Littérature scientifique sur le sujet « Parabailii »

Twenty-three yeast strains traditionally identified as Zygosaccharomyces bailii were studied in order to clarify their taxonomy and phylogenetic relationships. The molecular phylogeny from rRNA gene sequences showed that these yeasts were well divided into three major groups, and two of the groups could be clearly distinguished from the type strain of Z. bailii at the species level. Therefore, we propose Zygosaccharomyces parabailii sp. nov. (type strain ATCC 56075T = NBRC 1047T = NCYC 128T = CBS 12809T) and Zygosaccharomyces pseudobailii sp. nov. (type strain ATCC 56074T = NBRC 0488T = CBS 2856T) to accommodate the yeasts belonging to the two groups. By conventional physiological tests, Z. bailii and the two novel species are not clearly distinguished from one another, as variations exist more frequently between individual strains and are not species-specific. However, the conclusions from rRNA gene sequence analyses are well supported by genome fingerprinting patterns as well as other protein-coding gene sequence comparisons.

Kombucha is a fermented tea with a long history of production and consumption. It has been gaining popularity thanks to its refreshing taste and assumed beneficial properties. The microbial community responsible for tea fermentation—acetic acid bacteria (AAB), yeasts, and lactic acid bacteria (LAB)—is mainly found embedded in an extracellular cellulosic matrix located at the liquid–air interphase. To optimize the production process and investigate the contribution of individual strains, a collection of 26 unique strains was established from an artisanal-scale kombucha production; it included 13 AAB, 12 yeasts, and one LAB. Among these, distinctive strains, namely Novacetimonas hansenii T7SS-4G1, Brettanomyces bruxellensis T7SB-5W6, and Zygosaccharomyces parabailii T7SS-4W1, were used in mono- and co-culture fermentations. The monocultures highlighted important species-specific differences in the metabolism of sugars and organic acids, while binary co-cultures demonstrated the roles played by bacteria and yeasts in the production of cellulose and typical volatile acidity. Aroma complexity and sensory perception were comparable between reconstructed (with the three strains) and native microbial consortia. This study provided a broad picture of the strains’ metabolic signatures, facilitating the standardization of kombucha production in order to obtain a product with desired characteristics by modulating strains presence or abundance.

AbstractZygosaccharomyces parabailii (Z. parabailii) causes spoilage in salad dressings due to its tolerance to osmotic pressure. The objective of this study was to determine the effect of organic acids and storage temperatures (4, 10, and 25 °C) on Z. parabailii growth and salad dressing mechanical properties. Acetic, lactic, and gluconic acids were used alone and in combination to acidify salad dressing. Z. parabailii-challenged formulations containing acetic acid alone tended to have lower counts of Z. parabailii when compared to Z. parabailii-challenged formulations containing other acid combinations. Overall, storage temperature had the most impact on Z. parabailii growth over a 45-day storage. Acidulant type and combination impacted salad dressing mechanical properties. During the 45-day storage period, all formulations showed increased viscosity, a Herschel–Bulkley viscosity profile, and elastic-dominant viscoelastic behavior. The degree of change in rheological behaviors over time was dependent on the type of acid used in the formulation.

ABSTRACT Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH and a fermentative metabolism with a high growth rate. Here we used mRNA sequencing (RNA-seq) to analyze Z. parabailii 's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than under control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/Aft2, previously described as lactic acid responsive in Saccharomyces cerevisiae . Furthermore, formate dehydrogenase ( FDH ) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii in comparison to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production. IMPORTANCE Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression under conditions of interest and relating gene expression patterns to protein functions. However, hybrid organisms present a challenge to the standard use of mRNA sequencing (RNA-seq) to study transcriptional responses to stress, because their genomes contain two similar copies of almost every gene. Here we used stringent mapping methods and a high-quality genome sequence to study the transcriptional response to lactic acid stress in Zygosaccharomyces parabailii ATCC 60483, a natural interspecies hybrid yeast that contains two complete subgenomes that are approximately 7% divergent in sequence. Beyond the insights we gained into lactic acid tolerance in this study, the methods we developed will be broadly applicable to other yeast hybrid strains.

Entre las tecnologías emergentes para la optimización de la preservación de alimentos surge como novedosa alternativa el empleo de películas o recubrimientos comestibles autosoportados que confieren propiedades funcionales específicas. Los trabajos desarrollados en este tema, conducen a incorporar agentes antimicrobianos de fuentes naturales en la formulación de películas, ya que la inclusión de aditivos podría considerarse como una nueva tendencia de envasado activo. Los films o recubrimientos comestibles son láminas delgadas elaboradas con materiales aptos para el consumo y que actúan como una barrera entre el alimento y el ambiente que los rodea, incrementando así la vida útil del producto. Los films obtenidos a partir de polisacáridos, especialmente de almidones, presentan un alto potencial ya que tienen un bajo costo, son transparentes, no confieren olor ni color, poseen una baja permeabilidad al O2 y son biodegradables. Estos sistemas altamente prometedores son capaces de mejorar la calidad, vida útil, seguridad y funcionalidad de los alimentos, y además pueden actuar como carriers de ingredientes activos. En el presente trabajo, se expone la formulación de recubrimientos comestibles a base de almidón de mandioca y adicionados de aceites esenciales (AE), y su posterior evaluación del efecto antimicrobiano frente a la levadura Zygosaccharomyces parabailii, como antecedente de la investigación realizada en el marco de mi tesis de maestría. Sobre la base de los resultados obtenidos en la misma, se propone el actual plan de tesis doctoral que consiste en desarrollar un envase comestible, activo y ecofriendly a base de almidón de mandioca, glicerol y agua, y analizar la viabilidad de la incorporación de pulpas o concentrados de frutas regionales del Nordeste Argentino (mango, guayaba y mamón), estudiando su influencia en las propiedades físicas, mecánicas, de barrera y como fuente natural de polifenoles, antimicrobianos y antioxidantes.

L'obiettivo di questo lavoro di dottorato è di espandere il potenziale industriale di due lieviti non convenzionali, Zygosaccharomyces parabailii e Kluyveromyces marxianus, applicando approcci diretti e indiretti di ingegneria metabolica. Questi lieviti possiedono caratteristiche desiderabili. K. marxianus ha un'ampia specificità sia per gli zuccheri esosi che per quelli pentosi come fonte di carbonio e di energia. Oltre a questo, la termotolleranza, la rapida crescita e la capacità di crescere a pH inferiore a 3 lo rendono ideale per l'uso industriale. Tuttavia, la scarsa tolleranza agli acidi organici deboli liberati durante il pretrattamento di LCB, ne ostacola l'uso quando questa biomassa viene utilizzata come substrato. Sebbene negli ultimi anni lo sviluppo di tecniche di biologia sintetica stai facilitando lo studio e l’impiego di K. marxianus per la produzione di varie sostanze chimiche, i meccanismi relativi alla tolleranza agli acidi organici devono ancora essere decifrati. Per raggiungere questo obiettivo, abbiamo utilizzato una Adaptive Laboratory Evolution (ALE), un approccio di ingegneria indiretta dei ceppi, alternativo e spesso complementare all'ingegneria diretta. Nel capitolo 2, abbiamo mirato a migliorare la tolleranza di K. marxianus all'idrolizzato di polpa di barbabietola da zucchero (SBP) a pH 3,0 a due diverse temperature, 30 °C e 40 °C. Utilizzando l'approccio ALE, abbiamo selezionato isolati di K. marxianus evoluti con fenotipo robusto rispetto ai ceppi parentali, a 30 °C. A differenza di K. marxianus, il lievito ibrido Z. parabailii mostra resistenza agli acidi organici deboli (WOA) anche a pH bassi. La comprensione dei meccanismi coinvolti nella tolleranza a WOA può permettere di evitare la crescita di questo lievito nelle filiere di produzione alimentare, nonché promuoverne l'uso come cell factory per la produzione di acidi organici e altri bioprodotti. Nel capitolo 3 di questo studio, il nostro obiettivo era comprendere la correlazione fenotipo-genotipo coinvolta nella tolleranza a WOA, ed abbiamo concentrato l’attenzione sul trasportatore di membrana Pdr12, creando singoli e doppi mutanti.. Questo studio ha rivelato che Pdr12p è coinvolto nella tolleranza agli acidi acetico e butirrico e non nella tolleranza agli acidi sorbico e benzoico. Inoltre, l'analisi della sequenza delle due copie di Pdr12p ha fornito informazioni sulle differenze amminoacidiche. I progressi nell'ingegneria metabolica e nella biologia sintetica hanno spinto verso la necessità di sviluppare tecniche come CRISPR-Cas9 per una modifica del genoma più rapida ed efficiente. Nel capitolo 4 di questo studio il nostro obiettivo era quello di sviluppare un sistema CRISPR-Cas9 per l’inattivazione o la delezione simultanea di due alleli di un gene in Z. parabailii. Abbiamo valutato l'uso di quattro diversi sistemi di espressione di gRNA costituiti da combinazioni di tRNA, tRNA e ribozima o ribozimi come sequenze fiancheggianti di processamento. La funzionalità dei sistemi gRNA è stata testata analizzando l'inattivazione del gene ADE2 nel ceppo wild type ed è stato utilizzato il sistema gRNA più efficiente per costruire con successo un mutante Z. parabailii dnl4. Questo mutante ha mostrato ricombinazione omologa nell'eliminazione di entrambi gli alleli ADE2. L'analisi delle mutazioni nelle regioni bersaglio del gRNA di entrambi i geni ADE2 e DNL4 ha mostrato riarrangiamenti interallelici tra i due loci, nonché la perdita di estese regioni di cromosomi.
The aim of this PhD work is to expand the industrial potential of two non-conventional yeasts, Zygosaccharomyces parabailii and Kluyveromyces marxianus, by applying direct and indirect strain engineering approaches. These yeasts possess desirable characteristics. K. marxianus has broad specificity for both hexose and pentose sugars as carbon and energy source. Apart from this, its thermotolerance, fast growth and the ability to thrive at pH below 3 make it ideal for industrial use. However, the lack of tolerance of this yeast to inhibitory compounds, particularly weak organic acid produced during LCB pre-treatment, hinders its use when this biomass is used as substrates. Although the use of synthetic biology techniques has started to be employed to understand the robustness of K. marxianus and for the production of various chemicals, the mechanisms related to organic acid tolerance are yet to be deciphered. To match this goal, we used Adaptive Laboratory Evolution (ALE), an indirect strain engineering approach, alternative and often complementary to direct engineering. In chapter 2, we aimed to improve the tolerance of K. marxianus to sugar beet pulp (SBP) hydrolysate at pH 3.0 at two different temperatures, 30 oC and 40 oC. Using the ALE approach, we selected K. marxianus evolved isolates with robust phenotype compared to the parental strains, at 30 oC. Differently to K. marxianus, the hybrid yeast Z. parabailii exhibits resistance to weak organic acids (WOA) also at low pH. Understanding the mechanism involved in tolerance to WOA can be used for avoiding the growth of this yeast in food production pipelines as well as for promoting its use as a cell factory for the production of organic acids and other bio-products. In chapter 3 of this study, our aim was to understand the phenotype-genotype correlation involved in the WOA tolerance trait. Using direct engineering method we constructed and characterised single and double Z. parabailii pdr12 mutants. This study revealed that Pdr12p is involved in tolerance to acetic and butyric acids and not in tolerance towards sorbic and benzoic acids. Furthermore, analysis of the Pdr12p sequence provided insights in the amino acids differences. The pdr12 mutants were constructed by the classical tool of exploiting deletion cassettes. Advances in metabolic engineering and synthetic biology have increased the need for creating techniques such as CRISPR-Cas9 for faster and more efficient genome editing. In chapter 4 of this study our aim was to develop a CRISPR-Cas9 system for simultaneous disruption or deletion of two alleles of a gene in Z. parabailii. We evaluated the use of four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking. The functionality of the gRNA systems was tested by analysing the inactivation of the ADE2 gene in the wild type strain and the most efficient gRNA system was used to successfully construct a Z. parabailii dnl4 mutant. This mutant exhibited improved homologous recombination in the deletion of both ADE2 alleles. Analysis of mutations in the gRNA target regions of both ADE2 and DNL4 genes suggested inter-allelic rearrangements between the two gene loci, as well as absence of large regions of chromosomes. Overall, this work contributes to the vast array of studies that are shedding light on yeasts biodiversity, both as a way for understanding their natural potential and as an instrument for tailoring novel cell factories.

Al fine di rendere efficiente la produzione di biomolecole e biocarburanti tramite le bioraffinerie, è necessario avere a disposizione materie prime rinnovabili e cell factory microbiche robuste. Fra le diverse opzioni, la lignocellulosa e il siero di latte, sottoprodotti delle industrie forestali, agricole e casearie, rappresentano materie prime economiche e ricche di zuccheri. La conversione della lignocellulosa e del siero di latte nei prodotti desiderati tramite cell factory microbiche è un’opzione interessante per la sostituzione delle raffinerie petrolchimiche. Diversi batteri, alghe e lieviti sono già utilizzati come cell factory, ma è previsto che il loro numero aumenti nei prossimi anni. Scarse richieste nutrizionali e robustezza sono caratteristiche che hanno reso i lieviti una classe di microrganismi largamente utilizzata nelle biotecnologie industriali. Ciò è stato possibile grazie allo sfruttamento delle loro qualità naturali e all’ingegnerizzazione di pathway metabolici per la produzione di prodotti naturali o ricombinanti, tra i quali molecole come gli acidi organici. Lo scopo della tesi di ricerca è stato quello di valutare i meccanismi di risposta allo stress indotto da acidi deboli nei lieviti non-Saccaromiceti Zygosaccharomyces parabailii e Kluyveromyces marxianus. Per capire al meglio la risposta nei confronti dello stress indotto da acidi deboli di Z. parabailii, abbiamo ricapitolato tutti le recenti scoperte riguardo questa specie. Una volta venuti a conoscenza delle scoperte scientifiche più rilevanti, ci siamo focalizzati sull’effetto indotto da acido lattico nei confronti del ceppo di Z. parabailii utilizzato nel nostro laboratorio. Lo studio ha rivelato che le cellule sono in grado di sopportare fino a 40g/L di acido lattico senza mostrare una fase lag nelle cinetiche di crescita, ed una percentuale irrisoria di cellule morte. Ma ancor più importante è da sottolineare il fatto che durante l’esposizione all’acido lattico abbiamo osservato modificazioni strutturali a livello della parete e della membrana cellulare. Questi risultati hanno confermato la peculiare abilità di Z. parabailii di adattarsi agli acidi deboli tramite il rimodellamento di alcune componenti cellulari. La mancanza di un genoma di riferimento completo ci ha spinto a compiere il lavoro di sequenziamento, assemblaggio ed annotazione: questo lavoro, oltre a permetterci di evidenziare la natura ibrida del ceppo di Z. parabailii considerato, ha aperto la possibilità di ulteriori studi. I risultati hanno rivelato che Z. parabailii sta subendo un ripristino della fertilità, a seguito dell’evento di ibridazione interspecie, cosa che potrebbe chiarire il processo di duplicazione dell’intero genoma avvenuta in S. cerevisiae ed altri lieviti appartenenti al medesimo clade. Avere a disposizione le informazioni riguardo il genoma completo di Z. parabailii ci ha permesso di portare a termine la prima analisi di sequenziamento dell’RNA sulla specie, quando esposta allo stress da acido lattico. I risultati hanno mostrato l’up-regolazione di geni mitocondriali e connessi allo stress ossidativo, e la down-regolazione di una serie di geni codificanti per determinanti della parete cellulare, in aggiunta alle regolazioni specifiche riguardanti il bilanciamento redox e l’omeostasi di ioni, tra cui il Ferro. È degno di nota il fatto che molti geni sono regolati differentemente rispetto alla controparte di S. cerevisiae, o addirittura non sembrano possedere un omologo nel lievito di riferimento.
The efficient implementation of biorefinery to produce bio-based chemicals and fuels requires sustainable source of feedstock and robust microbial factories. Among others, lignocellulose and whey represent cheap and sugars-enriched feedstock, which are residual wastes deriving from wood/agriculture and dairy industry. The conversion of lignocellulose and whey into the desired products using microbial cell factories is a promising option to replace the fossil based petrochemical refinery. Different bacteria, algae and yeasts are currently used as microbial hosts, but their number is predicted to increase over next years. Minimum nutritional requirements and robustness have made yeasts a class of microbial hosts widely employed in industrial biotechnology, exploiting their natural abilities as well as genetically acquired pathways for production of natural and recombinant products, among which bulk chemicals such as organic acids. However, an efficient and economically viable production of organic acids has to face problems related to low productivity/titer and toxicity of the final product. Therefore, the exploration of yeast biodiversity to exploit unique native features and the understanding of mechanisms to endure harsh conditions are essential to develop ultra-efficient and robust industrial yeast with novel properties. The aim of the research thesis is to evaluate the mechanism of weak acid stress response in the non-Saccharomyces yeasts Zygosaccharomyces parabailii and Kluyveromyces marxianus. To better understand the weak acid stress response of Z. parabailii, we summarized recent finding on the species. Knowing the relevant scientific reports, next study was focused on the effect of lactic acid stress on Z. parabailii. The study revealed that cells are able to tolerate 40g/l of lactic acid without lag phase of growth and exhibiting a negligible percentage of dead cell. More importantly, during lactic acid exposure we observed structural modifications at the level of cell wall and membrane. These findings confirmed the peculiar ability of Z. parabailii to adapt to the weak acids via remodeling of cellular components. The lack of a complete genome assembly and annotation encouraged us to perform a genome sequencing and genome study of our Z. parabailii strain. The results revealed that Z. parabailii is undergoing fertility restoration after interspecies hybridization event, which may shed a light to the process of whole genome duplication. The availability of Z. parabailii complete genome information allowed us to perform the first RNA-sequencing analysis on the species exposed to lactic acid stress. The results showed upregulation of mitochondrial and oxidative stress genes, and downregulation of a subset of cell wall genes, in addition to other specific regulations related to redox balance and ion homeostasis. Remarkably, several differentially regulated genes differ significantly from the S. cerevisiae counterpart or even seems not to have a homologue. Increased interest of K. marxianus application in industrial biotechnology led us to study its multidrug resistance transporters during acetic and lactic acid stress. The results showed a strain specific response to weak organic acid stress, and a possible involvement of KmPDR12 in acetic and lactic acid stress resistance, opening possibility for future discoveries and novel studies.