Abstract
Overproduction of cellulolytic enzymes through conventional nucleartransformation approaches posed a major challenge as they can potentiallydegrade the cell wall components and thereby affect transgenic plant growth anddevelopment. In this study, we have tested the possibility to over produce analkali-thermostable xylanase gene from Bacillus sp. StrainNG-27 in tobacco plants through chloroplast expression. Our results showed thatthe xylanase expression can reach up to 6% of the total soluble protein, avaluecomparable to high level expression reported for several non-cellulolyticproteins in tobacco chloroplasts. The chloroplast-expressed xylanase retainedits activity even when the leaves were dried under sun or at 42°C, offering flexibility in the agricultural system intransport and storage. The recombinant enzyme was purified to homogeneity usingsingle step chromatography with more than 85% recovery. Most importantly,transgenic plants were indistinguishable from the control untransformed plantsin their morphology, growth and in seed setting. These results open up newavenues for large scale production of several other industrially usefulcellulolytic enzymes through chloroplast expression.
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References
Amato I. 1993. The crusade against chlorine. Science 261: 152–154.
Biely P. 1985. Microbial xylanolytic enzymes. Trends Biotechnol. 3: 286–290.
Bogorad L. 2000. Engineering chloroplasts: an alternative site for foreign genes, proteins, reactions and products. Trends Biotechnol. 18: 257–263.
Borisjuk N.V., Borisjuk L.G., Logendra S., Petersen F., Gleba Y. and Raskin I. 1999. Production of recombinant proteins in plant root exudates. Nat. Biotechnol. 17: 466–469.
Brauns F.E. and Brauns D.A. 1960. The Chemistry of Lignin Covering the Literature for the Years 1949-1958. Academic Press, New York.
Coruzzi G., Broglie R., Cashmore A. and Chua N.H. 1983. Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose 1,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide. J. Biol. Chem. 258: 1399–1402.
Daniell H., Datta R., Varma S., Gray S. and Lee S.B. 1998. Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nat. Biotechnol. 16: 345–348.
Daniell H., Lee S.B., Panchal T. and Wiebe P.O. 2001. Expression of the native cholora toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J. Mol. Biol. 311: 1001–1009.
Daniell H., Streatfield S.J. and Wycoff K. 2001. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 6: 219–226.
De Cosa B., Moar W., Lee S.B., Miller M. and Daniell H. 2001. Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat. Biotechnol. 19: 71–74.
Giddings G., Allison G., Brooks D. and Carter A. 2000. Transgenic plants as factories for biopharmaceuticals. Nat. Biotechnol. 18: 1151–1155.
Goddijn O.J.M. and Pen J. 1995. Pants as bioreactors. Trends Biotechnol. 13: 379–387.
Gupta N., Reddy V.S., Maiti S. and Ghosh A. 2000. Cloning, expression, and sequence analysis of the gene encoding the alkali-stable, thermostable endoxylanase from alkalophilic, mesophilic Bacillus sp. Strain NG-27. Appl. Environ. Microbiol. 66: 2631–2635.
Heifetz P.B. and Tuttle A.M. 2001. Protein expression in plastids. Curr. Opin. Plant Biol. 4: 157–161.
Herbers K., Filnt H.J. and Sonnewald U. 1996. Apoplastic expression of the xylanase and β (1-3, 1-4) glucanase domains of the xynD gene from Ruminococcus flavefaciens leads to functional polypeptides in transgenic tobacco plants. Mol. Breed. 2: 81–87.
Herbers K., Wilke I. and Sonnewald U.A. 1995. Thermostable xylanase from Clostridium thermocellum expressed at high levels in the apoplast of transgenic tobacco has no detrimental effects and is easily purified. Bio/Technology 13: 63–66.
Hood E.E. and Jilka J.M. 1999. Plant-based production of xenogenic proteins. Curr. Opin. Biotechnol. 10: 382–386.
Kota M., Daniell H., Varma S., Garczynski S.F., Gould F. and Moar W.J. 1999. Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proc. Natl. Acad. Sci. USA 96: 1840–1845.
Liu J.H., Selinger L.B., Cheng K.J., Beauchemin K.A. and Moloney M.M. 1997. Plant seed oil-bodies as an immobilization matrix for a recombinant xylanase from the rumen fungus neocallimastix patriciarum. Mol. Breed. 3: 463–470.
Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
McBride E., Svab Z., Schaaf D.J., Hogan P.S., Stalker D.M. and Maliga P. 1995. Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Bio/Technology 13: 362–365.
Miller G.L. 1959. The use of dinitrosalicylic acid reagent for the determination of reducing sugars. Anal. Chem. 31: 426–428.
Sambrook J., Fritsch E.F. and Maniatis J. 1989. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
Staub J.M., Garcia B., Graves J., Hajdukiewicz P.T., Hunter P., Nehra N. et al. 2000. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 18: 333–338.
Suurnakki A., Tenkanen M., Buchert J. and Viikari L. 1997. Hemicellulases in the bleaching of chemical pulps. Adv. Biochem. Eng. Biotechnol. 57: 261–2871.
Svab Z. and Maliga P. 1993. High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc. Natl. Acad. Sci. 90: 913–917.
Trevelyn W.E., Procter D.P. and Harrison J.S. 1950. Detection of sugars on paper chromatogram. Nature 166: 444–445.
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Leelavathi, S., Gupta, N., Maiti, S. et al. Overproduction of an alkali- and thermo-stable xylanase in tobacco chloroplasts and efficient recovery of the enzyme. Molecular Breeding 11, 59–67 (2003). https://doi.org/10.1023/A:1022168321380
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DOI: https://doi.org/10.1023/A:1022168321380