The core of the Revoluzion project is the manufacture of plastics containing specific enzymes for their degradation after the material’s useful life cycle. For this purpose, it is necessary to include specialized enzymes for this task in the manufacturing process, specifically in the extrusion phase, where temperatures above 130C are reached. Unfortunately, there are no such enzymes available in nature, so it is necessary to design them to withstand extreme temperatures, which can be embedded in the final plastic in a latent state, and activated a posteriori in order to promote the degradation of the discarded plastic, either in confined areas or in exsitu processes.

To this end, Prof. Miguel Alcalde’s group at ICP-CSIC is applying directed evolution tools to obtain more active and temperature-stable plastic-degrading enzymes that can withstand plastic manufacturing processes.

Directed evolution is a revolutionary synthetic biology tool that allows emulating the process of natural evolution, but at laboratory scale, in order to obtain more robust and active enzymes. Through iterative cycles of random mutation, DNA recombination and artificial selection, the time scale of natural evolution can be compressed from millions of years to just days of laboratory work. Through directed evolution, enzymes have been designed for biofuel production, CO2 capture, drug production or plastic degradation. The biotechnological potential of this tool is such that its pioneer, Prof. Frances H. Arnold of Caltech was awarded the 2018 Nobel Prize in Chemistry for this invention.

However, the challenge in the Revoluzion project is enormous given the extreme temperatures that the enzymes have to face, so the novelty of the project is that both modern and ancestral enzymes will be subjected to directed evolution experiments. The latter are recreations of enzymes that no longer exist on our planet because they belong to extinct organisms, but whose sequences have been inferred on the basis of phylogenetic prediction algorithms and produced in modern hosts; they have been “resurrected”. The enzyme resurrection, carried out by the pioneering group of Prof. José Manuel Sánchez Ruiz of the University of Granada, will allow Prof. Alcalde to have a new starting point for directed evolution, given that the ancestral enzymes are a priori highly thermostable because they are conditioned to periods of our planet (e.g. ancestral enzymes of Precambrian origin) where conditions were more extreme.

Schematic representation of a directed evolution process in Revoluzion

Genes coding for the starting enzymes (in the case of the Revoluzion project, modern and/or ancestral hydrolytic enzymes) are subjected to random mutagenesis and DNA recombination techniques to generate genetic diversity. The mutant gen libraries are introduced into microorganisms (yeast or bacteria), which facilitate the expression of the mutant enzymes, and finally an artificial selection or screening process is carried out at high temperatures, in search of the most thermostable mutants. The best variants are chosen as parents for a new generation of molecular evolution. This process is repeated in successive cycles until highly thermostable enzymes are reached, which will be used in the plastic extrusion process.