Miguel Alcalde Group
1. Fungal peroxygenases in synthetic chemistry
Fungal peroxygenases are highly promiscuous biocatalysts whose selective mono(per)oxygenase activity makes them extremely useful for many synthetic chemistry applications. We are combining a broad repertory of library creation methods, computational analysis and screening protocols for directing their evolution aimed at meeting these versatile biocatalysts with industrial standards, covering from the synthesis of agrochemicals to the cost-effective production of pharma compounds.
2. Engineering the ligninolytic secretome
The ligninolytic enzymatic consortium secreted by white-rot fungi is mostly composed of different high-redox potential oxidoreductases (laccases, peroxidases, peroxygenases and H2O2-supplying oxidases) and its natural function is the degradation of lignin during wood decay.
This lignin-degrading army places at the front line of green chemistry, with potential applications in the production of second generation biofuels, pulp biobleaching, the design of nanobiodevices (biosensors and biofuel cells), organic synthesis or bioremediation. In the last decade we have subjected this set of enzymes to laboratory evolution in order to tailor different properties of biotechnological relevance (e.g., enhanced kinetics and secretion levels, improved stability over a range of pHs and temperatures, the adaptation to the presence of non-natural environments ranging from human blood to organic co-solvents).
3. Laboratory evolution of iron-sulfur enzymes and carriers for isoprenoid production
Isoprenoids represent a vast family of natural products with an incredible number of practical uses, from commodities such as biofuels and rubber, to high-value compounds such as fragrances and pharmaceuticals. We are currently studying the directed evolution of FeS enzymes and carriers within artificial biosynthetic pathways for the production of a range of isoprenoids to be used as biofuels, solvents and fragances.
4. Travelling back and forth along the temporal scale of evolution
There is increasing interest in ancestral protein reconstruction and resurrection, travelling back in time by phylogenetic analysis and ancestral inference to recreate ancient enzymes with greater stability and broader substrate promiscuity than their extant counterparts. In our laboratory we are trying to meet ancestral resurrection with directed evolution in a drive towards more versatile biocatalysts.
Indeed, the interface between natural and artificial evolution can be explored by travelling back and forth along the evolutionary timeline with extant and ancestral enzymes. This approach is based on enzyme re-specialization and it promises to provide a deeper understanding of the principles underlying the evolution of new functions while rescuing latent promiscuous activities and stabilities.
5. Saccharomyces cerevisiae as a biomolecular tool-box for directed evolution and synthetic biology approaches
Of the heterologous hosts used in laboratory evolution experiments, the budding yeast Saccharomyces cerevisiae is the host of choice to express eukaryotic proteins with improved properties. S. cerevisiae not only allows the mutant enzymes to be secreted but also, thanks to its efficient DNA recombination apparatus it allows us to use a wide range of genetic manipulations, from in vivo cloning to the creation of increased molecular diversity.
This yeast provides a complete repertory of solutions to many bottlenecks encountered in synthetic biology and directed evolution experiments. We are continually learning how to further exploit this eukaryotic machinery to create DNA diversity as well as to assemble synthetic pathways.