# Impact of expression systems on the transcriptome of Bacillus subtilis: insights for enhanced production of glutaminase PrgA

## TL;DR

This study explores how different gene expression systems affect Bacillus subtilis' ability to produce proteins, revealing ways to improve production efficiency.

## Contribution

The study demonstrates how transcriptome analysis can reveal production bottlenecks and improve protein yields in industrial settings.

## Key findings

- Using the inducible Pxyl promoter affects more genes than the constitutive PamyQ promoter during protein overproduction.
- Transcriptome data helped improve glutaminase PrgA production by 2.7-fold through induction of Clp chaperone genes.
- Promoter choice significantly influences transcriptomic regulation and production efficiency in B. subtilis.

## Abstract

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.

The bacterium Bacillus subtilis is widely used in bioindustry to produce various proteins. However, not all proteins are efficiently produced. In this study, we examined how B. subtilis 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.