title: Advanced bioanalysis of light-controlled S. cerevisiae production strains
title: Bioanalyse von lichtgesteuerten S. cerevisiae-Produktionsstämmen
titleType: AlternativeTitle
Creators: name: Bezold, Filipp
Publisher: name: Philipps-Universität Marburg
Year: 2023
Subjects: Chemistry &amp; allied sciences
Chemie

description: Vast progress in the field of biotechnology enabled metabolic engineering to become a powerful tool for the production of numerous biogenic substances in the last decade. Alongside methods for editing biological pathways, bioanalytical techniques play a central role in metabolic engineering approaches, as they allow for monitoring of pathway modifications, identification of bottlenecks and quantification of target substances. The collaborative project "metabolic engineering with light controlled modules" (MELICOMO) aimed to harness optogenetic tools for the production of valuable secondary metabolites in Saccharomyces cerevisiae and to implement a light-inducible cell cycle arrest as the desired cellular production state. Within the present work a broad spectrum of bioanalytical methods was applied for the characterization of light-controlled S. cerevisiae cell cycle mutants (CCMs) and for the quantification of target molecules in the context of metabolic engineering. The first part of this thesis summarizes results of biomass composition for cell cycle arrested Clb2ΔDB-psd3 cells and the corresponding wild type (WT) strain. Here, growth-restricted Clb2ΔDB-psd3 cells were found to contain more protein and RNA and less carbohydrates in comparison to WT cells. Moreover, results of a proteomics experiment performed for the light-controlled CCM strains Cdc48-psd3, Clb2ΔDB-psd3 and bPAC were analyzed. Thereby, relative protein abundances comparing the restrictive growth conditions to the permissive growth conditions and to the WT strain were examined. Obtained results were processed using heatmaps, volcano plots, Venn diagrams and Gene Ontology enrichment analyses. Here, for each of the growth-restricted Cdc48-psd3 and bPAC strains an acetyl-CoA synthetase isoform was found to be high abundant in comparison to the permissive growth condition and to the WT strain. This finding might explain increased β-carotene yields observed for both growth-restricted strains and indicates beneĄcial production conditions for other isoprenoid-derived products generated from precursors provided by the mevalonate pathway. Furthermore, the phosphatase Pho8 was found to be present at low levels in growth-restricted Cdc48-psd3 cells compared with the WT strain, which may explain the observed increased cordycepin production of the growth-restricted CCM strain, since Pho8 is responsible for the degradation of the cordycepin precursor 3'AMP. In addition, enzymes of the respiratory chain and the tricarboxylic acid cycle were found to be upregulated for growth-restricted bPAC cells, which could imply an increased energy availability for this condition. Finally a selection of LC-MS and HPLC analyses is shown, which helped to establish proof of principle 3'AMP, cordycepin and GA4 production strains. This thesis provides a large toolbox of state of the art bioanalytical methods, which helped to improve and rationalize the metabolic engineering approaches established within the MELICOMO project.