An orthogonal system for heterologous expression of actinobacterial lasso peptides in Streptomyces hosts

Jimmy Mevaere,Séverine Zirah,Carlos Afonso,Sylvie Rebuffat,Sergey B. Zotchev,Olha Schneider,Olga N. Sekurova,Christophe Goulard,Yanyan Li,Haiyan Ma

doi:10.1038/s41598-018-26620-0

Abstract

Lasso peptides are ribosomally synthesized and post-translationally modified peptides produced by bacteria. They are characterized by an unusual lariat-knot structure. Targeted genome scanning revealed a wide diversity of lasso peptides encoded in actinobacterial genomes, but cloning and heterologous expression of these clusters turned out to be problematic. To circumvent this, we developed an orthogonal expression system for heterologous production of actinobacterial lasso peptides in Streptomyces hosts based on a newly-identified regulatory circuit from Actinoalloteichus fjordicus. Six lasso peptide gene clusters, mainly originating from marine Actinobacteria, were chosen for proof-of-concept studies. By varying the Streptomyces expression hosts and a small set of culture conditions, three new lasso peptides were successfully produced and characterized by tandem MS. The newly developed expression system thus sets the stage to uncover and bioengineer the chemo-diversity of actinobacterial lasso peptides. Moreover, our data provide some considerations for future bioprospecting efforts for such peptides.

Highlights

Microbial natural products have been the major source of human medicines and agrochemicals for hundreds of years
Recently-acquired genome sequences from several both known and newly isolated Actinobacteria were mined for putative lasso peptide biosynthetic gene clusters
The primary sequences of the predicted lasso peptides were quite different and diverse, representing a considerable chemo-diversity (Table 1). They appeared to display new features, including non-canonical residues involved in the macrolactam linkage, the presence of one disulphide bond and post-translational modifications decorating the lasso scaffold (Table 1)

Summary

Introduction

Microbial natural products have been the major source of human medicines and agrochemicals for hundreds of years. Most of the bioactive lasso peptides are isolated from Actinobacteria[7,9,10,11,12,13,14,15,16] Those displaying antibacterial activities are narrow-spectrum and frequently possess novel mode of action, including siamycins that attenuate the expression of virulence factors in Enterococcus faecalis by inhibiting a quorum sensing-related histidine kinase[17,18], lassomycin that displays anti-mycobacterial activity via inhibition of the Clp protease[11] and streptomonomicin, which potently inhibits the growth of Bacillus anthracis by targeting a vital two-component system[12]. Our results provide some guidelines in future bioprospecting efforts in Actinobacteria for lasso peptides

Methods

Results

Conclusion