Lignocellulose is the major structural component of woody and non-woody plants, representing a major potential source of renewable organic matter. Lignocellulose is primarily composed by two carbohydrate polymers, cellulose and hemicellulose and by lignin (an aromatic polymer). These complex polymers on the one hand often constitute a residual biomass of agro-food production chain, but at the same time they contain different sugar monomers and phenolic precursors, harbouring an enormous biotechnological value, since they can potentially be converted into different value-added products. In a scenario where the world population is increasing together with the generation of waste and pollution at the expenses of planet resources and human wellbeing, this project aims at proposing an example of circular bioeconomy and industrial symbiosis. More in detail, the project starts from the quali-quantitative evaluation of residual agricultural biomasses to the valorization of a subset of interest for our territory into folates, exploiting yeasts as microbial cell factory. Folate (Vitamin B9) is a water-soluble B vitamin with important roles in nucleic acid synthesis, repair and methylation, produced only by green plants and some microorganisms: for these reasons it represents an essential nutritional component for humans. Vitamin B9 commercially available is chemically synthetized as folic acid, suboptimal in terms of bioactivity for humans; the production of natural folates by microbial fermentation is therefore becoming a sustainable and desirable alternative for human supplementation. During the project the ENEA methodology for the analysis of resource flows and for the creation of possible synergies between the various companies present in the Lombardy region was acquired and applied. Thanks to this work it was possible to identify the main waste biomasses produced in the area in the agro-food sector and unfermented grape marcs was then selected for further studies in laboratory, and compared with previously utilised residual biomasses, deriving from sugar process of production. The non-conventional yeast Scheffersomyces stipitis was exploited as natural but never investigated host for the production of vitamin B9. The growth was optimized and folate production was assessed first in shake flasks and then in bioreactor in formulated media mimicking lignocellulose hydrolysates. The maximum folate production was 3.7 ± 0.07 mg/L, which to date is the highest reported when considering wild type microorganisms. Moreover, folate production was evaluated in shake flasks starting from three different residual biomasses: sugar beet molasses (SBM), sugar beet pulp (SBP) and unfermented grape marcs (UGM). S. stipitis was able to metabolize these biomasses, reaching folate titers of 188.2 ± 24.86 μg/L, 130.6 ± 1.34 μg/L and 101.9 ± 6.62 μg/L respectively. In parallel, the yeast Saccharomyces cerevisiae, amenable for genetic manipulation, was engineered into the anabolic pathway of folate production to acquire novel knowledge on possible targets for unlocking bottlenecks of production. Eight different genes were manipulated for the first time in the same genetic background and exploiting different engineering strategies. This was pivotal for testing the best strain in bioreactor and in bringing folate production and productivity up to 620.0 ± 12.30 μg/L and 41.33 μgfol/Lh respectively, among the highest in the literature. Overall, these results provide solid evidence of possible up-cycling microbial-based processes of lignocellulosic biomasses that characterize specific territory. The value in terms of circularity of the resources, minimization of management costs of wastes and generation of values in the logic of industrial symbiosis was demonstrated, matching the initial scope of the PhD project.

La lignocellulosa è il principale componente strutturale delle piante legnose e non, e rappresenta una delle principali fonti potenziali di materia organica rinnovabile. La lignocellulosa è composta principalmente da due polimeri di carboidrati, cellulosa ed emicellulosa e dalla lignina (un polimero aromatico). Questi polimeri complessi da un lato costituiscono spesso una biomassa residua della filiera agroalimentare, ma allo stesso tempo contengono differenti monomeri zuccherini e precursori fenolici, aventi un enorme valore biotecnologico, poiché potenzialmente possono essere convertiti in differenti prodotti ad elevato valore aggiunto. In uno scenario in cui la popolazione mondiale è in aumento insieme alla generazione di rifiuti ed inquinamento a scapito delle risorse del pianeta e del benessere umano, questo progetto mira a proporre un esempio di bioeconomia circolare e simbiosi industriale. Più in dettaglio, il progetto parte dalla valutazione quali-quantitativa delle biomasse agricole residue fino alla valorizzazione di un sottoinsieme di interesse per il nostro territorio in folati, sfruttando i lieviti come cell factory microbiche. Il folato (Vitamina B9) è una vitamina B idrosolubile con ruoli importanti nella sintesi, riparazione e metilazione degli acidi nucleici, prodotta solo dalle piante verdi e da alcuni microrganismi: per questi motivi rappresenta una componente nutritiva essenziale per l'uomo. La vitamina B9 disponibile in commercio è sintetizzata chimicamente come acido folico, non ottimale in termini di bioattività per l'uomo; la produzione di folati naturali mediante fermentazione microbica sta quindi diventando un'alternativa sostenibile e desiderabile per l'integrazione umana. Nel corso del progetto è stata acquisita ed applicata la metodologia ENEA per l'analisi dei flussi di risorse e per la creazione di possibili sinergie tra le varie aziende presenti nella regione Lombardia. Grazie a questo lavoro è stato possibile identificare le principali biomasse di scarto prodotte nell'area nel settore agroalimentare e le vinacce non fermentate sono state quindi selezionate per ulteriori studi in laboratorio, e confrontate con biomasse residue precedentemente utilizzate, derivanti dal processo di produzione dello zucchero. Il lievito non convenzionale Scheffersomyces stipitis è stato sfruttato come ospite naturale per la produzione di vitamina B9, per la prima volta in questo lavoro. La crescita è stata ottimizzata e la produzione di folati è stata valutata prima in beuta e successivamente in bioreattore in terreni formulati che imitano gli idrolizzati di lignocellulosa. La produzione massima di folati è stata di 3,7 ± 0,07 mg/L, che ad oggi è la più alta riportata se si considerano i microrganismi di tipo selvatico. Inoltre, è stata valutata la produzione di folati in beuta a partire da tre diverse biomasse residue: melassa di barbabietola da zucchero (SBM), polpa di barbabietola da zucchero (SBP) e vinacce non fermentate (UGM). S. stipitis è stato in grado di metabolizzare queste biomasse, raggiungendo titoli di folati rispettivamente di 188,2 ± 24,86 μg/L, 130,6 ± 1,34 μg/L e 101,9 ± 6,62 μg/L. Parallelamente, il lievito Saccharomyces cerevisiae, suscettibile di manipolazione genetica, è stato ingegnerizzato nel percorso anabolico della produzione di folati per acquisire nuove conoscenze sui possibili bersagli per sbloccare i precursori che ne limitano la produzione. Otto geni diversi sono stati manipolati per la prima volta nello stesso background genetico e sfruttando diverse strategie ingegneristiche. Questo è stato fondamentale per testare il miglior ceppo nel bioreattore e per portare la produzione e la produttività di folati rispettivamente a 620,0 ± 12,30 μg/L e 41,33 μgfol/Lh, tra i più alti in letteratura. Nel complesso, questi risultati forniscono una solida evidenza di possibili processi di upcycling a base microbica di biomasse lignocellulosiche.

(2023). PROCESS AND METABOLIC ENGINEERING FOR THE PRODUCTION OF VITAMIN B9 IN YEASTS AS EXAMPLE OF INDUSTRIAL SYMBIOSIS AND CIRCULAR ECONOMY. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2023).

PROCESS AND METABOLIC ENGINEERING FOR THE PRODUCTION OF VITAMIN B9 IN YEASTS AS EXAMPLE OF INDUSTRIAL SYMBIOSIS AND CIRCULAR ECONOMY

MASTELLA, LUCA
2023

Abstract

Lignocellulose is the major structural component of woody and non-woody plants, representing a major potential source of renewable organic matter. Lignocellulose is primarily composed by two carbohydrate polymers, cellulose and hemicellulose and by lignin (an aromatic polymer). These complex polymers on the one hand often constitute a residual biomass of agro-food production chain, but at the same time they contain different sugar monomers and phenolic precursors, harbouring an enormous biotechnological value, since they can potentially be converted into different value-added products. In a scenario where the world population is increasing together with the generation of waste and pollution at the expenses of planet resources and human wellbeing, this project aims at proposing an example of circular bioeconomy and industrial symbiosis. More in detail, the project starts from the quali-quantitative evaluation of residual agricultural biomasses to the valorization of a subset of interest for our territory into folates, exploiting yeasts as microbial cell factory. Folate (Vitamin B9) is a water-soluble B vitamin with important roles in nucleic acid synthesis, repair and methylation, produced only by green plants and some microorganisms: for these reasons it represents an essential nutritional component for humans. Vitamin B9 commercially available is chemically synthetized as folic acid, suboptimal in terms of bioactivity for humans; the production of natural folates by microbial fermentation is therefore becoming a sustainable and desirable alternative for human supplementation. During the project the ENEA methodology for the analysis of resource flows and for the creation of possible synergies between the various companies present in the Lombardy region was acquired and applied. Thanks to this work it was possible to identify the main waste biomasses produced in the area in the agro-food sector and unfermented grape marcs was then selected for further studies in laboratory, and compared with previously utilised residual biomasses, deriving from sugar process of production. The non-conventional yeast Scheffersomyces stipitis was exploited as natural but never investigated host for the production of vitamin B9. The growth was optimized and folate production was assessed first in shake flasks and then in bioreactor in formulated media mimicking lignocellulose hydrolysates. The maximum folate production was 3.7 ± 0.07 mg/L, which to date is the highest reported when considering wild type microorganisms. Moreover, folate production was evaluated in shake flasks starting from three different residual biomasses: sugar beet molasses (SBM), sugar beet pulp (SBP) and unfermented grape marcs (UGM). S. stipitis was able to metabolize these biomasses, reaching folate titers of 188.2 ± 24.86 μg/L, 130.6 ± 1.34 μg/L and 101.9 ± 6.62 μg/L respectively. In parallel, the yeast Saccharomyces cerevisiae, amenable for genetic manipulation, was engineered into the anabolic pathway of folate production to acquire novel knowledge on possible targets for unlocking bottlenecks of production. Eight different genes were manipulated for the first time in the same genetic background and exploiting different engineering strategies. This was pivotal for testing the best strain in bioreactor and in bringing folate production and productivity up to 620.0 ± 12.30 μg/L and 41.33 μgfol/Lh respectively, among the highest in the literature. Overall, these results provide solid evidence of possible up-cycling microbial-based processes of lignocellulosic biomasses that characterize specific territory. The value in terms of circularity of the resources, minimization of management costs of wastes and generation of values in the logic of industrial symbiosis was demonstrated, matching the initial scope of the PhD project.
BRANDUARDI, PAOLA
BELTRANI, TIZIANA
Economia Circolare; Bioraffinerie; Simbiosi Industriale; Vitamina B9; Lieviti
Circular Economy; Biorefineries; Industrial Symbiosis; Vitamin B9; Yeasts
CHIM/11 - CHIMICA E BIOTECNOLOGIA DELLE FERMENTAZIONI
English
23-gen-2023
TECNOLOGIE CONVERGENTI PER I SISTEMI BIOMOLECOLARI (TeCSBi)
35
2021/2022
open
(2023). PROCESS AND METABOLIC ENGINEERING FOR THE PRODUCTION OF VITAMIN B9 IN YEASTS AS EXAMPLE OF INDUSTRIAL SYMBIOSIS AND CIRCULAR ECONOMY. (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/10281/402373
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