# Library‐on‐Library Intercellular Labeling for Selection of Biotin Ligase and Acceptor Peptides

## TL;DR

A new method called library-on-library selection is developed to find enzyme and peptide pairs that work together efficiently.

## Contribution

The study introduces a sequential labeling platform for enzyme-substrate pair selection using LOL.

## Key findings

- A proof-of-concept selection was performed using Escherichia coli biotin ligase and its acceptor peptide.
- The method enables coevolution of enzyme-peptide pairs with tunable activity.

## Abstract

Library‐on‐library (LOL) selection screens combinatorial libraries to generate new protein pairs. Previously, LOL selection has only been applied to stable protein–protein interactions. To extend LOL to transient enzyme–substrate pairs, a generalizable sequential LOL trans‐ and cis‐labeling platform is developed, and a proof‐of‐concept selection is performed on Escherichia coli biotin ligase (BirA) and its acceptor peptide (AP). Using yeast surface display, AP mutant libraries are selected against BirA mutant libraries to identify AP variants trans‐biotinylated by BirA mutants. Matched BirA mutants are subsequently enriched via the SpyTag–SpyCatcher‐mediated cis‐labeling platform. This represents the first demonstration of enzyme–peptide substrate LOL selection and offers a versatile framework for engineering new enzyme–peptide substrate pairs with varied activities.

The library‐on‐library selection platform first identifies novel target peptides via intercellular trans‐biotinylation, followed by the selection of matched enzyme variants through SpyTag–SpyCatcher‐mediated cis‐biotinylation. Using Escherichia coli biotin ligase and its acceptor peptide as a model system for site‐specific conjugation, this method provides a proof of principle for the coevolution of enzyme–peptide pairs with tunable activity.© 2026 WILEY‐VCH GmbH