# Rapid Identification of Superior Endogenous Signal Peptides for Heterologous Protein Secretion by Corynebacterium glutamicum Through Modular Cloning and Automation

## TL;DR

A new method rapidly finds the best signal peptides in Corynebacterium glutamicum for efficient protein secretion, improving industrial enzyme production.

## Contribution

A modular cloning system and automation platform enable rapid screening of signal peptides for protein secretion in C. glutamicum.

## Key findings

- Several native signal peptides outperformed the Bacillus subtilis NprE reference in secreting a fungal cutinase.
- The workflow identified superior signal peptides for four PETases in addition to cutinase.
- The method enables high-throughput screening of hundreds of clones in parallel.

## Abstract

Secretory protein production by microbial hosts simplifies product recovery and is therefore preferred over intracellular production. Efficient secretion of heterologous proteins by bacteria requires the identification of optimal signal peptides (SPs), a step that often limits process development. Using
Corynebacterium glutamicum
as a model host, we established a modular cloning system enabling rapid assembly of expression plasmids for secretory protein production. Screening a library of 30 individually cloned endogenous SPs with a fungal cutinase as target protein demonstrated that several native SPs achieved substantially higher secretion levels than the widely used
Bacillus subtilis
NprE reference SP. To accelerate SP discovery, we developed a one‐pot approach in which
C. glutamicum
was directly transformed with a single modular cloning mixture containing all 30 SPs. Combined with the AutoBioTech high‐throughput platform for cultivation, harvesting, and protein quantification, this strategy enabled screening of several hundred clones in parallel. Superior SPs were rapidly identified not only for cutinase but also for four polyethylene terephthalate hydrolases (PETases). This streamlined workflow significantly reduces time and cost for selecting effective SPs and provides a versatile platform for advancing secretory protein production in
C. glutamicum
.

A modular cloning approach combined with an automated high‐throughput platform enabled rapid identification of superior endogenous signal peptides for the secretion of proteins of interest (POIs), such as industrial enzymes, by Corynebacterium glutamicum. This streamlined workflow accelerates discovery and development of efficient bacterial systems for secretory protein production.