Lasso peptides are a growing class of ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) produced in bacteria. Their knotted structure is defined by a seven to nine residue macrocycle, through which a nine to fifteen residue tail is threaded and locked in place. Two maturation enzymes are responsible for folding the peptide into the lasso shape and generating the ring. Lasso peptides hold exceptional promise as scaffolds for protein engineering because of their inherent protease resistance. To fulfill this promise, it is necessary to have a robust directed evolution system in order to engineer the peptides. Currently, no lasso peptide display system exists, because fusions to the N-termini of lasso peptides are impossible and C-terminal fusions have been unsuccessful for the model lasso peptide microcin J25 due to the inability of maturation enzymes to accept a larger, extended substrate.The recent discovery and characterization of astexin family lasso peptides, which contain exceptionally long tails, has opened up the possibility of fusing lassos to larger proteins. Here, we will present engineered fusion constructs of the astexin family lasso peptides to model proteins and discuss the implications that the maturation of these constructs has for lasso peptide biosynthesis.

Engineering a lasso peptide display system for directed evolution applications

249th National Meeting (2015)

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