32 Gilani, Mariam Dianat Sabet Blank, Lars M. Ebert, Birgitta Elisabeth 161710 160000 Apprimus Verlag Aachen 978-3-98555-246-7 English 1 Online-Ressource : Illustrationen 37 Triterpenoids are a diverse group of plant-derived secondary metabolites, which have been used in traditional medicine as antibacterial, antiviral, and anti-inflammatory agents, and show potential for application in the pharmaceutical, cosmetic, and food industries. The application of microbial factories for the production of triterpenoids is shown to be a promising alternative to the existing low-yield, complex, and environmentally-affecting processes. Up to now, most studies on triterpenoid production are still ongoing and need to be further developed to be economically feasible for large-scale production. A successfully achieved microbial process is the result of both metabolic and process engineering. The focus of this thesis is gaining insights into cell performance under production conditions for optimal process design. Specifically, the heterologous production of triterpenoids in Saccharomyces cerevisiae is investigated on the example of lupane-type pentacyclic triterpenoids. First, non-invasive monitoring of triterpenoid synthesis by non-linear microscopy was developed. Within a proof-of-principle study, it could be shown that the combination of coherent anti-Stokes Raman scattering (CARS) and second-harmonic-generation (SHG) microscopy techniques can be applied for monitoring of triterpenoid synthesis in both fixed yeast cells and cells growing in liquid culture. The SHG signal in triterpenoid-producing strains was significantly higher than in a non-producing reference strain, correlating with lipophile content as determined by classical Nile red staining. In growing cultures, both CARS and SHG signals changed over time, enabling new insights into the dynamics of triterpenoid production and storage by recombinant yeast. Up to this date, no transporters for triterpenoids in S. cerevisiae are known. The intracellular accumulation of triterpenoids limits the production capacity of the yeast. Moreover, intracellular accumulation of triterpenoids leads to the necessity of cell disruption in the downstream process (DSP). To address this challenge, second, an in situ extraction method allowing the extraction of intracellular triterpenoids from living cells was evaluated. A solvent screening based on the criteria of triterpenoid solubility, biocompatibility, and environmental impact, followed by solvent testing on yeast physiology and viability suggested isopropyl myristate (IPM) as a suitable solvent. In an experiment, where fresh IPM was added at the end of each batch, a significantly higher triterpenoid production was observed compared to a control group without IPM addition. In addition to IPM, also the effect of cyclodextrins (CD) on product secretion from the cells was shown to be effective. Subsequently, the developed extraction method was applied successfully in fed-batch experiments in a bioreactor, showing the possibility of in situ extraction of intracellular products without affecting cell viability. Third, glucose as carbon source for triterpenoid synthesis was revisited. The productivities of three triterpenoid-producing strains on glucose were compared. This was realized by glucose-limited fed-batch cultivations in shake flask. The thesis contributes novel analytical ideas, DSP simplification, and potentially a rapid production on the main biotech carbon source, glucose. The opportunities for triterpenoids seem endless, while the here envisaged lower cost production will facilitate future advances. Druckausgabe: 2024. - Auch veröffentlicht auf dem Publikationsserver der RWTH Aachen University 2025. - Weitere Reihe: Edition Wissenschaft Apprimus ; Dissertation, RWTH Aachen University, 2024 ; 2, ; PUB:(DE-HGF)11, ; PUB:(DE-HGF)3, ; Dissertation / PhD Thesis Dissertation RWTH Aachen University 2024 2024 RWTH-2025-00566 2024 https://publications.rwth-aachen.de/record/1002602