In the light of directed evolution: pathways of adaptive protein evolution

Abstract

Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. These experiments offer empirical lessons about how proteins evolve in the face of clearly-defined laboratory selection pressures. Directed evolution has revealed that single amino acid mutations can enhance properties such as catalytic activity or stability and that adaptation can often occur through pathways consisting of sequential beneficial mutations. When there are no single mutations that improve a particular protein property experiments always find a wealth of mutations that are neutral with respect to the laboratory-defined measure of fitness. These neutral mutations can open new adaptive pathways by at least 2 different mechanisms. Functionally-neutral mutations can enhance a protein's stability, thereby increasing its tolerance for subsequent functionally beneficial but destabilizing mutations. They can also lead to changes in "promiscuous" functions that are not currently under selective pressure, but can subsequently become the starting points for the adaptive evolution of new functions. These lessons about the coupling between adaptive and neutral protein evolution in the laboratory offer insight into the evolution of proteins in nature.

Fig. 1.

Schematic outline of a typical directed evolution experiment. The researcher begins with the gene for the parent protein. This parent gene is randomly mutagenized by using error-prone PCR or some similar technique. The library of mutant genes is then used to produce mutant proteins, which are screened or selected for the desired target property (e.g., improved enzymatic activity or increased stability). Mutants that fail to show improvements in the screening/selection are typically discarded, while the genes for the improved mutants are used as the parents for the next round of mutagenesis and screening. This procedure is repeated until the evolved protein exhibits the desired level of the target property (or until the student performing the experiments graduates).

Fig. 2.

Activity and stability changes during the directed evolution of a cytochrome P450 enzyme for activity on short-chain alkanes. (Upper) The changes in activity on propane (total turnover number, TTN) during steps along the directed evolution trajectory. (Lower) The changes in protein stability (measured as T₅₀ values for heat inactivation). During the steps of directed evolution, the protein was selected for activity on progressively shorter alkanes, without regard to stability. The exception is the step indicated by the red arrow, where stabilizing mutations were intentionally selected to recover some of the lost stability. Data are taken from ref. .

Fig. 3.

Fitness landscapes and neutral networks. (A) A fitness landscape in which a protein at a peak corresponding to activity on substrate 1 can only reach the peak corresponding to activity on substrate 2 by taking a downhill step corresponding to a deleterious mutation. (B) A neutral network in which a protein that is active on substrate 1 may initially be unable to achieve activity on substrate 2 with a single mutational step, but can reach activity on the latter substrate through a series of neutral steps. Although both fitness landscapes and neutral networks are conceptually valid views of evolution, fitness landscapes tend to emphasize the possibility of becoming trapped on peaks, whereas neutral networks emphasize the availability of neutral mutations and their potential coupling to adaptation. In the context of directed evolution, proteins have been found empirically to always have many possible neutral mutations.

Fig. 4.

The effect of a mutation can depend on the stability of the protein into which it is introduced. As shown here, proteins that are more stable than the threshold can fold and function, whereas those that are less stable than the threshold fail to fold and are therefore nonfunctional. A particular functionally beneficial but destabilizing mutation may therefore only be tolerated by a protein that has previously accumulated one or more stabilizing substitutions.