% IMPORTANT: The following is UTF-8 encoded. This means that in the presence
% of non-ASCII characters, it will not work with BibTeX 0.99 or older.
% Instead, you should use an up-to-date BibTeX implementation like “bibtex8” or
% “biber”.
@PHDTHESIS{Loevenich:781564,
author = {Loevenich, Johanna},
othercontributors = {Blank, Lars M. and Wierckx, Nick},
title = {{O}ptimization of itaconic acid production by {U}. maydis
through metabolic engineering $\&$ adaptive laboratory
evolution; 1. {A}uflage},
volume = {14},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {Apprimus Verlag},
reportid = {RWTH-2020-01372},
series = {Applied microbiology},
pages = {1 Online-Ressource (XIV, 123 Seiten) : Illustrationen,
Diagramme},
year = {2019},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University 2020; Dissertation, RWTH Aachen University, 2019},
abstract = {The incessant growth of the world population leads to an
already gigantic and still increasing demand for food,
energy, fuels, and chemicals. With the finiteness of fossil
resources as main feedstocks, a change from
petroleum-derived to sustainable, economically bio-based
production processes is indispensable to accomplish the
global needs. One of these processes is the production of
itaconic acid ranked as one of the top 12 value added
chemicals from biomass by the DoE. Nowadays, industrial
biotechnological production is performed by using the
filamentous fungus Aspergillus terreus. To circumvent the
challenges going along with such a filamentous production
host, alternatives are searched. In this context, the
Ustilaginaceae family including Ustilago maydis attracted
special attention. To establish an industrial itaconate
production host competitive to A. terreus and to
significantly improve the itaconate production performance
of U. maydis, two strategies were chased in this thesis:
metabolic engineering and adaptive laboratory evolution. By
the reduction of the diverse by-product spectrum of U.
maydis MB215 by the deletion of 2-hydroxy paraconate,
mannosyl-erythritol lipid, ustilagic acid and
triacylglycerol biosynthesis in combination with the
upregulation of ria1, the itaconate biosynthesis gene
cluster regulator, the flow of substrate could be
extensively pushed towards itaconate biosynthesis. This lead
to an itaconate titer increased by 10.2-fold compared to the
wildtype. Due to the upregulation of the
cis-aconitate/malate antiporter mtt1 as consequence of
ria1↑, the production of malate, another by-product, could
simultaneously be decreased by 84 $\%.In$ this by-product
reduced U. maydis strain, further metabolic engineering
steps were performed: filamentous growth prevention by fuz7
deletion and overexpression of mttA encoding for the A.
terreus mitochondrial tricarboxylate transporter. By
∆fuz7, the designed strain was able to produce itaconate
with improved production parameters, especially with an
$25\%$ increased yield from glucose. This could even be
outplayed by additional PetefmttA insertion. A clone with
three PetefmttA copies reached an itaconate titer of 54 g
L-1 and a maximal yield of 0.64 gITA gglu-1, which
corresponds to 89 $\%$ of the theoretical value. The great
itaconate production improvements imply a higher metabolic
and osmotic stress level for the cells. Adaptive laboratory
evolution was therefore used to generate a strain with
increased low pH and product resistance to itaconate. The
fitness of U. maydis could be significantly improved
represented by strains able to grow at pH 4 and in the
presents of 40 g L-1 itaconate. The consolidation of all
major modifications identified in this thesis in one strain,
though, resulted in a loss of this tolerance. Especially the
deletion of triacylglycerol production, the cells rely on as
main energy reserves, seems to destabilize the cells in the
long term. However, the results clearly illustrate that the
final engineered strains feature great, far optimized
itaconate production parameters close to the theoretical
maximum making U. maydis - besides A. terreus - an
industrial relevant production host for itaconic acid.},
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-2020-01372},
url = {https://publications.rwth-aachen.de/record/781564},
}
guest ::