Directed Evolution

The origin of new functions is central to innovation and ecological diversification. Early directed evolution experiments selected bacteria to grow on unusual carbon sources. The results were supported by comparative biochemical approaches. Substrate promiscuity, along with constitutive expression, gene duplication and subfunctionalization were identified as key to the origin of new functions. Enzymes might be recruited one at a time in a patchwork or entire pathways might be repurposed “en bloc.” Ohio’s dilemma, that duplicated genes must persist in the face of inactivating mutations before acquiring new functions, was resolved in favor of selection for higher promiscuous activities stabilizing the gene duplications. The advent of PCR enabled individual genes to be targeted for mutation, while advances in structural biology made visualizing the evolution of active sites a reality. Hard and soft trade-offs govern the evolution of specialist and generalist enzymes. The role of flexible active sites in the acquisition of new functions and the trade-offs at play are reviewed. The directed evolution of a synthetic enzyme and the evolutionary reversal of a repressor so that an inducer becomes a co-repressor reveal the ease with which dramatic changes in function can be acquired. Industrial applications of experimental evolution are briefly discussed.