Lasso Peptide Bioengineering and Bioprospecting - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 96

rational design of MccJ25-based antibacterials. Nevertheless, there was one report
of attempts to generate rationally designed active peptides based on the sequence
of MccJ25 (Soudy et al. 2012 ). It was hypothesized that an interlocked topology
could be acquired by a combination of intra-peptide disulphide bond formation and
electrostatic or hydrophobic interactions. Thus, related amino acids such as cyste-
ine, lysine, arginine and glutamine were introduced into the MccJ25 sequence, and
a total of six peptides were generated by chemical synthesis. Although two showed
some antibacterial activity, none of them had the lasso topology. This affirms that
engineering of the lasso scaffold can only be achieved by using the biosynthetic
enzymes.
The macrolactam ring sequence of RES-701-1, which is an antagonist of the
endothelin B receptor, has been exploited for peptide engineering. Hybrid peptides,
where the macrolactam ring of RES-701-1 was fused to the N-terminus of endothe-
lins, RGD-containing peptides and farnesyltransferase inhibitors, showed improved
biological activity and/or proteolytic stability (Shibata et al. 1998 , 2003 ). It was
suggested that the macrolactam ring of RES-701-1 can stabilize the solution con-
formation, particularly the β-turn structure, of the C-terminal peptide. However, this
was not demonstrated by NMR analysis of the hybrid peptides. Unrelated to the use
of the lasso topology, these studies nevertheless show the utility of the macrolactam
ring to impose conformational constraints on peptides.
A challenge of lasso peptide engineering is to generate chimeric lasso peptides
(or designer peptides) where the ring and the tail region can be changed indepen-
dently. In the case of MccJ25, these two regions are involved in different processes
of the mode of action. Thus, by modulating them, one may obtain novel bioactivi-
ties. Since the lasso scaffold is not accessible by chemical synthesis, this goal relies
essentially on enzymatic machineries. Understanding the substrate specificity of
lasso synthetases and the role of leader peptides is the key to success. Genome min-
ing provides a rich resource of lasso synthetases that can be applied to bioengineer-
ing. Reports are not yet available concerning this line of research.

5.2 Genome Mining for Lasso Peptide Discovery

MccJ25 is the first lasso peptide with a characterized gene cluster and has been
used as a model to study the biosynthetic mechanism (for details, see Chap. 3). It
has been confirmed in 2007 that the two maturation enzymes McjB and McjC are
sufficient to transform the precursor peptide McjA into MccJ25 (Clarke and Cam-
popiano 2007 ; Duquesne et al. 2007 ). Initial effort to find unknown lasso peptides
used McjB and McjC sequences to search available microbial genomes (Duquesne
et al. 2007 ; Severinov et al. 2007 ). The clustering of genes encoding McjB and
McjC homologues was considered as indicative of putative lasso gene clusters, and
adjacent mcjA -like genes were searched carefully. Phylogenetic analyses of McjB
and McjC homologues revealed that the overall branching pattern does not reflect
the species taxonomy, thus indicating a horizontal gene transfer mechanism of lasso