Chimeragenesis of distantly‐related proteins by noncontiguous recombination

Abstract

We introduce a method for identifying elements of a protein structure that can be shuffled to make chimeric proteins from two or more homologous parents. Formulating recombination as a graph-partitioning problem allows us to identify noncontiguous segments of the sequence that should be inherited together in the progeny proteins. We demonstrate this noncontiguous recombination approach by constructing a chimera of b-glucosidases from two different kingdoms of life. Although the protein's alpha-beta barrel fold has no obvious subdomains for recombination, noncontiguous SCHEMA recombination generated a functional chimera that takes approximately half its structure from each parent. The X-ray crystal structure shows that the structural blocks that make up the chimera maintain the backbone conformations found in their respective parental structures. Although the chimera has lower b-glucosidase activity than the parent enzymes, the activity was easily recovered by directed evolution. This simple method, which does not rely on detailed atomic models, can be used to design chimeras that take structural, and functional, elements from distantly-related proteins.

Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins

“…Two SCHEMA libraries were designed: contiguous (10, 23) and noncontiguous (24). Contiguous libraries are designed so that blocks are contiguous in the amino acid sequence, whereas noncontiguous libraries swap blocks in the 3D structure that are not necessarily contiguous in the primary structure.…”

“…SCHEMA was used to design 10-block contiguous and noncontiguous recombination libraries of the three parent ChRs that minimize the libraryaverage disruption of the ChR structure (10,23,24). Both recombination library designs were made using software packages for calculating SCHEMA energies openly available at cheme.che.caltech.edu/groups/fha/Software.…”

“…structure-guided recombination methods have been developedone restricts blocks to be contiguous in the polypeptide sequence (10,23), whereas the other allows for design of structural blocks that are noncontiguous in the polypeptide sequence but are contiguous in 3D space (24). SCHEMA has enabled successful recombination of parental sequences with as low as 34% identity (25), which is not possible using random DNA recombination methods such as DNA shuffling (26).…”

Advances in de novo strain design using integrated systems and synthetic biology tools

“…Furthermore, the systematic discovery and pro-active elimination of undesirable secondary enzymatic functions that could drain carbon flux away from the main product could help shorten the strain design cycle [68]. Both tasks require the reliable re-design of enzymes either using de novo [69] or evolutionary techniques [70]. Several computational design techniques have been proposed for improving enzyme turnover number, substrate specificity, reduced allosteric inhibition, among others [71][72][73], but reliable protein design remains elusive [74].…”

Noncontiguous SCHEMA Protein Recombination

“…These libraries are saved in the directory 'ncr/output' and listed in the text file 'library12_result_list.csv' (see Note 13). 4. Pick an NCR library (see Note 14).…”