Structure-Activity Analysis of Gram-positive Bacterium-producing Lasso Peptides with Anti-mycobacterial Activity

Junji Inokoshi,Hiroshi Tomoda,Yuji Shimizu,Midori Miyake,Nobuhiro Koyama

doi:10.1038/srep30375

Abstract

Lariatin A, an 18-residue lasso peptide encoded by the five-gene cluster larABCDE, displays potent and selective anti-mycobacterial activity. The structural feature is an N-terminal macrolactam ring, through which the C-terminal passed to form the rigid lariat-protoknot structure. In the present study, we established a convergent expression system by the strategy in which larA mutant gene-carrying plasmids were transformed into larA-deficient Rhodococcus jostii, and generated 36 lariatin variants of the precursor protein LarA to investigate the biosynthesis and the structure-activity relationships. The mutational analysis revealed that four amino acid residues (Gly1, Arg7, Glu8, and Trp9) in lariatin A are essential for the maturation and production in the biosynthetic machinery. Furthermore, the study on structure-activity relationships demonstrated that Tyr6, Gly11, and Asn14 are responsible for the anti-mycobacterial activity, and the residues at positions 15, 16 and 18 in lariatin A are critical for enhancing the activity. This study will not only provide a useful platform for genetically engineering Gram-positive bacterium-producing lasso peptides, but also an important foundation to rationally design more promising drug candidates for combatting tuberculosis.

Highlights

Analysis of lariatin non-producing R. jostii generated by transposon mutagenesis[11]
A convergent expression system to produce lariatin variants was established by the strategy in which larA mutant gene-carrying plasmids were transformed into larA-deficient R. jostii K01-B0171 (Fig. 2A), because heterologous expression strategy using R. erythropolis JCM3201 and R. jostii JCM11615 as hosts was not successful[23]
Our previous study has shown that a five-gene cluster is responsible for the biosynthesis and export of lariatin A in R. jostii; larA encodes a precursor peptide consisting of 46 amino acid residues, larB and larD encode maturation enzymes that convert the precursor peptide into the mature lariatin B, unspecific peptidase are probably relevant to hydrolyze the C terminus of lariatin B to yield lariatin A, and larE encodes an exporter of lariatin A23 (Fig. S1)

Summary

Introduction

Analysis of lariatin non-producing R. jostii generated by transposon mutagenesis[11]. The genetic determinants for only two lasso peptides produced by Gram-negative bacteria, microcin J2522 and capistruin[8], were firmly established. Capistruin was discovered from the genome mining approach using the biosynthesis gene cluster information of microcin J25. The mechanistic gene structure for biosynthesis of lariatins was found to be essentially similar to those from Gram-negative bacteria except for the presence of one extra ORF (larC for lariatins), which appears common in the clusters of lasso peptides produced by Gram-positive bacteria[23]. A number of lasso peptides were reported by the genome mining approach, they are mainly produced by proteobcateria, and the example from actinobacteria is limited. Few reports have addressed heterologous production of lasso peptides from Gram-positive bacteria. We focused on lariatins produced by Gram-positive R. jostii K01-B0171 with intriguing anti-mycobacterial activity. Our results concluded that four amino acid residues in lariatin A are essential for production and maturation in the biosynthetic machinery and that three amino acid residues of lariatin A are responsible for anti-mycobacterial activity

Methods

Results

Conclusion