title: Lasso peptides from proteobacteria: Genome mining employing heterologous expression and mass spectrometry.
abstract: Lasso peptides are natural products with a unique three dimensional structure resembling a lariat knot. They are from ribosomal origin and are post-translationally modified by two enzymes (B and C), one of which shares little similarity to enzymes outside of lasso peptide biosynthetic gene clusters and as such is a useful target for genome mining. In this study, we demonstrate a B protein-centric genome mining approach through which we were able to identify 102 putative lasso peptide biosynthetic gene clusters from a total of 87 different proteobacterial strains. Ten of these clusters were cloned into the pET41a expression vector, optimized through incorporation of a ribosomal binding site and heterologously expressed in Escherichia coli BL21(DE3). All 12 predicted lasso peptides (namely burhizin, caulonodin I, caulonodin II, caulonodin III, rhodanodin, rubrivinodin, sphingonodin I, sphingonodin II, syanodin I, sphingopyxin I, sphingopyxin II, and zucinodin) were detected by high-resolution Fourier transform mass spectrometry and their proposed primary structure was confirmed through tandem mass spectrometry. High yields (ranging from 0.4 to 5.2 mg/L) were observable for eight of these compounds, while thermostability assays revealed five new representatives of heat labile lasso peptides.
authors.affiliation: Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Strasse 4 and LOEWE-Center for Synthetic Microbiology, Marburg, D-35032, Germany.
authors.forename: Julian D
authors.initials: JD
authors.lastname: Hegemann
authors.forename: Marcel
authors.initials: M
authors.lastname: Zimmermann
authors.forename: Shaozhou
authors.initials: S
authors.lastname: Zhu
authors.forename: Dennis
authors.initials: D
authors.lastname: Klug
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D000595:Amino Acid Sequence; D001688:Biological Products; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D020560:Proteobacteria; D053719:Tandem Mass Spectrometry
chemical_list: D001688:Biological Products; D010455:Peptides
keywords: genome mining; lasso peptide; mass spectrometry; proteobacteria; thermal stability