Enriching productive mutational paths accelerates enzyme evolution.

Darwinian evolution has given rise to all the enzymes that enable life on Earth. Mimicking natural selection, scientists have learned to tailor these biocatalysts through recursive cycles of mutation, selection and amplification, often relying on screening large protein libraries to productively modulate the complex interplay between protein structure, dynamics and function. Here we show that by removing destabilizing mutations at the library design stage and taking advantage of recent advances in gene synthesis, we can accelerate the evolution of a computationally designed enzyme. In only five rounds of evolution, we generated a Kemp eliminase-an enzymatic model system for proton transfer from carbon-that accelerates the proton abstraction step >108-fold over the uncatalyzed reaction. Recombining the resulting variant with a previously evolved Kemp eliminase HG3.17, which exhibits similar activity but differs by 29 substitutions, allowed us to chart the topography of the designer enzyme's fitness landscape, highlighting that a given protein scaffold can accommodate several, equally viable solutions to a specific catalytic problem.

Advances in Noncanonical Amino Acid Incorporation for Enzyme Engineering Applications.

Incorporation of noncanonical amino acids (ncAAs) via genetic code expansion (GCE) opens up new possibilities for chemical biology. The technology has led to the development of novel xenobiotic enzymes with tailored properties which can serve as entry points into a multitude of applications, including protein conjugation, immobilization, or labeling. In this review, we discuss recent progress in the use of GCE to create biocatalysts possessing reaction repertoires that lie beyond what is achievable with canonical amino acids (cAAs). Furthermore, we highlight how GCE enables to gain mechanistic insights into protein function by the incorporation of judiciously selected ncAAs. As the amino acid alphabet continues to grow and improved tools for ncAA incorporation are being developed, we anticipate the creation of additional powerful biological catalysts for synthetic application which merge the chemical versatility of anthropogenic building blocks with the exquisite selectivities of enzymes.