Abstract
Ruminococcin A (RumA) is a lanthipeptide with high activity against pathogenic clostridia and is naturally produced by the strict anaerobic bacterium Ruminococcus gnavus E1, isolated from human intestine. Cultivating R. gnavus E1 is challenging, limiting high-quality production, further biotechnological development and therapeutic exploitation of RumA. To supply an alternative production system, the gene encoding RumA-modifying enzyme (RumM) and the gene encoding the unmodified precursor peptide (preRumA) were amplified from the chromosome of R. gnavus E1 and coexpressed in Escherichia coli. Our results show that the ruminococcin-A lanthionine synthetase RumM catalyzed dehydration of threonine and serine residues and subsequently installed thioether bridges into the core structure of a mutant version of preRumA (preRumA∗). These modifications were achieved when the peptide was expressed as a fusion protein together with green fluorescence protein (GFP), demonstrating that a larger attachment to the N-terminus of the leader peptide does not obstruct in vivo processivity of RumM in modifying the core peptide. The leader peptide serves as a docking sequence which the modifying enzyme recognizes and interacts with, enabling its catalytic role. We further investigated RumM catalysis in conjunction with the formation of complexes observed between RumM and the chimeric GFP fusion protein. Results obtained suggested some insights into the catalytic mechanisms of class II lanthipeptide synthetases. Our data further indicated the presence of three thioether bridges, contradicting a previous report whose findings ruled out the possibility of forming a third ring in RumA. Modified preRumA∗ was activated in vitro by removing the leader peptide using trypsin and biological activity was achieved against Bacillus subtilis ATCC 6633. A production yield of 6 mg of pure modified preRumA∗ per liter of E. coli culture was attained and considering the size ratio of the leader-to-core segments of preRumA∗, this amount would generate a final yield of approximately 1–2 mg of active RumA when the leader peptide is removed. The yield of our system exceeds that attainable in the natural producer by several 1000-fold. The system developed herein supplies useful tools for product optimization and for performing in vivo peptide engineering to generate new analogs with superior anti-infective properties.
Highlights
The healthcare sector of our society is currently facing a precarious situation that has been vividly described as a “new pre-antibiotic era” where it is estimated that within the few years, most of the commonly used anti-infective agents may become ineffective due to growing increase of antibiotic resistances provoked by improper use of antibiotics in human medication and animal farming (Rios et al, 2016)
The genes rumA and rumM were transferred to an E. coli host on individual plasmids with compatible origins of replication. rumM was encoded in three different plasmid versions pLEOrM, pLEOM1, and pLEOM1 together with a His-tag (His6-rumM which encodes the dualfunctional lanthionine synthetase (RumM)). rumA was encoded in three different plasmid versions pLEOrA, pLEOsrA∗ in which small ubiquitin-like modifier (SUMO) was fused to the N-terminus of preRumA∗, pLEOgrA and pLEOgrA∗
We have shown that fusing the structural gene for the precursor peptide preRumA to the gene encoding the fast folding green fluorescence protein (GFP) and co-expressing the chimeric construct simultaneously with the ruminococcin-A lanthionine synthetase RumM resulted in the biosynthesis and modification of the peptide in vivo
Summary
The healthcare sector of our society is currently facing a precarious situation that has been vividly described as a “new pre-antibiotic era” where it is estimated that within the few years, most of the commonly used anti-infective agents may become ineffective due to growing increase of antibiotic resistances provoked by improper use of antibiotics in human medication and animal farming (Rios et al, 2016) Owing to their special structural, physicochemical and functional characteristics; antimicrobial peptides (AMPs) of the lanthipeptides subgroup may be employed as alternative drugs (Dischinger et al, 2014). Lanthipeptide synthetases are enzymes that catalyze posttranslational modifications (PTMs) of lanthipeptides Those of class II lanthipeptides are dual-functional, possessing an N-terminal dehydratase domain and a C-terminal cyclase domain (Repka et al, 2017). Once all the PTMs are installed, the N-terminal domain of a dedicated bifunctional ABC-transporter maturation and secretory (AMS) protein cleaves off the leader peptide, activating the peptide which is exported by the same protein to the extracellular space (Repka et al, 2017)
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