Diseño de lacasas fúngicas activas en sangre mediante evolución molecular dirigida

• Autores: Diana Maté Mate
• Directores de la Tesis: Miguel Alcalde Galeote (dir. tes.), Francisco José Plou Gasca (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2013
• Idioma: español
• Tribunal Calificador de la Tesis: Vicente Fernández Herrero (presid.), Juan José Aragón Reyes (secret.), Ulrich Schwaneberg (voc.), Susana Camarero Fernandez (voc.), Antonio López de Lacey (voc.)
• Materias:
  • Química
    • Bioquímica
      • Enzimología
      • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • The high-redox potential laccase from basidiomycete PM1 is a highly active and stable oxidoreductase with a broad spectrum of biotechnological applications. In particular, the use of high-redox potential laccases for the engineering of 3D-nanobiodevices working in physiological fluids (blood, plasma, salive) is arousing great interest. Unfortunately, fungal laccases are not active under the specific conditions of mammal blood (pH 7.4; strong chloride concentration) hampering the successful development of this kind of devices for implants in human beings.

    Directed evolution is a suitable methodology to engineer enzymes beyond their natural limits. Through consecutives rounds of random mutation, DNA recombination and selection, new enzymes with improved or even novel properties can be created.

    We have subjected the PM1 laccase to a comprehensive directed evolution experiment to be functional in blood. Firstly, the enzyme was evolved for functional expression in Saccharomyces cerevisiae, establishing a reliable platform for further developments. The native signal sequence was replaced by the ¿-factor prepro-leader and the whole fusion gene was evolved enhancing the laccase total activity 34,000-fold over the parental type. A series of in vitro and in vivo DNA recombination and/or mutagenic methods were employed. After exploring over 50,000 clones in 8 rounds of directed evolution, 15 mutations were accumulated both in the prepro-leader and in the mature protein, whilst the stability of the protein was conserved by a combined strategy which included: i) a HTS assay for thermostability, ii) the recovery of beneficial mutations, and iii) a mutational exchange approach. The characteristic blue colour of the enzyme switched to yellow as side-consequence of the evolution process but its biochemical properties were kept intact or even improved: the final mutant of this process (OB-1 variant) was readily secreted by S. cerevisiae (8 mg/L) showing enhanced kinetics values for phenolic and non-phenolic compounds and being also stable in terms of temperature, pH and organic co-solvents.

    OB-1 was used as departure point to tailor a blood-tolerant laccase by 4 additional cycles of directed evolution. The laccase mutant was subjected to a customized directed evolution protocol in yeast, once again taking advantage of the physiology of S. cerevisiae to create DNA diversity. The inherent inhibition of laccases by the combined action of the high NaCl concentrations and the alkaline pH of blood was overcome with the help of an ad-hoc HTS assay based of these features in a buffer that simulated blood, albeit in the absence of coagulating agents and red blood cells. The ultimate mutant laccase (ChU-B variant) obtained through this evolutionary process was characterized comprehensively and its new features were tested on real human blood samples, revealing the mechanisms underlying this unprecedented improvement. Finally, ChU-B was cloned in Pichia pastoris and produced in a 42-L bioreactor (achieving production levels of 43 mg/L).

    Concluding the whole mutational pathway, the PM1 laccase was sculptured by 12 rounds of directed evolution accumulating 22 mutations (8 silent) in the whole fusion gene. Beneficial mutations enhancing secretion or activity were located at the signal prepro-leader (5 mutations) and at the mature protein (7 mutations), respectively. Significantly, only two mutations located at the second coordination sphere of the T1 copper site were responsible of the conferred tolerance to blood. Therefore, the re-specialization to adapt the PM1 laccase to such inclement conditions only required of 0.4% of the amino acid sequence.