A hybrid strategy for massive acceleration of directed evolution: meeting the need for high-turnover enzymes in industrial biotechnology.

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Enzymes are tiny machines that speed up chemical processes in living organisms; life would not exist without enzymes, because essential chemical reactions would not happen fast enough. Enzymes perform this miraculous function by first attaching to chemicals, changing their shapes temporarily and using this to elicit a chemical reaction, and then releasing the products. Some enzymes break down large chemicals into simpler parts, while some build smaller chemicals into more complex ones. All this happens at atmospheric pressure, room temperature, neutral acidity and all components are biodegradable! In contrast, current industrial chemistry needs high temperatures and pressures, and creates organic waste and pollutants. Consequently, industrial reactions are typically not very efficient and often lead to the formation of unwanted side products. With enzymes there is relatively little energy demand and side products can be eradicated. Enzymes, therefore, represent a huge opportunity in the 21st Century to revolutionise industrial chemical reactions and make them more cost-effective and environmentally friendly.Unfortunately, there is a catch: naturally occurring enzymes, perhaps unsurprisingly, are not suited to industrial processes. For example, an enzyme may not be naturally available for the chemical reaction at hand, they may be unstable in an industrial setting, or they may be too slow to be cost-effective (metabolic reactions don't demand such speed). Biology does, however, provide a potential solution to these shortcomings. Each enzyme is constructed as a string of 100s of smaller building blocks called amino acids. Furthermore, one enzyme can be converted to another by altering (or mutating) its amino acids. There are 20 possible amino acids and within a string of 100 there are vastly more combinations than stars in the universe and it is very clear that the natural world only uses the tiniest fraction of the possible gamut. The real opportunity is to mutate natural enzymes to make them more stable, faster and tailor their specificity so that they can be used in industry and make chemical processes much more cost-effective and environmentally friendly, which is at the heart of this research proposal.The active site of an enzyme is a space that only the right chemicals can slot into easily and perfectly, like a key in its lock, and it is the shape of this space and motions within that determine an enzyme's speed and specificity; mutations alter speed and specificity by affecting the active site. Scientists thought, naively, that randomly mutating a few amino acids around the active site would be sufficient to achieve their goals. In such a case there would be, say, only a million combinations to produce and test to identify a suitable one. However, it has now been found that mutations anywhere in an enzyme's string of amino acids may affect the active site and based on random mutations the number of combinations are truly astronomical. Some progress has been made in a process called directed evolution, where random mutations are made in an iterative cycle, but not nearly enough to satisfy industry since for some enzymes the process is predicted to take millennia.Our vision is to fundamentally change the way that directed evolution is performed, and reduce the timescales, not incrementally, but by potentially millions of times to facilitate rapid production of enzymes with industrially-relevant properties. We plan to use an enzyme, monoamine oxidase, which could be used in the manufacture of almost half of current developmental drugs, to show the validity of our new approach. The idea is to use very fast, but accurate, computer simulations of enzymes, leveraging hardware developed for rendering computer games, to understand how mutations throughout an enzyme affect the active site and use this to predict the optimal mutations for directed evolution, allowing the process to occur in weeks rather than millennia.