Chemo-enzymatic peptide synthesis (CEPS) using omniligases and selective peptiligases Efficient biocatalysts for assembling linear and cyclic peptides and protein conjugates

The large-scale chemical manufacture of peptides with a length exceeding ca. 30 amino acids is still a huge challenge. Using chemical approaches such as solid phase peptide synthesis (SPPS) synthetic yields decrease significantly with increasing peptide chain length. The crude purity and overall yield can be dramatically improved using a fragment condensation strategy. Unfortunately, chemical fragment condensation leads to epimerization (except of Gly and Pro residues) and native chemical ligation is often not feasible (no Cys present) and difficult to scale-up due to the thioester instability. Alternatively, enzymes can be used for peptide fragment condensation without any epimerization. To minimize the intrinsic hydrolytic activity of enzymes, peptide coupling enzymes can be improved by protein engineering. Recently, we described the discovery of peptiligase, an efficient biocatalyst for assembling linear and cyclic peptides. Herein, we describe the further engineering of peptiligase to improve the enzyme's synthetic efficiency, substrate scope and activity. Several peptiligase variants with a unique substrate specificity were found. By combining multiple positive mutations, a variant called omniligase was obtained that can couple virtually any peptide sequence. The application of specific peptiligases and omniligase for the synthesis of linear and cyclic peptides, and peptide-to-protein conjugates is discussed.