Renewable biomass-based processes are expected to contribute to the future supply of fuels, chemicals and materials due to the prospects of environmental friendliness and reducing the use of petrochemicals. The use of microorganisms to convert lignocellulosic biomass into desired products is a promising opportunity. The diverse nature of the biomass feedstocks, together with the biodiversity of microorganisms, has the potential to lead to the production of a wide array of molecules of industrial interest. Nonetheless, many microbial production processes fail to reach the requirements of industrial processes due to cellular inhibition. Inhibitory compounds released from the feedstock pretreatment or produced during fermentation can negatively interfere with the microbial cell factory metabolism and compromise the fermentation process. Organic acids often represent a major hurdle in industrial bio-based microbial processes. They can be released from lignocellulosic feedstocks pretreatment, inhibiting the fermentative processes, and, can also be the desirable products obtained by microbial fermentation with applications in different industrial sectors. However, when high titers are reached the product itself becomes an additional stress factor. The aim of the research described in this thesis was to evaluate the possibility of modifying the membrane lipid composition of Saccharomyces cerevisiae to obtain mutants with increased organic acid tolerance and also with improved for their production. We focused on ergosterol, an essential component of the plasma membrane and contributes to membrane integrity, fluidity and permeability, as well as to the sorting of lipid rafts, which are sterols and sphingolipids-enriched clusters that house several membrane proteins, among which the proton pump H+-ATPase Pma1. By combining in vitro and in vivo complementary assays, we demonstrated that modulation of ergosterol content is crucial for S. cerevisiae to gain tolerance towards organic acids. In the in vitro assay, we prepared synthetic lipid vesicles, from commercially available yeast lipids, enriched, or not, with different amounts of ergosterol. Liposomes were filled with a pH-responsive fluorescent dye, allowing to monitor pH and pH changes inside the liposomes upon exposure to organic acids. This assay, indirectly allowed to investigate the diffusion of organic acids through the liposomal membranes, independent of yeast metabolism. We observed changes in organic acids diffusion through the membrane as a function of ergosterol content. Next, we extended our approach to in vivo using S. cerevisiae. Given the ability of transcription factors to regulate multiple genes, they can be recruited to modulate entire metabolic pathways, as in the case of ergosterol biosynthesis in S. cerevisiae. We focused on the transcription factor ECM22, acting as the main regulator of ergosterol biosynthesis. The overexpression of ECM22 increased the ergosterol content of S. cerevisiae which revealed to be crucial for increased yeast tolerance towards lactic acid stress. We observed that ergosterol's effect on yeast resistance towards organic acid stress changes depending on the organic acid. The plasma membrane lipid composition is equally crucial when organic acids are being produced by S. cerevisiae, both for the proper export of the acids and also to avoid their re-entrance in the cell. In an attempt to increase lactic acid production of two S. cerevisiae lactic acid homofermentative production strains, the transcription factor ECM22 was overexpressed in these strains. Phenotypic characterization of the obtained strains revealed that ECM22 overexpression resulted in increased tolerance to lactic acid stress. However, the production of lactic acid was unaffected. Despite these findings, this research revealed that there is still much to learn about the role of plasma membrane composition in the production of organic acids.
I processi basati sulla biomassa rinnovabile potranno contribuire alla futura richiesta di combustibili, prodotti chimici e materiali date le prospettive di compatibilità ambientale e la riduzione dell'uso di prodotti petrolchimici. L'uso di microrganismi per convertire la biomassa lignocellulosica nei prodotti desiderati è un'opportunità promettente. La combinazione della diversa natura delle biomasse di scarto, insieme alla biodiversità dei microrganismi, rappresentano un’ottimo punto di partenza per la produzione di un'ampia gamma di molecole di interesse industriale. Tuttavia, molti processi di produzione microbica non riescono a soddisfare i requisiti dei processi industriali a causa dell'inibizione della crescita cellulare. Composti inibitori, come gli acidi organici, rilasciati dal pretrattamento della materia prima, come le biomasse lignocellulosiche, oppure prodotti durante la fermentazione interferiscono negativamente con il metabolismo dei microrganismi, compromettendo il processo fermentativo. Lo scopo della ricerca descritta e´ quello di valutare la possibilità di modificare la composizione lipidica della membrana di Saccharomyces cerevisiae per ottenere mutanti con una maggiore tolleranza agli acidi organici ed anche una migliore capacità di produzione di questi ultimi. Ci siamo concentrati sull'ergosterolo, un componente essenziale della membrana che contribuisce alla sua integrità, fluidità e permeabilità, nonché allo smistamento dei rafts lipidici, dei cluster arricchiti di steroli e sfingolipidi che ospitano diverse proteine di membrana, tra cui la pompa protonica ATPasica (Pma1). Dalla combinazione di saggi complementari in vitro e in vivo, abbiamo visto che la modulazione del contenuto di ergosterolo è fondamentale in S. cerevisiae per ottenere una maggiore tolleranza verso gli acidi organici. Nel saggio in vitro, abbiamo preparato vescicole lipidiche sintetiche contenenti diverse quantità di ergosterolo. I liposomi sono stati riempiti con un colorante fluorescente sensibile al pH, che consente di monitorarne i cambiamenti all'interno dei liposomi dopo l'esposizione agli acidi organici. Questo saggio, indirettamente, permette di indagare la diffusione di acidi organici attraverso le membrane liposomiali, indipendentemente dal metabolismo del lievito. Abbiamo osservato cambiamenti nella diffusione degli acidi organici attraverso la membrana, in funzione del contenuto di ergosterolo. Successivamente, abbiamo esteso il nostro approccio in vivo utilizzando S. cerevisiae. I fattori di trascrizione possono essere reclutati per modulare intere vie metaboliche, come nel caso della biosintesi dell'ergosterolo in S. cerevisiae. Ci siamo concentrati sul fattore di trascrizione ECM22, che funge da principale regolatore della biosintesi dell'ergosterolo. L’overespressione di ECM22 ha aumentato il contenuto di ergosterolo in S. cerevisiae, conducendo maggiore tolleranza allo stress da acido lattico. La composizione lipidica della membrana plasmatica influenza anche la produzione di acidi organici in S. cerevisiae. Il fattore di trascrizione ECM22 è stato quindi overespresso in due differenti ceppi di S. cerevisiae che producono acido lattico. La caratterizzazione fenotipica dei ceppi ottenuti ha rivelato che l’overespressione di ECM22 ha portato ad una maggiore tolleranza allo stress da acido lattico. Tuttavia, la produzione di acido lattico non è stata influenzata. Nonostante questi risultati, tale studio ha rivelato che c'è ancora molto da imparare sul ruolo della composizione della membrana plasmatica nella produzione di acidi organici.
(2022). Membrane stress caused by short chain fatty acids in Saccharomyces cerevisiae. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2022).
Membrane stress caused by short chain fatty acids in Saccharomyces cerevisiae
DE MATOS MELO FERRAZ, LUIS PEDRO
2022
Abstract
Renewable biomass-based processes are expected to contribute to the future supply of fuels, chemicals and materials due to the prospects of environmental friendliness and reducing the use of petrochemicals. The use of microorganisms to convert lignocellulosic biomass into desired products is a promising opportunity. The diverse nature of the biomass feedstocks, together with the biodiversity of microorganisms, has the potential to lead to the production of a wide array of molecules of industrial interest. Nonetheless, many microbial production processes fail to reach the requirements of industrial processes due to cellular inhibition. Inhibitory compounds released from the feedstock pretreatment or produced during fermentation can negatively interfere with the microbial cell factory metabolism and compromise the fermentation process. Organic acids often represent a major hurdle in industrial bio-based microbial processes. They can be released from lignocellulosic feedstocks pretreatment, inhibiting the fermentative processes, and, can also be the desirable products obtained by microbial fermentation with applications in different industrial sectors. However, when high titers are reached the product itself becomes an additional stress factor. The aim of the research described in this thesis was to evaluate the possibility of modifying the membrane lipid composition of Saccharomyces cerevisiae to obtain mutants with increased organic acid tolerance and also with improved for their production. We focused on ergosterol, an essential component of the plasma membrane and contributes to membrane integrity, fluidity and permeability, as well as to the sorting of lipid rafts, which are sterols and sphingolipids-enriched clusters that house several membrane proteins, among which the proton pump H+-ATPase Pma1. By combining in vitro and in vivo complementary assays, we demonstrated that modulation of ergosterol content is crucial for S. cerevisiae to gain tolerance towards organic acids. In the in vitro assay, we prepared synthetic lipid vesicles, from commercially available yeast lipids, enriched, or not, with different amounts of ergosterol. Liposomes were filled with a pH-responsive fluorescent dye, allowing to monitor pH and pH changes inside the liposomes upon exposure to organic acids. This assay, indirectly allowed to investigate the diffusion of organic acids through the liposomal membranes, independent of yeast metabolism. We observed changes in organic acids diffusion through the membrane as a function of ergosterol content. Next, we extended our approach to in vivo using S. cerevisiae. Given the ability of transcription factors to regulate multiple genes, they can be recruited to modulate entire metabolic pathways, as in the case of ergosterol biosynthesis in S. cerevisiae. We focused on the transcription factor ECM22, acting as the main regulator of ergosterol biosynthesis. The overexpression of ECM22 increased the ergosterol content of S. cerevisiae which revealed to be crucial for increased yeast tolerance towards lactic acid stress. We observed that ergosterol's effect on yeast resistance towards organic acid stress changes depending on the organic acid. The plasma membrane lipid composition is equally crucial when organic acids are being produced by S. cerevisiae, both for the proper export of the acids and also to avoid their re-entrance in the cell. In an attempt to increase lactic acid production of two S. cerevisiae lactic acid homofermentative production strains, the transcription factor ECM22 was overexpressed in these strains. Phenotypic characterization of the obtained strains revealed that ECM22 overexpression resulted in increased tolerance to lactic acid stress. However, the production of lactic acid was unaffected. Despite these findings, this research revealed that there is still much to learn about the role of plasma membrane composition in the production of organic acids.File | Dimensione | Formato | |
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Descrizione: Tesi di Ferraz Luis - 836892
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Doctoral thesis
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