# Myxarylin: Total In Vitro Biosynthesis, Expansion of Substrate Scope, and Bioengineered Thioamidated Biarylitides

## TL;DR

Scientists fully recreated the biosynthesis of a new type of peptide called myxarylin and expanded its chemical variety using engineered proteins.

## Contribution

The study achieved the first complete in vitro biosynthesis of myxarylin and demonstrated novel methods for expanding its chemical diversity.

## Key findings

- Cross-linking is the first and gatekeeping step in myxarylin biosynthesis.
- Precursor peptide engineering led to unexpected modification shifts in the biosynthetic pathway.
- The crystal structure of the methyltransferase guided engineering to expand substrate scope.

## Abstract

Biarylitides are
a new class of ribosomally synthesized
and post-translationally
modified peptides (RiPPs) featuring the smallest reported precursor
peptide and cytochrome P450-mediated cross-links. Here, we report
the complete in vitro reconstitution of the myxobacterial
biarylitide, myxarylin. We demonstrate that cross-linking is the first
step and acts as a gatekeeper for downstream processing. The cytochrome
P450 enzyme P450BytO from the myxarylin biosynthetic gene
cluster exhibits remarkable substrate tolerance, allowing biosynthesis
of new-to-nature thioamidated biarylitides through an unprecedented
modular precursor peptide engineering approach. Surprisingly, changes
in the precursor peptide sequence resulted in a shift in the installation
of the P450BytO-mediated modification from the expected
C- to the N-terminus. Leader peptide removal follows cross-linking
and is likely carried out by a prolyl oligopeptidase (POP), a member
of the serine protease family. The last step of the pathway involves
N-terminal methylation, which also prevents premature degradation
of the pathway intermediates by the POP. The crystal structure of
the methyltransferase in complex with SAH and myxarylin allowed us
to rationalize its substrate selectivity and guide protein engineering
to expand its substrate scope.