h1

%0 Thesis
%A Silva Neves, Dário Jorge
%T Pseudomonas taiwanensis VLB120 synthetic biology: parts, modules, and chassis; 1. Auflage
%V 31
%I RWTH Aachen University
%V Dissertation
%C Aachen
%M RWTH-2023-08705
%B Applied microbiology
%P 1 Online-Ressource : Illustrationen, Diagramme
%D 2023
%Z Veröffentlicht auf dem Publikationsserver der RWTH Aachen University
%Z Dissertation, RWTH Aachen University, 2023
%X 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.
%F PUB:(DE-HGF)11 ; PUB:(DE-HGF)3
%9 Dissertation / PhD ThesisBook
%R 10.18154/RWTH-2023-08705
%U https://publications.rwth-aachen.de/record/968643