Ultra-high throughput evolution of designer enzymes with extended amino acid alphabets

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With increasing societal and political acknowledgment of environment and climate issues, various industrial sectors are shifting their focus towards carbon neutral and environmentally benign technologies. As thus, biocatalysis is a rapidly expanding technology in chemical industry for the production of commodity chemicals and pharmaceuticals. In biocatalysis, enzymes, which are nature's catalysts, are repurposed for synthesizing chemicals in human devised processes. Enzymes can accelerate highly complex chemical reactions with speeds and specificities that are unrivalled by conventional chemical methods. In addition, reactions can be performed at low temperatures in aqueous solutions, unlike chemical processes that typically require high temperatures, toxic chemicals and large amounts of organic solvents. However, a major limitation for broad industrial exploitation of enzymes is that not for any desired reaction a suitable enzyme is available in nature's repertoire. As thus, enzymes that meet the specifications of organic chemists are urgently required. Rather than reengineering existing natural enzymes, bottom-up design of new enzymes is now becoming a feasible alternative. In fact, highly efficient artificial enzymes for simplistic reactions were successfully created through computation and experimental optimisation. The number of chemical mechanisms that can be designed is however inherently limited to natures repertoire of functional groups and amino acids. Through genetic code expansion, it is now possible to augment nature's amino acid alphabet with additional functionalities. In particular, through integration of amino acids that are fused to small molecule catalysts, which have been extensively explored by organic chemists, it is now possible to create enzymes with a whole new reaction scope that is unprecedented in nature. In order to design better enzymes with non-natural functionalities from scratch, it is essential to gain fundamental understanding of how artificial amino acids must be placed and further complemented within enzyme active sites. This project aims to address this by experimentally improving designer enzymes with non-natural amino acids, leading to a fundamental understanding of the true potential of augmenting nature with additional building blocks. This will be achieved through directed evolution, which is a mimic of Darwinian evolution on a laboratory time scale. Directed evolution allows for the discovery of mutations that improve enzyme activity but are rationally not predictable. Through iterative rounds of mutagenesis and selection, the activity levels of enzymes can be significantly improved. This is a laborious process as many enzyme variants have to be individually analysed, to identify rare mutations with beneficial effects. To accelerate this process, an ultra-high throughput assay will be implemented that utilises picolitre-sized droplets as reaction vessels that contain the enzyme variant and its coding gene. Droplets can be manipulated at high speeds of several thousand droplets per second and most importantly they can be sorted according to enzyme activity. With this technology at hand, it is now possible for the first time to explore the evolutionary limits of these designer enzymes for different unrelated reactions, including ester hydrolysis and an unnatural carbon-carbon bond forming reaction. In depth characterisation of the best performing enzymes will highlight functional and structural features that are essential for supporting catalysis by nonnatural amino acids. Recapitulating these findings by computational enzyme design will challenge our gained molecular understanding and give rise to new generations of improved designer enzymes. Overall, this research project will open up new avenues in development of highly active enzymes for abiological reactions with implications in biotechnology, biocatalysis and synthetic biology.