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

ABSTRACT: 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.

ORGANISM(S): Bacillus subtilis

PROVIDER: GSE293838 | GEO | 2025/04/17

REPOSITORIES: GEO

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Project description:Background. Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results. This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1beta -lactamase of Escherichia coli), membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus) and lipoproteins (MntA and YcdH of B. subtilis). Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes). Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 beta-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue) specifically under membrane proteins overproduction. Conclusions. The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host. Samples for transcriptome analyses were induced at the exponential-growth phase (OD600 = 0.7) with 0.1% subtilin (subtilin containing supernatant of subtilin producing B. subtilis strain ATCC 6633). Cells were harvested 30 min after induction. Three or four independent cultures of each strain (target strains and controls) were used, and cells were sampled for microarray experiment.

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

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