RNA sequencing of B. subtilis overexpressing GFPnb, XynA and PrgA

Bacillus subtilis is widely used for industrial protein production due to its high capacity and GRAS status. Despite extensive efforts to optimize the host, bottlenecks still limit its full production potential, particularly for secreted heterologous proteins. In this study, we employ RNA sequencing as an unbiased approach to investigate B. subtilis responses to the overexpression of three distinct secreted proteins: the native xylanase XynA, the protein glutaminase PrgA from C. proteolyticum, and a model nanobody targeting GFP. Our analyses reveal that most transcriptome changes occur regardless of the overexpressed construct. However, when comparing different expression systems, constitutive versus inducible, we observe that many transcriptional regulations are reversed. This highlights the importance of performing transcriptome analyses under conditions that closely mimic the industrial fermentation setting to optimize protein production. Overall design: RNA-seq profiling of Bacillus subtilis BWB143 cells harboring plasmids encoding GFPnb, XynA and PrgA under either PamyQ (constitutive) or Pxyl (xylose-inducible).