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    Effect of Nrg1 Repressor on NTH1 Transcription and Molecular Docking of Nrg1 on NTH1 Promoter
    (Univ Indonesia, 2019) Genc, Tulay Turgut; Dogan, Gamze
    The amount of intracellular trehalose increases in response to environmental stress in yeast (Saccharomyces cerevisiae). When that stress is terminated, the accumulated trehalose rapidly degrades into glucose rapidly. Synthesis of trehalose is fulfilled by the Trehalose Phosphate Synthase (TPS) enzyme complex, whereas the degradation of trehalose is done by the neutral trehalase enzyme. Under different stress conditions, transcription of the NTH1 gene is activated and Stress Response Elements (STRE) are required for this activation. Nrg1 protein can bind promoters including STRE and PDS elements. Because of the presence of three possible Nrg1 repressor binding sites on the NTH1 promoter, the NTH1 gene may be regulated by the Nrg1 repressor. In order to test this hypothesis,.nrg1 mutant yeast and its isogenic wildtype yeast strain were used to analyze the transcriptional activation of the NTH1 gene under nitrogen starving conditions. Nth1 transcription of the mutant yeast was seven-fold higher than that of the wild-type under growth conditions, and was not changed during nitrogen starvation. The protein-DNA docking analysis also supported the possibility of Nrg1 binding to the NTH1 promoter. These results revealed that NTH1 gene expression is constitutive in the absence of the Nrg1 repressor protein, hence the transcription of NTH1 is repressed by the Nrg1 protein.
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    Humoral immune response of Galleria mellonella after mono- and co-injection with Hypericum perforatum extract and Candida albicans
    (Wiley, 2024) Genc, Tulay Turgut; Kaya, Serhat; Gunay, Melih; Cakaloglu, Cagla
    Galleria mellonella is used as a model organism to study the innate immune response of insects. In this study, the humoral immune response was assessed by examining phenoloxidase activity, fungal burden, and the expression of phenoloxidase and antimicrobial peptide genes at different time point following separate and combined injections of Hypericum perforatum extract and a nonlethal dose of Candida albicans. The administration of a plant extract at low doses increased phenoloxidase activity, while higher doses had no effect. Similarly, co-injection of a low dose of the extract with the pathogen allowed half of the yeast cells to survive after 24 h. Co-injection of plant extract with the pathogen decreased the phenoloxidase activity at the end of 4 h compared to C. albicans mono-injection. The phenoloxidase gene expressions was reduced in all experimental conditions with respect to the control. When plant extracts and the pathogen were administered together, gallerimycin and hemolin gene expressions were considerably higher compared to mono-injections of plant extracts and the pathogen. The results of this study reveal that gene activation and regulatory mechanisms may change for each immune gene, and that recognition and signaling pathways may differ depending on the involved immunoregulator.
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    Internal transcribed spacer (ITS) sequence-based identification of yeast biota on pomegranate surface and determination of extracellular enzyme profile
    (Univ Sebelas Maret, 2020) Genc, Tulay Turgut; Gunay, Melih
    Yeasts are the most significant organisms to produce fermented products from different types of fruits such as grape, strawberry and pomegranate. The native yeasts on these fruits contribute to beverages' quality and aroma during fermentation. Pomegranate is used in fruit juice and wine production because of high antioxidant characteristic. In order to determine yeast microbiota on the pomegranate fruits collected from Gallipoli (Gelibolu), Canakkale-Turkey, ITS-5.8S rDNA gene region have been utilized. Also, phylogenetic relationships among identified yeast species were assigned by using sequences of ITS-5.8S rDNA gene region. In addition, extracellular enzyme activity of identified yeast strains was detected by using API-ZYM. Kluyveromyces lactis, Aureobasidium pullulans, Hanseniaspora uvarum, Candida zeylanoides, Kwoniella sp., and Metschnikowia pulcherrima and Metschnikowia ziziphicola yeast species were identified on pomegranate surface. Phylogenetic analysis, carried out in ITS-5.8S rDNA gene region of identified yeast strains, revealed the presence of five clades. Kwoniella sp., H. uvarum, M. pulcherrima, and Kl. lactis yeast strains revealed high leucine arylamidase activity. Also valine arylamidase activity was determined in M. pulcherrima and Kl. lactis yeast species. Acid phosphatase activity was determined in H. uvarum and K. lactis yeast species. Uncultured Kwoniella sp. and H. uvarum yeast species displayed high beta-galactosidase and beta-glucosidase activities, respectively.
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    Molecular identification of yeasts from Turkish traditional cheeses: Extracellular enzyme activities and physiological properties important for dairy industry
    (Univ Sebelas Maret, 2023) Gunay, Melih; Genc, Tulay Turgut
    Gunay M, Genc TT. 2023. Molecular identification of yeasts from Turkish traditional cheeses: Extracellular enzyme activities and physiological properties important for dairy industry. Nusantara Bioscience 15: 1-11. The determination of yeast microbiota in cheeses and the physiological properties of yeasts are very important for the dairy industry. In addition, the physiological features, proteolytic and lipolytic activities, and stress tolerance of yeasts have a significant role in the selection of starter yeast species for cheese ripening. This study aimed to determine industrially important yeasts isolated from cheese samples. Molecular techniques identified the isolated yeast strains. The yeast strains' extracellular enzyme activities, fermentation capacities, and thermotolerance and osmotolerance properties were also evaluated. A total of 81 yeast strains were isolated and characterized from three types of cheese samples. PCRRFLP determined the isolated yeast strains and sequence analysis of ITS1-5.8S-ITS2 and 26S rDNA regions. A maximum parsimony tree was constructed by MEGA X software to evaluate the phylogenetic relationship of identified yeast strains. Candida intermedia, Candida parapsilosis, Clavispora lusitaniae, Debaryomyces hansenii, Kluyveromyces marxianus, Pichia kudriavzevii, and Wickerhamomyces anomalus yeast species were identified on cheese samples. The distribution of identified yeast species on cheese samples was determined as 48.1% for W. anomalus, 17.3% for K. marxianus, 14.8% for C. parapsilosis, 8.6% for D. hansenii, 4.9% for Cl. lusitaniae, 3.7% for C. intermedia and 2.5% for P. kudriavzevii. The W. anomalus yeast species was common in three cheese types. All strains of W. anomalus and P. kudriavzevii yeast species, three C. parapsilosis, and two Cl. lusitaniae yeast strains have important physiological properties for industrial applications. These yeast strains have the potential to be used in combination as starter cultures to improve cheese maturation in the future. This comprehensive study identifies yeast species by ITS1-5.8S-ITS2 and 26S rDNA regions and determines industrially important yeast species using multiple criteria (extracellular enzyme activity, stress tolerance, and fermentation capacity).
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    Non-Saccharomyces yeasts diversity on grape berries and molecular characterization of Starmerella bacillaris yeasts strains having high invertase activity
    (Natl Inst Science Communication-Niscair, 2022) Genc, Tulay Turgut
    The diversity of yeast species on grape berries changes depending on various factors. Determination of indigenous yeast intensity and diversity on grape berries is important for increasing the sensory characteristics of wine as well as fermentation efficiency. The natural yeast biota of grape berries affects wine flavour and quality by producing some secondary metabolites and hydrolytic enzymes. Despite the application of different nonconventional yeasts in food and fermentation industries, many significant researches are conducted in finding and improving the new strains having industrially important enzyme activities. Invertase enzyme has a vital role in the food industry in which it increases the sweetness of food without crystallizing them. Here, we studied yeast diversity in grape berries from selected localities and also their invertase activity. We collected grape berries from Alphonse, Kmali Yapmcak, Cavus Efes Karasi, Cinsaut, Atasansi and Isabella grape varieties cultivated in Bozcaada island and Gelibolu peninsula. Twenty-one yeast species belonging to seven genera were identified. The yeast strains having high invertase activities were identified with 5.8S-ITS rDNA sequencing technique. The diversity of yeast biota on berries collected from Gelibolu was greater than that of Bozcaada. Metschnilcowia pulcherrima, Cryptococcus laurentii and Rhodotorula glutinis yeast species were dominant yeast species on grape berries. A total of 294 sucrose grown yeast strains showed growth on sucrose, and 19 of them exhibit the highest invertase activity that is not glucose repressible. These 19 yeast strains were identified as Starmerella bacillaris using 5.8S ITS rDNA region and the phylogenetic analysis was inferred with the Maximum Parsimony method.
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    Reserve Carbohydrate Metabolism in Crabtree-Negative and - Positive Yeasts at Different Carbon Sources
    (Centre Excellence Molecular Biology-Cemb, 2020) Genc, Tulay Turgut
    Background: The fermentation of sugars into ethanol even in the presence of oxygen is referred to as the Crabtree effect. The yeast cells displaying Crabtree effect are indicated as Crabtree-positive yeast. Saccharomyces cerevisiae is Crabtree positive and Debatyomyces occidentalis is Crabtree-negative yeast which does not have Crabtree effect. The reserve carbohydrate metabolism is different in Crabtree-positive and Crabtree-negative yeast cells. The present study aimed to determine the trehalose and glycogen accumulation patterns both in Crabtree-positive and Crabtree-negative yeast species. Methods: In this research, trehalose and glycogen contents of S. cerevisiae and D. occidentalis yeast species were examined in a time course manner in three different carbon sources: glucose, galactose and glycerol. Firstly, yeast cells were grown in rich media supplemented with glucose then all washed and switched to fresh cultures including glucose, galactose and glycerol. Results: In S. cerevisiae yeast cells the overnight accumulated trehalose degraded very rapidly after nonfermentable carbon source replenishment, but this took place in a long time, nearly two days, in D. occidentalis yeast cells. However, whenever D. occidentalis yeast cells shifted to glycerol, all the accumulated trehalose degraded within the twelve hours. Glycogen accumulation in D. occidentalis yeast cells is lower than S. cerevisiae yeast cells both in fermentable and non-fermentable carbon sources. Conclusion: Results indicated that glycogen and trehalose accumulation patterns are completely different in D. occidentalis than S. cerevisiae. Crabtree-negative yeast cells generally, prefer to accumulate glycogen instead of trehalose as reserve carbohydrate. But in our research we proved that Crabtree-negative yeast D. occidentalis, accumulates more trehalose than S. cerevisiae yeast cells in non-fermentable carbon sources.
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    THE SAGA COMPLEX IS ESSENTIAL FOR THE REGULATION OF GENES INVOLVED IN YEAST TREHALOSE METABOLISM
    (Trakya Univ Balkan Yerlesesi Enstituler Binasi, 2022) Genc, Tulay Turgut
    Saccharomyces cerevisiae accumulates trehalose as a stress metabolite in adverse environmental conditions. The trehalose synthesis and breakdown are important for the regulation of trehalose levels within the yeast cell. Therefore, TPS1 and NTH1 gene expressions are tightly regulated during transcription and also translation. Since both genes contain Stress Response Elements (STRE) in the promoter regions, they are co-activated under stress conditions. However, the presence of similar regulatory elements in the promoter of both genes shows that these genes undergo a different regulation at the transcriptional level. In our study, the role of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex in the transcriptional regulation of TPS1 and NTH1 genes was determined in nutrient-poor environment. For that purpose, the wild type and Delta ada] mutant yeast cells, where Ada1p is a member of the SAGA complex, were grown in normal and nitrogen starvation conditions. In addition, trehalose level was detected enzymatically in both wild type and mutant yeast cells. In silico promoter analysis of TPS1 and NTH1 promoters revealed that the STRE sequences required for binding of Msn2/4 transcription factors are closed by nucleosomes at the NTH1 promoter, but open at the TPS1 promoter. In the absence of Ada1p, stress-induced promoter activation in the TPS1 gene was observed, while NTH1 gene expression was not activated. According to these results, the nucleosomes spanning the STRE sequences could not be mobilized in the absence of Ada1 protein, and therefore the Msn2/4 transcription factors cannot bind to the promoter and activate the NTH1 gene expression under stress conditions. It was also observed that in the absence of Ada1p, trehalose accumulation was reduced regardless of stress conditions.