Matamouros_2026_Microb.Biotechnol_19_e70299

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.