Impact of expression systems on the transcriptome of : insights for enhanced production of glutaminase PrgA.

The gram-positive bacterium 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 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 P promoter or from the xylose-inducible P promoter. The use of the inducible P promoter influenced the expression of many more genes than when the constitutive P 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.IMPORTANCEThe bacterium is widely used in bioindustry to produce various proteins. However, not all proteins are efficiently produced. In this study, we examined how responds to the production of three different industrially relevant proteins. By analyzing gene activity using RNA sequencing, we identified factors that limit protein production and successfully improved the yield of one protein by 2.7-fold. However, our results also show that the choice of promoter used significantly affects transcriptomic regulation. This highlights the importance of testing production conditions that closely resemble relevant industrial settings to identify the best strategies for improving industrial protein yields.