Molecular, biochemical, and proteomic analyses of transplastomic tobacco plants expressing an endoglucanase support chloroplast-based molecular farming for industrial scale production of enzymes

  • M. Fumagalli
  • D. Gerace
  • M. Faè
  • P. Iadarola
  • S. Leelavathi
  • V. S. Reddy
  • Rino CellaEmail author
Biotechnologically relevant enzymes and proteins


The successful production of recombinant enzymes by tobacco transplastomic plants must maintain compatibility of the heterologous enzyme with chloroplast metabolism and its long-time enzyme stability. Based on previous reports, it has been taken for granted that following biolistic-transformation, homoplasticity could be obtained from the initially heteroplastic state following successive rounds of selection in the presence of the selection agent. However, several studies indicated that this procedure does not always ensure the complete elimination of unmodified wild-type plastomes. The present study demonstrates that CelK1 transplastomic plants, which were photosyntetically as active as untransformed ones, remain heteroplastomic even after repeated selection steps and that this state does not impair the relatively high-level production of the recombinant enzyme. In fact, even in the heteroplastomic state, the recombinant protein represented about 6% of the total soluble proteins (TSP). Moreover, our data also show that, while the recombinant endoglucanase undergoes phosphorylation, this post-translation modification does not have any significant impact on the enzymatic activity. Biomass storage might be required whenever the enzyme extraction process could not be performed immediately following the harvest of tobacco mature plants. In this respect, we have observed that enzyme activity in the detached leaves stored at 4 °C is maintained up to 20 weeks without significant loss of activity. These findings may have major implications in the future of chloroplast genetic engineering–based molecular farming to produce industrial enzymes in transplastomic plants.


Endoglucanase Homoplasticity Molecular farming Proteomics Tobacco Transplastomic plants 



We gratefully acknowledge Fondazione Bussolera-Branca (FBB) for generous financial support. Authors are indebted to Dr. Fabio Cei (President of FBB) and Prof. Roberto Schmid for their continuous support and interest in our work. Also, we are thankful to International Centre of Genetic Engineering and Biotechnology and Govt. of India (DBT) for financial support. We are also very grateful to Paolo Longoni for critically reading the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_10186_MOESM1_ESM.pdf (243 kb)
ESM 1 (PDF 242 kb).


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biology and BiotechnologyUniversity of PaviaPaviaItaly
  2. 2.Plant Transformation GroupInternational Center for Genetic Engineering and BiotechnologyNew DelhiIndia

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