Discovery of the Crescenodin-1 Lasso Peptide

Abstract

Recently in the Link Lab, a precursor genome mining approach to find novel lasso peptides from the known genomic sequences of bacteria had been shown accurate for a normal, Class II lasso peptide, astexin-1. The same technique hypothesized the presence of other new lasso peptides within the genomes of other common bacteria, including Caulobacter crescentus. The most distinguishing feature of this proposed lasso peptide is its uncommon core lasso peptide sequence. Instead of beginning with a glycine or cysteine amino acid, as most lasso peptide sequences do, the proposed lasso peptide begins with a serine amino acid and could have different characteristics than other lasso peptides. Three different DNA sequences of the proposed C. crescentus lasso peptide gene cluster were expressed under different conditions and tested for lasso peptide production: a wild-type, a negative control, and a no hairpin construct to increase peptide production. The lasso peptide, crescenodin-1, was confirmed to be produced with HPLC and mass spectrometry in the no hairpin construct after expression for three days at 20 °C in E. coli cells; however it is produced in very small quantities. Once the existence of the lasso peptide was confirmed, purified crescenodin-1 underwent thermostability and carboxypeptidase Y assays to determine some of its characteristics. The thermostability assays confirmed the security of the lasso structure for at least 1 hour at 95 °C and its delayed conversion after longer heat exposures up to 8 hours. The carboxypeptidase Y assay most likely confirmed the sensitivity of crescenodin-1 to the peptidase, but could not conclusively determine the location of the steric lock on the threaded lasso peptide.