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@ARTICLE{Jurischka:872957,
author = {Jurischka, Sarah and Bida, Astrid and Dohmen-Olma, Doris
and Kleine, Britta and Potzkei, Janko and Binder, Stephan
and Schaumann, Georg and Bakkes, Patrick J. and Freudl,
Roland},
title = {{A} secretion biosensor for monitoring {S}ec-dependent
protein export in {C}orynebacterium glutamicum},
journal = {Microbial cell factories},
volume = {19},
number = {1},
issn = {1475-2859},
address = {London},
publisher = {Biomed Central},
reportid = {FZJ-2020-00417},
pages = {11},
year = {2020},
note = {Biotechnologie 1},
abstract = {BackgroundIn recent years, the industrial workhorse
Corynebacterium glutamicum has gained increasing interest as
a host organism for the secretory production of heterologous
proteins. Generally, the yield of a target protein in the
culture supernatant depends on a multitude of interdependent
biological and bioprocess parameters which have to be
optimized. So far, the monitoring of such optimization
processes depends on the availability of a direct assay for
the respective target protein that can be handled also in
high throughput approaches. Since simple assays, such as
standard enzymatic activity assays, are not always at hand,
the availability of a general protein secretion biosensor is
highly desirable.ResultsHigh level secretion of proteins via
the Sec protein export pathway leads to secretion stress, a
phenomenon that is thought to be caused by the accumulation
of incompletely or misfolded proteins at the membrane-cell
envelope interface. We have analyzed the transcriptional
responses of C. glutamicum to the secretory production of
two different heterologous proteins and found that, in both
cases, the expression of the gene encoding a homologue of
the extracytosolic HtrA protease was highly upregulated.
Based on this finding, a C. glutamicum Sec secretion
biosensor strain was constructed in which the htrA gene on
the chromosome was replaced by the eyfp gene. The
fluorescence of the resulting reporter strain responded to
the secretion of different heterologous proteins (cutinase
from Fusarium solani pisi and alkaline phosphatase PhoA from
Escherichia coli) in a dose-dependent manner. In addition,
three differently efficient signal peptides for the
secretory production of the cutinase could be differentiated
by the biosensor signal. Furthermore, we have shown that an
efficient signal peptide can be separated from a poor signal
peptide by using the biosensor signal of the respective
cells in fluorescence activated cell sorting
experiments.ConclusionsWe have succeeded in the construction
of a C. glutamicum biosensor strain that allows for the
monitoring of Sec-dependent secretion of heterologous
proteins in a dose-dependent manner, independent of a direct
assay for the desired target protein.},
cin = {IBG-1},
ddc = {570},
cid = {I:(DE-Juel1)IBG-1-20101118},
pnm = {581 - Biotechnology (POF3-581)},
pid = {G:(DE-HGF)POF3-581},
typ = {PUB:(DE-HGF)16},
pubmed = {pmid:31964372},
UT = {WOS:000521188600001},
doi = {10.1186/s12934-019-1273-z},
url = {https://juser.fz-juelich.de/record/872957},
}