Cèl·lules vegetals com a bioreactor per a la producció de pèptids

• Autores: Tarik Ruiz Medina
• Directores de la Tesis: Juan José López Moya Gómez (dir. tes.), María Coca López (codir. tes.)
• Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2020
• Idioma: español
• ISBN: 9788449096983
• Tribunal Calificador de la Tesis: José Antonio Daròs Arnau (presid.), Ana Montserrat Martín Hernández (secret.), Rossana Andrea Novo Lopes Henriques (voc.)
• Programa de doctorado: Programa de Doctorado en Biología y Biotecnología Vegetal por la Universidad Autónoma de Barcelona
• Materias:
  • Química
    • Bioquímica
      • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: TDX
• Resumen

  • The production of proteins in plant cell cultures and whole plants represents great opportunities to develop products for commercial use. The main objective of this industrial thesis was to develop economic and efficient plant production systems to bring proteins of interest to the market. We explored two different systems, Daucus carota cell cultures and Nicotiana benthamiana leaves, each having advantages and drawbacks depending on the intended use of the products. As a proof of concept, both systems were applied in the production of the human insulin-like growth factor 1 (IGF1), a high value peptide for the cosmetic and therapeutic industries. Innovative strategies to enhance gene expression and to facilitate product purification were used to improve yields and to reduce costs. Moreover, the biological activity of the produced IGF1 and derivatives was evaluated and compared to the chemically synthesized peptides to demonstrate the usefulness of production systems.Our first approach to enhance gene expression and improve peptide yields was with RNA silencing suppressors (RSSs). Using transient expression assays and the green fluorescent protein (GFP) as reporter, we selected the P1b from the Cucumber vein yellowing virus (CVYV) Ipomovirus as the RSSs to enhance gene expression in carrot cell cultures. Our results demonstrated that transgenic lines overexpressing IGF1 or the derivative CPP-IGF1 (a variant tailored to enhance the delivery to human cells) reached up to 4-fold higher peptide yields in combination with P1b than without. The IGF1 or CPP-IGF1 was targeted to the culture media being easily purified by simple clarification of suspensions. Moreover, we found that the media containing the produced IGF1 or CPP-IGF1 stimulated the division of human fibroblasts. A cryopreservation process was applied to the transgenic lines to avoid the reduction in peptide production found over successive propagation cycles. This allowed us to recover the original yields, opening up the possibility of establishing master cell banks. We also developed a transient production system of IGF1 and CPP-IGF1 using N. benthamiana leaves and a derived tobacco mosaic virus vector. This system resulted in similar yields of active peptides to cell cultures with the main advantage of shortening production times, although requiring more complex downstream purification.Our innovative strategy to facilitate the purification of IGF1 from plant matrices was the use of oleosin fusion technology for lipid droplet (LDs) targeting. This technology has been previously used in LD-rich seeds, but unexplored in plant cell cultures or LD-poor tissues such as leaves. Our work showed that model cell cultures from Nicotiana tabacum or Arabidopsis thaliana were an exception, as many other plant cell cultures, including D. carota cells, do contain a large amount of LDs and are susceptible to produce oleosin fusion proteins. However, as the stable expression of oleosin fusions severely affected callus cell growth, we tested the technology in transient expression in leaves. Due to the low level of LDs in leaves, oleosin fusion proteins production was in combination with triacylglycerol (TAG) induction to increase LD content simultaneously. For this purpose, key components of the TAG biosynthetic pathway, A. thaliana derived elements such as the enzyme DGAT1 and the regulatory factor WRI1 were co-expressed with the IGF1 oleosin fusion proteins in N. benthamiana leaves. Using this strategy, we obtained yields up to 1 μg/g of IGF1 bound to LDs, easily purified and fully active.Our work provides evidence of the potential of plant matrices to produce valuable peptides. Also, the oleosin technology, the use of RSSs and viral vectors explored will serve to overcome some of the known limitations of plant systems to produce active products of industrial interest.