RESUMO
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-1 ß-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 ß-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.
Assuntos
Bacillus subtilis/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação para Baixo , Lipoproteínas/genética , Lipídeos de Membrana/genética , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Fator sigma/genética , Fator sigma/metabolismo , Transcriptoma , Regulação para CimaRESUMO
High-level production of secretory proteins in Bacillus subtilis leads to a stress response involving the two-component system CssRS and its target genes htrA and htrB. Here, we used this sensing system in a reporter strain in which gfp is under control of P(htrA) , the secretion stress responsive promoter of htrA. Overexpression of heterologous secretory proteins in this strain results in green fluorescent cells, which can be separated from non-secreting, low fluorescent cells using a fluorescence-activated cell sorter (FACS). Using this principle, genomic libraries of uncharacterized prokaryotic organisms, expressed in the reporter strain, can be screened for genes encoding secretory proteins.
