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@PHDTHESIS{SilvaNeves:968643,
author = {Silva Neves, Dário Jorge},
othercontributors = {Blank, Lars M. and Ebert, Birgitta},
title = {{P}seudomonas taiwanensis {VLB}120 synthetic biology:
parts, modules, and chassis; 1. {A}uflage},
volume = {31},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {Apprimus Verlag},
reportid = {RWTH-2023-08705},
series = {Applied microbiology},
pages = {1 Online-Ressource : Illustrationen, Diagramme},
year = {2023},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University; Dissertation, RWTH Aachen University, 2023},
abstract = {Climate change is a pressing global issue that is caused by
the consumption of fossil fuels, which releases greenhouse
gases into the atmosphere. One of the ways to reduce
dependency on fossil fuels and mitigate the effects of
climate change is by using cell factories. Cell factories
are biological systems that are engineered to produce a wide
range of products, such as biofuels, bioplastics, and
pharmaceuticals. These products can be produced using
renewable resources such as plant matter or algae, rather
than fossil fuels. Additionally, the production process of
these products in cell factories can be made more efficient
and sustainable by using advanced technologies such as
metabolic engineering and synthetic biology. Synthetic
biology aims to engineer biologically based systems with
novel functions by either applying a rational and systematic
approach or exploring the vast combinatorial potential of
DNA to create new-to nature molecular biology tools. Due to
the intrinsic complexity of DNA shuffling and the current
limitations in predicting accurate outcomes of synthetic
biology parts, it is crucial to properly standardize and
characterize synthetic biology tools to aid cell factory
developments. This thesis aimed to expand the genetic
toolbox of Pseudomonas taiwanensis VLB120 and implement them
for the generation of a chassis strain to enlarge the
product portfolio of this emerging industrial-relevant cell
factory. Sigma-70 dependent promoter libraries were
generated and integrated into the single genomic locus
attTn7 of P. taiwanensis VLB120 and E.coli TOP10. Each
promoter was characterized using a standardized promoter
strength unit developed within this work that calibrates
device-specific fluorescence output with fluorescein and
accounts for cell growth-specific differences. Such
characterization standards allow us to give an insight into
how a specific promoter behaves in each organism and create
sets of promoters relevant to metabolic engineering
purposes. This thesis also focused on the assessment of an
optimized gene expression architecture to achieve high gene
expression without relying on strong promoters. This module
achieved high gene expression across several expression
vectors of two fluorescent reporter genes by incorporating
mRNA stabilizing and translation-enhancing genetic parts.
This module was also applied to increase the productivities
of a short acetoin pathway and the relevance of mRNA
stability was proven through qPCR-based mRNA decay rates.
These tools were a component in the development of a P.
taiwanensis VLB120 propionyl-CoAchassis strain to expand the
portfolio of this pseudomonad to odd-chain products. The
successful incorporation of propionyl-CoA in the metabolism
of P. taiwanensis VLB120 was confirmed by the production of
propionate after identifying the deletion of the
methylcitrate synthase as a crucial factor. The
propionate-producing P. taiwanensis VLB120 was evaluated in
bioreactor fermentations under three different fed-batch
strategies to assess how feeding regimes and feast-famine
switches affect the production of propionyl-CoA-dependent
products. In summary, this thesis contributes to the
development of P. taiwanensis VLB120 as an emerging
industrial-relevant workhouse by expanding the available
genetic toolbox and setting the first stone to produce
odd-chain products in this organism. It also contributes to
the standardization of genetic tools characterization and
cross-species studies to aid the identification of the most
suitable microbe for specific biotechnological applications
and fasten the human independence of fossil fuels.},
cin = {161710 / 160000},
ddc = {570},
cid = {$I:(DE-82)161710_20140620$ / $I:(DE-82)160000_20140620$},
pnm = {BMBF-031A459 - ERASynBio - Runde 1 - SynPath - Synthetic
biochemical pathways for efficient production of novel
biofuels (031A459)},
pid = {G:(BMBF)031A459},
typ = {PUB:(DE-HGF)11 / PUB:(DE-HGF)3},
doi = {10.18154/RWTH-2023-08705},
url = {https://publications.rwth-aachen.de/record/968643},
}
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