Discovery of Borosin Catalytic Strategies and Function through Bioinformatic Profiling

Borosins are ribosomally synthesized and post-translationally
modified
peptides (RiPPs) containing backbone α-N-methylations.
These modifications confer favorable pharmacokinetic properties including
increased membrane permeability and resistance to proteolytic degradation.
Previous studies have biochemically and bioinformatically explored
several borosins, revealing (1) numerous domain architectures and
(2) diverse core regions lacking conserved sequence elements. Due
to these characteristics, large-scale computational identification
of borosin biosynthetic genes remains challenging and often requires
additional, time-intensive manual inspection. This work builds upon
previous findings and updates the genome-mining tool RODEO to automatically
evaluate borosin biosynthetic gene clusters (BGCs) and identify putative
precursor peptides. Using the new RODEO module, we provide an updated
analysis of borosin BGCs identified in the NCBI database. From our
data set, we bioinformatically predict and experimentally characterize
a new fused borosin domain architecture, in which the modified natural
product core is encoded N-terminal to the methyltransferase domain.
Additionally, we demonstrate that a borosin precursor peptide is a
native substrate of shewasin A, a reported aspartyl peptidase with
no previously identified substrates. Shewasin A requires post-translational
modification of the leader peptide for proteolytic maturation, a feature
not previously observed in RiPPs. Overall, this work provides a user-friendly
and open-access tool for the analysis of borosin BGCs and we demonstrate
its utility to uncover additional biosynthetic strategies within the
borosin class of RiPPs.