What happens in yeast during the Crabtree effect? : an investigation of S. cerevisiae’s volatile space

Halbfeld, Christoph; Blank, Lars Mathias (Thesis advisor); Baumbach, Jörg Ingo (Thesis advisor)

1. Auflage. - Aachen : Apprimus Verlag (2018)
Book, Dissertation / PhD Thesis

In: Applied microbiology ; Volume 8 8
Page(s)/Article-Nr.: 1 Online-Ressource (XV, 148 Seiten) : Illustrationen

Dissertation, RWTH Aachen University, 2018

Abstract

The baker’s yeast Saccharomyces cerevisiae is one of the best investigated organisms and widely used in science. This scientific interest is partially based on the yeast’s use in the industrial production of pharmaceuticals, beverages and food. Even though the taste of food and beverages is clearly influenced by volatiles, the yeast’s volatilome, i.e., the entirety of the volatile metabolites produced, is to date largely uncharted, with ethanol and acetaldehyde being prominent exceptions. In this thesis, in chapter 2.1, a metabolic model of S. cerevisiae was enhanced with the biochemistry of volatile metabolites that have been found by a literature investigation. Not only the volatile metabolites in question have been added, but also substances and reactions that connect them to metabolites already present in the model. In total, 225 metabolites and 219 reactions were added to the model. Furthermore, 12 metabolic reactions could be verified by physiological and enzyme assays of knockout mutants. These mutants were created using the CRISPR-Cas9 method and contained only one gene coding for a protein with alcohol dehydrogenase activity. In chapter 2.2, volatile metabolite dynamics during the induction of the Crabtree effect in a fully respiratory growing continuous culture of S. cerevisiae were explored with real-time analyses of the fermentation off-gas. SESI (secondary electrospray ionization)-Orbitrap-mass spectrometry (MS) was used for this endeavor. In these measurements, we detected about 2,500 signals of which 16 showed a response to the perturbation of the metabolic state prior to the detection of ethanol. These observations not only revealed the extent of the yeast’s volatilome, but also indicated that volatile metabolite dynamics correlate with the metabolic state of the cell culture and hence might be a noninvasive and fast online analytical method to monitor and later control fermentation processes. In chapter 2.3, to evaluate the possibility of online volatile metabolite monitoring, multi capillary column–ion mobility spectrometry (MCC-IMS) analysis of yeast fermentation off-gas was established. This analytical device was chosen because it runs at ambient temperature and pressure resulting in lower investment and operating costs compared to SESI-Orbitrap-MS. The MCC-IMS used was developed for the detection of volatiles in human breath, and several technical adaptations were required to allow robust detection of volatiles in the headspace of yeast fermentations. In chapter 2.4, the MCC-IMS in its optimized configuration was applied to monitor volatile metabolite changes of a laboratory and an industrial yeast strain during the transition from fully respiratory to respiro-fermentative metabolism (Crabtree effect). In addition, metabolic differences in this setting were examined on transcriptional level using a cDNA microarray. The metabolic shift could be observed in the volatile space of both strains and in all tested conditions. The transcriptome showed differences in the leucine and isoleucine pathways, as well as in genes related to the TCA cycle and the respiratory pathways. Interestingly, the expression data indicated that the industrial strain upregulated its respiration during the shift, while it was downregulated in the laboratory strain. Lastly, possible applications for the knowledge gained and methods developed in this work are discussed. This thesis provides a blueprint for studies of the volatile space in other organisms. The extended metabolic model could be used to generate yeast strains with special flavors or for the production of fragrances, perfumes or precursors of pharmaceuticals. Also, the knowledge gained about the changes in the volatilome during metabolic transitions could be used to online determine and potentially control the metabolic state of a yeast. Finally, the analytical methods developed in this work might be used for online flux analysis, if some more of the detected volatiles can be identified.

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