Directed evolution of protein enzymes using nonhomologous random recombination

Abstract

We recently reported the development of nonhomologous random recombination (NRR) as a method for nucleic acid diversification and applied NRR to the evolution of DNA aptamers. Here, we describe a modified method, protein NRR, that enables proteins to access diversity previously difficult or impossible to generate. We investigated the structural plasticity of protein folds and the ability of helical motifs to function in different contexts by applying protein NRR and in vivo selection to the evolution of chorismate mutase (CM) enzymes. Functional CM mutants evolved using protein NRR contained many insertions, deletions, and rearrangements. The distribution of these changes was not random but clustered in certain regions of the protein. Topologically rearranged but functional enzymes also emerged from these studies, indicating that multiple connectivities can accommodate a functional CM active site and demonstrating the ability to generate new domain connectivities through protein NRR. Protein NRR was also used to randomly recombine CM and fumarase, an unrelated but also alpha-helical protein. Whereas the resulting library contained fumarase fragments in many contexts before functional selection, library members surviving selection for CM activity invariably contained a CM core with fumarase sequences found only at the termini or in one loop. These results imply that internal helical fragments cannot be swapped between these proteins without the loss of nearly all CM activity. Our findings suggest that protein NRR will be useful in probing the functional requirements of enzymes and in the creation of new protein topologies.

Fig. 1.

Protein NRR. One or more parental genes are digested with DNase I. Fragments are blunt-ended with T4 DNA polymerase, size-selected, and ligated under conditions that favor intermolecular ligation. Two hairpin sequences are added in a defined stoichiometry to the ligation reaction to generate recombined products of the desired average size. The ends of the hairpins are removed by restriction digestion, and the PCR-amplified pool is cloned for protein expression and selection.

Fig. 2.

Sequence diversity created by NRR. Unselected (inactive) sequences were obtained from two libraries. Clones 1U–14U were derived from an average fragment size of 100 bp; clones 15U–29U were derived from an average fragment size of 50 bp. Numbering across the top corresponds to the residue position in the mMjCM protein. Each arrow represents a recombined fragment. The arrow positions indicate the origin of each fragment within the parental mMjCM gene. Arrow colors indicate the order of fragment reassembly (5′-red-orange-yellow-green-teal-blue-violet-3′). The direction of each arrow indicates the sense (Right) or antisense (Left) strand of mMjCM. Overlapping arrows indicate sequence that appears more than once in a clone.

Fig. 3.

The Claisen rearrangement catalyzed by CM during amino acid biosynthesis. Cells lacking CM activity are unable to grow on media lacking tyrosine.

Fig. 4.

Protein sequences of active NRR-diversified mMjCM clones. The labeling scheme is identical to that used in Fig. 2. Arrows outlined in black indicate out-of-frame protein fragments. The colored bar at the top indicates predicted helical (blue) and loop (pink) regions based on homology with EcCM (26). The type of mutation is indicated: overlapping arrows indicate a duplication of one or more residues; gaps indicate a deletion. Predicted active site residues are indicated at the top.

Fig. 6.

Structural models of CMs. (A) EcCM. Coloring proceeds in spectral order from the N to the C terminus. (B) Structural model of mMjCM based on homology between the MjCM dimer and the EcCM dimer. Numbering indicates the residue at the approximate start and the end of each helix. (C–F) Diagrammatic models of rearranged clones, reflecting the observed oligomeric states, that preserve the active-site region (indicated with the pink sphere). Coloring is maintained from B to illustrate crossovers. Numbers indicate amino acid residues at each nonhomologous crossover based on the numbering in B. Out-of-frame fumarase residues are gray and out-of-frame mMjCM residues are magenta.

Fig. 5.

Protein sequences of active CM-fumarase hybrids. The labeling scheme is identical to that used in Figs. 2 and 4. The amino acid positions of each fumarase fragment are indicated by their position in the gene as indicated at the top.