Directed evolution and rational design aproaches for the thermostabilisation o fan industrially relevant aldolase

Resumen

The use of enzymes in industrial processes often requires their improvement to make them suitable for their application. Among these conditions, thermostability allows the use of enzymes at high temperatures, which in turn entails a reduction of the medium viscosity and the risk of microbial contamination, as well as an increase in the solubility of substrates and products. Furthermore, thermostable enzymes present a longer lifespan, with the consequent reduction of process cost, besides a higher tolerance to proteolysis, to detergents and to organic solvents. Thermostable enzymes can be obtained from thermophilic organisms or through functional metagenomics. However, a thermophilic counterpart for each enzymatic activity cannot always be found. In this case, we can opt for protein engineering, which enables tailoring their properties for specific applications. Protein engineering includes a variety of strategies, among which directed evolution and rational design stand out. In directed evolution, mutations are inserted randomly generating a series of protein variants, which have to be screened to find individuals with improved fitness. In this work, we have used directed evolution to select variants of the hygromycin B phosphotransferase (Hph5) that confer hygromycin B resistance to T. thermophilus at higher temperature. Taking advantage of the evolved antibiotic resistance marker, Hph17, we fused variants of the pyruvate aldolase from E. coli, YfaU, to Hph17. The translational fusion expressed at high temperature in T. thermophilus allowed to select thermostable variants of YfaU due to the folding interference principle. Using this method, we identified two single replacements, Q107R and Q141L, with higher thermostability Alternatively to the selection of thermostable variants of YfaU using the folding interference principle, we have used different methods of rational design to thermostabilise YfaU. In this case, we localised the flexible regions of the protein and replaced those residues for the consensus residues, achieving six variants with higher thermodynamic stability, in particular mutants Q55R and Y58L.