The gram-positive bacterium Bacillus subtilis is widely used for enzyme production, especially due to its superior protein secretion capacity. In this study, we have investigated how efficient transcriptome analysis can identify general and protein-specific secretion stress. For this, we constructed B. subtilis strains overproducing different commercially relevant proteins, including a GFP-specific camelid nanobody (GFPnb), the xylanase XynA and the protein glutaminase PrgA, and expressed these proteins either from the strong constitutive PamyQ promoter or from the xylose-inducible Pxyl promoter. The use of the inducible Pxyl promoter influenced the expression of many more genes than when the constitutive PamyQ promoter was used to express the different proteins. Presumably, the constitutive expression gives cells time to adapt to the imposed secretion stress. These results underscore the importance of performing transcriptome analyses under conditions that most closely mimic industrial production settings. Finally, we tested whether the transcriptome data could provide clues to improve the production of PrgA. The secretion stress response upon PrgA expression appeared similar to that observed upon alpha-amylase overproduction. However, mutants that would normally improve alpha-amylase production did not result in better PrgA yields. Overexpression of PrgA also results in the downregulation of several Clp protein chaperones. Interestingly, induction of these genes, by inactivating their transcriptional repressor CtsR, strongly improved PrgA production. This analysis highlights the limitations, as well as the potential use of transcriptome comparisons to uncover production bottlenecks.