Every living organism needs to tightly regulate its growth depending on the environment that surrounds it. This is true from the single-celled microorganisms to the mammals. Failure to correctly respond to environmental changes can be dangerous for the correct homeostasis of the cell. For this reason, the molecular basis and the regulation of many signalling pathways are conserved from bacteria to man and understanding this regulation is critical both for basic biology and to its application, from fermentations to medical science. The work contained in this thesis is focused on the budding yeast Saccharomyces cerevisiae and on the analysis of its metabolism at the crossroad between carbon and nitrogen. S. cerevisiae is able to grow on different nitrogen sources, which can be classified as preferred or non-preferred. Not all the good nitrogen sources affect cellular processes in the same way. Glutamate is a very important amino acid and a good nitrogen source, being involved as nitrogen donor in several biosynthetic processes and possessing a carbon backbone (alpha-ketoglutarate), which can enter the TCA cycle. In the first chapter, we investigated growth, metabolism and transcriptional profiles of yeast cells grown in minimal media supplemented with either ammonium or glutamate, both considered good nitrogen sources. During the exponential phase, cells using glutamate as a nitrogen source have a larger cell size compared to cells grown in presence of ammonium. In stationary phase, glutamate-supplemented accumulate higher biomass levels. We refer to this behaviour as “enhanced growth”. In glutamate-grown cells, the deepest transcriptional and metabolic rearrangement takes place after glucose exhaustion and show a profound alteration of the metabolism of storage molecules during growth on glutamate with an accumulation of trehalose and fatty acids, which correlate with a higher stress resistance. Flux balance analysis simulations with a core model of yeast metabolism correctly predicted the optimal growth yield and computational analysis of the flux distribution identified a different allocation of oxygen as responsible for the observed interplay between ethanol and glutamate. Simulations using the model mentioned above suggest that growth on media containing glutamate as the only carbon and nitrogen source is possible. Despite these considerations, a wild-type S. cerevisiae strain is not able to grow in these conditions. To understand the reasons underlying this growth inability, in chapter 2 we applied a laboratory evolution approach and isolated 4 different mutants able to grow in the presence of just glutamate, vitamins and supplements. Sequencing of the mutant clones showed that they all carry mutations affecting the Ras/cAMP/PKA pathway. These results show that modification of the carbon sensing pathways can alter metabolism, allowing yeast to utilize glutamate in a way that the wild type strain cannot. Transcriptomics analysis of the more interesting mutant revealed a general up-regulation of the biosynthesis of amino acids and nucleotides, as well as an enhanced expression of plasma membrane transporter genes, including glutamate permeases. Together with the enhancement of energy producing pathways, like fatty acids β-oxidation, we propose that these changes are among the driving forces in the adaptation of the evolved mutants. In conclusion, our integrated analysis allowed us to demonstrate how glutamate cellular fate is strongly interlinked both with carbon and nitrogen metabolism and sensing and offers an example of combination of different techniques, which is able to deliver a system-level interpretation of biological data.

Ogni organismo vivente necessita di una regolazione stringente della propria crescita a seconda dell’ambiente che lo circonda. Ciò è vero a partire dai microorganismi unicellulari fino ad arrivare ai mammiferi. Comprendere questa regolazione è cruciale sia per la ricerca biologica di base che per le sue applicazioni, dalle fermentazioni industriali alla scienza medica. Il lavoro contenuto in questa tesi è focalizzato sul lievito gemmante Saccharomyces cerevisiae e sull’analisi del suo metabolismo all’interfaccia tra carbonio e azoto. S. cerevisiae è in grado di crescere utilizzando differenti fonti d’azoto, che vengono classificate in “preferite” e “non preferite”. Non tutte le buone fonti d’azoto hanno gli stessi effetti sui processi cellulari. Il glutammato è un amminoacido molto importante e una buona fonte d’azoto. Esso funge da donatore di gruppi amminici in svariati processi biosintetici. Inoltre, possiede uno scheletro carbonioso (alfa-ketoglutarato), che è un intermedio del ciclo degli acidi tricarbossilici (TCA). Nel primo capitolo vengono investigate la crescita, il metabolismo e il profilo trascrizionale di cellule di lievito cresciute in terreno minimo contenente ammonio solfato o glutammato. Durante la fase di crescita esponenziale su glucosio, le cellule in crescita con il glutammato come fonte d’azoto presentano una dimensione maggiore, rispetto a quelle cresciute in presenza di ammonio. Al termine della fase stazionaria, le cellule cresciute in glutammato mostrano un maggiore accumulo di biomassa. Abbiamo definito questo fenotipo come “crescita aumentata”. Nelle cellule cresciute in glutammato, il riarrangiamento trascrizionale più profondo avviene dopo l’esaurimento del glucosio e mostra una importante alterazione del metabolismo delle sostanze di riserva con accumulo di trealosio e acidi grassi, che correla con una maggiore resistenza gli stress. Simulazioni computazionali di un modello metabolico “core” del metabolismo di lievito tramite Flux Balance Analysis predicono correttamente il rendimento di crescita sulle due fonti. Analisi computazionali sulla distribuzione dei flussi metabolici identificano un uso differenziale dell’ossigeno come reponsabile dell’interazione osservata tra etanolo e glutammato. Ulteriori simulazioni suggeriscono che la crescita di lievito contenente glutammato come unica fonte di carbonio e di azoto sia teoricamente possibile. Nonostante ciò, ceppi selvatici di S. cerevisiae non sono in grado di crescere in queste condizioni. Per comprenderne le motivazioni, nel secondo capitolo abbiamo applicato un approccio di evoluzione diretta e abbiamo isolato 4 diversi mutanti in grado di crescere in presenza di glutammato, vitamine e supplementi. Il sequenziamento di questi mutanti ha mostrato che essi portano tutti mutazioni che alterano la via di segnalazione di Ras/cAMP/PKA. Questo risultato mostra che l’alterazione di vie deputate al sensing delle fonti di carbonio possono alterare il metabolismo, permettendo al lievito di utilizzare il glutammato in un modo diverso da un ceppo selvatico. Analisi di trascrittomica del più interessante tra i mutanti hanno rivelato una up-regolazione generale della biosintesi degli amminoacidi e dei nucleotidi, così come una aumentata espressione dei geni codificanti per i trasportatori di membrana, incluse permeasi che trasportano il glutammato. I risultati di questo lavoro suggeriscono che questi cambiamenti, insieme con un aumento delle vie metaboliche in grado di produrre energia siano tra le forze che hanno guidano l’adattamento di questi mutanti. In conclusione, la nostra analisi integrata ci ha permesso di dimostrare come il destino cellulare del glutammato sia fortemente interconnesso con il metabolismo del carbonio e dell’azoto e con le vie deputate al loro sensing.

(2019). GLUTAMATE, A NUTRIENT AT THE CROSS-ROAD OF CARBON AND NITROGEN ASSIMILATION. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).

GLUTAMATE, A NUTRIENT AT THE CROSS-ROAD OF CARBON AND NITROGEN ASSIMILATION

GNUGNOLI, MARCO
2019

Abstract

Every living organism needs to tightly regulate its growth depending on the environment that surrounds it. This is true from the single-celled microorganisms to the mammals. Failure to correctly respond to environmental changes can be dangerous for the correct homeostasis of the cell. For this reason, the molecular basis and the regulation of many signalling pathways are conserved from bacteria to man and understanding this regulation is critical both for basic biology and to its application, from fermentations to medical science. The work contained in this thesis is focused on the budding yeast Saccharomyces cerevisiae and on the analysis of its metabolism at the crossroad between carbon and nitrogen. S. cerevisiae is able to grow on different nitrogen sources, which can be classified as preferred or non-preferred. Not all the good nitrogen sources affect cellular processes in the same way. Glutamate is a very important amino acid and a good nitrogen source, being involved as nitrogen donor in several biosynthetic processes and possessing a carbon backbone (alpha-ketoglutarate), which can enter the TCA cycle. In the first chapter, we investigated growth, metabolism and transcriptional profiles of yeast cells grown in minimal media supplemented with either ammonium or glutamate, both considered good nitrogen sources. During the exponential phase, cells using glutamate as a nitrogen source have a larger cell size compared to cells grown in presence of ammonium. In stationary phase, glutamate-supplemented accumulate higher biomass levels. We refer to this behaviour as “enhanced growth”. In glutamate-grown cells, the deepest transcriptional and metabolic rearrangement takes place after glucose exhaustion and show a profound alteration of the metabolism of storage molecules during growth on glutamate with an accumulation of trehalose and fatty acids, which correlate with a higher stress resistance. Flux balance analysis simulations with a core model of yeast metabolism correctly predicted the optimal growth yield and computational analysis of the flux distribution identified a different allocation of oxygen as responsible for the observed interplay between ethanol and glutamate. Simulations using the model mentioned above suggest that growth on media containing glutamate as the only carbon and nitrogen source is possible. Despite these considerations, a wild-type S. cerevisiae strain is not able to grow in these conditions. To understand the reasons underlying this growth inability, in chapter 2 we applied a laboratory evolution approach and isolated 4 different mutants able to grow in the presence of just glutamate, vitamins and supplements. Sequencing of the mutant clones showed that they all carry mutations affecting the Ras/cAMP/PKA pathway. These results show that modification of the carbon sensing pathways can alter metabolism, allowing yeast to utilize glutamate in a way that the wild type strain cannot. Transcriptomics analysis of the more interesting mutant revealed a general up-regulation of the biosynthesis of amino acids and nucleotides, as well as an enhanced expression of plasma membrane transporter genes, including glutamate permeases. Together with the enhancement of energy producing pathways, like fatty acids β-oxidation, we propose that these changes are among the driving forces in the adaptation of the evolved mutants. In conclusion, our integrated analysis allowed us to demonstrate how glutamate cellular fate is strongly interlinked both with carbon and nitrogen metabolism and sensing and offers an example of combination of different techniques, which is able to deliver a system-level interpretation of biological data.
VANONI, MARCO ERCOLE
metabolismo; systems biology; trascrittomica; signalling; azoto
metabolism; systems biology; transcriptomics; signalling; azoto
BIO/10 - BIOCHIMICA
English
8-feb-2019
BIOLOGIA E BIOTECNOLOGIE - 93R
31
2017/2018
open
(2019). GLUTAMATE, A NUTRIENT AT THE CROSS-ROAD OF CARBON AND NITROGEN ASSIMILATION. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/241135
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