Biosynthesis of a Putative Lasso Peptide Produced by Sphingopyxis alaskensis

Abstract

Lasso peptides are a special class of peptides with a unique structure consisting of an 8- or 9-membered ring through which a peptide tail threads, giving them the appearance of a lasso. This structure renders them highly thermostable and resistant to cleavage by proteases, making them excellent candidates for use as bioengineering scaffolds. Lasso peptides also have a wide range of natural biologically relevant properties, such as antimicrobial activity. Two enzymes are required for the conversion of the lasso peptide precursor into the mature lasso peptide. The gene encoding the precursor is referred to as the A gene, and the maturation enzymes are referred to as the B and C genes. Previous work by the Link Lab has identified many putative lasso peptides using a genome-mining approach. Here, a putative lasso peptide produced by the marine bacterium Sphingopyxis alaskensis is examined. The gene cluster responsible for the peptide's production was amplified from the S. alaskensis genome and inserted into a plasmid for expression in Escherichia coli. Two separate constructs were expressed, but the putative lasso peptide was not produced. Phylogenetic analyses were conducted of the B and C genes of the putative lasso peptide clusters identified by the Link Lab's genome-mining algorithm and of the 16s rDNA of the organisms in which they are found. A comparison of these analyses suggests that lasso peptide clusters can move to new organisms via horizontal gene transfer and that the genes of a cluster are transferred together. Motifs were generated from each clade in the B gene phylogenetic tree, and the Link Lab's genome-mining algorithm was re-run using these new motifs. The identification of previously undiscovered putative lasso peptide clusters using these new motifs suggests that a model of lasso peptide gene clusters based solely on the microcin J25 and capistruin clusters may be too narrow. Furthermore, many lasso peptide clusters were identified using the new motifs that were also identified previously by the Link Lab. This double identification helps inform which putative lasso peptide clusters are the best candidates for future study.