Directed enzyme evolution: climbing fitness peaks one amino acid at a time

Abstract

Directed evolution can generate a remarkable range of new enzyme properties. Alternate substrate specificities and reaction selectivities are readily accessible in enzymes from families that are naturally functionally diverse. Activities on new substrates can be obtained by improving variants with broadened specificities or by step-wise evolution through a sequence of more and more challenging substrates. Evolution of highly specific enzymes has been demonstrated, even with positive selection alone. It is apparent that many solutions exist for any given problem, and there are often many paths that lead uphill, one step at a time.

Figure 1

Directed evolution of enzyme activity on a new substrate often proceeds via a `generalist' that shows weak activity on multiple substrates. Evolution of a specific enzyme from a generalist can be done with positive selection for the new activity and negative selection to remove those variants having the undesired activity. Specificity can also be achieved by positive selection alone, if the solution to high activity on one substrate precludes high activity on others.

Figure 2

Hypothetical evolutionary trajectories following an uphill walk to the desired fitness. Single amino acid substitutions corresponding to an increase in fitness are indicated by an arrow. (The much larger numbers of neutral and deleterious mutations are not shown.) More than one sequence can have the desired fitness, and multiple paths lead to those sequences. Restricting the number of amino acid positions that are varied or the number of different amino acids sampled at each position, however, may lead to dead ends (filled circles). A dead end is also reached when the protein is no longer stable enough to accept new mutations. Further improvements from these dead ends may become accessible once the protein is stabilized.