A novel signal peptide toolbox for optimized heterologous expression of yeast pheromones in Bacillus subtilis.

Optimising downstream processes and achieving high product yields are essential for cost-effective and scalable bioproduction, with efficient protein secretion being a critical factor in industrial applications. To address this, we leveraged the widely utilised Sec secretion system of Bacillus subtilis to enhance heterologous protein secretion. Given the lack of reliable in silico tools for predicting optimal combinations of Sec system signal peptides (SPs) with a protein of interest (POI), we aimed to optimise the secretion efficiency of SP-POI pairings by a toolbox approach. We developed an integrative evaluation vector in which the promoter, SPs, and a coding sequence (CDS) of the POI are easily exchangeable. Further, we generated a toolbox containing 74 SPs naturally present in B. subtilis that can easily be integrated into the evaluation vector and thereby fused to the POI. As proof-of-concept, two short peptides from yeast: α-pheromone from Saccharomyces cerevisiae and P-pheromone from Schizosaccharomyces pombe were chosen as POI and secretion efficiency was measured. Successful expression and secretion of both peptides in B. subtilis were verified by an indirect ELISA assay. Eight out of 74 SPs facilitated P-pheromone secretion, while just three effectively enabled the expression of α-pheromone. The maximum observed peptide secretion levels were 43 nM for P-pheromone and 8 nM for α-pheromone. This work demonstrates the necessity of versatile screening approaches to find a matching pairing of the Sec secretion system SP and a POI. We close this gap by providing a robust toolbox with easily exchangeable elements.