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@PHDTHESIS{Hemmerich:729768,
author = {Hemmerich, Johannes},
othercontributors = {Schwaneberg, Ulrich and Oldiges, Marco and Wiechert,
Wolfgang},
title = {{S}tudies on bioprocesses for protein secretion with
{C}orynebacterium glutamicum},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
reportid = {RWTH-2018-226510},
pages = {1 Online-Ressource (256 Seiten) : Illustrationen},
year = {2018},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University; Dissertation, RWTH Aachen University, 2018},
abstract = {After being discovered 60 years ago, Corynebacterium
glutamicum is a major industrial workhorse for the
production of amino acids like L-glutamate and L-lysine at
several million tons per year. Intense and still ongoing
basic and applied research fueled this great
biotechnological success story. Currently, C. glutamicum is
increasingly getting into focus as production host for
heterologous proteins of both technical and clinical
interest. Consequently, research is shifting towards the use
of C. glutamicum microbial cell factory for protein
production.To facilitate rapid and reliable characterization
of newly constructed strain variants from metabolic
engineering, microbioreactor (MBR) systems emerged as
versatile tools. Such systems provide an increased
experimental throughput with the ability to control
environmental cultivation parameters, as well as monitoring
of culture dynamics like biomass formation. In this study,
methods for MBR systems are developed to suite the demands
of a specific screening objective, namely the quantitative
phenotyping of C. glutamicum strains secreting green
fluorescent protein (GFP) and cutinase as heterologous model
proteins. This involves the careful selection of standard
operating conditions with respect to physiological demands
like oxygen-unlimited metabolization of glucose as main
carbon source, as well as determination of accuracy and
imprecision of online biomass monitoring. Furthermore, to
obtain time-resolved data on secretory protein formation and
substrate consumption, an improved method is presented and
validated that does not accompany a loss in MBR cultivation
throughput. The need for (semi-)automated data processing
from high-throughput MBR cultivations is also discussed on
the example of derived performance indicators (PIs) that
represent condensed evaluation metrics for rapid evaluation
of whole cultivation experiments. As application example, a
method for automated growth rate determination is presented
in detail, since this PI is probably the most often applied
characteristic in biological fitness testing of mutant
strain libraries. In addition, on the example of maximizing
secreted GFP titer, it is shown how MBR systems, integration
of laboratory automation and Kriging-based Design of
Experiments (DoE) complement each other in a synergistic
way. As a result, an iterative workflow is presented that
serves as blueprint for development of further
biotechnological applications. Unexpectedly, secreted GFP
titer could be doubled, showing that routinely applied
nutrition media designed for amino acid production with C.
glutamicum need to be carefully adapted and optimized with
changing screening objectives, that is here secretion of
heterologous proteins. To complement the current knowledge
on how to select the optimal signal peptide (SP) for
different expression hosts and different target proteins of
choice, the interrelation of bioprocess control strategy and
choice of SP to optimize cutinase secretion with C.
glutamicum is investigated in detail. Since the envisaged
degree of process control could not be realized with the
available MBR systems, a consistent data set was generated
relying on more than 150 bench scale bioreactor runs.
Furthermore, the results are discussed and interpreted in
the light of changing bioprocess optimization objectives,
which again highlights the need for careful definition of
optimization objectives. Representing a typical application
example of MBR systems, the quantitative microbial
phenotyping of a library of genome reduced C. glutamicum
strains for heterologous cutinase secretion was conducted.
The collected data comprise growth rates and cutinase yields
as extracellular phenotypes, as well as detailed analysis at
the transcriptome and proteome level for a small subset of
strains. Next to surprising phenotypes due to specific
genomic deletions, as well as differential analysis of
phenotypes from strains with overlapping genomic deletions,
attempts were made to explain the metabolic perturbations
from observed significantly differential regulation at the
protein level. Also, by incorporating all data on
extracellular phenotypes, a data-driven, phenomenological
multiple regression approach was used to identify the
minimum set of genomic deletions needed in terms of improved
cutinase secretion. Finally, a few future aspects
outreaching the scope of this work are presented as outlook.
These aspects concern the application of C. glutamicum as
potential alternative host for heterologous protein
production, the demand for further development of
microbioreactor systems and the need for smart solutions for
warehousing, (re-)processing and interpretation of
heterogeneous data sets to cope with the foreseeable
increase of information output generated from
high-throughput experimentation in combination with powerful
analytical methods.},
cin = {162610 / 160000},
ddc = {570},
cid = {$I:(DE-82)162610_20140620$ / $I:(DE-82)160000_20140620$},
typ = {PUB:(DE-HGF)11},
doi = {10.18154/RWTH-2018-226510},
url = {https://publications.rwth-aachen.de/record/729768},
}
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