BioEnergy Research

, Volume 6, Issue 2, pp 699–710 | Cite as

Enhanced Expression Levels of Cellulase Enzymes Using Multiple Transcription Units

  • Erin Egelkrout
  • Karen McGaughey
  • Todd Keener
  • Amberlyn Ferleman
  • Susan Woodard
  • Shivakumar Devaiah
  • Zivko Nikolov
  • Elizabeth Hood
  • John Howard


Transgenic cereals are an attractive option for the accumulation of foreign proteins when large volumes and low cost are required. Previous work has shown maize germ to be a particularly good location for accumulating enzymes that target cellulose for degradation. In this study, recently identified embryo-preferred promoters were used to investigate their ability to increase the accumulation of the enzymes endoglucanase E1 and cellobiohydrolase CBHI. The effect of increasing copy numbers of identical transcription units, as well as multiple copies of the enzyme driven by different promoters, was explored. Results show that accumulation of the E1 or CBHI enzymes can be significantly increased, particularly when using constructs with multiple copies of the transcription units. These findings demonstrate the highest levels of these enzymes obtained in a commercially relevant plant species observed thus far. The methodology described here may provide a low-cost plant-based source of enzymes enabling an economically viable solution for the conversion of cellulose to ethanol.


Cellulase Biofuels Maize Transcription units Embryo promoters Ethanol 



This work was supported by grant #DOE DE FG36 GO88025, Foundation, Walton Family Foundation, and Arkansas State University Biosciences Institute. We would also like to acknowledge the technical assistance of Aaron Harry, Mackenzie Tageson, and Raghavendra Rayadurg in this project.


  1. 1.
    Austin-Phillips S, Ziegelhoffer T, Will J (1999) Expression of bacterial cellulase genes in transgenic alfalfa (Medicago sativa L.), potato (Solanum tuberosum L.) and tobacco (Nicotiana tabacum L.). Mol Breeding 5(4):309–318. doi: 10.1023/A:1009646830403 CrossRefGoogle Scholar
  2. 2.
    Nigam PS, Singh A (2011) Production of liquid biofuels from renewable resources. Prog Energ Combust 37(1):52–68. doi: 10.1016/j.pecs.2010.01.003 CrossRefGoogle Scholar
  3. 3.
    Baker JO, Ehrman CI, Adney WS, Thomas SR, Himmel ME (1998) Hydrolysis of cellulose using ternary mixtures of purified celluloses. Appl Biochem Biotech 70(1):395–403. doi: 10.1007/BF02920154 CrossRefGoogle Scholar
  4. 4.
    Shoemaker S, Schweickart V, Ladner M, Gelfand D, Kwok S, Myambo K, Innis M (1983) Molecular cloning of exo–cellobiohydrolase I derived from Trichoderma reesei strain L27. Nat Biotechnol 1(8):691–696. doi: 10.1038/nbt1083-691 CrossRefGoogle Scholar
  5. 5.
    Mohagheghi A, Grohmann K, Wyman CE (1990) Production of cellulase on mixtures of xylose and cellulose in a fed-batch process. Biotechnol Bioeng 35(2):211–216. doi: 10.1002/bit.260350213 PubMedCrossRefGoogle Scholar
  6. 6.
    Adney WS, Tucker MP, Nieves RA, Thomas SR, Himmel ME (1995) Low molecular weight thermostable b-d-glucosidase from Acidothermus cellulolyticus. Biotechnol Lett 17(1):49–54. doi: 10.1007/BF00134195 CrossRefGoogle Scholar
  7. 7.
    Howard JA (2007) Commercialization of plant-based vaccines from research and development to manufacturing. Anim Health Res Rev 5(2):243–245. doi: 10.1079/AHR200476 CrossRefGoogle Scholar
  8. 8.
    Hood EE, Love R, Bray J, Lane J, Clough RC, Pappu K, Drees C, Hood KR, Yoon S, Ahmad A, Howard JA (2007) Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed. Plant Biotechnol J 5:709–719Google Scholar
  9. 9.
    Ziegelhoffer T, Will J, Austin-Phillips S (1999) Expression of bacterial cellulase genes in transgenic alfalfa (Medicago sativa L.), potato (Solanum tuberosum L.) and tobacco (Nicotiana tabacum L.). Molecular Breeding New Strategies in Plant Improvement 5(4):309–318Google Scholar
  10. 10.
    Tsai GJ, Wu ZY, Su WH (2000) Antibacterial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation. J Food Protect 63(6):747–752Google Scholar
  11. 11.
    Oraby H, Venkatesh B, Dale B, Ahmad R, Ransom C, Oehmke J, Sticklen M (2007) Enhanced conversion of plant biomass into glucose using transgenic rice-produced endoglucanase for cellulosic ethanol. Transgenic Res 16(6):739–749. doi: 10.1007/s11248-006-9064-9 PubMedCrossRefGoogle Scholar
  12. 12.
    Ziegler M, Thomas S, Danna K (2000) Accumulation of a thermostable endo-1,4-b-d-glucanase in the apoplast of Arabidopsis thaliana leaves. Mol Breeding 6(1):37–46. doi: 10.1023/A:1009667524690 CrossRefGoogle Scholar
  13. 13.
    Biswas G, Ransom C, Sticklen M (2006) Expression of biologically active Acidothermus cellulolyticus endoglucanase in transgenic maize plants. Plant Science 171(5):617–623CrossRefGoogle Scholar
  14. 14.
    Brunecky R, Selig MJ, Vinzant TB, Himmel ME, Lee D, Blaylock MJ, Decker SR (2011) In planta expression of A. cellulolyticus Cel5A endocellulase reduces cell wall recalcitrance in tobacco and maize. Biotechnol Biofuels 4(1):1–10, 1186/1754-6834-4-1PubMedCrossRefGoogle Scholar
  15. 15.
    Hood EE, Horn ME, Howard JA (2003) Production and Application of Proteins from Transgenic Plants. In: Vasil I (ed) Plant Biotechnology 2002 and Beyond, Proceedings of the 10th IAPTC&B Congress, Orlando, FL, Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 377–382Google Scholar
  16. 16.
    Workentine ML, Chang L, Ceri H, Turner RJ (2009) The GacS-GacA two-component regulatory system of Pseudomonas fluorescens: a bacterial two-hybrid analysis. FEMS Microbiol Lett 292(1):50–56. doi: 10.1111/j.1574-6968.2008.01445.x PubMedCrossRefGoogle Scholar
  17. 17.
    Ransom C, Balan V, Biswas G, Dale B, Crockett E, Sticklen M (2007) Heterologous Acidothermus cellulolyticus 1, 4-b-endoglucanase E1 produced within the corn biomass converts corn stover into glucose. Appli Biochem Biotech 137(1):207–219. doi: 10.1007/978-1-60327-181-3_20 CrossRefGoogle Scholar
  18. 18.
    Kusnadi AR, Evangelista RL, Hood EE, Howard JA, Nikolov ZL (1998) Processing of transgenic corn seed and its effect on the recovery of recombinant b-glucuronidase. Biotechnol Bioeng 60(1):44–52. doi:10.1002/(SICI)1097-0290(19981005)60:1<44::AID-BIT5>3.0.CO;2PubMedCrossRefGoogle Scholar
  19. 19.
    Howard JA, Hood E (2005) Bioindustrial and biopharmaceutical products produced in plants. Adv Agron 85:91–124CrossRefGoogle Scholar
  20. 20.
    Belanger FC, Kriz AL (1989) Molecular characterization of the major maize embryo globulin encoded by the glb1 gene. Plant Physiol 91(2):636–643. doi: 10.1104/pp.91.2.636 PubMedCrossRefGoogle Scholar
  21. 21.
    Streatfield SJ, Bray J, Love RT, Horn ME, Lane JR, Drees CF, Egelkrout EM, Howard JA (2010) Identification of maize embryo-preferred promoters suitable for high-level heterologous protein production. GM Crops 1(3):162–172. doi: 10.4161/gmcr.1.3.12816 PubMedCrossRefGoogle Scholar
  22. 22.
    Rogers JC (1985) Two barley alpha-amylase gene families are regulated differently in aleurone cells. J Biol Chem 260(6):3731–3738PubMedGoogle Scholar
  23. 23.
    Holwerda BC, Padgett HS, Rogers JC (1992) Proaleurain vacuolar targeting is mediated by short contiguous peptide interactions. Plant Cell 4(3):307–318. doi: 10.1105/tpc.4.3.307 PubMedGoogle Scholar
  24. 24.
    Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14(6):745–750. doi: 10.1038/nbt0696-745 PubMedCrossRefGoogle Scholar
  25. 25.
    Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6(2):271–282. doi: 10.1046/j.1365-313X.1994.6020271.x PubMedCrossRefGoogle Scholar
  26. 26.
    Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168(3):1291–1301PubMedGoogle Scholar
  27. 27.
    Armstrong C, Green C, Phillips R (1991) Development and availability of germplasm with high Type II culture formation response. Maize Genet Coop Newsletter 65:92–93Google Scholar
  28. 28.
    Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3):503–517PubMedCrossRefGoogle Scholar
  29. 29.
    Daniell H, Khan MS, Allison L (2002) Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci 7(2):84–91. doi: 10.1016/S1360-1385(01)02193-8 PubMedCrossRefGoogle Scholar
  30. 30.
    Padh H, Desai PN, Shrivastava N (2010) Production of heterologous proteins in plants: Strategies for optimal expression. Biotechnol Adv 28(4):427–435. doi: 10.1016/j.biotechadv.2010.01.005 PubMedCrossRefGoogle Scholar
  31. 31.
    Streatfield SJ, Love R, Bray J (2007) Globulin-1 promoter from maize and method of using same USA Patent 7,169,967, January 30, 2007Google Scholar
  32. 32.
    Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, Murray E, Morck D, Moloney MM (2010) Seed based expression systems for plant molecular farming. Plant Biotechnol J 8(5):588–606. doi: 10.1111/j.1467-7652.2010.00511.x PubMedCrossRefGoogle Scholar
  33. 33.
    Zheng W, Schingoethe DJ, Stegeman GA, Hippen AR, Treacher RJ (2000) Determination of when during the lactation cycle to start feeding a cellulase and xylanase enzyme mixture to dairy cows. J Dairy Sci 83(10):2319–2325. doi: 10.3168/jds.S0022-0302(00)75119-8 PubMedCrossRefGoogle Scholar
  34. 34.
    Gray BN, Ahner BA, Hanson MR (2009) High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions. Biotechnol Bioeng 102(4):1045–1054. doi: 10.1002/bit.22156 PubMedCrossRefGoogle Scholar
  35. 35.
    Gray BN, Bougri O, Carlson AR, Meissner J, Pan S, Parker MH, Zhang D, Samoylov V, Ekborg NA, Michael Raab R (2011) Global and grain specific accumulation of glycoside hydrolase family 10 xylanases in transgenic maize (Zea mays). Plant Biotechnol J 9(9):1100–1108. doi: 10.1111/j.1467-7652.2011.00632.x PubMedCrossRefGoogle Scholar
  36. 36.
    Petersen K, Bock R (2011) High-level expression of a suite of thermostable cell wall-degrading enzymes from the chloroplast genome. Plant Mol Biol 76(3–5):311–321. doi: 10.1007/s11103-011-9742-8 PubMedCrossRefGoogle Scholar
  37. 37.
    Gale SE, Westover EJ, Dudley N, Krishnan K, Merlin S, Scherrer DE, Han X, Zhai X, Brockman HL, Brown RE, Covey DF, Schaffer JE, Schlesinger P, Ory DS (2009) Side chain oxygenated cholesterol regulates cellular cholesterol homeostasis through direct sterol-membrane interactions. J Biol Chem 284(3):1755–1764. doi: 10.1074/jbc.M807210200 PubMedCrossRefGoogle Scholar
  38. 38.
    Halpin C (2005) Gene stacking in transgenic plants–the challenge for 21st century plant biotechnology. Plant Biotechnol J 3(2):141–155. doi: 10.1111/j.1467-7652.2004.00113.x PubMedCrossRefGoogle Scholar
  39. 39.
    Aluru M, Xu Y, Guo R, Wang Z, Li S, White W, Wang K, Rodermel S (2008) Generation of transgenic maize with enhanced provitamin A content. J Exp Bot 59(13):3551–3562. doi: 10.1093/jxb/ern212, 2008/08/30 ednGoogle Scholar
  40. 40.
    Naqvi S, Zhu C, Farre G, Ramessar K, Bassie L, Breitenbach J, Perez Conesa D, Ros G, Sandmann G, Capell T, Christou P (2009) Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc Natl Acad Sci USA 106(19):7762–7767. doi: 10.1073/pnas.090141210 PubMedCrossRefGoogle Scholar
  41. 41.
    Naqvi S, Farre G, Zhu C, Sandmann G, Capell T, Christou P (2010) Simultaneous expression of Arabidopsis r-hydroxyphenylpyruvate dioxygenase and MPBQ methyltransferase in transgenic corn kernels triples the tocopherol content. Transgenic Res 20(1):177–181. doi: 10.1007/s11248-010-9393-6 PubMedCrossRefGoogle Scholar
  42. 42.
    Hennegan K, Yang DC, Nguyen D, Wu LY, Goding J, Huang JM, Guo FL, Huang N, Watkins S (2005) Improvement of human lysozyme expression in transgenic rice grain by combining wheat (Triticum aestivum) puroindoline b and rice (Oryza sativa) Gt1 promoters and signal peptides. Transgenic Res 14(5):583–592. doi: 10.1007/s11248-004-6702-y PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Erin Egelkrout
    • 1
  • Karen McGaughey
    • 2
  • Todd Keener
    • 1
  • Amberlyn Ferleman
    • 1
  • Susan Woodard
    • 3
  • Shivakumar Devaiah
    • 4
  • Zivko Nikolov
    • 5
  • Elizabeth Hood
    • 4
  • John Howard
    • 1
  1. 1.Applied Biotechnology InstituteCal Poly State UniversitySan Luis ObispoUSA
  2. 2.Department of StatisticsCal Poly State UniversitySan Luis ObispoUSA
  3. 3.Kalon Biotherapeutics100 Discovery Drive, Suite 200College StationUSA
  4. 4.Arkansas Biosciences InstituteArkansas State UniversityJonesboroUSA
  5. 5.Department of Biological and Agricultural EngineeringTexas A&M UniversityCollege StationUSA

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