The role of industrial biotechnology is to provide game-changing solutions for some of the world’s greatest challenges. From climate change to alternative energy sources and to sustainable productions, industrial biotechnology is fighting to find new sustainable solutions. Despite the promising potential and the innovative techniques applied, bio-based biological processes still need further studies for becoming pervasive and therefore substituting the traditional processes of production. To make microbial processes economically feasible and environmentally friendly, one of the key factors resides in the choice of the starting biomass. In a logic of circular bioeconomy, by-products and residual biomasses have to be considered as starting feedstocks of the process. The use of these biomasses does not raise ethical issues and at the same time is economically advantageous and environment oriented. Indeed, they do not compete with the food industry, as they are usually production waste. Most of these residual biomasses are agricultural and forest residues, a family of biomasses characterised by a lignocellulosic structure. The problem related to their use in microbial-based biorefineries is to find an efficient pretreatment to convert them into fermentable sugars and other nutrients, while reducing to a minimum the release of inhibitors of microbial growth. Talking about microbial-based biorefinery as a substitute to petrol-based refinery, there are two main topics to keep in mind during the process design: the starting biomass and the microbial host. The chassis which will be involved in the final production process can be chosen following two complementary approaches: i) exploiting microbial biodiversity already present in nature by picking the final host depending on its innate characteristics, particularly advantageous in a specific production process; ii) working on a well-known cell factory by customising it as needed. In this thesis both principles were followed. In Chapter 2 a specific class of non-conventional yeasts, named oleaginous yeasts, was evaluated to obtain single cell oils (SCOs) for biodiesel production starting from wastes of the sugar beet industry. Lipomyces starkeyi was selected as cell factory for the conversion of sugar beet pulp and sugar beet molasses to maximise SCOs accumulation. With this applicative example we showed the possibility to take advantage of non-conventional microorganisms to achieve a more sustainable way to produce fuels. On the other hand, choosing Saccharomyces cerevisiae as final host has the major advantage of exploiting the wide knowledge around it, starting from its genome and physiology, and arriving at the tremendous number of synthetic biology approaches to engineer it and manipulate it in the desired final form. In Chapter 3 I introduce a novel toolkit: a new combination of synthetic biology approaches to accelerate the engineering procedures allowing the overexpression and the study of more and more complex biosynthetic heterologous pathways. Moreover, I show the application of this novel toolkit to the production of a selected plant secondary metabolite. In Chapter 4 I describe the design of a new vector to improve genome editing procedures in S. cerevisiae. Even in this second project the final goal was to speed up the design and build stages and laboratory procedures, standardising them as much as possible to simplify one part of scientists' work, to leave more space to the subsequent phases of testing and learning. In Chapter 5 I propose the concept of enzyme spatial co-localisation as a forefront field in synthetic biology to maximise the carbon flux toward the product of interest, exploiting the use of protein synthetic scaffolds and synthetic interaction domains. The presented thesis wants to pose itself as a practical example on how industrial biotechnology can be used as a powerful tool in the difficult transition to a more sustainable society.

Il ruolo principale delle biotecnologie industriali è quello di fornire soluzioni innovative per alcune delle più grandi sfide del mondo. Nonostante il potenziale e le tecniche innovative applicate, i processi microbiologici bio-based necessitano ancora di ulteriori studi per diventare pervasivi e quindi sostituire i processi di produzione tradizionali. Per rendere i processi microbici economicamente fattibili e rispettosi dell'ambiente, uno dei fattori chiave risiede nella scelta della biomassa di partenza. In una logica di bioeconomia circolare, i sottoprodotti e le biomasse residue devono essere considerati come materie prime di partenza del processo. L'uso di queste biomasse non solleva questioni etiche e allo stesso tempo è economicamente vantaggioso e orientato all'ambiente. La maggior parte di queste biomasse residue sono residui agricoli e forestali, una famiglia di biomasse caratterizzate da una struttura lignocellulosica. Il problema legato al loro utilizzo nelle bioraffinerie a base microbica è quello di trovare un pretrattamento efficiente per convertirli in zuccheri fermentabili e altri nutrienti, riducendo al minimo il rilascio di inibitori della crescita microbica. Parlando di bioraffinerie microbiche, ci sono due aspetti principali da tenere a mente durante la progettazione del processo: la biomassa di partenza e l'ospite microbico. L’host finale può essere scelto seguendo due approcci complementari: i) sfruttare la biodiversità microbica già presente in natura, scegliendo l'ospite finale in base alle sue caratteristiche innate, particolarmente vantaggiose in uno specifico processo produttivo; ii) lavorare su una cell factory già nota, customizzandola secondo le necessità. Nel Capitolo 2 è stata valutata una specifica classe di lieviti non convenzionali, denominata lieviti oleaginosi, per ottenere oli microbici (SCOs) per la produzione di biodiesel a partire da scarti dell'industria della barbabietola da zucchero. Lipomyces starkeyi è stato selezionato come cell factory per la conversione della polpa di barbabietola da zucchero e della melassa di barbabietola da zucchero per massimizzare l'accumulo di SCOs. Con questo esempio applicativo abbiamo dimostrato la possibilità di sfruttare microrganismi non convenzionali per ottenere bio-carburanti più sostenibili. D'altra parte, la scelta di Saccharomyces cerevisiae come ospite finale ha il grande vantaggio di sfruttare l'ampia conoscenza che lo circonda, compreso l’enorme numero di approcci di biologia sintetica per disegnarlo nella forma finale necessaria. Nel Capitolo 3 presento una nuova combinazione di approcci di biologia sintetica per accelerare le procedure di ingegnerizzazione, consentendo l’over-espressione e lo studio di vie biosintetiche eterologhe sempre più complesse. Inoltre, mostro l'applicazione di questo nuovo kit di strumenti alla produzione di un metabolita secondario di pianta. Nel capitolo 4 descrivo la progettazione di un nuovo vettore per migliorare le procedure di editing del genoma in S. cerevisiae. Anche in questo secondo progetto l'obiettivo finale è stato quello di velocizzare le fasi di progettazione e costruzione e le procedure di laboratorio, standardizzandole il più possibile per semplificare una parte del lavoro e lasciare più spazio alle fasi successive di test & learn. Nel Capitolo 5 propongo il concetto di co-localizzazione spaziale degli enzimi come campo d'avanguardia nella biologia sintetica per massimizzare il flusso di carbonio verso il prodotto di interesse, sfruttando l'uso di scaffold proteici sintetici e domini di interazione sintetici. La tesi qui presentata vuole porsi come esempio pratico di come le biotecnologie industriali possano essere utilizzate come potente strumento nella difficile transizione da una società basata sul petrolio e una più sostenibile.

(2023). TACKLING THE CHALLENGE OF BIO-BASED PRODUCTIONS BY LEVERAGING THE POTENTIAL OF YEAST BIODIVERSITY AND SYNTHETIC BIOLOGY. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2023).

TACKLING THE CHALLENGE OF BIO-BASED PRODUCTIONS BY LEVERAGING THE POTENTIAL OF YEAST BIODIVERSITY AND SYNTHETIC BIOLOGY

MAESTRONI, LETIZIA
2023

Abstract

The role of industrial biotechnology is to provide game-changing solutions for some of the world’s greatest challenges. From climate change to alternative energy sources and to sustainable productions, industrial biotechnology is fighting to find new sustainable solutions. Despite the promising potential and the innovative techniques applied, bio-based biological processes still need further studies for becoming pervasive and therefore substituting the traditional processes of production. To make microbial processes economically feasible and environmentally friendly, one of the key factors resides in the choice of the starting biomass. In a logic of circular bioeconomy, by-products and residual biomasses have to be considered as starting feedstocks of the process. The use of these biomasses does not raise ethical issues and at the same time is economically advantageous and environment oriented. Indeed, they do not compete with the food industry, as they are usually production waste. Most of these residual biomasses are agricultural and forest residues, a family of biomasses characterised by a lignocellulosic structure. The problem related to their use in microbial-based biorefineries is to find an efficient pretreatment to convert them into fermentable sugars and other nutrients, while reducing to a minimum the release of inhibitors of microbial growth. Talking about microbial-based biorefinery as a substitute to petrol-based refinery, there are two main topics to keep in mind during the process design: the starting biomass and the microbial host. The chassis which will be involved in the final production process can be chosen following two complementary approaches: i) exploiting microbial biodiversity already present in nature by picking the final host depending on its innate characteristics, particularly advantageous in a specific production process; ii) working on a well-known cell factory by customising it as needed. In this thesis both principles were followed. In Chapter 2 a specific class of non-conventional yeasts, named oleaginous yeasts, was evaluated to obtain single cell oils (SCOs) for biodiesel production starting from wastes of the sugar beet industry. Lipomyces starkeyi was selected as cell factory for the conversion of sugar beet pulp and sugar beet molasses to maximise SCOs accumulation. With this applicative example we showed the possibility to take advantage of non-conventional microorganisms to achieve a more sustainable way to produce fuels. On the other hand, choosing Saccharomyces cerevisiae as final host has the major advantage of exploiting the wide knowledge around it, starting from its genome and physiology, and arriving at the tremendous number of synthetic biology approaches to engineer it and manipulate it in the desired final form. In Chapter 3 I introduce a novel toolkit: a new combination of synthetic biology approaches to accelerate the engineering procedures allowing the overexpression and the study of more and more complex biosynthetic heterologous pathways. Moreover, I show the application of this novel toolkit to the production of a selected plant secondary metabolite. In Chapter 4 I describe the design of a new vector to improve genome editing procedures in S. cerevisiae. Even in this second project the final goal was to speed up the design and build stages and laboratory procedures, standardising them as much as possible to simplify one part of scientists' work, to leave more space to the subsequent phases of testing and learning. In Chapter 5 I propose the concept of enzyme spatial co-localisation as a forefront field in synthetic biology to maximise the carbon flux toward the product of interest, exploiting the use of protein synthetic scaffolds and synthetic interaction domains. The presented thesis wants to pose itself as a practical example on how industrial biotechnology can be used as a powerful tool in the difficult transition to a more sustainable society.
BRANDUARDI, PAOLA
Biomasse residuali; L. starkeyi; S. cerevisiae; Biologia sintetica; Yeast cell factory
residual biomass; L. starkeyi; S. cerevisiae; Synthetic biology; Yeast cell factory
BIO/11 - BIOLOGIA MOLECOLARE
Italian
23-gen-2023
TECNOLOGIE CONVERGENTI PER I SISTEMI BIOMOLECOLARI (TeCSBi)
35
2021/2022
embargoed_20260123
(2023). TACKLING THE CHALLENGE OF BIO-BASED PRODUCTIONS BY LEVERAGING THE POTENTIAL OF YEAST BIODIVERSITY AND SYNTHETIC BIOLOGY. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2023).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/402374
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