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Tobacco Plant: A Novel and Promising Heterologous Bioreactor for the Production of Recombinant Bovine Chymosin

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Abstract

The natural source of chymosin, a key enzyme in the dairy industry, is insufficient for rapidly growing cheese industries. Large-scale production of recombinant proteins in heterologous hosts provides an efficient alternative solution. Here, the codon-optimized synthetic prochymosin gene, which has a CAI index of 0.926, was subcloned from a cloning vector (pUC57-bCYM) into the pBI121 vector, resulting in the construct named pBI121-bCYM. CAI ranges from 0 to 1 and higher CAI improves gene expression in heterologous hosts. The overexpression of the prochymosin gene was under the control of constitutive CaMV 35S promoter and NOS terminator and was transferred into the tobacco via A. tumefaciens strain LBA4404. Explant type, regeneration method, inoculation temperature, cell density (OD600) of Agrobacterium for inoculation, and acetosyringone concentration were leaf explants, direct somatic embryogenesis, 19 °C, 0.1, and 100 µM, respectively. The successful integration and expression of the prochymosin gene, along with the bioactivity of recombinant chymosin, were confirmed by PCR, RT-PCR, and milk coagulation assay, respectively. Overall, this study reports the first successful overexpression of the codon-optimized prochymosin form of the bovine chymosin enzyme in the tobacco via indirect transformation. Production of recombinant bovine chymosin in plants can be an easy-to-scale-up, safe, and inexpensive platform.

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Authors and Affiliations

  1. Seed and Plant Certification and Registration Institute, Ardabil Agricultural and Natural Resources Research Center, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

    Shahnam Azizi-Dargahlou

  2. Faculty of Agricultural Sciences and Natural Resources, University of Mohaghegh Ardabili, Ardabi, Iran

    Mahin Pouresmaeil

  3. Department of Biotechnology, Azarbaijan Shahid Madani University, Tabriz, Iran

    Mohammad Ahmadabadi

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  3. Mohammad Ahmadabadi
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by the cooperation of all authors. All authors revised the manuscript critically and also read and approved the final version.

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Correspondence to Shahnam Azizi-Dargahlou.

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Azizi-Dargahlou, S., Pouresmaeil, M. & Ahmadabadi, M. Tobacco Plant: A Novel and Promising Heterologous Bioreactor for the Production of Recombinant Bovine Chymosin. Mol Biotechnol (2024). https://doi.org/10.1007/s12033-023-01043-z

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