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@PHDTHESIS{Lenzen:785128,
author = {Lenzen, Christoph},
othercontributors = {Blank, Lars M. and Wierckx, Nick},
title = {{M}etabolic engineering of {P}seudomonas taiwanensis
{VLB}120 for sustainable production of 4-{H}ydroxybenzoate;
1. {A}uflage},
volume = {17},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {Apprimus},
reportid = {RWTH-2020-02978},
series = {Applied microbiology},
pages = {1 Online-Ressource (XVIII, 153 Seiten) : Illustrationen,
Diagramme},
year = {2020},
note = {Auch veröffentlicht auf dem Publikationsserver der RWTH
Aachen University; Dissertation, RWTH Aachen University,
2019},
abstract = {The aromatic compound 4-hydroxybenzoate and its
derivatives, the parabens, find applications in everyday
life. Current production routes for aromatics such as
4-hydroxybenzoate are mainly based on chemical catalysis and
depend on the intensive use of energy and fossil resources.
Due to the decreasing availability of the latter, the rising
demand for aromatics and the non-ecofriendly way of
formation, there is an urgent need for finding more
efficient and sustainable syntheses. The present work
focused on the development of a Pseudomonas-based whole-cell
biocatalyst for the bioconversion of 4-hydroxybenzoate from
renewable substrates such as glucose or glycerol. Besides
the remarkable and versatile metabolism of this genus and
its native high tolerance towards toxic compounds such as
solvents, the species Pseudomonas taiwanensis VLB120 was
chosen as a production host, since it accepts five carbon
sugars such as xylose as sole carbon source and it is not
regarded as a pathogenic organism. Former attempts to
biotechnologically produce 4-hydroxybenzoate using different
Pseudomonads and other species were indeed successful, but
several studies resulted in only minor yields or required
the supplementation of additional metabolites due to
auxotrophies. Therefore, the objective was to enable
high-yield 4-hydroxybenzoate biosynthesis solely from one
specific carbon source. In order to exploit the full
potential of metabolic engineering, rational as well as
non-rational techniques were applied for host development.
By introducing a heterologous production pathway based on
tyrosine, eliminating and downregulating competing pathways
and overexpressing key genes, strain P. taiwanensis VLB120
CL4.3 produced 4-hydroxybenzoate with a C-mol yield of
$19.0\%$ on glucose, whereas strain P. taiwanensis VLB120
CL3.3 reached $29.6\%$ when grown on glycerol in batch mode,
and a titer of 9.9 g l-1 during pulsed fed-batch
fermentations. A non-rational approach for improved
4-hydroxybenzoate production was applied by random chemical
mutagenesis and subsequent high-throughput screening via
flow cytometry using a developed fluorescence-based
biosensor. The best identified strain, P. taiwanensis VLB120
CL1gfp2 P2H08, produced $31\%$ more 4-hydroxybenzoate than
the non-mutated strain. Although further rational
engineering of this strain did not result in better
production performance, the introduced mutations can still
give valuable insights into future engineering targets.
Further improvement of production performance was achieved
by exploiting the metabolic demand concept. In doing so, the
main metabolic routes from glucose to acetyl-CoA were
disrupted while leaving the acetyl-CoA generating
4-hydroxybenzoate production pathway intact in order to
establish a growth-coupled production. On glucose as sole
carbon source, stain P. taiwanensis VLB120 CL5.4 produced
4-hydroxybenzoate with a C-mol yield of $21.0\%,$ supporting
the assumption that the metabolic demand concept can be used
for more efficient aromatics production. The results gained
in this work underline the huge potential of P. taiwanensis
VLB120 as a host for sustainable industrial bioconversion of
aromatics and may be the basis for further engineering in
order to promote the biotechnological formation of valuable
compounds as a promising alternative to current chemical
production routes.},
cin = {161710 / 160000},
ddc = {570},
cid = {$I:(DE-82)161710_20140620$ / $I:(DE-82)160000_20140620$},
typ = {PUB:(DE-HGF)11 / PUB:(DE-HGF)3},
doi = {10.18154/RWTH-2020-02978},
url = {https://publications.rwth-aachen.de/record/785128},
}
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