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Chloroplast molecular farming: efficient production of a thermostable xylanase by Nicotiana tabacum plants and long-term conservation of the recombinant enzyme

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Abstract

The high cost of recombinant enzymes for the production of biofuel from ligno-cellulosic biomass is a crucial factor affecting the economic sustainability of the process. The use of plants as biofactories for the production of the suitable recombinant enzymes might be an alternative to microbial fermentation. In the case of enzyme accumulation in chloroplasts, it is fundamental to focus on the issue of full photosynthetic efficiency of transplastomic plants in the field where they might be exposed to abiotic stress such as high light intensity and high temperature. Xylanases (EC 3.2.1.8), a group of enzymes that hydrolyse linear polysaccharides of beta-1,4-xylan into xylose, find an application in the biofuel industry favouring biomass saccharification along with other cell-wall degrading enzymes. In the present study, we analysed how a high level of accumulation of a thermostable xylanase in tobacco chloroplasts does not impact on photosynthetic performance of transplastomic plants grown outdoors. The recombinant enzyme was found to be stable during plant development, ex planta and after long-term storage.

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Abbreviations

BSX:

Bacillus sp. xylanase

TSP:

Total soluble proteins

Chl:

Chlorophyll

Car:

Carotenoids

FM :

Maximum PSII fluorescence in the dark-adapted state

F0 :

Minimum fluorescence in the dark-adapted state

Fv :

Variable fluorescence

PAM:

Pulse amplitude modulated fluorimetry

PSII:

Photosystem II

References

  • Allahverdiyeva T, Aro E-M (2012) Photosynthetic responses of plants to excess light: mechanisms and conditions for photoinhibition, excess energy dissipation and repair. In: Eaton JJ, Tripathy BC, Sharkey TD (eds) Photosynthesis: plastid biology, energy conversion and carbon assimilation. Advances in photosynthesis and respiration. Springer, Dordrecht, pp 275–297

    Chapter  Google Scholar 

  • Anderson JM (1986) Photoregulation of the composition, function, and structure of thylakoid membranes. Annu Rev Plant Physiol Plant Mol Biol 37:93–136

    Article  CAS  Google Scholar 

  • Baldisserotto C, Ferroni L, Anfuso E, Pagnoni A, Fasulo MP, Pancaldi S (2007) Responses of Trapa natans L. floating laminae to high concentrations of manganese. Protoplasma 231:65–82

    Article  CAS  PubMed  Google Scholar 

  • Baldisserotto C, Ferroni L, Zanzi C, Marchesini R, Pagnoni A, Pancaldi S (2010) Morpho-physiological and biochemical responses in the floating lamina of Trapa natans exposed to molybdenum. Protoplasma 240:83–97

    Article  CAS  PubMed  Google Scholar 

  • Bally J, Nadai M, Vitel M, Rolland A, Dumain R, Dubald M (2009) Plant physiological adaptations to the massive foreign protein synthesis occurring in recombinant chloroplasts. Plant Physiol 150:1474–1481

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Bhardwaj A, Leelavathi S, Mazumdar-Leighton S, Ghosh A, Ramakumar S, Reddy VS (2010) The critical role of N- and C-terminal contact in protein stability and folding of a family 10 xylanase under extreme conditions. Plos One 5:e11347

    Article  PubMed Central  PubMed  Google Scholar 

  • Borisjuk NV, Borjsiuk LG, Logendra S, Petersen F, Gleba Y, Raskin I (1999) Production of recombinant proteins in plant root exudates. Nat Biotechnol 17:466–469

    Article  CAS  PubMed  Google Scholar 

  • Borkhardt B, Harholt J, Ulvskov P, Ahring BK, Jorgensen B, Brinch-Pedersen H (2010) Autohydrolysis of plant xylans by apoplastic expression of thermophilic bacterial endo-xylanases. Plant Biotechnol J 8:363–374

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Conley AJ, Joensuu JJ, Richman A, Manassa R (2011) Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants. Plant Biotechnol J 9:419–433

    Article  CAS  PubMed  Google Scholar 

  • Doran PM (2006) Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol 24:426–432

    Article  CAS  PubMed  Google Scholar 

  • Ghose TK (1987) Measurements of cellulase activities. Pure Appl Chem 59:257–268

    CAS  Google Scholar 

  • Gupta N, Reddy VS, Maiti S, 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hendrickson L, Furbank RT, Chow WS (2004) A simple alternative approach to assessing the fate of absorbed light energy using chlorophyll fluorescence. Photosynth Res 82:73–81

    Article  CAS  PubMed  Google Scholar 

  • Horton P, Ruban AV, Walters RG (1996) Regulation of light harvesting in green plants. Annu Rev Plant Physiol Plant Mol Biol 47:655–684

    Article  CAS  PubMed  Google Scholar 

  • Jahns P, Holzwarth AR (2012) The role of the xanthophyll cycle and of lutein in photoprotection of photosystem. Biochim Biophys Acta 1817:182–193

    Article  CAS  PubMed  Google Scholar 

  • Juturu V, Wu JC (2012) Microbial xylanases: engineering, production and industrial applications. Biotechnol Adv 30:1219–1227

    Article  CAS  PubMed  Google Scholar 

  • Kim JY, Kavas M, Fouad WM, Nong G, Preston JF, Altpeter F (2011) Production of hyperthermostable GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants enables complete hydrolysis of methylglucuronoxylan to fermentable sugars for biofuel production. Plant Mol Biol 76:357–369

    Article  CAS  PubMed  Google Scholar 

  • Klughammer C, Schreiber U (2008) Complementary PSII quantum yields calculated from simple fluorescence parameters measured byPAM fluorometry and the saturation pulse method. PAM Appl Notes 1:27–35

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Leelavathi S, Gupta N, Maiti S, Ghosh A, Reddy VS (2003) Overproduction of an alkali- and thermo-stable xylanase in tobacco chloroplasts and efficient recovery of the enzyme. Mol Breed 11:59–67

    Article  CAS  Google Scholar 

  • Lichtenthaler HK, Buschmann C, Knapp M (2005) How to correctly determine the different chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio R-Fd of leaves with the PAM fluorometer. Photosynthetica 43:379–393

    Article  CAS  Google Scholar 

  • Magee AM, Coyne S, Murphy D, Horvath EM, Medgyesy P, Kavanagh TA (2004) T7 RNA polymerase-directed expression of an antibody fragment transgene in plastids causes a semi-lethal pale-green seedling phenotype. Transgenic Res 13:325–337

    Article  CAS  PubMed  Google Scholar 

  • Matsubara S, Chow WS (2004) Populations of photo inactivated photosystem II reaction centers characterized by chlorophyll a fluorescence lifetime in vivo. PNAS 101:18234–18239

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Meyers B, Zaltsman A, Lacroix B, Kozlovsky SV, Krichevsky A (2010) Nuclear and plastid genetic engineering of plants: comparison of opportunities and challenges. Biotechnol Adv 28:747–756

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Obembe OO, Popoola JO, Leelavathi S, Reddy SV (2011) Advances in plant molecular farming. Biotechnol Adv 29:210–222

    Article  PubMed  Google Scholar 

  • Pauly M, Keegstra K (2010) Plant cell wall polymers as precursors for biofuels. Curr Opin Plant Biol 13:305–312

    Article  CAS  PubMed  Google Scholar 

  • Pogorelko G, Fursova O, Lin M, Pyle E, Jass J, Zabotina OA (2011) Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast. Plant Mol Biol 77:433–445

    Article  CAS  PubMed  Google Scholar 

  • Ruban AV, Johnson MP, Duffy CDP (2012) The photoprotective molecular switch in the photosystem II antenna. BBA Bioenergetics 1817:167–181

    Article  CAS  PubMed  Google Scholar 

  • Wagner R, Dietzel L, Braeutigam K, Fischer W, Pfannschmidt T (2008) The long-term response to fluctuating light quality is an important and distinct light acclimation mechanism that supports survival of Arabidopsis thaliana under low light conditions. Planta 228:573–587

    Article  CAS  PubMed  Google Scholar 

  • Wellburn AR (1994) The spectral determination of chlorophyll-a and chlorophyll-b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144:307–313

    Article  CAS  Google Scholar 

  • Xu J, Dolan MC, Medrano G, Cramer CL, Weathers PJ (2012) Green factory: plants as bioproduction platforms for recombinant proteins. Biotechnol Adv 30:1171–1184

    Article  CAS  PubMed  Google Scholar 

  • Zhang Z, Donaldson AA, Ma X (2012) Advancements and future directions in enzyme technology for biomass conversion. Biotechnol Adv 30:913–919

    Article  CAS  PubMed  Google Scholar 

  • Zieminski K, Romanowska I, Kowalska M (2012) Enzymatic pretreatment of lignocellulosic wastes to improve biogas production. Waste Manag 32:1131–1137

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Jenny Pamela Broncano Galeas for performing enzyme assays. This work was supported by a generous grant of Fondazione Bussolera-Branca. Authors acknowledge funding from Consorzio Universitario Italiano per l’Argentina (CUIA), Department of Biotechnology, India, and the internal support of ICGEB.

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

  1. Paolo Longoni

    Present address: Department of Botany and Plant Biology, University of Geneva, Sciences III, Quai E. Ansermet30, 1211, Geneve 4, Switzerland

Authors and Affiliations

  1. Department of Biology and Biotechnology, University of Pavia, Via Ferrata 9, 27100, Pavia, Italy

    Laura Pantaleoni, Paolo Longoni & Rino Cella

  2. Department of Life Sciences and Biotechnologies, University of Ferrara, C.so Ercole I d’Este 32, 44121, Ferrara, Italy

    Lorenzo Ferroni, Costanza Baldisserotto & Simonetta Pancaldi

  3. Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India

    Sadhu Leelavathi & Vanga Siva Reddy

Authors
  1. Laura Pantaleoni
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  2. Paolo Longoni
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  3. Lorenzo Ferroni
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  7. Simonetta Pancaldi
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Corresponding author

Correspondence to Rino Cella.

Additional information

Handling Editor: Peter Nick

L. Pantaleoni and P. Longoni equally contributed.

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Pantaleoni, L., Longoni, P., Ferroni, L. et al. Chloroplast molecular farming: efficient production of a thermostable xylanase by Nicotiana tabacum plants and long-term conservation of the recombinant enzyme. Protoplasma 251, 639–648 (2014). https://doi.org/10.1007/s00709-013-0564-1

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  • DOI: https://doi.org/10.1007/s00709-013-0564-1

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