Molecular, biochemical, and proteomic analyses of transplastomic tobacco plants expressing an endoglucanase support chloroplast-based molecular farming for industrial scale production of enzymes
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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.
KeywordsEndoglucanase 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.
This article does not contain studies with human participants or animals performed by any of the authors.
- Castiglia D, Sannino L, Marcolongo L, Ionata E, Tamburino R, De Stradis A, Cobucci-Ponzano B, Moracci M, La Cara F, Scotti N (2016) High-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated Arundo donax L. biomass. Biotechnol Biofuels 9:154CrossRefGoogle Scholar
- Lentz EM, Segretin ME, Morgenfeld MM, Wirth SA, Dus Santos MJ, Mozgovoj MV, Wigdorovitz A, Bravo-Almonacid FF (2010) High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants. Planta 231(2):387–395. https://doi.org/10.1007/s00425-009-1058-4 CrossRefPubMedGoogle Scholar
- Longoni P, Leelavathi S, Doria E, Reddy VS, Cella R (2015) Production by tobacco transplastomic plants of recombinant fungal and bacterial cell-wall degrading enzymes to be used for cellulosic biomass saccharification. BioMed Res Int:289759Google Scholar
- Lowry OH, Rosebroughr NJ, Farr AL, Randall RJ (1951) Protein measurement with folin phenol reagent. J Biol Chem 193:265–275Google Scholar
- Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x CrossRefGoogle Scholar
- Pagliano C, Bersanini L, Cella R, Longoni P, Pantaleoni L, Abhishek D, Leelavathi S, Reddy VS (2017) Use of Nicotiana tabacum transplastomic plants engineered to express a His-tagged CP47 for the isolation of functional photosystem II core complexes. Plant Physiol Biochem 111:266–273CrossRefGoogle Scholar
- Pantaleoni L, Longoni P, Ferroni L, Baldisserotto C, Leelavathi S, Reddy VS, Pancaldi S, Cella R (2014) Chloroplast molecular farming: efficient production of a thermostable xylanase by Nicotiana tabacum plants and long-term conservation of the recombinant enzyme. Protoplasma 251(3):639–648CrossRefGoogle Scholar
- Reddy VS, Leelavathi S, Selvapandiyan A, Raman R, Ferraiolo G, Shukla V, Bhatnagar RK (2002) Analysis of chloroplast transformed tobacco plants with cry1Ia5 under rice psbA transcriptional elements reveal high level expression of Bt toxin without imposing yield penalty and stable inheritance of transplastome. Mol Breeding 9(4):259–269CrossRefGoogle Scholar
- Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682CrossRefGoogle Scholar