Nowadays, for the production of energy and materials, our society mainly relies on fossil sources, but many concerns arise from their utilization, like greenhouse gases emission and non-renewability within the time of their consumption. Hence, biorefineries, which convert renewable biomasses into products and energy, could be a promising alternative. Despite some biorefineries are now at commercial scale, many challenges must be overcome to implement competitive bio-based industries, such as improving the performances of microorganisms, named cell factory, used during the production processes. Above all, the yeast Saccharomyces cerevisiae is the most prominent cell factory for producing bioethanol as biofuel. The main objective of this thesis was to engineer S. cerevisiae strains with biotechnological interesting traits, among which improved growth performances and increased thermotolerance. For this purpose, we investigated and applied a cellular rewiring, by selecting two targets: the glutamate synthase Glt1 that is an enzyme of the central nitrogen metabolism, CNM, and the main polyA binding protein Pab1, a master regulator of mRNA metabolism. Regarding GLT1, the physiological effects of its deletion and over-expression were assessed by growing yeasts in the presence of different nitrogen sources. Results showed that the supplementation of ammonium sulfate, glutamate or glutamine considerably affects growth, protein content, viability and reactive oxygen species, ROS, accumulation. Conversely, GLT1 modulation does not significantly influence these parameters. Overall, these data highlight the plasticity of the S. cerevisiae CNM in respect to the environment and confirm its robustness against internal perturbation. Moreover, even though the sole modulation of GLT1 expression might not reprogram the entire cell, the physiological characterization of this study might be helpful to guide the selection of other more promising candidate for the application of the rewiring approaches. Then, the induction of cellular reprogramming was assessed selecting Pab1. To this purpose, a strain carrying the unaltered PAB1 chromosomal allele was transformed with a PAB1 plasmid mutant library and then screened for isolating strains with high thermotolerance. The isolated clones showed growth improvement at both high temperatures and ethanol concentration by drop tests. Among all, the PAB1 S40.7 variant was further characterized because, strikingly, it dominantly confers higher thermotolerance by expressing just the first 20 amino acidic residues of Pab1. This improved phenotype was also confirmed in bioreactor at 40°C. Remarkably, the S40.7 strain accumulates less ROS compared to the control strain, thus possibly explaining its increased thermotolerance. Overall, these results demonstrated that Pab1 is a powerful candidate to evoke complex phenotypes with improved traits, among which higher thermotolerance. Finally, Pab1 was characterized to uncover the role of its domains in the recruitment of the protein within stress granules, aggregates of untranslated mRNPs that form during stressful conditions. This characterization shows that Pab1 association into these aggregates relies mainly on RNA recognition motives, RRM, whose number is important for an efficient recruitment. Although the P and C domains do not directly participate in Pab1 association to stress granules, their presence strengthens or decreases, respectively, the distribution of synthetic Pab1 variants lacking at least one RRM into these aggregates. As additional outcome, this part of the work is suggesting that Pab1 domains might be rationally exploited for synthetic biology purposes. Overall, the results of this thesis highlight and confirm the difficulty but at the same time the power of cellular rewiring in evoking industrially relevant phenotype. Pab1 is undoubtedly emerging as a pivotal element that deserves more attention for future strain design and tailoring.

Le attuali produzioni di energia e materiali si basano soprattutto sullo sfruttamento di fonti fossili, ma la loro non rinnovabilità e la necessità di una maggiore sostenibilità ambientale han contribuito ad aumentare l’interesse verso risorse alternative. In questo contesto si collocano le bioraffinerie, che sono volte alla conversione di biomasse rinnovabili in prodotti commerciabili. Per implementare bioindustrie competitive, molte sfide devono essere superate, tra cui il miglioramento delle prestazioni dei microrganismi, cell factory, utilizzati nei processi produttivi. Tra tutte, il lievito Saccharomyces cerevisiae è la principale cell factory per la produzione di bioetanolo come biocarburante. L'obiettivo principale di questa tesi è stato di ingegnerizzare ceppi di S. cerevisiae per ottenere una cell factory con caratteristiche rilevanti a livello industriale, tra cui aumentate performances di crescita e un incremento della termotolleranza. In particolare, è stato studiato e quindi indotto un profondo rewiring cellulare, selezionando due proteine: la glutammato sintasi Glt1, un enzima del metabolismo centrale dell'azoto, CNM e la principale polyA binding protein Pab1, un fattore chiave nel metabolismo dell'mRNA. Gli effetti fisiologici della delezione e overespressione di GLT1 sono stati valutati coltivando S. cerevisiae con diverse fonti di azoto. I risultati mostrano che l'aggiunta di solfato d’ammonio, glutammato o glutammina, ma non la modulazione dell’espressione di GLT1, influenza notevolmente la crescita, il contenuto proteico, la vitalità e l'accumulo di specie reattive dell'ossigeno ROS. Questi dati evidenziano la plasticità del CNM di S. cerevisiae rispetto a variazioni ambientale e confermano la sua robustezza contro perturbazioni interne. Inoltre, sebbene la modulazione dell'espressione di GLT1 potrebbe non indurre una riprogrammare cellulare, la caratterizzazione fisiologica descritta puo essere utile per selezionare altri target pio promettenti per riprogrammazioni cellulari. L’induzione del rewiring cellulare è stato poi valutato selezionando Pab1 come target. A tal scopo, un ceppo recante la copia endogena di PAB1 è stato trasformato con una mutant library plasmidica di PAB1 e poi sottoposto a screening per isolare ceppi con maggior termotolleranza. I cloni isolati han mostrato, in drop test, una maggior crescita ad alte temperature e alte concentrazioni di etanolo. Tra tutte, la variante PAB1 S40.7 conferisce una maggiore termotolleranza tramite l’espressione dei soli primi 20 amminoacidi di Pab1. Tale fenotipo è stato anche confermato in bioreattore a 40°C. Una possibile ragione della maggior termotolleranza del ceppo S40.7 potrebbe essere collegata ad un minor accumulo di ROS rispetto al ceppo di controllo. Nel complesso, questi risultati han dimostrato che Pab1 è un promettente target per indurre fenotipi complessi con tratti migliorati, tra cui una maggiore termotolleranza. Infine, Pab1 è stata caratterizzata per determinare il ruolo dei suoi domini nel suo reclutamento all'interno di stress granules, SG, aggregati di mRNP non tradotti che si formano in condizioni stressanti. Questa caratterizzazione ha mostrato che l'associazione di Pab1 negli SG è principalmente dovuta agli RNA Recognition Motives RRM, il cui numero è importante per un efficiente reclutamento. Sebbene i domini P e C non partecipino direttamente all'associazione di Pab1 negli SG, la loro presenza rafforza o diminuisce, rispettivamente, la localizzazione di varianti sintetiche di Pab1 prive di almeno un RRM in questi aggregati. Questo lavoro suggerisce inoltre che i domini di Pab1 potrebbero essere sfruttati razionalmente per scopi di biologia sintetica. Nel complesso, i risultati di questa tesi evidenziano la difficoltà ma allo stesso tempo la capacità del rewiring cellulare nell’indurre fenotipi industrialmente rilevanti e, in particolare, Pab1 è emerso come target promettente per questo approccio.

(2018). Rewiring yeast nitrogen and mRNA metabolism for eliciting industrially relevant phenotypes. The Saccharomyces cerevisiae glutamate synthase (Glt1) and the poly(A) binding protein (Pab1) as case studies. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2018).

Rewiring yeast nitrogen and mRNA metabolism for eliciting industrially relevant phenotypes. The Saccharomyces cerevisiae glutamate synthase (Glt1) and the poly(A) binding protein (Pab1) as case studies

BRAMBILLA, MARCO
2018

Abstract

Nowadays, for the production of energy and materials, our society mainly relies on fossil sources, but many concerns arise from their utilization, like greenhouse gases emission and non-renewability within the time of their consumption. Hence, biorefineries, which convert renewable biomasses into products and energy, could be a promising alternative. Despite some biorefineries are now at commercial scale, many challenges must be overcome to implement competitive bio-based industries, such as improving the performances of microorganisms, named cell factory, used during the production processes. Above all, the yeast Saccharomyces cerevisiae is the most prominent cell factory for producing bioethanol as biofuel. The main objective of this thesis was to engineer S. cerevisiae strains with biotechnological interesting traits, among which improved growth performances and increased thermotolerance. For this purpose, we investigated and applied a cellular rewiring, by selecting two targets: the glutamate synthase Glt1 that is an enzyme of the central nitrogen metabolism, CNM, and the main polyA binding protein Pab1, a master regulator of mRNA metabolism. Regarding GLT1, the physiological effects of its deletion and over-expression were assessed by growing yeasts in the presence of different nitrogen sources. Results showed that the supplementation of ammonium sulfate, glutamate or glutamine considerably affects growth, protein content, viability and reactive oxygen species, ROS, accumulation. Conversely, GLT1 modulation does not significantly influence these parameters. Overall, these data highlight the plasticity of the S. cerevisiae CNM in respect to the environment and confirm its robustness against internal perturbation. Moreover, even though the sole modulation of GLT1 expression might not reprogram the entire cell, the physiological characterization of this study might be helpful to guide the selection of other more promising candidate for the application of the rewiring approaches. Then, the induction of cellular reprogramming was assessed selecting Pab1. To this purpose, a strain carrying the unaltered PAB1 chromosomal allele was transformed with a PAB1 plasmid mutant library and then screened for isolating strains with high thermotolerance. The isolated clones showed growth improvement at both high temperatures and ethanol concentration by drop tests. Among all, the PAB1 S40.7 variant was further characterized because, strikingly, it dominantly confers higher thermotolerance by expressing just the first 20 amino acidic residues of Pab1. This improved phenotype was also confirmed in bioreactor at 40°C. Remarkably, the S40.7 strain accumulates less ROS compared to the control strain, thus possibly explaining its increased thermotolerance. Overall, these results demonstrated that Pab1 is a powerful candidate to evoke complex phenotypes with improved traits, among which higher thermotolerance. Finally, Pab1 was characterized to uncover the role of its domains in the recruitment of the protein within stress granules, aggregates of untranslated mRNPs that form during stressful conditions. This characterization shows that Pab1 association into these aggregates relies mainly on RNA recognition motives, RRM, whose number is important for an efficient recruitment. Although the P and C domains do not directly participate in Pab1 association to stress granules, their presence strengthens or decreases, respectively, the distribution of synthetic Pab1 variants lacking at least one RRM into these aggregates. As additional outcome, this part of the work is suggesting that Pab1 domains might be rationally exploited for synthetic biology purposes. Overall, the results of this thesis highlight and confirm the difficulty but at the same time the power of cellular rewiring in evoking industrially relevant phenotype. Pab1 is undoubtedly emerging as a pivotal element that deserves more attention for future strain design and tailoring.
BRANDUARDI, PAOLA
Yeast,; Glt1,; Pab1,; Cellular; rewiring
Yeast,; Glt1,; Pab1,; Cellular; rewiring
CHIM/11 - CHIMICA E BIOTECNOLOGIA DELLE FERMENTAZIONI
English
1-mar-2018
BIOLOGIA E BIOTECNOLOGIE - 93R
30
2016/2017
open
(2018). Rewiring yeast nitrogen and mRNA metabolism for eliciting industrially relevant phenotypes. The Saccharomyces cerevisiae glutamate synthase (Glt1) and the poly(A) binding protein (Pab1) as case studies. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2018).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/198932
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