DISCOVERY AND ENGINEERING OF LASSO PEPTIDES: MOLECULAR SWITCHES, POST-TRANSLATIONAL MODIFICATIONS, AND FUSION PROTEINS

Abstract

Lasso peptides are a unique class of natural products, characterized by a threaded topology and remarkable stability. In addition, lasso peptides exhibit a wide range of bioactivities. These properties make them strong candidates for peptide therapeutics and as peptide scaffolds. Despite progress made in the discovery of novel lasso peptides and engineering of lasso peptide based scaffolds, there is still much remaining for further exploration. Thus, this thesis aims 1) to discover and characterize novel lasso peptide gene clusters, 2) to develop an engineering platform for expanding the lasso peptide based scaffolds for potential therapeutic applications.

Chapter 2 presents the optimization of a previous developed precursor-centric genome mining algorithm using new lasso peptide precursor sequence features and new motifs of lasso peptide maturation enzymes. Applying the updated algorithm on DNA contig sets, complete and draft genomes from bacteria and archaea, I was able to identify 948 high confidence putative lasso peptide gene clusters. In addition, I discovered several new gene clusters with novel precursor sequence features and post-translational elements.

I found two lasso peptide gene clusters associated with isopeptidases from Gram-negative organism Asticcacaulis benevestitus through the genome mining. In chapter 3, along with Michelle Wu and Jason Qin, I successfully expressed both peptides, named beneondin-1 and benendin-2, in E. coli. The molecular structure of benenodin-1 was solved using solution NMR. I found benenodin-1 exhibits conformational switching between two distinct threaded conformers upon heat stimulation. This makes benenodin-1 the first natural peptide-based molecular switch. I also showed only the native conformer of benenodin-1 is cleaved by its associated isopeptidase, which may also be relevant to the biological function of these molecules.

Chapter 4 focuses on characterization of a lasso peptide, albusnodin, from Streptomyces albus DSM 41398. I found albusnodin is acetylated, the first example of a lasso peptide with this modification. Performing genetic studies with Wai Ling Cheung-Lee, I further showed that the acetyltransferse colocalized with the albusnodin gene cluster is required for the biosynthesis of this lasso peptide. In addition, bioinformatics analysis showed this type of lasso peptide is widespread in Actinobacteria, with 44 examples found in currently sequenced genomes.

Finally, in chapter 5, I developed a new engineering platform on the lasso peptide, astexin-1. I demonstrated a fusion of two model proteins, the artificial leucine zipper A1 and the superfolder variant of GFP, to the C-terminus of astexin-1. Moreover, along with Kenneth Hubbell and Shubham Chatterjee, I combined this fusion approach with cysteine bio-conjugation chemistry to graft two anticancer epitopes on astexin-1.

Overall, these exciting projects carried out in this thesis have enriched and opened new research areas in the field of lasso peptides.