Lasso peptides are natural products ribosomal origin with a unique knotted structural fold. Even though only a few of them are known, recent reports of newly isolated lasso peptides were scarce. In this work, we report the identification of a novel lasso peptide gene cluster from Caulobacter segnis, that produces three new lasso peptides (caulosegnins I, II, and III) using a single biosynthetic machinery. These lasso peptides possess different ring sizes and amino acid sequences. In this study, we have developed a system for enhanced lasso peptide production to allow isolation of these compounds through heterologous expression in Escherichia coli. We were able to elucidate the structure of the most abundant lasso peptide caulosegnin I via NMR spectroscopic analysis and performed a thorough mutational analysis that gave insight into their biosynthesis and revealed important factors affecting the stabilization of the lasso fold in general. The caulosegnins also show a diverse behavior when subjected to thermal denaturation, which is exceptional as all lasso peptides were believed to have an intrinsic high thermal stability.