Discovery and Engineering of Novel Lasso Peptides

Abstract

Lasso peptides are a class of ribosomal natural products, characterized by an unusual structural fold, remarkable stability, and high biotechnological potential. Despite progress made in the field, many members of the class await discovery and much remains to be learned about their biosynthesis and function. This thesis first describes a genome mining study to survey the distribution of lasso peptide biosynthesis clusters in Nature and reports 79 putative high ranking clusters identified across nine bacterial phyla. As validation, a lasso peptide - astexin-1 - was isolated from the organism Asticcacaulis excentricus CB48. Its molecular structure in dimethylsulfoxide was elucidated using NMR. A bioinformatics analysis identified a new class of enzymes present in a subgroup of lasso peptide gene clusters. One of these enzymes (AtxE2) was heterologously expressed and its activity as the first lasso peptide isopeptidase characterized. The structure of the astexin-3 peptide in dimethylsulfoxide was determined. Lasso maturation enzymes were organized into two distinct clades. A panel of alanine substituted variants of astexins-2 and -3 was used to investigate the specificity of AtxE2. The solution structures of all three astexin peptides in water were solved, enabling a fair comparison. Crystallization of AtxE2 is described and a preliminary structure presented. Finally, preliminary studies on engineering of the astexin system are described. A protocol for the expression and purification of cysteine variants of astexin-3 was developed. Gene deletion strains of A. excentricus were constructed to test the regulatory network of astexin-1. The astexins were the first examples of intracellular lasso peptides, which lacked an associated exporter and had an unexpected length and polar composition. Confirmation of the first lasso peptide isopeptidase challenged the paradigm that all lasso peptides are antimicrobial. Identification of conserved motifs within lasso peptide maturation enzymes, highlighted unique features of these enzymes. Similarly, phylogenetic analysis of maturation enzyme sequences allowed for a functional categorization of lasso peptide gene clusters. Overall, the astexin work presented here spawned many exciting projects, such as gene editing of A. excentricus and introduction of disulfide bonds into astexin-3, and will continue as a wellspring of fruitful research topics for years to come.