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Abstract

Lasso peptides are a subclass of RiPPs, ribosomally synthesized and posttranslationally modified peptides, characterized by a rigid rotaxane structure that is abundant across a variety of organisms. Due to their stable configuration, lasso peptides are less susceptible to proteases and thermal unfolding compared with other peptides, which makes them of a particular interest in drug discovery. In this thesis, the two lasso peptides cellulonodin-2 and thermonodin that belong to Gram-positive thermophiles Thermobifida cellulosilytica and Lihuaxuella thermophila, respectively, were studied. Furthermore, lasso peptide biosynthesis requires the involvement of at least three genes: A, B, C, but additional D genes (ABC transporter) or post-translational modifications (PTMs) can be found in biosynthetic gene clusters (BGCs). Cellulonodin-2 and thermonodin both contain protein L-isoaspartyl methyltransferase (PIMT) homologs, tceMT and lthMT in their BGCs. Preliminary liquid chromatography–mass spectrometry (LCMS) data suggests that an additional dehydration occurs during succinimide formation. Both gene clusters contain a conserved motif, where it is hypothesized that the tceMT and lthMT modify the residues in their corresponding BGCs. These two lasso peptides are unique not only because PIMT homologs in Grampositive bacteria are rare, but also because they are the first lasso peptides to contain PTMs in their rings. The biochemistry of the reaction in cellulonodin-2 was studied in vivo through principal point mutations to rule out hydrolysis between polar residues and provide further evidence for the formation of a stable succinimide. Thermonodin was expressed later with the primary purpose of serving as a comparison to cellulonodin-2. Multiple plasmids were constructed, followed by in vivo expressions to assess modified and unmodified peptide yield.