Abstract
BackgroundBacillus 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.ResultsThis 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-1 β-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 β-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.ConclusionsThe 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.
Highlights
The Gram-positive bacterium B. subtilis is widely used in large scale production of endogenous and heterologous proteins used in food- and other industries
It is favored as a production host since it has the capacity of secreting proteins to high levels into the medium enabling easy isolation and purification, it can be grown in large fermentations and is considered as a GRAS (Generally Recognized As Safe) organism by the US Food and Drug Administration
Transcriptome analysis of lipoprotein, membrane protein or secreted protein overproduction stress B. subtilis remains a powerful host for the production of secreted or membrane proteins but expression of heterologous proteins in particular has met limitations
Summary
The Gram-positive bacterium B. subtilis is widely used in large scale production of endogenous and heterologous proteins used in food- and other industries. It is favored as a production host since it has the capacity of secreting proteins to high levels into the medium enabling easy isolation and purification, it can be grown in large fermentations and is considered as a GRAS (Generally Recognized As Safe) organism by the US Food and Drug Administration. At the late stages, which include removal of the signal peptide, release from the translocase, folding and passing the cell wall, deficiency in signal peptidases, foldases, chaperones and presence of extracellular proteases resulting in incorrect folding of proteins and protein’s instability may set limits to the secretion efficiency [1,3].
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