hallett J. P.; Leak D. J.; Liotta C. L.; Mielenz J. R.; Murphy R.; Templer R.; Tschaplinski T. The path forward for biofuels and biomaterials. Science 311: 484–489; 2006. doi: 10.1126/science.1114736.PubMedCrossRefGoogle Scholar
  44. Ralph J.; Akiyama T.; Kim H.; Lu F.; Schatz P. F.; Marita J. M.; Ralph S. A.; Reddy M. S. S.; Chen F.; Dixon R. A. Effects of coumarate 3-hydroxylase down-regulation on lignin structure. J Biol Chem 281: 8843–8853; 2006. doi: 10.1074/jbc.M511598200.PubMedCrossRefGoogle Scholar
  45. Ransom C.; Venkatesh B.; Dale B.; Biswas G.; Sticklen M. B. Heterologous Acidothermus cellulolyticus 1,4-β-endoglucanase E1 produced within the corn biomass converts corn stover into glucose. Applied Biochem Biotech 140: 137–219; 2007. doi: 10.1007/s12010-007-9053-3.Google Scholar
  46. Reddy M. S.; Chen F.; Shadle G.; Jackson L.; Aljoe H.; Dixon R. A. Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa (Medicago sativa L.). Proc. Natl Acad. Sci. USA 102: 16573–16578; 2005. doi: 10.1073/pnas.0505749102.PubMedCrossRefGoogle Scholar
  47. Rogers L. A.; Campbell M. M. The genetic control of lignin deposition during plant growth and development.. New Phytologist 164: 17–30; 2004. doi: 10.1111/j.1469-8137.2004.01143.x.CrossRefGoogle Scholar
  48. Rook J. A.; Muller L. D.; Shank D. B. Intake and digestibility of brown-midrib corn silage by lactating dairy cows. J Dairy Sci 60: 1894–1904; 1977.Google Scholar
  49. Rubin E. M. Genomics of cellulosic biofuels. Nature 454: 841–844; 2008. doi: 10.1038/nature07190.PubMedCrossRefGoogle Scholar
  50. Sánchez O. J.; Cardona C. A. Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresour Technol 99: 5270–5295; 2007. doi: 10.1016/j.biortech.2007.11.013.PubMedCrossRefGoogle Scholar
  51. Schubert C. Can biofuels finally take center stage? Nature Biotechnol24: 777–784; 2006. . doi: 10.1038/nbt0706-777.CrossRefGoogle Scholar
  52. Shadle G.; Chen F.; Reddy M. S. S.; Jackson L.; Nakashima J.; Dixon R. A. Down-regulation of hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase in transgenic alfalfa affects lignification, development and forage quality. Phytochemistry 68: 1521–1529; 2007. doi: 10.1016/j.phytochem.2007.03.022.PubMedCrossRefGoogle Scholar
  53. Somleva M. N.; Tomaszewski Z.; Conger B. V. Agrobacterium-mediated genetic transformation of switchgrass.. Crop Sci 42: 2080–2087; 2002.CrossRefGoogle Scholar
  54. Stewart C. N. J. Biofuels and biocontainment. Nature Biotechnol 25: 283–284; 2007. doi: 10.1038/nbt0307-283.CrossRefGoogle Scholar
  55. Sticklen M. B. Plant genetic engineering to improve biomass characterization for biofuels. Curr Opin Biotechnol 17: 315–319; 2006. doi: 10.1016/j.copbio.2006.05.003.PubMedCrossRefGoogle Scholar
  56. Sticklen M. B. Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol. Nat Rev Genet 9: 433–443; 2008. doi: 10.1038/nrg2336.PubMedCrossRefGoogle Scholar
  57. Vignols F.; Rigau J.; Torres M. A.; Capellades M.; Puigdomenech P. The brown midrib3 (bm3) mutation in maize occurs in the gene encoding caffeic acid O-methyltransferase. Plant Cell 7: 407–416; 1995.PubMedCrossRefGoogle Scholar
  58. Wang Z. Y.; Ge Y. Recent advances in genetic transformation of forage and turf grasses. In Vitro Cell Develop. Biol Plant 42: 1–18; 2006. doi: 10.1079/IVP2005726.Google Scholar
  59. Weng J. K.; Li X.; Bonawitz N. D.; Chapple C. Emerging strategies of lignin engineering and degradation for cellulosic biofuel production. Curr Opin Biotechnol 19: 66–172; 2008. doi: 10.1016/j.copbio.2008.02.014.CrossRefGoogle Scholar
  60. Wesley S. V.; Helliwell C. A.; Smith N. A.; Wang M. B.; Rouse D. T.; Liu Q.; Gooding P. S.; Singh S. P.; Abbott D.; Stoutjesdijk P. A.; Robinson S. P.; Gleave A. P.; Green A. G.; Waterhouse P. M. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27: 581–590; 2001. doi: 10.1046/j.1365-313X.2001.01105.x.PubMedCrossRefGoogle Scholar
  61. Yuan J. S.; Tiller K. H.; Al-Ahmad H.; Stewart N. R.; Stewart N. C. Plants to power: bioenergy to fuel the future. Trends Plant Sci 13: 421–429; 2008. doi: 10.1016/j.tplants.2008.06.001.PubMedCrossRefGoogle Scholar
  62. Ziegelhoffer T.; Will J.; Austin-Phillips S. Expression of bacterial cellulase gene in transgenic alfalfa (Medicago sativa L), potato (Solanum tuberosum L) and tobacco (Nicotiana tobacum). Mol Breed 5: 309–318; 1999. doi: 10.1023/A:1009646830403.CrossRefGoogle Scholar
  63. Zhou J.; Lee C.; Zhong R.; Ye Z. H. MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. Plant Cell 21: 248–266; 2009. doi: 10.1105/tpc.108.063321.PubMedCrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2009

Authors and Affiliations

  • Hiroshi Hisano
    • 1
    • 2
  • Rangaraj Nandakumar
    • 1
    • 2
  • Zeng-Yu Wang
    • 1
    • 2
    Email author
  1. 1.Forage Improvement DivisionThe Samuel Roberts Noble FoundationArdmoreUSA
  2. 2.BioEnergy Science Center (BESC)Oak RidgeUSA

Personalised recommendations