S102: In vitro evolution as source for artificial biocatalysts

Novel enzymes are commonly discovered by tapping the wealth of proteins found in nature that have evolved over billions of years. An alternative route to new biocatalysts has been opened recently by rational protein design, which can generate enzymes if detailed knowledge of the underlying reaction mechanism is available. In contrast to the knowledge-driven rational design approach, we have developed a general strategy to create artificial enzymes by harnessing the functional diversity of very large libraries of randomized proteins. Using the formation of product as the only selection criterion, we isolated enzymes that catalyze a ligation reaction for which no natural enzymes are known. The starting library of 4 trillion mutants was based on a non-catalytic zinc finger scaffold. To our surprise, the new enzymes isolated by in vitro selection and evolution had entirely lost the starting scaffold and adopted a new fold instead. This example emphasizes the power of an evolutionary approach, which can yield novel enzymes that are unlikely to be found by rational design.

The methodology described here can be used to create a wide range of new enzymes as well as to optimize and modify known enzymes. We envision the in vitro-evolved artificial enzymes to eventually furnish living cells with novel functions.