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Genomic Response of Escherichia coli during coexpression of a bacteriocin release protein

ABSTRACT: Secretion of recombinant proteins to the culture medium after transport to the periplasm of gram-negative bacteria often facilitates downstream-processing. It could lead to more biological active and correct folded proteins and low contamination with host proteins, thereby reducing the costs of the production process. However, Escherichia coli K12 secrets only little amount and few substrates naturally. This is one of the major drawbacks for the applicability of this strain in the biotechnology industry, when large amounts of biological active proteins are desirable. Therefore, different attempts were made to enhance the level of secreted proteins. Coexpression of bacteriocin release proteins makes the outer membrane permeable for proteins. A detailed insight into the complex underlying regulatory mechanism and metabolic changes when such release proteins are expressed could be very useful for successful optimisation strategies. The identification of potential optimisation targets can be achieved by DNA-microarray-technology, as proven in many cases before. In this work DNA-microarrays were used for the identification of differentially expressed genes of an inducible E.coli secretion strain expressing reporter proteins bacterial alkaline phosphatase, PhoA and beta-lactamase, Bla and their release to the culture medium by coexpression of the BRP of CloDF13. Based on the data of whole genome experiments and on the different databases genes which are regulated after induction of BRP expression are identified and discussed. BRP activity was found to cause a substantial cellular response and considerable changes in global gene expression. Additionally, performed cluster-analysis using k-means algorithm identified clusters containing promising candidates for new optimisation strategies aiming for an enhanced protein secretion.

ORGANISM(S): Escherichia coli Escherichia coli K-12

PROVIDER: GSE16946 | GEO | 2009/08/01

SECONDARY ACCESSION(S): PRJNA117305

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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Genomic Response of Escherichia coli during coexpression of a bacteriocin release protein

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