High-glutathione producing yeasts obtained by genetic improvement strategies: a focus on adaptive evolution approaches for novel wine strains

Glutathione (GSH) is the most abundant non-protein thiol in living organisms. Due to its important antioxidant role, it is widely used in medicine, as a food additive, and in the cosmetic industry. Recently, GSH has received growing attention in winemaking because of its ability to control oxidative spoilage damage and to protect various aromatic compounds. Indeed, GSH concentration in wine is highly variable and several factors are involved in its regulation, ranging from grape must to yeast fermentation activity. This short review aims at highlighting the common genetic strategies, useful for obtaining wine yeasts with enhanced GSH production, paying particular attention to the adaptive evolution approaches. Moreover, other strategies, such as random mutagenesis, metabolic engineering and hybridization have been briefly reviewed with a stress on both their strengths and weaknesses in terms of actual feasibility and acceptance by wine consumers

Molecular two-step strategy to select inter-species Saccharomyces hybrids

Hybridization is a common tool to improve yeast for wine industry. Using non-GE hybridization techniques a lot of attempts lead to failed mating which is mainly caused by low spore viability and haplo-selfing. This work proposed a two-step molecular strategy to validate inter-species Saccharomyces hybrids rapidly

Screening dell\u2019attivit\ue0 \u3b2-glucosidasica in ceppi di Saccharomyces spp. isolati da mosto

Nei lieviti, l'attivit\ue0 della \u3b2-glucosidasi \ue8 stata ampiamente descritta, specialmente nelle specie non-Saccharomyces, mentre questa attivit\ue0 risulta essere poco evidente nei ceppi appartenente al genere Saccharomyces. In enologia l'attivit\ue0 \u3b2-glucosidasica \ue8 un carattere di interesse, in quanto correlata al miglioramento dell'espressione aromatica dei vini. Infatti, il suddetto enzima va ad agire sulla classe dei composti aromatici terpenici. Tali composti si trovano sotto forma di coniugati \u3b2-glucosidici nelle variet\ue0 d'uva aromatiche come Moscato e Gew\ufcrztraminer. La forma coniugata \ue8 tuttavia non volatile e, conseguentemente, non aromatica. La presente ricerca fornisce nuove conoscenze sull'attivit\ue0 della \u3b2-glucosidasi in ceppi di interesse enologico appartenenti alle specie Saccharomyces cerevisiae e Saccharomyces uvarum. In particolare, 75 ceppi, isolati da mosto d\u2019uva refrigerato e appartenenti alle due specie suddette, sono stati valutati relativamente all\u2019attivit\ue0 \u3b2-glucosidasica, sia dal punto di vista qualitativo che quantitativo. Lo screening qualitativo in piastra, mediante l\u2019uso di substrati cromogenici, quali esculina e arbutina, ha messo in evidenza un maggior grado di positivit\ue0 dei test provati per i ceppi della specie S. uvarum rispetto a quelli della specie S. cerevisiae. Successivamente, sui ceppi positivi ad entrambi i due precedenti test, \ue8 stato condotto un test quantitativo dell\u2019attivit\ue0 \u3b2-glucosidasica, mediante il metodo spettrofotomentrico basato sull\u2019idrolisi del p\u2010nitrophenyl\u2010\u3b2\u2010D\u2010glucopyranoside (p\u2010NPG). La valutazione \ue8 stata effettuata su tre diverse frazioni dei campioni analizzati: il surnatante, il lisato cellulare e le cellule intere. I risultati ottenuti hanno messo in evidenza che l'attivit\ue0 enzimatica \ue8 principalmente associata alle cellule intere. Inoltre, tre ceppi di S. uvarum, CRY14, VA42 and GRAS14, hanno mostrato una pronunciata attivit\ue0 \u3b2-glucosidasica (Bonciani et al., 2018). Questi ceppi potrebbero essere sfruttati per la loro potenziale capacit\ue0 di migliorare i profili aromatici del vino. Pi\uf9 in generale, lo studio apre un nuovo e promettente campo della ricerca dell'attivit\ue0 \u3b2-glucosidasica in ceppi appartenenti alla specie S. uvarum, fin'ora poco esplorata. Reference Bonciani T., De Vero L., Giannuzzi E., Verspohl A. and Giudici P. (2018). Qualitative and quantitative screening of the \u3b2\u2010glucosidase activity in Saccharomyces cerevisiae and Saccharomyces uvarum strains isolated from refrigerated must. Lett. Appl. Microbiol. Accepted Manuscript. doi:10.1111/lam.1289

Lieviti enologici alto-produttori di glutatione ottenuti mediante strategia basata sull'evoluzione adattativa

Le strategie di evoluzione adattativa per il miglioramento genetico dei lieviti, sono ampiamente utilizzate in campo enologico. I vantaggi di queste strategie sono quelli di non introdurre geni ricombinanti estranei nel genoma dei microrganismi e di non richiedere una pregressa conoscenza dei geni coinvolti nell’espressione dei fenotipi desiderati. Infatti, sottoponendo i microrganismi a colture seriali o continue per diverse generazioni, in presenza di una specifica pressione selettiva, si selezionano i ceppi evoluti esprimenti il carattere d’interesse. Tuttavia, poiché lo screening dei microrganismi evoluti richiede necessariamente l’espressione di fenotipi selezionabili, ossia facilmente riconoscibili, tali strategie non sono applicabili per l’ottenimento di caratteristiche enologiche legate a variazioni e ricombinazioni genetiche non direttamente selezionabili, come nel caso della elevata produzione di glutatione (GSH), o di composti sensorialmente attivi. Ciò nonostante, questo limite è superabile individuando, nella pathway biosintetica del metabolita di interesse, uno o più step in cui applicare una pressione selettiva in modo da selezionare i ceppi evoluti potenzialmente in grado di esprimere il fenotipo desiderato. Questa strategia “evolution-based” è stata recentemente messa a punto e applicata per la selezione di ceppi di lievito alto produttori di GSH (De Vero et al., 2017). La strategia descritta si basa sulla formazione di ricombinanti attraverso la riproduzione sessuale e sulla successiva individuazione dei ricombinanti di interesse attraverso uno screening rapido e altamente selettivo basato sulla resistenza ad elevate concentrazioni di molibdato, tossico per le cellule nella forma ionica esavalente. Infatti, il molibdato applicato come pressione selettiva, consente di selezionare i ceppi evoluti con un metabolismo alterato relativamente alla pathway di assimilazione dei solfati e della biosintesi del GSH. Tale strategia ha permesso di individuare, tra i ceppi evoluti, resistenti a elevate concentrazioni di molibdato, quelli migliorati per la formazione di GSH (Mezzetti et al., 2014, 2017; Bonciani et al., 2018). Reference Bonciani, T., De Vero, L., Mezzetti, F., Fay, J.C. and Giudici, P. (2018) A multi-phase approach to select new wine yeast strains with enhanced fermentative fitness and glutathione production. Appl Microbiol Biotechnol 102, 2269–2278. De Vero, L., Bonciani, T., Verspohl, A., Mezzetti, F. and Giudici, P. (2017) High-glutathione producing yeasts obtained by genetic improvement strategies: a focus on adaptive evolution approaches for novel wine strains. AIMS Microbiol 3, 155–170. Mezzetti, F., De Vero, L. and Giudici, P. (2014) Evolved Saccharomyces cerevisiae wine strains with enhanced glutathione production obtained by an evolution-based strategy. FEMS Yeast Res 14, 977–87. Mezzetti, F., Fay, J.C., Giudici, P. and De Vero, L. (2017) Genetic variation and expression changes associated with molybdate resistance from a glutathione producing wine strain of Saccharomyces cerevisiae. PLoS One 12, e0180814

Fast method for identifying inter- and intra-species Saccharomyces hybrids in extensive genetic improvement programs based on yeast breeding

Aims: The present work proposes a two-step molecular strategy to select inter- and intra-species Saccharomyces hybrids obtained by spore-to-spore mating, one of the most used methods for generating improved hybrids from homothallic wine yeasts. Methods and Results: As low spore viability and haplo-selfing are the main causes of failed mating, at first, we used colony screening PCR (csPCR) of discriminative gene markers to select hybrids directly on dissection plate and discard homozygous diploid colonies arisen from one auto-diploidized progenitor. Then, pre-selected candidates were submitted to recursive streaking and conventional PCR in order to discriminate between the hybrids with stable genomic background and the false-positive admixtures of progenitor cells both undergone haplo-selfing. csPCRs of internal transcribed spacer (ITS) 1 or 2, and the subsequent digestion with diagnostic endonucleases HaeIII and RsaI, respectively, were efficient to select six new Saccharomyces cerevisiae × Saccharomyces uvarum hybrids from 64 crosses. Intragenic minisatellite regions in PIR3, HSP150, and DAN4 genes showed high inter-strain size variation detectable by cost-effective agarose gel electrophoresis and were successful to validate six new intra-species S. cerevisiae hybrids from 34 crosses. Conclusions: Both protocols reduce significantly the number of massive DNA extractions, prevent misinterpretations caused by one or both progenitors undergone haplo-selfing, and can be easily implemented in yeast labs without any specific instrumentation. Significance and Impact of the Study: The study provides a method for the marker-assisted selection of several inter- and intra-species yeast hybrids in a cost-effective, rapid and reproducible manner

Ectopic recombination in sex-determination system as a source of genetic variation in the diploid yeast Zygosaccharomyces sapae.

Sexual reproduction increases genetic variation, which is strongly advantageous under harsh environmental conditions, as it allows natural selection to proceed more effectively. The yeasts of the Zygosaccharomyces rouxii complex are relevant in food elaboration and spoilage due to their ability to cope with low water activity environments and are characterized by gene copy number variation, genome instability, and aneuploidy/allodiploidy (Solieri et al. 2013). The mating-type locus (MAT) is a hotspot for chromosome rearrangement in yeasts. Here, we investigated the genetic architecture of sex determinants, including MAT loci and HO endonuclease in Zygosaccharomyces sapae diploid strain ABT301T, belonging to the Z. rouxii complex. We cloned these genes through a DNA walking strategy and a characterization of the flanking regions, while the chromosome assignment was performed combining Southern blotting and PFGE-karyotyping. We identified three divergent mating type-like (MTL) α-idiomorph sequences, designated as ZsMTLα copies 1, 2, and 3, which encoded homologues of Z. rouxii CBS 732T MATα2 (aa sequence identity from 67.0 to 99.5%) and MATα1 (identity 81.5-99.5%). Cloning of MATa-idiomorph yielded one ZsMTLa locus encoding two Z. rouxii-like proteins, MATa1 and MATa2. ABT301T possesses two divergent HO genes encoding distinct endonucleases. Based on the cloned ZsMTLα and ZsMTLa idiomorphs flanking regions we discovered that Z. sapae ABT301T displays an aααα genotype lacking the HMR silent cassette. Additionally, four putative HML cassettes were identified, two harbouring the ZsMTLα copy 1 and the remaining containing ZsMTLα copies 2 and 3. In conclusion, our results show that the mating-type switching is responsible for hyper-mutation in Z. rouxii complex. The ectopic recombination underlying this process is an error-prone mechanism, which represents a possible source of genetic variation providing yeast progeny with phenotypic variability and adaptation to hostile environments

Bacteriological investigation of milk samples and biopsies from the mammary gland of healthy sows

Objective: Milk samples from clinically healthy sows often contain a similar bacterial content as samples from mastitic sows. To verify whether contamination during sample collection is a possible reason for this or bacterial content in the mammary gland postpartum is a regular finding, the aim of the present study was to compare the suitability of milk samples and mammary gland biopsies for assessing the bacteriological status of healthy sows. Material and methods: Twenty-five clinically healthy sows of different parities were selected. The mammary skin and teats were cleaned and disinfected before biopsies and milk samples were taken from the second mammary gland on the left udder side one day postpartum. Needle biopsies were performed after local anaesthesia. Samples were investigated bacteriologically for aerobic bacteria and a semi-quantitative classification of bacterial growth was carried out. Additionally, formalin-fixed paraffin-embedded biopsies from 18 of the healthy sows were stained and scored for inflammatory cells. Results: A low bacterial content could be found in 96 % of milk samples and in 92 % of biopsies from the healthy sows. Both Gram-positive (mostly streptococci and staphylococci) and Gram-negative bacteria (Escherichia coli) were detected. Histopathological examination revealed mild inflammatory cell infiltration with mainly plasma cells and lymphocytes, in rare cases neutrophilic granulocytes. Conclusion: Biopsies of the mammary gland provided similar results with regard to bacteriological investigation compared to milk sampling although these were collected under aseptic conditions. Therefore, it is assumed that ubiquitous bacteria are regularly present in the colostrum and in the mammary gland tissue of clinically healthy sows after parturition. Histopathological findings were not entirely uneventful. Clinical relevance: Milk samples and biopsies from the thoroughly cleaned and disinfected mammary gland indicate a regular bacterial load of the gland postpartum; biopsies do not provide advantages with regard to hygienic conditions

Unimore Microbial Culture Collection: a source for providing novel yeasts for winemaking.

The University of Modena and Reggio Emilia (UNIMORE) Microbial Culture Collection (UMCC) supplies authenticated strains and fundamental biological data for research and biotechnological application (www.umcc.unimore.it). More than 1600 yeast strains, isolated from must, wine, beer vinegar, sourdough and other fermented products are included in UMCC and are maintained by techniques that ensure preservation and long term genetic stability

The Canine Vaginal Flora: A Large-Cohort Retrospective Study

Microbiological examinations are frequently performed as part of breeding management examinations in the bitch, but also in case of (suspected) reproductive tract problems. As most bacteria are opportunistic pathogens, evaluation of bacterial findings is challenging for veterinarians. Besides, breeders might request antimicrobial treatment in breeding bitches, fearing conception failure—even without medical indication. Considering the rising threat of antimicrobial resistance, gaining deeper insights into the bacterial findings from the vagina of healthy and (suspected) reproductive-diseased bitches might contribute to the knowledge of the canine aerobic vaginal flora and consequently improve the responsible use of antibiotics. We analyzed results from bacteriological cultures of 23,254 vaginal swabs sent in to three commercial laboratories in Germany between 2015 and 2021, where standard aerobic microbiological examination was carried out. We found a variety of 319 bacterial species that mostly grew in mixed cultures of two or more bacterial species. Commonly found species were Escherichia coli, beta-hemolytic Streptococci, coagulase-positive Staphylococci, Pasteurellales, and aerobic sporulators, as well as other Streptococcus spp. Our results showed a large diversity of the canine vaginal flora in healthy and (suspected) reproductive-diseased bitches. They largely support earlier findings of small studies on the physiological canine vaginal flora, emphasizing that solely the results of a bacterial evaluation should not be the base for antimicrobial treatment. Instead, bacterial findings should be evaluated with the results of a clinical gynecological examination