Plant Molecular Biology

, Volume 76, Issue 3–5, pp 311–321 | Cite as

High-level expression of a suite of thermostable cell wall-degrading enzymes from the chloroplast genome

Article

Abstract

The biological conversion of plant biomass into fermentable sugars is key to the efficient production of biofuels and other renewable chemicals from plants. As up to more than 90% of the dry weight of higher plants is fixed in the cell wall, this will require the low-cost production of large amounts of cell wall-degrading enzymes. Transgenic plants can potentially provide an unbeatably cheap production platform for industrial enzymes. Transgene expression from the plastid genome is particularly attractive, due to high-level foreign protein accumulation in chloroplasts, absence of epigenetic gene silencing and improved transgene containment. Here, we have explored the potential of transplastomic plants to produce large amounts of thermostable cell wall-degrading enzymes from the bacterium Thermobifida fusca. We show that a set of four enzymes that are required for efficient degradation of cellulose (and the hemicellulose xyloglucan) could be expressed successfully in transplastomic tobacco plants. However, overexpression of the enzymes (to between approximately 5 and 40% of the plant’s total soluble protein) resulted in pigment-deficient mutant phenotypes. We demonstrate that the chloroplast-produced cellulolytic enzymes are highly active. Although further optimization is needed, our data indicate that transgenic plastids offer great potential for the production of enzyme cocktails for the bioconversion of cellulosic biomass.

Keywords

Chloroplast Plastid transformation Nicotiana tabacum Cellulose Cell wall degradation Cellulose degradation Biofuel Cellulosic ethanol Thermostable enzyme 

References

  1. Bayer EA, Lamed R, Himmel ME (2007) The potential of cellulases and cellulosomes for cellulosic waste management. Curr Op Biotechnol 18:237–245CrossRefGoogle Scholar
  2. Birch-Machin I, Newell CA, Hibberd JM, Gray JC (2004) Accumulation of rotavirus VP6 protein in chloroplasts of transplastomic tobacco is limited by protein stability. Plant Biotechnol J 2:261–270PubMedCrossRefGoogle Scholar
  3. Bock R (2001) Transgenic chloroplasts in basic research and plant biotechnology. J Mol Biol 312:425–438PubMedCrossRefGoogle Scholar
  4. Bock R (2007) Plastid biotechnology: prospects for herbicide and insect resistance, metabolic engineering and molecular farming. Curr Opin Biotechnol 18:100–106PubMedCrossRefGoogle Scholar
  5. Bohne A-V, Ruf S, Börner T, Bock R (2007) Faithful transcription initiation from a mitochondrial promoter in transgenic plastids. Nucleic Acids Res 35:7256–7266PubMedCrossRefGoogle Scholar
  6. Cahoon EB, Shanklin J, Ohlrogge JB (1992) Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco. Proc Natl Acad Sci USA 89:11184–11188PubMedCrossRefGoogle Scholar
  7. Chen S, Wilson DB (2007) Proteomic and transcriptomic analysis of extracellular proteins and mRNA levels in Thermobifida fusca grown on cellobiose and glucose. J Bacteriol 189:6260–6265PubMedCrossRefGoogle Scholar
  8. da Costa Sousa L, Chundawat SPS, Balan V, Dale BE (2009) ‘Cradle-to-grave’ assessment of existing lignocellulose pretreatment technologies. Curr Op Biotechnol 20:339–347CrossRefGoogle Scholar
  9. Daniell H, Singh ND, Mason H, Streatfield SJ (2009) Plant-made vaccine antigens and biopharmaceuticals. Trends Plant Sci 14:669–679PubMedCrossRefGoogle Scholar
  10. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  11. Glenz K, Bouchon B, Stehle T, Wallich R, Simon MM, Warzecha H (2006) Production of a recombinant bacterial lipoprotein in higher plant chloroplasts. Nature Biotechnol 24:76–77CrossRefGoogle Scholar
  12. Gomez LD, Steele-King CG, McQueen-Mason SJ (2008) Sustainable liquid biofuels from biomass: the writing’s on the walls. New Phytol 178:473–485PubMedCrossRefGoogle Scholar
  13. Gray BN, Ahner BA, Hanson MR (2009) High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions. Biotechnol Bioeng 102:1045–1054PubMedCrossRefGoogle Scholar
  14. Hager M, Biehler K, Illerhaus J, Ruf S, Bock R (1999) Targeted inactivation of the smallest plastid genome-encoded open reading frame reveals a novel and essential subunit of the cytochrome b6f complex. EMBO J 18:5834–5842PubMedCrossRefGoogle Scholar
  15. Hager M, Hermann M, Biehler K, Krieger-Liszkay A, Bock R (2002) Lack of the small plastid-encoded PsbJ polypeptide results in a defective water-splitting apparatus of photosystem II, reduced photosystem I levels, and hypersensitivity to light. J Biol Chem 277:14031–14039PubMedCrossRefGoogle Scholar
  16. Herz S, Füßl M, Steiger S, Koop H-U (2005) Development of novel types of plastid transformation vectors and evaluation of factors controlling expression. Transgenic Res 14:969–982PubMedCrossRefGoogle Scholar
  17. Irwin DC, Cheng M, Xiang B, Rose JKC, Wilson DB (2003) Cloning, expression and characterization of a family-74 xyloglucanase from Thermobifida fusca. Eur J Biochem 270:3083–3091PubMedCrossRefGoogle Scholar
  18. Jung S, Kim S, Bae H, Lim HS, Bae HJ (2010) Expression of thermostable bacterial beta-glucosidase (BglB) in transgenic tobacco plants. Bioresour Technol 101:7155–7161PubMedGoogle Scholar
  19. Kuroda H, Maliga P (2001) Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs. Nucleic Acids Res 29:970–975PubMedCrossRefGoogle Scholar
  20. Lever M (1972) A new reaction for colorimetric determination of carbohydrates. Anal Biochem 47:273–279PubMedCrossRefGoogle Scholar
  21. Li X, Weng J-K, Chapple C (2008) Improvement of biomass through lignin modification. Plant J 54:569–581PubMedCrossRefGoogle Scholar
  22. Martínez AT, Ruiz-Duenas FJ, Martínez MJ, del Río JC, Gutiérrez A (2009) Enzymatic delignification of plant cell wall: from nature to mill. Curr Op Biotechnol 20:348–357CrossRefGoogle Scholar
  23. McCabe MS, Klaas M, Gonzalez-Rabade N, Poage M, Badillo-Corona JA, Zhou F, Karcher D, Bock R, Gray JC, Dix PJ (2008) Plastid transformation of high-biomass tobacco variety Maryland Mammoth for production of human immunodeficiency virus type 1 (HIV-1) p24 antigen. Plant Biotechnol J 6:914–929PubMedCrossRefGoogle Scholar
  24. Mühlbauer SK, Koop H-U (2005) External control of transgene expression in tobacco plastids using the bacterial lac repressor. Plant J 43:941–946PubMedCrossRefGoogle Scholar
  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  26. Oey M, Lohse M, Kreikemeyer B, Bock R (2009a) Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J 57:436–445PubMedCrossRefGoogle Scholar
  27. Oey M, Lohse M, Scharff LB, Kreikemeyer B, Bock R (2009b) Plastid production of protein antibiotics against pneumonia via a new strategy for high-level expression of antimicrobial proteins. Proc Natl Acad Sci USA 106:6579–6584PubMedCrossRefGoogle Scholar
  28. Rogalski M, Ruf S, Bock R (2006) Tobacco plastid ribosomal protein S18 is essential for cell survival. Nucleic Acids Res 34:4537–4545PubMedCrossRefGoogle Scholar
  29. Rogalski M, Schöttler MA, Thiele W, Schulze WX, Bock R (2008) Rpl33, a nonessential plastid-encoded ribosomal protein in tobacco, is required under cold stress conditions. Plant Cell 20:2221–2237PubMedCrossRefGoogle Scholar
  30. Ruf S, Hermann M, Berger IJ, Carrer H, Bock R (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nature Biotechnol 19:870–875CrossRefGoogle Scholar
  31. Ruf S, Karcher D, Bock R (2007) Determining the transgene containment level provided by chloroplast transformation. Proc Natl Acad Sci USA 104:6998–7002PubMedCrossRefGoogle Scholar
  32. Spiridonov NA, Wilson DB (2001) Cloning and biochemical characterization of BglC, a beta-glucosidase from the cellulolytic actinomycete Thermobifida fusca. Curr Microbiol 42:295–301PubMedGoogle Scholar
  33. Staub JM, Maliga P (1994) Translation of the psbA mRNA is regulated by light via the 5′-untranslated region in tobacco plastids. Plant J 6:547–553PubMedCrossRefGoogle Scholar
  34. Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917PubMedCrossRefGoogle Scholar
  35. Svab Z, Maliga P (2007) Exceptional transmission of plastids and mitochondria from the transplastomic pollen parent and its impact on transgene containment. Proc Natl Acad Sci USA 104:7003–7008PubMedCrossRefGoogle Scholar
  36. Taylor LE II, Dai Z, Decker SR, Brunecky R, Adney WS, Ding S-Y, Himmel ME (2008) Heterologous expression of glycosyl hydrolases in planta: a new departure for biofuels. Trends Biotechnol 26:413–424PubMedCrossRefGoogle Scholar
  37. Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F, Fairweather N, Ytterberg J, van Wijk KJ, Dougan G, Maliga P (2003) Expression of tetanus toxin fragment C in tobacco chloroplasts. Nucleic Acids Res 31:1174–1179PubMedCrossRefGoogle Scholar
  38. Verhounig A, Karcher D, Bock R (2010) Inducible gene expression from the plastid genome by a synthetic riboswitch. Proc Natl Acad Sci USA 107:6204–6209PubMedCrossRefGoogle Scholar
  39. Verma D, Kanagaraj A, Jin S, Singh ND, Kolattukudy PE, Daniell H (2010) Chloroplast-derived enzyme cocktails hydrolyse lignocellulosic biomass and release fermentable sugars. Plant Biotechnol J 8:332–350PubMedCrossRefGoogle Scholar
  40. Wei H, Xu Q, Taylor LE II, Baker JO, Tucker MP, Ding S-Y (2009) Natural paradigms of plant cell wall degradation. Curr Op Biotechnol 20:330–338CrossRefGoogle Scholar
  41. Weng J-K, Li X, Bonawitz ND, Chapple C (2008) Emerging strategies of lignin engineering and degradation for cellulosic biofuel production. Curr Op Biotechnol 19:166–172CrossRefGoogle Scholar
  42. Wurbs D, Ruf S, Bock R (2007) Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome. Plant J 49:276–288PubMedCrossRefGoogle Scholar
  43. Ye G-N, Hajdukiewicz PTJ, Broyles D, Rodriguez D, Xu CW, Nehra N, Staub JM (2001) Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J 25:261–270PubMedCrossRefGoogle Scholar
  44. Yu L-X, Gray BN, Rutzke CJ, Walker LP, Wilson DB, Hanson MR (2007) Expression of thermostable microbial cellulases in the chloroplasts of nicotine-free tobacco. J Biotechnol 131:362–369PubMedCrossRefGoogle Scholar
  45. Zhou F, Badillo-Corona JA, Karcher D, Gonzalez-Rabade N, Piepenburg K, Borchers A-MI, Maloney AP, Kavanagh TA, Gray JC, Bock R (2008) High-level expression of HIV antigens from the tobacco and tomato plastid genomes. Plant Biotechnol J 6:897–913PubMedCrossRefGoogle Scholar
  46. Ziegelhoffer T, Raasch JA, Austin-Phillips S (2009) Expression of Acidothermus cellulolyticus E1 endo-β-1, 4-glucanase catalytic domain in transplastomic tobacco. Plant Biotechnol J 7:527–536PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Max-Planck-Institut für Molekulare PflanzenphysiologiePotsdam-GolmGermany

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