Etablierung der Biosynthese von Gibberellinsäurederivaten in Saccharomyces cerevisiae und Anwendung optogenetischer Module in der Grundlagenforschung und Biotechnologie

Die Proteindegradation ist ein zentraler Bestandteil der Regulationsmechanismen in Saccharomyces cerevisiae zur Bewerkstelligung der Proteinhomöostase. Hierbei ist das Ubiquitin-Proteasom-System von fundamentaler Bedeutung und reguliert den Abbau von fehlerhaften Proteinen, sowie solchen, die aufgru...

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Bibliographic Details
Main Author: Scheffer, Johannes
Contributors: Taxis, Christof (Dr. Priv. Doz.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2020
Online Access:PDF Full Text
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Protein degradation is a central component of the regulatory circuits in Saccharomyces cerevisiae to obtain protein homeostasis. The ubiquitin proteasome system is a crucial component within these regulatory circuits and controls the degradation of faulty proteins, as well as those which have been targeted for degradation through cell cycle signalling and other influences. The targeting of such proteins happens through polyubiquitination by a cascade of three enzymes (E1-E2-E3). The ERAD-system (ER associated degradation) is part of the protein quality control for proteins of the endoplasmatic reticulum (ER) and secretory proteins. There are three ERAD-pathways, whose involvement depends on the localisation of the substrate protein or the damaged domain. ERAD-L is responsible for ER-luminal proteins, ERAD-M for membrane integrated substrates and ERAD-C for membrane anchored proteins with domains in the cytosol. Three different ER-membrane bound proteins with C-terminal cytosolic domains (Sec61, Sec62, Sec66) were analysed in this thesis. The degradation of these proteins, mediated by the C-terminally fused photosensitive degron (psd) was to be examined closer, in order to check for a dependency of the proteolysis on the ERAD-C machinery. It could be demonstrated, that the psd-dependant degradation of Sec61, Sec62 and Sec66 required ubiquitination. The necessary ubiquitination enzymes were the ubiquitin activating E1 Uba1, the ubiquitin conjugating E2s Ubc6/Ubc7 and the E3 ubiquitin ligase Ssm4, which are all part of the ERAD-C pathway. The cell cycle of S. cerevisiae is controlled by an interplay of several regulatory proteins. In additional investigations, the heterologous production of secondary plant metabolites was successfully increased through optogenetic control of several cell cycle regulators. By psd-dependent degradation of the AAA-ATPase Cdc48 and the dominant negative mutants ΔNSic1-psd3 and ClbΔDB-psd3, cells were arrested in the G1- and G2-phase of the cell cycle, which led to elevated production rate of β-carotene from the mevalonate pathway. The effectiveness of the constructs was additionally demonstrated by the increased production of the fungal metabolite Cordycepin. In the course of this thesis the complex biosynthesis pathway of gibberellic-acid-4 (GA4) was established and tested in S. cerevisiae. This facilitates the production of gibberellins. Since the GA4-pathway is branching off the mevalonate pathway, the previously tested optogenetic modules are also applicable for the increased production of gibberellic-acid-4.