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Sample records for kluyveromyces marxianus imb3

  1. Simultaneous saccharification and fermentation of Kanlow switchgrass by thermotolerant Kluyveromyces marxianus IMB3: the effect of enzyme loading, temperature and higher solid loadings.

    PubMed

    Pessani, Naveen K; Atiyeh, Hasan K; Wilkins, Mark R; Bellmer, Danielle D; Banat, Ibrahim M

    2011-11-01

    Switchgrass (Panicum virgatum) was subjected to hydrothermolysis pretreatment and then used to study the effect of enzyme loading and temperature in a simultaneous saccharification and fermentation (SSF) with the thermotolerant yeast strain Kluyveromyces marxianus IMB3 at 8% solid loading. Various loadings of Accellerase 1500 between 0.1 and 1.1 mL g(-1) glucan were tested in SSF at 45 °C (activity of enzyme was 82.2 FPU mL(-1)). The optimum enzyme loading was 0.7 mL g(-1) glucan based on the six different enzyme loadings tested. SSFs were performed at 37, 41 and 45 °C with an enzyme loading of 0.7 mL g(-1) glucan. The highest ethanol concentration of 22.5 g L(-1) was obtained after 168 h with SSF at 45 °C, which was equivalent to 86% yield. Four different batch and fed-batch strategies were evaluated using a total solid loading of 12% (dry basis). About 32 g L(-1) ethanol was produced with the four strategies, which was equivalent to 82% yield.

  2. Ethanol production through simultaneous saccharification and fermentation of switchgrass using Saccharomyces cerevisiae D(5)A and thermotolerant Kluyveromyces marxianus IMB strains.

    PubMed

    Faga, Brian A; Wilkins, Mark R; Banat, Ibrahim M

    2010-04-01

    Hydrothermolysis pretreated switchgrass at 200 degrees C for 10min was used in a simultaneous saccharification and fermentation (SSF) process using five thermotolerant yeast strains Kluyveromyces marxianus IMB 1, IMB 2, IMB 3, IMB 4, and IMB 5 at 45 degrees C and Saccharomyces cerevisiae D(5)A at 37 degrees C. SSF was carried out for 7d using 5, 10, and 15FPU/g glucan to determine the effect of decreasing cellulase loading on ethanol yield. The effect of initial pH on SSF by S. cerevisiae D(5)A was also investigated. Fermentation by K. marxianus IMB 1, IMB 2, IMB 4, and IMB 5 ceased by 72 h and fermentation by K. marxianus IMB 3 ceased by 96 h, while fermentation S. cerevisiae D(5)A continued for 7d. At 96 and 120 h, IMB 3 and S. cerevisiae D(5)A had similar ethanol yields while the other K. marxianus strains were lower at a 95% confidence level. Final ethanol yields for IMB 3, IMB 1, IMB 5 strains were similar to one another, however, ethanol yield for S. cerevisiae D(5)A (92% maximum theoretical) was greater than all of the IMB strains except IMB 3 at a 95% confidence level. Reducing enzyme loading reduced ethanol yields for both K. marxianus IMB 3 and S. cerevisiae D(5)A. Reducing buffer pH from 5.5 to 4.8 reduced ethanol yields for S. cerevisiae D(5)A. This study shows that K. marxianus IMB 3 has potential for commercial use for ethanol production from cellulose in SSF processes with further improvement of its thermotolerance.

  3. Characterizing yeast promoters used in Kluyveromyces marxianus.

    PubMed

    Yang, Chun; Hu, Shenglin; Zhu, Songli; Wang, Dongmei; Gao, Xiaolian; Hong, Jiong

    2015-10-01

    Fermentation at higher temperatures can potentially reduce the cooling cost in large-scale fermentation and reduce the contamination risk. Thus, the thermotolerant yeast, Kluyveromyces marxianus, which can grow and ferment at elevated temperatures, is a promising biotechnological tool for future applications. However, the promoters used in K. marxianus are not well characterized, especially at elevated temperatures, which is important in efficient metabolic pathway construction. In this study, six constitutive promoters (P(TDH3), P(PGK), and P(ADH1) from both Saccharomyces cerevisiae and K. marxianus) were evaluated in K. marxianus through the heterologous expression of the KlLAC4, GUSA, and SH BLE genes at various temperatures, with various carbon sources and oxygen conditions. The expression was evaluated at the transcription and protein level using real-time PCR and protein activity determination to eliminate the effect of heterologous protein stability. While the transcription of all the promoters decreased at higher temperatures, the order of their promoting strength at various temperatures with glucose as the carbon source was P(KmPGK) > P(KmTDH3) > P(ScPGK) > P(ScTDH3) > P(KmADH1) > P(ScADH1). When glycerol or xylose was supplied as the carbon source at 42 °C, the order of promoter strength was P(KmPGK) > P(ScPGK) > P(KmADH1) > P(ScADH1) > P(ScTDH3) > P(KmTDH3). The promoter activity of P TDH3 decreased significantly, while the promoter activity of both of the P(ADH1) promoters increased. Oxygen conditions had non-significant effect. The results of this study provide important information for fine-tuned pathway construction for the metabolic engineering of K. marxianus.

  4. Kluyveromyces marxianus as a host for heterologous protein synthesis.

    PubMed

    Gombert, Andreas K; Madeira, José Valdo; Cerdán, María-Esperanza; González-Siso, María-Isabel

    2016-07-01

    The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

  5. Production of inulinase from Kluyveromyces marxianus using dahlia tuber extract

    PubMed Central

    Jain, Sumat Chand; Jain, P.C.; Kango, Naveen

    2012-01-01

    Various carbon sources were evaluated for production of inulinase by yeast, Kluyveromyces marxianus MTCC 3995. Highest inulinase activity was observed with Dahlia extract (25.3 nkat mL-1) as carbon source. The enzyme activity was 1.4 folds higher than that observed in media containing pure chicory inulin (17.8 nkat mL-1). The yeast showed good growth on a simple medium containing dahlia extract (20% w/v) and yeast extract (2%w/v) as carbon and nitrogen source respectively, in 96 h. at 28°C and 120 rpm. Lowest inulinase yield (4.8 nkat mL-1) was seen in the medium containing glucose as C-source. Although varied inulinase levels were noticed on different C- sources, Inulinase: Sucrase (I/S) ratios were noticed to be similar. Among various protein sources tested, yeast extract was found to be the best source followed by beef extract (17.9 nkat mL-1) and peptone (13.8 nkat mL-1). The enzyme was optimally active at pH (4.0) and 50°C. TLC analysis of end product revealed that inulinase hydrolyzed inulin exclusively into fructose. Results suggest that the dahlia extract induced exoinulinase synthesis in Kluyveromyces marxianus and can be utilized as a potential substrate for inulinase production. PMID:24031804

  6. Thermal adaptability of Kluyveromyces marxianus in recombinant protein production

    PubMed Central

    2013-01-01

    Background Kluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5°C and 40°C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins. Results The recombinant strains were able to grow up to 40°C and, along the tested temperature interval (5-40°C), the specific growth rates (μ) were generally lower as compared to those of the untransformed strain. Biomass yields were slightly affected by temperature, with the highest values reached at 15°C and 30°C. The secretion of the endogenous β-fructofuranosidase, used as an internal control, was efficient in the range of the tested temperature, as evaluated by assaying the enzyme activity in the culture supernatants. The endogenous β-fructofuranosidase production was temperature dependent, with the highest yield at 30°C. The heterologous proteins HSA, GAA and Sod1p were all successfully produced and secreted between 5°C and 40°C, albeit each one presented a different optimal production temperature (15, 40, 5-30°C for HSA, GAA and Sod1p, respectively). Conclusions K. marxianus L3 has been identified as a promising and flexible cell factory. In a sole host, the optimization of growth temperatures for the efficient secretion of each individual protein can be carried out over a wide range of temperatures. PMID:23587421

  7. Xylitol production at high temperature by engineered Kluyveromyces marxianus.

    PubMed

    Zhang, Jia; Zhang, Biao; Wang, Dongmei; Gao, Xiaolian; Hong, Jiong

    2014-01-01

    Several recombinant Kluyveromyces marxianus strains were constructed through overexpressing the Neurospora crassa xylose reductase genes. YZJ015, which maintained the original xylitol dehydrogenase gene, produced xylitol with the highest productivity (1.49 g L(-1) h(-1)) from 100 g L(-1) xylose at 42 °C. Even at 45 °C, YZJ015 was still able to produce 60.03 g L(-1) xylitol from 100 g L(-1) xylose with a productivity of 1.25 g L(-1)h(-1). In addition, for 20 rounds of cell recycling at 42 °C, YZJ015 produced 71.35 g L(-1) xylitol from 100 g L(-1) xylose with a productivity of 4.43 g L(-1) h(-1) per cycle. YZJ017, in which the xylitol dehydrogenase gene was disrupted, produced 100.02 g L(-1) xylitol at a yield of 1.01 g g(-1) from 100 g L(-1) xylose with 40 g L(-1) glycerol as co-substrate at 42 °C. These engineered strains provide an excellent foundation for xylitol production at elevated temperatures.

  8. Draft Genome Sequence of the Probiotic Yeast Kluyveromyces marxianus fragilis B0399

    PubMed Central

    Quarella, Sara; Lovrovich, Paola; Scalabrin, Simone; Campedelli, Ilenia; Backovic, Ana; Gatto, Veronica; Cattonaro, Federica; Turello, Alessandro; Felis, Giovanna E.

    2016-01-01

    Here, we report the draft genome sequence of Kluyveromyces marxianus fragilis B0399, the first yeast approved as a probiotic for human consumption not belonging to the genus Saccharomyces. The genome is composed of 8 chromosomes, with a total size of 11.44 Mb, including mitochondrial DNA. PMID:27587830

  9. Volatile metabolites produced from agro-industrial wastes by Na-alginate entrapped Kluyveromyces marxianus.

    PubMed

    Güneşer, Onur; Karagül-Yüceer, Yonca; Wilkowska, Agnieszka; Kregiel, Dorota

    The aim of this study was to evaluate the effects of alginate entrapment on fermentation metabolites of Kluyveromyces marxianus grown in agrowastes that served as the liquid culture media. K. marxianus cells entrapped in Na-alginate were prepared using the traditional liquid-droplet-forming method. Whey and pomaces from processed tomatoes, peppers, and grapes were used as the culture media. The changes in the concentrations of sugar, alcohol, organic acids, and flavor compounds were analyzed using gas chromatography-mass spectrometry (GC-MS) and high pressure liquid chromatography (HPLC). Both free and entrapped, K. marxianus were used individually to metabolize sugars, organic acids, alcohols, and flavor compounds in the tomato, pepper, grape, and acid whey based media. Marked changes in the fermentation behaviors of entrapped and free K. marxianus were observed in each culture. A 1.45-log increase was observed in the cell numbers of free K. marxianus during fermentation. On the contrary, the cell numbers of entrapped K. marxianus remained the same. Both free and entrapped K. marxianus brought about the fermentation of sugars such as glucose, fructose, and lactose in the agrowaste cultures. The highest volume of ethanol was produced by K. marxianus in the whey based media. The concentrations of flavor compounds such as ethyl acetate, isoamyl alcohol, isoamyl acetate, 2-phenylethyl isobutyrate, phenylethyl acetate, and phenylethyl alcohol were higher in fermented agrowaste based media compared to the control.

  10. Combination of Kluyveromyces marxianus and sodium bicarbonate for controlling green mold of citrus fruit.

    PubMed

    Geng, Peng; Chen, Shaohua; Hu, Meiying; Rizwan-Ul-Haq, Muhammad; Lai, Kaiping; Qu, Fei; Zhang, Yanbo

    2011-12-02

    Biocontrol efficacy of an antagonistic yeast Kluyveromyces marxianus was evaluated individually or in combination with sodium bicarbonate (SBC) against green mold of citrus fruit caused by Penicillium digitatum. Their effects on postharvest quality of citrus fruit were also investigated. The results indicated that the antagonistic activity of K. marxianus at 1×10⁸ CFU/mL on green mold of citrus fruit was enhanced by 2% SBC treatment. In artificial inoculation trials, disease control after 3 and 6 days, respectively, with the mixture of K. marxianus and 2% SBC (18.33%, 58.33%) was significantly improved over that obtained with K. marxianus (41.67%, 70.00%) or SBC (43.33%, 81.67%) alone. The combination of K. marxianus with SBC was as effective as the imazalil treatment in natural infection trials, which gave about 90% control of green mold. Addition of 2% SBC significantly stimulated the growth of K. marxianus in citrus fruit wounds after 72 h. Moreover, K. marxianus, SBC and their combination did not impair quality parameters including weight loss, fruit firmness, total soluble solids, titratable acidity and ascorbic acid at 4 °C for 30 days followed by 20 °C for 15 days. These results suggested that the use of SBC is a useful approach to improve the efficacy of K. marxianus for the postharvest green mold of citrus fruit.

  11. Encapsulated whey-native yeast Kluyveromyces marxianus as a feed additive for animal production.

    PubMed

    Díaz-Vergara, Ladislao; Pereyra, Carina Maricel; Montenegro, Mariana; Pena, Gabriela Alejandra; Aminahuel, Carla Ayelen; Cavaglieri, Lilia R

    2017-03-06

    Whey is the main byproduct of the cheese industry. While the composition is variable, it retains up to 55% of milk nutrients. The beneficial features of whey indicates a promising source of new potentially probiotic strains for the development of food additives destined for animal production. The aim of this study was to identify Kluyveromyces spp. isolated from whey, to study some probiotic properties and to select the best strain to be encapsulated using derivatised chitosan. Kluyveromyces marxianus strains (VM003, VM004 and VM005) were isolated from whey and identified by phenotypic and molecular techniques. These three yeast strains were able to survive under gastrointestinal conditions. Moreover, they exhibited weak auto-aggregation and co-aggregation with pathogenic bacteria (Salmonella sp., Serratia sp., Escherichia coli and Salmonella typhimurium). In general the K. marxianus strains had a strong antimicrobial activity against pathogenic bacteria. The potential probiotic K. marxianus VM004 strain was selected for derivatised-chitosan encapsulation. Material treated with native chitosan exhibited a strong antimicrobial activity of K. marxianus, showing a total growth inhibition at 10 min exposure. However, derivatised-chitosan encapsulation showed a reduced antimicrobial activity. This is the first study to show some probiotic properties of whey-native K. marxianus, in vitro. An encapsulation strategy was applied using derivatised chitosan.

  12. Bioflavour production from tomato and pepper pomaces by Kluyveromyces marxianus and Debaryomyces hansenii.

    PubMed

    Güneşer, Onur; Demirkol, Aslı; Karagül Yüceer, Yonca; Özmen Toğay, Sine; İşleten Hoşoğlu, Müge; Elibol, Murat

    2015-06-01

    Bioflavours are called natural flavour and/or fragrance compounds which are produced using metabolic pathway of the microorganism and/or plant cells or their enzyme systems with bioengineering approaches. The aim of this study was to investigate bioflavour production from tomato and red pepper pomaces by Kluyveromyces marxianus and Debaryomyces hansenii. Obtained specific growth rates of K. marxianus and D. hansenii in tomato pomace were 0.081/h and 0.177/h, respectively. The bioflavour profile differed between the yeasts. Both yeasts can produce esters and alcohols such as phenyl ethyl alcohol, isoamyl alcohol, isoamyl acetate, phenyl ethyl acetate and isovaleric acid. "Tarhana" and "rose" were descriptive flavour terms for tomato and pepper pomaces fermented by K. marxianus, respectively. Tomato pomace fermented by D. hansenii had the most intense "green bean" flavour while "fermented vegetable" and "storage/yeast" were defined as characteristic flavour terms for pepper pomaces fermented by D. hansenii.

  13. Production of bioethanol from organic whey using Kluyveromyces marxianus.

    PubMed

    Christensen, Anne Deen; Kádár, Zsófia; Oleskowicz-Popiel, Piotr; Thomsen, Mette Hedegaard

    2011-02-01

    Ethanol production by K. marxianus in whey from organic cheese production was examined in batch and continuous mode. The results showed that no pasteurization or freezing of the whey was necessary and that K. marxianus was able to compete with the lactic acid bacteria added during cheese production. The results also showed that, even though some lactic acid fermentation had taken place prior to ethanol fermentation, K. marxianus was able to take over and produce ethanol from the remaining lactose, since a significant amount of lactic acid was not produced (1-2 g/l). Batch fermentations showed high ethanol yield (~0.50 g ethanol/g lactose) at both 30°C and 40°C using low pH (4.5) or no pH control. Continuous fermentation of nonsterilized whey was performed using Ca-alginate-immobilized K. marxianus. High ethanol productivity (2.5-4.5 g/l/h) was achieved at dilution rate of 0.2/h, and it was concluded that K. marxianus is very suitable for industrial ethanol production from whey.

  14. Interactions between Kluyveromyces marxianus and Saccharomyces cerevisiae in tequila must type medium fermentation.

    PubMed

    Lopez, Claudia Lorena Fernandez; Beaufort, Sandra; Brandam, Cédric; Taillandier, Patricia

    2014-08-01

    Traditional tequila fermentation is a complex microbial process performed by different indigenous yeast species. Usually, they are classified in two families: Saccharomyces and Non-Saccharomyces species. Using mixed starter cultures of several yeasts genera and species is nowadays considered to be beneficial to enhance the sensorial characteristics of the final products (taste, odor). However, microbial interactions occurring in such fermentations need to be better understood to improve the process. In this work, we focussed on a Saccharomyces cerevisiae/Kluyveromyces marxianus yeast couple. Indirect interactions due to excreted metabolites, thanks to the use of a specific membrane bioreactor, and direct interaction due to cell-to-cell contact have been explored. Comparison of pure and mixed cultures was done in each case. Mixed cultures in direct contact showed that both yeast were affected but Saccharomyces rapidly dominated the cultures whereas Kluyveromyces almost disappeared. In mixed cultures with indirect contact the growth of Kluyveromyces was decreased compared to its pure culture but its concentration could be maintained whereas the growth of Saccharomyces was enhanced. The loss of viability of Kluyveromyces could not be attributed only to ethanol. The sugar consumption and ethanol production in both cases were similar. Thus the interaction phenomena between the two yeasts are different in direct and indirect contact, Kluyveromyces being always much more affected than Saccharomyces.

  15. Performance evaluation of Pichia kluyveri, Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation.

    PubMed

    Amaya-Delgado, L; Herrera-López, E J; Arrizon, Javier; Arellano-Plaza, M; Gschaedler, A

    2013-05-01

    Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.

  16. Opuntia ficus-indica cladodes as feedstock for ethanol production by Kluyveromyces marxianus and Saccharomyces cerevisiae.

    PubMed

    Kuloyo, Olukayode O; du Preez, James C; García-Aparicio, Maria del Prado; Kilian, Stephanus G; Steyn, Laurinda; Görgens, Johann

    2014-12-01

    The feasibility of ethanol production using an enzymatic hydrolysate of pretreated cladodes of Opuntia ficus-indica (prickly pear cactus) as carbohydrate feedstock was investigated, including a comprehensive chemical analysis of the cladode biomass and the effects of limited aeration on the fermentation profiles and sugar utilization. The low xylose and negligible mannose content of the cladode biomass used in this study suggested that the hemicellulose structure of the O. ficus-indica cladode was atypical of hardwood or softwood hemicelluloses. Separate hydrolysis and fermentation and simultaneous saccharification and fermentation procedures using Kluyveromyces marxianus and Saccharomyces cerevisiae at 40 and 35 °C, respectively, gave similar ethanol yields under non-aerated conditions. In oxygen-limited cultures K. marxianus exhibited almost double the ethanol productivity compared to non-aerated cultures, although after sugar depletion utilization of the produced ethanol was evident. Ethanol concentrations of up to 19.5 and 20.6 g l(-1) were obtained with K. marxianus and S. cerevisiae, respectively, representing 66 and 70 % of the theoretical yield on total sugars in the hydrolysate. Because of the low xylan content of the cladode biomass, a yeast capable of xylose fermentation might not be a prerequisite for ethanol production. K. marxianus, therefore, has potential as an alternative to S. cerevisiae for bioethanol production. However, the relatively low concentration of fermentable sugars in the O. ficus-indica cladode hydrolysate presents a technical constraint for commercial exploitation.

  17. Improved xylose fermentation of Kluyveromyces marxianus at elevated temperature through construction of a xylose isomerase pathway.

    PubMed

    Wang, Rongliang; Li, Lulu; Zhang, Biao; Gao, Xiaolian; Wang, Dongmei; Hong, Jiong

    2013-08-01

    To improve the xylose fermentation ability of Kluyveromyces marxianus, a xylose assimilation pathway through xylose isomerase was constructed. The genes encoding xylose reductase (KmXyl1) and xylitol dehydrogenase (KmXyl2) were disrupted in K. marxianus YHJ010 and the resultant strain was named YRL002. A codon-optimized xylose isomerase gene from Orpinomyces was transformed into K. marxianus YRL002 and expressed under GAPDH promoter. The transformant was adapted in the SD medium containing 1 % casamino acid with 2 % xylose as sole carbon source. After 32 times of trans-inoculation, a strain named YRL005, which can grow at a specific growth rate of 0.137/h with xylose as carbon source, was obtained. K. marxianus YRL005 could ferment 30.15 g/l of xylose and produce 11.52 g/l ethanol with a yield of 0.38 g/g, production rate of 0.069 g/l/h at 42 °C, and also could ferment 16.60 g/l xylose to produce 5.21 g/l ethanol with a yield of 0.31 g/g, and production rate of 0.054 g/l h at 45 °C. Co-fermentation with 2 % glucose could not improve the amount and yield of ethanol fermented from xylose obviously, but it could improve the production rate. Furthermore, K. marxianus YRL005 can ferment with the corn cob hydrolysate, which contained 20.04 g/l xylose to produce 8.25 g/l ethanol. It is a good platform to construct thermo-tolerant xylose fermentation yeast.

  18. Impact of high temperature on ethanol fermentation by Kluyveromyces marxianus immobilized on banana leaf sheath pieces.

    PubMed

    Le, Hoang Du; Thanonkeo, Pornthap; Le, Van Viet Man

    2013-10-01

    Ethanol fermentation was carried out with Kluyveromyces marxianus cells at various temperatures (30, 35, 40, and 45 °C). Fermentation performance of the immobilized yeast on banana leaf sheath pieces and the free yeast were evaluated and compared. Generally, ethanol production of the immobilized and free yeast was stable in a temperature range of 30-40 °C. Temperature of 45 °C restricted yeast growth and lengthened the fermentation. The immobilized yeast demonstrated faster sugar assimilation and higher ethanol level in the fermentation broth in comparison with the free yeast at all fermentation temperatures. Change in fatty acid level in cellular membrane was determined to clarify the response of the free and immobilized yeast to thermal stress. The free cells of K. marxianus responded to temperature increase by increasing saturated fatty acid (C16:0 and C18:0) level and by decreasing unsaturated fatty acid (C18:1 and C18:2) level in cellular membrane. For fermentation at 40 °C with immobilized cells of K. marxianus, however, the changes were not observed in both saturated fatty acid (C16:0) and unsaturated fatty acid (C18:1 and C18:2) level.

  19. Simultaneous saccharification and fermentation of Agave tequilana fructans by Kluyveromyces marxianus yeasts for bioethanol and tequila production.

    PubMed

    Flores, Jose-Axel; Gschaedler, Anne; Amaya-Delgado, Lorena; Herrera-López, Enrique J; Arellano, Melchor; Arrizon, Javier

    2013-10-01

    Agave tequilana fructans (ATF) constitute a substrate for bioethanol and tequila industries. As Kluyveromyces marxianus produces specific fructanases for ATF hydrolysis, as well as ethanol, it can perform simultaneous saccharification and fermentation. In this work, fifteen K. marxianus yeasts were evaluated to develop inoculums with fructanase activity on ATF. These inoculums were added to an ATF medium for simultaneous saccharification and fermentation. All the yeasts, showed exo-fructanhydrolase activity with different substrate specificities. The yeast with highest fructanase activity in the inoculums showed the lowest ethanol production level (20 g/l). Five K. marxianus strains were the most suitable for the simultaneous saccharification and fermentation of ATF. The volatile compounds composition was evaluated at the end of fermentation, and a high diversity was observed between yeasts, nevertheless all of them produced high levels of isobutyl alcohol. The simultaneous saccharification and fermentation of ATF with K. marxianus strains has potential for industrial application.

  20. Respiratory capacity of the Kluyveromyces marxianus yeast isolated from the mezcal process during oxidative stress.

    PubMed

    Arellano-Plaza, Melchor; Gschaedler-Mathis, Anne; Noriega-Cisneros, Ruth; Clemente-Guerrero, Mónica; Manzo-Ávalos, Salvador; González-Hernández, Juan Carlos; Saavedra-Molina, Alfredo

    2013-07-01

    During the mezcal fermentation process, yeasts are affected by several stresses that can affect their fermentation capability. These stresses, such as thermal shock, ethanol, osmotic and growth inhibitors are common during fermentation. Cells have improved metabolic systems and they express stress response genes in order to decrease the damage caused during the stress, but to the best of our knowledge, there are no published works exploring the effect of oxidants and prooxidants, such as H2O2 and menadione, during growth. In this article, we describe the behavior of Kluyveromyces marxianus isolated from spontaneous mezcal fermentation during oxidative stress, and compared it with that of Saccharomyces cerevisiae strains that were also obtained from mezcal, using the W303-1A strain as a reference. S. cerevisiae strains showed greater viability after oxidative stress compared with K. marxianus strains. However, when the yeast strains were grown in the presence of oxidants in the media, K. marxianus exhibited a greater ability to grow in menadione than it did in H2O2. Moreover, when K. marxianus SLP1 was grown in a minibioreactor, its behavior when exposed to menadione was different from its behavior with H2O2. The yeast maintained the ability to consume dissolved oxygen during the 4 h subsequent to the addition of menadione, and then stopped respiration. When exposed to H2O2, the yeast stopped consuming oxygen for the following 8 h, but began to consume oxygen when stressors were no longer applied. In conclusion, yeast isolated from spontaneous mezcal fermentation was able to resist oxidative stress for a long period of time.

  1. Automated UV-C mutagenesis of Kluyveromyces marxianus NRRL Y-1109 and selection for microaerophilic growth and ethanol production at elevated temperature on biomass sugars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The yeast Kluyveromyces marxianus is a potential microbial catalyst for producing ethanol from lignocellulosic substrates at elevated temperatures. To improve its growth and ethanol yield under anaerobic conditions, K. marxianus NRRL Y-1109 was irradiated with UV-C, and surviving cells were grown a...

  2. Hydrolysis of Agave fourcroydes Lemaire (henequen) leaf juice and fermentation with Kluyveromyces marxianus for ethanol production

    PubMed Central

    2014-01-01

    Background Carbon sources for biofuel production are wide-ranging and their availability depends on the climate and soil conditions of the land where the production chain is located. Henequen (Agave fourcroydes Lem.) is cultivated in Yucatán, Mexico to produce natural fibers from the leaves, and a juice containing fructans is produced during this process. Fructans can be hydrolyzed to fructose and glucose and metabolized into ethanol by appropriate yeasts. In Mexico, different Agave species provide the carbon source for (distilled and non-distilled) alcoholic beverage production using the stem of the plant, whilst the leaves are discarded. In this work, we investigated the effect of thermal acid and enzymatic hydrolysis of the juice on the amount of reducing sugars released. Growth curves were generated with the yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus and fermentations were then carried out with Kluyveromyces marxianus to determine alcohol yields. Results With thermal acid hydrolysis, the greatest increase in reducing sugars (82.6%) was obtained using 5% H2SO4 at 100°C with a 30 min reaction time. Statistically similar results can be obtained using the same acid concentration at a lower temperature and with a shorter reaction time (60°C, 15 min), or by using 1% H2SO4 at 100°C with a 30 min reaction time. In the case of enzymatic hydrolysis, the use of 5.75, 11.47 and 22.82 U of enzyme did not produce significant differences in the increase in reducing sugars. Although both hydrolysis processes obtained similar results, the difference was observed after fermentation. Ethanol yields were 50.3 ± 4 and 80.04 ± 5.29% of the theoretical yield respectively. Conclusions Final reducing sugars concentrations obtained with both thermal acid and enzymatic hydrolysis were similar. Saccharomyces cerevisiae, a good ethanol producer, did not grow in the hydrolysates. Only Kluyveromyces marxianus was able to grow in them, giving a higher ethanol

  3. A growth kinetic model of Kluyveromyces marxianus cultures on cheese whey as substrate.

    PubMed

    Longhi, Luís G S; Luvizetto, Débora J; Ferreira, Luciane S; Rech, Rosane; Ayub, Marco A Z; Secchi, Argimiro R

    2004-01-01

    This work presents a multi-route, non-structured kinetic model for determination of microbial growth and substrate consumption in an experimental batch bioreactor in which beta-galactosidase is produced by Kluyveromyces marxianus growing on cheese whey. The main metabolic routes for lactose, and oxygen consumption, cell growth, and ethanol production are derived based on experimental data. When these individual rates are combined into a single growth rate, by rewriting the model equations, the model re-interpretation has a complexity similar to that of the usual variations of the Monod kinetic model, available in the literature. Furthermore, the proposed model is in good agreement with the experimental data for different growth temperatures, being acceptable for dynamic simulations, processes optimization, and implementations of model-based control technologies.

  4. Cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025.

    PubMed

    Rocha, Maria Valderez Ponte; Rodrigues, Tigressa Helena Soares; Melo, Vania M M; Gonçalves, Luciana R B; de Macedo, Gorete Ribeiro

    2011-08-01

    The potential of cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025 was evaluated in this work. This strain was preliminarily cultivated in a synthetic medium containing glucose and xylose and was able to produce ethanol and xylitol at pH 4.5. Next, cashew apple bagasse hydrolysate (CABH) was prepared by a diluted sulfuric acid pretreatment and used as fermentation media. This hydrolysate is rich in glucose, xylose, and arabinose and contains traces of formic acid and acetic acid. In batch fermentations of CABH at pH 4.5, the strain produced only ethanol. The effects of temperature on the kinetic parameters of ethanol fermentation by K. marxianus CE025 using CABH were also evaluated. Maximum specific growth rate (μ(max)), overall yields of ethanol based on glucose consumption [Formula: see text] and based on glucose + xylose consumption (Y ( P/S )), overall yield of ethanol based on biomass (Y ( P/X )), and ethanol productivity (P (E)) were determined as a function of temperature. Best results of ethanol production were achieved at 30°C, which is also quite close to the optimum temperature for the formation of biomass. The process yielded 12.36 ± 0.06 g l(-1) of ethanol with a volumetric production rate of 0.257 ± 0.002 g l(-1) h(-1) and an ethanol yield of 0.417 ± 0.003 g g(-1) glucose.

  5. Improving xylitol production at elevated temperature with engineered Kluyveromyces marxianus through over-expressing transporters.

    PubMed

    Zhang, Jia; Zhang, Biao; Wang, Dongmei; Gao, Xiaolian; Hong, Jiong

    2015-01-01

    Three transporter genes including Kluyveromyces marxianus aquaglyceroporin gene (KmFPS1), Candida intermedia glucose/xylose facilitator gene (CiGXF1) or glucose/xylose symporter gene (CiGXS1) were over-expressed in K. marxianus YZJ017 to improve xylitol production at elevated temperatures. The xylitol production of YZJ074 that harbored CiGXF1 was improved to 147.62g/L in Erlenmeyer flask at 42°C. In fermenter, 99.29 and 149.60g/L xylitol were produced from 99.55 and 151.91g/L xylose with productivity of 4.14 and 3.40g/L/h respectively at 42°C. Even at 45°C, YZJ074 could produce 101.30g/L xylitol from 101.41g/L xylose with productivity of 2.81g/L/h. Using fed-batch fermentation through repeatedly adding non-sterilized substrate directly, YZJ074 could produce 312.05g/L xylitol which is the highest yield reported to date. The engineered strains YZJ074 which can produce xylitol at elevated temperatures is an excellent foundation for xylitol bioconversion.

  6. Trichoderma sp. Spores and Kluyveromyces marxianus Cells Magnetic Separation: Immobilization on Chitosan-Coated Magnetic Nanoparticles.

    PubMed

    Palacios-Ponce, Sócrates; Ramos-González, Rodolfo; Ruiz, Héctor A; Aguilar, Miguel A; Martínez-Hernández, José L; Segura-Ceniceros, Elda P; Aguilar, Cristóbal N; Michelena, Georgina; Ilyina, Anna

    2016-12-29

    In the present study, the interactions between chitosan-coated magnetic nanoparticles (C-MNP) and Trichoderma sp. spores as well as Kluyveromyces marxianus cells were studied. By means of Plackett-Burman design, it was demonstrated that factors which directly influenced on yeast cells immobilization and magnetic separation were: inoculum and C-MNP quantity, stirring speed, interaction time, and volume of medium, while in the case of fungal spores, the temperature also was disclosed as an influencing factor. Langmuir and Freundlich models were applied for the mathematical analysis of adsorption isotherms at 30 °C. For Trichoderma sp. spores adsorption isotherm, the highest correlation coefficient was observed for lineal function of Langmuir model with a maximum adsorption capacity at 5.00E+09 spores (C-MNP g(-1)). Adsorption isotherm of K. marxianus cells was better adjusted to Freundlich model with a constant (Kf) estimated as 2.05E+08 cells (C-MNP g(-1)). Both systems may have a novel application in fermentation processes assisted with magnetic separation of biomass.

  7. Formation of ethyl acetate from whey by Kluyveromyces marxianus on a pilot scale.

    PubMed

    Löser, Christian; Urit, Thanet; Stukert, Anton; Bley, Thomas

    2013-01-10

    Whey arising in huge amounts during milk processing is a valuable renewable resource in the field of White Biotechnology. Kluyveromyces marxianus is able to convert whey-borne lactose into ethyl acetate, an environmentally friendly solvent. Formation of ethyl acetate as a bulk product is triggered by iron (Fe). K. marxianus DSM 5422 was cultivated aerobically in whey-borne medium originally containing 40 μg/L Fe, supplemented with 1, 3 or 10 mg/L Fe in the pre-culture, using an 1 L or 70 L stirred reactor. The highest Fe content in the pre-culture promoted yeast growth in the main culture causing a high sugar consumption for growth and dissatisfactory formation of ethyl acetate, while the lowest Fe content limited yeast growth and promoted ester synthesis but slowed down the process. An intermediate Fe dose (ca. 0.5 μg Fe/g sugar) lastly represented a compromise between some yeast growth, a quite high yield of ethyl acetate and an acceptable duration of the process. The mass of ethyl acetate related to the sugar consumed amounted to 0.113, 0.265 and 0.239 g/g in the three processes corresponding to 21.9%, 51.4% and 46.3% of the theoretically maximum yield. The performance on a pilot scale was somewhat higher than on lab scale.

  8. Studies on the mechanism of synthesis of ethyl acetate in Kluyveromyces marxianus DSM 5422.

    PubMed

    Löser, Christian; Urit, Thanet; Keil, Peter; Bley, Thomas

    2015-02-01

    Kluyveromyces marxianus converts whey-borne sugar into ethyl acetate, an environmentally friendly solvent with many applications. K. marxianus DSM 5422 presumably synthesizes ethyl acetate from acetyl-SCoA. Iron limitation as a trigger for this synthesis is explained by a diminished aconitase and succinate dehydrogenase activity (both enzymes depend on iron) causing diversion of acetyl-SCoA from the tricarboxic acid cycle to ester synthesis. Copper limitation as another trigger for ester synthesis in this yeast refers to involvement of the electron transport chain (all ETC complexes depend on iron and complex IV requires copper). This hypothesis was checked by using several ETC inhibitors. Malonate was ineffective but carboxin partially inhibited complex II and initiated ester synthesis. Antimycin A and cyanide as complexes III and IV inhibitors initiated ester synthesis only at moderate levels while higher concentrations disrupted all respiration and caused ethanol formation. A restricted supply of oxygen (the terminal electron acceptor) also initiated some ester synthesis but primarily forced ethanol production. A switch from aerobic to anaerobic conditions nearly stopped ester synthesis and induced ethanol formation. Iron-limited ester formation was compared with anaerobic ethanol production; the ester yield was lower than the ethanol yield but a higher market price, a reduced number of process stages, a faster process, and decreased expenses for product recovery by stripping favor biotechnological ester production.

  9. Fermentative production of ribonucleotides from whey by Kluyveromyces marxianus: effect of temperature and pH.

    PubMed

    Húngaro, Humberto Moreira; Calil, Natalia Oliveira; Ferreira, Aline Siqueira; Chandel, Anuj Kumar; da Silva, Silvio Silvério

    2013-10-01

    Ribonucleotides have shown many promising applications in food and pharmaceutical industries. The aim of the present study was to produce ribonucleotides (RNA) by Kluyveromyces marxianus ATCC 8,554 utilizing cheese whey, a dairy industry waste, as a main substrate under batch fermentation conditions. The effects of temperature, pH, aeration rate, agitation and initial cellular concentration were studied simultaneously through factorial design for RNA, biomass production and lactose consumption. The maximum RNA production (28.66 mg/g of dry biomass) was observed at temperature 30°C, pH 5.0 and 1 g/l of initial cellular concentration after 2 h of fermentation. Agitation and aeration rate did not influence on RNA concentration (p > 0.05). Maximum lactose consumption (98.7%) and biomass production (6.0 g/l) was observed after 12 h of incubation. This study proves that cheese whey can be used as an adequate medium for RNA production by K. marxianus under the optimized conditions at industrial scale.

  10. High-Efficiency Carbohydrate Fermentation to Ethanol at Temperatures above 40°C by Kluyveromyces marxianus var. marxianus Isolated from Sugar Mills

    PubMed Central

    Anderson, P. J.; McNeil, K.; Watson, K.

    1986-01-01

    A number of yeast strains, isolated from sugar cane mills and identified as strains of Kluyveromyces marxianus var. marxianus, were examined for their ability to ferment glucose and cane syrup to ethanol at high temperatures. Several strains were capable of rapid fermentation at temperatures up to 47°C. At 43°C, >6% (wt/vol) ethanol was produced after 12 to 14 h of fermentation, concurrent with retention of high cell viability (>80%). Although the type strain (CBS 712) of K. marxianus var. marxianus produced up to 6% (wt/vol) ethanol at 43°C, cell viability was low, 30 to 50%, and the fermentation time was 24 to 30 h. On the basis of currently available strains, we suggest that it may be possible by genetic engineering to construct yeasts capable of fermenting carbohydrates at temperatures close to 50°C to produce 10 to 15% (wt/vol) ethanol in 12 to 18 h with retention of cell viability. PMID:16347089

  11. Hypocholesterolemic effects of Kluyveromyces marxianus M3 isolated from Tibetan mushrooms on diet-induced hypercholesterolemia in rat

    PubMed Central

    Xie, Yuanhong; Zhang, Hongxing; Liu, Hui; Xiong, Lixia; Gao, Xiuzhi; Jia, Hui; Lian, Zhengxing; Tong, Nengsheng; Han, Tao

    2015-01-01

    To investigate the effects of Kluyveromyces marxianus M3 isolated from Tibetan mushrooms on diet-induced hypercholesterolemia in rats, female Wistar rats were fed a high-cholesterol diet (HCD) for 28 d to generate hyperlipidemic models. Hyperlipidemic rats were assigned to four groups, which were individually treated with three different dosages of K. marxianus M3+HCD or physiological saline+HCD via oral gavage for 28 d. The total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels in the serum and liver of the rats were measured using commercially available enzyme kits. In addition, the liver morphology was also examined using hematoxylin and eosin staining and optical microscopy. According to our results, the serum and liver TC, TG, LDL-C levels and atherogenic index (AI) were significantly decreased in rats orally administered K. marxianus M3 (p <0.01), and the HDL-C levels and anti atherogenic index (AAI) were significantly increased (p <0.01) compared to the control group. Moreover, K. marxianus M3 treatment also reduced the build-up of lipid droplets in the liver and exhibited normal hepatocytes, suggesting a protective effect of K. marxianus M3 in hyperlipidemic rats. PMID:26273253

  12. Production of bioethanol from effluents of the dairy industry by Kluyveromyces marxianus.

    PubMed

    Zoppellari, Francesca; Bardi, Laura

    2013-09-25

    Whey and scotta are effluents coming from cheese and ricotta processing respectively. Whey contains minerals, lipids, lactose and proteins; scotta contains mainly lactose. Whey can be reused in several ways, such as protein extraction or animal feeding, while nowadays scotta is just considered as a waste; moreover, due to very high volumes of whey produced in the world, it poses serious environmental and disposal problems. Alternative destinations of these effluents, such as biotechnological transformations, can be a way to reach both goals of improving the added value of the agroindustrial processes and reducing their environmental impact. In this work we investigated the way to produce bioethanol from lactose of whey and scotta and to optimize the fermentation yields. Kluyveromyces marxianus var. marxianus was chosen as lactose-fermenting yeast. Batch, aerobic and anaerobic, fermentations and semicontinuous fermentations in dispersed phase and in packed bed reactor were carried out of row whey, scotta and mix 1:1 whey:scotta at a laboratory scale. Different temperatures (28-40°C) were also tested to check whether the thermotolerance of the chosen yeast could be useful to improve the ethanol yield. The best performances were reached at low temperatures (28°C); high temperatures are also compatible with good ethanol yields in whey fermentations, but not in scotta fermentations. Semicontinuous fermentations in dispersed phase gave the best fermentation performances, particularly with scotta. Then both effluents can be considered suitable for ethanol production. The good yields obtained from scotta allow us to transform this waste in a source.

  13. Oxidative stress response of Kluyveromyces marxianus to hydrogen peroxide, paraquat and pressure.

    PubMed

    Pinheiro, R; Belo, I; Mota, M

    2002-05-01

    The aim of this work was to study the oxidative stress response of Kluyveromyces marxianus to hydrogen peroxide (50 mM), paraquat (1 mM), an increase in air pressure (120 kPa, 600 kPa) and pure oxygen pressure (120-600 kPa) in a pressurized bioreactor. The effect of these oxidants on metabolism and on the induction of antioxidant enzymes was investigated. The exposure for 1 h of K. marxianus at exponential growth phase with either H(2)O(2) or paraquat, under air pressure of 120 kPa or 600 kPa, induced an increase in both superoxide dismutase (SOD) and glutathione reductase (GR) content. SOD induction by the chemical oxidants was independent of the air pressure values used. A 2-fold increase in SOD activity was observed after 1 h of exposure to H(2)O(2) and a 3-fold increase was obtained by the presence of paraquat, with both air pressures studied. In contrast, GR activity was raised 1.7-fold by the exposure to both chemicals with 120 kPa, but a 2.4-fold GR induction was obtained with 600 kPa. As opposed to Saccharomyces cerevisiae, catalase was not induced and was even lower than the normal basal levels. This antioxidant enzyme seemed to be inhibited under increasing oxygen partial pressure. The cells showed a significant increase in SOD and GR activity levels, 4.7-fold and 4.4-fold, when exposed for 24 h to 120 kPa pure oxygen pressure. This behaviour was even more patent with 400 kPa. However, whenever cells were previously exposed to low air pressures, low enzymatic activity levels were measured after subsequent exposure to pure oxygen pressure.

  14. Bioethanol production from taro waste using thermo-tolerant yeast Kluyveromyces marxianus K21.

    PubMed

    Wu, Wei-Hao; Hung, Wei-Chun; Lo, Kai-Yin; Chen, Yen-Hui; Wan, Hou-Peng; Cheng, Kuan-Chen

    2016-02-01

    In the present study, evaluation and optimization of taro waste (TW), which was mainly composed of taro peels that contain many starch residues, as the main carbon source in medium were studied. The flask studies showed the optimal medium was using 170g/L of TW which is about 100g/L of glucose and 9g/L of CGM as alternative nitrogen source. Simultaneous saccharification and fermentation (SSF) exhibited higher bioethanol productivity toward separation hydrolysis and fermentation (SHF). The optimal condition of SSF was 5% of Kluyveromyces marxianus K21 inoculum at 40°C resulting in the maximum ethanol concentration (48.98g/L) and productivity (2.23g/L/h) after 22h of cultivation. The scaling up experiment in a 5L bioreactor demonstrated that K21 can still maintain its capability. After 20h of cultivation, 43.78g/L of ethanol (2.19g/L/h of productivity) was achieved corresponding to a 94.2% theoretical ethanol yield.

  15. Polygalacturonase and ethanol production in Kluyveromyces marxianus: potential use of polygalacturonase in foodstuffs.

    PubMed

    Serrat, Manuel; Bermúdez, Rosa C; Villa, Tomás G

    2004-04-01

    The coproduction of ethanol and polygalacturonase (PG) in a pilot-scale batch fermentor using yeast extract--glucose (YD)--and sugar beet molasses (SBM)-based media was implemented utilizing a new high-PG-producing strain of Kluyveromyces marxianus. A certain growth inhibition was observed in SBM medium, causing ethanol and PG production to be lower. Ethanol productivity and accumulation values of 1.94 g/(L x h) and 40 g/L, respectively, were attained in YD, whereas the best fermentation efficiency (95.1%) was achieved with SBM medium. Maximal PG synthesis occurred at the end of cell growth, with values of 1.08 and 0.46 U/(mg x h) for the YD and SBM media, respectively. When the cultures reached stationary phase, PG production stopped. The highest accumulation level (17 U/mL) occurred in YD medium, in agreement with previous laboratory-scale studies carried out for this strain. The potential applications of the crude enzyme preparations were evaluated with different fruit juices and vegetable slices. The enzyme was able to increase the filtration rate of orange, pear, and apple juices by twofold. Additionally, complete clarification of apple juice was readily accomplished, whereas cucumber, carrot, and banana tissues were macerated to a lesser extent.

  16. Ethanol production using whole plant biomass of Jerusalem artichoke by Kluyveromyces marxianus CBS1555.

    PubMed

    Kim, Seonghun; Park, Jang Min; Kim, Chul Ho

    2013-03-01

    Jerusalem artichoke is a low-requirement sugar crop containing cellulose and hemicellulose in the stalk and a high content of inulin in the tuber. However, the lignocellulosic component in Jerusalem artichoke stalk reduces the fermentability of the whole plant for efficient bioethanol production. In this study, Jerusalem artichoke stalk was pretreated sequentially with dilute acid and alkali, and then hydrolyzed enzymatically. During enzymatic hydrolysis, approximately 88 % of the glucan and xylan were converted to glucose and xylose, respectively. Batch and fed-batch simultaneous saccharification and fermentation of both pretreated stalk and tuber by Kluyveromyces marxianus CBS1555 were effectively performed, yielding 29.1 and 70.2 g/L ethanol, respectively. In fed-batch fermentation, ethanol productivity was 0.255 g ethanol per gram of dry Jerusalem artichoke biomass, or 0.361 g ethanol per gram of glucose, with a 0.924 g/L/h ethanol productivity. These results show that combining the tuber and the stalk hydrolysate is a useful strategy for whole biomass utilization in effective bioethanol fermentation from Jerusalem artichoke.

  17. Engineered Kluyveromyces marxianus for pyruvate production at elevated temperature with simultaneous consumption of xylose and glucose.

    PubMed

    Zhang, Biao; Zhu, Yelin; Zhang, Jia; Wang, Dongmei; Sun, Lianhong; Hong, Jiong

    2017-01-01

    Xylose and glucose from lignocellulose are sustainable sources for production of pyruvate, which is the starting material for the synthesis of many drugs and agrochemicals. In this study, the pyruvate decarboxylase gene (KmPDC1) and glycerol-3-phosphate dehydrogenase gene (KmGPD1) of Kluyveromyces marxianus YZJ051 were disrupted to prevent ethanol and glycerol accumulation. The deficient growth of PDC disruption was rescued by overexpressing mutant KmMTH1-ΔT. Then pentose phosphate pathway and xylitol dehydrogenase SsXYL2-ARS genes were overexpressed to obtain strain YZB053 which produced pyruvate with xylose other than glucose. It produced 24.62g/L pyruvate from 80g/L xylose with productivity of 0.51g/L/h at 42°C. Then, xylose-specific transporter ScGAL2-N376F was overexpressed to obtain strain YZB058, which simultaneously consumed 40g/L glucose and 20g/L xylose and produced 29.21g/L pyruvate with productivity of 0.81g/L/h at 42°C. Therefore, a platform for pyruvate production from glucose and xylose at elevated temperature was developed.

  18. Metals sorption from aqueous solutions by Kluyveromyces marxianus: process optimization, equilibrium modeling and chemical characterization.

    PubMed

    Pal, Rama; Tewari, Saumyata; Rai, Jai P N

    2009-10-01

    The dead Kluyveromyces marxianus biomass, a fermentation industry waste, was used to explore its sorption potential for lead, mercury, arsenic, cobalt, and cadmium as a function of pH, biosorbent dosage, contact time, agitation speed, and initial metal concentration. The equilibrium data fitted the Langmuir model better for cobalt and cadmium, but Freundlich isotherm for all metals tested. At equilibrium, the maximum uptake capacity (Qmax) was highest for lead followed by mercury, arsenic, cobalt, and cadmium. The RL values ranged between 0-1, indicating favorable sorption of all test metals by the biosorbent. The maximum Kf value of Pb showed its efficient removal from the solution. However, multi-metal analysis depicted that sorption of all metals decreased except Pb. The potentiometric titration of biosorbent revealed the presence of functional groups viz. amines, carboxylic acids, phosphates, and sulfhydryl group involved in heavy metal sorption. The extent of contribution of functional groups and lipids to biosorption was in the order: carboxylic>lipids>amines>phosphates. Blocking of sulfhydryl group did not have any significant effect on metal sorption.

  19. Production of extracellular exoinulinase from Kluyveromyces marxianus YS-1 using root tubers of Asparagus officinalis.

    PubMed

    Singh, R S; Bhermi, H K

    2008-10-01

    Root tubers of Asparagus officinalis were used as a source of raw inulin for the production of exoinulinase (EC 3.2.1.7) from Kluyveromyces marxianus YS-1. Root extract prepared at 10kg/cm2 pressure for 10min showed maximum inulinase production. Medium components and process parameters were standardized to improve the enzyme production. Inulinase yield of 40.2IU/mL in a medium containing raw inulin (3.5%), beef extract (2%), SDS (0.001%), Mn2+ (2.0mM), Mg2+ (1.5mM), Co2+ (2mM) and pH 6.5 has been obtained under agitation (150rpm) after 60h of incubation at 30 degrees C at shake flask level. After optimization, the enzyme production was 4.8 times more than the basal medium. To test the feasibility of raw inulin from A. officinalis for the production of inulinase, trials were also made in a bioreactor (1.5L). Inulinase activity of 50.2IU/mL was obtained from raw inulin (4.0%) under agitation (200rpm) and aeration (0.75vvm) at 30 degrees C after 60h of fermentation. Inulinase yield in bioreactor was almost six times higher than the basal medium used initially in shake flask.

  20. Protein enrichment of an Opuntia ficus-indica cladode hydrolysate by cultivation of Candida utilis and Kluyveromyces marxianus

    PubMed Central

    Akanni, Gabriel B; du Preez, James C; Steyn, Laurinda; Kilian, Stephanus G

    2015-01-01

    BACKGROUND The cladodes of Opuntia ficus-indica (prickly pear cactus) have a low protein content; for use as a balanced feed, supplementation with other protein sources is therefore desirable. We investigated protein enrichment by cultivation of the yeasts Candida utilis and Kluyveromyces marxianus in an enzymatic hydrolysate of the cladode biomass. RESULTS Dilute acid pretreatment and enzymatic hydrolysis of sun-dried cladodes resulted in a hydrolysate containing (per litre) 45.5 g glucose, 6.3 g xylose, 9.1 g galactose, 10.8 g arabinose and 9.6 g fructose. Even though K. marxianus had a much higher growth rate and utilized l-arabinose and d-galactose more completely than C. utilis, its biomass yield coefficient was lower due to ethanol and ethyl acetate production despite aerobic cultivation. Yeast cultivation more than doubled the protein content of the hydrolysate, with an essential amino acid profile superior to sorghum and millet grains. CONCLUSIONS This K. marxianus strain was weakly Crabtree positive. Despite its low biomass yield, its performance compared well with C. utilis. This is the first report showing that the protein content and quality of O. ficus-indica cladode biomass could substantially be improved by yeast cultivation, including a comparative evaluation of C. utilis and K. marxianus. © 2014 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:25371280

  1. Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing.

    PubMed

    Hu, Nan; Yuan, Bo; Sun, Juan; Wang, Shi-An; Li, Fu-Li

    2012-09-01

    Thermotolerant inulin-utilizing yeast strains are desirable for ethanol production from Jerusalem artichoke tubers by consolidated bioprocessing (CBP). To obtain such strains, 21 naturally occurring yeast strains isolated by using an enrichment method and 65 previously isolated Saccharomyces cerevisiae strains were investigated in inulin utilization, extracellular inulinase activity, and ethanol fermentation from inulin and Jerusalem artichoke tuber flour at 40 °C. The strains Kluyveromyces marxianus PT-1 (CGMCC AS2.4515) and S. cerevisiae JZ1C (CGMCC AS2.3878) presented the highest extracellular inulinase activity and ethanol yield in this study. The highest ethanol concentration in Jerusalem artichoke tuber flour fermentation (200 g L(-1)) at 40 °C achieved by K. marxianus PT-1 and S. cerevisiae JZ1C was 73.6 and 65.2 g L(-1), which corresponded to the theoretical ethanol yield of 90.0 and 79.7 %, respectively. In the range of 30 to 40 °C, temperature did not have a significant effect on ethanol production for both strains. This study displayed the distinctive superiority of K. marxianus PT-1 and S. cerevisiae JZ1C in the thermotolerance and utilization of inulin-type oligosaccharides reserved in Jerusalem artichoke tubers. It is proposed that both K. marxianus and S. cerevisiae have considerable potential in ethanol production from Jerusalem artichoke tubers by a high temperature CBP.

  2. Sulfuric acid hydrolysis and detoxification of red alga Pterocladiella capillacea for bioethanol fermentation with thermotolerant yeast Kluyveromyces marxianus.

    PubMed

    Wu, Chien-Hui; Chien, Wei-Chen; Chou, Han-Kai; Yang, Jungwoo; Lin, Hong-Ting Victor

    2014-09-01

    One-step sulfuric acid saccharification of the red alga Pterocladiella capillacea was optimized, and various detoxification methods (neutralization, overliming, and electrodialysis) of the acid hydrolysate were evaluated for fermentation with the thermotolerant yeast Kluyveromyces marxianus. A proximate composition analysis indicated that P. capillacea was rich in carbohydrates. A significant galactose recovery of 81.1 ± 5% was also achieved under the conditions of a 12% (w/v) biomass load, 5% (v/v) sulfuric acid, 121°C, and hydrolysis for 30 min. Among the various detoxification methods, electrodialysis was identified as the most suitable for fermentable sugar recovery and organic acid removal (100% reduction of formic and levulinic acids), even though it failed to reduce the amount of the inhibitor 5-HMF. As a result, K. marxianus fermentation with the electrodialyzed acid hydrolysate of P. capillacea resulted in the best ethanol levels and fermentation efficiency.

  3. The predominance, biodiversity and biotechnological properties of Kluyveromyces marxianus in the production of Pecorino di Farindola cheese.

    PubMed

    Tofalo, Rosanna; Fasoli, Giuseppe; Schirone, Maria; Perpetuini, Giorgia; Pepe, Alessia; Corsetti, Aldo; Suzzi, Giovanna

    2014-09-18

    Pecorino di Farindola is a handicraft cheese made by farmers on small scale using raw ewes' milk and pig rennet. In this study, yeast consortia were evaluated during Pecorino di Farindola making and ripening. Molecular identification of 156 isolates was achieved by a combination of PCR-RFLP of the 5.8S ITS rRNA region and sequencing of the D1/D2 domain of the 26S rRNA gene. Kluyveromyces marxianus was the predominant species, while other species (Pichia kudriavzevii, Candida parapsilosis, Candida glaebosa and Candida zeylanoides) were present only during the early weeks of ripening. Moreover, the isolates were differentiated both by RAPD-PCR and a sequence alignment of D1/D2 26S rRNA gene, revealing different K. marxianus profiles and variants, and suggesting the role of local selective pressure as the origin of distinctive K. marxianus populations. The strains were characterized also on the basis of different dairy properties such as growth temperature, lactose, galactose, lactate and citrate assimilation at different NaCl concentrations, as well as lipolytic and caseinolytic activities. Moreover, 39 selected K. marxianus strains were inoculated in pasteurized whey to evaluate their growth kinetics, besides lactose, lactate and free amino acids metabolism. The growth kinetics distinguished different biotypes and different metabolic behavior were determined. The general picture of K. marxianus population from Pecorino di Farindola shows a high biodiversity at genetic and phenotypic levels that potentially offers many opportunities for new and advanced knowledge at species level, providing in the meantime a good basis to study the relationship between genetic variability and functional diversities.

  4. UV-C mutagenesis of Kluyveromyces marxianus NRRL Y-1109 strain for improved anaerobic growth at elevated temperature on pentose and hexose sugars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    More robust industrial yeast strains from Kluyveromyces marxianus NRRL Y-1109 and have been produced using UV-C irradiation specifically for anaerobic conversion of lignocellulosic sugar streams to fuel ethanol at elevated temperature (45°C). This type of random mutagenesis offers the possibility o...

  5. Fermentation of cacao (Theobroma cacao L.) seeds with a hybrid Kluyveromyces marxianus strain improved product quality attributes.

    PubMed

    Leal, Gildemberg Amorim; Gomes, Luiz Humberto; Efraim, Priscilla; de Almeida Tavares, Flavio Cesar; Figueira, Antonio

    2008-08-01

    Fermentation of Theobroma cacao (cacao) seeds is an absolute requirement for the full development of chocolate flavor precursors. An adequate aeration of the fermenting cacao seed mass is a fundamental prerequisite for a satisfactory fermentation. Here, we evaluated whether a controlled inoculation of cacao seed fermentation using a Kluyveromyces marxianus hybrid yeast strain, with an increased pectinolytic activity, would improve an earlier liquid drainage ('sweatings') from the fermentation mass, developing a superior final product quality. Inoculation with K. marxianus increased by one third the volume of drained liquid and affected the microorganism population structure during fermentation, which was detectable up to the end of the process. Introduction of the hybrid yeast affected the profile of total seed protein degradation evaluated by polyacrylamide gel electrophoresis, with improved seed protein degradation, and reduction of titrable acidity. Sensorial evaluation of the chocolate obtained from beans fermented with the K. marxianus inoculation was more accepted by analysts in comparison with the one from cocoa obtained through natural fermentation. The increase in mass aeration during the first 24 h seemed to be fundamental for the improvement of fermentation quality, demonstrating the potential application of this improved hybrid yeast strain with superior exogenous pectinolytic activity.

  6. Extraction of inulinase obtained by solid state fermentation of sugarcane bagasse by Kluyveromyces marxianus NRRL Y-7571.

    PubMed

    Bender, João Paulo; Mazutti, Marcio Antônio; Di Luccio, Marco; Treichel, Helen

    2008-06-01

    Production of inulinase by solid state fermentation always involves an extraction step, which dictates enzyme recovery yield and is related to cultivation conditions and control of process parameters. This work is focused on the study of extraction conditions aiming to maximize yield of an inulinase obtained by solid state fermentation of sugar cane bagasse and Kluyveromyces marxianus NRRL Y-7571. Kinetics of extraction was followed varying the kind of solvent used. After determining the best solvent, an experimental design was carried out to study the effect of the solid/liquid ratio (1:10-1:20), extraction temperature (20-53 degrees C), and stirring rate (50-177 rpm). Results showed that maximum yield was obtained when sodium acetate buffer 0.1 M pH 4.8 was used, using a solid/liquid ratio of 1:10, at 53 degrees C and 150 rpm for 40 min.

  7. Effect of lignocellulosic degradation compounds from steam explosion pretreatment on ethanol fermentation by thermotolerant yeast Kluyveromyces marxianus.

    PubMed

    Oliva, Jose Miguel; Sáez, Felicia; Ballesteros, Ignacio; González, Alberto; Negro, Maria José; Manzanares, Paloma; Ballesteros, Mercedes

    2003-01-01

    The filtrate from steam-pretreated poplar was analyzed to identify degradation compounds. The effect of selected compounds on growth and ethanolic fermentation of the thermotolerant yeast strain Kluyveromyces marxianus CECT 10875 was tested. Several fermentations on glucose medium, containing individual inhibitory compounds found in the hydrolysate, were carried out. The degree of inhibition on yeast strain growth and ethanolic fermentation was determined. At concentrations found in the prehy-drolysate, none of the individual compounds significantly affected the fermentation. For all tested compounds, growth was inhibited to a lesser extent than ethanol production. Lower concentrations of catechol (0.96 g/L) and 4-hydroxybenzaldehyde (1.02 g/L) were required to produce the 50% reduction in cell mass in comparison to other tested compounds.

  8. Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal.

    PubMed

    Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

    2011-02-01

    A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight® and compared with the activity of Fructozyme®. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme®, which is important for its potential application in the tequila industry.

  9. Fed-batch fermentation for production of Kluyveromyces marxianus FII 510700 cultivated on a lactose-based medium.

    PubMed

    Lukondeh, Tredwell; Ashbolt, Nicholas J; Rogers, Peter L

    2005-07-01

    A strain of Kluyveromyces marxianus was grown in batch culture in lactose-based media at varying initial lactose concentrations (10-60 g L(-1)) at 30 degrees C, pH 5.0, dissolved oxygen concentrations greater than 20%. Increasing the concentration of mineral salts three-fold at 40 g L(-1) and 60 g L(-1) initial lactose concentration showed only a small increase in the yield of biomass, from 0.38 g g(-1) to 0.41 g g(-1), indicating that the initial batch cultures were not significantly nutrient- (mineral salts)-limited. A relatively high biomass concentration (105 g L(-1)) was obtained in fed-batch culture following extended lactose feeding. An average specific growth rate (0.27 h(-1)), biomass yield (0.38 g g(-1)) and overall productivity (2.9 g L(-1) h(-1)) were obtained for these fed-batch conditions. This fed-batch protocol provides a strategy for achieving relatively high concentrations and productivities of K. marxianus on other lactose-based substrate streams (e.g., whey) from the dairy industry.

  10. Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes.

    PubMed

    Yanase, Shuhei; Hasunuma, Tomohisa; Yamada, Ryosuke; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2010-09-01

    To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous saccharification-fermentation has been required. However, a major drawback is the optimum temperature for the saccharification and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 degrees C and 37 degrees C, while the activity of cellulolytic enzymes is highest at around 50 degrees C and significantly decreases with a decrease in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus beta-glucosidase on the cell surface, which successfully converts a cellulosic beta-glucan to ethanol directly at 48 degrees C with a yield of 4.24 g/l from 10 g/l within 12 h. The yield (in grams of ethanol produced per gram of beta-glucan consumed) was 0.47 g/g, which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface.

  11. Formation of ethyl acetate by Kluyveromyces marxianus on whey during aerobic batch cultivation at specific trace element limitation.

    PubMed

    Urit, Thanet; Stukert, Anton; Bley, Thomas; Löser, Christian

    2012-12-01

    Kluyveromyces marxianus is able to transform lactose into ethyl acetate as a bulk product which offers a chance for an economical reuse of whey-borne sugar. Ethyl acetate is highly volatile and allows its process-integrated recovery by stripping from the aerated bioreactor. Extensive formation of ethyl acetate by K. marxianus DSM 5422 required restriction of yeast growth by a lack of trace elements. Several aerobic batch processes were done in a 1-L stirred reactor using whey-borne culture medium supplemented with an individual trace element solution excluding Mn, Mo, Fe, Cu, or Zn for identifying the trace element(s) crucial for the observed ester synthesis. Only a lack of Fe, Cu, or Zn restricted yeast growth while exclusion of Mn and Mo did not exhibit any effect due to a higher amount of the latter in the used whey. Limitation of growth by Fe or Cu caused significant production of ethyl acetate while limitation by Zn resulted in formation of ethanol. A lack of Fe or Cu obviously makes the respiratory chain inefficient resulting in an increased mitochondrial NADH level followed by a reduced metabolic flux of acetyl-SCoA into the citrate cycle. Synthesis of ethyl acetate from acetyl-SCoA and ethanol by alcoholysis is thus interpreted as an overflow metabolism.

  12. Immobilized Kluyveromyces marxianus cells in carboxymethyl cellulose for production of ethanol from cheese whey: experimental and kinetic studies.

    PubMed

    Roohina, Fatemeh; Mohammadi, Maedeh; Najafpour, Ghasem D

    2016-09-01

    Cheese whey fermentation to ethanol using immobilized Kluyveromyces marxianus cells was investigated in batch and continuous operation. In batch fermentation, the yeast cells were immobilized in carboxymethyl cellulose (CMC) polymer and also synthesized graft copolymer of CMC with N-vinyl-2-pyrrolidone, denoted as CMC-g-PVP, and the efficiency of the two developed cell entrapped beads for lactose fermentation to ethanol was examined. The yeast cells immobilized in CMC-g-PVP performed slightly better than CMC with ethanol production yields of 0.52 and 0.49 g ethanol/g lactose, respectively. The effect of supplementation of cheese whey with lactose (42, 70, 100 and 150 g/l) on fermentative performance of K. marxianus immobilized in CMC beads was considered and the results were used for kinetic studies. The first order reaction model was suitable to describe the kinetics of substrate utilization and modified Gompertz model was quite successful to predict the ethanol production. For continuous ethanol fermentation, a packed-bed immobilized cell reactor (ICR) was operated at several hydraulic retention times; HRTs of 11, 15 and 30 h. At the HRT of 30 h, the ethanol production yield using CMC beads was 0.49 g/g which implies that 91.07 % of the theoretical yield was achieved.

  13. Gene expression analysis using strains constructed by NHEJ-mediated one-step promoter cloning in the yeast Kluyveromyces marxianus.

    PubMed

    Suzuki, Ayako; Fujii, Hiroshi; Hoshida, Hisashi; Akada, Rinji

    2015-09-01

    Gene expression analysis provides valuable information to evaluate cellular state. Unlike quantitative mRNA analysis techniques like reverse-transcription PCR and microarray, expression analysis using a reporter gene has not been commonly used for multiple-gene analysis, probably due to the difficulty in preparing multiple reporter-gene constructs. To circumvent this problem, we developed a novel one-step reporter-gene construction system mediated by non-homologous end joining (NHEJ) in the yeast Kluyveromyces marxianus. As a selectable reporter gene, the ScURA3 selection marker was fused in frame with a red fluorescent gene yEmRFP (ScURA3:yEmRFP). The N-terminally truncated ScURA3:yEmRFP fragment was prepared by PCR. Promoter sequences were also prepared by PCR using primers containing the sequence of the deleted ScURA3 N-terminus to attach at their 3(') ends. The two DNA fragments were used for the transformation of a ura3(-) strain of K. marxianus, in which two DNA fragments are randomly joined and integrated into the chromosome through NHEJ. Only the correctly aligned fragments produced transformants on uracil-deficient medium and expressed red fluorescence under the control of the introduced promoters. A total of 36 gene promoters involved in glycolysis and other pathways were analyzed. Fluorescence measurements of these strains allowed real-time gene expression analysis in different culture conditions.

  14. Co-expression of two heterologous lactate dehydrogenases genes in Kluyveromyces marxianus for l-lactic acid production.

    PubMed

    Lee, Jae Won; In, Jung Hoon; Park, Joon-Bum; Shin, Jonghyeok; Park, Jin Hwan; Sung, Bong Hyun; Sohn, Jung-Hoon; Seo, Jin-Ho; Park, Jin-Byoung; Kim, Soo Rin; Kweon, Dae-Hyuk

    2017-01-10

    Lactic acid (LA) is a versatile compound used in the food, pharmaceutical, textile, leather, and chemical industries. Biological production of LA is possible by yeast strains expressing a bacterial gene encoding l-lactate dehydrogenase (LDH). Kluyveromyces marxianus is an emerging non-conventional yeast with various phenotypes of industrial interest. However, it has not been extensively studied for LA production. In this study, K. marxianus was engineered to express and co-express various heterologous LDH enzymes that were reported to have different pH optimums. Specifically, three LDH enzymes originating from Staphylococcus epidermidis (SeLDH; optimal at pH 5.6), Lactobacillus acidophilus (LaLDH; optimal at pH 5.3), and Bos taurus (BtLDH; optimal at pH 9.8) were functionally expressed individually and in combination in K. marxianus, and the resulting strains were compared in terms of LA production. A strain co-expressing SeLDH and LaLDH (KM5 La+SeLDH) produced 16.0g/L LA, whereas the strains expressing those enzymes individually produced only 8.4 and 6.8g/L, respectively. This co-expressing strain produced 24.0g/L LA with a yield of 0.48g/g glucose in the presence of CaCO3. Our results suggest that co-expression of LDH enzymes with different pH optimums provides sufficient LDH activity under dynamic intracellular pH conditions, leading to enhanced production of LA compared to individual expression of the LDH enzymes.

  15. Comparing cell viability and ethanol fermentation of the thermotolerant yeast Kluyveromyces marxianus and Saccharomyces cerevisiae on steam-exploded biomass treated with laccase.

    PubMed

    Moreno, Antonio D; Ibarra, David; Ballesteros, Ignacio; González, Alberto; Ballesteros, Mercedes

    2013-05-01

    In this study, the thermotolerant yeast Kluyveromyces marxianus CECT 10875 was compared to the industrial strain Saccharomyces cerevisiae Ethanol Red for lignocellulosic ethanol production. For it, whole slurry from steam-exploded wheat straw was used as raw material, and two process configurations, simultaneous saccharification and fermentation (SSF) and presaccharification and simultaneous saccharification and fermentation (PSSF), were evaluated. Compared to S. cerevisiae, which was able to produce ethanol in both process configurations, K. marxianus was inhibited, and neither growth nor ethanol production occurred during the processes. However, laccase treatment of the whole slurry removed specifically lignin phenols from the overall inhibitory compounds present in the slurry and triggered the fermentation by K. marxianus, attaining final ethanol concentrations and yields comparable to those obtained by S. cerevisiae.

  16. High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042▿

    PubMed Central

    Nonklang, Sanom; Abdel-Banat, Babiker M. A.; Cha-aim, Kamonchai; Moonjai, Nareerat; Hoshida, Hisashi; Limtong, Savitree; Yamada, Mamoru; Akada, Rinji

    2008-01-01

    We demonstrate herein the ability of Kluyveromyces marxianus to be an efficient ethanol producer and host for expressing heterologous proteins as an alternative to Saccharomyces cerevisiae. Growth and ethanol production by strains of K. marxianus and S. cerevisiae were compared under the same conditions. K. marxianus DMKU3-1042 was found to be the most suitable strain for high-temperature growth and ethanol production at 45°C. This strain, but not S. cerevisiae, utilized cellobiose, xylose, xylitol, arabinose, glycerol, and lactose. To develop a K. marxianus DMKU3-1042 derivative strain suitable for genetic engineering, a uracil auxotroph was isolated and transformed with a linear DNA of the S. cerevisiae ScURA3 gene. Surprisingly, Ura+ transformants were easily obtained. By Southern blot hybridization, the linear ScURA3 DNA was found to have inserted randomly into the K. marxianus genome. Sequencing of one Lys− transformant confirmed the disruption of the KmLYS1 gene by the ScURA3 insertion. A PCR-amplified linear DNA lacking K. marxianus sequences but containing an Aspergillus α-amylase gene under the control of the ScTDH3 promoter together with an ScURA3 marker was subsequently used to transform K. marxianus DMKU3-1042 in order to obtain transformants expressing Aspergillus α-amylase. Our results demonstrate that K. marxianus DMKU3-1042 can be an alternative cost-effective bioethanol producer and a host for transformation with linear DNA by use of S. cerevisiae-based molecular genetic tools. PMID:18931291

  17. Effect of Evolutionary Adaption on Xylosidase Activity in Thermotolerant Yeast Isolates Kluyveromyces marxianus NIRE-K1 and NIRE-K3.

    PubMed

    Behera, Shuvashish; Sharma, Nilesh K; Arora, Richa; Kumar, Sachin

    2016-08-01

    Efficient use of xylose along with glucose is necessary for the economic production of lignocellulosic based biofuels. Xylose transporters play an important role in the microorganisms for efficient utilization of xylose. In the present study, a novel method has been developed for a rapid assay of xylose transport activity in the xylose-utilizing isolates and other known yeasts. An assay was conducted to compare the activity of β-xylosidase using p-nitrophenyl-β-D-xylopyranoside (pNPX) in the intact, intracellular, and extracellular yeasts cells showing xylose transporter. Saccharomyces cerevisiae (MTCC 170) showed no xylosidase activity, while little growth was observed in the xylose-containing medium. Although other yeasts, i.e., Kluyveromyces marxianus NIRE-K1 (MTCC 5933), K. marxianus NIRE-K3 (MTCC 5934), and Candida tropicalis (MTCC 230), showed xylosidase activity in intact, intracellular, and extracellular culture. The xylosidase activity in intact cell was higher than that of extracellular and intracellular activity in all the yeast cells. The enzyme activity was higher in case of K. marxianus NIRE-K1 and K. marxianus NIRE-K3 rather than the C. tropicalis. Further, better xylosidase activity was observed in adapted K. marxianus cells which were 2.79-28.46 % higher than that of native (non-adapted) strains, which indicates the significant improvement in xylose transportation.

  18. Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae.

    PubMed

    Yarimizu, Tohru; Nonklang, Sanom; Nakamura, Junpei; Tokuda, Shuya; Nakagawa, Takaaki; Lorreungsil, Sasithorn; Sutthikhumpha, Surasit; Pukahuta, Charida; Kitagawa, Takao; Nakamura, Mikiko; Cha-Aim, Kamonchai; Limtong, Savitree; Hoshida, Hisashi; Akada, Rinji

    2013-12-01

    The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

  19. Development of mutated Kluyveromyces marxianus strains for ethanol production at elevated temperature from biomass hydrolysate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The yeast K. marxianus has advantages over the most commonly used industrial ethanologen, Saccharomyces cerevisiae, such as the ability to grow at 47°C, to produce ethanol at temperatures above 40°C, and to grow on a wide variety of substrates, including starch, sucrose, pectins, and cellulosic biom...

  20. Direct fermentation of raw starch using a Kluyveromyces marxianus strain that expresses glucoamylase and alpha-amylase to produce ethanol.

    PubMed

    Wang, Rongliang; Wang, Dongmei; Gao, Xiaolian; Hong, Jiong

    2014-01-01

    Raw starch and raw cassava tuber powder were directly and efficiently fermented at elevated temperatures to produce ethanol using the thermotolerant yeast Kluyveromyces marxianus that expresses α-amylase from Aspergillus oryzae as well as α-amylase and glucoamylase from Debaryomyces occidentalis. Among the constructed K. marxianus strains, YRL 009 had the highest efficiency in direct starch fermentation. Raw starch from corn, potato, cassava, or wheat can be fermented at temperatures higher than 40°C. At the optimal fermentation temperature 42°C, YRL 009 produced 66.52 g/L ethanol from 200 g/L cassava starch, which was the highest production among the selected raw starches. This production increased to 79.75 g/L ethanol with a 78.3% theoretical yield (with all cassava starch were consumed) from raw cassava starch at higher initial cell densities. Fermentation was also carried out at 45 and 48°C. By using 200 g/L raw cassava starch, 137.11 and 87.71 g/L sugar were consumed with 55.36 and 32.16 g/L ethanol produced, respectively. Furthermore, this strain could directly ferment 200 g/L nonsterile raw cassava tuber powder (containing 178.52 g/L cassava starch) without additional nutritional supplements to produce 69.73 g/L ethanol by consuming 166.07 g/L sugar at 42°C. YRL 009, which has consolidated bioprocessing ability, is the best strain for fermenting starches at elevated temperatures that has been reported to date.

  1. Non-homologous end joining-mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus.

    PubMed

    Hoshida, Hisashi; Murakami, Nobutada; Suzuki, Ayako; Tamura, Ryoko; Asakawa, Jun; Abdel-Banat, Babiker M A; Nonklang, Sanom; Nakamura, Mikiko; Akada, Rinji

    2014-01-01

    The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date.

  2. Optimizing promoters and secretory signal sequences for producing ethanol from inulin by recombinant Saccharomyces cerevisiae carrying Kluyveromyces marxianus inulinase.

    PubMed

    Hong, Soo-Jeong; Kim, Hyo Jin; Kim, Jin-Woo; Lee, Dae-Hee; Seo, Jin-Ho

    2015-02-01

    Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from Kluyveromyces marxianus (KmINU), with the ability of converting inulin to fructose, was introduced into S. cerevisiae D452-2. The inulinase gene was fused to three different types of promoter (GPD, PGK1, truncated HXT7) and secretory signal sequence (KmINU, MFα1, SUC2) to generate nine expression cassettes. The inulin fermentation performance of the nine transformants containing different promoter and signal sequence combinations for inulinase production were compared to select an optimized expression system for efficient inulin fermentation. Among the nine inulinase-producing transformants, the S. cerevisiae carrying the PGK1 promoter and MFα1 signal sequence (S. cerevisiae D452-2/p426PM) showed not only the highest specific KmINU activity, but also the best inulin fermentation capability. Finally, a batch fermentation of the selected S. cerevisiae D452-2/p426PM in a bioreactor with 188.2 g/L inulin was performed to produce 80.2 g/L ethanol with 0.43 g ethanol/g inulin of ethanol yield and 1.22 g/L h of ethanol productivity.

  3. The modeling of ethanol production by Kluyveromyces marxianus using whey as substrate in continuous A-Stat bioreactors.

    PubMed

    Gabardo, Sabrina; Pereira, Gabriela Feix; Rech, Rosane; Ayub, Marco Antônio Záchia

    2015-09-01

    We investigated the kinetics of whey bioconversion into ethanol by Kluyveromyces marxianus in continuous bioreactors using the "accelerostat technique" (A-stat). Cultivations using free and Ca-alginate immobilized cells were evaluated using two different acceleration rates (a). The kinetic profiles of these systems were modeled using four different unstructured models, differing in the expressions for the specific growth (μ) and substrate consumption rates (r s), taking into account substrate limitation and product inhibition. Experimental data showed that the dilution rate (D) directly affected cell physiology and metabolism. The specific growth rate followed the dilution rate (μ≈D) for the lowest acceleration rate (a = 0.0015 h(-2)), condition in which the highest ethanol yield (0.52 g g(-1)) was obtained. The highest acceleration rate (a = 0.00667 h(-2)) led to a lower ethanol yield (0.40 g g(-1)) in the system where free cells were used, whereas with immobilized cells ethanol yields increased by 23 % (0.49 g g(-1)). Among the evaluated models, Monod and Levenspiel combined with Ghose and Tyagi models were found to be more appropriate for describing the kinetics of whey bioconversion into ethanol. These results may be useful in scaling up the process for ethanol production from whey.

  4. Kluyveromyces marxianus and Saccharomyces boulardii Induce Distinct Levels of Dendritic Cell Cytokine Secretion and Significantly Different T Cell Responses In Vitro

    PubMed Central

    Smith, Ida M.; Baker, Adam; Christensen, Jeffrey E.; Boekhout, Teun; Frøkiær, Hanne; Arneborg, Nils; Jespersen, Lene

    2016-01-01

    Interactions between members of the intestinal microbiota and the mucosal immune system can significantly impact human health, and in this context, fungi and food-related yeasts are known to influence intestinal inflammation through direct interactions with specialized immune cells in vivo. The aim of the present study was to characterize the immune modulating properties of the food-related yeast Kluyveromyces marxianus in terms of adaptive immune responses indicating inflammation versus tolerance and to explore the mechanisms behind the observed responses. Benchmarking against a Saccharomyces boulardii strain with probiotic effects documented in clinical trials, we evaluated the ability of K. marxianus to modulate human dendritic cell (DC) function in vitro. Further, we assessed yeast induced DC modulation of naive T cells toward effector responses dominated by secretion of IFNγ and IL-17 versus induction of a Treg response characterized by robust IL-10 secretion. In addition, we blocked relevant DC surface receptors and investigated the stimulating properties of β-glucan containing yeast cell wall extracts. K. marxianus and S. boulardii induced distinct levels of DC cytokine secretion, primarily driven by Dectin-1 recognition of β-glucan components in their cell walls. Upon co-incubation of yeast exposed DCs and naive T cells, S. boulardii induced a potent IFNγ response indicating TH1 mobilization. In contrast, K. marxianus induced a response dominated by Foxp3+ Treg cells, a characteristic that may benefit human health in conditions characterized by excessive inflammation and positions K. marxianus as a strong candidate for further development as a novel yeast probiotic. PMID:27898740

  5. Increased ethanol production with UV-C mutagenized Kluyveromyces marxianus capable of anaerobic growth at elevated temperature on pentose and hexose sugars using fermentation strategies with corn pericarp hydrolysates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several novel Kluyveromyces marxianus strains were obtained by irradiation with UV-C (UV-C 234nm) to achieve an 80% mortality rate. The surviving cells were subsequently grown anaerobically for 5 months at 46C and resulted in two mutagenized strains that were able to grow anaerobically at elevated ...

  6. Ethanol production from Jerusalem artichoke tubers at high temperature by newly isolated thermotolerant inulin-utilizing yeast Kluyveromyces marxianus using consolidated bioprocessing.

    PubMed

    Charoensopharat, Kanlayani; Thanonkeo, Pornthap; Thanonkeo, Sudarat; Yamada, Mamoru

    2015-07-01

    Thermotolerant inulin-utilizing yeast strains were successfully isolated in this study. Among the isolated strains, Kluyveromyces marxianus DBKKU Y-102 was found to be the most effective strain for direct ethanol fermentation at high temperature from fresh Jerusalem artichoke (JA) tubers without inulin hydrolysis under consolidated bioprocessing (CBP). The maximum ethanol concentrations produced by this strain under the optimum culture conditions were 104.83 and 97.46 g L(-1) at 37 and 40 °C, respectively. Data from this study clearly demonstrated that the use of thermotolerant inulin-utilizing yeast K. marxianus for ethanol production from fresh JA tubers in the CBP process not only provided high levels of ethanol, but also could eliminate the addition of external enzyme for inulin hydrolysis, which might lead to the reduction of operating costs. The expression of genes involved in carbohydrate metabolism in K. marxianus DBKKU Y-102 during ethanol fermentation was investigated by real-time RT-PCR, and the results revealed that expression levels were distinctive depending on the growth phase and growth conditions. However, among the genes tested, adh4 and tdh2 were highly expressed under high temperature conditions in both exponential- and stationary-growth phases, suggesting that these genes might play a crucial role in acquiring thermotolerance ability in this organism under stress conditions.

  7. VNTR fingerprinting of Kluyveromyces marxianus strains WT, 7-1, and 8-1 by using different primer types to give best results in PCR and on electrophorese gel in order to find differentiation of the DNA of the yeast strains.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using mutagenized Kluyveromyces marxianus strains (WT, 7-1, 8-1) we wish to find out the variable numbered tandem repeats (VNTR) of each of the DNA strains from the different mutagenized K. marxianus strains. To do this we used Phusion HF Buffer Pack to try and give a clear picture of the VNTR by u...

  8. Lactase production by solid-state cultivation of Kluyveromyces marxianus CDBBL 278 on an inert support: effect of inoculum, buffer, and nitrogen source.

    PubMed

    Tovar-Castro, Luz; García-Garibay, Mariano; Saucedo-Castañeda, Gerardo

    2008-12-01

    A study was carried out to select the conditions for cultivation of Kluyveromyces marxianus CDBBL 278 in solid-state culture (SSC) using polyurethane foam (PUF) as an inert support. PUF was impregnated with culture media containing lactose (50 g/L) as the carbon and energy source. Evaluation of culture parameters during different growth phases was carried out by respirometry. The effect of inoculum level, buffer capacity of the medium, and nitrogen source upon the yield of biomass on lactose (Yx/s) and production of lactase and inulinase was investigated. The highest lactase titre was achieved with an inoculum level of 1 x 10(7) cells per gram of wet matter (gwm) and 20% of the total nitrogen source provided as urea. The best biomass yield (0.37) was obtained when less than 40% of the total nitrogen was provided as urea. Using potassium phosphate allowed 90% substrate consumption in 30 h. In the best conditions, intracellular lactase and extracellular inulinase activities of 1147.7 IU/gX and 241.6 IU/gX were obtained, respectively, with a lag phase of 13.8 h and a rate of respiratory activity (microCO2) of 0.23 +/- 0.01 h(-1). To our knowledge, this is the first report on lactase production by K. marxianus CDBBL 278 in SSC. This study gives basic information about biomass yield and enzyme production using lactose as the sole carbon source in SSC on an inert support.

  9. High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources.

    PubMed

    Löbs, Ann-Kathrin; Lin, Jyun-Liang; Cook, Megan; Wheeldon, Ian

    2016-10-01

    Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z-factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.

  10. Growth and ethanol fermentation ability on hexose and pentose sugars and glucose effect under various conditions in thermotolerant yeast Kluyveromyces marxianus.

    PubMed

    Rodrussamee, Nadchanok; Lertwattanasakul, Noppon; Hirata, Katsushi; Suprayogi; Limtong, Savitree; Kosaka, Tomoyuki; Yamada, Mamoru

    2011-05-01

    Ethanol fermentation ability of the thermotolerant yeast Kluyveromyces marxianus, which is able to utilize various sugars including glucose, mannose, galactose, xylose, and arabinose, was examined under shaking and static conditions at high temperatures. The yeast was found to produce ethanol from all of these sugars except for arabinose under a shaking condition but only from hexose sugars under a static condition. Growth and sugar utilization rate under a static condition were slower than those under a shaking condition, but maximum ethanol yield was slightly higher. Even at 40°C, a level of ethanol production similar to that at 30°C was observed except for galactose under a static condition. Glucose repression on utilization of other sugars was observed, and it was more evident at elevated temperatures. Consistent results were obtained by the addition of 2-deoxyglucose. The glucose effect was further examined at a transcription level, and it was found that KmGAL1 for galactokinase and KmXYL1 for xylose reductase for galactose and xylose/arabinose utilization, respectively, were repressed by glucose at low and high temperatures, but KmHXK2 for hexokinase was not repressed. We discuss the possible mechanism of glucose repression and the potential for utilization of K. marxianus in high-temperature fermentation with mixed sugars containing glucose.

  11. Differential RNA-seq, Multi-Network Analysis and Metabolic Regulation Analysis of Kluyveromyces marxianus Reveals a Compartmentalised Response to Xylose

    PubMed Central

    Schabort, Du Toit W. P.; Letebele, Precious K.; Steyn, Laurinda; Kilian, Stephanus G.; du Preez, James C.

    2016-01-01

    We investigated the transcriptomic response of a new strain of the yeast Kluyveromyces marxianus, in glucose and xylose media using RNA-seq. The data were explored in a number of innovative ways using a variety of networks types, pathway maps, enrichment statistics, reporter metabolites and a flux simulation model, revealing different aspects of the genome-scale response in an integrative systems biology manner. The importance of the subcellular localisation in the transcriptomic response is emphasised here, revealing new insights. As was previously reported by others using a rich medium, we show that peroxisomal fatty acid catabolism was dramatically up-regulated in a defined xylose mineral medium without fatty acids, along with mechanisms to activate fatty acids and transfer products of β-oxidation to the mitochondria. Notably, we observed a strong up-regulation of the 2-methylcitrate pathway, supporting capacity for odd-chain fatty acid catabolism. Next we asked which pathways would respond to the additional requirement for NADPH for xylose utilisation, and rationalised the unexpected results using simulations with Flux Balance Analysis. On a fundamental level, we investigated the contribution of the hierarchical and metabolic regulation levels to the regulation of metabolic fluxes. Metabolic regulation analysis suggested that genetic level regulation plays a major role in regulating metabolic fluxes in adaptation to xylose, even for the high capacity reactions, which is unexpected. In addition, isozyme switching may play an important role in re-routing of metabolic fluxes in subcellular compartments in K. marxianus. PMID:27315089

  12. Simultaneous fermentation of glucose and xylose at elevated temperatures co-produces ethanol and xylitol through overexpression of a xylose-specific transporter in engineered Kluyveromyces marxianus.

    PubMed

    Zhang, Biao; Zhang, Jia; Wang, Dongmei; Han, Ruixiang; Ding, Rui; Gao, Xiaolian; Sun, Lianhong; Hong, Jiong

    2016-09-01

    Engineered Kluyveromyces marxianus strains were constructed through over-expression of various transporters for simultaneous co-fermentation of glucose and xylose. The glucose was converted into ethanol, whereas xylose was converted into xylitol which has higher value than ethanol. Over-expressing xylose-specific transporter ScGAL2-N376F mutant enabled yeast to co-ferment glucose and xylose and the co-fermentation ability was obviously improved through increasing ScGAL2-N376F expression. The production of glycerol was blocked and acetate production was reduced by disrupting gene KmGPD1. The obtained K. marxianus YZJ119 utilized 120g/L glucose and 60g/L xylose simultaneously and produced 50.10g/L ethanol and 55.88g/L xylitol at 42°C. The yield of xylitol from consumed xylose was over 98% (0.99g/g). Through simultaneous saccharification and co-fermentation at 42°C, YZJ119 produced a maximal concentration of 44.58g/L ethanol and 32.03g/L xylitol or 29.82g/L ethanol and 31.72g/L xylitol, respectively, from detoxified or non-detoxified diluted acid pretreated corncob.

  13. Comparison of a pectinolytic extract of Kluyveromyces marxianus and a commercial enzyme preparation in the production of Ives (Vitis labrusca) grape juice.

    PubMed

    Piemolini-Barreto, Luciani Tatsch; Antônio, Regina Vasconcellos; Echeverrigaray, Sergio

    2015-05-01

    This study analyses the effect of the crude enzymatic extract produced by Kluyveromyces marxianus (EEB) in the maceration and clarification of juice produced from Ives (Vitis labrusca) grapes compared to the commercial enzyme preparation Pectinex(®)Ultra Color (PEC). Treatments were conducted with a total pectinolytic activity of 1 U/mL of fruit juice, at 40 °C, for 60 min. After the enzymatic treatment, the juices were evaluated with respect to yield, viscosity, and degree of clarification, as well as the effect of the enzymes on polyphenol concentration, anthocyanins, and juice color. The results showed that both EEB and PEC increase yield, reduce viscosity and contribute to the clarification of grape juice. After enzyme treatment with the EEB preparation, the extraction yield increased 28.02 % and decreased 50.70 % in viscosity during the maceration of the pulp. During the juice production process clarification increased 11.91 %. With PEC, higher values for these parameters: 42.36, 63.20, and 26.81 % respectively, were achieved. The addition of EEB resulted in grape juice with better color intensity and extraction of phenolic compounds and anthocyanins. Considering all comparison criteria, the enzymatic extract of K. marxianus NRRL-Y-7571 can potentially be used in the production of juice.

  14. Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production.

    PubMed

    López-Alvarez, Arnoldo; Díaz-Pérez, Alma Laura; Sosa-Aguirre, Carlos; Macías-Rodríguez, Lourdes; Campos-García, Jesús

    2012-05-01

    In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the baker's yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 baker's yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae baker's yeast used in tequila production.

  15. Novel technology development through thermal drying of encapsulated Kluyveromyces marxianus in micro- and nano-tubular cellulose in lactose fermentation and its evaluation for food production.

    PubMed

    Papapostolou, Harris; Servetas, Yiannis; Bosnea, Loulouda A; Kanellaki, Maria; Koutinas, Athanasios A

    2012-12-01

    A novel technology development based on the production of a low-cost starter culture for ripening of cheeses and baking is reported in the present study. The starter culture comprises thermally dried cells of Kluyveromyces marxianus encapsulated in micro- and nano-tubular cellulose. For production of a low-cost and effective biocatalyst, whey was used as raw material for biomass production and thermal drying methods (convective, conventional, and vacuum) were applied and evaluated at drying temperatures ranging from 35 to 60 °C. The effect of drying temperature of biocatalysts on fermentability of lactose and whey was evaluated. Storage stability and suitability of biocatalysts as a commercial starter cultures was also assessed and evaluated. All thermally dried biocatalysts were found to be active in lactose and whey fermentation. In all cases, there was sugar conversion ranging from 92 to 100 %, ethanol concentration of up to 1.47 % (v/v), and lactic acid concentrations ranged from 4.1 to 5.5 g/l. However, convective drying of the encapsulated cells of K. marxianus in micro- and nano-tubular cellulose was faster and a more effective drying method while drying at 42 °C appear to be the best drying temperature in terms of cell activity, ethanol, and lactic acid formation. Storage of the biocatalysts for 3 months at 4 °C proved maintenance of its activity even though fermentation times increased by 50-100 % compared with the fresh dried ones.

  16. Effect of temperature and pH on ethanol production by free and immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract

    SciTech Connect

    Bajpai, P.; Margaritis, A.

    1987-01-01

    The effect of temperature and pH on the kinetics of ethanol production by free and calcium alginate immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract was investigated. With the free cells, the ethanol and biomass yields were relatively constant over the temperature range 25-35 degrees C, but dropped sharply beyond 35 degrees C. Other kinetic parameters, specific growth rate, specific ethanol production rate, and specific total sugar uptake rate were maximum at 35 degrees C. However, with the immobilized cells, ethanol yield remained almost constant in the temperatue range 25-45 degrees C, and the specific ethanol production rate and specific total sugar uptake rate attained their maximum values at 40 degrees C. For the pH range between 3 and 7, the free-cell optimum for growth and product formation was found to be circa pH 5. At this pH, the specific growth rate was 0.35/h and specific ethanol production rate was 2.83 g/g/h. At values higher or lower than pH 5, a sharp decrease in specific ethanol production rate as well as specific growth rate was observed. In comparison, the immobilized cells showed a broad optimum pH profile. The best ethanol production rates were observed between pH 4 and 6. (Refs. 22).

  17. Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant beta-glucosidase from synthetic medium by Kluyveromyces marxianus.

    PubMed

    Rajoka, M I; Khan, Samia; Latif, Farooq; Shahid, Riaz

    2004-05-01

    The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on beta-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and beta-glucosidase production rate. The highest product yield, specific product yield, and productivity of beta-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved beta-glucosidase production measured as product yield (YP/S) and volumetric productivity (QP) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L.h]) of beta-glucosidase occurred on cellobiose in the presence of CSL at 35 degrees C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of beta-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60 degrees C and may find application in some industrial processes.

  18. Identification of hexose kinase genes in Kluyveromyces marxianus and thermo-tolerant one step producing glucose-free fructose strain construction

    PubMed Central

    Zhang, Guorong; Lu, Min; Wang, Jichao; Wang, Dongmei; Gao, Xiaolian; Hong, Jiong

    2017-01-01

    In yeast, the hexose assimilation is started at hexose phosphorylation. However, in Kluyveromyces marxianus, the hexokinase (HXK) and glucokinase (GLK) genes were not identified by experiments. Meanwhile, the glucose-free fructose product requires more cost-efficient method. In this study, the KmHXK1 and KmGLK1 genes were functionally identified through gene disruption, over-expression and recombinant enzymes characterization. Both glucose and fructose assimilation ability decreased significantly in KmHXK1 disrupted strain YLM001, however, this ability was not changed obviously in KmGLK1 disrupted strain YLM002. When over-expressing KmGLK1 in YLM001, only the glucose assimilation ability was recovered in obtained strain (YLM005). The kinetic constant analysis of recombinant enzymes also proved that KmHXK1 could phosphorylate glucose (Vmax 553.01 U/mg, Km 0.83 mM) and fructose (Vmax 609.82 U/mg, Km 0.52 mM), and KmGLK1 only phosphorylate glucose with a Vmax of 0.73 U/mg and a Km 4.09 mM. A thermo-tolerant strain YGR003 which produced glucose-free fructose from Jerusalem artichoke tuber in one step was constructed based on the obtained information. The highest production and fastest productivity were 234.44 g/L and 10.26 g/L/h, respectively, which were several folds of the results in previous reports. PMID:28338054

  19. Search for nucleon decay using the IMB-3 detector

    SciTech Connect

    McGrew, C.; Breault, J.L.; Gajewski, W.; Halverson, P.G.; Kropp, W.R.; Price, L.R.; Reines, F.; Schultz, J.; Sobel, H.W.; Becker-Szendy, R.; Dye, S.T.; Learned, J.G.; Matsuno, S.; McGrath, G.; Bratton, C.B.; Cady, D.R.; LoSecco, J.M.; Casper, D.; Stone, J.L.; Sulak, L.R.; Ganezer, K.S.; Goldhaber, M.; Haines, T.J.; Miller, R.; Kielczewska, D.; Matthews, J.; Sinclair, D.; van der Velde, J.C.; Svoboda, R.

    1999-03-01

    The IMB-3 experiment was a large water Cherenkov ring imaging detector with a fiducial mass of 3.3 kton. During a 7.6-kton-year exposure ({approximately}4.6{times}10{sup 33}thinspnucleonthinspyr) 935 contained events were observed. The observed rate and characteristics are consistent with the expected backgrounds from atmospheric neutrinos. Lower limits on the nucleon lifetime are set for a wide variety of proposed decay modes. {copyright} {ital 1999} {ital The American Physical Society}

  20. Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on L-arabinose and D-xylose.

    PubMed

    Knoshaug, Eric P; Vidgren, Virve; Magalhães, Frederico; Jarvis, Eric E; Franden, Mary Ann; Zhang, Min; Singh, Arjun

    2015-10-01

    Genes encoding L-arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on L-arabinose was dependent on a functioning L-arabinose transporter, or by screening a differential display library, respectively. These transporters also transport D-xylose and were designated KmAXT1 (arabinose-xylose transporter) and PgAXT1, respectively. Transport assays using L-arabinose showed that KmAxt1p has K(m) 263 mM and V(max) 57 nM/mg/min, and PgAxt1p has K(m) 0.13 mM and V(max) 18 nM/mg/min. Glucose, galactose and xylose significantly inhibit L-arabinose transport by both transporters. Transport assays using D-xylose showed that KmAxt1p has K(m) 27 mM and V(max) 3.8 nM/mg/min, and PgAxt1p has K(m) 65 mM and V(max) 8.7 nM/mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K(m) 371 mM and V(max) 341 nM/mg/min for L-arabinose, and K(m) 25 mM and V(max) 76 nM/mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both L-arabinose and D-xylose, one scenario for the complete usage of biomass-derived pentose sugars would require only the low-affinity, high-throughput transporter Gal2p and one additional high-affinity general pentose transporter, rather than dedicated D-xylose or L-arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering.

  1. Particle Type Identification with the Kek and IMB3 Detectors

    NASA Astrophysics Data System (ADS)

    Breault, John Louis, IV

    1997-12-01

    One of the outstanding problems in particle physics today is the discrepancy between the predicted and observed ratios of non-showering particles to all particles that are observed in earth-based detectors. These particles are produced by electron and muon neutrino interactions. The IMB3 detector (1) recorded the ratio of non-showering particles to total particles (the 'non-showering fraction') as being 0.36 ± 0.02(stat) ± 0.02(syst) (see Ref. (3)). The IMB group's Monte Carlo simulation gives the expected ratio of 0.51 ± 0.01(stat) ± 0.05(syst). The analysis contained in this dissertation results in a value of 0.281 ± 0.022(stat) ± 0.050(syst).

  2. Measurement of atmospheric neutrino composition with the IMB-3 detector

    SciTech Connect

    Casper, D.; Becker-Szendy, R.; Bratton, C.B.; Cady, D.R.; Claus, R.; Dye, S.T.; Gajewski, W.; Goldhaber, M.; Haines, T.J.; Halverson, P.G.; Jones, T.W.; Kielczewska, D.; Kropp, W.R.; Learned, J.G.; LoSecco, J.M.; McGrew, C.; Matsuno, S.; Matthews, J.; Mudan, M.S.; Price, L.; Reines, F.; Schultz, J.; Sinclair, D.; Sobel, H.W.; Stone, J.L.; Sulak, L.R.; Svoboda, R.; Thornton, G.; van der Velde, J.C. The University of Michigan, Ann Arbor, Michigan 48109 Brookhaven National Laboratory, Upton, New York 11973 Boston University, Boston, Massachusetts 02215 The University of Hawaii, Honolulu, Hawaii 96822 University College, London, WC1E F6BT, United Kingdom Warsaw University, Warsaw, Poland Cleveland State University, Cleveland, Ohio 44115 The University of Notre Dame, Notre Dame, Indiana 46556 Lousiana State University, Baton Rouge, Lousisiana 70803 The University of Maryland, College Park, Maryland 20742)

    1991-05-20

    The atmospheric neutrino flux is measured using a 3.4-kt yr exposure of the IMB-3 detector. Single-ring events are classified as showering or nonshowering using the geometry of the {hacek C}erenkov pattern. A simulation of neutrino interactions and three models of atmospheric neutrino production are used to predict the composition of the sample. Showering-nonshowering character is strongly correlated with the flavor of the neutrino parent. In the lepton momentum range {ital p}{lt}1500 MeV/{ital c}, we find that nonshowering events comprise (41{plus minus}3{plus minus}2syst)% of the total. The fraction expected is (51{plus minus}5(syst))%.

  3. Preliminary results from IMB3 muon/electron identification tests at KEK

    SciTech Connect

    Bratton, C.B.; Breault, J.; Conner, Z.

    1995-09-01

    A test has been conducted at KEK, Japan using beams of electrons and muons in a 1 kiloton water Cherenkov detector instrumented with IMB3 phototubes and electronics to evaluate IMB`s algorithms for identifying electrons and muons. This identification is important because the IMB3 detector`s results on the atmospheric neutrino anomaly depend on the proper identification of the electrons and muons produced in neutrino charged-current interactions. Preliminary results are presented.

  4. a Search for Baryon Non-Conservation Using the IMB-3 Detector

    NASA Astrophysics Data System (ADS)

    McGrew, Clark Duane

    The IMB-3 Detector is a large water Cerenkov ring imaging detector with a fiducial mass of 3.3 kton. During a 7.6 kton-year exposure (about 4.6times10 ^{33} nucleon-years) 935 contained events were observed. The observed rate and characteristics are consistent with the expected backgrounds from the atmospheric neutrino flux. Partial lifetime limits are set for a wide variety of proposed decay modes.

  5. Structure of glycosylated Cu/Zn-superoxide dismutase from Kluyveromyces yeast NBIMCC 1984

    NASA Astrophysics Data System (ADS)

    Dolashka-Angelova, Pavlina; Moshtanska, Vesela; Kujumdzieva, Anna; Atanasov, Boris; Petrova, Vencislava; Voelter, Wolfgang; Beeumen, Jozef Van

    2010-09-01

    The primary structure of Cu/Zn-superoxide dismutase from Kluyveromyces marxianus NBIMCC 1984 was elucidated by N-terminal sequence analysis of the intact protein and by determination of the amino acid sequences of tryptic peptides by MALDI-TOF-TOF tandem mass spectrometry. The molecular mass of one subunit of the homodimer SOD, containing 152 amino acid residues, was calculated to be 15858.3 Da while a value of 17096.63 Da was obtained by MALDI-TOF MS. This difference is explained by the presence of N-glycosylation of one linkage site, -Asn-Ile/Leu-Thr-, and a glycan chain with the structure Hex 5 GlcNAc 2. Glycosylation of K.marxianus superoxide dismutase is a post-translational modification. Recent developments in mass spectrometry have enabled detailed structural analyses of covalent modifications of proteins. Therefore, in this paper, we introduce a covalent modification of Cu/Zn-SOD from K. marxianus NBIMCC 1984, by analysis of the enzymatic liberated N-glycan from the enzyme using MALDI-TOF and tandem mass spectrometry on a Q-Trap mass spectrometer. This is the first report of the structure of the oligosaccharide of a naturally-glycosylated superoxide dismutase, determined by mass spectrometry.

  6. A biochemically structured model for ethanol fermentation by Kluyveromyces marxianus: A batch fermentation and kinetic study.

    PubMed

    Sansonetti, S; Hobley, T J; Calabrò, V; Villadsen, J; Sin, G

    2011-08-01

    Anaerobic batch fermentations of ricotta cheese whey (i.e. containing lactose) were performed under different operating conditions. Ethanol concentrations of ca. 22g L(-1) were found from whey containing ca. 44g L(-1) lactose, which corresponded to up to 95% of the theoretical ethanol yield within 15h. The experimental data could be explained by means of a simple knowledge-driven biochemically structured model that was built on bioenergetics principles applied to the metabolic pathways through which lactose is converted into major products. Use of the model showed that the observed concentrations of ethanol, lactose, biomass and glycerol during batch fermentation could be described within a ca. 6% deviation, as could the yield coefficients for biomass and ethanol produced on lactose. The model structure confirmed that the thermodynamics considerations on the stoichiometry of the system constrain the metabolic coefficients within a physically meaningful range thereby providing valuable and reliable insight into fermentation processes.

  7. Kluyveromyces lactis: An emerging tool in biotechnology.

    PubMed

    Spohner, Sebastian C; Schaum, Vivienne; Quitmann, Hendrich; Czermak, Peter

    2016-03-20

    Kluyveromyces lactis has emerged as one of the most important yeast species for research and industrial biotechnology. This Crabtree-negative species is suitable for the production of metabolites and heterologous proteins, and its ability to achieve high levels of protein secretion makes it an attractive alternative for industrial protein production. Since 1991, almost 100 recombinant proteins have been expressed in K. lactis, 20% of which have been produced in the last 2 years. This review provides an overview of the genetic modifications used to accomplish heterologous gene expression in K. lactis, as well as fermentation techniques, and recent examples of industrial proteins produced in this species.

  8. Cellulosic ethanol production on temperature-shift simultaneous saccharification and fermentation using the thermostable yeast Kluyveromyces marxianus CHY1612.

    PubMed

    Kang, Hyun-Woo; Kim, Yule; Kim, Seung-Wook; Choi, Gi-Wook

    2012-01-01

    In cellulosic ethanol production, use of simultaneous saccharification and fermentation (SSF) has been suggested as the favorable strategy to reduce process costs. Although SSF has many advantages, a significant discrepancy still exists between the appropriate temperature for saccharification (45-50 °C) and fermentation (30-35 °C). In the present study, the potential of temperature-shift as a tool for SSF optimization for bioethanol production from cellulosic biomass was examined. Cellulosic ethanol production of the temperature-shift SSF (TS-SSF) from 16 w/v% biomass increased from 22.2 g/L to 34.3 g/L following a temperature shift from 45 to 35 °C compared with the constant temperature of 45 °C. The glucose conversion yield and ethanol production yield in the TS-SSF were 89.3% and 90.6%, respectively. At higher biomass loading (18 w/v%), ethanol production increased to 40.2 g/L with temperature-shift time within 24 h. These results demonstrated that the temperature-shift process enhances the saccharification ratio and the ethanol production yield in SSF, and the temperature-shift time for TS-SSF process can be changed according to the fermentation condition within 24 h.

  9. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Lactase enzyme preparation from Kluyveromyces... Specific Substances Affirmed as GRAS § 184.1388 Lactase enzyme preparation from Kluyveromyces lactis. (a) This enzyme preparation is derived from the nonpathogenic, nontoxicogenic yeast Kluyveromyces...

  10. Genome-wide metabolic (re-) annotation of Kluyveromyces lactis

    PubMed Central

    2012-01-01

    Background Even before having its genome sequence published in 2004, Kluyveromyces lactis had long been considered a model organism for studies in genetics and physiology. Research on Kluyveromyces lactis is quite advanced and this yeast species is one of the few with which it is possible to perform formal genetic analysis. Nevertheless, until now, no complete metabolic functional annotation has been performed to the proteins encoded in the Kluyveromyces lactis genome. Results In this work, a new metabolic genome-wide functional re-annotation of the proteins encoded in the Kluyveromyces lactis genome was performed, resulting in the annotation of 1759 genes with metabolic functions, and the development of a methodology supported by merlin (software developed in-house). The new annotation includes novelties, such as the assignment of transporter superfamily numbers to genes identified as transporter proteins. Thus, the genes annotated with metabolic functions could be exclusively enzymatic (1410 genes), transporter proteins encoding genes (301 genes) or have both metabolic activities (48 genes). The new annotation produced by this work largely surpassed the Kluyveromyces lactis currently available annotations. A comparison with KEGG’s annotation revealed a match with 844 (~90%) of the genes annotated by KEGG, while adding 850 new gene annotations. Moreover, there are 32 genes with annotations different from KEGG. Conclusions The methodology developed throughout this work can be used to re-annotate any yeast or, with a little tweak of the reference organism, the proteins encoded in any sequenced genome. The new annotation provided by this study offers basic knowledge which might be useful for the scientific community working on this model yeast, because new functions have been identified for the so-called metabolic genes. Furthermore, it served as the basis for the reconstruction of a compartmentalized, genome-scale metabolic model of Kluyveromyces lactis, which is

  11. Lipid-Enhanced Ethanol Production by Kluyveromyces fragilis

    PubMed Central

    Janssens, Jacques H.; Burris, Neil; Woodward, Anne; Bailey, Richard B.

    1983-01-01

    The fermentation ability of a strain of Kluyveromyces fragilis, already selected for rapid lactose-fermenting capability, was improved dramatically by the addition of unsaturated fatty acids and ergosterol to the medium. The fermentation time of a 20% whey-lactose medium was decreased from over 90 h to less than 60 h. The lipids were shown to be taken up by the organism, and the effects on specific growth rate and biomass production were determined. PMID:16346208

  12. Lipid-enhanced ethanol production by Kluyveromyces fragilis

    SciTech Connect

    Janssens, J.H.; Burris, N.; Woodward, A.; Bailey, R.B.

    1983-02-01

    The fermentation ability of a strain of Kluyveromyces fragilis, already selected for rapid lactose-fermenting capability, was improved dramatically by the addition of unsaturated fatty acids and ergosterol to the medium. The fermentation time of a 20% whey-lactose medium was decreased from over 90 h to less than 60 h. The lipids were shown to be taken up by the organism, and the effects on specific growth rate and biomass production was determined.

  13. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Lactase enzyme preparation from Kluyveromyces... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1388 Lactase enzyme preparation from Kluyveromyces lactis. (a) This enzyme preparation is derived from the...

  14. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Lactase enzyme preparation from Kluyveromyces... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1388 Lactase enzyme preparation from Kluyveromyces lactis. (a) This enzyme preparation is derived from the...

  15. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Lactase enzyme preparation from Kluyveromyces... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1388 Lactase enzyme preparation from Kluyveromyces lactis. (a) This enzyme preparation is derived from the...

  16. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Lactase enzyme preparation from Kluyveromyces... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1388 Lactase enzyme preparation from Kluyveromyces lactis. (a) This enzyme preparation is derived from the...

  17. Melanin determination by high performance liquid chromatography (HPLC) for K. marxianus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ultraviolet light (UV) mutated K. marxianus was found to turn dark brown during a growth assay. This brown color was hypothesized to be melanin overproduction influenced by the UV exposure. Cell cultures were oxidized and HPLC analyzed to determine melanin concentrations. The resulting melanin con...

  18. Accumulation of selenium and changes in the activity of inulinase and catalase in the cells of Kluyveromyces marxianus on pulsed electric field treatment.

    PubMed

    Pankiewicz, Urszula; Jamroz, Jerzy

    2010-07-01

    Pulsed electric field (PEF) of 1Hz, 1.5 kV, and 1 ms increased the activities of catalase and inulinase over the whole range of applied Se concentrations compared with the non-treated cultures. A significant effect of selenium concentration (in the range of 5-14 microg/ml) on both intra- and extracellular enzyme activities was noted. At a Se concentration of 10 microg/ml, the activities of intra- and extracellular inulinases and extracellular catalase in the PEF-treated cultures reached the maximum of 71 U/g d.m., 46 U/g d.m., and approx. 8 U/ml, respectively. The maximum activity of intracellular catalase of approx. 6 U/ ml (with and without PEF) was recorded at 5 microg Se/ml. Further increasing of selenium concentration caused a decrease in the activity of the enzymes.

  19. Growth, ethanol production, and inulinase activity on various inulin substrates by mutant kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Economically important plants contain large amounts of inulin. Disposal of waste resulting from their processing presents environmental issues. Finding microorganisms capable of converting inulin waste to biofuel and valuable co-products in a biorefinery at the processing site would have significant...

  20. [Kluyveromyces blattae sp. n., a new multispored yeast for Blatta orientalis (author's transl)].

    PubMed

    Henninger, W; Windisch, S

    1976-08-01

    A new hitherto undescribed species of yeast of the genus Kluyveromyces has been isolated for the intestinal tract of the oriental cockroach (Blatta orientalis). A description of the new species including latin diagnosis is given.

  1. Electroinduced extraction of beta-galactosidase from Kluyveromyces lactis.

    PubMed

    Ganeva, V; Galutzov, B; Eynard, N; Teissié, J

    2001-08-01

    A new methodology for the extraction of beta-galactosidase from the yeast Kluyveromyces lactis was obtained by electropulsation. The application of a series of electric pulses (2 ms duration, 1 Hz frequency, and 4-4.5 kV/cm field strength) to fresh cells suspended in deionized water, followed by incubation in PBS, led to a spontaneous slow release of enzyme at a yield of 75-80% without any further treatment. Most of the enzyme was extracted within 8 h after electropulsation. This release was dependent on the growth phase. The specific activity of beta-galactosidase in the supernatant of pulsed cells was higher by a factor of 1.5-1.7 in comparison with crude extract.

  2. A Preliminary Study of Europium Uptake by Yeast Cells. The Case of Kluveromyces Marxianus

    NASA Astrophysics Data System (ADS)

    Anagnostopoulos, V.; Symeopoulos, B.

    2008-08-01

    The objective of the present work is an exploration of a cost effective recovery of lanthanides, either for minimizing the industrial processes losses, or for reasons related to Radioactive Waste Management. Specifically, the uptake of europium from aqueous solutions by Kluveromyces marxianus cells was studied. Moreover, this biotechnological approach turns out to be environmental friendly, considering that cells of Kluveromyces marxianus are readily available as wastes from food fermentation industries. Europium [152Eu+154Eu]-labelled solutions were used providing better accuracy and reproducibility of measurements, mainly in low concentration range. The effect of pH, contact time and europium initial concentration were investigated. Adsorption data were fitted to Langmuir and Freundlich sorption models and Scatchard plots were used to reveal the existence of at least two types of binding sites.

  3. A Preliminary Study of Europium Uptake by Yeast Cells. The Case of Kluveromyces Marxianus

    SciTech Connect

    Anagnostopoulos, V.; Symeopoulos, B.

    2008-08-14

    The objective of the present work is an exploration of a cost effective recovery of lanthanides, either for minimizing the industrial processes losses, or for reasons related to Radioactive Waste Management. Specifically, the uptake of europium from aqueous solutions by Kluveromyces marxianus cells was studied. Moreover, this biotechnological approach turns out to be environmental friendly, considering that cells of Kluveromyces marxianus are readily available as wastes from food fermentation industries. Europium [{sup 152}Eu+{sup 154}Eu]-labelled solutions were used providing better accuracy and reproducibility of measurements, mainly in low concentration range. The effect of pH, contact time and europium initial concentration were investigated. Adsorption data were fitted to Langmuir and Freundlich sorption models and Scatchard plots were used to reveal the existence of at least two types of binding sites.

  4. Genome wide distribution of illegitimate recombination events in Kluyveromyces lactis

    PubMed Central

    Kegel, Andreas; Martinez, Paula; Carter, Sidney D.; Åström, Stefan U.

    2006-01-01

    Illegitimate recombination (IR) is the process by which two DNA molecules not sharing homology to each other are joined. In Kluyveromyces lactis, integration of heterologous DNA occurred very frequently therefore constituting an excellent model organism to study IR. IR was completely dependent on the nonhomologous end-joining (NHEJ) pathway for DNA double strand break (DSB) repair and we detected no other pathways capable of mediating IR. NHEJ was very versatile, capable of repairing both blunt and non-complementary ends efficiently. Mapping the locations of genomic IR-events revealed target site preferences, in which intergenic regions (IGRs) and ribosomal DNA were overrepresented six-fold compared to open reading frames (ORFs). The IGR-events occurred predominantly within transcriptional regulatory regions. In a rad52 mutant strain IR still preferentially occurred at IGRs, indicating that DSBs in ORFs were not primarily repaired by homologous recombination (HR). Introduction of ectopic DSBs resulted in the efficient targeting of IR to these sites, strongly suggesting that IR occurred at spontaneous mitotic DSBs. The targeting efficiency was equal when ectopic breaks were introduced in an ORF or an IGR. We propose that spontaneous DSBs arise more frequently in transcriptional regulatory regions and in rDNA and such DSBs can be mapped by analyzing IR target sites. PMID:16549875

  5. Optimizing alcohol production from whey using computer technology. [Kluyveromyces fragilis

    SciTech Connect

    Zertuche, L.; Zall, R.R.

    1985-01-01

    This study was undertaken with the major goal of optimizing the ethanol production from whey using computer technology. To reach this goal, a mathematical model that would describe the fermentation and that could be used for the optimization was developed. Kluyveromyces fragilis was the microorganism used to ferment the lactose in the whey into ethanol. Preliminary studies showed that K. fragilis produced about 90% of the theoretical ethanol yield when grown in whey-complemented media. However, when this yeast is grown in nonsupplemented whey media, it does not produce more than 32% of that yield. Comparative batch fermentations of lactose and whey-complemented media showed that whey possibly contains enhancing components for yeast growth and ethanol production. To obtain the mathematical model, the one-to-one effect of the process variables (lactose and yeast extract concentrations, air flow rate, pH, and dilution rate) on the ethanol production were first investigated. Experiments on the pH effect showed that a decrease in pH from 7 to 4 produced an increase in ethanol concentration from 16.5 to 26.5 g/L (50 g/L initial lactose). The results obtained from modeling of the continuous fermentation using the previously listed variables showed that air flow rate, pH, and dilution rate were the process variables that most influence the production of ethanol.

  6. Genome wide distribution of illegitimate recombination events in Kluyveromyces lactis.

    PubMed

    Kegel, Andreas; Martinez, Paula; Carter, Sidney D; Aström, Stefan U

    2006-01-01

    Illegitimate recombination (IR) is the process by which two DNA molecules not sharing homology to each other are joined. In Kluyveromyces lactis, integration of heterologous DNA occurred very frequently therefore constituting an excellent model organism to study IR. IR was completely dependent on the nonhomologous end-joining (NHEJ) pathway for DNA double strand break (DSB) repair and we detected no other pathways capable of mediating IR. NHEJ was very versatile, capable of repairing both blunt and non-complementary ends efficiently. Mapping the locations of genomic IR-events revealed target site preferences, in which intergenic regions (IGRs) and ribosomal DNA were overrepresented six-fold compared to open reading frames (ORFs). The IGR-events occurred predominantly within transcriptional regulatory regions. In a rad52 mutant strain IR still preferentially occurred at IGRs, indicating that DSBs in ORFs were not primarily repaired by homologous recombination (HR). Introduction of ectopic DSBs resulted in the efficient targeting of IR to these sites, strongly suggesting that IR occurred at spontaneous mitotic DSBs. The targeting efficiency was equal when ectopic breaks were introduced in an ORF or an IGR. We propose that spontaneous DSBs arise more frequently in transcriptional regulatory regions and in rDNA and such DSBs can be mapped by analyzing IR target sites.

  7. RAG4 gene encodes a glucose sensor in Kluyveromyces lactis.

    PubMed Central

    Betina, S; Goffrini, P; Ferrero, I; Wésolowski-Louvel, M

    2001-01-01

    The rag4 mutant of Kluyveromyces lactis was previously isolated as a fermentation-deficient mutant, in which transcription of the major glucose transporter gene RAG1 was affected. The wild-type RAG4 was cloned by complementation of the rag4 mutation and found to encode a protein homologous to Snf3 and Rgt2 of Saccharomyces cerevisiae. These two proteins are thought to be sensors of low and high concentrations of glucose, respectively. Rag4, like Snf3 and Rgt2, is predicted to have the transmembrane structure of sugar transporter family proteins as well as a long C-terminal cytoplasmic tail possessing a characteristic 25-amino-acid sequence. Rag4 may therefore be expected to have a glucose-sensing function. However, the rag4 mutation was fully complemented by one copy of either SNF3 or RGT2. Since K. lactis appears to have no other genes of the SNF3/RGT2 type, we suggest that Rag4 of K. lactis may have a dual function of signaling high and low concentrations of glucose. In rag4 mutants, glucose repression of several inducible enzymes is abolished. PMID:11404320

  8. Impact on growth and aflatoxin B1 accumulation by Kluyveromyces isolates at different water activity conditions.

    PubMed

    Penna, Mariángeles La; Etcheverry, Miriam

    2006-11-01

    This study showed the impact on germination, mycelial growth and aflatoxin B(1) accumulation when interacting Aspergillus aflatoxigenic strains with Kluyveromyces isolates and the effect of water activity on this relationship. Isolates Y(14) and Y(16) reduced the percentage of germination of all Aspergillus strains and decrease germ tube elongation rate at majority of water activity assayed. Similarly they produced an increase of germination lag phase and lag phase of growth beside decreased growth rate of all Aspergillus strains. At water activities 0.994, 0.982, 0.955 and 0.937, no aflatoxins were produced in paired cultures with isolates Y(25,) Y(22), Y(16), and Y(14), and Kluyveromyces isolates Y(14) and Y(16) impact both growth and aflatoxin accumulation at wide range of water activity.

  9. Crystallization and preliminary X-ray crystallographic analysis of β-galactosidase from Kluyveromyces lactis

    PubMed Central

    Pereira-Rodríguez, Ángel; Fernández-Leiro, Rafael; González Siso, M. Isabel; Cerdán, M. Esperanza; Becerra, Manuel; Sanz-Aparicio, Julia

    2010-01-01

    β-Galactosidase from Kluyveromyces lactis catalyses the hydrolysis of the β-­galactosidic linkage in lactose. Owing to its many industrial applications, the biotechnological potential of this enzyme is substantial. This protein has been expressed in yeast and purified for crystallization trials. However, optimization of the best crystallization conditions yielded crystals with poor diffraction quality that precluded further structural studies. Finally, the crystal quality was improved using the streak-seeding technique and a complete diffraction data set was collected at 2.8 Å resolution. PMID:20208165

  10. Yeast on the milky way: genetics, physiology and biotechnology of Kluyveromyces lactis.

    PubMed

    Rodicio, Rosaura; Heinisch, Jürgen J

    2013-05-01

    The milk yeast Kluyveromyces lactis has a life cycle similar to that of Saccharomyces cerevisiae and can be employed as a model eukaryote using classical genetics, such as the combination of desired traits, by crossing and tetrad analysis. Likewise, a growing set of vectors, marker cassettes and tags for fluorescence microscopy are available for manipulation by genetic engineering and investigating its basic cell biology. We here summarize these applications, as well as the current knowledge regarding its central metabolism, glucose and extracellular stress signalling pathways. A short overview on the biotechnological potential of K. lactis concludes this review.

  11. The utilization of 4-aminobutylphosphonate as sole nitrogen source by a strain of Kluyveromyces fragilis.

    PubMed

    Ternan, N G; McMullan, G

    2000-03-15

    A strain of the yeast Kluyveromyces fragilis was screened for its ability to utilize a range of synthetic and natural organophosphonate compounds as the sole source of phosphorus, nitrogen or carbon. Only 4-aminobutylphosphonate was utilized as sole nitrogen source with protein yields increasing proportionally with substrate concentrations up to 10 mM. No 4-aminobutylphosphonate metabolizing enzyme activity was detectable in cell-free extracts prepared from K. fragilis pregrown on 2.5 mM 4-aminobutylphosphonate. None of the organophosphonates tested served as a source of carbon or phosphorus for K. fragilis.

  12. Kluyveromyces aestuarii, a potential environmental quality indicator yeast for mangroves in the State of Rio de Janeiro, Brazil

    PubMed Central

    Araujo, F.V.; Hagler, A. N.

    2011-01-01

    Kluyveromyces aestuarii was found in sediments from 7 of 8 mangroves in Rio de Janeiro; and absent only at one site with heavy plastic bag pollution. Its presence suggests influence in other habitats from a mangrove and its absence in a mangrove suggests some non- fecal pollution or other habitat alteration. PMID:24031711

  13. [Production and partial characterization of beta-galactosidase from Kluyveromyces lactis grown in deproteinized whey].

    PubMed

    Ramírez Matheus, Alejandra O; Rivas, Nilo

    2003-06-01

    The purpose of this work was to optimize the beta-galactosidase production by Kluyveromyces lactis, applying the Surface Response Methodology (SRM) and using deproteinized whey as fermentation medium. An Orthogonal Central Compound Design (OCCD) was used without repetition, with four factors: temperature, pH, agitation speed and fermentation time. Then, enzyme activity (U/ml) as response variable was used. Thirty trials in twenty-five treatments, with six repetitions at the central point, were carried out, in a New Brunswick Bioflo 2000 fermentor with a volume of 2 liters. The deproteinized whey obtained by thermocoagulation was chemically analyzed. The results were: moisture 93.83%, total solids 6.17%, protein 0.44%, lactose 4.85%, acidity 0.43% and pH 4.58. The best conditions in the enzyme production were: temperature 30.3 degrees C, pH 4.68, agitation speed 191 r.p.m. and fermentation time 18.5 h. with an enzyme production of 8.3 U/ml. The degree of purification obtained was 7.4 times and the yield was 50.8%. The purified enzyme had an optimum temperature of 60 degrees C and a pH of 6.2. This work shows that the yeast Kluyveromyces lactis grown in deproteinized whey is able to produce the enzyme beta-galactosidase and SRM can be used in the fermentology processes, specifically in determining the best suitable operation conditions.

  14. Alcohol and single-cell protein production by Kluyveromyces in concentrated whey permeates with reduced ash

    SciTech Connect

    Mahmoud, M.M.; Kosikowski, F.V.

    1982-01-01

    Five Kluyveromyces yeasts were grown in concentrated whey permeates under aerobic and anaerobic conditions to produce single-cell protein and ethanol. K. fragilis NRRL Y2415 produced the highest yield of alcohol, 9.1%, and K. bulgaricus ATCC 1605 gave the highest yield of biomass, 13.5 mg/mL. High ash, apparently through Na and K effects, inhibited production of biomass and alcohol. A 0.77% ash was optimum. Lactose utilization was more rapid under aerobic than anaerobic conditions. (NH/sub 4/)/sub 2/SO/sub 4/ and urea supplementation were without effect on yeast growth or were slightly inhibitory. A 1% peptone inclusion gave the highest biomass yield with minimum alcohol production.

  15. The yeast Kluyveromyces lactis as an efficient host for heterologous gene expression.

    PubMed

    Swinkels, B W; van Ooyen, A J; Bonekamp, F J

    Several different yeast species have been developed into systems for efficient heterologous gene expression. In this paper we review foreign gene expression in the dairy yeast Kluyveromyces lactis. This yeast presents several advantageous properties in comparison to other yeast species. These include its impressive secretory capacities, its excellent fermentation characteristics on large scale, its food grade status and the availability of both episomal and integrative expression vectors. Moreover, in contrast to the methylotrophic yeasts that are frequently used for the expression of foreign genes, K. lactis does not require explosion-proof fermentation equipment. Here, we present an overview of the available tools for heterologous gene expression in K. lactis (available promoters, vector systems, etc). Also, the production of prochymosin, human serum albumin and pancreatic phospholipase by K. lactis is discussed in more detail, and used to rate the achievements of K. lactis with respect to other micro-organisms in which these proteins have been produced.

  16. Inducible Amplification of Gene Copy Number and Heterologous Protein Production in the Yeast Kluyveromyces lactis

    PubMed Central

    Morlino, Giovanni B.; Tizzani, Lorenza; Fleer, Reinhard; Frontali, Laura; Bianchi, Michele M.

    1999-01-01

    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1β, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system. PMID:10543790

  17. Inducible amplification of gene copy number and heterologous protein production in the yeast Kluyveromyces lactis.

    PubMed

    Morlino, G B; Tizzani, L; Fleer, R; Frontali, L; Bianchi, M M

    1999-11-01

    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1beta, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.

  18. Attraction of Coffee Bean Weevil, Araecerus fasciculatus, to Volatiles from the Industrial Yeast Kluyveromyces lactis.

    PubMed

    Yang, Shuai; Mei, Xiang-Dong; Zhang, Xiao-Fang; Li, Yao-Fa; She, Dongmei; Zhang, Tao; Ning, Jun

    2017-02-01

    The coffee bean weevil (CBW), Araecerus fasciculatus (De Geer, 1775) (Coleoptera: Anthribidae) is an important pest of stored products such as grains, coffee beans, cassava, and traditional Chinese medicine materials. In China, CBW causes large losses of Daqu, a traditional Chinese liquor fermentation starter, and, unfortunately, the use of conventional insecticides against CBW is not suitable in Daqu storage. We found CBW to be highly attracted to fermenting yeast cultures, such as Kluyveromyces lactis. Eight volatile compounds, produced by fermenting cultures and not by sterile samples, were identified by gas chromatography coupled with mass spectrometry. Five of these substances elicited significant responses in Y-tube behavioral bioassays. Field trapping experiments revealed 2-phenylethanol and 2-phenylethyl acetate to be crucial for attraction of CBW. Results show that yeast volatiles play an important role in host location, and that 2-phenylethanol and 2-phenylethyl acetate could be utilized as potential attractants in monitoring and control systems against this important pest.

  19. Kinetics and regulation of lactose transport and metabolism in Kluyveromyces lactis JA6.

    PubMed

    Santos, A M; Silveira, W B; Fietto, L G; Brandão, R L; Castro, I M

    2014-07-01

    Kluyveromyces lactis strains are able to assimilate lactose. They have been used industrially to eliminate this sugar from cheese whey and in other industrial products. In this study, we investigated specific features and the kinetic parameters of the lactose transport system in K. lactis JA6. In lactose grown cells, lactose was transported by a system transport with a half-saturation constant (K s) of 1.49 ± 0.38 mM and a maximum velocity (V max) of 0.96 ± 0.12 mmol. (g dry weight)(-1) h(-1) for lactose. The transport system was constitutive and energy-dependent. Results obtained by different approaches showed that the lactose transport system was regulated by glucose at the transcriptional level and by glucose and other sugars at a post-translational level. In K. lactis JA6, galactose metabolization was under glucose control. These findings indicated that the regulation of lactose-galactose regulon in K. lactis was similar to the regulation of galactose regulon in Saccharomyces cerevisiae.

  20. Ethanol tolerance and membrane fatty acid adaptation in adh multiple and null mutants of Kluyveromyces lactis.

    PubMed

    Heipieper, H J; Isken, S; Saliola, M

    2000-11-01

    The effects of ethanol and 1-octanol on growth and fatty acid composition of different strains of Kluyveromyces lactis containing a mutation in the four different alcohol dehydrogenase (KlADH) genes were investigated. In the presence of ethanol and 1-octanol K. lactis reduced the fluidity of its lipids by decreasing the unsaturation index (UI) of its membrane fatty acids. In this way, a direct correlation between nonlethal ethanol concentrations and the decrease in the UI could be observed. At concentrations which totally inhibited cell growth no reaction occurred. These adaptive modifications of the fatty acid pattern of K. lactis to ethanol contrasted with those reported for Saccharomyces cerevisiae and Schizosaccharomyces pombe. Whereas these two yeasts increased the fluidity of their membrane lipids in the presence of ethanol, K. lactis reduced the fluidity (UI) of its lipids. Among the different isogenic adh negative strains tested, the strain containing no ADH (adh0) and that containing only KlADH1 were the most alcohol-sensitive. The strain with only KlADH2 showed nearly the same tolerance as reference strain CBS 2359/152 containing all four ADH genes. This suggests that the KlADH2 product could play an important role in the adaptation/detoxification reactions of K. lactis to high ethanol concentrations.

  1. Feedback Regulation of Glucose Transporter Gene Transcription in Kluyveromyces lactis by Glucose Uptake

    PubMed Central

    Milkowski, C.; Krampe, S.; Weirich, J.; Hasse, V.; Boles, E.; Breunig, K. D.

    2001-01-01

    In the respirofermentative yeast Kluyveromyces lactis, only a single genetic locus encodes glucose transporters that can support fermentative growth. This locus is polymorphic in wild-type isolates carrying either KHT1 and KHT2, two tandemly arranged HXT-like genes, or RAG1, a low-affinity transporter gene that arose by recombination between KHT1 and KHT2. Here we show that KHT1 is a glucose-induced gene encoding a low-affinity transporter very similar to Rag1p. Kht2p has a lower Km (3.7 mM) and a more complex regulation. Transcription is high in the absence of glucose, further induced by low glucose concentrations, and repressed at higher glucose concentrations. The response of KHT1 and KHT2 gene regulation to high but not to low concentrations of glucose depends on glucose transport. The function of either Kht1p or Kht2p is sufficient to mediate the characteristic response to high glucose, which is impaired in a kht1 kht2 deletion mutant. Thus, the KHT genes are subject to mutual feedback regulation. Moreover, glucose repression of the endogenous β-galactosidase (LAC4) promoter and glucose induction of pyruvate decarboxylase were abolished in the kht1 kht2 mutant. These phenotypes could be partially restored by HXT gene family members from Saccharomyces cerevisiae. The results indicate that the specific responses to high but not to low glucose concentrations require a high rate of glucose uptake. PMID:11514503

  2. Selection of strain, growth conditions, and extraction procedures for optimum production of lactase from Kluyveromyces fragilis.

    PubMed

    Mahoney, R R; Nickerson, T A; Whitaker, J R

    1975-11-01

    Forty-one strains of Kluyveromyces fragilis (Jörgensen) van der Walt 1909 varied 60-fold in ability to produce lactase (beta-galactosidase). The four best strains were UCD No. 55-31 (Northern Regional Research Center NRRL Y-1196), UCD No. C21(-), UCD No. 72-297(-), and UCD No. 55-61 (NRRL Y-1109). Biosynthesis of lactase during the growth of K. fragilis strain UCD No. 55-61 was followed on both lactose and sweet whey media. Maximum enzyme yield was obtained at the beginning of the stationary phase of growth. Bets lactase yields from K. fragilis UCD No. 55-61 were obtained with 15% lactose and an aeration rate of at least .2 mmol oxygen/liter per min. Supplementary growth factors were unneccessary for good lactase yeilds when yeast was grown on whey media. Best extraction of lactase from fresh yeast cells was obtained by toluene autolysis (2% vol/vol) at 37 C in .1 M potassium phosphate buffer, pH 7.0, containing .1 mM manganese chloride and .5 mM magnesium sulfate. The enzyme was concentrated and purified partially by acetone precipitation. At least 95% of the enzyme activity of the concentrated solution was retained after storage for 7 days at 22 C, for 3 wk at 4 C, and for 6 wk at -20 C.

  3. The major facilitator superfamily transporter Knq1p modulates boron homeostasis in Kluyveromyces lactis.

    PubMed

    Svrbicka, Alexandra; Toth Hervay, Nora; Gbelska, Yvetta

    2016-03-01

    Boron is an essential micronutrient for living cells, yet its excess causes toxicity. To date, the mechanisms of boron toxicity are poorly understood. Recently, the ScATR1 gene has been identified encoding the main boron efflux pump in Saccharomyces cerevisiae. In this study, we analyzed the ScATR1 ortholog in Kluyveromyces lactis--the KNQ1 gene, to understand whether it participates in boron stress tolerance. We found that the KNQ1 gene, encoding a permease belonging to the major facilitator superfamily, is required for K. lactis boron tolerance. Deletion of the KNQ1 gene led to boron sensitivity and its overexpression increased K. lactis boron tolerance. The KNQ1 expression was induced by boron and the intracellular boron concentration was controlled by Knq1p. The KNQ1 promoter contains two putative binding motifs for the AP-1-like transcription factor KlYap1p playing a central role in oxidative stress defense. Our results indicate that the induction of the KNQ1 expression requires the presence of KlYap1p and that Knq1p like its ortholog ScAtr1p in S. cerevisiae functions as a boron efflux pump providing boron resistance in K. lactis.

  4. Evaluation of the performance of Torulaspora delbrueckii, Williopsis saturnus, and Kluyveromyces lactis in lychee wine fermentation.

    PubMed

    Chen, Dai; Yap, Zhi Yin; Liu, Shao-Quan

    2015-08-03

    This study evaluated the effects of three non-Saccharomyces yeasts, namely Torulaspora delbrueckii PRELUDE, Williopsis saturnus NCYC22, and Kluyveromyces lactis KL71 on lychee juice fermentation. The fermentation performance of these non-Saccharomyces yeasts was significantly different. T. delbrueckii PRELUDE had the fastest rate of growth and high sugar consumption. W. saturnus NCYC22 used the lowest amount of sugars, but consumed the highest amount of nitrogen. Correspondingly, strain PRELUDE produced the highest level of ethanol (7.6% v/v), followed by strain KL71 (3.4% v/v) and strain NCYC22 (0.8% v/v). Aroma character-impact terpenes and terpenoids could be partially retained in all lychee wines, with higher odour activity values (OAVs) of geraniol and citronellol in strain KL71. However, strain KL71 and strain NCYC22 over-produced ethyl acetate. Strain PRELUDE had a better ability to generate high levels of ethanol, isoamyl alcohol, 2-phenylethyl alcohol, ethyl octanoate, and ethyl decanoate and retained high OAVs of lychee aroma-character compounds cis-rose oxide (16.5) and linalool (3.5). Thus, it is deemed to be a promising non-Saccharomyces yeast for lychee wine fermentation.

  5. Saccharomyces cerevisiae Elongator mutations confer resistance to the Kluyveromyces lactis zymocin

    PubMed Central

    Frohloff, Frank; Fichtner, Lars; Jablonowski, Daniel; Breunig, Karin D.; Schaffrath, Raffael

    2001-01-01

    Kluyveromyces lactis killer strains secrete a zymocin complex that inhibits proliferation of sensitive yeast genera including Saccharomyces cerevisiae. In search of the putative toxin target (TOT), we used mTn3:: tagging to isolate zymocin-resistant tot mutants from budding yeast. Of these we identified the TOT1, TOT2 and TOT3 genes (isoallelic with ELP1, ELP2 and ELP3, respectively) coding for the histone acetyltransferase (HAT)-associated Elongator complex of RNA polymerase II holoenzyme. Other than the typical elp ts-phenotype, tot phenocopies hypersensitivity towards caffeine and Calcofluor White as well as slow growth and a G1 cell cycle delay. In addition, TOT4 and TOT5 (isoallelic with KTI12 and IKI1, respectively) code for components that associate with Elongator. Intriguingly, strains lacking non-Elongator HATs (gcn5Δ, hat1Δ, hpa3Δ and sas3Δ) or non-Elongator transcription elongation factors TFIIS (dst1Δ) and Spt4p (spt4Δ) cannot confer resistance towards the K.lactis zymocin, thus providing evidence that Elongator equals TOT and that Elongator plays an important role in signalling toxicity of the K.lactis zymocin. PMID:11296232

  6. The Role Of Nonhomologous End-Joining Components in Telomere Metabolism in Kluyveromyces lactis

    PubMed Central

    Carter, Sidney D.; Iyer, Shilpa; Xu, Jianing; McEachern, Michael J.; Åström, Stefan U.

    2007-01-01

    The relationship between telomeres and nonhomologous end-joining (NHEJ) is paradoxical, as NHEJ proteins are part of the telomere cap, which serves to differentiate telomeres from DNA double-strand breaks. We explored these contradictory functions for NHEJ proteins by investigating their role in Kluyveromyces lactis telomere metabolism. The ter1-4LBsr allele of the TER1 gene resulted in the introduction of sequence altered telomeric repeats and subsequent telomere–telomere fusions (T–TFs). In this background, Lig4 and Ku80 were necessary for T–TFs to form. Nej1, essential for NHEJ at internal positions, was not. Hence, T–TF formation was mediated by an unusual NHEJ mechanism. Rad50 and mre11 strains exhibited stable short telomeres, suggesting that Rad50 and Mre11 were required for telomerase recruitment. Introduction of the ter1-4LBsr allele into these strains failed to result in telomere elongation as normally observed with the ter1-4LBsr allele. Thus, the role of Rad50 and Mre11 in the formation of T–TFs was unclear. Furthermore, rad50 and mre11 mutants had highly increased subtelomeric recombination rates, while ku80 and lig4 mutants displayed moderate increases. Ku80 mutant strains also contained extended single-stranded 3′ telomeric overhangs. We concluded that NHEJ proteins have multiple roles at telomeres, mediating fusions of mutant telomeres and ensuring end protection of normal telomeres. PMID:17237517

  7. Two mitochondrial alcohol dehydrogenase activities of Kluyveromyces lactis are differently expressed during respiration and fermentation.

    PubMed

    Saliola, M; Falcone, C

    1995-12-20

    The lactose-utilizing yeast Kluyveromyces lactis is an essentially aerobic organism in which both respiration and fermentation can coexist depending on the sugar concentration. Despite a low fermentative capacity as compared to Saccharomyces cerevisiae, four structural genes encoding alcohol dehydrogenase (ADH) activities are present in this yeast. Two of these activities, namely K1ADH III and K1ADH IV, are located within mitochondria and their presence is dependent on the carbon sources in the medium. In this paper we demonstrate by transcription and activity analysis that KlADH3 is expressed in the presence of low glucose concentrations and in the presence of respiratory carbon sources other than ethanol. Indeed ethanol acts as a strong repressor of this gene. On the other hand, KlADH4 is induced by the presence of ethanol and not by other respiratory carbon sources. We also demonstrate that the presence of KLADH III and KLADH IV in K. lactis cells is dependent on glucose concentration, glucose uptake and the amount of ethanol produced. As a consequence, these activities can be used as markers for the onset of respiratory and fermentative metabolism in this yeast.

  8. Nutritional profile of food yeast Kluyveromyces fragilis biomass grown on whey.

    PubMed

    Paul, Deepen; Mukhopadhyay, Rupak; Chatterjee, Bishnu P; Guha, Arun K

    2002-03-01

    Biomass of food yeast Kluyveromyces fragilis (MTCC 188) grown on deproteinized whey supplemented with 0.8% diammonium hydrogen phosphate and 10 ppm indole-3-acetic acid, had a crude protein content of 37%. The true protein content based on nitrogen fractionation procedure was 28.1%. Total nucleic acid content was 4.82%. This amount does not appear to be toxicologically offensive. Crude fiber, ash, and lipid content of K.fragilis dry cells were found to be 4.9%, 16%, and 7.8%, respectively. Essential fatty acids of both omega-3 and omega-6 series were found present in the fat of the yeast and represented 21.5% of the total fatty acids. All the essential amino acids were present in the proteins of K. fragilis; however, sulfur containing amino acids were found in lower amounts. Calculated protein scores indicate moderate biological value. B vitamins in the biomass were present as expected, but folic acid and pyridoxine were present in high concentration.

  9. Expression and secretion of recombinant ovine beta-lactoglobulin in Saccharomyces cerevisiae and Kluyveromyces lactis.

    PubMed Central

    Rocha, T L; Paterson, G; Crimmins, K; Boyd, A; Sawyer, L; Fothergill-Gilmore, L A

    1996-01-01

    High expression and secretion of recombinant ovine beta-lactoglobulin has been achieved in the yeast Kluyveromyces lactis. The yield of beta-lactoglobulin is 40-50 mg per litre of culture supernatant and accounts for approx. 72% of the total secreted protein. Constitutive expression is under the control of the Saccharomyces cerevisiae phosphoglycerate kinase promoter from an intronless version of the beta-lactoglobulin gene. Secretion is specified by the ovine protein's own signal sequence. this system, coupled to an efficient and novel recovery protocol, allows 30 mg of pure protein to be isolated from a typical 1 litre culture. The protein is virtually indistinguishable from beta-lactoglobulin conventionally purified from sheep milk by its behaviour in native PAGE and SDS/PAGE, reactivity to antibodies, CD, fluorescence spectroscopy and N-terminal sequencing. Attempts to achieve a similar expression and secretion system in the yeast S. cerevisiae met with only limited success, although it was found that heat-shock treatment modestly increased the yield up to approx. 3-4 mg per litre of culture supernatant. Site-directed mutagenesis showed that secretion in S. cerevisiae depended upon correct formation of the two disulphide bonds present in beta-lactoglobulin. PMID:8611177

  10. Sugar metabolism, redox balance and oxidative stress response in the respiratory yeast Kluyveromyces lactis

    PubMed Central

    González-Siso, M Isabel; García-Leiro, Ana; Tarrío, Nuria; Cerdán, M Esperanza

    2009-01-01

    A lot of studies have been carried out on Saccharomyces cerevisiae, an yeast with a predominant fermentative metabolism under aerobic conditions, which allows exploring the complex response induced by oxidative stress. S. cerevisiae is considered a eukaryote model for these studies. We propose Kluyveromyces lactis as a good alternative model to analyse variants in the oxidative stress response, since the respiratory metabolism in this yeast is predominant under aerobic conditions and it shows other important differences with S. cerevisiae in catabolic repression and carbohydrate utilization. The knowledge of oxidative stress response in K. lactis is still a developing field. In this article, we summarize the state of the art derived from experimental approaches and we provide a global vision on the characteristics of the putative K. lactis components of the oxidative stress response pathway, inferred from their sequence homology with the S. cerevisiae counterparts. Since K. lactis is also a well-established alternative host for industrial production of native enzymes and heterologous proteins, relevant differences in the oxidative stress response pathway and their potential in biotechnological uses of this yeast are also reviewed. PMID:19715615

  11. Environmental effects on growth and ethanol fermentation of immobilized Kluyveromyces fragilis

    SciTech Connect

    Chen, C.

    1988-01-01

    A trickle flow, short column reactor system using a natural sponge as a carrier was used to study the response of the immobilized yeast, Kluyveromyces fragilis NRRL 2415, to environmental and nutritional factors. The specific growth rate of adsorbed K. fragilis using a complete medium as feed during the start-up period was 0.0152 hr{sup {minus}1} which was much lower than 0.60 hr{sup {minus}1}, the value for free cells. The transient responses of immobilized cells to changes in lactose concentration and temperature were quick, reaching new steady state values within half an hour. There was little or no detectable CO{sub 2} evolved if the lactose concentration was below 3.8 g/l. The immobilized K. fragilis did not perform well at temperatures over 40{degree}C, although free cells maintained their viability at 40{degree}C. The inhibitory effect of added ethanol on immobilized K. fragilis was a nonlinear relationship. The thermotolerance of K. fragilis was significantly reduced by the presence of ethanol. A kinetic model was developed to determine the steady state productivity based upon growth and death rates as a function of temperature and ethanol concentration.

  12. Enzymatic synthesis and characterization of hydroquinone galactoside using Kluyveromyces lactis lactase.

    PubMed

    Kim, Go-Eun; Lee, Jin-Ha; Jung, Sun-Hwa; Seo, Eun-Seong; Jin, Sheng-De; Kim, Ghahyun J; Cha, Jaeho; Kim, Eui-Joong; Park, Ki-Deok; Kim, Doman

    2010-09-08

    Hydroquinone galactoside (HQ-Gal) as a potential skin whitening agent was synthesized by the reaction of lactase (beta-galactosidase) from Kluyveromyces lactis, Aspergillus oryzae, Bacillus circulans, and Thermus sp. with lactose as a donor and HQ as an acceptor. Among these lactases, the acceptor reaction involving HQ and lactose with K. lactis lactase showed a higher conversion ratio to HQ-Gal (60.27%). HQ-Gal was purified using butanol partitioning and silica gel column chromatography. The structure of the purified HQ-Gal was determined by nuclear magnetic resonance, and the ionic product was observed at m/z 295 (C12H16O7Na)+ using matrix assisted laser desorption ionization time-of-flight mass spectrometry. HQ-Gal was identified as 4-hydroxyphenyl-beta-d-galactopyranoside. The optimum conditions for HQ-Gal synthesis by K. lactis determined using response surface methodology were 50 mM HQ, 60 mM lactose, and 20 U mL(-1) lactase. These conditions produced a yield of 2.01 g L(-1) HQ-Gal. The half maximal inhibitory concentration (IC50) of diphenylpicrylhydrazyl scavenging activity was 3.31 mM, indicating a similar antioxidant activity compared to beta-arbutin (IC50=3.95 mM). The Ki value of HQ-Gal (0.75 mM) against tyrosinase was smaller than that of beta-arbutin (Ki=1.97 mM), indicating its superiority as an inhibitor. HQ-Gal inhibited (23%) melanin synthesis without being significantly toxic to the cells, while beta-arbutin exhibited only 8% reduction of melanin synthesis in B16 melanoma cells compared with the control. These results indicate that HQ-Gal may be a suitable functional component in the cosmetics industry.

  13. Proteomic and functional consequences of hexokinase deficiency in glucose-repressible Kluyveromyces lactis.

    PubMed

    Mates, Nadia; Kettner, Karina; Heidenreich, Falk; Pursche, Theresia; Migotti, Rebekka; Kahlert, Günther; Kuhlisch, Eberhard; Breunig, Karin D; Schellenberger, Wolfgang; Dittmar, Gunnar; Hoflack, Bernard; Kriegel, Thomas M

    2014-03-01

    The analysis of glucose signaling in the Crabtree-positive eukaryotic model organism Saccharomyces cerevisiae has disclosed a dual role of its hexokinase ScHxk2, which acts as a glycolytic enzyme and key signal transducer adapting central metabolism to glucose availability. In order to identify evolutionarily conserved characteristics of hexokinase structure and function, the cellular response of the Crabtree-negative yeast Kluyveromyces lactis to rag5 null mutation and concomitant deficiency of its unique hexokinase KlHxk1 was analyzed by means of difference gel electrophoresis. In total, 2,851 fluorescent spots containing different protein species were detected in the master gel representing all of the K. lactis proteins that were solubilized from glucose-grown KlHxk1 wild-type and mutant cells. Mass spectrometric peptide analysis identified 45 individual hexokinase-dependent proteins related to carbohydrate, short-chain fatty acid and tricarboxylic acid metabolism as well as to amino acid and protein turnover, but also to general stress response and chromatin remodeling, which occurred as a consequence of KlHxk1 deficiency at a minimum 3-fold enhanced or reduced level in the mutant proteome. In addition, three proteins exhibiting homology to 2-methylcitrate cycle enzymes of S. cerevisiae were detected at increased concentrations, suggesting a stimulation of pyruvate formation from amino acids and/or fatty acids. Experimental validation of the difference gel electrophoresis approach by post-lysis dimethyl labeling largely confirmed the abundance changes detected in the mutant proteome via the former method. Taking into consideration the high proportion of identified hexokinase-dependent proteins exhibiting increased proteomic levels, KlHxk1 is likely to have a repressive function in a multitude of metabolic pathways. The proteomic alterations detected in the mutant classify KlHxk1 as a multifunctional enzyme and support the view of evolutionary conservation of

  14. Quantitative comparison of transient growth of Saccharomyces cerevisiae, Saccharomyces kluyveri, and Kluyveromyces lactis.

    PubMed

    Herwig, Christoph; Von Stockar, Urs

    2003-03-30

    A multitude of metabolic regulations occur in yeast, particularly under dynamic process conditions, such as under sudden glucose excess. However, quantification of regulations and classification of yeast strains under these conditions have yet to be elucidated, which requires high-frequency and consistent quantification of the metabolic response. The present study aimed at quantifying the dynamic regulation of the central metabolism of strains Saccharomyces cerevisiae, S. kluyveri, and Kluyveromyces lactis upon sudden glucose excess, accomplished by a shift-up in dilution rate inside of the oxidative region using a small metabolic flux model. It was found that, under transient growth conditions, S. kluyveri behaved like K. lactis, while classification using steady-state conditions would position S. kluyveri close to S. cerevisiae. For transient conditions and based on the observation whether excess glucose is initially used for catabolism (energy) or anabolism (carbon), we propose to classify strains into energy-driven, such as S. cerevisiae, and carbon-driven, such as S. kluyveri and K. lactis, strains. Furthermore, it was found that the delayed onset of fermentative catabolism in carbon-driven strains is a consequence of low catabolic flux and the initial shunt of glucose in non-nitrogen-containing biomass constituents. The MFA model suggests that energy limitation forced the cell to ultimately increase catabolic flux, while the capacity of oxidative catabolism is not sufficient to process this flux oxidatively. The combination of transient experiments and its exploitation with reconciled intrinsic rates using a small metabolic model could corroborate earlier findings of metabolic regulations, such as tight glucose control in carbon-driven strains and transient changes in biomass composition, as well as explore new regulations, such as assimilation of ethanol before glucose. The benefit from using small metabolic flux models is the richness of information and the

  15. SOD1, a New Kluyveromyces lactis Helper Gene for Heterologous Protein Secretion▿

    PubMed Central

    Raimondi, S.; Zanni, E.; Talora, C.; Rossi, M.; Palleschi, C.; Uccelletti, D.

    2008-01-01

    Bottlenecks in protein expression and secretion often limit the development of industrial processes. By manipulating chaperone and foldase levels, improvements in yeast secretion were found for a number of proteins. Recently, sustained endoplasmic reticulum stress, occurring due to recombinant protein production, was reported to cause oxidative stress in yeast. Saccharomyces cerevisiae cells are able to trigger an adaptive response to oxidative-stress conditions, resulting in the upregulation of both primary and secondary antioxidant defenses. SOD1 encodes for a superoxide dismutase that catalyzes the dismutation of superoxide anions (O2−) into oxygen and hydrogen peroxide. It is a Cu2+/Zn2+ metalloenzyme and represents an important antioxidant defense in nearly all aerobic and aerotolerant organisms. We found that overexpression of the Kluyveromyces lactis SOD1 (KlSOD1) gene was able to increase the production of two different heterologous proteins, human serum albumin (HSA) and glucoamylase from Arxula adeninivorans. In addition, KlSOD1 overexpression led to a significant decrease in the amount of reactive oxygen species (ROS) that originated during protein production. The yield of HSA also increased when K. lactis cells were grown in the presence of the antioxidant agent ascorbic acid and decreased when cells were challenged with menadione, a ROS generator compound. Moreover, we observed that, in high-osmolarity medium, cells overexpressing KlSOD1 showed higher growth rates than control cells. Our results thus further support the notion that the production of some heterologous proteins may be improved by manipulating genes involved in general stress responses. PMID:18836000

  16. Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis.

    PubMed

    Bozzi, A; Saliola, M; Falcone, C; Bossa, F; Martini, F

    1997-04-25

    The cytosolic and mitochondrial alcohol dehydrogenases from Kluyveromyces lactis (KlADHs) were purified and characterised. Both the N-terminally blocked cytosolic isozymes, KlADH I and KlADH II, were strictly NAD-dependent and exhibited catalytic properties similar to those previously reported for other yeast ADHs. Conversely, the mitochondrial isozymes, KlADH III and KlADH IV, displayed Ala and Asn, respectively, as N-termini and were able to oxidise at an increased rate primary alcohols with aliphatic chains longer than ethanol, such as propanol, butanol, pentanol and hexanol. Interestingly, the mitochondrial KlADHs, at variance with cytosolic isozymes and the majority of ADHs from other sources, were capable of accepting as a cofactor, and in some case almost equally well, either NAD or NADP. Since Asp-223 of horse liver ADH, thought to be responsible for the selection of NAD as coenzyme, is strictly conserved in all the KlADH isozymes, this amino-acid residue should not be considered critical for the coenzyme discrimination with respect to the other residues lining the coenzyme binding pocket of the mitochondrial isozymes. The relatively low specificity of the mitochondrial KlADHs both toward the alcohols and the cofactor could be explained on the basis of an enhanced flexibility of the corresponding catalytic pockets. An involvement of the mitochondrial KlADH isozymes in the physiological reoxidation of the cytosolic NADPH was also hypothesized. Moreover, both cytosolic and KlADH IV isozymes have an additional cysteine, not involved in zinc binding, that could be responsible for the increased activity in the presence of 2-mercaptoethanol.

  17. Comparison between immobilized Kluyveromyces fragilis and saccharomyces cerevisiae coimmobilized with. beta. -galactosidase, with respect to continuous ethanol production from concentrated whey permeate

    SciTech Connect

    Hahn-Haegerdal, B.

    1985-06-01

    Kluyveromyces fragilis immobilized in calcium alginate gel was compared to Saccharomyces cerevisiae coimmobilized with ..beta..-galactosidase, for continuous ethanol production from whey permeate in packed-bed-type columns. Four different whey concentrations were studied, equivalent to 4.5, 10, 15 and 20% lactose, respectively. In all cases the coimmobilized preparation produced more ethanol than Kluyveromyces fragilis. The study went on for more than 5 weeks. Kluyveromyces fragilis showed a decline in activity after 20 days, while the coimmobilized preparation was stable during the entire investigation. Under experimental conditions theoretical yields of ethanol were obtained from 4.5 and 10% lactose substrates with the coimmobilized system. Using 15% lactose substrate, theoretical yields were only obtained when a galactose-adapted immobilized Saccharomyces cerevisiae column was run in series with the coimmobilized column. Then a maximum of 71 g/l ethanol was produced with a productivity of 2.5 g/l hour. The coimmobilized column alone gave a maximum ethanol concentration of 52 g/l with a productivity of 4.5 g/l hour, whereas immobilized Kluyveromyces fragilis only produced 13 g/l ethanol with a productivity of 1.1 g/l hour. It was not possible to obtain theoretical yields of ethanol from the highest substrate concentration. 13 references.

  18. Use of continuous lactose fermentation for ethanol production by Kluveromyces marxianus for verification and extension of a biochemically structured model.

    PubMed

    Sansonetti, S; Hobley, T J; Curcio, S; Villadsen, J; Sin, G

    2013-02-01

    A biochemically structured model has been developed to describe the continuous fermentation of lactose to ethanol by Kluveromyces marxianus and allowed metabolic coefficients to be determined. Anaerobic lactose-limited chemostat fermentations at different dilution rates (0.02-0.35h(-1)) were performed. Species specific rates of consumption/formation, as well as yield coefficients were determined. Ethanol yield (0.655 C-mol ethanol(∗)C-mol lactose(-1)) was as high as 98% of theoretical. The modeling procedure allowed calculation of maintenance coefficients for lactose consumption and ethanol production of m(s)=0.6029 and m(e)=0.4218 (C-mol) and (C-molh)(-1), respectively. True yield coefficients for biomass, ethanol and glycerol production were calculated to be Y(true)(sx)=0.114, Y(true)(ex)=0.192 and Y(sg)=2.250 (C-mol) and (C-mol)(-1), respectively. Model calculated maintenance and true yield coefficients agreed very closely with those determined by regressions of the experimental data. The model developed provides a solid basis for the rational design of optimised fermentation of cheese whey.

  19. Proteomic and Functional Consequences of Hexokinase Deficiency in Glucose-repressible Kluyveromyces lactis

    PubMed Central

    Mates, Nadia; Kettner, Karina; Heidenreich, Falk; Pursche, Theresia; Migotti, Rebekka; Kahlert, Günther; Kuhlisch, Eberhard; Breunig, Karin D.; Schellenberger, Wolfgang; Dittmar, Gunnar; Hoflack, Bernard; Kriegel, Thomas M.

    2014-01-01

    The analysis of glucose signaling in the Crabtree-positive eukaryotic model organism Saccharomyces cerevisiae has disclosed a dual role of its hexokinase ScHxk2, which acts as a glycolytic enzyme and key signal transducer adapting central metabolism to glucose availability. In order to identify evolutionarily conserved characteristics of hexokinase structure and function, the cellular response of the Crabtree-negative yeast Kluyveromyces lactis to rag5 null mutation and concomitant deficiency of its unique hexokinase KlHxk1 was analyzed by means of difference gel electrophoresis. In total, 2,851 fluorescent spots containing different protein species were detected in the master gel representing all of the K. lactis proteins that were solubilized from glucose-grown KlHxk1 wild-type and mutant cells. Mass spectrometric peptide analysis identified 45 individual hexokinase-dependent proteins related to carbohydrate, short-chain fatty acid and tricarboxylic acid metabolism as well as to amino acid and protein turnover, but also to general stress response and chromatin remodeling, which occurred as a consequence of KlHxk1 deficiency at a minimum 3-fold enhanced or reduced level in the mutant proteome. In addition, three proteins exhibiting homology to 2-methylcitrate cycle enzymes of S. cerevisiae were detected at increased concentrations, suggesting a stimulation of pyruvate formation from amino acids and/or fatty acids. Experimental validation of the difference gel electrophoresis approach by post-lysis dimethyl labeling largely confirmed the abundance changes detected in the mutant proteome via the former method. Taking into consideration the high proportion of identified hexokinase-dependent proteins exhibiting increased proteomic levels, KlHxk1 is likely to have a repressive function in a multitude of metabolic pathways. The proteomic alterations detected in the mutant classify KlHxk1 as a multifunctional enzyme and support the view of evolutionary conservation of

  20. Evaluation of pKD1-based plasmid systems for heterologous protein production in Kluyveromyces lactis.

    PubMed

    Panuwatsuk, W; Da Silva, N A

    2002-02-01

    The stability of pKD1-based vectors was evaluated during the synthesis of intracellular and extracellular gene products in the yeast Kluyveromyces lactis. The Escherichia coli lacZ and MFalpha1 leader-BPTI (bovine pancreatic trypsin inhibitor) cassettes were placed under the control of the inducible K. lactis LAC4 promoter and inserted into the pKD1-based plasmids. To induce gene expression while maintaining inducer level, a gratuitous gal1-209 K. lactis strain was employed. Selective medium containing 5 g glucose/l and 0.5 g galactose (inducer)/l allowed optimum expression and secretion of heterologous products without a significant effect on the growth of the recombinant cells. During long-term sequential batch cultures (60 generations), plasmid instability was mainly the result of structural instability. The expression and secretion of BPTI resulted in greater structural instability relative to the intracellular beta-galactosidase. For both products, vectors carrying the pKD1 replication origin and the cis-acting stability locus (partial-pKD1 vectors) were more stable than vectors carrying the full pKD1 sequence (full-pKD1 vectors). However, after 55 generations, the beta-galactosidase and BPTI activities were still higher with the full-pKD1 vectors. This was due to the significantly higher initial beta-galactosidase and BPTI activities for the full-pKD1 vectors (approximately 85% and 47% higher, respectively) relative to the partial-pKDI vectors. Southern blots confirmed that these increases were due to the higher copy number of the vectors carrying the full pKD1 sequence. In contrast to our previously reported results for the secretion of invertase, full-pKD1 vectors were preferred for the expression/secretion of beta-galactosidase and BPTI for at least 55 generations. Due to their structural stability, partial-pKD1 vectors will be advantageous for very long cultivation times.

  1. Evaluation of yeast strains for production of fuel ethanol from biomass hydrolysates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Robust industrial yeast strains are needed for profitable production of fuel ethanol from mixed biomass waste. USDA, ARS, NCAUR, RPT has been evaluating ethanol-producing yeasts, including Saccharomyces cerevisiae, engineered GMAX Saccharomyces cerevisiae, irradiated Kluyveromyces marxianus, and Pi...

  2. CTAB-mediated, single-step preparation of competent Escherichia coli, Bifidobacterium sp. and Kluyveromyces lactis cells.

    PubMed

    Rajagopal, Kammara; Singh, Praveen Kumar; Kumar, Rajesh; Siddiqui, Kaneez Fatima

    2014-12-01

    An efficient and reproducible method for transformation depends on the competency of the organism. We have developed a simple method for the preparation of competent Escherichia coli, Kluyveromyces lactis, and Bifidobacterium sp. by using a buffer containing cetyl trimethyl ammonium bromide (CTAB) and permits efficient uptake of plasmid DNA and ligation-reaction products. Cells are harvested, washed, mixed with 1-10 μg/ml CTAB, incubated, and followed by a buffer wash. For long-term storage of competent cells, bacteria may be frozen in 10% glycerol without the addition of other components. The transformation process is very simple; plasmid DNA and the cells are mixed and incubated for 5-60 min at 4 °C; no heat pulse is required, and the duration of incubation at 4 °C is not crucial.

  3. Ethanol fermentation from molasses at high temperature by thermotolerant yeast Kluyveromyces sp. IIPE453 and energy assessment for recovery.

    PubMed

    Dasgupta, Diptarka; Ghosh, Prasenjit; Ghosh, Debashish; Suman, Sunil Kumar; Khan, Rashmi; Agrawal, Deepti; Adhikari, Dilip K

    2014-10-01

    High temperature ethanol fermentation from sugarcane molasses B using thermophilic Crabtree-positive yeast Kluyveromyces sp. IIPE453 was carried out in batch bioreactor system. Strain was found to have a maximum specific ethanol productivity of 0.688 g/g/h with 92 % theoretical ethanol yield. Aeration and initial sugar concentration were tuning parameters to regulate metabolic pathways of the strain for either cell mass or higher ethanol production during growth with an optimum sugar to cell ratio 33:1 requisite for fermentation. An assessment of ethanol recovery from fermentation broth via simulation study illustrated that distillation-based conventional recovery was significantly better in terms of energy efficiency and overall mass recovery in comparison to coupled solvent extraction-azeotropic distillation technique for the same.

  4. A positive regulatory element is involved in the induction of the beta-galactosidase gene from Kluyveromyces lactis.

    PubMed Central

    Das, S; Breunig, K D; Hollenberg, C P

    1985-01-01

    The regulation of the LAC4 gene encoding beta-galactosidase in the yeast Kluyveromyces lactis has been studied. The expression of cloned LAC4 gene present on autonomously replicating plasmids was normally regulated by lactose or galactose as inducers. The LAC4 transcription initiation sites were mapped on two plasmids, PTY75-LAC4 and pKL2. The sites were found to be dependent on the level of gene expression and on the plasmid used. Under induced conditions, the normal cluster of initiation sites was used on both plasmids, whereas under non-induced conditions LAC4 on pKL2 showed additional sites. Deletion mapping of the 5' regulatory region of the LAC4 gene revealed a DNA element required for induction, presumably for the binding of a positive regulator. Images Fig. 2. Fig. 3. Fig. 6. PMID:3924596

  5. A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in Kluyveromyces lactis

    PubMed Central

    2012-01-01

    Background The Crabtree-negative yeast species Kluyveromyces lactis has been established as an attractive microbial expression system for recombinant proteins at industrial scale. Its LAC genes allow for utilization of the inexpensive sugar lactose as a sole source of carbon and energy. Lactose efficiently induces the LAC4 promoter, which can be used to drive regulated expression of heterologous genes. So far, strain manipulation of K. lactis by homologous recombination was hampered by the high rate of non-homologous end-joining. Results Selection for growth on lactose was applied to target the insertion of heterologous genes downstream of the LAC4 promoter into the K. lactis genome and found to yield high numbers of positive transformants. Concurrent reconstitution of the β-galactosidase gene indicated the desired integration event of the expression cassette, and β-galactosidase activity measurements were used to monitor gene expression for strain improvement and fermentation optimization. The system was particularly improved by usage of a cell lysis resistant strain, VAK367-D4, which allowed for protein accumulation in long-term fermentation. Further optimization was achieved by increased gene dosage of KlGAL4 encoding the activator of lactose and galactose metabolic genes that led to elevated transcription rates. Pilot experiments were performed with strains expressing a single-chain antibody fragment (scFvox) and a viral envelope protein (BVDV-E2), respectively. scFvox was shown to be secreted into the culture medium in an active, epitope-binding form indicating correct processing and protein folding; the E2 protein could be expressed intracellularly. Further data on the influence of protein toxicity on batch fermentation and potential post-transcriptional bottlenecks in protein accumulation were obtained. Conclusions A novel Kluyveromyces lactis host-vector system was developed that places heterologous genes under the control of the chromosomal LAC4 promoter

  6. Pyrimidine motif triple helix in the Kluyveromyces lactis telomerase RNA pseudoknot is essential for function in vivo.

    PubMed

    Cash, Darian D; Cohen-Zontag, Osnat; Kim, Nak-Kyoon; Shefer, Kinneret; Brown, Yogev; Ulyanov, Nikolai B; Tzfati, Yehuda; Feigon, Juli

    2013-07-02

    Telomerase is a ribonucleoprotein complex that extends the 3' ends of linear chromosomes. The specialized telomerase reverse transcriptase requires a multidomain RNA (telomerase RNA, TER), which includes an integral RNA template and functionally important template-adjacent pseudoknot. The structure of the human TER pseudoknot revealed that the loops interact with the stems to form a triple helix shown to be important for activity in vitro. A similar triple helix has been predicted to form in diverse fungi TER pseudoknots. The solution NMR structure of the Kluyveromyces lactis pseudoknot, presented here, reveals that it contains a long pyrimidine motif triple helix with unexpected features that include three individual bulge nucleotides and a C(+)•G-C triple adjacent to a stem 2-loop 2 junction. Despite significant differences in sequence and base triples, the 3D shape of the human and K. lactis TER pseudoknots are remarkably similar. Analysis of the effects of nucleotide substitutions on cell growth and telomere lengths provides evidence that this conserved structure forms in endogenously assembled telomerase and is essential for telomerase function in vivo.

  7. An alternative, arginase-independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme

    PubMed Central

    Romagnoli, G; Verhoeven, M D; Mans, R; Fleury Rey, Y; Bel-Rhlid, R; van den Broek, M; Maleki Seifar, R; Ten Pierick, A; Thompson, M; Müller, V; Wahl, S A; Pronk, J T; Daran, J M

    2014-01-01

    Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi. PMID:24912400

  8. Immobilization of β-d-galactosidase from Kluyveromyces lactis on functionalized silicon dioxide nanoparticles: characterization and lactose hydrolysis.

    PubMed

    Verma, Madan Lal; Barrow, Colin James; Kennedy, J F; Puri, Munish

    2012-03-01

    β-D-Galactosidase (BGAL) from Kluyveromyces lactis was covalently immobilized to functionalized silicon dioxide nanoparticles (10-20 nm). The binding of the enzyme to the nanoparticles was confirmed by Fourier transform-infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Functionalized nanoparticles showed 87% immobilization yield. Soluble and immobilized enzyme preparation exhibited pH-optima at pH 6.5 and 7.0, respectively, with temperature optima at 35 and 40°C, respectively. Michaelis constant (K(m)) was 4.77 and 8.4mM for free and immobilized BGAL, respectively. V(max) for the soluble and immobilized enzyme was 12.25 and 13.51 U/ml, respectively. Nanoparticle immobilized BGAL demonstrated improved stability after favoring multipoint covalent attachment. Thermal stability of the immobilized enzyme was enhanced at 40, 50 and 65°C. Immobilized nanoparticle-enzyme conjugate retained more than 50% enzyme activity up to the eleventh cycle. Maximum lactose hydrolysis by immobilized BGAL was achieved at 8h.

  9. Protective Vaccination against Infectious Bursal Disease Virus with Whole Recombinant Kluyveromyces lactis Yeast Expressing the Viral VP2 Subunit

    PubMed Central

    Arnold, Marina; Durairaj, Vijay; Mundt, Egbert; Schulze, Katja; Breunig, Karin D.; Behrens, Sven-Erik

    2012-01-01

    Here we report on vaccination approaches against infectious bursal disease (IBD) of poultry that were performed with complete yeast of the species Kluyveromyces lactis (K. lactis). Employing a genetic system that enables the rapid production of stably transfected recombinant K. lactis, we generated yeast strains that expressed defined quantities of the virus capsid forming protein VP2 of infectious bursal disease virus (IBDV). Both, subcutaneous as well as oral vaccination regiments with the heat-inactivated but otherwise untreated yeast induced IBDV-neutralizing antibodies in mice and chickens. A full protection against a subsequent IBDV infection was achieved by subcutaneous inoculation of only milligram amounts of yeast per chicken. Oral vaccination also generated protection: while mortality was observed in control animals after virus challenge, none of the vaccinees died and ca. one-tenth were protected as indicated by the absence of lesions in the bursa of Fabricius. Recombinant K. lactis was thus indicated as a potent tool for the induction of a protective immune response by different applications. Subcutaneously applied K. lactis that expresses the IBDV VP2 was shown to function as an efficacious anti-IBD subunit vaccine. PMID:23024743

  10. Expression of the transcriptional activator LAC9 (KlGAL4) in Kluyveromyces lactis is controlled by autoregulation.

    PubMed Central

    Zachariae, W; Breunig, K D

    1993-01-01

    The concentration of the transcriptional activator LAC9 (KlGAL4) of Kluyveromyces lactis is moderately regulated by the carbon source as is the case for GAL4, its homolog in Saccharomyces cerevisiae. Expression of the LAC9 gene is induced about twofold in galactose. This induction is due to autoregulation. The LAC9 gene product binds to a low-affinity binding site in the LAC9 promoter and moderately activates transcription in response to galactose above a basal level. As for the LAC9-controlled metabolic genes, induction of LAC9 is inhibited in the presence of glucose. This inhibition of induction is a prerequisite for glucose repression of the lactose-galactose metabolic pathway. On the other hand, induced LAC9 levels are required for optimal growth on galactose, since mutating the LAC9 binding site in the LAC9 promoter resulted in poor growth and reduced expression of LAC9-controlled genes. Thus, in addition to the GAL80-dependent regulation by protein-protein interaction, the regulation of LAC9 gene expression is an important parameter in determining carbon source control of the LAC-GAL regulon. Although the mode of control is different, the pattern of LAC9 gene regulation resembles that of the S. cerevisiae GAL4 gene, being lower in glucose and glucose-galactose than in galactose. Images PMID:8474461

  11. Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respiratory to fermentative metabolism by deletion of NDI1.

    PubMed

    González-Siso, María Isabel; Touriño, Alba; Vizoso, Ángel; Pereira-Rodríguez, Ángel; Rodríguez-Belmonte, Esther; Becerra, Manuel; Cerdán, María Esperanza

    2015-03-01

    In this paper, we report the metabolic engineering of the respiratory yeast Kluyveromyces lactis by construction and characterization of a null mutant (Δklndi1) in the single gene encoding a mitochondrial alternative internal dehydrogenase. Isolated mitochondria of the Δklndi1 mutant show unaffected rate of oxidation of exogenous NADH, but no oxidation of matrix NADH; this confirms that KlNdi1p is the only internal NADH dehydrogenase in K. lactis mitochondria. Permeabilized cells of the Δklndi1 mutant do not show oxidation of matrix NADH, which suggests that shuttle systems to transfer the NADH from mitochondrial matrix to cytosol, for being oxidized by external dehydrogenases, are not functional. The Δklndi1 mutation decreases the chronological life span in absence of nutrients. The expression of KlNDI1 is increased by glutathione reductase depletion. The Δklndi1 mutation shifts the K. lactis metabolism from respiratory to fermentative: the Δklndi1 strain shows reduced respiration rate and increased ethanol production from glucose, while it does not grow in non-fermentable carbon sources such as lactate. The biotechnological benefit of the Δklndi1 mutant for bioethanol production from waste cheese whey lactose was proved.

  12. Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respiratory to fermentative metabolism by deletion of NDI1

    PubMed Central

    González-Siso, María Isabel; Touriño, Alba; Vizoso, Ángel; Pereira-Rodríguez, Ángel; Rodríguez-Belmonte, Esther; Becerra, Manuel; Cerdán, María Esperanza

    2015-01-01

    In this paper, we report the metabolic engineering of the respiratory yeast Kluyveromyces lactis by construction and characterization of a null mutant (Δklndi1) in the single gene encoding a mitochondrial alternative internal dehydrogenase. Isolated mitochondria of the Δklndi1 mutant show unaffected rate of oxidation of exogenous NADH, but no oxidation of matrix NADH; this confirms that KlNdi1p is the only internal NADH dehydrogenase in K. lactis mitochondria. Permeabilized cells of the Δklndi1 mutant do not show oxidation of matrix NADH, which suggests that shuttle systems to transfer the NADH from mitochondrial matrix to cytosol, for being oxidized by external dehydrogenases, are not functional. The Δklndi1 mutation decreases the chronological life span in absence of nutrients. The expression of KlNDI1 is increased by glutathione reductase depletion. The Δklndi1 mutation shifts the K. lactis metabolism from respiratory to fermentative: the Δklndi1 strain shows reduced respiration rate and increased ethanol production from glucose, while it does not grow in non-fermentable carbon sources such as lactate. The biotechnological benefit of the Δklndi1 mutant for bioethanol production from waste cheese whey lactose was proved. PMID:25186243

  13. Hypoxia and iron requirements are the main drivers in transcriptional adaptation of Kluyveromyces lactis during wine aerobic fermentation.

    PubMed

    Tronchoni, Jordi; Rodrigues, Alda J; Curiel, Jose Antonio; Morales, Pilar; Gonzalez, Ramon

    2017-04-04

    The respiratory metabolism of yeast species alternative to Saccharomyces cerevisiae has been explored in recent years as a tool to reduce ethanol content in grape wine. The efficacy of this strategy has been previously proven for mixed cultures of non-Saccharomyces and S. cerevisiae strains. In this work, we perform a transcriptomic analysis of the Crabtree-negative yeast Kluyveromyces lactis under tightly controlled growth conditions in order to better understand physiology of non-Saccharomyces yeasts during the fermentation of grape must under aerated conditions. Transcriptional changes in K. lactis are mainly driven by oxygen limitation, iron requirement, and oxidative stress. Oxidative stress appears as a consequence of the hypoxic conditions achieved by K. lactis once oxygen supply is no longer sufficient to sustain fully respiratory metabolism. This species copes with low oxygen and iron availability by repressing iron consuming pathways and activating iron transport mechanisms. Most of the physiological and transcriptomic features of K. lactis in aerobic wine fermentation are not shared with the Crabtree-positive yeast S. cerevisiae.

  14. Efficient Homolactic Fermentation by Kluyveromyces lactis Strains Defective in Pyruvate Utilization and Transformed with the Heterologous LDH Gene†

    PubMed Central

    Bianchi, Michele M.; Brambilla, Luca; Protani, Francesca; Liu, Chi-Li; Lievense, Jefferson; Porro, Danilo

    2001-01-01

    A high yield of lactic acid per gram of glucose consumed and the absence of additional metabolites in the fermentation broth are two important goals of lactic acid production by microrganisms. Both purposes have been previously approached by using a Kluyveromyces lactis yeast strain lacking the single pyruvate decarboxylase gene (KlPDC1) and transformed with the heterologous lactate dehydrogenase gene (LDH). The LDH gene was placed under the control the KlPDC1 promoter, which has allowed very high levels of lactate dehydrogenase (LDH) activity, due to the absence of autoregulation by KlPdc1p. The maximal yield obtained was 0.58 g g−1, suggesting that a large fraction of the glucose consumed was not converted into pyruvate. In a different attempt to redirect pyruvate flux toward homolactic fermentation, we used K. lactis LDH transformant strains deleted of the pyruvate dehydrogenase (PDH) E1α subunit gene. A great process improvement was obtained by the use of producing strains lacking both PDH and pyruvate decarboxylase activities, which showed yield levels of as high as 0.85 g g−1 (maximum theoretical yield, 1 g g−1), and with high LDH activity. PMID:11722915

  15. An alkali-thermostable xylanase from Bacillus pumilus functionally expressed in Kluyveromyces lactis and evaluation of its deinking efficiency.

    PubMed

    Thomas, Leya; Ushasree, Mrudula V; Pandey, Ashok

    2014-08-01

    This work aimed at studying the recombinant expression of an alkali- and thermo-stable xylanase from Bacillus pumilus in Kluyveromyces lactis and its use in deinking of civic paper waste. Efficient expression with a 3-fold increase in the activity than the native organism was achieved. An inducer concentration of 2.5% and medium pH of 9.0 was the best for enzyme expression. Purified enzyme showed an optimum activity at temperatures 50 and 60°C and pH 9.0 and 10.0, respectively. At pH 12.0, enzyme retained 74% and 26% activity after 2 and 3h of incubation, respectively. After incubation at 50 and 60°C for 1h, the enzyme showed 100% retention of activity, and remained active for 4h at 60°C retaining 23% residual activity. Partially purified recombinant enzyme showed higher deinking efficiency (273%) of laser print waste paper than crude xylanase from Bacillus and commercial acidic enzyme. This xylanase with superior stability characteristics could be a suitable candidate in paper and pulp industries.

  16. Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation.

    PubMed

    Yamaoka, Chizuru; Kurita, Osamu; Kubo, Tomoko

    2014-12-01

    The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.

  17. Autoregulation of GAL4 transcription is essential for rapid growth of Kluyveromyces lactis on lactose and galactose.

    PubMed Central

    Czyz, M; Nagiec, M M; Dickson, R C

    1993-01-01

    Transcriptional induction of genes in the lactose-galactose regulon of the yeast Kluyveromyces lactis requires the GAL4 transcription activator protein. Previous data indicated that the concentration of GAL4 was tightly regulated under basal, inducing, and glucose repressing conditions but the mechanisms were unknown. In this paper we demonstrate that transcription of the GAL4 gene (KI-GAL4) increases 3- to 4-fold during induction of the regulon. This increase requires a KI-GAL4 binding site, UASG, in front of the KI-GAL4 gene, indicating that the KI-GAL4 protein autoregulates transcription of its own gene. Our data demonstrate that the autoregulatory circuit is essential for full induction of the lactose-galactose regulon and, hence, for rapid growth on lactose or galactose. Other data indicate that basal transcription of the KI-GAL4 gene is governed by unidentified promoter elements. The existence of the autoregulatory circuit reveals an important difference between the lactose-galactose regulon and its homologue in Saccharomyces cerevisiae, the melibiose-galactose regulon. This difference may have evolved in response to different selective pressures encountered by the two organisms. PMID:8414996

  18. Cloning, Production, and Functional Expression of the Bacteriocin Enterocin A, Produced by Enterococcus faecium T136, by the Yeasts Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Arxula adeninivorans

    PubMed Central

    Borrero, Juan; Kunze, Gotthard; Jiménez, Juan J.; Böer, Erik; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M.

    2012-01-01

    The bacteriocin enterocin A (EntA) produced by Enterococcus faecium T136 has been successfully cloned and produced by the yeasts Pichia pastoris X-33EA, Kluyveromyces lactis GG799EA, Hansenula polymorpha KL8-1EA, and Arxula adeninivorans G1212EA. Moreover, P. pastoris X-33EA and K. lactis GG799EA produced EntA in larger amounts and with higher antimicrobial and specific antimicrobial activities than the EntA produced by E. faecium T136. PMID:22685156

  19. Role of the Sln1-phosphorelay pathway in the response to hyperosmotic stress in the yeast Kluyveromyces lactis.

    PubMed

    Rodríguez-González, Miriam; Kawasaki, Laura; Velázquez-Zavala, Nancy; Domínguez-Martín, Eunice; Trejo-Medecigo, Abraham; Martagón, Natalia; Espinoza-Simón, Emilio; Vázquez-Ibarra, Araceli; Ongay-Larios, Laura; Georgellis, Dimitris; de Nadal, Eulàlia; Posas, Francesc; Coria, Roberto

    2017-03-13

    The Kluyveromyces lactis SLN1 phosphorelay system includes the osmosensor histidine kinase Sln1, the phosphotransfer protein Ypd1 and the response regulator Ssk1. Here we show that K. lactis has a functional phosphorelay system. In vitro assays, using a heterolougus histidine kinase, show that the phosphate group is accepted by KlYpd1 and transfer to KlSsk1. Upon hyperosmotic stress the phosphorelay is inactivated, KlYpd1 is dephosphorylated in a KlSln1 dependent manner, and only the version of KlSsk1 that lacks the phosphate group interacts with the MAPKKK KlSsk2. Interestingly, inactivation of the KlPtp2 phosphatase in a ΔKlsln1 mutant did not lead to KlHog1 constitutive phosphorylation. KlHog1 can replace ScHog1p and activate the hyperosmotic response in S. cereviseae, and when ScSln1 is inactivated, KlHog1 becomes phosphorylated and induces cell lethality. All these observations indicate that the phosphorelay negatively regulates KlHog1. Nevertheless, in the absence of KlSln1 or KlYpd1, no constitutive phosphorylation is detected and cells are viable, suggesting that a strong negative feedback that is independent of KlPtp2 operates in K. lactis. Compared to S. cereviseae, K. lactis has only a moderate accumulation of glycerol and fails to produce trehalose under hyperosmotic stress, indicating that regulation of osmolyte production is different in K. lactis. This article is protected by copyright. All rights reserved.

  20. Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis.

    PubMed

    Liu, Bo; Gong, Xin; Chang, Shaohong; Yang, Yili; Song, Miao; Duan, Demin; Wang, Lina; Ma, Qingjun; Wu, Jun

    2009-08-20

    Glycoproteins secreted by the yeast Kluyveromyces lactis are usually modified by the addition at asparagines-linked glycosylation sites of heterogeneous mannan residues. The secreted glycoproteins in K. lactis that become hypermannosylated will bear a non-human glycosylation pattern and can adversely affect the half-life, tissue distribution and immunogenicity of a therapeutic protein. Here, we describe engineering a K. lactis strain to produce non-hypermannosylated glycoprotein, decreasing the outer-chain mannose residues of N-linked oligosaccharides. We investigated and developed the method of two-step homologous recombination to knockout the OCH1 gene, encoding alpha1,6-mannosyltransferase and MNN1 gene, which is homologue of Saccharomyces cerevisiae MNN1, encoding a putative alpha1,3-mannosyltransferase. We found the Kloch1 mutant strain has a defect in hyperglycosylation, inability in adding mannose to the core oligosaccharide. The N-linked oligosaccharides assembled on a secretory glycoprotein, HSA/GM-CSF in Kloch1 mutant, contained oligosaccharide Man(13-14)GlcNAc(2), and in Kloch1 mnn1 mutant, contained oligosaccharide Man(9-11)GlcNAc(2), whereas those in the wild-type strain, consisted of oligosaccharides with heterogeneous sizes, Man(>30)GlcNAc(2). Taken together, these results indicated that KlOch1p plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in K. lactis. The KlMnn1p, was proved to be certain contribution to the outer hypermannosylation, most possibly encodes alpha1,3-mannosyltransferase. Therefore, the Kloch1 and Kloch1 mnn1 mutants can be used as a foundational host to produce glycoproteins lacking the outer-chain hypermannoses and further maybe applicable to be a promising system for yeast therapeutic protein production.

  1. A catalytic and non-catalytic role for the Yen1 nuclease in maintaining genome integrity in Kluyveromyces lactis.

    PubMed

    Chen, Jiang; Aström, Stefan U

    2012-10-01

    Yen1 is a nuclease identified in Saccharomyces cerevisiae that cleaves the Holliday junction (HJ) intermediate formed during homologous recombination. Alternative routes to disjoin HJs are performed by the Mus81/Mms4- and Sgs1/Top3/Rmi1-complexes. Here, we investigate the role of the Yen1 protein in the yeast Kluyveromyces lactis. We demonstrate that both yen1 mus81 and yen1 sgs1 double mutants displayed negative genetic interactions in the presence of DNA-damaging chemicals. To test if these phenotypes required the catalytic activity of Yen1, we introduced point mutations targeting the catalytic site of Yen1, which abolished the nuclease activity in vitro. Remarkably, catalytically inactive Yen1 did not exacerbate the hydroxyurea sensitivity of the sgs1Δ strain, which the yen1Δ allele did. In addition, overexpression of catalytically inactive Yen1 partially rescued the DNA damage sensitivity of both mus81 and sgs1 mutant strains albeit less efficiently than WT Yen1. These results suggest that Yen1 serves both a catalytic and non-catalytic role in its redundant function with Mus81 and Sgs1. Diploids lacking Mus81 had a severe defect in sporulation efficiency and crossover frequency, but diploids lacking both Mus81 and Yen1 showed no further reduction in spore formation. Hence, Yen1 had no evident role in meiosis. However, overexpression of WT Yen1, but not catalytically inactive Yen1 partially rescued the crossover defect in mus81/mus81 mutant diploids. Yen1 is a member of the RAD2/XPG-family of nucleases, but genetic analyses revealed no genetic interaction between yen1 and other family members (rad2, exo1 and rad27). In addition, yen1 mutants had normal nonhomologous end-joining efficiency. We discuss the similarities and differences between K. lactis Yen1 and Yen1/GEN1 from other organisms.

  2. Ume6 Is Required for the MATa/MATα Cellular Identity and Transcriptional Silencing in Kluyveromyces lactis

    PubMed Central

    Barsoum, E.; Sjöstrand, J. O. O.; Åström, S. U.

    2010-01-01

    To explore the similarities and differences of regulatory circuits among budding yeasts, we characterized the role of the unscheduled meiotic gene expression 6 (UME6) gene in Kluyveromyces lactis. We found that Ume6 was required for transcriptional silencing of the cryptic mating-type loci HMLα and HMRa. Chromatin immunoprecipitation (ChIP) suggested that Ume6 acted directly by binding the cis-regulatory silencers of these loci. Unexpectedly, a MATa ume6 strain was mating proficient, whereas a MATα ume6 strain was sterile. This observation was explained by the fact that ume6 derepressed HMLα2 only weakly, but derepressed HMRa1 strongly. Consistently, two a/α-repressed genes (MTS1 and STE4) were repressed in the MATα ume6 strain, but were expressed in the MATa ume6 strain. Surprisingly, ume6 partially suppressed the mating defect of a MATa sir2 strain. MTS1 and STE4 were repressed in the MATa sir2 ume6 double-mutant strain, indicating that the suppression acted downstream of the a1/α2-repressor. We show that both STE12 and the MATa2/HMRa2 genes were overexpressed in the MATa sir2 ume6 strain. Consistent with the idea that this deregulation suppressed the mating defect, ectopic overexpression of Ste12 and a2 in a MATa sir2 strain resulted in efficient mating. In addition, Ume6 served as a block to polyploidy, since ume6/ume6 diploids mated as pseudo a-strains. Finally, Ume6 was required for repression of three meiotic genes, independently of the Rpd3 and Sin3 corepressors. PMID:20139343

  3. Differences in the hydrolysis of lactose and other substrates by beta-D-galactosidase from Kluyveromyces lactis.

    PubMed

    Kim, S H; Lim, K P; Kim, H S

    1997-10-01

    The hydrolysis of o-nitrophenyl galactopyranoside and lactose by beta-D-galactosidase from Kluyveromyces lactis was enhanced by the addition of Mg2+ and Mn2+, but the rates of activation by each metal on both substrates were not the same. The Co2+, Zn2+, and Ni2+ activated the o-nitrophenyl galactopyranoside-hydrolyzing activity of the enzyme, but these same metals inhibited the lactose-hydrolyzing activity. The addition of Mg2+ and EDTA to the assay buffer increased the hydrolysis of o-nitrophenyl galactopyranoside and lactose at different rates. The responses of o-nitrophenyl galactopyranoside and lactose to the enzyme activity were different as a function of pH. The hydrolyzing activity toward both substrates also was influenced by the concentration of the phosphate in the assay buffer. However, the profile of the enzyme activity toward each substrate was different as a function of concentration. Because the assay of beta-galactosidase using o-nitrophenyl galactopyranoside is fast and convenient, the estimation of lactose-hydrolyzing activity of the enzyme has frequently been made based on the assay of o-nitrophenyl galactopyranoside hydrolysis. As shown in this study, a slight change in the conditions of the assay system and the enzyme application may cause changes in the ability of the enzyme to hydrolyze both lactose and o-nitrophenyl galactopyranoside. The change in o-nitrophenyl galactopyranoside-hydrolyzing activity is not always consistent with that of the lactose-hydrolyzing activity under the given condition, which may cause an inaccurate estimation of the enzyme activity in the enzyme preparation as well as in actual applications of the enzyme.

  4. Cloning and characterization of Kluyveromyces lactis SEC14, a gene whose product stimulates Golgi secretory function in Saccharomyces cerevisiae.

    PubMed Central

    Salama, S R; Cleves, A E; Malehorn, D E; Whitters, E A; Bankaitis, V A

    1990-01-01

    The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for secretory protein movement from the Golgi complex. That some conservation of SEC14p function may exist was initially suggested by experiments that revealed immunoreactive polypeptides in cell extracts of the divergent yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. We have cloned and characterized the K. lactis SEC14 gene (SEC14KL). Immunoprecipitation experiments indicated that SEC14KL encoded the K. lactis structural homolog of SEC14p. In agreement with those results, nucleotide sequence analysis of SEC14KL revealed a gene product of 301 residues (Mr, 34,615) and 77% identity to SEC14p. Moreover, a single ectopic copy of SEC14KL was sufficient to render S. cerevisiae sec14-1(Ts) mutants, or otherwise inviable sec14-129::HIS3 mutant strains, completely proficient for secretory pathway function by the criteria of growth, invertase secretion, and kinetics of vacuolar protein localization. This efficient complementation of sec14-129::HIS3 was observed to occur when the rates of SEC14pKL and SEC14p synthesis were reduced by a factor of 7 to 10 with respect to the wild-type rate of SEC14p synthesis. Taken together, these data provide evidence that the high level of structural conservation between SEC14p and SEC14pKL reflects a functional identity between these polypeptides as well. On the basis of the SEC14p and SEC14pKL primary sequence homology to the human retinaldehyde-binding protein, we suggest that the general function of these SEC14p species may be to regulate the delivery of a hydrophobic ligand to Golgi membranes so that biosynthetic secretory traffic can be supported. Images PMID:2198263

  5. Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications

    PubMed Central

    Rico-Díaz, Agustín; Álvarez-Cao, María-Efigenia; Escuder-Rodríguez, Juan-José; González-Siso, María-Isabel; Cerdán, M. Esperanza; Becerra, Manuel

    2017-01-01

    Kluyveromyces lactis β-galactosidase (Kl-β-Gal) is one of the most important enzymes in the dairy industry. The poor stability of this enzyme limits its use in the synthesis of galactooligosaccharides (GOS) and other applications requiring high operational temperature. To obtain thermoresistant variants, a rational mutagenesis strategy by introducing disulphide bonds in the interface between the enzyme subunits was used. Two improved mutants, R116C/T270C and R116C/T270C/G818C, had increased half-lives at 45 °C compared to Kl-β-Gal (2.2 and 6.8 fold increases, respectively). Likewise, Tm values of R116C/T270C and R116C/T270C/G818C were 2.4 and 8.5 °C, respectively, higher than Kl-β-Gal Tm. Enrichment in enzymatically active oligomeric forms in these mutant variants also increased their catalytic efficiency, due to the reinforcement of the interface contacts. In this way, using an artificial substrate (p-nitrophenyl-β-D-galactopyranoside), the Vmax values of the mutants were ~1.4 (R116C/T270C) and 2 (R116C/T270C/G818C) fold higher than that of native Kl-β-Gal. Using the natural substrate (lactose) the Vmax for R116C/T270C/G818C almost doubled the Vmax for Kl-β-Gal. Validation of these mutant variants of the enzyme for their use in applications that depend on prolonged incubations at high temperatures was achieved at the laboratory scale by monitoring their catalytic activity in GOS synthesis. PMID:28361909

  6. Secretion of Human Serum Albumin by Kluyveromyces lactis Overexpressing KlPDI1 and KlERO1

    PubMed Central

    Lodi, Tiziana; Neglia, Barbara; Donnini, Claudia

    2005-01-01

    The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast Kluyveromyces lactis and evaluated the secretion of heterologous proteins. KlERO1, an orthologue of Saccharomyces cerevisiae ERO1, was cloned by functional complementation of the ts phenotype of an Scero1 mutant. The expression of KlERO1 was induced by treatment of the cells with dithiothreitol and by overexpression of human serum albumin (HSA), a disulfide bond-rich protein. Duplication of either PDI1 or ERO1 led to a similar increase in HSA yield. Duplication of both genes accelerated the secretion of HSA and improved cell growth rate and yield. Increasing the dosage of KlERO1 did not affect the production of human interleukin 1β, a protein that has no disulfide bridges. The results confirm that the ERO1 genes of S. cerevisiae and K. lactis are functionally similar even though portions of their coding sequence are quite different and the phenotypes of mutants overexpressing the genes differ. The marked effects of KlERO1 copy number on the expression of heterologous proteins with a high number of disulfide bridges suggests that control of KlERO1 and KlPDI1 is important for the production of high levels of heterologous proteins of this type. PMID:16085825

  7. Trehalose-mediated inhibition of the plasma membrane H+-ATPase from Kluyveromyces lactis: dependence on viscosity and temperature.

    PubMed

    Sampedro, José G; Muñoz-Clares, Rosario A; Uribe, Salvador

    2002-08-01

    The effect of increasing trehalose concentrations on the kinetics of the plasma membrane H+-ATPase from Kluyveromyces lactis was studied at different temperatures. At 20 degrees C, increasing concentrations of trehalose (0.2 to 0.8 M) decreased V(max) and increased S(0.5) (substrate concentration when initial velocity equals 0.5 V(max)), mainly at high trehalose concentrations (0.6 to 0.8 M). The quotient V(max)/S(0.5) decreased from 5.76 micromol of ATP mg of protein(-1) x min(-1) x mM(-1) in the absence of trehalose to 1.63 micromol of ATP mg of protein(-1) x min(-1) x mM(-1) in the presence of 0.8 M trehalose. The decrease in V(max) was linearly dependent on solution viscosity (eta), suggesting that inhibition was due to hindering of protein domain diffusional motion during catalysis and in accordance with Kramer's theory for reactions in solution. In this regard, two other viscosity-increasing agents, sucrose and glycerol, behaved similarly, exhibiting the same viscosity-enzyme inhibition correlation predicted. In the absence of trehalose, increasing the temperature up to 40 degrees C resulted in an exponential increase in V(max) and a decrease in enzyme cooperativity (n), while S(0.5) was not modified. As temperature increased, the effect of trehalose on V(max) decreased to become negligible at 40 degrees C, in good correlation with the temperature-mediated decrease in viscosity. The trehalose-mediated increase in S(0.5) was similar at all temperatures tested, and thus, trehalose effects on V(max)/S(0.5) were always observed. Trehalose increased the activation energy for ATP hydrolysis. Trehalose-mediated inhibition of enzymes may explain why yeast rapidly hydrolyzes trehalose when exiting heat shock.

  8. Role of Snf1p in Regulation of Intracellular Sorting of the Lactose and Galactose Transporter Lac12p in Kluyveromyces lactis†

    PubMed Central

    Wiedemuth, Christian; Breunig, Karin D.

    2005-01-01

    The protein kinase Snf1/AMPK plays a central role in carbon and energy homeostasis in yeasts and higher eukaryotes. To work out which aspects of the Snf1-controlled regulatory network are conserved in evolution, the Snf1 requirement in galactose metabolism was analyzed in the yeast Kluyveromyces lactis. Whereas galactose induction was only delayed, K. lactis snf1 mutants failed to accumulate the lactose/galactose H+ symporter Lac12p in the plasma membran,e as indicated by Lac12-green fluorescent protein fusions. In contrast to wild-type cells, the fusion protein was mostly intracellular in the mutant. Growth on galactose and galactose uptake could be restored by the KHT3 gene, which encodes a new transporter of the HXT subfamily of major facilitators These findings indicate a new role of Snf1p in regulation of sugar transport in K. lactis. PMID:15821131

  9. A new regulatory element mediates ethanol repression of KlADH3, a Kluyveromyces lactis gene coding for a mitochondrial alcohol dehydrogenase.

    PubMed

    Saliola, Michele; Getuli, Claudia; Mazzoni, Cristina; Fantozzi, Ivana; Falcone, Claudio

    2007-08-01

    KlADH3 is a Kluyveromyces lactis alcohol dehydrogenase gene induced in the presence of all respiratory carbon sources except ethanol, which specifically represses this gene. Deletion analysis of the KlADH3 promoter revealed the presence of both positive and negative elements. However, by site-directed mutagenesis and gel retardation experiments, we identified a 15-bp element responsible for the transcriptional repression of this gene by ethanol. In particular, this element showed putative sites required for the sequential binding of ethanol-induced factors responsible for the repressed conditions, and the binding of additional factors relieved repression. In addition, we showed that the ethanol element was required for in vivo repression of KlAdh3 activity.

  10. Study of the influence of yeast inoculum concentration (Yarrowia lipolytica and Kluyveromyces lactis) on blue cheese aroma development using microbiological models.

    PubMed

    Price, Elliott J; Linforth, Robert S T; Dodd, Christine E R; Phillips, Carol A; Hewson, Louise; Hort, Joanne; Gkatzionis, Konstantinos

    2014-02-15

    Yarrowia lipolytica and Kluyveromyces lactis occur as part of Stilton cheese microflora yet are not controlled during production. This study investigated the influence of their inoculum concentration on aroma production. Models of Y. lipolytica and K. lactis, with Penicillium roqueforti, were analysed using instrumental and sensory analysis. Different concentrations of Y. lipolytica produced important changes in the aroma profiles of microbiological models, analysed by solid-phase microextraction (SPME GC-MS). Sensory analysis with discrimination tests showed differences were detectable via human perception but did not concern the similarity to blue cheese odour. Increasing the inoculum concentration of K. lactis resulted in decreased variation between replicates. Partial least squares (PLS) regression on Flash profile data showed models inoculated with low concentrations of K. lactis exhibited blue cheese-related attributes, associated with increased ketone production. Results suggest that controlling the amount of Y. lipolytica and K. lactis during production offers potential to manipulate blue cheese aroma development.

  11. Establishing the Yeast Kluyveromyces lactis as an Expression Host for Production of the Saposin-Like Domain of the Aspartic Protease Cirsin

    PubMed Central

    Curto, Pedro; Lufrano, Daniela; Pinto, Cátia; Custódio, Valéria; Gomes, Ana Catarina; Trejo, Sebastián A.; Bakás, Laura; Vairo-Cavalli, Sandra; Faro, Carlos

    2014-01-01

    Typical plant aspartic protease zymogens comprise a characteristic and plant-specific insert (PSI). PSI domains can interact with membranes, and a role as a defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet due to problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI, as well as its nonglycosylated mutant. A purification protocol with protein yields of ∼4.0 mg/liter was established for both wild-type and nonglycosylated PSIs, which represents the highest reported yield for a nontagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its antifungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins. PMID:24123748

  12. Simultaneous saccharification and fermentation of delignified lignocellulosic biomass at high solid loadings by a newly isolated thermotolerant Kluyveromyces sp. for ethanol production.

    PubMed

    Narra, Madhuri; James, Jisha P; Balasubramanian, Velmurugan

    2015-03-01

    Simultaneous saccharification and fermentation studies were carried out using thermotolerant newly isolated Kluyveromyces sp. with three different delignified lignocellulosic biomass viz. rice straw, wheat straw and sugarcane bagasse at 5-15% solid loading and 6-12 FPU g(-1) substrate enzyme loading for different time intervals 0-72 h at 42°C. Maximum ethanol achieved from rice straw, wheat straw and sugarcane bagasse with in-house crude cellulases from Aspergillus terreus was 23.23, 18.29 and 17.91 mg mL(-1) at 60 h with 10% solid load and 9 FPU g(-1) substrate enzyme loading. Tween 80 1% (v/v) enhanced the ethanol yield by 8.39%, 9.26% and 8.14% in rice straw, wheat straw and sugarcane bagasse, respectively. External supplementation of β-glucosidase to the crude as well commercial cellulases produced maximum theoretical ethanol yield of 71.76%, 63.77%, 57.15% and 84.56%, 72.47%, 70.55% from rice straw, wheat straw and sugarcane bagasse, respectively.

  13. Glucose repression of lactose/galactose metabolism in Kluyveromyces lactis is determined by the concentration of the transcriptional activator LAC9 (K1GAL4) [corrected

    PubMed Central

    Zachariae, W; Kuger, P; Breunig, K D

    1993-01-01

    In the budding yeast Kluyveromyces lactis glucose repression of genes involved in lactose and galactose metabolism is primarily mediated by LAC9 (or K1GAL4) the homologue of the well-known Saccharomyces cerevisiae transcriptional activator GAL4. Phenotypic difference in glucose repression existing between natural strains are due to differences in the LAC9 gene (Breunig, 1989, Mol.Gen.Genet. 261, 422-427). Comparison between the LAC9 alleles of repressible and non-repressible strains revealed that the phenotype is a result of differences in LAC9 gene expression. A two-basepair alteration in the LAC9 promoter region produces a promoter-down effect resulting in slightly reduced LAC9 protein levels under all growth conditions tested. In glucose/galactose medium any change in LAC9 expression drastically affects expression of LAC9 controlled genes e.g. those encoding beta-galactosidase or galactokinase revealing a strong dependence of the kinetics of induction on the LAC9 concentration. We propose that in tightly repressible strains the activator concentration drops below a critical threshold that is required for induction to occur. A model is presented to explain how small differences in activator levels are amplified to produce big changes in expression levels of metabolic genes. Images PMID:8441621

  14. Molecular cloning of the plasma membrane H(+)-ATPase from Kluyveromyces lactis: a single nucleotide substitution in the gene confers ethidium bromide resistance and deficiency in K+ uptake.

    PubMed Central

    Miranda, M; Ramírez, J; Peña, A; Coria, R

    1995-01-01

    A Kluyveromyces lactis strain resistant to ethidium bromide and deficient in potassium uptake was isolated. Studies on the proton-pumping activity of the mutant strain showed that a decreased H(+)-ATPase specific activity was responsible for the observed phenotypes. The putative K. lactis PMA1 gene encoding the plasma membrane H(+)-ATPase was cloned by its ability to relieve the potassium transport defect of this mutant and by reversing its resistance to ethidium bromide. Its deduced amino acid sequence predicts a protein 899 residues long that is structurally colinear in its full length to H(+)-ATPases cloned from different yeasts, except for the presence of a variable N-terminal domain. By PCR-mediated amplification, we identified a transition from G to A that rendered the substitution of the fully conserved methionine at position 699 by isoleucine. We attribute to this amino acid change the low capacity of the mutant H(+)-ATPase to pump out protons. PMID:7730265

  15. Heterologous Protein Secretion Directed by a Repressible Acid Phosphatase System of Kluyveromyces lactis: Characterization of Upstream Region-Activating Sequences in the KIPHO5 Gene

    PubMed Central

    Fermiñán, Encarnación; Domínguez, Angel

    1998-01-01

    Transcription of the repressible acid phosphatase gene (KIPHO5) in Kluyveromyces lactis is strongly regulated in response to the level of inorganic phosphate (Pi) present in the growth medium. We have begun a study of the promoter region of this gene in order to identify sequences involved in the phosphate control of KIPHO5 expression and to design new expression-secretion systems in K. lactis. Deletion analysis and directed mutagenesis revealed two major identical upstream activating sequences (UAS) CACGTG at positions −430 (UAS1) and −192 (UAS2) relative to the ATG initiation codon. These sequences are identical to those described for Saccharomyces cerevisiae for the binding of Pho4p. Deletion or directed mutagenesis of either one or both UAS reduce KIPHO5 expression by the same amount (approximately 80%). When fused to the coding region of trout growth hormone cDNA (tGH-II), the promoter and signal peptide-encoding region of the phosphate-repressible KIPHO5 gene drives the expression of this gene and the secretion of the tGHII protein. Synthesis of tGHIIp in K. lactis transformants carrying this construct was found to be regulated by the Pi present in the medium; derepression of heterologous protein expression can therefore be achieved by lowering the Pi concentration. PMID:9647807

  16. Bioethanol production from sodium hydroxide/hydrogen peroxide-pretreated water hyacinth via simultaneous saccharification and fermentation with a newly isolated thermotolerant Kluyveromyces marxianu strain.

    PubMed

    Yan, Jinping; Wei, Zhilei; Wang, Qiaoping; He, Manman; Li, Shumei; Irbis, Chagan

    2015-10-01

    In this study, bioethanol production from NaOH/H2O2-pretreated water hyacinth was investigated. Pretreatment of water hyacinth with 1.5% (v/v) H2O2 and 3% (w/v) NaOH at 25 °C increased the production of reducing sugars (223.53 mg/g dry) and decreased the cellulose crystallinity (12.18%), compared with 48.67 mg/g dry and 22.80% in the untreated sample, respectively. The newly isolated Kluyveromyces marxianu K213 showed greater ethanol production from glucose (0.43 g/g glucose) at 45 °C than did the control Saccharomyces cerevisiae angel yeast. The maximum ethanol concentration (7.34 g/L) achieved with K. marxianu K213 by simultaneous saccharification and fermentation (SSF) from pretreated water hyacinth at 42 °C was 1.78-fold greater than that produced by angel yeast S. cerevisiae at 30 °C. The present work demonstrates that bioethanol production achieved via SSF of NaOH/H2O2-pretreated water hyacinth with K. marxianu K213 is a promising strategy to utilize water hyacinth biomass.

  17. Maintenance of very long telomeres by recombination in the Kluyveromyces lactis stn1-M1 mutant involves extreme telomeric turnover, telomeric circles, and concerted telomeric amplification.

    PubMed

    Xu, Jianing; McEachern, Michael J

    2012-08-01

    Some cancers utilize the recombination-dependent process of alternative lengthening of telomeres (ALT) to maintain long heterogeneous telomeres. Here, we studied the recombinational telomere elongation (RTE) of the Kluyveromyces lactis stn1-M1 mutant. We found that the total amount of the abundant telomeric DNA in stn1-M1 cells is subject to rapid variation and that it is likely to be primarily extrachromosomal. Rad50 and Rad51, known to be required for different RTE pathways in Saccharomyces cerevisiae, were not essential for the production of either long telomeres or telomeric circles in stn1-M1 cells. Circles of DNA containing telomeric repeats (t-circles) either present at the point of establishment of long telomeres or introduced later into stn1-M1 cells each led to the formation of long tandem arrays of the t-circle's sequence, which were incorporated at multiple telomeres. These tandem arrays were extraordinarily unstable and showed evidence of repeated rounds of concerted amplification. Our results suggest that the maintenance of telomeres in the stn1-M1 mutant involves extreme turnover of telomeric sequences from processes including both large deletions and the copying of t-circles.

  18. A NADP-glutamate dehydrogenase mutant of the petit-negative yeast Kluyveromyces lactis uses the glutamine synthetase-glutamate synthase pathway for glutamate biosynthesis.

    PubMed

    Valenzuela, L; Guzmán-León, S; Coria, R; Ramírez, J; Aranda, C; González, A

    1995-10-01

    The activities of the enzymes involved in ammonium assimilation and glutamate biosynthesis were determined in wild-type and NADP-glutamate dehydrogenase (GDH) null mutant strains of Kluyveromyces lactis. The specific NADP-GDH activity from K. lactis was fivefold lower than that found in Saccharomyces cerevisiae. The glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were similar to those reported in S. cerevisiae. The NADP-GDH null mutant was obtained by transforming the uraA strain MD2/1 with a linearized integrative yeast vector harbouring a 390 bp fragment of the NADP-GDH structural gene. This mutant grew as well as the parent strain on ammonium, but showed GS and GOGAT activities higher that those found in the wild-type strain, implying that the GS-GOGAT pathway could play a leading role in glutamate biosynthesis in K. lactis. Southern blotting analysis of K. lactis chromosomes separated by contour-clamped homogeneous electric field electrophoresis, indicated that the NADP-GDH structural gene is localized on chromosome VI.

  19. Constitutive expression in gal7 mutants of Kluyveromyces lactis is due to internal production of galactose as an inducer of the Gal/Lac regulon.

    PubMed Central

    Cardinali, G; Vollenbroich, V; Jeon, M S; de Graaf, A A; Hollenberg, C P

    1997-01-01

    The induction process of the galactose regulon has been intensively studied, but until now the nature of the inducer has remained unknown. We have analyzed a delta gal7 mutant of the yeast Kluyveromyces lactis, which lacks the galactotransferase activity and is able to express the genes of the Gal/Lac regulon also in the absence of galactose. We found that this expression is semiconstitutive and undergoes a strong induction during the stationary phase. The gal1-209 mutant, which has a reduced kinase activity but retains its positive regulatory function, also shows a constitutive expression of beta-galactosidase, suggesting that galactose is the inducer. A gal10 deletion in delta gal7 or gal1-209 mutants reduces the expression to under wild-type levels. The presence of the inducer could be demonstrated in both delta gal7 crude extracts and culture medium by means of a bioassay using the induction in gal1-209 cells. A mutation in the transporter gene LAC12 decreases the level of induction in gal7 cells, indicating that galactose is partly released into the medium and then retransported into the cells. Nuclear magnetic resonance analysis of crude extracts from delta gal7 cells revealed the presence of 50 microM galactose. We conclude that galactose is the inducer of the Gal/Lac regulon and is produced via UDP-galactose through a yet-unknown pathway. PMID:9032299

  20. Glucose represses the lactose-galactose regulon in Kluyveromyces lactis through a SNF1 and MIG1- dependent pathway that modulates galactokinase (GAL1) gene expression.

    PubMed Central

    Dong, J; Dickson, R C

    1997-01-01

    Expression of the lactose-galactose regulon in Kluyveromyces lactis is induced by lactose or galactose and repressed by glucose. Some components of the induction and glucose repression pathways have been identified but many remain unknown. We examined the role of the SNF1 (KlSNF1) and MIG1 (KlMIG1) genes in the induction and repression pathways. Our data show that full induction of the regulon requires SNF1; partial induction occurs in a Klsnf1 -deleted strain, indicating that a KlSNF1 -independent pathway(s) also regulates induction. MIG1 is required for full glucose repression of the regulon, but there must be a KlMIG1 -independent repression pathway also. The KlMig1 protein appears to act downstream of the KlSnf1 protein in the glucose repression pathway. Most importantly, the KlSnf1-KIMig repression pathway operates by modulating KlGAL1 expression. Regulating KlGAL1 expression in this manner enables the cell to switch the regulon off in the presence of glucose. Overall, our data show that, while the Snf1 and Mig1 proteins play similar roles in regulating the galactose regulon in Saccharomyces cerevisiae and K.lactis , the way in which these proteins are integrated into the regulatory circuits are unique to each regulon, as is the degree to which each regulon is controlled by the two proteins. PMID:9278487

  1. Production of volatile compounds by cheese-ripening yeasts: requirement for a methanethiol donor for S-methyl thioacetate synthesis by Kluyveromyces lactis.

    PubMed

    Arfi, K; Spinnler, H E; Tache, R; Bonnarme, P

    2002-03-01

    Five cheese-ripening yeasts (Geotrichum candidum, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica and Debaryomyces hansenii) were compared with respect to their ability to generate volatile aroma compounds. K. lactis produced a variety of esters - ethylacetate (EA) being the major one - and relatively limited amounts of volatile sulphur compounds (VSCs). Conversely, G. candidum produced significant amounts of VSCs [with the thioester S-methyl thioacetate (MTA) being the most prevalent] and lower quantities of non-sulphur volatile compounds than K. lactis. We suspect that K. lactis is able to produce and/or accumulate acetyl CoA - a common precursor of MTA and EA - but that it produces limited amounts of methanethiol (MTL); both acetyl CoA and MTL are precursors for MTA synthesis. When supplemented with exogenous MTL, MTA production greatly increased in K. lactis cultures whereas it was unchanged in G. candidum cultures, suggesting that MTL is a limiting factor for MTA synthesis in K. lactis but not in G. candidum. Our results are discussed with respect to L-methionine catabolism.

  2. The nuclear genes encoding the internal (KlNDI1) and external (KlNDE1) alternative NAD(P)H:ubiquinone oxidoreductases of mitochondria from Kluyveromyces lactis.

    PubMed

    Tarrío, N; Díaz Prado, S; Cerdán, M E; González Siso, M I

    2005-01-01

    Cloning, sequence and functional analyses of the Kluyveromyces lactis genes KlNDI1 and KlNDE1 are reported. These genes encode for proteins with high homology to the mitochondrial internal (Ndi1p) and external (Nde1p) alternative NADH:ubiquinone oxidoreductases from Saccharomyces cerevisiae and complement the respective mutations. Analysis of KlNDI1 transcriptional regulation showed that expression of this gene is lower in 2% glucose than in 0.5% glucose or non-fermentable carbon sources. Beta-galactosidase activity values, shown by lacZ fusions of KlNDI1 promoter deletions, suggested that two Adr1p binding sites mediate this carbon source regulation of KlNDI1. The expression of the KlNDE1 gene in S. cerevisiae mutant strains and measurement of respiration with isolated mitochondria showed that the protein encoded by KlNDE1 oxidizes NADPH, this being an important difference with respect to the conventional yeast S. cerevisiae. Moreover, Northern blot experiments using a phosphoglucose isomerase mutant showed that KlNDE1 gene transcription increases with glucose metabolism through the pentose phosphate pathway.

  3. Gal80 proteins of Kluyveromyces lactis and Saccharomyces cerevisiae are highly conserved but contribute differently to glucose repression of the galactose regulon.

    PubMed Central

    Zenke, F T; Zachariae, W; Lunkes, A; Breunig, K D

    1993-01-01

    We cloned the GAL80 gene encoding the negative regulator of the transcriptional activator Gal4 (Lac9) from the yeast Kluyveromyces lactis. The deduced amino acid sequence of K. lactis GAL80 revealed a strong structural conservation between K. lactis Gal80 and the homologous Saccharomyces cerevisiae protein, with an overall identity of 60% and two conserved blocks with over 80% identical residues. K. lactis gal80 disruption mutants show constitutive expression of the lactose/galactose metabolic genes, confirming that K. lactis Gal80 functions in essentially in the same way as does S. cerevisiae Gal80, blocking activation by the transcriptional activator Lac9 (K. lactis Gal4) in the absence of an inducing sugar. However, in contrast to S. cerevisiae, in which Gal4-dependent activation is strongly inhibited by glucose even in a gal80 mutant, glucose repressibility is almost completely lost in gal80 mutants of K. lactis. Indirect evidence suggests that this difference in phenotype is due to a higher activator concentration in K. lactis which is able to overcome glucose repression. Expression of the K. lactis GAL80 gene is controlled by Lac9. Two high-affinity binding sites in the GAL80 promoter mediate a 70-fold induction by galactose and hence negative autoregulation by Gal80. Gal80 in turn not only controls Lac9 activity but also has a moderate influence on its rate of synthesis. Thus, a feedback control mechanism exists between the positive and negative regulators. By mutating the Lac9 binding sites of the GAL80 promoter, we could show that induction of GAL80 is required to prevent activation of the lactose/galactose regulon in glycerol or glucose plus galactose, whereas the noninduced level of Gal80 is sufficient to completely block Lac9 function in glucose. Images PMID:8246973

  4. Key Role of Ser562/661 in Snf1-Dependent Regulation of Cat8p in Saccharomyces cerevisiae and Kluyveromyces lactis

    PubMed Central

    Charbon, Godefroid; Breunig, Karin D.; Wattiez, Ruddy; Vandenhaute, Jean; Noël-Georis, Isabelle

    2004-01-01

    Utilization of nonfermentable carbon sources by Kluyveromyces lactis and Saccharomyces cerevisiae requires the Snf1p kinase and the Cat8p transcriptional activator, which binds to carbon source-responsive elements of target genes. We demonstrate that KlSnf1p and KlCat8p from K. lactis interact in a two-hybrid system and that the interaction is stronger with a kinase-dead mutant form of KlSnf1p. Of two putative phosphorylation sites in the KlCat8p sequence, serine 661 was identified as a key residue governing KlCat8p regulation. Serine 661 is located in the middle homology region, a regulatory domain conserved among zinc cluster transcription factors, and is part of an Snf1p consensus phosphorylation site. Single mutations at this site are sufficient to completely change the carbon source regulation of the KlCat8p transactivation activity observed. A serine-to-glutamate mutant form mimicking constitutive phosphorylation results in a nearly constitutively active form of KlCat8p, while a serine-to-alanine mutation has the reverse effect. Furthermore, it is shown that KlCat8p phosphorylation depends on KlSNF1. The Snf1-Cat8 connection is evolutionarily conserved: mutation of corresponding serine 562 of ScCat8p gave similar results in S. cerevisiae. The enhanced capacity of ScCat8S562E to suppress the phenotype caused by snf1 strengthens the hypothesis of direct phosphorylation of Cat8p by Snf1p. Unlike that of S. cerevisiae ScCAT8, KlCAT8 transcription is not carbon source regulated, illustrating the prominent role of posttranscriptional regulation of Cat8p in K. lactis. PMID:15121831

  5. Growth kinetics and physiological behavior of co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis, fermenting carob sugars extracted with whey.

    PubMed

    Rodrigues, B; Lima-Costa, M E; Constantino, A; Raposo, S; Felizardo, C; Gonçalves, D; Fernandes, T; Dionísio, L; Peinado, J M

    2016-10-01

    Alcoholic fermentation of carob waste sugars (sucrose, glucose and fructose) extracted with cheese whey, by co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis has been analyzed. Growth and fermentation of S. cerevisiae in the carob-whey medium showed an inhibition of about 30% in comparison with water-extracted carob. The inhibition of K. lactis on carob-whey was greater (70%) when compared with the whey medium alone, due to osmolarity problems. Oxygen availability was a very important factor for K. lactis, influencing its fermentation performance. When K. lactis was grown alone on carob-whey medium, lactose was always consumed first, and glucose and fructose were consumed afterwards, only at high aeration conditions. In co-culture with S. cerevisiae, K. lactis was completely inhibited and, at low aeration, died after 3 days; at high aeration this culture could survive but growth and lactose fermentation were only recovered after S. cerevisiae became stationary. To overcome the osmolarity and K. lactis' oxygen problems, the medium had to be diluted and a sequential fermentative process was designed in a STR-3l reactor. K. lactis was inoculated first and, with low aeration (0.13vvm), consumed all the lactose in 48h. Then S. cerevisiae was inoculated, consuming the total of the carob sugars, and producing ethanol in a fed-batch regime. The established co-culture with K. lactis increased S. cerevisiae ethanol tolerance. This fermentation process produced ethanol with good efficiency (80g/l final concentration and a conversion factor of 0.4g ethanol/g sugar), eliminating all the sugars of the mixed waste. These efficient fermentative results pointed to a new joint treatment of agro-industrial wastes which may be implemented successfully, with economic and environmental sustainability for a bioethanol industrial proposal.

  6. RAS/Cyclic AMP and Transcription Factor Msn2 Regulate Mating and Mating-Type Switching in the Yeast Kluyveromyces lactis ▿

    PubMed Central

    Barsoum, E.; Rajaei, N.; Åström, S. U.

    2011-01-01

    In response to harsh environmental conditions, ascomycetes produce stress-resistant spores to promote survival. As sporulation requires a diploid DNA content, species with a haploid lifestyle, such as Kluyveromyces lactis, first induce mating in response to stress. In K. lactis, mating and mating-type switching are induced by the DNA-binding protein Mts1. Mts1 expression is known to be upregulated by nutrient limitation, but the mechanism is unknown. We show that a ras2 mutation results in a hyperswitching phenotype. In contrast, strains lacking the phosphodiesterase Pde2 had lower switching rates compared to that of the wild type (WT). As Ras2 promotes cyclic AMP (cAMP) production and Pde2 degrades cAMP, these data suggest that low cAMP levels induce switching. Because the MTS1 regulatory region contains several Msn2 binding sites and Msn2 is a transcription factor that is activated by low cAMP levels, we investigated if Msn2 regulates MTS1 transcription. Consistently with this idea, an msn2 mutant strain displayed lower switching rates than the WT strain. The transcription of MTS1 is highly induced in the ras2 mutant strain. In contrast, an msn2 ras2 double mutant strain displays WT levels of the MTS1 transcript, showing that Msn2 is a critical inducer of MTS1 transcription. Strains lacking Msn2 and Pde2 also exhibit mating defects that can be complemented by the ectopic expression of Mts1. Finally, we show that MTS1 is subjected to negative autoregulation, presumably adding robustness to the mating and switching responses. We suggest a model in which Ras2/cAMP/Msn2 mediates the stress-induced mating and mating-type switching responses in K. lactis. PMID:21890818

  7. Inactivation of the Kluyveromyces lactis KlPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobic alcoholic fermentation.

    PubMed

    Zeeman, A M; Luttik, M A; Thiele, C; van Dijken, J P; Pronk, J T; Steensma, H Y

    1998-12-01

    The KlPDA1 gene, encoding the E1alpha subunit of the mitochondrial pyruvate-dehydrogenase (PDH) complex was isolated from a Kluyveromyces lactis genomic library by screening with a 1.1 kb internal fragment of the Saccharomyces cerevisiae PDA1 gene. The predicted amino acid sequence encoded by KlPDA1 showed 87% similarity and 79% identity to its S. cerevisiae counterpart. Disruption of KIPDA1 resulted in complete absence of PDH activity in cell extracts. The maximum specific growth rate on glucose of null mutants was 3.5-fold lower than that of the wild-type, whereas growth on ethanol was unaffected. Wild-type K. lactis CBS 2359 exhibits a Crabtree-negative phenotype, i.e. no ethanol was produced in aerobic batch cultures grown on glucose. In contrast, substantial amounts of ethanol and acetaldehyde were produced in aerobic cultures of an isogenic Klpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KlPDA1 gene but not, as previously found for S. cerevisiae pda1 mutants, by cultivation in the presence of leucine. The occurrence of aerobic fermentation and slow growth of the Klpda1 null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only at relatively low growth rates (D = 0.10 h(-1)) in aerobic, glucose-limited chemostat cultures, could the PDH bypass completely replace the PDH complex, thus allowing fully respiratory growth. This resulted in a lower biomass yield [g biomass (g glucose)-1] than in the wild-type due to a higher consumption of ATP in the PDH bypass compared to the formation of acetyl-CoA via the PDH complex.

  8. Use of synthetic genes for cloning, production and functional expression of the bacteriocins enterocin A and bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis.

    PubMed

    Jiménez, Juan J; Borrero, Juan; Gútiez, Loreto; Arbulu, Sara; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

    2014-06-01

    The use of synthetic genes may constitute a successful approach for the heterologous production and functional expression of bacterial antimicrobial peptides (bacteriocins) by recombinant yeasts. In this work, synthetic genes with adapted codon usage designed from the mature amino acid sequence of the bacteriocin enterocin A (EntA), produced by Enterococcus faecium T136, and the mature bacteriocin E 50-52 (BacE50-52), produced by E. faecium NRRL B-32746, were synthesized. The synthetic entA and bacE50-52 were cloned into the protein expression vectors pPICZαA and pKLAC2 for transformation of derived vectors into Pichia pastoris X-33 and Kluyveromyces lactis GG799, respectively. The recombinant vectors were linearized and transformed into competent cells selecting for P. pastoris X-33EAS (entA), P. pastoris X-33BE50-52S (bacE50-52), K. lactis GG799EAS (entA), and K. lactis GG799BE50-52S (bacE50-52). P. pastoris X-33EAS and K. lactis GG799EAS, but not P. pastoris X-33BE50-52S and K. lactis GG799BE50-52S, showed antimicrobial activity in their supernatants. However, purification of the supernatants of the producer yeasts permitted recovery of the bacteriocins EntA and BacE50-52. Both purified bacteriocins were active against Gram-positive bacteria such as Listeria monocytogenes but not against Gram-negative bacteria, including Campylobacter jejuni.

  9. The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene.

    PubMed Central

    Kuzhandaivelu, N; Jones, W K; Martin, A K; Dickson, R C

    1992-01-01

    Induction of the lactose-galactose regulon is strongly repressed by glucose in some but not all strains of Kluyveromyces lactis. We show here that in strongly repressed strains, two to three times less Kl-GAL4 mRNA is synthesized and that expression of structural genes in the regulon such as LAC4, the structural gene for beta-galactosidase, is down regulated 40-fold or more. Comparative analysis of strains having a strong or weak repression phenotype revealed a two-base difference in the promoter of the Kl-GAL4 (also called LAC9) positive regulatory gene. This two-base difference is responsible for the strong versus the weak repression phenotype. The two base changes are symmetrically located in a DNA sequence having partial twofold rotational symmetry (14 of 21 bases). We hypothesize that this region functions as a sensitive regulatory switch, an upstream repressor sequence (URS). According to our model, the presence of glucose in the culture medium signals, by an unidentified pathway, a repressor protein to bind the URS. Binding reduces transcription of the Kl-GAL4 gene so that the concentration of the Kl-GAL4 protein falls below the level needed for induction of LAC4 and other genes in the regulon. For strains showing weak glucose repression, we hypothesize that the two base changes in the URS reduce repressor binding so that the regulon is not repressed. Our results illustrate an important principle of genetic regulation: a small (2- to 3-fold) change in the concentration of a regulatory protein can produce a large (40-fold or greater) change in expression of structural genes. This mechanism of signal amplification could play a role in many biological phenomena that require regulated transcription. Images PMID:1569929

  10. 21 CFR 184.1685 - Rennet (animal-derived) and chymosin preparation (fermentation-derived).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ..., from a nonpathogenic and nontoxigenic strain of Escherichia coli K-12 containing the prochymosin gene... nontoxigenic strain of Kluyveromyces marxianus variety lactis, containing the prochymosin gene. The prochymosin... prochymosin gene. Chymosin is recovered from the fermentation broth after acid treatment. All materials...

  11. Ineffective Phosphorylation of Mitogen-Activated Protein Kinase Hog1p in Response to High Osmotic Stress in the Yeast Kluyveromyces lactis

    PubMed Central

    Velázquez-Zavala, Nancy; Rodríguez-González, Miriam; Navarro-Olmos, Rocío; Ongay-Larios, Laura; Kawasaki, Laura; Torres-Quiroz, Francisco

    2015-01-01

    When treated with a hyperosmotic stimulus, Kluyveromyces lactis cells respond by activating the mitogen-activated protein kinase (MAPK) K. lactis Hog1 (KlHog1) protein via two conserved branches, SLN1 and SHO1. Mutants affected in only one branch can cope with external hyperosmolarity by activating KlHog1p by phosphorylation, except for single ΔKlste11 and ΔKlste50 mutants, which showed high sensitivity to osmotic stress, even though the other branch (SLN1) was intact. Inactivation of both branches by deletion of KlSHO1 and KlSSK2 also produced sensitivity to high salt. Interestingly, we have observed that in ΔKlste11 and ΔKlsho1 ΔKlssk2 mutants, which exhibit sensitivity to hyperosmotic stress, and contrary to what would be expected, KlHog1p becomes phosphorylated. Additionally, in mutants lacking both MAPK kinase kinases (MAPKKKs) present in K. lactis (KlSte11p and KlSsk2p), the hyperosmotic stress induced the phosphorylation and nuclear internalization of KlHog1p, but it failed to induce the transcriptional expression of KlSTL1 and the cell was unable to grow in high-osmolarity medium. KlHog1p phosphorylation via the canonical HOG pathway or in mutants where the SHO1 and SLN1 branches have been inactivated requires not only the presence of KlPbs2p but also its kinase activity. This indicates that when the SHO1 and SLN1 branches are inactivated, high-osmotic-stress conditions activate an independent input that yields active KlPbs2p, which, in turn, renders KlHog1p phosphorylation ineffective. Finally, we found that KlSte11p can alleviate the sensitivity to hyperosmotic stress displayed by a ΔKlsho1 ΔKlssk2 mutant when it is anchored to the plasma membrane by adding the KlSho1p transmembrane segments, indicating that this chimeric protein can substitute for KlSho1p and KlSsk2p. PMID:26150414

  12. Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food

    PubMed Central

    Mahor, Durga; Priyanka, Anu; Prasad, Gandham S; Thakur, Krishan Gopal

    2016-01-01

    Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a

  13. Positive regulation of the beta-galactosidase gene from Kluyveromyces lactis is mediated by an upstream activation site that shows homology to the GAL upstream activation site of Saccharomyces cerevisiae.

    PubMed Central

    Ruzzi, M; Breunig, K D; Ficca, A G; Hollenberg, C P

    1987-01-01

    In contrast to the Escherichia coli lac operon, the yeast beta-galactosidase gene is positively regulated. In the 5'-noncoding region of the Kluyveromyces lactis LAC4 gene, we mapped an upstream activation site (UAS) that is required for induction. This sequence, located between positions -435 and -326 from the start of translation, functions irrespective of its orientation and can confer lactose regulation to the heterologous CYC1 promoter. It is composed of at least two subsequences that must act in concert. One of these subsequences showed a strong homology to the UAS consensus sequence of the Saccharomyces cerevisiae GAL genes (E. Giniger, S. M. Varnum, and M. Ptashne, Cell 40:767-774, 1985). We propose that this region of homology located at about position -426 is a binding site for the product of the regulatory gene LAC9 which probably induces transcription of the LAC4 gene in a manner analogous to that of the GAL4 protein. PMID:3104772

  14. Analysis of a eukaryotic beta-galactosidase gene: the N-terminal end of the yeast Kluyveromyces lactis protein shows homology to the Escherichia coli lacZ gene product.

    PubMed Central

    Breunig, K D; Dahlems, U; Das, S; Hollenberg, C P

    1984-01-01

    The LAC4 gene of Kluyveromyces lactis, encoding the enzyme beta-galactosidase was mapped on a cloned DNA fragment and the sequence of the 5' end was determined. This sequence includes the 5' regulatory region involved in the induction by lactose and the N-terminal end of the protein coding region. Comparison of the deduced amino acid sequence of this eukaryotic enzyme with the N-terminal end of the Escherichia coli beta-galactosidase revealed substantial homology. Two major RNA initiation sites were mapped at -115 and -105. A number of structural peculiarities of the 5'non-coding region are discussed as in comparison to Saccharomyces cerevisiae genes. Images PMID:6324114

  15. Extranuclear expression of the bacterial xylose isomerase (xylA) and the UDP-glucose dehydrogenase (hasB) genes in yeast with Kluyveromyces lactis linear killer plasmids as vectors.

    PubMed

    Schründer, J; Gunge, N; Meinhardt, F

    1996-11-01

    On the basis of the linear killer plasmid pGKL1 from Kluyveromyces lactis, two new linear hybrid plasmids were constructed. One of these, pRSC126, carried the xylA gene from Streptomyces rubiginosus encoding the xylose isomerase. The other linear hybrid molecule, pRSC128, carried the hasB gene of Streptococcus pyogenes encoding the UDP glucose dehydrogenase. Construction was performed in a way that the putative cytoplasmic promoter element of ORF5 of pGKL2 was fused to the coding region of the heterologous genes. After transformation, in vivo recombination led to the establishment of linear hybrid vectors. Though efficiency of expression was low when compared with bacterial systems, cytoplasmic expression of both genes was clearly demonstrated.

  16. Galactokinase encoded by GAL1 is a bifunctional protein required for induction of the GAL genes in Kluyveromyces lactis and is able to suppress the gal3 phenotype in Saccharomyces cerevisiae.

    PubMed Central

    Meyer, J; Walker-Jonah, A; Hollenberg, C P

    1991-01-01

    We have analyzed a GAL1 mutant (gal1-r strain) of the yeast Kluyveromyces lactis which lacks the induction of beta-galactosidase and the enzymes of the Leloir pathway in the presence of galactose. The data show that the K. lactis GAL1 gene product has, in addition to galactokinase activity, a function required for induction of the lactose system. This regulatory function is not dependent on galactokinase activity, as it is still present in a galactokinase-negative mutant (gal1-209). Complementation studies in Saccharomyces cervisiae show that K. lactis GAL1 and gal1-209, but not gal1-r, complement the gal3 mutation. We conclude that the regulatory function of GAL1 in K. lactis soon after induction is similar to the function of GAL3 in S. cerevisiae. PMID:1922058

  17. KlAft, the Kluyveromyces lactis Ortholog of Aft1 and Aft2, Mediates Activation of Iron-Responsive Transcription Through the PuCACCC Aft-Type Sequence

    PubMed Central

    Conde e Silva, Natalia; Gonçalves, Isabelle R.; Lemaire, Marc; Lesuisse, Emmanuel; Camadro, Jean Michel; Blaiseau, Pierre Louis

    2009-01-01

    Iron homeostasis in fungi is regulated at the transcriptional level by two different mechanisms. It is mediated by a conserved GATA-type repressor in most fungi except in the yeast Saccharomyces cerevisiae, where it is controlled by the transcription activators Aft1 and Aft2. These activators are encoded by the paralogous genes AFT1 and AFT2, which result from the whole-genome duplication. Here, we explore regulation of iron homeostasis in the yeast Kluyveromyces lactis that diverged from S. cerevisiae before this event. We identify an ortholog of AFT1/AFT2, designated KlAFT, whose deletion leads to the inability to grow under iron limitation. We show with quantitative real-time PCR analysis that KlAft activates the transcription of all homologs of the Aft1-target genes involved in the iron transport at the cell surface in response to iron limitation. However, homologs of Aft2-specific target genes encoding intracellular iron transporters are regulated neither by KlAft nor by iron. Both bioinformatic and DNA binding and transcription analyses demonstrate that KlAft activates iron-responsive gene expression through the PuCACCC Aft-type sequence. Thus, K. lactis is the first documented species with a positive iron-transcriptional control mediated by only one copy of the Aft-type regulator. This indicates that this function was acquired before the whole-genome duplication and was then diversified into two regulators in S. cerevisiae. PMID:19581449

  18. Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.

    PubMed Central

    Breunig, K D; Kuger, P

    1987-01-01

    As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription. Images PMID:2830492

  19. The Golgi α-1,6 mannosyltransferase KlOch1p of Kluyveromyces lactis is required for Ca2+/calmodulin-based signaling and for proper mitochondrial functionality

    PubMed Central

    2009-01-01

    Background Protein N-glycosylation is a relevant metabolic pathway in eukaryotes and plays key roles in cell processes. In yeasts, outer chain branching is initiated in the Golgi apparatus by the alpha-1,6-mannosyltransferase Och1p. Results Here we report that, in Kluyveromyces lactis, this glycosyltransferase is also required to maintain functional mitochondria and calcium homeostasis. Cells carrying a mutation in KlOCH1 gene showed altered mitochondrial morphology, increased accumulation of ROS and reduced expression of calcium signalling genes such as calmodulin and calcineurin. Intracellular calcium concentration was also reduced in the mutant cells with respect to the wild type counterparts. Phenotypes that occur in cells lacking the alpha-1,6-mannosyltransferase, including oxidative stress and impaired mitochondria functionality, were suppressed by increased dosage of KlCmd1p. This, in turn, acts through the action of calcineurin. Conclusions Proper functioning of the alpha-1,6-mannosyltransferase in the N-glycosylation pathway of K. lactis is required for maintaining normal calcium homeostasis; this is necessary for physiological mitochondria dynamics and functionality. PMID:20003441

  20. Unsaturated fatty acids-dependent linkage between respiration and fermentation revealed by deletion of hypoxic regulatory KlMGA2 gene in the facultative anaerobe-respiratory yeast Kluyveromyces lactis.

    PubMed

    Ottaviano, Daniela; Montanari, Arianna; De Angelis, Lorenzo; Santomartino, Rosa; Visca, Andrea; Brambilla, Luca; Rinaldi, Teresa; Bello, Cristiano; Reverberi, Massimo; Bianchi, Michele M

    2015-08-01

    In the yeast Kluyveromyces lactis, the inactivation of structural or regulatory glycolytic and fermentative genes generates obligate respiratory mutants which can be characterized by sensitivity to the mitochondrial drug antimycin A on glucose medium (Rag(-) phenotype). Rag(-) mutations can occasionally be generated by the inactivation of genes not evidently related to glycolysis or fermentation. One such gene is the hypoxic regulatory gene KlMGA2. In this work, we report a study of the many defects, in addition to the Rag(-) phenotype, generated by KlMGA2 deletion. We analyzed the fermentative and respiratory metabolism, mitochondrial functioning and morphology in the Klmga2Δ strain. We also examined alterations in the regulation of the expression of lipid biosynthetic genes, in particular fatty acids, ergosterol and cardiolipin, under hypoxic and cold stress and the phenotypic suppression by unsaturated fatty acids of the deleted strain. Results indicate that, despite the fact that the deleted mutant strain had a typical glycolytic/fermentative phenotype and KlMGA2 is a hypoxic regulatory gene, the deletion of this gene generated defects linked to mitochondrial functions suggesting new roles of this protein in the general regulation and cellular fitness of K. lactis. Supplementation of unsaturated fatty acids suppressed or modified these defects suggesting that KlMga2 modulates membrane functioning or membrane-associated functions, both cytoplasmic and mitochondrial.

  1. Coregulation of the Kluyveromyces lactis lactose permease and beta-galactosidase genes is achieved by interaction of multiple LAC9 binding sites in a 2.6 kbp divergent promoter.

    PubMed Central

    Gödecke, A; Zachariae, W; Arvanitidis, A; Breunig, K D

    1991-01-01

    The coregulated genes LAC4 and LAC12 encoding beta-galactosidase and lactose permease, respectively, are responsible for the ability of the milk yeast Kluyveromyces lactis to utilise lactose. They are divergently transcribed and separated by an unusually large intergenic region of 2.6 kbp. Mapping of the upstream border of the beta-galactosidase gene (LAC4) promoter by introduction of mutations at the chromosomal locus showed that LAC4 and LAC12 share the same upstream activation sites (UAS). The UASs represent binding sites for the trans-activator LAC9, a K. lactis homologue of GAL4, conforming to the consensus sequence 5'-CGG(N5)A/T(N5)CCG-3'. Two binding sites are located in front of each of the genes at almost symmetrical positions. beta-galactosidase activity measurements as well as quantitation of LAC4 and LAC12 mRNA levels demonstrated that all four sites are required for full induction. LAC4 proximal and a LAC12 proximal sites cooperate in activating transcription of both genes. These sites are more than 1.7 kbp apart and the distal site is located more than 2.3 kbp upstream of the respective start of transcription. Thus, the distance between interacting sites is larger than in any of the well characterised yeast promoters. The contribution to gene activation differs for individual binding sites and correlates with the relative affinity of LAC9 for these sites in vitro suggesting that LAC9 binding is a rate limiting step for LAC promoter function. Images PMID:1923819

  2. A new search for thermotolerant yeasts, its characterization and optimization using response surface methodology for ethanol production

    PubMed Central

    Arora, Richa; Behera, Shuvashish; Sharma, Nilesh K.; Kumar, Sachin

    2015-01-01

    The progressive rise in energy crisis followed by green house gas (GHG) emissions is serving as the driving force for bioethanol production from renewable resources. Current bioethanol research focuses on lignocellulosic feedstocks as these are abundantly available, renewable, sustainable and exhibit no competition between the crops for food and fuel. However, the technologies in use have some drawbacks including incapability of pentose fermentation, reduced tolerance to products formed, costly processes, etc. Therefore, the present study was carried out with the objective of isolating hexose and pentose fermenting thermophilic/thermotolerant ethanologens with acceptable product yield. Two thermotolerant isolates, NIRE-K1 and NIRE-K3 were screened for fermenting both glucose and xylose and identified as Kluyveromyces marxianus NIRE-K1 and K. marxianus NIRE-K3. After optimization using Face-centered Central Composite Design (FCCD), the growth parameters like temperature and pH were found to be 45.17°C and 5.49, respectively for K. marxianus NIRE-K1 and 45.41°C and 5.24, respectively for K. marxianus NIRE-K3. Further, batch fermentations were carried out under optimized conditions, where K. marxianus NIRE-K3 was found to be superior over K. marxianus NIRE-K1. Ethanol yield (Yx∕s), sugar to ethanol conversion rate (%), microbial biomass concentration (X) and volumetric product productivity (Qp) obtained by K. marxianus NIRE-K3 were found to be 9.3, 9.55, 14.63, and 31.94% higher than that of K. marxianus NIRE-K1, respectively. This study revealed the promising potential of both the screened thermotolerant isolates for bioethanol production. PMID:26388844

  3. Yeast diversity in a traditional French cheese "Tomme d'orchies" reveals infrequent and frequent species with associated benefits.

    PubMed

    Ceugniez, Alexandre; Drider, Djamel; Jacques, Philippe; Coucheney, Françoise

    2015-12-01

    This study is aimed at unrevealing the yeast diversity of handmade cheese, Tomme d'orchies, produced and marketed in the north of France. A total of 185 yeast colonies were isolated from the surface and core of this cheese. From these, 80 morphologically different colonies were selected and subjected to rep-PCR analysis. The isolates were clustered into six distinct groups based on their DNA fingerprints. From each group, at least 30% of isolates were selected and identified to species level by biochemical characteristics (ID32C Api system) and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA regions. The isolates belonged to Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Kluyveromyces marxianus, frequently isolated, and less frequently isolated Saturnispora mendoncae and Clavispora lusitaniae. Two isolates designated as Kluyveromyces lactis (isolate S-3-05) and Kluyveromyces marxianus (isolate S-2-05) were non-hemolytic, sensitive to antifungal compounds and able to inhibit the growth of pathogens including Candida albicans, Listeria monocytogenes and some bacilli.

  4. Dynamics of yeast immobilized-cell fluidized-bed bioreactors systems in ethanol fermentation from lactose-hydrolyzed whey and whey permeate.

    PubMed

    Gabardo, Sabrina; Pereira, Gabriela Feix; Klein, Manuela P; Rech, Rosane; Hertz, Plinho F; Ayub, Marco Antônio Záchia

    2016-01-01

    We studied the dynamics of ethanol production on lactose-hydrolyzed whey (LHW) and lactose-hydrolyzed whey permeate (LHWP) in batch fluidized-bed bioreactors using single and co-cultures of immobilized cells of industrial strains of Saccharomyces cerevisiae and non-industrial strains of Kluyveromyces marxianus. Although the co-culture of S. cerevisiae CAT-1 and K. marxianus CCT 4086 produced two- to fourfold the ethanol productivity of single cultures of S. cerevisiae, the single cultures of the K. marxianus CCT 4086 produced the best results in both media (Y EtOH/S = 0.47-0.49 g g(-1) and Q P = 1.39-1.68 g L(-1) h(-1), in LHW and LHWP, respectively). Ethanol production on concentrated LHWP (180 g L(-1)) reached 79.1 g L(-1), with yields of 0.46 g g(-1) for K. marxianus CCT 4086 cultures. Repeated batches of fluidized-bed bioreactor on concentrated LHWP led to increased ethanol productivity, reaching 2.8 g L(-1) h(-1).

  5. Comparison of enzymatic antioxidant defence systems in different metabolic types of yeasts.

    PubMed

    Koleva, Dafinka I; Petrova, Ventsislava Y; Kujumdzieva, Anna V

    2008-11-01

    The enzymatic defence system in the 2 yeasts Kluyveromyces marxianus and Rhodotorula glutinis, differing in their mode of oxygen uptake and energy generation, was characterized and compared with the well-studied facultatively fermentative Crabtree-positive Saccharomyces cerevisiae strain. Twofold higher superoxide dismutase (SOD) and catalase activities were detected in K. marxianus and R. glutinis when cells were cultured on glucose. Further increases of 10%-15% in SOD activity and 30%-50% in catalase were measured in all studied yeasts strains after transfer to media containing ethanol. An evaluation of the ratio of Cu/Zn SOD / Mn SOD was performed as a measure of the oxidative metabolism. A 20% decrease was observed when the respiratory source of energy was ethanol, with the lowest ratio being observed for the oxidative type of K. marxianus yeasts. Electrophoretic analysis revealed that all tested strains possess active Cu/Zn and Mn SODs. A reverse electrophoretic mobility pattern of K. marxianus and R. glutinis SOD enzymes was observed in comparison with the same couple in S. cerevisiae. The investigation of electrophoretic profile of catalase enzymes showed that alongside their different taxonomic status and fermentative capacity, all tested strains possess 2 separate catalases. The role of antioxidant enzymes in preventing oxidant-induced cytotoxicity (treatment with hydrogen peroxide, paraquat, and menadione) was shown.

  6. Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol.

    PubMed

    Ballesteros, I; Ballesteros, M; Cabañas, A; Carrasco, J; Martín, C; Negro, M J; Saez, F; Saez, R

    1991-01-01

    A total of 27 yeast strains belonging to the groups Candida, Saccharomyces, and Kluyveromyces were screened for their ability to grow and ferment glucose at temperatures ranging 32-45 degrees C. K. marxianus and K. fragilis were found to be the best ethanol producing organisms at the higher temperature tested and, so, were selected for subsequent simultaneous saccharification and fermentation (SSF) studies. SSF experiments were performed at 42 and 45 degrees C, utilizing Solkafloc (10%) as cellulose substrate and a cellulase loading of 15 FPU/g substrate. Best results were achieved at 42 degrees C with K. marxianus L. G. and K. fragilis L. G., both of which produced close to 38 g/L ethanol and 0.5 ethanol yield, in 78 h.

  7. Identification and characterization of yeast isolated from the elaboration of seasoned green table olives.

    PubMed

    Hernández, Alejandro; Martín, Alberto; Aranda, Emilio; Pérez-Nevado, Francisco; Córdoba, María G

    2007-06-01

    The purpose of this study was to investigate the yeast population during the processing of green table olives. In the fresh olives, yeast were found at concentrations of around 3.0 log cfu/g, with Cryptococcus spp. being predominant. In the brine, the yeast concentrations were greater than 4.9 log cfu/ml, with Pichia anomala, Kluyveromyces marxianus, and Saccharomyces cerevisiae being the predominant species. Unlike the yeast isolated from the fresh olives, the strains obtained from the olive brine mostly showed low pectolytic but high catalase activities. Some of these strains also exhibited other biochemical desirable properties for the fermentation of green table olives, including their lipolytic activities and their assimilation or production of organic acids in the brine. Seven strains in particular of P. anomala, K. marxianus, S. cerevisiae, and Candida maris showed the best properties for use in trials as starter culture in pilot fermenters.

  8. Fructanase and fructosyltransferase activity of non-Saccharomyces yeasts isolated from fermenting musts of Mezcal.

    PubMed

    Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

    2012-04-01

    Fructanase and fructosyltransferase are interesting for the tequila process and prebiotics production (functional food industry). In this study, one hundred thirty non-Saccharomyces yeasts isolated from "Mezcal de Oaxaca" were screened for fructanase and fructosyltransferase activity. On solid medium, fifty isolates grew on Agave tequilana fructans (ATF), inulin or levan. In liquid media, inulin and ATF induced fructanase activities of between 0.02 and 0.27U/ml depending of yeast isolate. High fructanase activity on sucrose was observed for Kluyveromyces marxianus and Torulaspora delbrueckii, while the highest fructanase activity on inulin and ATF was observed for Issatchenkia orientalis, Cryptococcus albidus, and Candida apicola. Zygosaccharomyces bisporus and Candida boidinii had a high hydrolytic activity on levan. Sixteen yeasts belonging to K. marxianus, T. delbrueckii and C. apicola species were positive for fructosyltransferase activity. Mezcal microbiota proved to showed to be a source for new fructanase and fructosyltransferases with potential application in the tequila and food industry.

  9. Hydrolysis of whey lactose using CTAB-permeabilized yeast cells.

    PubMed

    Kaur, Gurpreet; Panesar, Parmjit S; Bera, Manav B; Kumar, Harish

    2009-01-01

    Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. The enzymatic hydrolysis of whey lactose to glucose and galactose by beta-galactosidase constitutes the basis of the most biotechnological processes currently developed to exploit the sugar content of whey. Keeping this in view, lactose hydrolysis in whey was performed using CTAB permeabilized Kluyveromyces marxianus cells. Permeabilization of K. marxianus cells in relation to beta-galactosidase activity was carried out using cetyltrimethyl ammonium bromide (CTAB) to avoid the problem of enzyme extraction. Different process parameters (biomass load, pH, temperature, and incubation time) were optimized to enhance the lactose hydrolysis in whey. Maximum hydrolysis (90.5%) of whey lactose was observed with 200 mg DW yeast biomass after 90 min of incubation period at optimum pH of 6.5 and temperature of 40 degrees C.

  10. Solid state fermentation of food waste mixtures for single cell protein, aroma volatiles and fat production.

    PubMed

    Aggelopoulos, Theodoros; Katsieris, Konstantinos; Bekatorou, Argyro; Pandey, Ashok; Banat, Ibrahim M; Koutinas, Athanasios A

    2014-02-15

    Growth of selected microorganisms of industrial interest (Saccharomyces cerevisiae, Kluyveromyces marxianus and kefir) by solid state fermentation (SSF) of various food industry waste mixtures was studied. The fermented products were analysed for protein, and nutrient minerals content, as well as for aroma volatile compounds by GC/MS. The substrate fermented by K. marxianus contained the highest sum of fat and protein concentration (59.2% w/w dm) and therefore it could be considered for utilisation of its fat content and for livestock feed enrichment. Regarding volatiles, the formation of high amounts of ε-pinene was observed only in the SSF product of kefir at a yield estimated to be 4 kg/tn of SSF product. A preliminary design of a biorefinery-type process flow sheet and its economic analysis, indicated potential production of products (enriched livestock feed, fat and ε-pinene) of significant added value.

  11. The Bioconversion of Pretreated Cashew Apple Bagasse into Ethanol by SHF and SSF Processes.

    PubMed

    Rodrigues, Tigressa Helena S; de Barros, Emanuel Meneses; de Sá Brígido, Jeferson; da Silva, Winne M; Rocha, Maria Valderez P; Gonçalves, Luciana Rocha B

    2016-03-01

    Ethanol production from acidic-alkaline pretreated cashew apple bagasse (CAB-OH) was investigated using separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes. First, a screening of Kluyveromyces strains was conducted by SHF and a maximum ethanol concentration of 24.1 g L(-1) was obtained using Kluyveromyces marxianus ATCC36907, which presented similar profiles when compared to results obtained by a Saccharomyces strain. The effect of temperature on ethanol production conducted by SHF using K. marxianus ATCC36907 was investigated, and the maximum ethanol yield (YE/G) was obtained at 40 °C (0.46 g g(-1)) using a synthetic medium. In the SHF using CAB-OH hydrolysate, the maximum ethanol concentration obtained was 24.9 g L(-1), 5.92 g L(-1) h(-1) of productivity, and ethanol yield of 0.43 g g(-1) at 40 °C. Afterwards, K. marxianus ATCC36907 was used in the bioconversion of CAB-OH by SSF, and an ethanol concentration of 41.41 ± 0.2 g L(-1) was obtained using 10 % CAB-OH at 40 °C, 150 rpm and 24 h, resulting in a Y'E/G of 0.50 gE gG (-1) and an efficiency of 98.4 %, in the process conducted with cellobiase supplementation. SHF and SSF processes using CAB-OH and K. marxianus ATCC36907 can be used to ethanol production, but the SSF process required only one step to achieve the same production.

  12. Optimization of the simultaneous saccharification and fermentation process using thermotolerant yeasts.

    PubMed

    Ballesteros, I; Oliva, J M; Ballesteros, M; Carrasco, J

    1993-01-01

    Different treatments to improve the thermotolerance of fermenting yeasts for simultaneous ethanol saccharification and fermentation process of cellulosic materials have been examined. Yeasts of the genera Saccharomyces and Kluyveromyces were tested for growth and fermentation at progressively higher temperatures in the range of 42-47 degrees C. The best results were obtained with K. marxianus LG, which was then submitted to different treatments in order to achieve thermotolerant clones. A total of 35 new clones were obtained that dramatically improved the SSF of 10% Solka-floc substrate at 45 degrees C when compared to the original strain, some with ethanol concentrations as high as 33 g/L.

  13. [Specific features of fermentation of D-xylose and D-glucose by xylose-assimilating yeasts].

    PubMed

    Iablochkova, E N; Bolotnikova, O I; Mikhaĭlova, N P; Nemova, N N; Ginak, A I

    2003-01-01

    The ability to assimilate D-glucose and D-xylose was studied in 21 yeast species of the following genera: Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis. All the cultures fermented D-glucose with the formation of ethanol. During the assimilation of D-xylose, ethanol was produced by P. stipitis and C. shehatae, whereas xylitol was produced by C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, K. fragilis, K. marxianus, P. guillermondii, and T. molishiama. The yeast P. tannophilus produced comparable amounts of both alcohols. The possible use of xylose-assimilating yeasts for the production of xylitol and ethanol is discussed.

  14. A new method to determine optimum temperature and activation energies for enzymatic reactions.

    PubMed

    Wojcik, M; Miłek, J

    2016-08-01

    A new method for determination of the optimum temperature and activation energies based on an idea of the average rate of enzymatic reaction has been developed. A mathematical model describing the effect of temperature on a dimensionless activity for enzyme deactivation following the first-order kinetics has been derived. The necessary condition for existence of the function extreme of the optimal temperature has been applied in the model. The developed method has been verified using the experimental data for inulinase from Kluyveromyces marxianus.

  15. Microbial biogeography of the transnational fermented milk matsoni.

    PubMed

    Bokulich, Nicholas A; Amiranashvili, Lia; Chitchyan, Karine; Ghazanchyan, Narine; Darbinyan, Karen; Gagelidze, Nino; Sadunishvili, Tinatin; Goginyan, Vigen; Kvesitadze, Giorgi; Torok, Tamas; Mills, David A

    2015-09-01

    The fermented milk matsoni is a traditional, national food product of both Georgia and Armenia. Little is known about the effects of biogeography and milk type on the microbial biodiversity of matsoni or the fungal composition of matsoni fermentations. High-throughput marker-gene sequencing was used to survey the bacterial and fungal communities of matsoni from different milk types and regions throughout Armenia and Georgia. Results demonstrate that both production region and milk type influence matsoni microbiota, suggesting that the traditional production methods preserve the transfer of unique regional microbiota from batch to batch. Bacterial profiles were dominated by Lactobacillus and Streptococcus species. Yeast profiles varied dramatically, with Kluyveromyces marxianus, Candida famata, Saccharomyces cerevisiae, Lodderomyces elongisporus, and Kluyveromyces lactis being the most important species distinguishing production regions and milk types. This survey will enable more detailed capture and characterization of specific microbiota detected within these fermentations.

  16. Evaluation of Galactose Adapted Yeasts for Bioethanol Fermentation from Kappaphycus alvarezii Hydrolyzates.

    PubMed

    Nguyen, Trung Hau; Ra, Chae Hun; Sunwoo, In Yung; Jeong, Gwi-Taek; Kim, Sung-Koo

    2016-07-28

    Bioethanol was produced from Kappaphycus alvarezii seaweed biomass using separate hydrolysis and fermentation (SHF). Pretreatment was evaluated for 60 min at 121°C using 12% (w/v) biomass slurry with 364 mM H2SO4. Enzymatic saccharification was then carried out at 45°C for 48 h using Celluclast 1.5 L. Ethanol fermentation with 12% (w/v) K. alvarezii hydrolyzate was performed using the yeasts Saccharomyces cerevisiae KCTC1126, Kluyveromyces marxianus KCTC7150, and Candida lusitaniae ATCC42720 with or without prior adaptation to high concentrations of galactose. When non-adapted S. cerevisiae, K. marxianus, and C. lusitaniae were used, 11.5 g/l, 6.7 g/l, and 6.0 g/l of ethanol were produced, respectively. When adapted S. cerevisiae, K. marxianus, and C. lusitaniae were used, 15.8 g/l, 11.6 g/l, and 13.4 g/l of ethanol were obtained, respectively. The highest ethanol concentration was 15.8 g/l, with YEtOH = 0.43 and YT% = 84.3%, which was obtained using adapted S. cerevisiae.

  17. Physiological characterization of thermotolerant yeast for cellulosic ethanol production.

    PubMed

    Costa, Daniela A; de Souza, Carlos J A; Costa, Patrícia S; Rodrigues, Marina Q R B; dos Santos, Ancély F; Lopes, Mariana R; Genier, Hugo L A; Silveira, Wendel B; Fietto, Luciano G

    2014-04-01

    The conversion of lignocellulose into fermentable sugars is considered a promising alternative for increasing ethanol production. Higher fermentation yield has been achieved through the process of simultaneous saccharification and fermentation (SSF). In this study, a comparison was performed between the yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus for their potential use in SSF process. Three strains of S. cerevisiae were evaluated: two are widely used in the Brazilian ethanol industry (CAT-1 and PE-2), and one has been isolated based on its capacity to grow and ferment at 42 °C (LBM-1). In addition, we used thermotolerant strains of K. marxianus. Two strains were obtained from biological collections, ATCC 8554 and CCT 4086, and one strain was isolated based on its fermentative capacity (UFV-3). SSF experiments revealed that S. cerevisiae industrial strains (CAT-1 and PE-2) have the potential to produce cellulosic ethanol once ethanol had presented yields similar to yields from thermotolerant strains. The industrial strains are more tolerant to ethanol and had already been adapted to industrial conditions. Moreover, the study shows that although the K. marxianus strains have fermentative capacities similar to strains of S. cerevisiae, they have low tolerance to ethanol. This characteristic is an important target for enhancing the performance of this yeast in ethanol production.

  18. The color of Brevibacterium linens depends on the yeast used for cheese deacidification.

    PubMed

    Leclercq-Perlat, M N; Corrieu, G; Spinnler, H E

    2004-05-01

    The color of smear cheeses (Muenster) is traditionally thought to be due to the bacterial flora, e.g., Brevibacterium linens. This study was carried out to evaluate indirect effects of yeast on the color of B. linens. A 60% cheese medium was desacidified with Debaryomyces hansenii or Kluyveromyces marxianus until pH 5.8 was reached. After inactivation of the yeast and addition of agar-NaCl, B. linens was inoculated on the medium surface and incubated at 12 degrees C from d 2 to 28. For each bacterial biofilm, color was evaluated by L*C*h(degrees) (brightness, chroma, hue angle) spectrocolorimetry. After d 14 (D. hansenii deacidification) and d 21 (K marxianus desacidification), the color level (as a function of all 3 factors) of B. linens biofilms became maximal and remained so until d 28. Debaryomyces hansenii 304 (LGMPA) was less efficient for deacidification than K. marxianus Laf5. However, color intensity (function of chroma only) was higher when D. hansenii was used. The yeast used had an effect on the composition of the cheese medium in relation to production and consumption of metabolites during deacidification. The results concerning color are discussed with respect to this cheese medium composition.

  19. Lipid-enhanced ethanol production by Kluyveromyces fragilis

    SciTech Connect

    Janssens, J.H.; Burris, N.; Woodward, A.; Bailey, R.B.

    1983-02-01

    The fermentation ability of a strain of Kluyveromyes fragilis, already selected for rapid lactose-fermenting capability, was improved dramatically by the addition of unsaturated fatty acids and ergosterol to the medium. The fermentation time of a 20% whey-lactose medium was decreased from over 90 hours to less than 60 hours. The lipids were shown to be taken up by the organism, and the effects on specific growth rate and biomass production were determined. (Refs. 23).

  20. Recombinant Production of an Inulinase in a Saccharomyces cerevisiae gal80 Strain.

    PubMed

    Lim, Seok-Hwan; Lee, Hongweon; Sok, Dai-Eun; Choi, Eui-Sung

    2010-11-01

    The inulinase gene (INU1) from Kluyveromyces marxianus NCYC2887 strain was overexpressed by using GAL10 promotor in a △gal80 strain of Saccharomyces cerevisiae. The inulinase gene lacking the original signal sequence was fused in-frame to mating factor alpha signal sequence for secretory expression. Use of the △gal80 strain allowed the galactose-free induction of inulinase expression using a glucose-only medium. Shake flask cultivation in YPD medium produced 34.6 U/ml of the recombinant inulinase, which was approximately 13-fold higher than that produced by K. marxianus NCYC2887. It was found that the use of the △gal80 strain improved the expression of inulinase in the recombinant S. cerevisiae in both the aerobic and the anaerobic condition by about 2.9- and 1.7-fold, respectively. 5 L fed-batch fermentation using YPD medium was performed under aerobic condition with glucose feeding, which resulted in the inulinase production of 31.7 U/ml at OD600 of 67. Ethanol fermentation of dried powder of Jerusalem artichoke, an inulin-rich biomass, was also performed using the recombinant S. cerevisiae expressing INU1 and K. marxianus NCYC2887. Fermentation in a 5L scale fermentor was carried out at an aeration rate of 0.2 vvm, an agitation rate of 300 rpm, and the pH was controlled at 5.0. The temperature was maintained at 30degrees C and 37degrees C, respectively, for the recombinant S. cerevisiae and K. marxianus. The maximum productivities of ethanol were 59.0 and 53.5 g/L, respectively.

  1. Phytase-producing capacity of yeasts isolated from traditional African fermented food products and PHYPk gene expression of Pichia kudriavzevii strains.

    PubMed

    Greppi, Anna; Krych, Łukasz; Costantini, Antonella; Rantsiou, Kalliopi; Hounhouigan, D Joseph; Arneborg, Nils; Cocolin, Luca; Jespersen, Lene

    2015-07-16

    Phytate is known as a strong chelate of minerals causing their reduced uptake by the human intestine. Ninety-three yeast isolates from traditional African fermented food products, belonging to nine species (Pichia kudriavzevii, Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces marxianus, Millerozyma farinosa, Candida glabrata, Wickerhamomyces anomalus, Hanseniaspora guilliermondii and Debaryomyces nepalensis) were screened for phytase production on solid and liquid media. 95% were able to grow in the presence of phytate as sole phosphate source, P. kudriavzevii being the best growing species. A phytase coding gene of P. kudriavzevii (PHYPk) was identified and its expression was studied during growth by RT-qPCR. The expression level of PHYPk was significantly higher in phytate-medium, compared to phosphate-medium. In phytate-medium expression was seen in the lag phase. Significant differences in gene expression were detected among the strains as well as between the media. A correlation was found between the PHYPk expression and phytase extracellular activity.

  2. Alcohol production from cheese whey permeate using genetically modified flocculent yeast cells.

    PubMed

    Domingues, L; Lima, N; Teixeira, J A

    2001-03-05

    Alcoholic fermentation of cheese whey permeate was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus enabling for lactose metabolization. Data on yeast fermentation and growth on cheese whey permeate from a Portuguese dairy industry is presented. For cheese whey permeate having a lactose concentration of 50 gL(-1), total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. Using a continuously operating 5.5-L bioreactor, ethanol productivity near 10 g L(-1) h(-1) (corresponding to 0.45 h(-1) dilution rate) was obtained, which raises new perspectives for the economic feasibility of whey alcoholic fermentation. The use of 2-times concentrated cheese whey permeate, corresponding to 100 gL(-1) of lactose concentration, was also considered allowing for obtaining a fermentation product with 5% (w/v) alcohol.

  3. Influence of temperature, pH and water activity on "in vitro" inhibition of Penicillium glabrum (Wehmer) Westling by yeasts.

    PubMed

    Sinigaglia, M; Corbo, M R; Ciccarone, C

    1998-08-01

    Four different yeast species (Metschnikowia pulcherrima, Saccharomycopsis vini, Kluyveromyces marxianus, Cryptococcus albidus), isolated from surface of grapes, were evaluated for biocontrol potential against Penicillium glabrum. In order to investigate the influence of temperature, pH, water activity and yeast cell concentration on Penicillium glabrum inhibition, the individual effects and the interaction of these factors were analyzed by means of a Central Composite Design (CCD). All yeast species tested showed antagonistic effects which were more pronounced at high cell concentrations. The other variables affected the antagonistic effect differentially depending on the yeast species. Results of the experimental design showed that the selective success of a competitive microflora is under environmental control; moreover, when microbial cells are subjected to multiple factors, the effects and the reciprocal interactions of the individual variables cannot be independently evaluated.

  4. Ethanol-based organosolv treatment with trace hydrochloric acid improves the enzymatic digestibility of Japanese cypress (Chamaecyparis obtusa) by exposing nanofibers on the surface.

    PubMed

    Hideno, Akihiro; Kawashima, Ayato; Endo, Takashi; Honda, Katsuhisa; Morita, Masatoshi

    2013-03-01

    The effects of adding trace acids in ethanol based organosolv treatment were investigated to increase the enzymatic digestibility of Japanese cypress. A high glucose yield (60%) in the enzymatic hydrolysis was obtained by treating the sample at 170 °C for 45 min in 50% ethanol liquor containing 0.4% hydrochloric acid. Moreover, the enzymatic digestibility of the treated sample was improved to ∼70% by changing the enzyme from acremonium cellulase to Accellerase1500. Field emission scanning electron microscopy revealed the presence of lignin droplets and partial cellulose nanofibers on the surface of the treated sample. Simultaneous saccharification and fermentation of the treated samples using thermotolerant yeast (Kluyveromyces marxianus NBRC1777) was tested. A high ethanol concentration (22.1 g/L) was achieved using the EtOH50/W50/HCl0.4-treated sample compared with samples from other treatments.

  5. Role of hydrophobicity in adhesion of wild yeast isolated from the ultrafiltration membranes of an apple juice processing plant.

    PubMed

    Tarifa, María Clara; Brugnoni, Lorena Inés; Lozano, Jorge Enrique

    2013-01-01

    The role of cell surface hydrophobicity in the adhesion to stainless steel (SS) of 11 wild yeast strains isolated from the ultrafiltration membranes of an apple juice processing plant was investigated. The isolated yeasts belonged to four species: Candida krusei (5 isolates), Candida tropicalis (2 isolates), Kluyveromyces marxianus (3 isolates) and Rhodotorula mucilaginosa (1 isolate). Surface hydrophobicity was measured by the microbial adhesion to solvents method. Yeast cells and surfaces were incubated in apple juice and temporal measurements of the numbers of adherent cells were made. Ten isolates showed moderate to high hydrophobicity and 1 strain was hydrophilic. The hydrophobicity expressed by the yeast surfaces correlated positively with the rate of adhesion of each strain. These results indicated that cell surface hydrophobicity governs the initial attachment of the studied yeast strains to SS surfaces common to apple juice processing plants.

  6. Application of Fenton's reaction to steam explosion prehydrolysates from poplar biomass.

    PubMed

    Oliva, J M; Manzanares, P; Ballesteros, I; Negro, M J; González, A; Ballesteros, M

    2005-01-01

    The application of Fenton's reaction to enhance the fermentability of prehydrolysates obtained from steam explosion pretreatment of poplar biomass was studied. Reaction conditions of temperature and H2O2 and Fe(II) concentrations were studied. The fermentability of prehydrolysate treated by Fenton's reaction was tested by using different inoculum sizes of thermotolerant strain Kluyveromyces marxianus CECT 10875. The highest percentages of toxic compound degradation (ranging from 71 to 93% removal) were obtained at the highest H2O2 concentration tested (50 mM). However, a negative effect on fermentability was observed at this H2O2 concentration at the lower inoculum loading. An increase in inoculum size to 0.6 g/L resulted in an enhanced ethanol fermentation yield of 95% relative to control.

  7. Evaluation of the Biolog system for the identification of food and beverage yeasts.

    PubMed

    Praphailong, W; Van Gestel, M; Fleet, G H; Heard, G M

    1997-06-01

    The inconvenience of conventional yeast identification methods has resulted in the development of rapid, commercial systems, mainly for clinical yeast species. The Biolog system (Biolog Inc., Hayward, CA, USA) is a new semi-automated, computer-linked technology for rapid identification of clinical and non-clinical yeasts. The system is based around a microtitre tray and includes assimilation and oxidation tests. This paper evaluates the Biolog system for the identification of 21 species (72 strains) of yeasts of food and wine origin. Species correctly identified included Saccharomyces cerevisiae, Debaryomyces hansenii, Yarrowia lipolytica, Kluyveromyces marxianus, Kloeckera apiculata, Dekkera bruxellensis and Schizosaccharomyces pombe. Zygosaccharomyces bailii and Zygosaccharomyces rouxii were identified correctly 50% of the time and Pichia membranaefaciens 20% of the time.

  8. High-temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast?

    PubMed

    Abdel-Banat, Babiker M A; Hoshida, Hisashi; Ano, Akihiko; Nonklang, Sanom; Akada, Rinji

    2010-01-01

    The process of ethanol fermentation has a long history in the production of alcoholic drinks, but much larger scale production of ethanol is now required to enable its use as a substituent of gasoline fuels at 3%, 10%, or 85% (referred to as E3, E10, and E85, respectively). Compared with fossil fuels, the production costs are a major issue for the production of fuel ethanol. There are a number of possible approaches to delivering cost-effective fuel ethanol production from different biomass sources, but we focus in our current report on high-temperature fermentation using a newly isolated thermotolerant strain of the yeast Kluyveromyces marxianus. We demonstrate that a 5 degrees C increase only in the fermentation temperature can greatly affect the fuel ethanol production costs. We contend that this approach may also be applicable to the other microbial fermentations systems and propose that thermotolerant mesophilic microorganisms have considerable potential for the development of future fermentation technologies.

  9. Short communication: Identification and technological characterization of yeast strains isolated from samples of water buffalo Mozzarella cheese.

    PubMed

    Aponte, M; Pepe, O; Blaiotta, G

    2010-06-01

    Sixty yeast cultures were isolated from samples of water buffalo Mozzarella, a popular "pasta filata" cheese, originating on 16 farms located in the provinces of Salerno, Caserta, and Frosinone (Italy). Strains were identified by means of 5.8S internal transcribed spacer rDNA PCR-RFLP combined with 26S rRNA gene partial sequencing and characterized for their ability to exert biochemical properties of technological interest. The recorded dominance of fermenting yeasts such as the lactose-fermenting Kluyveromyces marxianus (38.3% of the total isolates) and the galactose-fermenting Saccharomyces cerevisiae (21.6% of the total isolates) suggests that these yeasts contribute to the organoleptic definition of the water buffalo Mozzarella. The speciographic analysis revealed the presence of 7 other species rarely or never reported in a dairy environment belonging to the genera Pichia and Candida, whose role in Mozzarella cheese organoleptic properties need to be further investigated.

  10. Development of a hybrid fermentation-enzymatic bioprocess for the production of ethyl lactate from dairy waste.

    PubMed

    Koutinas, Michalis; Menelaou, Maria; Nicolaou, Evrydiki N

    2014-08-01

    This work explores the potential for the development of a hybrid fermentation-enzymatic process for the production of ethyl lactate from dairy waste. Cheese whey was used in Kluyveromyces marxianus and Lactobacillus bulgaricus batch cultures to produce ethanol and lactic acid respectively. Subsequently, the fermentation products were transferred into an organic phase through liquid-liquid extraction and ethyl lactate was formed in an esterification reaction catalyzed by lipases. The production of ethanol and lactic acid achieved under different conditions was 23gL(-1) and 29gL(-1), respectively. Furthermore, the efficiency of various organic solvents for the esterification reaction was evaluated and toluene was chosen for application in the process. The effect of water content was determined aiming to maximize the product yield and 40mgml(-1) was the optimal enzyme concentration. The bioprocess achieved maximum conversion of 33% constituting a valuable alternative to the application of energy demanding chemically derived methods.

  11. Bioconversion of renewable resources into ethanol: An economic evaluation of selected hydrolysis, fermentation, and membrane technologies

    SciTech Connect

    Qureshi, N.; Manderson, G.J.

    1995-03-01

    Four renewable agricultural resources were considered in a process design analysis for the industrial production of ethanol. Raw materials considered were wood, molasses, whey permeate, and starch. Final fermentation substrates were diluted and/or concentrated to give equivalent sugar concentrations for each case. Renewable resource costs were expressed as $/kg of sugar rather than /kg of the raw material. Molasses sugars were cheaper than sugars derived from the other raw materials. Various fermentation technologies were considered, including continuous culture and cell recycle. Ethanol recovery was examined using pervaporation and costs compared with distillation. The effects on ethanol prices of raw material costs, fermentation technology, product recovery, tax, plant size, and Lang factor are presented. Cultures of Candida shehatae, Zymomonas mobilis, Kluyveromyces marxianus var. lactis and Saccharomyces cerevisiae (with Zymomonas mobilis) were used, depending on the substrate. The report identifies the most appropriate technologies in terms of final ethanol price.

  12. Preparation and characterization of whey protein hydrolysates: applications in industrial whey bioconversion processes.

    PubMed

    Perea, A; Ugalde, U; Rodriguez, I; Serra, J L

    1993-05-01

    A whey protein hydrolysate was prepared by incubation of reconstituted whey or a whey protein concentrate with Alcalase 0.6L. The proteolytic degradation of alpha-lactalbumin and beta-lactoglobulin initially resulted in 6-kDa and, later, 2.5-kDa degradation products, quickly followed by the appearance of multiple peptides of 1 kDa or smaller. The hydrolysate showed a steady increase in solubility and a biphasic change in foaming characteristics with decreasing peptide size. At the highest degree of hydrolysis achieved (22%), the majority of the peptides were smaller than 1 kDa and could be efficiently assimilated by the yeast Kluyveromyces marxianus growing in a defined medium.

  13. Continuous ethanol fermentation of lactose by a recombinant flocculating Saccharomyces cerevisiae strain

    SciTech Connect

    Domingues, L.; Dantas, M.M.; Lima, N.; Teixeira, J.A.

    1999-09-20

    Alcohol fermentation of lactose was investigated using a recombinant flocculating Saccharomyces cetevisiae, expressing the LAC4 (coding the {beta}-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus. Data on yeast fermentation and growth on a medium containing lactose as the sole carbon source are presented. In the range of studied lactose concentrations, total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. For the continuously operating bioreactor, an ethanol productivity of 11 g L{sup {minus}1} h{sup {minus}1} (corresponding to a feed lactose concentration of 50 g L{sup {minus}1} and a dilution rate of 0.55 h{sup {minus}1}) was obtained, which is 7 times larger than the continuous conventional systems. The system stability was confirmed by keeping it in operation for 6 months.

  14. A mathematical model of ethanol fermentation from cheese whey. I: Model development and parameter estimation

    SciTech Connect

    Wang, Chen-Jen; Bajpai, R.K.

    1997-12-31

    The cybernetic approach to modeling of microbial kinetics in a mixed-substrate environment has been used in this work for the fermentative production of ethanol from cheese whey. In this system, the cells grow on multiple substrates and generate metabolic energy during product formation. This article deals with the development of a mathematical model in which the concept of cell maintenance was modified in light of the specific nature of product formation. Continuous culture data for anaerobic production of ethanol by Kluyveromyces marxianus CBS 397 on glucose and lactose were used to estimate the kinetic parameters for subsequent use in predicting the behavior of microbial growth and product formation in new situations. 28 refs., 4 figs., 2 tabs.

  15. Isolation, Identification and Characterization of Yeasts from Fermented Goat Milk of the Yaghnob Valley in Tajikistan.

    PubMed

    Qvirist, Linnea A; De Filippo, Carlotta; Strati, Francesco; Stefanini, Irene; Sordo, Maddalena; Andlid, Thomas; Felis, Giovanna E; Mattarelli, Paola; Cavalieri, Duccio

    2016-01-01

    The geographically isolated region of the Yaghnob Valley, Tajikistan, has allowed its inhabitants to maintain a unique culture and lifestyle. Their fermented goat milk constitutes one of the staple foods for the Yaghnob population, and is produced by backslopping, i.e., using the previous fermentation batch to inoculate the new one. This study addresses the yeast composition of the fermented milk, assessing genotypic, and phenotypic properties. The 52 isolates included in this study revealed small species diversity, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces cerevisiae, and one Kazachstania unispora. The K. marxianus strains showed two different genotypes, one of which never described previously. The two genetically different groups also differed significantly in several phenotypic characteristics, such as tolerance toward high temperatures, low pH, and presence of acid. Microsatellite analysis of the S. cerevisiae strains from this study, compared to 350 previously described strains, attributed the Yaghnobi S. cerevisiae to two different ancestry origins, both distinct from the wine and beer strains, and similar to strains isolated from human and insects feces, suggesting a peculiar origin of these strains, and the existence of a gut reservoir for S. cerevisiae. Our work constitutes a foundation for strain selection for future applications as starter cultures in food fermentations. This work is the first ever on yeast diversity from fermented milk of the previously unexplored area of the Yaghnob Valley.

  16. Yeasts associated with Sardinian ewe's dairy products.

    PubMed

    Cosentino, S; Fadda, M E; Deplano, M; Mulargia, A F; Palmas, F

    2001-09-19

    In the present work, the occurrence of yeasts in different types of typical Sardinian ewe's cheeses (32 samples of pecorino, 32 of caciotta, 40 of feta, 56 of ricotta) was determined. For the strains isolated the following properties were studied: proteolytic and lipolytic activities, the ability to grow at different temperatures, different concentrations of salt, and to assimilate and/or ferment compounds like lactate, citrate, lactose, glucose, galactose, lactic acid. Of 160 samples analysed, 76.2% yielded growth of yeasts. Yeast counts showed a certain variability among the samples. The highest levels were observed in caciotta and feta cheeses. A total of 281 strains belonging to 16 genera and 25 species were identified. In general, Debaryomyces hansenii was the dominant species, representing 28.8% of the total isolates. Other frequently appearing species were Geotrichum candidum, Kluyveromyces lactis and K. marxianus. Other genera encountered were Pichia, Candida, Dekkera, Yarrowia and Rhodotorula. With regard to the biochemical and technological properties of the yeasts, only K. lactis, K. marxianus and Dek. anomala assimilated and fermented lactose, whereas the majority of the species assimilated lactic acid. The assimilation of citrate was a characteristic of D. hansenii, R. rubra and Y. lipolytica. On the whole, the yeasts were weakly proteolytic while lipolytic activity was present in several species. A high percentage of strains showed a certain tolerance to low temperatures while only some strains of D. hansenii and K. lactis were able to grow at a 10% NaCl concentration.

  17. Yeast communities associated with artisanal mezcal fermentations from Agave salmiana.

    PubMed

    Verdugo Valdez, A; Segura Garcia, L; Kirchmayr, M; Ramírez Rodríguez, P; González Esquinca, A; Coria, R; Gschaedler Mathis, A

    2011-11-01

    The aims of this work were to characterize the fermentation process of mezcal from San Luis Potosi, México and identify the yeasts present in the fermentation using molecular culture-dependent methods (RFLP of the 5.8S-ITS and sequencing of the D1/D2 domain) and also by using a culture-independent method (DGGE). The alcoholic fermentations of two separate musts obtained from Agave salmiana were analyzed. Sugar, ethanol and major volatile compounds concentrations were higher in the first fermentation, which shows the importance of having a quality standard for raw materials, particularly in the concentration of fructans, in order to produce fermented Agave salmiana must with similar characteristics. One hundred ninety-two (192) different yeast colonies were identified, from those present on WL agar plates, by RFLP analysis of the ITS1-5.8S- ITS2 from the rRNA gene, with restriction endonucleases, HhaI, HaeIII and HinfI. The identified yeasts were: Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Clavispora lusitaniae, Torulaspora delbrueckii, Candida ethanolica and Saccharomyces exiguus. These identifications were confirmed by sequencing the D1-D2 region of the 26S rRNA gene. With the PCR-DGGE method, bands corresponding to S. cerevisiae, K. marxianus and T. delbrueckii were clearly detected, confirming the results obtained with classic techniques.

  18. Discarded oranges and brewer's spent grains as promoting ingredients for microbial growth by submerged and solid state fermentation of agro-industrial waste mixtures.

    PubMed

    Aggelopoulos, Theodoros; Bekatorou, Argyro; Pandey, Ashok; Kanellaki, Maria; Koutinas, Athanasios A

    2013-08-01

    The exploitation of various agro-industrial wastes for microbial cell mass production of Kluyveromyces marxianus, kefir, and Saccharomyces cerevisiae is reported in the present investigation. Specifically, the promotional effect of whole orange pulp on cell growth in mixtures consisting of cheese whey, molasses, and potato pulp in submerged fermentation processes was examined. A 2- to 3-fold increase of cell mass was observed in the presence of orange pulp. Likewise, the promotional effect of brewer's spent grains on cell growth in solid state fermentation of mixtures of whey, molasses, potato pulp, malt spent rootlets, and orange pulp was examined. The cell mass was increased by 3-fold for K. marxianus and 2-fold for S. cerevisiae in the presence of these substrates, proving their suitability for single-cell protein production without the need for extra nutrients. Cell growth kinetics were also studied by measurements of cell counts at various time intervals at different concentrations of added orange pulp. The protein content of the fermented substrates was increased substantially, indicating potential use of mixed agro-industrial wastes of negligible cost, as protein-enriched livestock feed, achieving at the same time creation of added value and waste minimization.

  19. Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and Saccharomyces cerevisiae Cells in Dual Matrices.

    PubMed

    Kokkiligadda, Anusha; Beniwal, Arun; Saini, Priyanka; Vij, Shilpa

    2016-08-01

    Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.

  20. Enhanced enzymatic hydrolysis and ethanol production from cashew apple bagasse pretreated with alkaline hydrogen peroxide.

    PubMed

    da Costa, Jessyca Aline; Marques, José Edvan; Gonçalves, Luciana Rocha Barros; Rocha, Maria Valderez Ponte

    2015-03-01

    The effect of combinations and ratios between different enzymes has been investigated in order to assess the optimal conditions for hydrolysis of cashew apple bagasse pretreated with alkaline hydrogen peroxide (the solids named CAB-AHP). The separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes were evaluated in the ethanol production. The enzymatic hydrolysis conducted with cellulase complex and β-glucosidase in a ratio of 0.61:0.39, enzyme loading of 30FPU/g(CAB-AHP) and 66CBU/g(CAB-AHP), respectively, using 4% cellulose from CAB-AHP, turned out to be the most effective conditions, with glucose and xylose yields of 511.68 mg/g(CAB-AHP) and 237.8 mg/g(CAB-AHP), respectively. Fermentation of the pure hydrolysate by Kluyveromyces marxianus ATCC 36907 led to an ethanol yield of 61.8kg/ton(CAB), corresponding to 15 g/L ethanol and productivity of 3.75 g/( Lh). The ethanol production obtained for SSF process using K. marxianus ATCC 36907 was 18 g/L corresponding to 80% yield and 74.2kg/ton(CAB).

  1. Isolation, Identification and Characterization of Yeasts from Fermented Goat Milk of the Yaghnob Valley in Tajikistan

    PubMed Central

    Qvirist, Linnea A.; De Filippo, Carlotta; Strati, Francesco; Stefanini, Irene; Sordo, Maddalena; Andlid, Thomas; Felis, Giovanna E.; Mattarelli, Paola; Cavalieri, Duccio

    2016-01-01

    The geographically isolated region of the Yaghnob Valley, Tajikistan, has allowed its inhabitants to maintain a unique culture and lifestyle. Their fermented goat milk constitutes one of the staple foods for the Yaghnob population, and is produced by backslopping, i.e., using the previous fermentation batch to inoculate the new one. This study addresses the yeast composition of the fermented milk, assessing genotypic, and phenotypic properties. The 52 isolates included in this study revealed small species diversity, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces cerevisiae, and one Kazachstania unispora. The K. marxianus strains showed two different genotypes, one of which never described previously. The two genetically different groups also differed significantly in several phenotypic characteristics, such as tolerance toward high temperatures, low pH, and presence of acid. Microsatellite analysis of the S. cerevisiae strains from this study, compared to 350 previously described strains, attributed the Yaghnobi S. cerevisiae to two different ancestry origins, both distinct from the wine and beer strains, and similar to strains isolated from human and insects feces, suggesting a peculiar origin of these strains, and the existence of a gut reservoir for S. cerevisiae. Our work constitutes a foundation for strain selection for future applications as starter cultures in food fermentations. This work is the first ever on yeast diversity from fermented milk of the previously unexplored area of the Yaghnob Valley. PMID:27857705

  2. Inhibition of Yeast Growth by Octanoic and Decanoic Acids Produced during Ethanolic Fermentation

    PubMed Central

    Viegas, Cristina A.; Rosa, M. Fernanda; Sá-Correia, Isabel; Novais, Júlio M.

    1989-01-01

    The inhibition of growth by octanoic or decanoic acids, two subproducts of ethanolic fermentation, was evaluated in Saccharomyces cerevisiae and Kluyveromyces marxianus in association with ethanol, the main product of fermentation. In both strains, octanoic and decanoic acids, at concentrations up to 16 and 8 mg/liter, respectively, decreased the maximum specific growth rate and the biomass yield at 30°C as an exponential function of the fatty acid concentration and increased the duration of growth latency. These toxic effects increased with a decrease in pH in the range of 5.4 to 3.0, indicating that the undissociated form is the toxic molecule. Decanoic acid was more toxic than octanoic acid. The concentrations of octanoic and decanoic acids were determined during the ethanolic fermentation (30°C) of two laboratory media (mineral and complex) by S. cerevisiae and of Jerusalem artichoke juice by K. marxianus. Based on the concentrations detected (0.7 to 23 mg/liter) and the kinetics of growth inhibition, the presence of octanoic and decanoic acids cannot be ignored in the evaluation of the overall inhibition of ethanolic fermentation. PMID:16347826

  3. Utilization of inulin-containing waste in industrial fermentations to produce biofuels and bio-based chemicals.

    PubMed

    Hughes, Stephen R; Qureshi, Nasib; López-Núñez, Juan Carlos; Jones, Marjorie A; Jarodsky, Joshua M; Galindo-Leva, Luz Ángela; Lindquist, Mitchell R

    2017-04-01

    Inulins are polysaccharides that belong to an important class of carbohydrates known as fructans and are used by many plants as a means of storing energy. Inulins contain 20 to several thousand fructose units joined by β-2,1 glycosidic bonds, typically with a terminal glucose unit. Plants with high concentrations of inulin include: agave, asparagus, coffee, chicory, dahlia, dandelion, garlic, globe artichoke, Jerusalem artichoke, jicama, onion, wild yam, and yacón. To utilize inulin as its carbon and energy source directly, a microorganism requires an extracellular inulinase to hydrolyze the glycosidic bonds to release fermentable monosaccharides. Inulinase is produced by many microorganisms, including species of Aspergillus, Kluyveromyces, Penicillium, and Pseudomonas. We review various inulinase-producing microorganisms and inulin feedstocks with potential for industrial application as well as biotechnological efforts underway to develop sustainable practices for the disposal of residues from processing inulin-containing crops. A multi-stage biorefinery concept is proposed to convert cellulosic and inulin-containing waste produced at crop processing operations to valuable biofuels and bioproducts using Kluyveromyces marxianus, Yarrowia lipolytica, Rhodotorula glutinis, and Saccharomyces cerevisiae as well as thermochemical treatments.

  4. Taggiasca extra virgin olive oil colonization by yeasts during the extraction process.

    PubMed

    Ciafardini, G; Cioccia, G; Zullo, B A

    2017-04-01

    The opalescent appearance of the newly produced olive oil is due to the presence of solid particles and microdrops of vegetation water in which the microorganisms from the olives' carposphere are trapped. Present research has demonstrated that the microbiota of the fresh extracted olive oil, produced in the mills, is mainly composed of yeasts and to a lesser extent of molds. The close link between the composition of the microbiota of the olives' carposphere undergoing to processing, and that of the microbiota of the newly produced olive oil, concerns only the yeasts and molds, given that the bacterial component is by and large destroyed mainly in the kneaded paste during the malaxation process. Six physiologically homogenous yeast groups were highlighted in the wash water, kneaded paste and newly produced olive oil from the Taggiasca variety which had been collected in mills located in the Liguria region. The more predominant yeasts of each group belonged to a single species called respectively: Kluyveromyces marxianus, Candida oleophila, Candida diddensiae, Candida norvegica, Wickerhamomyces anomalus and Debaryomyces hansenii. Apart from K. marxianus, which was found only in the wash water, all the other species were found in the wash water and in the kneaded paste as well as in the newly produced olive oil, while in the six-month stored olive oil, was found only one physiologically homogeneous group of yeast represented by the W. anomalus specie. These findings in according to our previous studies carried out on other types of mono varietal olive oils, confirms that the habitat of the Taggiascas' extra virgin olive oil, had a strong selective pressure on the yeast biota, allowing only to a few member of yeast species, contaminating the fresh product, to survive and reproduce in it during storage.

  5. Characteristics of microbial biofilm on wooden vats ('gerles') in PDO Salers cheese.

    PubMed

    Didienne, Robert; Defargues, Catherine; Callon, Cécile; Meylheuc, Thierry; Hulin, Sophie; Montel, Marie-Christine

    2012-05-15

    The purpose of this study was to characterize microbial biofilms from 'gerles' (wooden vats for making PDO Salers cheese) and identify their role in milk inoculation and in preventing pathogen development. Gerles from ten farms producing PDO Salers cheese were subjected to microbial analysis during at least 4 periods spread over two years. They were distinguished by their levels of Lactobacillus (between 4.50 and 6.01 log CFU/cm(2)), Gram negative bacteria (between 1.45 and 4.56 log CFU/cm(2)), yeasts (between 2.91 and 5.57 log CFU/cm(2)), and moulds (between 1.72 and 4.52 log CFU/cm(2)). They were then classed into 4 groups according their microbial characteristics. These 4 groups were characterized by different milk inoculations (with either sour whey or starter culture, daily or not), and different washing procedures (with water or whey from cheese making). The farm gerles were not contaminated by Salmonella, Listeria monocytogenes or Staphylococcus aureus. Only one slight, punctual contamination was found on one gerle among the ten studied. Even when the milk was deliberately contaminated with L. monocytogenes and S. aureus in the 40 L experimental gerles, these pathogens were found neither on the gerle surfaces nor in the cheeses. Using 40 L experimental gerles it was shown that the microbial biofilms on the gerle surfaces formed in less than one week and then remained stable. They were mainly composed of a great diversity of lactic acid bacteria (Leuconostoc pseudomesenteroides, Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus hilgardii,…), Gram positive catalase positive bacteria (Curtobacterium flaccumfaciens, Curtobacterium oceanosedimentum Citrococcus spp., Brachybacterium rhamnosum, Kocuria rhizophila, Arthrobacter spp.…) and yeast (Kluyveromyces lactis, Kluyveromyces marxianus). In less than 1 min, even in a 500 L farm gerle, the gerle's microbial biofilm can inoculate pasteurized milk with micro-organisms at levels

  6. Fine Structure of Tibetan Kefir Grains and Their Yeast Distribution, Diversity, and Shift

    PubMed Central

    Lu, Man; Wang, Xingxing; Sun, Guowei; Qin, Bing; Xiao, Jinzhou; Yan, Shuling; Pan, Yingjie; Wang, Yongjie

    2014-01-01

    Tibetan kefir grains (TKGs), a kind of natural starter for fermented milk in Tibet, China, host various microorganisms of lactic acid bacteria, yeasts, and occasionally acetic acid bacteria in a polysaccharide/protein matrix. In the present study, the fine structure of TKGs was studied to shed light on this unusual symbiosis with stereomicroscopy and thin sections. The results reveal that TKGs consist of numerous small grain units, which are characterized by a hollow globular structure with a diameter between 2.0 and 9.0 mm and a wall thickness of approximately 200 µm. A polyhedron-like net structure, formed mainly by the bacteria, was observed in the wall of the grain units, which has not been reported previously to our knowledge. Towards the inside of the grain unit, the polyhedron-like net structures became gradually larger in diameter and fewer in number. Such fine structures may play a crucial role in the stability of the grains. Subsequently, the distribution, diversity, and shift of yeasts in TKGs were investigated based on thin section, scanning electron microscopy, cloning and sequencing of D1/D2 of the 26S rRNA gene, real-time quantitative PCR, and in situ hybridization with specific fluorescence-labeled oligonucleotide probes. These show that (i) yeasts appear to localize on the outer surface of the grains and grow normally together to form colonies embedded in the bacterial community; (ii) the diversity of yeasts is relatively low on genus level with three dominant species – Saccharomyces cerevisiae, Kluyveromyces marxianus, and Yarrowia lipolytica; (iii) S. cerevisiae is the stable predominant yeast species, while the composition of Kluyveromyces and Yarrowia are subject to change over time. Our results indicate that TKGs are relatively stable in structure, and culture conditions to some extent shape the microbial community and interaction in kefir grains. These findings pave the way for further study of the specific symbiotic associations between S

  7. Coexistence of Lactic Acid Bacteria and Potential Spoilage Microbiota in a Dairy Processing Environment.

    PubMed

    Stellato, Giuseppina; De Filippis, Francesca; La Storia, Antonietta; Ercolini, Danilo

    2015-11-01

    Microbial contamination in food processing plants can play a fundamental role in food quality and safety. In this study, the microbiota in a dairy plant was studied by both 16S rRNA- and 26S rRNA-based culture-independent high-throughput amplicon sequencing. Environmental samples from surfaces and tools were studied along with the different types of cheese produced in the same plant. The microbiota of environmental swabs was very complex, including more than 200 operational taxonomic units with extremely variable relative abundances (0.01 to 99%) depending on the species and sample. A core microbiota shared by 70% of the samples indicated a coexistence of lactic acid bacteria with a remarkable level of Streptococcus thermophilus and possible spoilage-associated bacteria, including Pseudomonas, Acinetobacter, and Psychrobacter, with a relative abundance above 50%. The most abundant yeasts were Kluyveromyces marxianus, Yamadazyma triangularis, Trichosporon faecale, and Debaryomyces hansenii. Beta-diversity analyses showed a clear separation of environmental and cheese samples based on both yeast and bacterial community structure. In addition, predicted metagenomes also indicated differential distribution of metabolic pathways between the two categories of samples. Cooccurrence and coexclusion pattern analyses indicated that the occurrence of potential spoilers was excluded by lactic acid bacteria. In addition, their persistence in the environment can be helpful to counter the development of potential spoilers that may contaminate the cheeses, with possible negative effects on their microbiological quality.

  8. Selection of enhanced antimicrobial activity posing lactic acid bacteria characterised by (GTG)5-PCR fingerprinting.

    PubMed

    Šalomskienė, Joana; Abraitienė, Asta; Jonkuvienė, Dovilė; Mačionienė, Irena; Repečkienė, Jūratė

    2015-07-01

    The aim of the study was a detail evaluation of genetic diversity among the lactic acid bacteria (LAB) strains having an advantage of a starter culture in order to select genotypically diverse strains with enhanced antimicrobial effect on some harmfull and pathogenic microorganisms. Antimicrobial activity of LAB was performed by the agar well diffusion method and was examined against the reference strains and foodborne isolates of Bacillus cereus, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium. Antifungal activity was tested against the foodborne isolates of Candida parapsilosis, Debaromyces hansenii, Kluyveromyces marxianus, Pichia guilliermondii, Yarowia lipolytica, Aspergillus brasiliensis, Aspergillus versicolor, Cladosporium herbarum, Penicillium chrysogenum and Scopulariopsis brevicaulis. A total 40 LAB strains representing Lactobacillus (23 strains), Lactococcus (13 strains) and Streptococcus spp. (4 strains) were characterised by repetitive sequence based polymerase chain reaction fingerprinting which generated highly discriminatory profiles, confirmed the identity and revealed high genotypic heterogeneity among the strains. Many of tested LAB demonstrated strong antimicrobial activity specialised against one or few indicator strains. Twelve LAB strains were superior in suppressing growth of the whole complex of pathogenic bacteria and fungi. These results demonstrated that separate taxonomic units offered different possibilities of selection for novel LAB strains could be used as starter cultures enhancing food preservation.

  9. Oxidative stress and antioxidant response in a thermotolerant yeast.

    PubMed

    Mejía-Barajas, Jorge A; Montoya-Pérez, Rocío; Salgado-Garciglia, Rafael; Aguilera-Aguirre, Leopoldo; Cortés-Rojo, Christian; Mejía-Zepeda, Ricardo; Arellano-Plaza, Melchor; Saavedra-Molina, Alfredo

    Stress tolerance is a key attribute that must be considered when using yeast cells for industrial applications. High temperature is one factor that can cause stress in yeast. High environmental temperature in particular may exert a natural selection pressure to evolve yeasts into thermotolerant strains. In the present study, three yeasts (Saccharomyces cerevisiae, MC4, and Kluyveromyces marxianus, OFF1 and SLP1) isolated from hot environments were exposed to increased temperatures and were then compared with a laboratory yeast strain. Their resistance to high temperature, oxidative stress, and antioxidant response were evaluated, along with the fatty acid composition of their cell membranes. The SLP1 strain showed a higher specific growth rate, biomass yield, and biomass volumetric productivity while also showing lower duplication time, reactive oxygen species (ROS) production, and lipid peroxidation. In addition, the SLP1 strain demonstrated more catalase activity after temperature was increased, and this strain also showed membranes enriched in saturated fatty acids. It is concluded that the SLP1 yeast strain is a thermotolerant yeast with less oxidative stress and a greater antioxidant response. Therefore, this strain could be used for fermentation at high temperatures.

  10. Strategy for biotechnological process design applied to the enzymatic hydrolysis of agave fructo-oligosaccharides to obtain fructose-rich syrups.

    PubMed

    García-Aguirre, Mauricio; Sáenz-Alvaro, Victor A; Rodríguez-Soto, Mayra A; Vicente-Magueyal, Francisco J; Botello-Alvarez, Enrique; Jimenez-Islas, Hugo; Cárdenas-Manríquez, Marcela; Rico-Martínez, Ramiro; Navarrete-Bolaños, Jose L

    2009-11-11

    A strategy to optimize biotechnological process design is illustrated for the production of fructose-rich syrups via enzymatic hydrolysis of agave fructo-oligosaccharides. The optimization process includes ecological studies from natural fermentations leading to the selection of a strain with capacity for inulinase synthesis, and variable optimization for the synthesis, and enzymatic hydrolysis using the response surface methodology. The results lead to the selection of Kluyveromyces marxianus , endogenous strains isolated from aguamiel (natural fermented sugary sap from agave plants), as the main strain with high capacity for enzyme synthesis with inulinase activity. Production optimization at bioreactor level revealed that operation at 30.6 degrees C, 152 rpm, 1.3 VVM of aeration, and pH 6.3 leads to maximum inulinase synthesis, whereas 31 degrees C, 50 rpm, and pH 6.2 leads to maximum hydrolysis of agave fructo-oligosaccharides. HPLC analysis of the fructose-rich syrups obtained at these optimal conditions showed an average composition of 95% of fructose and 5% of glucose and the absence of sucrose. The analysis also revealed that the syrups are free of residues and toxic compounds, an undesirable occurrence often present when traditional methods based on thermal or acid hydrolysis are applied for their obtainment. Therefore, the product may be suitable for use as additive in many applications in the food and beverage industries.

  11. Cellulose Biorefinery Based on a Combined Catalytic and Biotechnological Approach for Production of 5-HMF and Ethanol.

    PubMed

    Sorokina, Ksenia N; Taran, Oxana P; Medvedeva, Tatiana B; Samoylova, Yuliya V; Piligaev, Alexandr V; Parmon, Valentin N

    2017-02-08

    In this study, a combination of catalytic and biotechnological processes was proposed for the first time for application in a cellulose biorefinery for the production of 5-hydroxymethylfurfural (5-HMF) and bioethanol. Hydrolytic dehydration of the mechanically activated microcrystalline cellulose over a carbon-based mesoporous Sibunt-4 catalyst resulted in moderate yields of glucose and 5-HMF (21.1-25.1 and 6.6-9.4 %). 5-HMF was extracted from the resulting mixture with isobutanol and subjected to ethanol fermentation. A number of yeast strains were isolated that also revealed high thermotolerance (up to 50 °C) and resistance to inhibitors found in the hydrolysates. The strains Kluyveromyces marxianus C1 and Ogataea polymorpha CBS4732 were capable of producing ethanol from processed catalytic hydrolysates of cellulose at 42 °C, with yields of 72.0±5.7 and 75.2±4.3 % from the maximum theoretical yield of ethanol, respectively.

  12. Looking beyond Saccharomyces: the potential of non-conventional yeast species for desirable traits in bioethanol fermentation.

    PubMed

    Radecka, Dorota; Mukherjee, Vaskar; Mateo, Raquel Quintilla; Stojiljkovic, Marija; Foulquié-Moreno, María R; Thevelein, Johan M

    2015-09-01

    Saccharomyces cerevisiae has been used for millennia in the production of food and beverages and is by far the most studied yeast species. Currently, it is also the most used microorganism in the production of first-generation bioethanol from sugar or starch crops. Second-generation bioethanol, on the other hand, is produced from lignocellulosic feedstocks that are pretreated and hydrolyzed to obtain monomeric sugars, mainly D-glucose, D-xylose and L-arabinose. Recently, S. cerevisiae recombinant strains capable of fermenting pentose sugars have been generated. However, the pretreatment of the biomass results in hydrolysates with high osmolarity and high concentrations of inhibitors. These compounds negatively influence the fermentation process. Therefore, robust strains with high stress tolerance are required. Up to now, more than 2000 yeast species have been described and some of these could provide a solution to these limitations because of their high tolerance to the most predominant stress conditions present in a second-generation bioethanol reactor. In this review, we will summarize what is known about the non-conventional yeast species showing unusual tolerance to these stresses, namely Zygosaccharomyces rouxii (osmotolerance), Kluyveromyces marxianus and Ogataea (Hansenula) polymorpha (thermotolerance), Dekkera bruxellensis (ethanol tolerance), Pichia kudriavzevii (furan derivatives tolerance) and Z. bailii (acetic acid tolerance).

  13. Bioethanol production from Scenedesmus obliquus sugars: the influence of photobioreactors and culture conditions on biomass production.

    PubMed

    Miranda, J R; Passarinho, P C; Gouveia, L

    2012-10-01

    A closed-loop vertical tubular photobioreactor (PBR), specially designed to operate under conditions of scarce flat land availability and irregular solar irradiance conditions, was used to study the potential of Scenedesmus obliquus biomass/sugar production. The results obtained were compared to those from an open-raceway pond and a closed-bubble column. The influence of the type of light source and the regime (natural vs artificial and continuous vs light/dark cycles) on the growth of the microalga and the extent of the sugar accumulation was studied in both PBRs. The best type of reactor studied was a closed-loop PBR illuminated with natural light/dark cycles. In all the cases, the relationship between the nitrate depletion and the sugar accumulation was observed. The microalga Scenedesmus was cultivated for 53 days in a raceway pond (4,500 L) and accumulated a maximum sugar content of 29 % g/g. It was pre-treated for carrying out ethanol fermentation assays, and the highest ethanol concentration obtained in the hydrolysate fermented by Kluyveromyces marxianus was 11.7 g/L.

  14. Molecular identification of yeasts associated with traditional Egyptian dairy products.

    PubMed

    El-Sharoud, W M; Belloch, C; Peris, D; Querol, A

    2009-09-01

    This study aimed to examine the diversity and ecology of yeasts associated with traditional Egyptian dairy products employing molecular techniques in yeast identification. A total of 120 samples of fresh and stored Domiati cheese, kariesh cheese, and "Matared" cream were collected from local markets and examined. Forty yeast isolates were cultured from these samples and identified using the restriction-fragment length polymorphism (RFLPs) of 5.8S-ITS rDNA region and sequencing of the domains D1 and D2 of the 26S rRNA gene. Yeasts were identified as Issatchenkia orientalis (13 isolates), Candida albicans (4 isolates), Clavispora lusitaniae (Candida lusitaniae) (9 isolates), Kodamaea ohmeri (Pichia ohmeri) (1 isolate), Kluyveromyces marxianus (6 isolates), and Candida catenulata (7 isolates). With the exception of C. lusitaniae, the D1/D2 26S rRNA gene sequences were 100% identical for the yeast isolates within the same species. Phylogenetic reconstruction of C. lusitaniae isolates grouped them into 3 distinguished clusters. Kariesh cheese was found to be the most diverse in its yeast floras and contained the highest total yeast count compared with other examined dairy products. This was linked to the acidic pH and lower salt content of this cheese, which favor the growth and survival of yeasts in foodstuffs. Stored Domiati cheese also contained diverse yeast species involving isolates of the pathogenic yeast C. albicans. This raises the possibility of dairy products being vehicles of transmission of pathogenic yeasts.

  15. Yeast Biomass Production in Brewery's Spent Grains Hemicellulosic Hydrolyzate

    NASA Astrophysics Data System (ADS)

    Duarte, Luís C.; Carvalheiro, Florbela; Lopes, Sónia; Neves, Ines; Gírio, Francisco M.

    Yeast single-cell protein and yeast extract, in particular, are two products which have many feed, food, pharmaceutical, and biotechnological applications. However, many of these applications are limited by their market price. Specifically, the yeast extract requirements for culture media are one of the major technical hurdles to be overcome for the development of low-cost fermentation routes for several top value chemicals in a biorefinery framework. A potential biotechnical solution is the production of yeast biomass from the hemicellulosic fraction stream. The growth of three pentose-assimilating yeast cell factories, Debaryomyces hansenii, Kluyveromyces marxianus, and Pichia stipitis was compared using non-detoxified brewery's spent grains hemicellulosic hydrolyzate supplemented with mineral nutrients. The yeasts exhibited different specific growth rates, biomass productivities, and yields being D. hansenii as the yeast species that presented the best performance, assimilating all sugars and noteworthy consuming most of the hydrolyzate inhibitors. Under optimized conditions, D. hansenii displayed a maximum specific growth rate, biomass yield, and productivity of 0.34 h-1, 0.61 g g-1, and 0.56 g 1-1 h-1, respectively. The nutritional profile of D. hansenii was thoroughly evaluated, and it compares favorably to others reported in literature. It contains considerable amounts of some essential amino acids and a high ratio of unsaturated over saturated fatty acids.

  16. The influence of presaccharification, fermentation temperature and yeast strain on ethanol production from sugarcane bagasse.

    PubMed

    de Souza, Carlos J A; Costa, Daniela A; Rodrigues, Marina Q R B; dos Santos, Ancély F; Lopes, Mariana R; Abrantes, Aline B P; dos Santos Costa, Patrícia; Silveira, Wendel Batista; Passos, Flávia M L; Fietto, Luciano G

    2012-04-01

    Ethanol can be produced from cellulosic biomass in a process known as simultaneous saccharification and fermentation (SSF). The presence of yeast together with the cellulolytic enzyme complex reduces the accumulation of sugars within the reactor, increasing the ethanol yield and saccharification rate. This paper reports the isolation of Saccharomyces cerevisiae LBM-1, a strain capable of growth at 42 °C. In addition, S. cerevisiae LBM-1 and Kluyveromyces marxianus UFV-3 were able to ferment sugar cane bagasse in SSF processes at 37 and 42 °C. Higher ethanol yields were observed when fermentation was initiated after presaccharification at 50°C than at 37 or 42° C. Furthermore, the volumetric productivity of fermentation increased with presaccharification time, from 0.43 g/L/h at 0 h to 1.79 g/L/h after 72 h of presaccharification. The results suggest that the use of thermotolerant yeasts and a presaccharification stage are key to increasing yields in this process.

  17. Steam explosion treatment for ethanol production from branches pruned from pear trees by simultaneous saccharification and fermentation.

    PubMed

    Sasaki, Chizuru; Okumura, Ryosuke; Asada, Chikako; Nakamura, Yoshitoshi

    2014-01-01

    This study investigated the production of ethanol from unutilized branches pruned from pear trees by steam explosion pretreatment. Steam pressures of 25, 35, and 45 atm were applied for 5 min, followed by enzymatic saccharification of the extracted residues with cellulase (Cellic CTec2). High glucose recoveries, of 93.3, 99.7, and 87.1%, of the total sugar derived from the cellulose were obtained from water- and methanol-extracted residues after steam explosion at 25, 35, and 45 tm, respectively. These values corresponded to 34.9, 34.3, and 27.1 g of glucose per 100 g of dry steam-exploded branches. Simultaneous saccharification and fermentation experiments were done on water-extracted residues and water- and methanol-extracted residues by Kluyveromyces marxianus NBRC 1777. An overall highest theoretical ethanol yield of 76% of the total sugar derived from cellulose was achieved when 100 g/L of water- and methanol-washed residues from 35 atm-exploded pear branches was used as substrate.

  18. Molecular cloning of alcohol dehydrogenase genes of the yeast Pichia stipitis and identification of the fermentative ADH.

    PubMed

    Passoth, V; Schäfer, B; Liebel, B; Weierstall, T; Klinner, U

    1998-10-01

    Two Pichia stipitis ADH genes (PsADH1 and PsADH2) were isolated by complementation of a Saccharomyces cerevisiae Adh(-)-mutant. The genes enabled the transformants to grow in the presence of antimycin A on glucose, to use ethanol as sole carbon source and made them sensitive to allylalcohol. The sequences of the genes showed similarities of 70-77% to sequences of ADH genes of Candida albicans, Kluyveromyces lactis, K. marxianus, and S. cerevisiae and about 60% homology to those of Schizosaccharomyces pombe and Aspergillus flavus. Southern hybridization experiments suggested that P. stipitis has only these two ADH genes. Both genes are located on the largest chromosome of P. stipitis. PsADH2 encodes for the ADH activity that is responsible for ethanol formation at oxygen limitation. The gene is regulated at the transcriptional level. Moreover, also in cells grown on ethanol, only PsADH2 transcript was found. PsADH1 transcript was detected under aerobic conditions on fermentable carbon sources.

  19. Whey valorisation: a complete and novel technology development for dairy industry starter culture production.

    PubMed

    Koutinas, Athanasios A; Papapostolou, Harris; Dimitrellou, Dimitra; Kopsahelis, Nikolaos; Katechaki, Eleftheria; Bekatorou, Argyro; Bosnea, Loulouda A

    2009-08-01

    Whey is the major by-product of the dairy industry, produced in large quantities and usually disposed off causing major environmental pollution, due to its high organic load that makes treatment cost prohibitive. This paper comprises a contribution on the valorisation of this high polluting liquid waste of the dairy industry, based on research for the production of novel dairy starter cultures using whey as raw material. Starter cultures are used for cheese ripening in order to: (i) accelerate ripening, (ii) improve quality and (iii) increase shelf-life. The developed technology involves biomass production from whey followed by thermal drying of cultures. Specifically, Kluyveromyces marxianus, Lactobacillus bulgaricus and kefir yeasts were thermally dried, and their efficiency in lactose and milk whey fermentations was studied. The most suitable culture regarding its technological properties was kefir, which was used for cheese ripening in freeze-dried and thermally dried form. Besides the reduction of production cost, which is an essential requirement for the food industry, the use of thermally dried kefir displayed several other advantages such as acceleration of ripening, increase of shelf-life, and improvement of hard-type cheese quality.

  20. Coexistence of Lactic Acid Bacteria and Potential Spoilage Microbiota in a Dairy Processing Environment

    PubMed Central

    Stellato, Giuseppina; De Filippis, Francesca; La Storia, Antonietta

    2015-01-01

    Microbial contamination in food processing plants can play a fundamental role in food quality and safety. In this study, the microbiota in a dairy plant was studied by both 16S rRNA- and 26S rRNA-based culture-independent high-throughput amplicon sequencing. Environmental samples from surfaces and tools were studied along with the different types of cheese produced in the same plant. The microbiota of environmental swabs was very complex, including more than 200 operational taxonomic units with extremely variable relative abundances (0.01 to 99%) depending on the species and sample. A core microbiota shared by 70% of the samples indicated a coexistence of lactic acid bacteria with a remarkable level of Streptococcus thermophilus and possible spoilage-associated bacteria, including Pseudomonas, Acinetobacter, and Psychrobacter, with a relative abundance above 50%. The most abundant yeasts were Kluyveromyces marxianus, Yamadazyma triangularis, Trichosporon faecale, and Debaryomyces hansenii. Beta-diversity analyses showed a clear separation of environmental and cheese samples based on both yeast and bacterial community structure. In addition, predicted metagenomes also indicated differential distribution of metabolic pathways between the two categories of samples. Cooccurrence and coexclusion pattern analyses indicated that the occurrence of potential spoilers was excluded by lactic acid bacteria. In addition, their persistence in the environment can be helpful to counter the development of potential spoilers that may contaminate the cheeses, with possible negative effects on their microbiological quality. PMID:26341209

  1. Isolation and characterisation of lactic acid bacteria from donkey milk.

    PubMed

    Soto Del Rio, Maria de Los Dolores; Andrighetto, Christian; Dalmasso, Alessandra; Lombardi, Angiolella; Civera, Tiziana; Bottero, Maria Teresa

    2016-08-01

    During the last years the interest in donkey milk has increased significantly mainly because of its compelling functional elements. Even if the composition and nutritional properties of donkey milk are known, its microbiota is less studied. This Research Communication aimed to provide a comprehensive characterisation of the lactic acid bacteria in raw donkey milk. RAPD-PCR assay combined with 16S rDNA sequencing analysis were used to describe the microbial diversity of several donkey farms in the North West part of Italy. The more frequently detected species were: Lactobacillus paracasei, Lactococcus lactis and Carnobacterium maltaromaticum. Less abundant genera were Leuconostoc, Enterococcus and Streptococcus. The yeast Kluyveromyces marxianus was also isolated. The bacterial and biotype distribution notably diverged among the farms. Several of the found species, not previously detected in donkey milk, could have an important probiotic activity and biotechnological potential. This study represents an important insight to the ample diversity of the microorganisms present in the highly selective ecosystem of raw donkey milk.

  2. Immobilization of yeast inulinase on chitosan beads for the hydrolysis of inulin in a batch system.

    PubMed

    Singh, R S; Singh, R P; Kennedy, J F

    2017-02-01

    An extracellular inulinase was partially purified by ethanol precipitation and gel exclusion chromatography from a cell free extract of Kluyveromyces marxianus. Partially purified inulinase exhibited 420 IU/mg specific activity and it was immobilized on chitosan beads. Activity yield of immobilized inulinase was optimized with glutaraldehyde concentration (1-5%), glutaraldehyde treatment time (30-240min), enzyme coupling-time (2-16h) and enzyme loading (5-30 IU) as functions. Under the optimized conditions maximum yield 65.5% of immobilized inulinase was obtained. Maximum hydrolysis of inulin 84.5% and 78.2% was observed at 125rpm after 4h by immobilized and free enzyme, respectively. A retention-time of 4h and 5h was found optimal for the hydrolysis of inulin under agitation (125rpm) by free and immobilized enzyme, respectively. The recycling of the developed immobilized biocatalyst was carried out after 5h of inulin hydrolysis in a batch system. The developed immobilized biocatalyst was successfully used for the hydrolysis of inulin for 14 batches. This is the first report on the immobilization of yeast inulinase on chitosan beads for the hydrolysis of inulin in a batch system.

  3. Evaluation of hyper thermal acid hydrolysis of Kappaphycus alvarezii for enhanced bioethanol production.

    PubMed

    Ra, Chae Hun; Nguyen, Trung Hau; Jeong, Gwi-Taek; Kim, Sung-Koo

    2016-06-01

    Hyper thermal (HT) acid hydrolysis of Kappaphycus alvarezii, a red seaweed, was optimized to 12% (w/v) seaweed slurry content, 180mM H2SO4 at 140°C for 5min. The maximum monosaccharide concentration of 38.3g/L and 66.7% conversion from total fermentable monosaccharides of 57.6g/L with 120gdw/L K. alvarezii slurry were obtained from HT acid hydrolysis and enzymatic saccharification. HT acid hydrolysis at a severity factor of 0.78 efficiently converted the carbohydrates of seaweed to monosaccharides and produced a low concentration of inhibitory compounds. The levels of ethanol production by separate hydrolysis and fermentation with non-adapted and adapted Kluyveromyces marxianus to high concentration of galactose were 6.1g/L with ethanol yield (YEtOH) of 0.19 at 84h and 16.0g/L with YEtOH of 0.42 at 72h, respectively. Development of the HT acid hydrolysis process and adapted yeast could enhance the overall ethanol fermentation yields of K. alvarezii seaweed.

  4. Yeasts are essential for cocoa bean fermentation.

    PubMed

    Ho, Van Thi Thuy; Zhao, Jian; Fleet, Graham

    2014-03-17

    Cocoa beans (Theobroma cacao) are the major raw material for chocolate production and fermentation of the beans is essential for the development of chocolate flavor precursors. In this study, a novel approach was used to determine the role of yeasts in cocoa fermentation and their contribution to chocolate quality. Cocoa bean fermentations were conducted with the addition of 200ppm Natamycin to inhibit the growth of yeasts, and the resultant microbial ecology and metabolism, bean chemistry and chocolate quality were compared with those of normal (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii and Kluyveromyces marxianus, the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in the control fermentation. In fermentations with the presence of Natamycin, the same bacterial species grew but yeast growth was inhibited. Physical and chemical analyses showed that beans fermented without yeasts had increased shell content, lower production of ethanol, higher alcohols and esters throughout fermentation and lesser presence of pyrazines in the roasted product. Quality tests revealed that beans fermented without yeasts were purplish-violet in color and not fully brown, and chocolate prepared from these beans tasted more acid and lacked characteristic chocolate flavor. Beans fermented with yeast growth were fully brown in color and gave chocolate with typical characters which were clearly preferred by sensory panels. Our findings demonstrate that yeast growth and activity were essential for cocoa bean fermentation and the development of chocolate characteristics.

  5. Yeast derived from lignocellulosic biomass as a sustainable feed resource for use in aquaculture.

    PubMed

    Øverland, Margareth; Skrede, Anders

    2017-02-01

    The global expansion in aquaculture production implies an emerging need of suitable and sustainable protein sources. Currently, the fish feed industry is dependent on high-quality protein sources of marine and plant origin. Yeast derived from processing of low-value and non-food lignocellulosic biomass is a potential sustainable source of protein in fish diets. Following enzymatic hydrolysis, the hexose and pentose sugars of lignocellulosic substrates and supplementary nutrients can be converted into protein-rich yeast biomass by fermentation. Studies have shown that yeasts such as Saccharomyces cerevisiae, Candida utilis and Kluyveromyces marxianus have favourable amino acid composition and excellent properties as protein sources in diets for fish, including carnivorous species such as Atlantic salmon and rainbow trout. Suitable downstream processing of the biomass to disrupt cell walls is required to secure high nutrient digestibility. A number of studies have shown various immunological and health benefits from feeding fish low levels of yeast and yeast-derived cell wall fractions. This review summarises current literature on the potential of yeast from lignocellulosic biomass as an alternative protein source for the aquaculture industry. It is concluded that further research and development within yeast production can be important to secure the future sustainability and economic viability of intensive aquaculture. © 2016 Society of Chemical Industry.

  6. Batch and fed-batch simultaneous saccharification and fermentation of primary sludge from pulp and paper mills.

    PubMed

    Mendes, Cátia Vanessa Teixeira; Rocha, Jorge Manuel Dos Santos; de Menezes, Fabrícia Farias; Carvalho, Maria da Graça Videira Sousa

    2016-09-26

    Primary sludge from a Portuguese pulp and paper mill, containing 60% of carbohydrates, and unbleached pulp (as reference material), with 93% of carbohydrates, were used to produce ethanol by simultaneous saccharification and fermentation (SSF). SSF was performed in batch or fed-batch conditions without the need of a pretreatment. Cellic(®) CTec2 was the cellulolytic enzymatic complex used and Saccharomyces cerevisiae (baker's yeast or ATCC 26602 strain) or the thermotolerant yeast Kluyveromyces marxianus NCYC 1426 were employed. Primary sludge was successfully converted to ethanol and the best results in SSF efficiency were obtained with S. cerevisiae. An ethanol concentration of 22.7 g L(-1) was produced using a content of 50 g L(-1) of carbohydrates from primary sludge, in batch conditions, with a global conversion yield of 81% and a production rate of 0.94 g L(-1) h(-1). Fed-batch operation enabled higher solids content (total carbohydrate concentration of 200 g L(-1), equivalent to a consistency of 33%) and a reduction of three-quarters of cellulolytic enzyme load, leading to an ethanol concentration of 40.7 g L(-1), although with lower yield and productivity. Xylitol with a concentration up to 7 g L(-1) was also identified as by-product in the primary sludge bioconversion process.

  7. Ethanol-induced leakage in Saccharomyces cerevisiae: kinetics and relationship to yeast ethanol tolerance and alcohol fermentation productivity

    SciTech Connect

    Salgueiro, S.P.; Sa-Correia, I.; Novais, J.M.

    1988-04-01

    Ethanol stimulated the leakage of amino acids and 260-nm-light-absorbing compounds from cells of Saccharomyces cerevisiae. The efflux followed first-order kinetics over an initial period. In the presence of lethal concentrations of ethanol, the efflux rates at 30 and 36/sup 0/C were an exponential function of ethanol concentration. At 36/sup 0/C, as compared with the corresponding values at 30/sup 0/C, the efflux rates were higher and the minimal concentration of ethanol was lower. The exponential constants for the enhancement of the rate of leakage had similar values at 30 or 36/sup 0/C and were of the same order of magnitude as the corresponding exponential constants for ethanol-induced death. Under isothermic conditions (30/sup 0/C) and up to 22% (vol/vol) ethanol, the resistance to ethanol-induced leakage of 260-nm-light-absorbing compounds was found to be closely related with the ethanol tolerance of three strains of yeasts, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Saccharomyces bayanus. The resistance to ethanol-induced leakage indicates the possible adoption of the present method for the rapid screening of ethanol-tolerant strains. The addition to a fermentation medium of the intracellular material obtained by ethanol permeabilization of yeast cells led to improvements in alcohol fermentation by S. cerevisiae and S. bayanus. The action of the intracellular material, by improving yeast ethanol tolerance, and the advantages of partially recycling the fermented medium after distillation were discussed.

  8. Optimization of ricotta cheese whey (RCW) fermentation by response surface methodology.

    PubMed

    Sansonetti, Sascha; Curcio, Stefano; Calabrò, Vincenza; Iorio, Gabriele

    2010-12-01

    A central composite design (CCD) was performed to evaluate the effects of four factors, i.e. temperature (T), pH, agitation rate (K) and initial lactose concentration (L), on ricotta cheese whey batch fermentation and to optimize the process leading to the formation of bio-ethanol. Anaerobic batch fermentation experiments were carried out by using the yeast Kluyveromyces marxianus. After a preliminary experimental analysis, the values of the chosen factors were 32 and 40 degrees C for T, 4 and 6 for pH, 100 and 300 rpm for K, 40 and 80 g L(-1) for L. Response surface methodology (RSM) was used to optimize the fermentation process and an empirical polynomial model was used to fit the experimental data. The best operating conditions resulted to be T=32.35 degrees C, pH 5.41, K=195.56 rpm and L=40 g L(-1) and the model ensured a good fitting of the observed data.

  9. Yeast communities in a natural tequila fermentation.

    PubMed

    Lachance, M A

    1995-08-01

    Fresh and cooked agave, Drosophila spp., processing equipment, agave molasses, agave extract, and fermenting must at a traditional tequila distillery (Herradura, Amatitan, Jalisco, México) were studied to gain insight on the origin of yeasts involved in a natural tequila fermentations. Five yeast communities were identified. (1) Fresh agave contained a diverse mycobiota dominated by Clavispora lusitaniae and an endemic species, Metschnikowia agaveae. (2) Drosophila spp. from around or inside the distillery yielded typical fruit yeasts, in particular Hanseniaspora spp., Pichia kluyveri, and Candida krusei. (3) Schizosaccharomyces pombe prevailed in molasses. (4) Cooked agave and extract had a considerable diversity of species, but included Saccharomyces cerevisiae. (5) Fermenting juice underwent a gradual reduction in yeast heterogeneity. Torulaspora delbrueckii, Kluyveromyces marxianus, and Hanseniaspora spp. progressively ceded the way to S. cerevisiae, Zygosaccharomyces bailii, Candida milleri, and Brettanomyces spp. With the exception of Pichia membranaefaciens, which was shared by all communities, little overlap existed. That separation was even more manifest when species were divided into distinguishable biotypes based on morphology or physiology. It is concluded that crushing equipment and must holding tanks are the main source of significant inoculum for the fermentation process. Drosophila species appear to serve as internal vectors. Proximity to fruit trees probably contributes to maintaining a substantial Drosophila community, but the yeasts found in the distillery exhibit very little similarity to those found in adjacent vegetation. Interactions involving killer toxins had no apparent direct effects on the yeast community structure.

  10. Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey.

    PubMed

    Guimarães, Pedro M R; Teixeira, José A; Domingues, Lucília

    2010-01-01

    Cheese whey, the main dairy by-product, is increasingly recognized as a source of many bioactive valuable compounds. Nevertheless, the most abundant component in whey is lactose (ca. 5% w/v), which represents a significant environmental problem. Due to the large lactose surplus generated, its conversion to bio-ethanol has long been considered as a possible solution for whey bioremediation. In this review, fermentation of lactose to ethanol is discussed, focusing on wild lactose-fermenting yeasts, particularly Kluyveromyces marxianus, and recombinant Saccharomyces cerevisiae strains. The early efforts in the screening and characterization of the fermentation properties of wild lactose-consuming yeasts are reviewed. Furthermore, emphasis is given on the latter advances in engineering S. cerevisiae strains for efficient whey-to-ethanol bioprocesses. Examples of industrial implementation are briefly discussed, illustrating the viability of whey-to-ethanol systems. Current developments on strain engineering together with the growing market for biofuels will likely boost the industrial interest in such processes.

  11. Overexpression of the trichodiene synthase gene tri5 increases trichodermin production and antimicrobial activity in Trichoderma brevicompactum.

    PubMed

    Tijerino, Anamariela; Cardoza, R Elena; Moraga, Javier; Malmierca, Mónica G; Vicente, Francisca; Aleu, Josefina; Collado, Isidro G; Gutiérrez, Santiago; Monte, Enrique; Hermosa, Rosa

    2011-03-01

    Trichoderma brevicompactum produces trichodermin, a simple trichothecene-type toxin that shares the first steps of the sesquiterpene biosynthetic pathway with other phytotoxic trichothecenes from Fusarium spp. Trichodiene synthase catalyses the conversion of farnesyl pyrophosphate to trichodiene and it is encoded by the tri5 gene that was cloned and analysed functionally by homologous overexpression in T. brevicompactum. tri5 expression was up-regulated in media with glucose, H(2)O(2) or glycerol. tri5 repression was observed in cultures supplemented with the antioxidants ferulic acid and tyrosol. Acetone extracts of tri5-overexpressing transformants displayed higher antifungal activity than those from the wild-type. Chromatographic and spectroscopic analyses revealed that tri5 overexpression led to an increased production of trichodermin and tyrosol. Agar diffusion assays with these two purified metabolites from the tri5-overexpressing transformant T. brevicompactum Tb41tri5 showed that only trichodermin had antifungal activity against Saccharomyces cerevisiae, Kluyveromyces marxianus, Candida albicans, Candida glabrata, Candida tropicalis and Aspergillus fumigatus, in most cases such activity being higher than that observed for amphotericin B and hygromycin. Our results point to the significant role of tri5 in the production of trichodermin and in the antifungal activity of T. brevicompactum.

  12. Microbial flora and some chemical properties of ersho, a starter for teff (Eragrostis tef) fermentation.

    PubMed

    Ashenafi, M

    1994-01-01

    Ersho is a clear, yellow liquid that accumulates on the surface of fermenting teff-flour batter and is collected to serve as an inoculum for the next fermentation. The pH of ersho samples was about 3.5 and titratable acidity ranged between 3.1% and 5.7%. The mean aerobic mesophilic counts from four households varied between 6.9×10(6) and 1.3×10(8) c.f.u./ml and the aerobic bacterial flora consisted of Bacillus spp. Mean yeast counts ranged between 5.2×10(5) and 1.8×10(6) c.f.u./ml and comprised, in order of abundance, Candida milleri, Rhodotorula mucilaginosa, Kluyveromyces marxianus, Pichia naganishii and Debaromyces hansenii. Candida milleri was the most dominant isolate in all samples. About 90% of the teff flour samples had aerobic mesophilic counts ≥10(5) c.f.u./g and Gram-positive bacteria constituted about 71% of the total isolates. About 80% of samples had Enterobacteriaceae counts of 10(4) c.f.u./g.

  13. Structural insights into Atg10-mediated formation of the autophagy-essential Atg12-Atg5 conjugate.

    PubMed

    Yamaguchi, Masaya; Noda, Nobuo N; Yamamoto, Hayashi; Shima, Takayuki; Kumeta, Hiroyuki; Kobashigawa, Yoshihiro; Akada, Rinji; Ohsumi, Yoshinori; Inagaki, Fuyuhiko

    2012-07-03

    The Atg12-Atg5 conjugate, which is formed by an ubiquitin-like conjugation system, is essential to autophagosome formation, a central event in autophagy. Despite its importance, the molecular mechanism of the Atg12-Atg5 conjugate formation has not been elucidated. Here, we report the solution and crystal structures of Atg10 and Atg5 homologs from Kluyveromyces marxianus (Km), a thermotolerant yeast. KmAtg10 comprises an E2-core fold with characteristic accessories, including two β strands, whereas KmAtg5 has two ubiquitin-like domains and a helical domain. The nuclear magnetic resonance experiments, mutational analyses, and crosslinking experiments showed that KmAtg10 directly recognizes KmAtg5, especially its C-terminal ubiquitin-like domain, by its characteristic two β strands. Kinetic analysis suggests that Tyr56 and Asn114 of KmAtg10 may place the side chain of KmAtg5 Lys145 into the optimal orientation for its conjugation reaction with Atg12. These structural features enable Atg10 to mediate the formation of the Atg12-Atg5 conjugate without a specific E3 enzyme.

  14. Utilization and Transport of L-Arabinose by Non-Saccharomyces Yeasts

    SciTech Connect

    Knoshaug, E. P.; Franden, M. A.; Stambuk, B. U.; Zhang, M.; Singh, A.

    2009-01-01

    L-Arabinose is one of the sugars found in hemicellulose, a major component of plant cell walls. The ability to convert L-arabinose to ethanol would improve the economics of biomass to ethanol fermentations. One of the limitations for L-arabinose fermentation in the current engineered Saccharomyces cerevisiae strains is poor transport of the sugar. To better understand L-arabinose transport and use in yeasts and to identify a source for efficient L-arabinose transporters, 165 non-Saccharomyces yeast strains were studied. These yeast strains were arranged into six groups based on the minimum time required to utilize 20 g/L of L-arabinose. Initial transport rates of L-arabinose were determined for several species and a more comprehensive transport study was done in four selected species. Detailed transport kinetics in Arxula adeninivorans suggested both low and high affinity components while Debaryomyces hansenii var. fabryii, Kluyveromyces marxianus and Pichia guilliermondii possessed a single component, high affinity active transport systems.

  15. Enhancement of survival of probiotic and non-probiotic lactic acid bacteria by yeasts in fermented milk under non-refrigerated conditions.

    PubMed

    Liu, Shao-Quan; Tsao, Marlene

    2009-09-30

    The effects of yeasts on the survival of probiotic and non-probiotic lactic acid bacteria (LAB) were studied in fermented milk under non-refrigerated conditions (30 degrees C) with a view to develop ambient-stable fermented milk with live LAB. Five yeasts tested (Saccharomyces bayanus, Williopsis saturnus var. saturnus, Yarrowia lipolytica, Candida kefyr and Kluyveromyces marxianus) enhanced the survival of Lactobacillus bulgaricus (but not Streptococcus thermophilus) in a mixed yoghurt culture in yoghurt by approximately 10(2) to 10(5)-fold. Seven yeasts examined (Candida krusei, Geotrichum candidum, Pichia subpelliculosa, Kloeckera apiculata, Pichia membranifaciens, Schizosaccharomyces pombe and Y. lipolytica) improved the survival of Lactobacillus rhamnosus in fermented milk by approximately10(3) to 10(6)-fold. W. saturnus var. saturnus enhanced the survival of Lactobacillus acidophilus, L. rhamnosus (probiotic) and Lactobacillus reuteri by up to 10(6)-fold, but the same yeast failed to improve the survival of Lactobacillus johnsonii (probiotic), S. thermophilus and L. bulgaricus in fermented milk. These results provide definitive evidence that yeasts possess stability-enhancing effects on LAB and that the specific effects of yeasts on LAB stability vary with yeasts as well as with LAB. However, the molecular mechanism of such interaction of yeasts with LAB remains to be found.

  16. Inhibition of Listeria monocytogenes by Food-Borne Yeasts†

    PubMed Central

    Goerges, Stefanie; Aigner, Ulrike; Silakowski, Barbara; Scherer, Siegfried

    2006-01-01

    Many bacteria are known to inhibit food pathogens, such as Listeria monocytogenes, by secreting a variety of bactericidal and bacteriostatic substances. In sharp contrast, it is unknown whether yeast has an inhibitory potential for the growth of pathogenic bacteria in food. A total of 404 yeasts were screened for inhibitory activity against five Listeria monocytogenes strains. Three hundred and four of these yeasts were isolated from smear-ripened cheeses. Most of the yeasts were identified by Fourier transform infrared spectroscopy. Using an agar-membrane screening assay, a fraction of approximately 4% of the 304 red smear cheese isolates clearly inhibited growth of L. monocytogenes. Furthermore, 14 out of these 304 cheese yeasts were cocultivated with L. monocytogenes WSLC 1364 on solid medium to test the antilisterial activity of yeast in direct cell contact with Listeria. All yeasts inhibited L. monocytogenes to a low degree, which is most probably due to competition for nutrients. However, one Candida intermedia strain was able to reduce the listerial cell count by 4 log units. Another four yeasts, assigned to C. intermedia (three strains) and Kluyveromyces marxianus (one strain), repressed growth of L. monocytogenes by 3 log units. Inhibition of L. monocytogenes was clearly pronounced in the cocultivation assay, which simulates the conditions and contamination rates present on smear cheese surfaces. We found no evidence that the unknown inhibitory molecule is able to diffuse through soft agar. PMID:16391059

  17. A novel acid-stable, acid-active beta-galactosidase potentially suited to the alleviation of lactose intolerance.

    PubMed

    O'Connell, Shane; Walsh, Gary

    2010-03-01

    Extracellular beta-galactosidase produced by a strain of Aspergillus niger van Tiegh was purified to homogeneity using a combination of gel filtration, ion-exchange, chromatofocusing, and hydrophobic interaction chromatographies. The enzyme displayed a temperature optimum of 65 degrees C and a low pH optimum of between 2.0 and 4.0. The monomeric glycosylated enzyme displayed a molecular mass of 129 kDa and an isoelectric point of 4.7. Protein database similarity searching using mass spectrometry-derived sequence data indicate that the enzyme shares homology with a previously sequenced A. niger beta-galactosidase. Unlike currently commercialised products, the enzyme displayed a high level of stability when exposed to simulated gastric conditions in vitro, retaining 68+/-2% of original activity levels. This acid-stable, acid-active beta-galactosidase was formulated, along with a neutral beta-galactosidase from Kluyveromyces marxianus DSM5418, in a novel two-segment capsule system designed to ensure delivery of enzymes of appropriate physicochemical properties to both stomach and small intestine. When subjected to simulated full digestive tract conditions, the twin lactase-containing capsule hydrolyzed, per unit activity, some 3.5-fold more lactose than did the commercial supplemental enzyme. The acid-stable, acid-active enzyme, along with the novel two-segment delivery system, may prove beneficial in the more effective treatment of lactose intolerance.

  18. Alcohol from whey permeate: strain selection, temperature, and medium optimization. [Candida pseudotropicalis, Kluyveromyces fragilis, and K. lactis

    SciTech Connect

    Vienne, P.; Von Stockar, U.

    1983-01-01

    A comparative study of shaken flask cultures of some yeast strains capable of fermenting lactose showed no significant differences in alcohol yield among the four best strains. Use of whey permeate concentrated three times did not affect the yields. An optimal growth temperature of 38/sup 0/C was determined for K. fragilis NRRL 665. Elemental analysis of both the permeate and the dry cell mass of two strains indicated the possibility of a stoichiometric limitation by nitrogen. Batch cultures in laboratory fermentors confirmed this finding and revealed in addition the presence of a limitation due to growth factors. Both types of limitations could be overcome by adding yeast extract. The maximum productivity of continuous cultures could thus be improved to 5.1 g/l-h. The maximum specific growth rate was of the order of 0.310 h/sup -1/. 15 references, 10 figures, 9 tables.

  19. Indigenous Georgian Wine-Associated Yeasts and Grape Cultivars to Edit the Wine Quality in a Precision Oenology Perspective

    PubMed Central

    Vigentini, Ileana; Maghradze, David; Petrozziello, Maurizio; Bonello, Federica; Mezzapelle, Vito; Valdetara, Federica; Failla, Osvaldo; Foschino, Roberto

    2016-01-01

    In Georgia, one of the most ancient vine-growing environment, the homemade production of wine is still very popular in every rural family and spontaneous fermentation of must, without addition of chemical preservatives, is the norm. The present work investigated the yeast biodiversity in five Georgian areas (Guria, Imereti, Kakheti, Kartli, Ratcha-Lechkhumi) sampling grapes and wines from 22 different native cultivars, in 26 vineyards and 19 family cellars. One hundred and eighty-two isolates were ascribed to 15 different species by PCR-ITS and RFLP, and partial sequencing of D1/D2 domain 26S rDNA gene. Metschnikowia pulcherrima (F’ = 0.56, I’ = 0.32), Hanseniaspora guilliermondii (F’ = 0.49, I’ = 0.27), and Cryptococcus flavescens (F’ = 0.31, I’ = 0.11) were the dominant yeasts found on grapes, whereas Saccharomyces cerevisiae showed the highest prevalence into wine samples. Seventy four isolates with fermentative potential were screened for oenological traits such as ethanol production, resistance to SO2, and acetic acid, glycerol and H2S production. Three yeast strains (Kluyveromyces marxianus UMY207, S. cerevisiae UMY255, Torulaspora delbrueckii UMY196) were selected and separately inoculated in vinifications experiments at a Georgian cellar. Musts were prepared from healthy grapes of local varieties, Goruli Mtsvane (white berry cultivar) and Saperavi (black berry cultivar). Physical (°Brix) and microbial analyses (plate counts) were performed to monitor the fermentative process. The isolation of indigenous S. cerevisiae yeasts beyond the inoculated strains indicated that a co-presence occurred during the vinification tests. Results from quantitative GC-FID analysis of volatile compounds revealed that the highest amount of fermentation flavors, such as 4-ethoxy-4-oxobutanoic acid (monoethyl succinate), 2-methylpropan-1-ol, ethyl 2-hydroxypropanoate, and 2-phenylethanol, were significantly more produced in fermentation conducted in Saperavi variety

  20. Indigenous Georgian Wine-Associated Yeasts and Grape Cultivars to Edit the Wine Quality in a Precision Oenology Perspective.

    PubMed

    Vigentini, Ileana; Maghradze, David; Petrozziello, Maurizio; Bonello, Federica; Mezzapelle, Vito; Valdetara, Federica; Failla, Osvaldo; Foschino, Roberto

    2016-01-01

    In Georgia, one of the most ancient vine-growing environment, the homemade production of wine is still very popular in every rural family and spontaneous fermentation of must, without addition of chemical preservatives, is the norm. The present work investigated the yeast biodiversity in five Georgian areas (Guria, Imereti, Kakheti, Kartli, Ratcha-Lechkhumi) sampling grapes and wines from 22 different native cultivars, in 26 vineyards and 19 family cellars. One hundred and eighty-two isolates were ascribed to 15 different species by PCR-ITS and RFLP, and partial sequencing of D1/D2 domain 26S rDNA gene. Metschnikowia pulcherrima (F' = 0.56, I' = 0.32), Hanseniaspora guilliermondii (F' = 0.49, I' = 0.27), and Cryptococcus flavescens (F' = 0.31, I' = 0.11) were the dominant yeasts found on grapes, whereas Saccharomyces cerevisiae showed the highest prevalence into wine samples. Seventy four isolates with fermentative potential were screened for oenological traits such as ethanol production, resistance to SO2, and acetic acid, glycerol and H2S production. Three yeast strains (Kluyveromyces marxianus UMY207, S. cerevisiae UMY255, Torulaspora delbrueckii UMY196) were selected and separately inoculated in vinifications experiments at a Georgian cellar. Musts were prepared from healthy grapes of local varieties, Goruli Mtsvane (white berry cultivar) and Saperavi (black berry cultivar). Physical (°Brix) and microbial analyses (plate counts) were performed to monitor the fermentative process. The isolation of indigenous S. cerevisiae yeasts beyond the inoculated strains indicated that a co-presence occurred during the vinification tests. Results from quantitative GC-FID analysis of volatile compounds revealed that the highest amount of fermentation flavors, such as 4-ethoxy-4-oxobutanoic acid (monoethyl succinate), 2-methylpropan-1-ol, ethyl 2-hydroxypropanoate, and 2-phenylethanol, were significantly more produced in fermentation conducted in Saperavi variety inoculated

  1. Temperature and relative humidity influence the microbial and physicochemical characteristics of Camembert-type cheese ripening.

    PubMed

    Leclercq-Perlat, M-N; Sicard, M; Trelea, I C; Picque, D; Corrieu, G

    2012-08-01

    To evaluate the effects of temperature and relative humidity (RH) on microbial and biochemical ripening kinetics, Camembert-type cheeses were prepared from pasteurized milk seeded with Kluyveromyces marxianus, Geotrichum candidum, Penicillium camemberti, and Brevibacterium aurantiacum. Microorganism growth and biochemical changes were studied under different ripening temperatures (8, 12, and 16°C) and RH (88, 92, and 98%). The central point runs (12°C, 92% RH) were both reproducible and repeatable, and for each microbial and biochemical parameter, 2 kinetic descriptors were defined. Temperature had significant effects on the growth of both K. marxianus and G. candidum, whereas RH did not affect it. Regardless of the temperature, at 98% RH the specific growth rate of P. camemberti spores was significantly higher [between 2 (8°C) and 106 times (16°C) higher]. However, at 16°C, the appearance of the rind was no longer suitable because mycelia were damaged. Brevibacterium aurantiacum growth depended on both temperature and RH. At 8°C under 88% RH, its growth was restricted (1.3 × 10(7) cfu/g), whereas at 16°C and 98% RH, its growth was favored, reaching 7.9 × 10(9) cfu/g, but the rind had a dark brown color after d 20. Temperature had a significant effect on carbon substrate consumption rates in the core as well as in the rind. In the rind, when temperature was 16°C rather than 8°C, the lactate consumption rate was approximately 2.9 times higher under 88% RH. Whatever the RH, temperature significantly affected the increase in rind pH (from 4.6 to 7.7 ± 0.2). At 8°C, an increase in rind pH was observed between d 6 and 9, whereas at 16°C, it was between d 2 and 3. Temperature and RH affected the increasing rate of the underrind thickness: at 16°C, half of the cheese thickness appeared ripened on d 14 (wrapping day). However, at 98% RH, the underrind was runny. In conclusion, some descriptors, such as yeast growth and the pH in the rind, depended solely on

  2. The effect of lactic acid bacteria on cocoa bean fermentation.

    PubMed

    Ho, Van Thi Thuy; Zhao, Jian; Fleet, Graham

    2015-07-16

    Cocoa beans (Theobroma cacao L.) are the raw material for chocolate production. Fermentation of cocoa pulp by microorganisms is crucial for developing chocolate flavor precursors. Yeasts conduct an alcoholic fermentation within the bean pulp that is essential for the production of good quality beans, giving typical chocolate characters. However, the roles of bacteria such as lactic acid bacteria and acetic acid bacteria in contributing to the quality of cocoa bean and chocolate are not fully understood. Using controlled laboratory fermentations, this study investigated the contribution of lactic acid bacteria to cocoa bean fermentation. Cocoa beans were fermented under conditions where the growth of lactic acid bacteria was restricted by the use of nisin and lysozyme. The resultant microbial ecology, chemistry and chocolate quality of beans from these fermentations were compared with those of indigenous (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii, Kluyveromyces marxianus and Saccharomyces cerevisiae, the lactic acid bacteria Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in control fermentations. In fermentations with the presence of nisin and lysozyme, the same species of yeasts and acetic acid bacteria grew but the growth of lactic acid bacteria was prevented or restricted. These beans underwent characteristic alcoholic fermentation where the utilization of sugars and the production of ethanol, organic acids and volatile compounds in the bean pulp and nibs were similar for beans fermented in the presence of lactic acid bacteria. Lactic acid was produced during both fermentations but more so when lactic acid bacteria grew. Beans fermented in the presence or absence of lactic acid bacteria were fully fermented, had similar shell weights and gave acceptable chocolates with no differences

  3. Diversity of fungal flora in raw milk from the Italian Alps in relation to pasture altitude.

    PubMed

    Panelli, Simona; Brambati, Eva; Bonacina, Cesare; Feligini, Maria

    2013-01-01

    The present paper explores the diversity of mycobiota inhabiting raw milk sampled at different altitudes (1400 m, 1800 m, 2200 m) from cows grazing Alpine pastures of Valle d'Aosta (North-Western Italian Alps). To this aim, multilocus sequencing was performed at barcodes commonly used for fungal identification (ITS1, D1/D2 domains of the 26S rRNA gene, and part of the β-tubulin gene). A total of 31 species were detected, most of them yeasts, followed by moulds and by 2 sequences of macroscopic fungi. Several yeasts and moulds were well-characterized inhabitants of the dairy environment, known to positively contribute to cheesemaking. Among these, Candida was the most represented genus with a tendency to cluster at the highest altitudes (6 over 8 observations at ≥ 1800 m), and Kluyveromyces marxianus the most abundant single species, retrieved at all altitudes. The environmental ascomycetous Atrotorquata lineata, never put in relation with food nor described outside North-America, was another species among those most frequently retrieved and was detected in 6 milks at 1400 and 1800 m. The remaining fungi, in general never reported in milk, were mostly environmental. Many of them resulted associated with plants as pathogens or symbionts. Finally, the highest sampled altitude yielded a significant fungal diversity (17 species). This work enlarges the knowledge of fungal consortia inhabiting raw milk and introduces microbial ecology among the altitude-dependent factors, in the composition of Alpine pastures, with the potential of shaping the properties of milks and cheeses, together with the already described physical, chemical and botanical variables.

  4. Transforming sugars into fat-lipid biosynthesis using different sugars in Yarrowia lipolytica.

    PubMed

    Hapeta, Piotr; Rakicka, Magdalena; Dulermo, Remi; Gamboa-Meléndez, Heber; Crutz-Le Coq, Anne-Marie; Nicaud, Jean-Marc; Lazar, Zbigniew

    2017-03-17

    In an era of ever-increasing energy demands, a promising technology is being developed: the use of oleaginous microorganisms such as Yarrowia lipolytica to convert waste materials into biofuels. Here, we constructed two Y. lipolytica strains that displayed both increased lipid accumulation and a more efficient use of biomass-derived sugars, including glucose, fructose, galactose, and inulin. The first strain, Y. lipolytica YLZ150, was derived from the French wild-type strain W29. It had inhibited triacylglycerol mobilization (∆tgl4) and β-oxidation (∆pox1-6), and it overexpressed GPD1, DGA2, HXK1, the native Leloir pathway, SUC2 from Saccharomyces cerevisiae, and INU1 from Kluyveromyces marxianus. The second strain, Y. lipolytica Y4779, was derived from the Polish A-101 strain. It had inhibited β-oxidation (∆mfe2) and overexpressed GPD1, DGA1, HXK1, YHT3, SUC2, and INU1. In the first experiment, strain YLZ150 was batch-cultured in media containing different hexoses; the highest values for lipid concentration and yield of lipids from the substrate were obtained using fructose (20.3 g dm(-3) and 0.14 g g(-1) , respectively). In the second experiment, we grew the two strains in fed-batch cultures to examine lipid biosynthesis from inulin (a fructose polymer). For Y4779, the lipid concentration was 10.3 g dm(-3) and the yield of lipids from substrate was 0.07 g g(-1) ; in contrast, for YLZ150, these values were 24 g dm(-3) and 0.16 g g(-1) , respectively. The YLZ150 strain is thus able to efficiently exploit glucose, fructose, galactose, sucrose, and inulin for lipid biosynthesis.

  5. The structure of DesR from Streptomyces venezuelae, a β-glucosidase involved in macrolide activation.

    PubMed

    Zmudka, Matthew W; Thoden, James B; Holden, Hazel M

    2013-07-01

    Antibiotics have, indeed, altered the course of human history as is evidenced by the increase in human life expectancy since the 1940s. Many of these natural compounds are produced by bacteria that, by necessity, must have efficient self-resistance mechanisms. The methymycin/pikromycin producing species Streptomyces venezuelae, for example, utilizes β-glucosylation of its macrolide products to neutralize their effects within the confines of the cell. Once released into the environment, these compounds are activated by the removal of the glucose moiety. In S. venezuelae, the enzyme responsible for removal of the sugar from the parent compound is encoded by the desR gene and referred to as DesR. It is a secreted enzyme containing 828 amino acid residues, and it is known to be a retaining glycosidase. Here, we describe the structure of the DesR/D-glucose complex determined to 1.4-Å resolution. The overall architecture of the enzyme can be envisioned in terms of three regions: a catalytic core and two auxiliary domains. The catalytic core harbors the binding platform for the glucose ligand. The first auxiliary domain adopts a "PA14 fold," whereas the second auxiliary domain contains an immunoglobulin-like fold. Asp 273 and Glu 578 are in the proper orientation to function as the catalytic base and proton donor, respectively, required for catalysis. The overall fold of the core region places DesR into the GH3 glycoside hydrolase family of enzymes. Comparison of the DesR structure with the β-glucosidase from Kluyveromyces marxianus shows that their PA14 domains assume remarkably different orientations.

  6. Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.

    PubMed

    Zhou, Shun-Hua; Liu, Yuan; Zhao, Yu-Juan; Chi, Zhe; Chi, Zhen-Ming; Liu, Guang-Lei

    2016-09-01

    In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

  7. C- and N-catabolic utilization of tricarboxylic acid cycle-related amino acids by Scheffersomyces stipitis and other yeasts.

    PubMed

    Freese, Stefan; Vogts, Tanja; Speer, Falk; Schäfer, Bernd; Passoth, Volkmar; Klinner, Ulrich

    2011-05-01

    Scheffersomyces stipitis and the closely related yeast Candida shehatae assimilated the L-amino acids glutamate, aspartate and proline as both carbon and nitrogen sole sources. We also found this rarely investigated ability in ascomycetous species such as Candida glabrata, C. reukaufii, C. utilis, Debaryomyces hansenii, Kluyveromyces lactis, K. marxianus, Candida albicans, L. elongisporus, Meyerozyma guilliermondii, C. maltosa, Pichia capsulata and Yarrowia lipolytica and in basidiomycetous species such as Rhodotorula rubra and Trichosporon beigelii. Glutamate was a very efficient carbon source for Sc. stipitis, which enabled a high biomass yield/mole, although the growth rate was lower when compared to growth on glucose medium. The cells secreted waste ammonium during growth on glutamate alone. In Sc. stipitis cultures grown in glucose medium containing glutamate as the nitrogen source the biomass yield was maximal, and ethanol concentration and specific ethanol formation rate were significantly higher than in glucose medium containing ammonium as the nitrogen source. Mainly C-assimilation of glutamate but also N-assimilation in glucose-containing medium correlated with enhanced activity of the NAD-dependent glutamate dehydrogenase 2 (GDH2). A Δgdh2 disruptant was unable to utilize glutamate as either a carbon or a nitrogen source; moreover, this disruptant was also unable to utilize aspartate as a carbon source. The mutation was complemented by retransformation of the GDH2 ORF into the Δgdh2 strain. The results show that Gdh2p plays a dual role in Sc. stipitis as both C- and N-catabolic enzyme, which indicates its role as an interface between the carbon and nitrogen metabolism of this yeast.

  8. Characterization of Osmotolerant Yeasts and Yeast-Like Molds from Apple Orchards and Apple Juice Processing Plants in China and Investigation of Their Spoilage Potential.

    PubMed

    Wang, Huxuan; Hu, Zhongqiu; Long, Fangyu; Niu, Chen; Yuan, Yahong; Yue, Tianli

    2015-08-01

    Yeasts and yeast-like fungal isolates were recovered from apple orchards and apple juice processing plants located in the Shaanxi province of China. The strains were evaluated for osmotolerance by growing them in 50% (w/v) glucose. Of the strains tested, 66 were positive for osmotolerance and were subsequently identified by 26S or 5.8S-ITS ribosomal RNA (rRNA) gene sequencing. Physiological tests and RAPD-PCR analysis were performed to reveal the polymorphism of isolates belonging to the same species. Further, the spoilage potential of the 66 isolates was determining by evaluating their growth in 50% to 70% (w/v) glucose and measuring gas generation in 50% (w/v) glucose. Thirteen osmotolerant isolates representing 9 species were obtained from 10 apple orchards and 53 target isolates representing 19 species were recovered from 2 apple juice processing plants. In total, members of 14 genera and 23 species of osmotolerant isolates including yeast-like molds were recovered from all sources. The commonly recovered osmotolerant isolates belonged to Kluyveromyces marxianus, Hanseniaspora uvarum, Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Candida tropicalis, and Pichia kudriavzevii. The polymorphism of isolates belonging to the same species was limited to 1 to 3 biotypes. The majority of species were capable of growing within a range of glucose concentration, similar to sugar concentrations found in apple juice products with a lag phase from 96 to 192 h. Overall, Z. rouxii was particularly the most tolerant to high glucose concentration with the shortest lag phase of 48 h in 70% (w/v) glucose and the fastest gas generation rate in 50% (w/v) glucose.

  9. Bioprospecting of thermo- and osmo-tolerant fungi from mango pulp-peel compost for bioethanol production.

    PubMed

    Dandi, N D; Dandi, B N; Chaudhari, A B

    2013-04-01

    The persistent edaphic stress on microbial succession due to dynamic changes during composting was explored for selection of multi-stress tolerant microbe(s) desirable for ethanol production. A total of 23 strains were isolated from mango compost using four successive enrichments in YP broth (g l(-1)): glucose, 100; 150; 250 with ethanol (40) and cycloheximide (0.4) at 40 °C, pH 6.0. Based on multi-gene ribotyping, 14 yeasts (61 %) of Saccharomycetaceae, 2 filamentous fungi (8.6 %) and 7 bacteria (30.4 %) were obtained. Phenetic and phylogenetic analysis of the 14 yeasts revealed 64.3 % tolerant to 500 g l(-1) glucose, growth at 45 °C and resemblance to Candida sp. (14.3 %), Kluyveromyces marxianus (35.7 %), Pichia kudriavzevii (21.4 %) and Saccharomyces cerevisiae (28.6 %). Assessment of the 14 yeasts in glucose fermentation medium (pH 4.5 at 40 °C) showed ethanol productivity of ≥92 % by 12 yeasts with theoretical yields of 90-97 %. Fermentation of molasses (150 g l(-1) glucose equivalent) by P. kudriavzevii D1C at 40 °C resulted in 73.70 ± 0.02 g l(-1) ethanol and productivity of 4.91 ± 0.01 g l(-1) h(-1). Assessment of P. kudriavzevii D1C revealed multi-stress tolerance towards 5-hydroxymethyl furfural, ethanol (20 %, v/v), high gravity and H2O2 (0.3 M) indicating suitability for ethanol production using high gravity molasses and pre-treated lignocellulosic biomass fermentation.

  10. Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

    PubMed Central

    dos Santos, Bruna Silveira Lamanes; Gomes, Arthur Filipe Sousa; Franciscon, Emanuele Giuliane; de Oliveira, Jean Maikon; Baffi, Milla Alves

    2015-01-01

    Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors. PMID:26413077

  11. Diversity of culturable yeasts in phylloplane of sugarcane in Thailand and their capability to produce indole-3-acetic acid.

    PubMed

    Limtong, Savitree; Kaewwichian, Rungluk; Yongmanitchai, Wichien; Kawasaki, Hiroko

    2014-06-01

    Yeasts were isolated by the enrichment technique from the phylloplane of 94 samples of sugarcane leaf collected from seven provinces in Thailand. All sugarcane leaf samples contained yeasts and 158 yeast strains were obtained. On the basis of the D1/D2 domain of the large subunit rRNA gene sequence analysis, 144 strains were identified to 24 known species in 14 genera belonging to the Ascomycota viz. Candida akabanensis, Candida dendronema, Candida mesorugosa, Candida michaelii, Candida nivariensis, Candida rugosa, Candida orthopsilosis, Candida quercitrusa, Candida tropicalis, Candida xylopsoci, Cyberlindnera fabianii, Cyberlindnera rhodanensis, Debaryomyces nepalensis, Hannaella aff. coprosmaensis, Hanseniaspora guilliermondii, Kluyveromyces marxianus, Lachancea thermotolerans, Lodderomyces elongisporus, Metschnikowia koreensis, Meyerozyma caribbica, Millerozyma koratensis, Pichia kudriavzevii, Torulaspora delbrueckii and Wickerhamomyces edaphicus, and 12 species in six genera of the Basidiomycota viz . Cryptococcus flavescens, Cryptococcus laurentii, Cryptococcus rajasthanensis, Kwoniella heveanensis, Rhodosporidium fluviale, Rhodosporidium paludigenum, Rhodotorula mucilaginosa, Rhodotorula sesimbrana, Rhodotorula taiwanensis, Sporidiobolus ruineniae, Sporobolomyces carnicolor and Sporobolomyces nylandii. Seven strains were identical or similar to four undescribed species. Another seven strains represented four novels species in the genus Metschnikowia, Nakazawaea, Wickerhamomyces and Yamadazyma. The results revealed 69 % of the isolated strains were ascomycete yeasts and 31 % were basidiomycete yeast. The most prevalent species was M. caribbica with a 23 % frequency of occurrence followed by Rh. taiwanensis (11 %) and C. tropicalis (10 %). All strains were assessed for indole-3-acetic acid (IAA) producing capability showing that 69 strains had the capability of producing IAA when cultivated in yeast extract peptone dextrose broth supplemented with 1

  12. Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production

    PubMed Central

    2013-01-01

    Background Many microorganisms possess enzymes that can efficiently degrade lignocellulosic materials, but do not have the capability to produce a large amount of ethanol. Thus, attempts have been made to transform such enzymes into fermentative microbes to serve as hosts for ethanol production. However, an efficient host for a consolidated bioprocess (CBP) remains to be found. For this purpose, a synthetic biology technique that can transform multiple genes into a genome is instrumental. Moreover, a strategy to select cellulases that interact synergistically is needed. Results To engineer a yeast for CBP bio-ethanol production, a synthetic biology technique, called “promoter-based gene assembly and simultaneous overexpression” (PGASO), that can simultaneously transform and express multiple genes in a kefir yeast, Kluyveromyces marxianus KY3, was recently developed. To formulate an efficient cellulase cocktail, a filter-paper-activity assay for selecting heterologous cellulolytic enzymes was established in this study and used to select five cellulase genes, including two cellobiohydrolases, two endo-β-1,4-glucanases and one beta-glucosidase genes from different fungi. In addition, a fungal cellodextrin transporter gene was chosen to transport cellodextrin into the cytoplasm. These six genes plus a selection marker gene were one-step assembled into the KY3 genome using PGASO. Our experimental data showed that the recombinant strain KR7 could express the five heterologous cellulase genes and that KR7 could convert crystalline cellulose into ethanol. Conclusion Seven heterologous genes, including five cellulases, a cellodextrin transporter and a selection marker, were simultaneously transformed into the KY3 genome to derive a new strain, KR7, which could directly convert cellulose to ethanol. The present study demonstrates the potential of our strategy of combining a cocktail formulation protocol and a synthetic biology technique to develop a designer yeast host

  13. Ethanol-Induced Leakage in Saccharomyces cerevisiae: Kinetics and Relationship to Yeast Ethanol Tolerance and Alcohol Fermentation Productivity.

    PubMed

    Salgueiro, S P; Sá-Correia, I; Novais, J M

    1988-04-01

    Ethanol stimulated the leakage of amino acids and 260-nm-light-absorbing compounds from cells of Saccharomyces cerevisiae. The efflux followed first-order kinetics over an initial period. In the presence of lethal concentrations of ethanol, the efflux rates at 30 and 36 degrees C were an exponential function of ethanol concentration: k(e) = k(e)e, where k(e) and k(e) are the efflux rate constants, respectively, in the presence of a concentration X of ethanol or the minimal concentration of ethanol, X(m), above which the equation was applicable, coincident with the minimal lethal concentration of ethanol. E is the enhancement constant. At 36 degrees C, as compared with the corresponding values at 30 degrees C, the efflux rates were higher and the minimal concentration of ethanol (X(m)) was lower. The exponential constants for the enhancement of the rate of leakage (E) had similar values at 30 or 36 degrees C and were of the same order of magnitude as the corresponding exponential constants for ethanol-induced death. Under isothermic conditions (30 degrees C) and up to 22% (vol/vol) ethanol, the resistance to ethanol-induced leakage of 260-nm-light-absorbing compounds was found to be closely related with the ethanol tolerance of three strains of yeasts, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Saccharomyces bayanus. The resistance to ethanol-induced leakage indicates the possible adoption of the present method for the rapid screening of ethanol-tolerant strains. The addition to a fermentation medium of the intracellular material obtained by ethanol permeabilization of yeast cells led to improvements in alcohol fermentation by S. cerevisiae and S. bayanus. The action of the intracellular material, by improving yeast ethanol tolerance, and the advantages of partially recycling the fermented medium after distillation were discussed.

  14. Ethanol-Induced Leakage in Saccharomyces cerevisiae: Kinetics and Relationship to Yeast Ethanol Tolerance and Alcohol Fermentation Productivity

    PubMed Central

    Salgueiro, Sancha P.; Sá-Correia, Isabel; Novais, Júlio M.

    1988-01-01

    Ethanol stimulated the leakage of amino acids and 260-nm-light-absorbing compounds from cells of Saccharomyces cerevisiae. The efflux followed first-order kinetics over an initial period. In the presence of lethal concentrations of ethanol, the efflux rates at 30 and 36°C were an exponential function of ethanol concentration: keX = keXmeE (X-Xm), where keX and keXm are the efflux rate constants, respectively, in the presence of a concentration X of ethanol or the minimal concentration of ethanol, Xm, above which the equation was applicable, coincident with the minimal lethal concentration of ethanol. E is the enhancement constant. At 36°C, as compared with the corresponding values at 30°C, the efflux rates were higher and the minimal concentration of ethanol (Xm) was lower. The exponential constants for the enhancement of the rate of leakage (E) had similar values at 30 or 36°C and were of the same order of magnitude as the corresponding exponential constants for ethanol-induced death. Under isothermic conditions (30°C) and up to 22% (vol/vol) ethanol, the resistance to ethanol-induced leakage of 260-nm-light-absorbing compounds was found to be closely related with the ethanol tolerance of three strains of yeasts, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Saccharomyces bayanus. The resistance to ethanol-induced leakage indicates the possible adoption of the present method for the rapid screening of ethanol-tolerant strains. The addition to a fermentation medium of the intracellular material obtained by ethanol permeabilization of yeast cells led to improvements in alcohol fermentation by S. cerevisiae and S. bayanus. The action of the intracellular material, by improving yeast ethanol tolerance, and the advantages of partially recycling the fermented medium after distillation were discussed. PMID:16347612

  15. Manufacture of a beverage from cheese whey using a "tea fungus" fermentation.

    PubMed

    Belloso-Morales, Genette; Hernández-Sánchez, Humberto

    2003-01-01

    Kombucha is a sour beverage reported to have potential health effects prepared from the fermentation of black tea and sugar with a "tea fungus", a symbiotic culture of acetic acid bacteria and yeasts. Although black tea is the preferred substrate for Kombucha fermentation, other beverages have also been tested as substrates with fair results. Cheese whey is a by-product with a good amount of fermentable lactose that has been used before in the production of beverages, so the objective of this study was to test three types of whey (fresh sweet, fresh acid and reconstituted sweet) in the elaboration of a fermented beverage using a kombucha culture as inoculum. The isolation and identification of bacteria and yeasts from the fermented tea and wheys was done along with the study of the rates of change in sugar consumption, acid production and pH decrease. Several species of acetic acid bacteria (Acetobacter aceti subsp. aceti, Gluconobacter oxydans subsp. industrius, subsp. oxydans and Gluconoacetobacter xylinus) were isolated from the different kombuchas along with the yeasts Saccharomyces cerevisiae, Kluyveromyces marxianus, and Brettanomyces bruxelensis. The main metabolic products in the fermented wheys included ethanol, lactic and acetic acids. A good growth was obtained in both sweet wheys in which a pH of 3.3 and a total acid content (mainly lactic and acetic acids) of 0.07 mol/l was reached after 96 h. The sweet whey fermented beverages contained a relatively low lactose concentration (< 12 g/l). The final ethanol content was low (5 g/l) in all the fermented wheys. The whey products were strongly sour and salty non sparkling beverages.

  16. Quantifying the individual effects of ethanol and temperature on the fitness advantage of Saccharomyces cerevisiae.

    PubMed

    Salvadó, Z; Arroyo-López, F N; Barrio, E; Querol, A; Guillamón, J M

    2011-09-01

    The presence of Saccharomyces cerevisiae in grape berries and fresh musts is usually very low. However, as fermentation progresses, the population levels of this species considerably increase. In this study, we use the concept of fitness advantage to measure how increasing ethanol concentrations (0-25%) and temperature values (4-46 °C) in wine fermentations affects competition between S. cerevisiae and several non-Saccharomyces yeasts (Hanseniaspora uvarum, Torulaspora delbrueckii, Candida zemplinina, Pichia fermentans and Kluyveromyces marxianus). We used a mathematical approach to model the hypothetical time needed for S. cerevisiae to impose itself on a mixed population of the non-Saccharomyces species described above. This approach also took into consideration the influence of environmental factors and the initial population levels of S. cerevisiae (0.1, 1.0 and 10.0%). Our results suggest that Saccharomyces niche construction via ethanol production does not provide a clear ecological advantage (at least not until the ethanol concentration exceeds 9%), whereas a temperature rise (above 15 °C) does give S. cerevisiae a considerable advantage. The initial frequency of S. cerevisiae considerably influences the time it needs to impose itself (until it reaches a final frequency of 99% in the mixed culture), the lowest time values being found at the highest initial frequency. In light of these results, the application of low temperatures in the wine industry could favor the growth and survival of non-Saccharomyces species for a longer period of time.

  17. Sustainable conversion of coffee and other crop wastes to biofuels and bioproducts using coupled biochemical and thermochemical processes in a multi-stage biorefinery concept.

    PubMed

    Hughes, Stephen R; López-Núñez, Juan Carlos; Jones, Marjorie A; Moser, Bryan R; Cox, Elby J; Lindquist, Mitch; Galindo-Leva, Luz Angela; Riaño-Herrera, Néstor M; Rodriguez-Valencia, Nelson; Gast, Fernando; Cedeño, David L; Tasaki, Ken; Brown, Robert C; Darzins, Al; Brunner, Lane

    2014-10-01

    The environmental impact of agricultural waste from the processing of food and feed crops is an increasing concern worldwide. Concerted efforts are underway to develop sustainable practices for the disposal of residues from the processing of such crops as coffee, sugarcane, or corn. Coffee is crucial to the economies of many countries because its cultivation, processing, trading, and marketing provide employment for millions of people. In coffee-producing countries, improved technology for treatment of the significant amounts of coffee waste is critical to prevent ecological damage. This mini-review discusses a multi-stage biorefinery concept with the potential to convert waste produced at crop processing operations, such as coffee pulping stations, to valuable biofuels and bioproducts using biochemical and thermochemical conversion technologies. The initial bioconversion stage uses a mutant Kluyveromyces marxianus yeast strain to produce bioethanol from sugars. The resulting sugar-depleted solids (mostly protein) can be used in a second stage by the oleaginous yeast Yarrowia lipolytica to produce bio-based ammonia for fertilizer and are further degraded by Y. lipolytica proteases to peptides and free amino acids for animal feed. The lignocellulosic fraction can be ground and treated to release sugars for fermentation in a third stage by a recombinant cellulosic Saccharomyces cerevisiae, which can also be engineered to express valuable peptide products. The residual protein and lignin solids can be jet cooked and passed to a fourth-stage fermenter where Rhodotorula glutinis converts methane into isoprenoid intermediates. The residues can be combined and transferred into pyrocracking and hydroformylation reactions to convert ammonia, protein, isoprenes, lignins, and oils into renewable gas. Any remaining waste can be thermoconverted to biochar as a humus soil enhancer. The integration of multiple technologies for treatment of coffee waste has the potential to

  18. Microbial diversity and dynamics during the production of May bryndza cheese.

    PubMed

    Pangallo, Domenico; Saková, Nikoleta; Koreňová, Janka; Puškárová, Andrea; Kraková, Lucia; Valík, Lubomír; Kuchta, Tomáš

    2014-01-17

    paradoxus. The diversity of yeasts and fungi encompassed Alternaria alternata, "Ascomycete sp.", Aspergillus fumigatus, Beauveria brongniartii, Candida xylopsoci, C. inconspicua, Cladosporium cladosporioides, Debaromyces hansenii, Fomes fomentarius, Galactomyces candidus, Gymnoascus reesii, Chaetomium globosum, Kluyveromyces marxianus, Metarhizium anisopliae, Penicillium aurantiogriseum, P. camemberti, P. freii, P. polonicum, P. viridicatum, Pichia kudriavzevii, Sordaria alcina, Trichosporon lactis and Yarrowia lipolytica.

  19. Studies on antimicrobial activities of solvent extracts of different spices.

    PubMed

    Keskin, Dilek; Toroglu, Sevil

    2011-03-01

    The antimicrobial activities of the ethyl acetate, acetone and methanol extract of 12 plant species were studied. The extract of Capsicum annuum (red pepper) (fruit) Zingiber officinale (ginger) (root), Cuminum cyminum (cumin), Alpinia ficinarum (galingale), Coriandrum sativum (coriander), Cinnamomun zeylanicum Nees (cinnamomun), Origanum onites L. (thyme), Folium sennae (senna), Eugenia caryophyllata (cloves), Flos tiliae (lime), Folium menthae crispae (peppermint) and Piper nigrum (blackpepper) were tested in vitro against 2 fungi and 8 bacterial species by the disc diffusion method. Klebsiella pneumonia 13883, Bacillus megaterium NRS, Pseudomonas aeroginosa ATCC 27859, Staphylococcus aureus 6538 P, Escherichia coil ATCC 8739, Enterobacter cloaca ATCC 13047, Corynebacterium xerosis UC 9165, Streptococcus faecalis DC 74, Kluyveromyces marxianus, Rhodotorula rubra were used in this investigation. The results indicated that extracts of different spices has shown antibacterial activity in the range of 7-24 mm 30 microl(-1) inhibition zone Eugenia caryophyllata (clove), 7-20 mm 30microl(-1) inhibition zone Capsicum annum (red pepper) and Cinnamomun zeylanicum (cinnamon) bark, 7-18 mm 30microl(-1) inhibition zone Folium sennae (senna) leaves, 7-16 mm 30 microl(-1) inhibition zone Zingiber officinale (ginger) root, 7-15 mm 30microl(-1) inhibition zone Cuminum cyminum (cumin) seed, 7-14 mm 30 microl(-1) inhibition zone Folium menthae crispae (peppermint), Origanum onites (thyme) leaves and Alpinia ficinarum (galingale) root, 7-12 mm 30 microl(-1) inhibiton zone Piper nigrum (blackpepper), 7-11 mm 30microl(-1) inhibition zone Flos tiliae (lime) leaves, 7-8 mm 30microl(-1) inhibition zone Coriandrum sativum (coriander) to the microorganisms tested.

  20. Contributions from the SuperKamiokande Collaboration

    SciTech Connect

    Wilkes, R.J.; Fukuda, Y.; Inoue, K.

    1995-09-01

    This document consists of two reports contributed to the XXIV International Cosmic Ray Conference (Rome, Italy, August 28--September 8, 1995) from the SuperKamiokande Collaboration: one on the SuperKamiokande outer data acquisition system, and one on preliminary results from muon/electron identification tests at KEK of IMB3-detector phototubes and electronics.

  1. Yeasts associated with fresh and frozen pulps of Brazilian tropical fruits.

    PubMed

    Trindade, Rita C; Resende, Maria Aparecida; Silva, Claudia M; Rosa, Carlos A

    2002-08-01

    The occurrence of yeasts on ripe fruits and frozen pulps of pitanga (Eugenia uniflora L), mangaba (Hancornia speciosa Gom.), umbu (Spondias tuberosa Avr. Cam.), and acerola (Malpighia glaba L) was verified. The incidence of proteolytic, pectinolytic, and mycocinogenic yeasts on these communities was also determined. A total of 480 colonies was isolated and grouped in 405 different strains. These corresponded to 42 ascomycetous and 28 basidiomycetous species. Candida sorbosivorans, Pseudozyma antarctica, C. spandovensis-like, C. spandovensis, Kloeckera apis, C. parapsilosis, Rhodotorula graminis, Kluyveromyces marxianus, Cryptococcus laurentii, Metchnikowia sp (isolated only from pitanga ripe fruits), Issatchenkia occidentalis and C. krusei (isolated only from mangaba frozen pulps), were the most frequent species. The yeast communities from pitanga ripe fruits exhibited the highest frequency of species, followed by communities from acerola ripe fruits and mangaba frozen pulps. Yeast communities from frozen pulp and ripe fruits of umbu had the lowest number of species. Except the yeasts from pitanga, yeast communities from frozen pulp exhibited higher number of yeasts than ripe fruit communities. Mycocinogenic yeasts were found in all of the substrates studied except in communities from umbu ripe fruits and pitanga frozen pulps. Most of the yeasts found to produce mycocins were basidiomycetes and included P. antarctica, Cryptococcus albidus, C. bhutanensis-like, R. graminis and R. mucilaginosa-like from pitanga ripe fruits as well as black yeasts from pitanga and acerola ripe fruits. The umbu frozen pulps community had the highest frequency of proteolytic species. Yeasts able to hydrolyse casein at pH 5.0 represented 38.5% of the species isolated. Thirty-seven percent of yeast isolates were able to hydrolyse casein at pH 7.0. Pectinolytic yeasts were found in all of the communities studied, excepted for those of umbu frozen pulps. The highest frequency of

  2. A highly efficient β-glucosidase from the buffalo rumen fungus Neocallimastix patriciarum W5

    PubMed Central

    2012-01-01

    Background Cellulose, which is the most abundant renewable biomass on earth, is a potential bio-resource of alternative energy. The hydrolysis of plant polysaccharides is catalyzed by microbial cellulases, including endo-β-1,4-glucanases, cellobiohydrolases, cellodextrinases, and β-glucosidases. Converting cellobiose by β-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production. Results In this study, a cDNA encoding β-glucosidase was isolated from the buffalo rumen fungus Neocallimastix patriciarum W5 and is named NpaBGS. It has a length of 2,331 bp with an open reading frame coding for a protein of 776 amino acid residues, corresponding to a theoretical molecular mass of 85.1 kDa and isoelectric point of 4.4. Two GH3 catalytic domains were found at the N and C terminals of NpaBGS by sequence analysis. The cDNA was expressed in Pichia pastoris and after protein purification, the enzyme displayed a specific activity of 34.5 U/mg against cellobiose as the substrate. Enzymatic assays showed that NpaBGS was active on short cello-oligosaccharides from various substrates. A weak activity in carboxymethyl cellulose (CMC) digestion indicated that the enzyme might also have the function of an endoglucanase. The optimal activity was detected at 40°C and pH 5 ~ 6, showing that the enzyme prefers a weak acid condition. Moreover, its activity could be enhanced at 50°C by adding Mg2+ or Mn2+ ions. Interestingly, in simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae BY4741 or Kluyveromyces marxianus KY3 as the fermentation yeast, NpaBGS showed advantages in cell growth, glucose production, and ethanol production over the commercial enzyme Novo 188. Moreover, we showed that the KY3 strain engineered with the NpaNGS gene can utilize 2 % dry napiergrass as the sole carbon source to produce 3.32 mg/ml ethanol when Celluclast 1.5 L was

  3. Kefir-isolated bacteria and yeasts inhibit Shigella flexneri invasion and modulate pro-inflammatory response on intestinal epithelial cells.

    PubMed

    Bolla, P A; Abraham, A G; Pérez, P F; de Los Angeles Serradell, M

    2016-02-01

    The aim of this work was to evaluate the ability of a kefir-isolated microbial mixture containing three bacterial and two yeast strains (MM) to protect intestinal epithelial cells against Shigella flexneri invasion, as well as to analyse the effect on pro-inflammatory response elicited by this pathogen. A significant decrease in S. flexneri strain 72 invasion was observed on both HT-29 and Caco-2 cells pre-incubated with MM. Pre-incubation with the individual strains Saccharomyces cerevisiae CIDCA 8112 or Lactococcus lactis subsp. lactis CIDCA 8221 also reduced the internalisation of S. flexneri into HT-29 cells although in a lesser extent than MM. Interestingly, Lactobacillus plantarum CIDCA 83114 exerted a protective effect on the invasion of Caco-2 and HT-29 cells by S. flexneri. Regarding the pro-inflammatory response on HT-29 cells, S. flexneri infection induced a significant activation of the expression of interleukin 8 (IL-8), chemokine (C-C motif) ligand 20 (CCL20) and tumour necrosis factor alpha (TNF-α) encoding genes (P<0.05), whereas incubation of cells with MM did not induce the expression of any of the mediators assessed. Interestingly, pre-incubation of HT-29 monolayer with MM produced an inhibition of S. flexneri-induced IL-8, CCL20 and TNF-α mRNA expression. In order to gain insight on the effect of MM (or the individual strains) on this pro-inflammatory response, a series of experiments using a HT-29-NF-κB-hrGFP reporter system were performed. Pre-incubation of HT-29-NF-κB-hrGFP cells with MM significantly dampened Shigella-induced activation. Our results showed that the contribution of yeast strain Kluyveromyces marxianus CIDCA 8154 seems to be crucial in the observed effect. In conclusion, results presented in this study demonstrate that pre-treatment with a microbial mixture containing bacteria and yeasts isolated from kefir, resulted in inhibition of S. flexneri internalisation into human intestinal epithelial cells, along with the

  4. Production of 2-phenylethanol and 2-phenylethylacetate from L-phenylalanine by coupling whole-cell biocatalysis with organophilic pervaporation.

    PubMed

    Etschmann, Maria M W; Sell, Dieter; Schrader, Jens

    2005-12-05

    An integrated bioprocess for the production of the natural rose-like aroma compounds, 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEAc), from L-phenylalanine (L-phe) with yeasts was investigated. The hydrophobicity of the products leads to product inhibition, which can be compensated by in situ product removal (ISPR). An organophilic pervaporation unit, equipped with a polyoctylmethylsiloxane (POMS) membrane, was coupled via a bypass to a bioreactor and proved to be a suitable technique for the in situ removal of high-boiling products from culture broth. With batch cultures of the thermotolerant yeast Kluyveromyces marxianus CBS 600 in a standard medium at 35 degrees C, the use of pervaporation resulted in a double 2-PE concentration (2.2 g/L) and 1.3 g/L 2-PEAc, which only accumulated transiently in low concentrations during cultivation without ISPR. Using a previously optimized medium, the variation of the temperature from 30 degrees C to 40 degrees C caused an increase in the total conversion yield from 63% to 79%, corresponding to total product concentrations of 5.23 and 5.85 g/L, respectively. In the 40 degrees C batch experiment, the volumetric productivity (2-PE + 2-PEAc) during the exponential phase was 5.2 mmol/L h. While for 2-PE, there is still potential for further optimization, the more hydrophobic 2-PEAc was nearly completely removed from the aqueous culture broth (enrichment factor >400), resulting in highly aroma-enriched permeates. Due to the temperature-correlated performance of the pervaporation, the bioconversion was still efficient even at 45 degrees C (conversion yield: 69%). Surprisingly, at 45 degrees C, the molar ratio of the two products inverted and 2-PEAc turned out to be the main product (4.0 g/L), which opens easy control of the reaction's selectivity by external means. Retrofitting the process with interim heating and cooling equipment to use different temperature levels for cultivation and pervaporation resulted in a decreased

  5. Construction of lactose-consuming Saccharomyces cerevisiae for lactose fermentation into ethanol fuel.

    PubMed

    Zou, Jing; Guo, Xuewu; Shen, Tong; Dong, Jian; Zhang, Cuiying; Xiao, Dongguang

    2013-04-01

    Two lactose-consuming diploid Saccharomyces cerevisiae strains, AY-51024A and AY-51024M, were constructed by expressing the LAC4 and LAC12 genes of Kluyveromyces marxianus in the host strain AY-5. In AY-51024A, both genes were targeted to the ATH1 and NTH1 gene-encoding regions to abolish the activity of acid/neutral trehalase. In AY-51024M, both genes were respectively integrated into the MIG1 and NTH1 gene-encoding regions to relieve glucose repression. Physiologic studies of the two transformants under anaerobic cultivations in glucose and galactose media indicated that the expression of both LAC genes did not physiologically burden the cells, except for AY-51024A in glucose medium. Galactose consumption was initiated at higher glucose concentrations in the MIG1 deletion strain AY-51024M than in the corresponding wild-type strain and AY-51024A, wherein galactose was consumed until glucose was completely depleted in the mixture. In lactose medium, the Sp. growth rates of AY-51024A and AY-51024M under anaerobic shake-flasks were 0.025 and 0.067 h(-1), respectively. The specific lactose uptake rate and ethanol production of AY-51024M were 2.50 g lactose g CDW(-1) h(-1) and 23.4 g l(-1), respectively, whereas those of AY-51024A were 0.98 g lactose g CDW(-1) h(-1) and 24.3 g lactose g CDW(-1) h(-1), respectively. In concentrated cheese whey powder solutions, AY-51024M produced 63.3 g l(-1) ethanol from approximately 150 g l(-1) initial lactose in 120 h, conversely, AY-51024A consumed 63.7 % of the initial lactose and produced 35.9 g l(-1) ethanol. Therefore, relieving glucose repression is an effective strategy for constructing lactose-consuming S. cerevisiae.

  6. Effects of glucose, ethanol and acetic acid on regulation of ADH2 gene from Lachancea fermentati

    PubMed Central

    Yaacob, Norhayati; Salleh, Abu Bakar; Abdul Rahman, Nor Aini

    2016-01-01

    Background. Not all yeast alcohol dehydrogenase 2 (ADH2) are repressed by glucose, as reported in Saccharomyces cerevisiae. Pichia stipitis ADH2 is regulated by oxygen instead of glucose, whereas Kluyveromyces marxianus ADH2 is regulated by neither glucose nor ethanol. For this reason, ADH2 regulation of yeasts may be species dependent, leading to a different type of expression and fermentation efficiency. Lachancea fermentati is a highly efficient ethanol producer, fast-growing cells and adapted to fermentation-related stresses such as ethanol and organic acid, but the metabolic information regarding the regulation of glucose and ethanol production is still lacking. Methods. Our investigation started with the stimulation of ADH2 activity from S. cerevisiae and L. fermentati by glucose and ethanol induction in a glucose-repressed medium. The study also embarked on the retrospective analysis of ADH2 genomic and protein level through direct sequencing and sites identification. Based on the sequence generated, we demonstrated ADH2 gene expression highlighting the conserved NAD(P)-binding domain in the context of glucose fermentation and ethanol production. Results. An increase of ADH2 activity was observed in starved L. fermentati (LfeADH2) and S. cerevisiae (SceADH2) in response to 2% (w/v) glucose induction. These suggest that in the presence of glucose, ADH2 activity was activated instead of being repressed. An induction of 0.5% (v/v) ethanol also increased LfeADH2 activity, promoting ethanol resistance, whereas accumulating acetic acid at a later stage of fermentation stimulated ADH2 activity and enhanced glucose consumption rates. The lack in upper stream activating sequence (UAS) and TATA elements hindered the possibility of Adr1 binding to LfeADH2. Transcription factors such as SP1 and RAP1 observed in LfeADH2 sequence have been implicated in the regulation of many genes including ADH2. In glucose fermentation, L. fermentati exhibited a bell-shaped ADH2

  7. Search for Cygnus X-3 in underground muons during the 1989 radio outbursts using the IMB detector

    SciTech Connect

    Becker-Szendy, R.; Bratton, C.B.; Cady, R.; Casper, D.; Dye, S.T.; Gajewski, W.; Goldhaber, M.; Haines, T.J.; Halverson, P.G.; Jones, T.W.; Kielczewska, D.; Kropp, W.R.; Learned, J.G.; LoSecco, J.M.; Matsuno, S.; McGrew, C.; Mudan, M.S.; Price, L.; Reines, F.; Schultz, J.; Sobel, H.W.; Stone, J.L.; Sulak, L.R.; Svoboda, R.; Wittel, F. The University of Michigan, Ann Arbor, Michigan 48019 Brookhaven National Laboratory, Upton, New York 11973 Boston University, Boston, Massachusetts 02215 The University of Hawaii, Honolulu, Hawaii 96822 University College, London, WC1 E6BT, United Kingdom Warsaw University, Warsaw, Poland Cleveland State University, Cleveland, Ohio 44115 The University of Notre Dame, Notre Dame, Indiana 46556 Louisiana State University, Baton Rouge, Lousiana 70803 The University of Maryland, College Park, Maryland 20742)

    1991-02-15

    A search is made for underground muons from Cygnus X-3 with the Irvine-Michigan-Brookhaven IMB-3 detector around the time of the 1989 radio outbursts. No long-term pulsed signal is found in a sample of 11 117 underground muons collected from the direction of Cygnus X-3. We place an upper limit of 3{times}10{sup {minus}10} cm{sup {minus}2} sec{sup {minus}1} on the flux of underground muons from Cygnus X-3.

  8. Whole cells in enantioselective reduction of benzyl acetoacetate

    PubMed Central

    Ribeiro, Joyce Benzaquem; Ramos, Aline de Souza; Lopes, Raquel de Oliveira; da Silva, Gabriela Veloso Vieira; de Souza, Rodrigo Octavio Mendonça Alves

    2014-01-01

    The β-ketoester benzyl acetoacetate was enantioselectively reduced to benzyl (S)-3-hydroxybutanoate by seven microorganism species. The best result using free cells was obtained with the yeast Hansenula sp., which furnished 97% ee and 85% of conversion within 24 h. After immobilization in calcium alginate spheres, K.marxianus showed to be more stable after 2 cycles of reaction. PMID:25477927

  9. Antimycotic activity of 4-thioisosteres of flavonoids towards yeast and yeast-like microorganisms.

    PubMed

    Buzzini, Pietro; Menichetti, Stefano; Pagliuca, Chiara; Viglianisi, Caterina; Branda, Eva; Turchetti, Benedetta

    2008-07-01

    Different substituted methoxy- and hydroxy-4-thioisosteres of flavonoids were prepared and their in vitro antimycotic activity towards yeast (Candida spp., Clavispora spp., Cryptococcus spp., Filobasidiella spp., Issatchenkia spp., Pichia spp., Kluyveromyces spp., Saccharomyces spp. and Yarrowia spp.) and yeast-like (Prototheca spp.) microorganisms was tested. Further insights in the biological activities of these antioxidant, oestrogenic and antimicrobial biomimetic derivatives were obtained.

  10. 76 FR 27653 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-12

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines... Kluyveromyces lactis as a host-vector 1 system has been reviewed by the NIH ] Recombinant DNA Advisory...

  11. Geotrichum candidum dominates in yeast population dynamics in Livarot, a French red-smear cheese.

    PubMed

    Larpin, Sandra; Mondoloni, Céline; Goerges, Stefanie; Vernoux, Jean-Paul; Guéguen, Micheline; Desmasures, Nathalie

    2006-12-01

    The diversity and dynamics of yeast populations in four batches of Livarot cheese at three points of ripening were determined. Nine different species were identified by Fourier transform infrared spectroscopy and/or sequencing, and each batch had its own unique yeast community. A real-time PCR method was developed to quantify the four main yeast species: Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces sp. and Yarrowia lipolytica. Culture and molecular approaches showed that G. candidum was the dominant yeast in Livarot cheese. When D. hansenii was added as a commercial strain, it codominated with G. candidum. Kluyveromyces lactis was present only at the start of ripening. Yarrowia lipolytica appeared primarily at the end of ripening. We propose a scheme for the roles and dynamics of the principal Livarot yeasts.

  12. The secretion of human serum albumin from the yeast Saccharomyces cerevisiae using five different leader sequences.

    PubMed

    Sleep, D; Belfield, G P; Goodey, A R

    1990-01-01

    We demonstrate the secretion of human serum albumin into the culture supernatant from the yeast Saccharomyces cerevisiae. Studies with five KEX2 processed leader sequences, namely the S. cerevisiae alpha factor, the natural human serum albumin, the Kluyveromyces lactis killer, a natural human serum albumin/alpha factor fusion, and a Kluyveromyces lactis killer/alpha factor fusion leader, are described. We show that the leader sequence used to direct secretion influences the quantity and quality of the secreted product. In designing secretion systems for heterologous proteins, one aims to maximise both the yield and fidelity of the product. Our results indicate that the choice of leader sequence and its relationship to the structural protein under study are crucial to the success of this process.

  13. The occurrence of killer activity in yeasts isolated from natural habitats.

    PubMed

    Wójcik, Monika; Kordowska-Wiater, Monika

    2015-01-01

    Yeast's ability to restrict the growth and kill other yeasts, fungi and bacteria has been known for over 50 years. Killer activity was detected in yeasts deposited in the world collections or isolated from natural habitats. In this study, isolates from the forest environment, leaves of fruit trees, flower petals, cereals and frozen fruit have been screened in terms of their killer activities. Killer activity was tested on strains belonging to six yeast species: Candida, Rhodotorula, Pichia, Pachysolen, Yarrowia, Trichosporon. The reference strains were Kluyveromyces lactis Y-6682 and Kluyveromyces marxinanus Y-8281, well-known to be sensitive to yeast killer toxins. Among one hundred and two tested strains, 24 (23.5% of isolates) showed positive killer action, and 10 (9.8% of the isolates) a weak killer action against at least one sensitive reference strain. The highest killer activity was observed among isolates from forest soil and flowers.

  14. Production of alcohol from Jerusalem artichokes by yeasts

    SciTech Connect

    Duvnjak, Z.; Kosaric, N.; Kliza, S.; Hayes, D.

    1982-11-01

    Various yeasts such as several strains of Saccharomyces diastaticus, S. cerevisiae, and Kluyveromyces fragilis were investigated for their ability to ferment the carbohydrates from Jerusalem artichokes to alcohol. Juice extracted from the artichokes was used as the fermentation substrate with and without prior hydrolysis of the carbohydrates. Fermentation was also carried out with raw artichokes without prior juice extraction. Results indicate that this raw material has good potential for fuel alcohol production by fermentation. (Refs. 15).

  15. Extracellular Proteinases of Yeasts and Yeastlike Fungi1

    PubMed Central

    Ahearn, D. G.; Meyers, S. P.; Nichols, R. A.

    1968-01-01

    Approximately 800 yeasts and other fungi, representing over 70 species, were tested for extracellular caseinolysis. Isolates of a variety of genera, including Aureobasidium, Cephalosporium, Endomycopsis, Kluyveromyces, and numerous sporobolomycetes, demonstrated significant proteolytic activity. Caseinolysis was not necessarily correlated with gelatin liquefaction or with albuminolysis. Numerous fungi showed significant proteolysis at 5 C. The most active organisms were isolates of Candida lipolytica, Aureobasidium pullulans, Candida punicea, and species of Cephalosporium. Taxonomic and ecological implications of proteolytic activity are discussed. Images Fig. 1 PMID:5692110

  16. Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication.

    PubMed

    Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

    2016-01-01

    Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under

  17. Runaway telomere elongation caused by telomerase RNA gene mutations.

    PubMed

    McEachern, M J; Blackburn, E H

    1995-08-03

    The ribonucleoprotein enzyme telomerase adds telomeric DNA onto chromosome ends and is normally regulated so that telomeric DNA lengths are kept within defined bounds. In the telomerase RNA gene from the yeast Kluyveromyces lactis, specific mutations that alter telomeric DNA sequences result in telomeres elongating to up to 100 times their normal length and impair cell growth. Some mutations cause immediate elongation whereas others behave like genetic time bombs, causing elongation only after a latent period of hundreds of generations.

  18. Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication*

    PubMed Central

    Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

    2016-01-01

    Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under

  19. Study of atmospheric neutrino interactions and search for nucleon decay in Soudan 2

    SciTech Connect

    Leeson, William R.

    1995-12-14

    Contained event samples, including 30 single-track muon-like events, 35 single-shower electron-like events, and 34 multiprong events, have been obtained from a 1.0 kiloton-year exposure of the Soudan 2 detector. A sample of 15 multiprong events which are partially contained has also been isolated. Properties of these events are used to examine the verity of the atmospheric neutrino flavor ratio anomaly as reported by the Kamiokande and IMB-3 water Cherenkov experiments. The compatibility of the Soudan data with each of two `new physics` explanations for the anomaly, namely proton decay and neutrino oscillations, is investigated. We examine background processes which have not been explicitly treated by the water Cherenkov detectors. Chapters discuss underground non-accelerator particle physics, the atmospheric neutrino anomaly and its interpretation, the Soudan 2 detector and event selection, reconstruction of neutrino events, rock event contamination in Soudan `quasi-elastic` samples, contained multiprong events in Soudan 2, neutrino flavor composition of the multiprong sample, partially contained events in Soudan 2, nucleon decay in Soudan 2, and a summary and discussion.

  20. Functional Diversity of Silencers in Budding Yeasts

    PubMed Central

    Sjöstrand, Jimmy O. O.; Kegel, Andreas; Åström, Stefan U.

    2002-01-01

    We studied the silencing of the cryptic mating-type loci HMLα and HMRa in the budding yeast Kluyveromyces lactis. A 102-bp minimal silencer fragment was defined that was both necessary and sufficient for silencing of HMLα. Mutagenesis of the silencer revealed three distinct regions (A, B, and C) that were important for silencing. Recombinant K. lactis ribosomal DNA enhancer binding protein 1 (Reb1p) could bind the silencer in vitro, and point mutations in the B box abolished both Reb1p binding and silencer function. Furthermore, strains carrying temperature-sensitive alleles of the REB1 gene derepressed the transcription of the HMLα1 gene at the nonpermissive temperature. A functional silencer element from the K. lactis cryptic HMRa locus was also identified, which contained both Reb1p binding sites and A boxes, strongly suggesting a general role for these sequences in K. lactis silencing. Our data indicate that different proteins bind to Kluyveromyces silencers than to Saccharomyces silencers. We suggest that the evolution of silencers is rapid in budding yeasts and discuss the similarities and differences between silencers in Saccharomyces and Kluyveromyces. PMID:12456003

  1. Level-set surface segmentation and registration for computing intrasurgical deformations

    NASA Astrophysics Data System (ADS)

    Audette, Michel A.; Peters, Terence M.

    1999-05-01

    We propose a method for estimating intrasurgical brain shift for image-guided surgery. This method consists of five stages: the identification of relevant anatomical surfaces within the MRI/CT volume, range-sensing of the skin and cortex in the OR, rigid registration of the skin range image with its MRI/CT homologue, non-rigid motion tracking over time of cortical range images, and lastly, interpolation of this surface displacement information over the whole brain volume via a realistically valued finite element model of the head. This paper focuses on the anatomical surface identification and cortical range surface tracking problems. The surface identification scheme implements a recent algorithm which imbeds 3D surface segmentation as the level- set of a 4D moving front. A by-product of this stage is a Euclidean distance and closest point map which is later exploited to speed up the rigid and non-rigid surface registration. The range-sensor uses both laser-based triangulation and defocusing techniques to produce a 2D range profile, and is linearly swept across the skin or cortical surface to produce a 3D range image. The surface registration technique is of the iterative closest point type, where each iteration benefits from looking up, rather than searching for, explicit closest point pairs. These explicit point pairs in turn are used in conjunction with a closed-form SVD-based rigid transformation computation and with fast recursive splines to make each rigid and non-rigid registration iteration essentially instantaneous. Our method is validated with a novel deformable brain-shaped phantom, made of Polyvinyl Alcohol Cryogel.

  2. Split-Doa10: a naturally split polytopic eukaryotic membrane protein generated by fission of a nuclear gene.

    PubMed

    Stuerner, Elisabeth; Kuraku, Shigehiro; Hochstrasser, Mark; Kreft, Stefan G

    2012-01-01

    Large polytopic membrane proteins often derive from duplication and fusion of genes for smaller proteins. The reverse process, splitting of a membrane protein by gene fission, is rare and has been studied mainly with artificially split proteins. Fragments of a split membrane protein may associate and reconstitute the function of the larger protein. Most examples of naturally split membrane proteins are from bacteria or eukaryotic organelles, and their exact history is usually poorly understood. Here, we describe a nuclear-encoded split membrane protein, split-Doa10, in the yeast Kluyveromyces lactis. In most species, Doa10 is encoded as a single polypeptide with 12-16 transmembrane helices (TMs), but split-KlDoa10 is encoded as two fragments, with the split occurring between TM2 and TM3. The two fragments assemble into an active ubiquitin-protein ligase. The K. lactis DOA10 locus has two ORFs separated by a 508-bp intervening sequence (IVS). A promoter within the IVS drives expression of the C-terminal KlDoa10 fragment. At least four additional Kluyveromyces species contain an IVS in the DOA10 locus, in contrast to even closely related genera, allowing dating of the fission event to the base of the genus. The upstream Kluyveromyces Doa10 fragment with its N-terminal RING-CH and two TMs resembles many metazoan MARCH (Membrane-Associated RING-CH) and related viral RING-CH proteins, suggesting that gene splitting may have contributed to MARCH enzyme diversification. Split-Doa10 is the first unequivocal case of a split membrane protein where fission occurred in a nuclear-encoded gene. Such a split may allow divergent functions for the individual protein segments.

  3. Investigation of bacterial and fungal diversity in tarag using high-throughput sequencing.

    PubMed

    Sun, Zhihong; Liu, Wenjun; Bao, Qiuhua; Zhang, Jiachao; Hou, Qiangchuan; Kwok, Laiyu; Sun, Tiansong; Zhang, Heping

    2014-10-01

    This is the first study on the bacterial and fungal community diversity in 17 tarag samples (naturally fermented dairy products) through a metagenomic approach involving high-throughput pyrosequencing. Our results revealed the presence of a total of 47 bacterial and 43 fungal genera in all tarag samples, in which Lactobacillus and Galactomyces were the predominant genera of bacteria and fungi, respectively. The number of some microbial genera, such as Lactococcus, Acetobacter, Saccharomyces, Trichosporon, and Kluyveromyces, among others, was found to vary between different samples. Altogether, our results showed that the microbial flora in different samples may be stratified by geographic region.

  4. Microbial terroir and food innovation: The case of yeast biodiversity in wine.

    PubMed

    Capozzi, Vittorio; Garofalo, Carmela; Chiriatti, Maria Assunta; Grieco, Francesco; Spano, Giuseppe

    2015-12-01

    Saccharomyces and non-Saccharomyces represents a heterogeneous class in the grape/must/wine environments including several yeast genera (e.g., Saccharomyces, Hanseniaspora, Pichia, Candida, Metschnikowia, Kluyveromyces, Zygosaccharomyces, Torulaspora, Dekkera and Schizosaccharomyces) and species. Since, each species may differently contribute to the improvement/depreciation of wine qualities, it appears clear the reason why species belong to non-Saccharomyces are also considered a biotechnological resource in wine fermentation. Here, we briefly review the oenological significance of this specific part of microbiota associated with grapes/musts/wine. Moreover, the diversity of cultivable non-Saccharomyces genera and their contribute to typical wines fermentations will be discussed.

  5. Fluid-phase endocytosis in yeasts other than Saccharomyces cerevisiae.

    PubMed

    Fernandez, N; Puente, P; Leal, F

    1990-05-01

    A FITC-dextran internalization assay with Saccharomyces cerevisiae as positive control was used to determine whether fluid-phase endocytosis is a general characteristic of yeasts. Schizosaccharomyces pombe, Pichia polymorpha, Kluyveromyces phaseolosporus, Yarrowia lipolytica and Candida albicans were clearly positive, whereas results obtained with Debaryomyces marama were inconclusive. In all cases internalized FITC-dextran was found to be localized in the vacuoles and the process was always time- and temperature-dependent. Lower eucaryotes, particularly yeasts, appear to have the ability to incorporate substances from the extracellular medium through fluid-phase endocytosis.

  6. A recombinant Saccharomyces cerevisiae strain for efficient conversion of lactose in salted and unsalted cheese whey into ethanol.

    PubMed

    Tahoun, M K; el-Nemr, T M; Shata, O H

    2002-10-01

    For utilization of lactose in salted and unsalted cheese whey, intergeneric protoplast fusion between lactose nonfermenting, salt-tolerant Saccharomyces cerevisiae ATCC4126 and lactose fermenting Kluyveromyces lactis CBS683 was carried out. The fusion process gave rise to new hybrid yeast strains that revealed higher significant DNA contents than parental strains. The recombinants showed growth on either lactose or sucrose. The ethanol yields by some recombinants were 5.55% from sweet whey and 4.66% from salted whey containing up to 6% sodium chloride compared to 4.15 and 2.86% for parental K. lactis CBS683, respectively.

  7. PMAA-stabilized ferrofluid/chitosan/yeast composite for bioapplications

    NASA Astrophysics Data System (ADS)

    Baldikova, Eva; Prochazkova, Jitka; Stepanek, Miroslav; Hajduova, Jana; Pospiskova, Kristyna; Safarikova, Mirka; Safarik, Ivo

    2017-04-01

    A simple, one-pot process for the preparation of magnetically responsive yeast-based biocatalysts was developed. Saccharomyces cerevisiae, Candida utilis and Kluyveromyces lactis cells were successfully incorporated into chitosan gel magnetically modified with poly(methacrylic acid)-stabilized magnetic fluid (PMAA-FF) during its formation. Magnetic PMAA-FF/chitosan/yeast composites were efficiently employed for invert sugar production. The dependence of invertase activity on used yeast, amount of magnetic biocatalyst, agitation time and after reuse was studied in detail. The tested magnetic biocatalysts retained at least 69% of their initial activity after 8 reuse cycles.

  8. The RNA polymerase III-dependent family of genes in hemiascomycetes: comparative RNomics, decoding strategies, transcription and evolutionary implications

    PubMed Central

    Marck, Christian; Kachouri-Lafond, Rym; Lafontaine, Ingrid; Westhof, Eric; Dujon, Bernard; Grosjean, Henri

    2006-01-01

    We present the first comprehensive analysis of RNA polymerase III (Pol III) transcribed genes in ten yeast genomes. This set includes all tRNA genes (tDNA) and genes coding for SNR6 (U6), SNR52, SCR1 and RPR1 RNA in the nine hemiascomycetes Saccharomyces cerevisiae, Saccharomyces castellii, Candida glabrata, Kluyveromyces waltii, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica and the archiascomycete Schizosaccharomyces pombe. We systematically analysed sequence specificities of tRNA genes, polymorphism, variability of introns, gene redundancy and gene clustering. Analysis of decoding strategies showed that yeasts close to S.cerevisiae use bacterial decoding rules to read the Leu CUN and Arg CGN codons, in contrast to all other known Eukaryotes. In D.hansenii and C.albicans, we identified a novel tDNA-Leu (AAG), reading the Leu CUU/CUC/CUA codons with an unusual G at position 32. A systematic ‘p-distance tree’ using the 60 variable positions of the tRNA molecule revealed that most tDNAs cluster into amino acid-specific sub-trees, suggesting that, within hemiascomycetes, orthologous tDNAs are more closely related than paralogs. We finally determined the bipartite A- and B-box sequences recognized by TFIIIC. These minimal sequences are nearly conserved throughout hemiascomycetes and were satisfactorily retrieved at appropriate locations in other Pol III genes. PMID:16600899

  9. Yeast Diversity and Persistence in Botrytis-Affected Wine Fermentations

    PubMed Central

    Mills, David A.; Johannsen, Eric A.; Cocolin, Luca

    2002-01-01

    Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected (“botrytized”) wine fermentations carried out at high (∼30°C) and ambient (∼20°C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by Saccharomyces. In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>106 cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of Candida. Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations. PMID:12324335

  10. Production of Palmitoleic and Linoleic Acid in Oleaginous and Nonoleaginous Yeast Biomass

    PubMed Central

    Kolouchová, Irena; Maťátková, Olga; Sigler, Karel; Masák, Jan; Řezanka, Tomáš

    2016-01-01

    We investigated the possibility of utilizing both oleaginous yeast species accumulating large amounts of lipids (Yarrowia lipolytica, Rhodotorula glutinis, Trichosporon cutaneum, and Candida sp.) and traditional biotechnological nonoleaginous ones (Kluyveromyces polysporus, Torulaspora delbrueckii, and Saccharomyces cerevisiae) as potential producers of dietetically important major fatty acids. The main objective was to examine the cultivation conditions that would induce a high ratio of dietary fatty acids and biomass. Though genus-dependent, the type of nitrogen source had a higher influence on biomass yield than the C/N ratio. The nitrogen source leading to the highest lipid accumulation was potassium nitrate, followed by ammonium sulfate, which is an ideal nitrogen source supporting, in both oleaginous and nonoleaginous species, sufficient biomass growth with concomitantly increased lipid accumulation. All yeast strains displayed high (70–90%) content of unsaturated fatty acids in total cell lipids. The content of dietary fatty acids of interest, namely, palmitoleic acid and linoleic acid, reached in Kluyveromyces and Trichosporon strains over 50% of total fatty acids and the highest yield, over 280 mg per g of dry cell weight of these fatty acids, was observed in Trichosporon with ammonium sulfate as nitrogen source at C/N ratio 70. PMID:27022398

  11. Bioprocessing of bagasse hydrolysate for ethanol and xylitol production using thermotolerant yeast.

    PubMed

    Kumar, Sachin; Dheeran, Pratibha; Singh, Surendra P; Mishra, Indra M; Adhikari, Dilip K

    2015-01-01

    Fermentation of xylose-rich and glucose-rich bagasse hydrolysates, obtained from the two-stage acid hydrolysis was studied using the thermotolerant yeast Kluyveromyces sp. IIPE453. The yeast could grow on xylose-rich hydrolysate at 50 °C with the dry cell weight, cell mass yield and maximum specific growth rate of 5.35 g l(-1), 0.58 g g(-1) and 0.13 h(-1), respectively. The yeast was found to be very promising for ethanol as well as xylitol production from the sugars obtained from the lignocellulosic biomass. Batch fermentations of xylose-rich and glucose-rich hydrolysates yielded 0.61 g g(-1) xylitol and 0.43 g g(-1) ethanol in the broth, respectively based on the sugars present in the hydrolysate. Overall ethanol yield of 165 g (210 ml) and 183 g xylitol per kg of bagasse was obtained, when bagasse hydrolysate was used as a substrate. Utilization of both the glucose and xylose sugars makes the process most economical by producing both ethanol and xylitol based on biorefinery concept. On validating the experimental data of ethanol fermentation, the modified Luong kinetic model for product inhibition as well as inhibition due to inhibitory compounds present in hydrolysate, the model was found to be the best fit for ethanol formation from bagasse hydrolysate using Kluyveromyces sp. IIPE453.

  12. The yeast regulator of transcription protein Rtr1 lacks an active site and phosphatase activity.

    PubMed

    Xiang, Kehui; Manley, James L; Tong, Liang

    2012-07-10

    The activity of RNA polymerase II (Pol II) is controlled in part by the phosphorylation state of the C-terminal domain (CTD) of its largest subunit. Recent reports have suggested that yeast regulator of transcription protein, Rtr1, and its human homologue RPAP2, possess Pol II CTD Ser5 phosphatase activity. Here we report the crystal structure of Kluyveromyces lactis Rtr1, which reveals a new type of zinc finger protein and does not have any close structural homologues. Importantly, the structure does not show evidence of an active site, and extensive experiments to demonstrate its CTD phosphatase activity have been unsuccessful, suggesting that Rtr1 has a non-catalytic role in CTD dephosphorylation.

  13. Influence of the soft X-ray plasma focus radiation on live microorganisms

    NASA Astrophysics Data System (ADS)

    Zapryanov, S.; Goltsev, V.; Galutsov, B.; Gelev, M.; Blagoev, A.

    2012-04-01

    A 3 kJ plasma focus device was used to study the influence of the soft X-ray on live microorganisms. When Saccharomyces cerevisiae - (yeast) was treated with a dose of 65 mSv of the X-ray radiation (14 shots), no difference in the fertility activity between the control probe and the sample was observed. Also no change in the survival enzyme activity was found after irradiation through a 100 μm Al foil of another type of yeast - Kluyveromyces marxiamus. The irradiation of the Chlamydomonas reinhardtii samples by the PF-X-ray emission through 20 μm Al foil with a dose of 11 mSv produces a considerable change of the photosynthesis parameters. This result is similar to the results of previous studies with plasma focus radiation where strong effects were derived with low doses but with a high dose power.

  14. Controlled Microbial Cenoses in Closed Spaces

    NASA Astrophysics Data System (ADS)

    Somova, Lydia; Mikheeva, Galina

    Controlled microbial cenoses have good prospects in closed spaces: for air treatment in LSS and cellars industrial premises; for sewage treatment in LSS; for increase of productivity and protect of plants from infections in LSS. Possible methods of formation of microbiocenoses are: selection, autoselection, artificial formation taking into account their biochemical properties and metabolic interactions. Experimental microbiocenoses, has been produced on the basis of natural association of microorganisms by long cultivation on specially developed medium. Dominating groups are bacteria of genera: Lactobacillus, Streptococcus, Leuconostoc, Bidobac-terium, Rhodopseudomonas and yeast of genera: Kluyveromyces, Saccharomyces and Torulop-sis. Microbiocenoses do not contain pathogenic and conditionally pathogenic microorganisms, they possess opposing and probiotic properties. Different examples of microbial cenoses actions are to be presented in the paper.

  15. Magnetic studies of ferrofluid-modified microbial cells.

    PubMed

    Mosiniewicz-Szablewska, Ewa; Safarikova, Mirka; Safarik, Ivo

    2010-04-01

    Microbial cells (Kluyveromyces fragilis and Chlorella vulgaris) efficiently interacted with maghemite nanoparticles stabilized as low-pH ionic magnetic fluid, leading to the formation of magnetically labeled cells. This simple procedure allows to use the prepared materials as new cheap and easy to get magnetic affinity adsorbents to the removal of water-soluble dyes from polluted water sources using magnetic separation techniques. Magnetically modified cells were investigated by means of electron spin resonance spectroscopy and conventional magnetic methods over the temperature range 4-300 K. The magnetic behavior of these materials was dominated by the superparamagnetic relaxation of isolated single domain maghemite particles although a little amount of agglomerates was also present on the cell surface. However, these agglomerates were sufficiently small to show at static conditions the superparamagnetic behavior at room temperature. Therefore, the ferrofluid-modified microbial cells represent new interesting magnetic affinity adsorbents which could be applied for large-scale magnetic separation processes.

  16. A mutation in the Zn-finger of the GAL4 homolog LAC9 results in glucose repression of its target genes.

    PubMed Central

    Kuger, P; Gödecke, A; Breunig, K D

    1990-01-01

    The transcriptional activator LAC9, a GAL4 homolog of Kluyveromyces lactis which mediates lactose and galactose-dependent activation of genes involved in the utilization of these sugars can also confer glucose repression to those genes. Here we report on the isolation and characterization of LAC9-2, an allele which encodes a glucose-sensitive activator in contrast to the one previously cloned. A single amino acid exchange of leu-104 to tryptophan is responsible for the glucose-insensitive phenotype. The mutation is located within the Zn-finger-like DNA binding domain which is highly conserved between LAC9 and GAL4. Glucose repression is also eliminated by duplication of the LAC9-2 allele. The data indicate that LAC9 is a limiting factor for beta-galactosidase gene expression under all growth conditions and that glucose reduces the activity of the activator. Images PMID:2107531

  17. Synthetic biology and molecular genetics in non-conventional yeasts: Current tools and future advances.

    PubMed

    Wagner, James M; Alper, Hal S

    2016-04-01

    Coupling the tools of synthetic biology with traditional molecular genetic techniques can enable the rapid prototyping and optimization of yeast strains. While the era of yeast synthetic biology began in the well-characterized model organism Saccharomyces cerevisiae, it is swiftly expanding to include non-conventional yeast production systems such as Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. These yeasts already have roles in the manufacture of vaccines, therapeutic proteins, food additives, and biorenewable chemicals, but recent synthetic biology advances have the potential to greatly expand and diversify their impact on biotechnology. In this review, we summarize the development of synthetic biological tools (including promoters and terminators) and enabling molecular genetics approaches that have been applied in these four promising alternative biomanufacturing platforms. An emphasis is placed on synthetic parts and genome editing tools. Finally, we discuss examples of synthetic tools developed in other organisms that can be adapted or optimized for these hosts in the near future.

  18. Metabolic engineering of Saccharomyces cerevisiae for lactose/whey fermentation

    PubMed Central

    Guimarães, Pedro MR; Oliveira, Carla

    2010-01-01

    Lactose is an interesting carbon source for the production of several bio-products by fermentation, primarily because it is the major component of cheese whey, the main by-product of dairy activities. However, the microorganism more widely used in industrial fermentation processes, the yeast Saccharomyces cerevisiae, does not have a lactose metabolization system. Therefore, several metabolic engineering approaches have been used to construct lactose-consuming S. cerevisiae strains, particularly involving the expression of the lactose genes of the phylogenetically related yeast Kluyveromyces lactis, but also the lactose genes from Escherichia coli and Aspergillus niger, as reviewed here. Due to the existing large amounts of whey, the production of bio-ethanol from lactose by engineered S. cerevisiae has been considered as a possible route for whey surplus. Emphasis is given in the present review on strain improvement for lactose-to-ethanol bioprocesses, namely flocculent yeast strains for continuous high-cell-density systems with enhanced ethanol productivity. PMID:21326922

  19. Yeast cell factories for fine chemical and API production

    PubMed Central

    Pscheidt, Beate; Glieder, Anton

    2008-01-01

    This review gives an overview of different yeast strains and enzyme classes involved in yeast whole-cell biotransformations. A focus was put on the synthesis of compounds for fine chemical and API (= active pharmaceutical ingredient) production employing single or only few-step enzymatic reactions. Accounting for recent success stories in metabolic engineering, the construction and use of synthetic pathways was also highlighted. Examples from academia and industry and advances in the field of designed yeast strain construction demonstrate the broad significance of yeast whole-cell applications. In addition to Saccharomyces cerevisiae, alternative yeast whole-cell biocatalysts are discussed such as Candida sp., Cryptococcus sp., Geotrichum sp., Issatchenkia sp., Kloeckera sp., Kluyveromyces sp., Pichia sp. (including Hansenula polymorpha = P. angusta), Rhodotorula sp., Rhodosporidium sp., alternative Saccharomyces sp., Schizosaccharomyces pombe, Torulopsis sp., Trichosporon sp., Trigonopsis variabilis, Yarrowia lipolytica and Zygosaccharomyces rouxii. PMID:18684335

  20. [Fructose transporter in yeasts].

    PubMed

    Lazar, Zbigniew; Dobrowolski, Adam; Robak, Małgorzata

    2014-01-01

    Study of hexoses transporter started with discovery of galactose permease in Saccharomyces cerevisiae. Glucose, fructose and mannose assimilation is assumed by numerous proteins encoded by different genes. To date over 20 hexoses transporters, belonging to Sugar Porter family and to Major Facilitator Superfamily, were known. Genome sequence analysis of Candida glabrata, Kluyveromyces lactis, Yarrowia lipolytica, S. cerevisaie and Debaryomyces hansenii reveled potential presence of 17-48 sugar porter proteins. Glucose transporters in S. cerevisiae have been already characterized. In this paper, hexoses transporters, responsible for assimilation of fructose by cells, are presented and compared. Fructose specific transporter are described for yeasts: Zygosaccharomyces rouxii, Zygosaccharomyces bailli, K. lactis, Saccharomyces pastorianus, S. cerevisiae winemaking strain and for fungus Botritys cinerea and human (Glut5p). Among six yeasts transporters, five are fructose specific, acting by facilitated diffusion or proton symport. Yeasts monosaccharides transporter studies allow understanding of sugars uptake and metabolism important aspects, even in higher eukaryotes cells.

  1. Metabolic engineering of Saccharomyces cerevisiae for lactose/whey fermentation.

    PubMed

    Domingues, Lucília; Guimarães, Pedro M R; Oliveira, Carla

    2010-01-01

    Lactose is an interesting carbon source for the production of several bio-products by fermentation, primarily because it is the major component of cheese whey, the main by-product of dairy activities. However, the microorganism more widely used in industrial fermentation processes, the yeast Saccharomyces cerevisiae, does not have a lactose metabolization system. Therefore, several metabolic engineering approaches have been used to construct lactose-consuming S. cerevisiae strains, particularly involving the expression of the lactose genes of the phylogenetically related yeast Kluyveromyces lactis, but also the lactose genes from Escherichia coli and Aspergillus niger, as reviewed here. Due to the existing large amounts of whey, the production of bio-ethanol from lactose by engineered S. cerevisiae has been considered as a possible route for whey surplus. Emphasis is given in the present review on strain improvement for lactose-to-ethanol bioprocesses, namely flocculent yeast strains for continuous high-cell-density systems with enhanced ethanol productivity.

  2. Production of a bioactive sweetener steviolbioside via specific hydrolyzing ester linkage of stevioside with a β-galactosidase.

    PubMed

    Chen, Jun-ming; Ding, Li; Sui, Xiao-chen; Xia, Yong-mei; Wan, Hui-da; Lu, Tong

    2016-04-01

    A β-galactosidase from Kluyveromyces lactis was found to specifically catalyze hydrolysis of the glycosyl ester linkage of stevioside to yield steviolbioside, a rare sweetener that also exists in Stevia rebaudiana leaves. In a packed bed reactor, a reaction coupling separation was realized and a production yield of steviolbioside reached 90% in 6 h. The hydrolysis product steviolbioside presented higher cytoxicity on human normal cells (hepatocytes cell L02 and intestinal epithelial cell T84) than stevioside did. Comparing to the typical chemotherapy agent, 5-fluorouracil (5-FU), steviolbioside presents much lower cytotoxicity on all assayed human normal cells; it presented notable inhibition on human hepatocarcinoma cell Hep3B, human breast cancer cell MDA-MB-231 and human pancreatic cancer cell BxPC-3. The remarkable inhibition on MDA-MB-231 cells makes steviolbioside a potential remedy for human breast cancer, when steviolbioside is served as a natural sweetener.

  3. Ethanol from whey: Continuous fermentation with cell recycle.

    PubMed

    Janssens, J H; Bernard, A; Bailey, R B

    1984-01-01

    The production of ethanol from cheese whey lactose has been demonstrated using a single-stage continuous culture fermentation with 100% cell recycle. In a two-step process, an aerobic fed batch operation was used initially to allow biomass buildup in the absence of inhibitory ethanol concentrations. In the anaerobic ethanol-producing second step, a strain of Kluyveromyces fragilis selected on the basis of batch fermentation data had a maximum productivity of 7.1 g ethanol/L/h at a dilution rate of 0.15 h(-1), while achieving the goal of zero residual sugar concentration. The fermentation productivity diminished when the feed sugar concentation exceeded 120 g/L despite the inclusion of a lipid mixture previous shown to enhance batch fermentation productivities.

  4. Ethanol from whey: continuous fermentation with cell recycle

    SciTech Connect

    Janssens, J.H.; Bernard, A.; Bailey, R.B.

    1984-01-01

    The production of ethanol from cheese whey lactose has been demonstrated using a single-stage continuous culture fermentation with 100% cell recycle. In a two-step process, an aerobic fed batch operation was used initially to allow biomass buildup in the absence of inhibitory ethanol concentrations. In the anaerobic ethanol-producing second step, a strain of Kluyveromyces fragilis selected on the basis of batch fermentation data had a maximum productivity of 7.1 g ethanol/L/h at a dilution rate of 0.15 h/sup -1/, while achieving the goal of zero residual sugar concentration. The fermentation productivity diminished when the feed sugar concentration exceeded 120 g/L despite the inclusion of a lipid mixture previous shown to enhance batch fermentation productivities.

  5. Alcohol from membrane processed concentrated cheese whey

    SciTech Connect

    Rajagopalan, K.; Kosikowskik, F.V.

    1982-01-01

    A fermentable whey substrate in the form of a high solids permeate was obtained by reconstituting spray-dried whey powder to 36% total solids followed by ultrafiltration to separate the protein. The high solids permeate was demineralized to permit rapid yeast growth. The final permeate with 24% lactose and at pH 4.8 gave high yields of EtOH rapidly upon inoculation with lactose-fermenting yeasts. One yeast species, Kluyveromyces fragilis NRRL Y 2415, yielded 108.8 g of EtOH/L, giving 84.3% of the theoretical maximum. Batch EtOH productivity was 3.2 g/L/h. The cost analysis of the ultrafiltration-fermentation process is highly favorable, if evaporation instead of the widely used reverse osmosis is employed for preconcentration of whey.

  6. Galactose-inducible expression systems in Candida maltosa using promoters of newly-isolated GAL1 and GAL10 genes.

    PubMed

    Park, S M; Ohkuma, M; Masuda, Y; Ohta, A; Takagi, M

    1997-01-01

    The GAL1 and GAL10 gene cluster encoding the enzymes of galactose utilization was isolated from an asporogenic yeast, Candida maltosa. The structure of the gene cluster in which both genes were divergently transcribed from the central promoter region resembled those of some other yeasts. The expression of both genes was strongly induced by galactose and repressed by glucose in the medium. Galactose-inducible expression vectors in C. maltosa were constructed on low- and high-copy number plasmids using the promoter regions of both genes. With these vectors and the beta-galactosidase gene from Kluyveromyces lactis as a reporter, galactose-inducible expression was confirmed. Homologous overexpression of members of the cytochrome P-450 gene family in C. maltosa was also successful by using a high-copy-number vector under the control of these promoters.

  7. Fuel alcohol from whey

    SciTech Connect

    Lyons, T.P.; Cunningham, J.D.

    1980-01-01

    According to the 'Report on alcohol fuels policy review', published in 1979 by the US Department of Energy, cheese whey had a very low net feedstock cost/gal of ethanol produced ($0.22) and the production potential in the USA is 90 million gal ethanol/yr. Three processes are described, i.e. the Milbrew whey fermentation process using Kluyveromyces fragilis with whey of 10-15% TS under sterile or non-sterile conditions and in batch, semi-continuous or continuous operation (primarily, designed for the production of single-cell protein), the continuous Carbery process in commercial operation in Ireland (DSA 42, 7856) and the Danish process (Dansk Gaerings-industri, Copenhagen) producing edible alcohol from whey permeate, and methane from distillation wastes for use as fuel for heating the distillation units.

  8. Relaxation of yeast mitochondrial functions after whole-genome duplication

    PubMed Central

    Jiang, Huifeng; Guan, Wenjun; Pinney, David; Wang, Wen; Gu, Zhenglong

    2008-01-01

    Mitochondria are essential for cellular energy production in most eukaryotic organisms. However, when glucose is abundant, yeast species that underwent whole-genome duplication (WGD) mostly conduct fermentation even under aerobic conditions, and most can survive without a functional mitochondrial genome. In this study, we show that the rate of evolution for the nuclear-encoded mitochondrial genes was greater in post-WGD species than pre-WGD species. Furthermore, codon usage bias was relaxed for these genes in post-WGD yeast species. The codon usage pattern and the distribution of a particular transcription regulatory element suggest that the change to an efficient aerobic fermentation lifestyle in this lineage might have emerged after WGD between the divergence of Kluyveromyces polysporus and Saccharomyces castellii from their common ancestor. This new energy production strategy could have led to the relaxation of mitochondrial function in the relevant yeast species. PMID:18669479

  9. Identification and characterization of antimicrobial activity in two yeast genera.

    PubMed Central

    Bilinski, C A; Innamorato, G; Stewart, G G

    1985-01-01

    A general screening test for the expression of antibacterial activity was performed on over 400 cultures belonging to 31 yeast genera. Of these cultures, only two, Kluyveromyces thermotolerans and Kloeckera apiculata, were found to produce zones of inhibition of bacterial growth on Diagnostic Sensitivity Test Agar medium supplemented with 0.002% methylene blue. Of nine bacteria used as test organisms, only Lactobacillus plantarum and Bacillus megaterium were inhibited. No antibacterial activity was evident against four gram-negative bacteria used in this study. Optimal activities were found to be expressed after yeasts were grown at pH 6. A requirement for cultivation in the presence of methylene blue added to culture media for the expression of apparent antibacterial activity was demonstrated. Images PMID:3937494

  10. Fermentation of lactose in direct-acid-set cottage cheese whey

    SciTech Connect

    Demott, B.J.; Draughon, F.A.; Herald, P.J.

    1981-01-01

    Kluyveromyces fragilis was more suitable than Candida pseudotropicalis or K. lactis for production of EtOH from whey. Direct-acid-set cottage cheese whey and the supernatant fluid resulting from heat treatment of the whey at 95 degrees for 20 min showed similar rates of fermentation when inoculated with K. fragilis. Inoculation rates of 10, 12 and 14 mL of active K. fragilis culture/100 mL of media were not different in rate of EtOH production. Samples incubated with K. fragilis at 35, 37, 40 and 42 degrees showed more rapid reduction in specific gravity than samples incubated at room temperature or 30 degrees. Lactose conversion in whey was 83% complete and in whey supernatant fluid, 77%.

  11. Emergence of Species-Specific Transporters During Evolution of the Hemiascomycete Phylum

    PubMed Central

    De Hertogh, Benoît; Hancy, Frédéric; Goffeau, André; Baret, Philippe V.

    2006-01-01

    We have traced the evolution patterns of 2480 transmembrane transporters from five complete genome sequences spanning the entire Hemiascomycete phylum: Saccharomyces cerevisiae, Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica. The use of nonambiguous functional and phylogenetic criteria derived from the TCDB classification system has allowed the identification within the Hemiascomycete phylum of 97 small phylogenetic transporter subfamilies comprising a total of 355 transporters submitted to four distinct evolution patterns named “ubiquitous,” “species specific,” “phylum gains and losses,” or “homoplasic.” This analysis identifies the transporters that contribute to the emergence of species during the evolution of the Hemiascomycete phylum and may aid in establishing novel phylogenetic criteria for species classification. PMID:16118182

  12. A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae

    PubMed Central

    Huang, Bo; Lu, Jian; Byström, Anders S.

    2008-01-01

    We recently showed that the γ-subunit of Kluyveromyces lactis killer toxin (γ-toxin) is a tRNA endonuclease that cleaves , , and 3′ of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The 5-methoxycarbonylmethyl (mcm5) side chain was important for efficient cleavage by γ-toxin, and defects in mcm5 side-chain synthesis correlated with resistance to γ-toxin. Based on this correlation, a genome-wide screen was performed to identify gene products involved in the formation of the mcm5 side chain. From a collection of 4826 homozygous diploid Saccharomyces cerevisiae strains, each with one nonessential gene deleted, 63 mutants resistant to Kluyveromyces lactis killer toxin were identified. Among these, eight were earlier identified to have a defect in formation of the mcm5 side chain. Analysis of the remaining mutants and other known γ-toxin resistant mutants revealed that sit4, kti14, and KTI5 mutants also have a defect in the formation of mcm5. A mutant lacking two of the Sit4-associated proteins, Sap185 and Sap190, displays the same modification defect as a sit4-null mutant. Interestingly, several mutants were found to be defective in the synthesis of the 2-thio (s2) group of the mcm5s2U nucleoside. In addition to earlier described mutants, formation of the s2 group was also abolished in urm1, uba4, and ncs2 mutants and decreased in the yor251c mutant. Like the absence of the mcm5 side chain, the lack of the s2 group renders less sensitive to γ-toxin, reinforcing the importance of the wobble nucleoside mcm5s2U for tRNA cleavage by γ-toxin. PMID:18755837

  13. Diversification of the kinetic properties of yeast NADP-glutamate-dehydrogenase isozymes proceeds independently of their evolutionary origin.

    PubMed

    Campero-Basaldua, Carlos; Quezada, Héctor; Riego-Ruíz, Lina; Márquez, Dariel; Rojas, Erendira; González, James; El-Hafidi, Mohammed; González, Alicia

    2016-11-19

    In the yeast Saccharomyces cerevisiae, the ScGDH1 and ScGDH3 encoded glutamate dehydrogenases (NADP-GDHs) catalyze the synthesis of glutamate from ammonium and α-ketoglutarate (α-KG). Previous kinetic characterization showed that these enzymes displayed different allosteric properties and respectively high or low rate of α-KG utilization. Accordingly, the coordinated action of ScGdh1 and ScGdh3, regulated balanced α-KG utilization for glutamate biosynthesis under either fermentative or respiratory conditions, safeguarding energy provision. Here, we have addressed the question of whether there is a correlation between the regulation and kinetic properties of the NADP-GDH isozymes present in S. cerevisiae (ScGdh1 and ScGdh3), Kluyveromyces lactis (KlGdh1), and Lachancea kluyveri (LkGdh1) and their evolutionary history. Our results show that the kinetic properties of K. lactis and L. kluyveri single NADP-GDHs are respectively similar to either ScGDH3 or ScGDH1, which arose from the whole genome duplication event of the S. cerevisiae lineage, although, KlGDH1 and LkGDH1 originated from a GDH clade, through an ancient interspecies hybridization event that preceded the divergence between the Saccharomyces clade and the one containing the genera Kluyveromyces, Lachancea, and Eremothecium. Thus, the kinetic properties which determine the NADP-GDHs capacity to utilize α-KG and synthesize glutamate do not correlate with their evolutionary origin.

  14. Domesticated transposase Kat1 and its fossil imprints induce sexual differentiation in yeast.

    PubMed

    Rajaei, Naghmeh; Chiruvella, Kishore K; Lin, Feng; Aström, Stefan U

    2014-10-28

    Transposable elements (TEs) have had a major influence on shaping both prokaryotic and eukaryotic genomes, largely through stochastic events following random or near-random insertions. In the mammalian immune system, the recombination activation genes1/2 (Rag1/2) recombinase has evolved from a transposase gene, demonstrating that TEs can be domesticated by the host. In this study, we uncovered a domesticated transposase, Kluyveromyces lactis hobo/Activator/Tam3 (hAT) transposase 1 (Kat1), operating at the fossil imprints of an ancient transposon, that catalyzes the differentiation of cell type. Kat1 induces mating-type switching from mating type a (MATa) to MATα in the yeast K. lactis. Kat1 activates switching by introducing two hairpin-capped DNA double-strand breaks (DSBs) in the MATa1-MATa2 intergenic region, as we demonstrate both in vivo and in vitro. The DSBs stimulate homologous recombination with the cryptic hidden MAT left alpha (HMLα) locus resulting in a switch of the cell type. The sites where Kat1 acts in the MATa locus most likely are ancient remnants of terminal inverted repeats from a long-lost TE. The KAT1 gene is annotated as a pseudogene because it contains two overlapping ORFs. We demonstrate that translation of full-length Kat1 requires a programmed -1 frameshift. The frameshift limited Kat1 activity, because restoring the zero frame causes switching to the MATα genotype. Kat1 also was transcriptionally activated by nutrient limitation via the transcription factor mating type switch 1 (Mts1). A phylogenetic analysis indicated that KAT1 was domesticated specifically in the Kluyveromyces clade of the budding yeasts. We conclude that Kat1 is a highly regulated transposase-derived endonuclease vital for sexual differentiation.

  15. Domesticated transposase Kat1 and its fossil imprints induce sexual differentiation in yeast

    PubMed Central

    Rajaei, Naghmeh; Chiruvella, Kishore K.; Lin, Feng; Åström, Stefan U.

    2014-01-01

    Transposable elements (TEs) have had a major influence on shaping both prokaryotic and eukaryotic genomes, largely through stochastic events following random or near-random insertions. In the mammalian immune system, the recombination activation genes1/2 (Rag1/2) recombinase has evolved from a transposase gene, demonstrating that TEs can be domesticated by the host. In this study, we uncovered a domesticated transposase, Kluyveromyces lactis hobo/Activator/Tam3 (hAT) transposase 1 (Kat1), operating at the fossil imprints of an ancient transposon, that catalyzes the differentiation of cell type. Kat1 induces mating-type switching from mating type a (MATa) to MATα in the yeast K. lactis. Kat1 activates switching by introducing two hairpin-capped DNA double-strand breaks (DSBs) in the MATa1–MATa2 intergenic region, as we demonstrate both in vivo and in vitro. The DSBs stimulate homologous recombination with the cryptic hidden MAT left alpha (HMLα) locus resulting in a switch of the cell type. The sites where Kat1 acts in the MATa locus most likely are ancient remnants of terminal inverted repeats from a long-lost TE. The KAT1 gene is annotated as a pseudogene because it contains two overlapping ORFs. We demonstrate that translation of full-length Kat1 requires a programmed −1 frameshift. The frameshift limited Kat1 activity, because restoring the zero frame causes switching to the MATα genotype. Kat1 also was transcriptionally activated by nutrient limitation via the transcription factor mating type switch 1 (Mts1). A phylogenetic analysis indicated that KAT1 was domesticated specifically in the Kluyveromyces clade of the budding yeasts. We conclude that Kat1 is a highly regulated transposase-derived endonuclease vital for sexual differentiation. PMID:25313032

  16. IBS-associated phylogenetic unbalances of the intestinal microbiota are not reverted by probiotic supplementation.

    PubMed

    Maccaferri, Simone; Candela, Marco; Turroni, Silvia; Centanni, Manuela; Severgnini, Marco; Consolandi, Clarissa; Cavina, Piero; Brigidi, Patrizia

    2012-01-01

    IBS is a prevalent functional gastrointestinal disorder, in which the microbiota has been demonstrated to play a role. An increasing number of studies have suggested how probiotics may alleviate IBS symptoms and several mechanisms of action have been proposed.   In the present study we characterized the intestinal microbiota of 19 subjects suffering from diagnosed IBS using a fully validated High Taxonomic Fingerprint Microbiota Array (HTF-Microbi.Array). We demonstrated that the IBS microbiota is different from that of healthy individuals due to an unbalance in a number of commensal species, with an increase in relative abundance of lactobacilli, B. cereus and B. clausii, bifidobacteria, Clostridium cluster IX and E. rectale, and a decrease in abundance of Bacteroides/Prevotella group and Veillonella genus. Additionally, we demonstrated that some bacterial groups of the human intestinal microbiota, recently defined as pathobionts, are increased in concentration in the IBS microbiota. Furthermore, we aimed at investigating if the daily administration of a novel probiotic yogurt containing B. animalis subsp lactis Bb12 and K. marxianus B0399, recently demonstrated to have beneficial effects in the management of IBS symptoms, could impact on the biostructure of IBS microbiota, modulating its composition to counteract putative dysbiosis found in IBS subjects. Notably, we demonstrated that the beneficial effects associated to the probiotic preparation are not related to significant modifications in the composition of the human intestinal microbiota.

  17. Recovery of residual soluble protein by two-step precipitation process with concomitant COD reduction from the yeast-cultivated cheese whey.

    PubMed

    Yadav, J S S; Yan, S; More, T T; Tyagi, R D; Surampalli, R Y

    2014-09-01

    The present study was conducted to recover the residual soluble protein after cultivation of yeast (K. marxianus) in cheese whey. Cheese whey continuous fermentation with cell recycle system was carried out at 40 °C and pH 3.5. The yeast biomass was separated from the fermented broth by centrifugation and residual soluble protein from fermented whey supernatant was precipitated by heat treatment (at 100 °C, pH 4.5 and 10 min incubation). The maximum soluble protein recovery up to 53 % was achieved at pH 4.5 with 54 % residual COD removal. However, gravity sedimentable precipitates were obtained at pH 3.5 with 47 % protein recovery. Therefore, the reactor (scale up) study was conducted at pH 3.5 with agitation, which resulted in 68 % of residual soluble protein recovery and simultaneously residual COD removal of 62 %. Further precipitation/coagulation of soluble protein was also evaluated using carboxymethylcellulose (CMC) and then two precipitation (thermal followed by CMC precipitation) processes were combined to increase the protein precipitation, which finally reached up to 81 % of total soluble protein recovery from the supernatant. This optimized process could be applied to recover the residual protein left after fermentation of cheese whey without centrifugation.

  18. Novel high-performance metagenome β-galactosidases for lactose hydrolysis in the dairy industry.

    PubMed

    Erich, Sarah; Kuschel, Beatrice; Schwarz, Thilo; Ewert, Jacob; Böhmer, Nico; Niehaus, Frank; Eck, Jürgen; Lutz-Wahl, Sabine; Stressler, Timo; Fischer, Lutz

    2015-09-20

    The industrially utilised β-galactosidases from Kluyveromyces spp. and Aspergillus spp. feature undesirable kinetic properties in praxis, such as an unsatisfactory lactose affinity (KM) and product inhibition (KI) by galactose. In this study, a metagenome library of about 1.3 million clones was investigated with a three-step activity-based screening strategy in order to find new β-galactosidases with more favourable kinetic properties. Six novel metagenome β-galactosidases (M1-M6) were found with an improved lactose hydrolysis performance in original milk when directly compared to the commercial β-galactosidase from Kluyveromyces lactis (GODO-YNL2). The best metagenome candidate, called "M1", was recombinantly produced in Escherichia coli BL21(DE3) in a bioreactor (volume 35 L), resulting in a total β-galactosidase M1 activity of about 1100 μkatoNPGal,37 °C L(-1). Since milk is a sensitive and complex medium, it has to be processed at 5-10 °C in the dairy industry. Therefore, the β-galactosidase M1 was tested at 8 °C in milk and possessed a good stability (t1/2=21.8 d), a desirably low apparent KM,lactose,8 °C value of 3.8±0.7 mM and a high apparent KI,galactose,8 °C value of 196.6±55.5 mM. A lactose hydrolysis process (milk, 40 nkatlactose mLmilk,8 °C(-1)) was conducted at a scale of 0.5L to compare the performance of M1 with the commercial β-galactosidase from K. lactis (GODO-YNL2). Lactose was completely (>99.99%) hydrolysed by M1 and to 99.6% (w/v) by K. lactis β-galactosidase after 25 h process time. Thus, M1 was able to achieve the limit of <100 mg lactose per litre milk, which is recommended for dairy products labelled as "lactose-free".

  19. Effect of magnesium cations on the activity and stability of β-galactosidases

    NASA Astrophysics Data System (ADS)

    Atyaksheva, L. F.; Pilipenko, O. S.; Poltorak, O. M.; Chukhrai, E. S.

    2007-07-01

    It was shown that the presence of magnesium cations in the reaction mixture increases, approximately twofold, the activity of bacterial Escherichia coli and yeast Kluyveromyces lactis β-galactosidases but does not affect the activity of bovine liver and fungous Penicillium canescens β-galactosidases. The catalytic constants for E. coli and yeast K. lactis β-galactosidases in the presence of 0.01 M and in the absence of Mg2+ cations were determined (490 and 220 s-1 and 59.8 and 37.4 s-1, respectively). It was shown that the Michaelis constants for these two enzymes are higher in the presence of Mg2+ cations, that the thermal stability of E. coli and K. Lactis β-galactosidases is higher in the presence of 0.01 M Mg2+, and that the effective rate constants of thermal inactivation of the enzymes are two-to eightfold lower, depending on conditions, in the presence of Mg2+ cations. The maximum stabilizing effect of magnesium cations was observed at weak alkaline pH values (7.5-8.5).

  20. Influencing fatty acid composition of yeasts by lanthanides.

    PubMed

    Kolouchova, Irena; Sigler, Karel; Zimola, Michal; Rezanka, Tomas; Matatkova, Olga; Masak, Jan

    2016-08-01

    The growth of microorganisms is affected by cultivation conditions, concentration of carbon and nitrogen sources and the presence of trace elements. One of the new possibilities of influencing the production of cell mass or lipids is the use of lanthanides. Lanthanides are biologically non-essential elements with wide applications in technology and industry and their concentration as environmental contaminants is therefore increasing. Although non-essential, lanthanides have been proposed (and even used) to produce beneficial effects in plants but their mechanisms of action are unclear. Recently, it was suggested that they may replace essential elements or operate as potent blockers of Ca(2+) channels. We tested the effect of low concentrations of lanthanides on traditional biotechnologically useful yeast species (Kluyveromyces polysporus, Saccharomyces cerevisiae, Torulospora delbrueckii), and species capable of high accumulation of lipids (Rhodotorula glutinis, Trichosporon cutaneum, Candida sp., Yarrowia lipolytica). Low concentrations of lanthanum and monazite were conducive to an increase in cell mass and lipids and also higher production of palmitoleic acid, commonly used in cosmetics and medicine, and ω6-linoleic acid which is a precursor of thromboxanes, prostaglandins and leucotrienes.

  1. Potential benefits of the application of yeast starters in table olive processing.

    PubMed

    Arroyo-López, Francisco N; Romero-Gil, Verónica; Bautista-Gallego, Joaquín; Rodríguez-Gómez, Francisco; Jiménez-Díaz, Rufino; García-García, Pedro; Querol, Amparo; Garrido-Fernández, Antonio

    2012-01-01

    Yeasts play an important role in the food and beverage industry, especially in products such as bread, wine, and beer, among many others. However, their use as a starter in table olive processing has not yet been studied in detail. The candidate yeast strains should be able to dominate fermentation, together with lactic acid bacteria, but should also provide a number of beneficial advantages. Technologically, yeasts should resist low pH and high salt concentrations, produce desirable aromas, improve lactic acid bacteria growth, and inhibit spoilage microorganisms. Nowadays, they are being considered as probiotic agents because many species are able to resist the passage through the gastrointestinal tract and show favorable effects on the host. In this way, yeasts may improve the health of consumers by means of the degradation of non-assimilated compounds (such as phytate complexes), a decrease in cholesterol levels, the production of vitamins and antioxidants, the inhibition of pathogens, an adhesion to intestinal cell line Caco-2, and the maintenance of epithelial barrier integrity. Many yeast species, usually found in table olive processing (Wickerhamomyces anomalus, Saccharomyces cerevisiae, Pichia membranifaciens, and Kluyveromyces lactis, among others), have exhibited some of these properties. Thus, the selection of the most appropriate strains to be used as starters in this fermented vegetable, alone or in combination with lactic acid bacteria, is a promising research line to develop in the near future.

  2. Lactose Hydrolysis in Milk and Dairy Whey Using Microbial β-Galactosidases

    PubMed Central

    Dutra Rosolen, Michele; Gennari, Adriano; Volpato, Giandra; Volken de Souza, Claucia Fernanda

    2015-01-01

    This work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial β-galactosidases of microbial origins. We used Aspergillus oryzae (at temperatures of 10 and 55°C) and Kluyveromyces lactis (at temperatures of 10 and 37°C) β-galactosidases, both in 3, 6, and 9 U/mL concentrations. In the temperature of 10°C, the K. lactis β-galactosidase enzyme is more efficient in the milk, cheese whey, and whey permeate lactose hydrolysis when compared to A. oryzae. However, in the enzyme reaction time and concentration conditions evaluated, 100% lactose hydrolysis was not reached using the K. lactis β-galactosidase. The total lactose hydrolysis in whey and permeate was obtained with the A. oryzae enzyme, when using its optimum temperature (55°C), at the end of a 12 h reaction, regardless of the enzyme concentration used. For the lactose present in milk, this result occurred in the concentrations of 6 and 9 U/mL, with the same time and temperature conditions. The studied parameters in the lactose enzymatic hydrolysis are critical for enabling the application of β-galactosidases in the food industry. PMID:26587283

  3. Lactose Hydrolysis in Milk and Dairy Whey Using Microbial β-Galactosidases.

    PubMed

    Dutra Rosolen, Michele; Gennari, Adriano; Volpato, Giandra; Volken de Souza, Claucia Fernanda

    2015-01-01

    This work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial β-galactosidases of microbial origins. We used Aspergillus oryzae (at temperatures of 10 and 55°C) and Kluyveromyces lactis (at temperatures of 10 and 37°C) β-galactosidases, both in 3, 6, and 9 U/mL concentrations. In the temperature of 10°C, the K. lactis β-galactosidase enzyme is more efficient in the milk, cheese whey, and whey permeate lactose hydrolysis when compared to A. oryzae. However, in the enzyme reaction time and concentration conditions evaluated, 100% lactose hydrolysis was not reached using the K. lactis β-galactosidase. The total lactose hydrolysis in whey and permeate was obtained with the A. oryzae enzyme, when using its optimum temperature (55°C), at the end of a 12 h reaction, regardless of the enzyme concentration used. For the lactose present in milk, this result occurred in the concentrations of 6 and 9 U/mL, with the same time and temperature conditions. The studied parameters in the lactose enzymatic hydrolysis are critical for enabling the application of β-galactosidases in the food industry.

  4. An Effective Big Data Supervised Imbalanced Classification Approach for Ortholog Detection in Related Yeast Species

    PubMed Central

    Galpert, Deborah; del Río, Sara; Herrera, Francisco; Ancede-Gallardo, Evys; Antunes, Agostinho; Agüero-Chapin, Guillermin

    2015-01-01

    Orthology detection requires more effective scaling algorithms. In this paper, a set of gene pair features based on similarity measures (alignment scores, sequence length, gene membership to conserved regions, and physicochemical profiles) are combined in a supervised pairwise ortholog detection approach to improve effectiveness considering low ortholog ratios in relation to the possible pairwise comparison between two genomes. In this scenario, big data supervised classifiers managing imbalance between ortholog and nonortholog pair classes allow for an effective scaling solution built from two genomes and extended to other genome pairs. The supervised approach was compared with RBH, RSD, and OMA algorithms by using the following yeast genome pairs: Saccharomyces cerevisiae-Kluyveromyces lactis, Saccharomyces cerevisiae-Candida glabrata, and Saccharomyces cerevisiae-Schizosaccharomyces pombe as benchmark datasets. Because of the large amount of imbalanced data, the building and testing of the supervised model were only possible by using big data supervised classifiers managing imbalance. Evaluation metrics taking low ortholog ratios into account were applied. From the effectiveness perspective, MapReduce Random Oversampling combined with Spark SVM outperformed RBH, RSD, and OMA, probably because of the consideration of gene pair features beyond alignment similarities combined with the advances in big data supervised classification. PMID:26605337

  5. Thermodynamic characterization of the Saccharomyces cerevisiae telomerase RNA pseudoknot domain in vitro.

    PubMed

    Liu, Fei; Kim, Yoora; Cruickshank, Charmion; Theimer, Carla A

    2012-05-01

    Recent structural and functional characterization of the pseudoknot in the Saccharomyces cerevisiae telomerase RNA (TLC1) has demonstrated that tertiary structure is present, similar to that previously described for the human and Kluyveromyces lactis telomerase RNAs. In order to biophysically characterize the identified pseudoknot secondary and tertiary structures, UV-monitored thermal denaturation experiments, nuclear magnetic resonance spectroscopy, and native gel electrophoresis were used to investigate various potential conformations in the pseudoknot domain in vitro, in the absence of the telomerase protein. Here, we demonstrate that alternative secondary structures are not mutually exclusive in the S. cerevisiae telomerase RNA, tertiary structure contributes 1.5 kcal mol(-1) to the stability of the pseudoknot (≈ half the stability observed for the human telomerase pseudoknot), and identify additional base pairs in the 3' pseudoknot stem near the helical junction. In addition, sequence conservation in an adjacent overlapping hairpin appears to prevent dimerization and alternative conformations in the context of the entire pseudoknot-containing region. Thus, this work provides a detailed in vitro characterization of the thermodynamic features of the S. cerevisiae TLC1 pseudoknot region for comparison with other telomerase RNA pseudoknots.

  6. Structure of yeast Argonaute with guide RNA

    SciTech Connect

    Nakanishi, Kotaro; Weinberg, David E.; Bartel, David P.; Patel, Dinshaw J.

    2012-06-26

    The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 {angstrom} crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded and processed by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1-8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2{prime}-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide-target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage.

  7. Genetic Modification of the Marine-Isolated Yeast Aureobasidium melanogenum P16 for Efficient Pullulan Production from Inulin.

    PubMed

    Ma, Zai-Chao; Liu, Nan-Nan; Chi, Zhe; Liu, Guang-Lei; Chi, Zhen-Ming

    2015-08-01

    In this study, in order to directly and efficiently convert inulin into pullulan, the INU1 gene from Kluyveromyces maximum KM was integrated into the genomic DNA and actively expressed in the high pullulan producer Aureobasidium melanogenum P16 isolated from the mangrove ecosystem. After the ability to produce pullulan from inulin by different transformants was examined, it was found that the recombinant strain EI36, one of the transformants, produced 40.92 U/ml of inulinase activity while its wild-type strain P16 only yielded 7.57 U/ml of inulinase activity. Most (99.27 %) of the inulinase produced by the recombinant strain EI36 was secreted into the culture. During the 10-l fermentation, 70.57 ± 1.3 g/l of pullulan in the fermented medium was attained from inulin (138.0 g/l) within 108 h, high inulinase activity (42.03 U/ml) was produced within 60 h, the added inulin was actively hydrolyzed by the secreted inulinase, and most of the reducing sugars were used by the recombinant strain EI36. This confirmed that the genetically engineered yeast of A. melanogenum strain P16 was suitable for direct pullulan production from inulin.

  8. Selection of non-Saccharomyces yeast strains for reducing alcohol levels in wine by sugar respiration.

    PubMed

    Quirós, Manuel; Rojas, Virginia; Gonzalez, Ramon; Morales, Pilar

    2014-07-02

    Respiration of sugars by non-Saccharomyces yeasts has been recently proposed for lowering alcohol levels in wine. Development of industrial fermentation processes based on such an approach requires, amongst other steps, the identification of yeast strains which are able to grow and respire under the relatively harsh conditions found in grape must. This work describes the characterization of a collection of non-Saccharomyces yeast strains in order to identify candidate yeast strains for this specific application. It involved the estimation of respiratory quotient (RQ) values under aerated conditions, at low pH and high sugar concentrations, calculation of yields of ethanol and other relevant metabolites, and characterization of growth responses to the main stress factors found during the first stages of alcoholic fermentation. Physiological features of some strains of Metschnikowia pulcherrima or two species of Kluyveromyces, suggest they are suitable for lowering ethanol yields by respiration. The unsuitability of Saccharomyces cerevisiae strains for this purpose was not due to ethanol yields (under aerated conditions they are low enough for a significant reduction in final ethanol content), but to the high acetic acid yields under these growth conditions. According to results from controlled aeration fermentations with one strain of M. pulcherrima, design of an aeration regime allowing for lowering ethanol yields though preserving grape must components from excessive oxidation, would be conceivable.

  9. One-step separation of β-galactosidase from β-lactoglobulin using water-in-oil microemulsions.

    PubMed

    Mazı, Bekir G; Hamamcı, Haluk; Dungan, Stephanie R

    2012-05-01

    Solubilisation of β-galactosidase from Kluyveromyces lactis in Aerosol-OT water-in-isooctane microemulsions was measured as a function of buffer type and concentration, pH, and protein concentration. For buffer concentrations above ∼40mM, the enzyme was largely excluded from the droplets. Based on these results, a one-step separation was developed. A protein-containing aqueous feed was injected into an AOT/isooctane solution, with the feed volume slightly in excess of the predicted water solubility. Impurity proteins were entrapped inside the microemulsion droplets that then formed in the organic phase, while the high MW target protein was excluded and entered a newly formed, excess aqueous phase. The separation of β-galactosidase from the test protein β-lactoglobulin was most complete at 100mM KCl salt concentration, where the droplets were large enough to carry β-lactoglobulin but too small for β-galactosidase. At 100mM [KCl], 92% of the total enzyme activity was recovered in a concentrated and virtually pure form.

  10. The composition of Camembert cheese-ripening cultures modulates both mycelial growth and appearance.

    PubMed

    Lessard, Marie-Hélène; Bélanger, Gaétan; St-Gelais, Daniel; Labrie, Steve

    2012-03-01

    The fungal microbiota of bloomy-rind cheeses, such as Camembert, forms a complex ecosystem that has not been well studied, and its monitoring during the ripening period remains a challenge. One limitation of enumerating yeasts and molds on traditional agar media is that hyphae are multicellular structures, and colonies on a petri dish rarely develop from single cells. In addition, fungi tend to rapidly invade agar surfaces, covering small yeast colonies and resulting in an underestimation of their number. In this study, we developed a real-time quantitative PCR (qPCR) method using TaqMan probes to quantify a mixed fungal community containing the most common dairy yeasts and molds: Penicillium camemberti, Geotrichum candidum, Debaryomyces hansenii, and Kluyveromyces lactis on soft-cheese model curds (SCMC). The qPCR method was optimized and validated on pure cultures and used to evaluate the growth dynamics of a ripening culture containing P. camemberti, G. candidum, and K. lactis on the surface of the SCMC during a 31-day ripening period. The results showed that P. camemberti and G. candidum quickly dominated the ecosystem, while K. lactis remained less abundant. When added to this ecosystem, D. hansenii completely inhibited the growth of K. lactis in addition to reducing the growth of the other fungi. This result was confirmed by the decrease in the mycelium biomass on SCMC. This study compares culture-dependent and qPCR methods to successfully quantify complex fungal microbiota on a model curd simulating Camembert-type cheese.

  11. In vivo continuous evolution of genes and pathways in yeast

    PubMed Central

    Crook, Nathan; Abatemarco, Joseph; Sun, Jie; Wagner, James M.; Schmitz, Alexander; Alper, Hal S.

    2016-01-01

    Directed evolution remains a powerful, highly generalizable approach for improving the performance of biological systems. However, implementations in eukaryotes rely either on in vitro diversity generation or limited mutational capacities. Here we synthetically optimize the retrotransposon Ty1 to enable in vivo generation of mutant libraries up to 1.6 × 107 l−1 per round, which is the highest of any in vivo mutational generation approach in yeast. We demonstrate this approach by using in vivo-generated libraries to evolve single enzymes, global transcriptional regulators and multi-gene pathways. When coupled to growth selection, this approach enables in vivo continuous evolution (ICE) of genes and pathways. Through a head-to-head comparison, we find that ICE libraries yield higher-performing variants faster than error-prone PCR-derived libraries. Finally, we demonstrate transferability of ICE to divergent yeasts, including Kluyveromyces lactis and alternative S. cerevisiae strains. Collectively, this work establishes a generic platform for rapid eukaryotic-directed evolution across an array of target cargo. PMID:27748457

  12. A novel killer protein from Pichia kluyveri isolated from an Algerian soil: purification and characterization of its in vitro activity against food and beverage spoilage yeasts.

    PubMed

    Labbani, Fatima-Zohra Kenza; Turchetti, Benedetta; Bennamoun, Leila; Dakhmouche, Scheherazad; Roberti, Rita; Corazzi, Lanfranco; Meraihi, Zahia; Buzzini, Pietro

    2015-04-01

    A novel killer protein (Pkkp) secreted by a Pichia kluyveri strain isolated from an Algerian soil was active against food and beverage spoilage yeasts of the genera Dekkera, Kluyveromyces, Pichia, Saccharomyces, Torulaspora, Wickerhamomyces and Zygosaccharomyces. After purification by gel filtration chromatography Pkkp revealed an apparent molecular mass of 54 kDa with SDS-PAGE. Minimum inhibitory concentrations (MICs) of purified Pkkp exhibited a high in vitro activity against Dekkera bruxellensis (MICs from 64,000- to 256,000-fold lower than that exhibited by potassium metabisulphite) and Saccharomyces cerevisiae (MICs from 32,000- to 64,000- fold lower than potassium sorbate). No in vitro synergistic interactions (calculated by FIC index - Σ FIC) were observed when Pkkp was used in combination with potassium metabisulphite, potassium sorbate, or ethanol. Pkkp exhibited a dose-response effect against D. bruxellensis and S. cerevisiae in a low-alcoholic drink and fruit juice, respectively. The results of the present study suggest that Pkkp could be proposed as a novel food-grade compound useful for the control of food and beverage spoilage yeasts.

  13. The Yeast Elongator Histone Acetylase Requires Sit4-dependent Dephosphorylation for Toxin-Target Capacity

    PubMed Central

    Jablonowski, Daniel; Fichtner, Lars; Stark, Michael J.R.; Schaffrath, Raffael

    2004-01-01

    Kluyveromyces lactis zymocin, a heterotrimeric toxin complex, imposes a G1 cell cycle block on Saccharomyces cerevisiae that requires the toxin-target (TOT) function of holo-Elongator, a six-subunit histone acetylase. Here, we demonstrate that Elongator is a phospho-complex. Phosphorylation of its largest subunit Tot1 (Elp1) is supported by Kti11, an Elongator-interactor essential for zymocin action. Tot1 dephosphorylation depends on the Sit4 phosphatase and its associators Sap185 and Sap190. Zymocin-resistant cells lacking or overproducing Elongator-associator Tot4 (Kti12), respectively, abolish or intensify Tot1 phosphorylation. Excess Sit4·Sap190 antagonizes the latter scenario to reinstate zymocin sensitivity in multicopy TOT4 cells, suggesting physical competition between Sit4 and Tot4. Consistently, Sit4 and Tot4 mutually oppose Tot1 de-/phosphorylation, which is dispensable for integrity of holo-Elongator but crucial for the TOT-dependent G1 block by zymocin. Moreover, Sit4, Tot4, and Tot1 cofractionate, Sit4 is nucleocytoplasmically localized, and sit4Δ-nuclei retain Tot4. Together with the findings that sit4Δ and totΔ cells phenocopy protection against zymocin and the ceramide-induced G1 block, Sit4 is functionally linked to Elongator in cell cycle events targetable by antizymotics. PMID:14718557

  14. Development of a novel integrated process for co-production of β-galactosidase and ethanol using lactose as substrate.

    PubMed

    You, Shengping; Zhang, Jianye; Yin, Qingdian; Qi, Wei; Su, Rongxin; He, Zhimin

    2017-04-01

    A novel integrated process was developed successfully for co-production of β-galactosidase and ethanol using lactose as substrate, containing fermentation (Kluyveromyces lactis), isolation, permeabilization (a new recycling process) and spray drying. Firstly, a new fed-batch strategy optimized co-produced β-galactosidase at 105.91U/mL and ethanol at 32.16mg/mL, 4.40-fold and 10.82-fold increase over the results from initial conditions, respectively. Then a new mathematic model for the recycling permeabilization was established successfully. As expected, the total cells sediment from isolation of the fed-batch culture was permeabilized completely by distilled ethanol from broth supernatant. More amazedly, the specific activity of β-galactosidase product by spray drying the permeabilized cells reached 2.61U/mg, meeting the demand of commercial products. Furthermore, the ethanol product at 33.8% (v/v) was obtained from the novel integrated process, which could be applied for various applications. To conclude, the novel integrated process might be a feasible strategy to scale up for industrialization.

  15. A novel pseudoknot element is essential for the action of a yeast telomerase.

    PubMed

    Tzfati, Yehuda; Knight, Zachary; Roy, Jagoree; Blackburn, Elizabeth H

    2003-07-15

    Telomerase contains an essential RNA, which includes the template sequence copied by the reverse transcription action of telomerase into telomeric DNA. Using phylogenetic comparison, we identified seven conserved sequences in telomerase RNAs from Kluyveromyces budding yeasts. We show that two of these sequences, CS3 and CS4, are essential for normal telomerase function and can base-pair to form a putative long-range pseudoknot. Disrupting this base-pairing was deleterious to cell growth, telomere maintenance, and telomerase activity. Restoration of the base-pairing potential alleviated these phenotypes. Mutating this pseudoknot caused a novel mode of shifting of the boundaries of the RNA template sequence copied by telomerase. A phylogenetically derived model of yeast TER structure indicates that these RNAs can form two alternative predicted core conformations of similar stability: one brings the CS3/CS4 pseudoknot spatially close to the template; in the other, CS3 and CS4 move apart and the conformation of the template is altered. We propose that such disruption of the pseudoknot, and potentially the predicted telomerase RNA conformation, affects polymerization to cause the observed shifts in template usage.

  16. Sporadic Distribution of Prion-Forming Ability of Sup35p from Yeasts and Fungi

    PubMed Central

    Edskes, Herman K.; Khamar, Hima J.; Winchester, Chia-Lin; Greenler, Alexandria J.; Zhou, Albert; McGlinchey, Ryan P.; Gorkovskiy, Anton; Wickner, Reed B.

    2014-01-01

    Sup35p of Saccharomyces cerevisiae can form the [PSI+] prion, an infectious amyloid in which the protein is largely inactive. The part of Sup35p that forms the amyloid is the region normally involved in control of mRNA turnover. The formation of [PSI+] by Sup35p’s from other yeasts has been interpreted to imply that the prion-forming ability of Sup35p is conserved in evolution, and thus of survival/fitness/evolutionary value to these organisms. We surveyed a larger number of yeast and fungal species by the same criteria as used previously and find that the Sup35p from many species cannot form prions. [PSI+] could be formed by the Sup35p from Candida albicans, Candida maltosa, Debaromyces hansenii, and Kluyveromyces lactis, but orders of magnitude less often than the S. cerevisiae Sup35p converts to the prion form. The Sup35s from Schizosaccharomyces pombe and Ashbya gossypii clearly do not form [PSI+]. We were also unable to detect [PSI+] formation by the Sup35ps from Aspergillus nidulans, Aspergillus fumigatus, Magnaporthe grisea, Ustilago maydis, or Cryptococcus neoformans. Each of two C. albicans SUP35 alleles can form [PSI+], but transmission from one to the other is partially blocked. These results suggest that the prion-forming ability of Sup35p is not a conserved trait, but is an occasional deleterious side effect of a protein domain conserved for another function. PMID:25081567

  17. Sporadic distribution of prion-forming ability of Sup35p from yeasts and fungi.

    PubMed

    Edskes, Herman K; Khamar, Hima J; Winchester, Chia-Lin; Greenler, Alexandria J; Zhou, Albert; McGlinchey, Ryan P; Gorkovskiy, Anton; Wickner, Reed B

    2014-10-01

    Sup35p of Saccharomyces cerevisiae can form the [PSI+] prion, an infectious amyloid in which the protein is largely inactive. The part of Sup35p that forms the amyloid is the region normally involved in control of mRNA turnover. The formation of [PSI+] by Sup35p's from other yeasts has been interpreted to imply that the prion-forming ability of Sup35p is conserved in evolution, and thus of survival/fitness/evolutionary value to these organisms. We surveyed a larger number of yeast and fungal species by the same criteria as used previously and find that the Sup35p from many species cannot form prions. [PSI+] could be formed by the Sup35p from Candida albicans, Candida maltosa, Debaromyces hansenii, and Kluyveromyces lactis, but orders of magnitude less often than the S. cerevisiae Sup35p converts to the prion form. The Sup35s from Schizosaccharomyces pombe and Ashbya gossypii clearly do not form [PSI+]. We were also unable to detect [PSI+] formation by the Sup35ps from Aspergillus nidulans, Aspergillus fumigatus, Magnaporthe grisea, Ustilago maydis, or Cryptococcus neoformans. Each of two C. albicans SUP35 alleles can form [PSI+], but transmission from one to the other is partially blocked. These results suggest that the prion-forming ability of Sup35p is not a conserved trait, but is an occasional deleterious side effect of a protein domain conserved for another function.

  18. The use of controlled microbial cenoses in producers' link to increase steady functioning of artificial ecosystems

    NASA Astrophysics Data System (ADS)

    Somova, Lydia; Mikheeva, Galina; Somova, Lydia

    The life support systems (LSS) for long-term missions are to use cycling-recycling systems, including biological recycling. Simple ecosystems include 3 links: producers (plants), consumers (man, animals) and reducers (microorganisms). Microorganisms are substantial component of every link of LSS. Higher plants are the traditional regenerator of air and producer of food. They should be used in many successive generations of their reproduction in LSS. Controlled microbiocenoses can increase productivity of producer's link and protect plants from infections. The goal of this work was development of methodological bases of formation of stable, controlled microbiocenoses, intended for increase of productivity of plants and for obtaining ecologically pure production of plants. Main results of our investigations: 1. Experimental microbiocenoses, has been produced in view of the developed methodology on the basis of natural association of microorganisms by long cultivation on specially developed medium. Dominating groups are bacteria of genera: Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium, Rhodopseudomonas and yeast of genera: Kluyveromyces, Saccharomyces, Torulopsis. 2. Optimal parameters of microbiocenosis cultivation (t, pH, light exposure, biogenic elements concentrations) were experimentally established. Conditions of cultivation on which domination of different groups of microbiocenosis have been found. 3. It was shown, that processing of seeds of wheat, oats, bulbs and plants Allium cepa L. (an onions) with microbial association raised energy of germination of seeds and bulbs and promoted the increase (on 20-30 %) of growth green biomass and root system of plants in comparison with the control. This work is supported by grant, Yenissey , 07-04-96806

  19. Advances in Gene Expression in Non-Conventional Yeasts

    NASA Astrophysics Data System (ADS)

    Nel, Sanet; Labuschagne, Michel; Albertyn, Jacobus

    Yeast has been a favoured lower eukaryotic system for the expression and production of recombinant proteins for both basic research and practical applications, and the demand for foreign-gene expression systems is increasing rapidly. Despite the vast amount of information on the molecular biology and physiology of Saccharomyces cerevisiae, which has consequently been the first choice as host system for recombinant protein production in the past, several limitations have been identified in this expression system. These limitations have recently been relieved by the development of expression systems in other yeast species known as ‘ non-conventional yeasts’ or ‘non-Saccharomyces ’ yeasts. With the increasing interest in the biotechnological applications of these yeasts in applied and fundamental studies and processes, the term ‘ non-conventional ’ yeast may well soon become redundant. As there is no universal expression system for heterologous protein production, it is necessary to recognize the merits and demerits of each system in order to make a right choice. This chapter will evaluate the competitive environment of non-conventional expression platforms represented by some of the best-known alternative yeasts systems including Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris and more recently, Arxula adeninivorans.

  20. From grape berries to wine: population dynamics of cultivable yeasts associated to "Nero di Troia" autochthonous grape cultivar.

    PubMed

    Garofalo, Carmela; Tristezza, Mariana; Grieco, Francesco; Spano, Giuseppe; Capozzi, Vittorio

    2016-04-01

    The aim of this work was to study the biodiversity of yeasts isolated from the autochthonous grape variety called "Uva di Troia", monitoring the natural diversity from the grape berries to wine during a vintage. Grapes were collected in vineyards from two different geographical areas and spontaneous alcoholic fermentations (AFs) were performed. Different restriction profiles of ITS-5.8S rDNA region, corresponding to Saccharomyces cerevisiae, Issatchenkia orientalis, Metschnikowia pulcherrima, Hanseniaspora uvarum, Candida zemplinina, Issatchenkia terricola, Kluyveromyces thermotolerans, Torulaspora delbrueckii, Metschnikowia chrysoperlae, Pichia fermentans, Hanseniaspora opuntiae and Hanseniaspora guilliermondii, were observed. The yeast occurrences varied significantly from both grape berries and grape juices, depending on the sampling location. Furthermore, samples collected at the end of AF revealed the great predominance of Saccharomyces cerevisiae, with a high intraspecific biodiversity. This is the first report on the population dynamics of 'cultivable' microbiota diversity of "Uva di Troia" cultivar from the grape to the corresponding wine ("Nero di Troia"), and more general for Southern Italian oenological productions, allowing us to provide the basis for an improved management of wine yeasts (with both non-Saccharomyces and Saccharomyces) for the production of typical wines with desired unique traits. A certain geographical-dependent variability has been reported, suggesting the need of local based formulation for autochthonous starter cultures, especially in the proportion of the different species/strains in the design of mixed microbial preparations.

  1. Signaling pathways and posttranslational modifications of tau in Alzheimer's disease: the humanization of yeast cells

    PubMed Central

    Heinisch, Jürgen J.; Brandt, Roland

    2016-01-01

    In the past decade, yeast have been frequently employed to study the molecular mechanisms of human neurodegenerative diseases, generally by means of heterologous expression of genes encoding the relevant hallmark proteins. However, it has become evident that substantial posttranslational modifications of many of these proteins are required for the development and progression of potentially disease relevant changes. This is exemplified by the neuronal tau proteins, which are critically involved in a class of neuro-degenerative diseases collectively called tauopathies and which includes Alz-heimer’s disease (AD) as its most common representative. In the course of the disease, tau changes its phosphorylation state and becomes hyperphosphory-lated, gets truncated by proteolytic cleavage, is subject to O-glycosylation, sumoylation, ubiquitinylation, acetylation and some other modifications. This poses the important question, which of these posttranslational modifications are naturally occurring in the yeast model or can be reconstituted by heterol-ogous gene expression. Here, we present an overview on common modifica-tions as they occur in tau during AD, summarize their potential relevance with respect to disease mechanisms and refer to the native yeast enzyme orthologs capable to perform these modifications. We will also discuss potential approaches to humanize yeast in order to create modification patterns resembling the situation in mammalian cells, which could enhance the value of Saccharomyces cerevisiae and Kluyveromyces lactis as disease models. PMID:28357346

  2. The binary response of the GAL/MEL genetic switch of Saccharomyces cerevisiae is critically dependent on Gal80p-Gal4p interaction.

    PubMed

    Das Adhikari, Akshay Kumar; Bhat, Paike Jayadeva

    2016-09-01

    Studies on the Saccharomyces cerevisiae GAL/MEL genetic switch have revealed that its bistability is dependent on ultrasensitivity that can be altered or abolished by disabling different combinations of nested feedback loops. In contrast, we have previously demonstrated that weakening of the interaction between Gal80p and Gal4p alone is sufficient to abolish the ultrasensitivity (Das Adhikari et al. 2014). Here, we demonstrate that altering the epistatic interaction between Gal80p and Gal4p also abolishes the bistability, and the switch response to galactose becomes graded instead of binary. However, the GAL/MEL switch of wild-type and epistatically altered strains responded in a graded fashion to melibiose. The properties of the epistatically altered strain resemble Kluyveromyces lactis, which separated from the Saccharomyces lineage 100 mya before whole-genome duplication (WGD). Based on the results reported here, we propose that epistatic interactions played a crucial role in the evolution of the fine regulation of S. cerevisiae GAL/MEL switch following WGD.

  3. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase.

    PubMed

    Irani, Seema; Yogesha, S D; Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L; Nie, Grace; Prescott, Nicholas A; Zhang, Yan Jessie

    2016-03-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.

  4. An ancient oxidoreductase making differential use of its cofactors.

    PubMed

    Blüher, Doreen; Reinhardt-Tews, Annekathrin; Hey, Martin; Lilie, Hauke; Golbik, Ralph; Breunig, Karin D; Anders, Alexander

    2014-07-01

    Abstract Many transcription factors contribute to cellular homeostasis by integrating multiple signals. Signaling via the yeast Gal80 protein, a negative regulator of the prototypic transcription activator Gal4, is primarily regulated by galactose. ScGal80 from Saccharomyces cerevisiae has been reported to bind NAD(P). Here, we show that the ability to bind these ligands is conserved in KlGal80, a Gal80 homolog from the distantly related yeast Kluyveromyces lactis. However, the homologs apparently have diverged with respect to response to the dinucleotide. Strikingly, ScGal80 binds NAD(P) and NAD(P)H with more than 50-fold higher affinity than KlGal80. In contrast to ScGal80, where NAD is neutral, NAD and NADP have a negative effect in KlGal80 on its interaction with a KlGal4-peptide in vitro. Swapping a loop in the NAD(P) binding Rossmann-fold of ScGal80 into KlGal80 increases the affinity for NAD(P) and has a significant impact on KlGal4 regulation in vivo. Apparently, dinucleotide binding allows coupling of the metabolic state of the cell to regulation of the GAL/LAC genes. The particular sequences involved in binding determine how exactly the metabolic state is sensed and integrated by Gal80 to regulate Gal4.

  5. Structure of the Kti11/Kti13 heterodimer and its double role in modifications of tRNA and eukaryotic elongation factor 2.

    PubMed

    Glatt, Sebastian; Zabel, Rene; Vonkova, Ivana; Kumar, Amit; Netz, Daili J; Pierik, Antonio J; Rybin, Vladimir; Lill, Roland; Gavin, Anne-Claude; Balbach, Jochen; Breunig, Karin D; Müller, Christoph W

    2015-01-06

    The small, highly conserved Kti11 alias Dph3 protein encoded by the Kluyveromyces lactis killer toxin insensitive gene KTI11/DPH3 is involved in the diphthamide modification of eukaryotic elongation factor 2 and, together with Kti13, in Elongator-dependent tRNA wobble base modifications, thereby affecting the speed and accuracy of protein biosynthesis through two distinct mechanisms. We have solved the crystal structures of Saccharomyces cerevisiae Kti13 and the Kti11/Kti13 heterodimer at 2.4 and 2.9 Å resolution, respectively, and validated interacting residues through mutational analysis in vitro and in vivo. We show that metal coordination by Kti11 and its heterodimerization with Kti13 are essential for both translational control mechanisms. Our structural and functional analyses identify Kti13 as an additional component of the diphthamide modification pathway and provide insight into the molecular mechanisms that allow the Kti11/Kti13 heterodimer to coregulate two consecutive steps in ribosomal protein synthesis.

  6. Isolation and identification of yeast flora from genital tract in healthy female camels (Camelus dromedarius).

    PubMed

    Shokri, Hojjatollah; Khosravi, Alireza; Sharifzadeh, Aghil; Tootian, Zahra

    2010-07-29

    Yeasts are commensal organisms found in the skin, genital and gastrointestinal tracts, and other mucosal sites in mammalians. The purposes of this study were to identify yeast flora and to determine the number of colony forming units (CFUs) in genital tract of healthy female dromedary camels, establishing their connection in both mated and unmated conditions. The samples were taken from different parts of genital tract including vestibule, vagina, cervix, uterine body, and uterine horns of 50 camels using sterilized cotton swabs. They were cultured onto Sabouraud glucose agar containing chloramphenicol and incubated at 30 degrees C for 7-10 days. A total of 454 yeast colonies were obtained from genital tract. Yeast isolates belonged to 8 genera: Candida (73.1%), Trichosporon (10.1%), Geotrichum (7.5%), Kluyveromyces (3.5%), Rhodotorula (2.4%), Aureobasidium (1.4%), Cryptococcus (1.1%) and Prototheca (0.8%). Among different Candida species, C. zeylanoides was the most common isolated species, representing significant difference with other Candida species (P<0.05). The mean number of yeasts found in the vestibule (46%) was significantly higher than the results obtained from other parts (P<0.05). In addition, the mean value of CFUs from unmated females (71.1%) was significantly higher than mated females (P<0.05). The results showed that C. zeylanoides was a common component of healthy camel females' genital mycoflora and the number of yeasts varied between mated and unmated females.

  7. Lst4, the yeast Fnip1/2 orthologue, is a DENN-family protein.

    PubMed

    Pacitto, Angela; Ascher, David B; Wong, Louise H; Blaszczyk, Beata K; Nookala, Ravi K; Zhang, Nianshu; Dokudovskaya, Svetlana; Levine, Tim P; Blundell, Tom L

    2015-12-01

    The folliculin/Fnip complex has been demonstrated to play a crucial role in the mechanisms underlying Birt-Hogg-Dubé (BHD) syndrome, a rare inherited cancer syndrome. Lst4 has been previously proposed to be the Fnip1/2 orthologue in yeast and therefore a member of the DENN family. In order to confirm this, we solved the crystal structure of the N-terminal region of Lst4 from Kluyveromyces lactis and show it contains a longin domain, the first domain of the full DENN module. Furthermore, we demonstrate that Lst4 through its DENN domain interacts with Lst7, the yeast folliculin orthologue. Like its human counterpart, the Lst7/Lst4 complex relocates to the vacuolar membrane in response to nutrient starvation, most notably in carbon starvation. Finally, we express and purify the recombinant Lst7/Lst4 complex and show that it exists as a 1 : 1 heterodimer in solution. This work confirms the membership of Lst4 and the Fnip proteins in the DENN family, and provides a basis for using the Lst7/Lst4 complex to understand the molecular function of folliculin and its role in the pathogenesis of BHD syndrome.

  8. Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae.

    PubMed

    Comitini, Francesca; Gobbi, Mirko; Domizio, Paola; Romani, Cristina; Lencioni, Livio; Mannazzu, Ilaria; Ciani, Maurizio

    2011-08-01

    Non-Saccharomyces yeasts are metabolically active during spontaneous and inoculated must fermentations, and by producing a plethora of by-products, they can contribute to the definition of the wine aroma. Thus, use of Saccharomyces and non-Saccharomyces yeasts as mixed starter cultures for inoculation of wine fermentations is of increasing interest for quality enhancement and improved complexity of wines. We initially characterized 34 non-Saccharomyces yeasts of the genera Candida, Lachancea (Kluyveromyces), Metschnikowia and Torulaspora, and evaluated their enological potential. This confirmed that non-Saccharomyces yeasts from wine-related environments represent a rich sink of unexplored biodiversity for the winemaking industry. From these, we selected four non-Saccharomyces yeasts to combine with starter cultures of Saccharomyces cerevisiae in mixed fermentation trials. The kinetics of growth and fermentation, and the analytical profiles of the wines produced indicate that these non-Saccharomyces strains can be used with S. cerevisiae starter cultures to increase polysaccharide, glycerol and volatile compound production, to reduce volatile acidity, and to increase or reduce the total acidity of the final wines, depending on yeast species and inoculum ratio used. The overall effects of the non-Saccharomyces yeasts on fermentation and wine quality were strictly dependent on the Saccharomyces/non-Saccharomyces inoculum ratio that mimicked the differences of fermentation conditions (natural or simultaneous inoculated fermentation).

  9. Spectroscopic studies, antimicrobial activities and crystal structures of N-(2-hydroxy-3-methoxybenzalidene)1-aminonaphthalene

    NASA Astrophysics Data System (ADS)

    Ünver, Hüseyin; Yıldız, Mustafa; Dülger, Başaran; Özgen, Özen; Kendi, Engin; Durlu, Tahsin Nuri

    2005-03-01

    Schiff base N-(2-hydroxy-3-methoxybenzalidene)1-aminonaphthalene has been synthesized from the reaction of 2-hydroxy-3-methoxybenzaldehyde with 1-aminonaphthalene. The compound were characterized by elemental analysis, FT-IR, 1H NMR, 13C NMR and UV-visible techniques. The UV-visible spectra of the Schiff base were studied in polar and nonpolar solvents in acidic and basic media. The structure of the compound has been examined cyrstallographically. There are two independent molecules in the asymmetric unit. It crystallizes in the monoclinic space group P21/c, with unit cell parameters: a=14, 602(2), b=5,800(1), c=16, 899(1) Å, V=1394.4(2) Å 3, Dx=1.321 g cm -3 and Z=4. The crystal structure was solved by direct methods and refined by full-matrix least squares to a find R=0.041 of for 1179 observed reflections. The title compound's antimicrobial activities also have been studied. The antimicrobial activities of the ligand has been screened in vitro against the organisms Escherichia coli ATCC 11230, Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae UC57, Micrococcus luteus La 2971, Proteus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 27853, Mycobacterium smegmatis CCM 2067, Bacillus cereus ATCC 7064 and Listeria monocytogenes ATCC 15313, the yeast cultures Candida albicans ATCC 10231, Kluyveromyces fragilis NRRL 2415, Rhodotorula rubra DSM 70403, Debaryomyces hansenii DSM 70238 and Hanseniaspora guilliermondii DSM 3432.

  10. Alpha3, a transposable element that promotes host sexual reproduction.

    PubMed

    Barsoum, Emad; Martinez, Paula; Aström, Stefan U

    2010-01-01

    Theoretical models predict that selfish DNA elements require host sex to persist in a population. Therefore, a transposon that induces sex would strongly favor its own spread. We demonstrate that a protein homologous to transposases, called alpha3, was essential for mating type switch in Kluyveromyces lactis. Mutational analysis showed that amino acids conserved among transposases were essential for its function. During switching, sequences in the 5' and 3' flanking regions of the alpha3 gene were joined, forming a DNA circle, showing that alpha3 mobilized from the genome. The sequences encompassing the alpha3 gene circle junctions in the mating type alpha (MATalpha) locus were essential for switching from MATalpha to MATa, suggesting that alpha3 mobilization was a coupled event. Switching also required a DNA-binding protein, Mating type switch 1 (Mts1), whose binding sites in MATalpha were important. Expression of Mts1 was repressed in MATa/MATalpha diploids and by nutrients, limiting switching to haploids in low-nutrient conditions. A hairpin-capped DNA double-strand break (DSB) was observed in the MATa locus in mre11 mutant strains, indicating that mating type switch was induced by MAT-specific DSBs. This study provides empirical evidence for selfish DNA promoting host sexual reproduction by mediating mating type switch.

  11. Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching

    PubMed Central

    Chiruvella, Kishore K.; Rajaei, Naghmeh; Jonna, Venkateswara Rao; Hofer, Anders; Åström, Stefan U.

    2016-01-01

    Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MATα). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons. PMID:26902909

  12. Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching.

    PubMed

    Chiruvella, Kishore K; Rajaei, Naghmeh; Jonna, Venkateswara Rao; Hofer, Anders; Åström, Stefan U

    2016-02-23

    Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MATα). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons.

  13. α3, a transposable element that promotes host sexual reproduction

    PubMed Central

    Barsoum, Emad; Martinez, Paula; Åström, Stefan U.

    2010-01-01

    Theoretical models predict that selfish DNA elements require host sex to persist in a population. Therefore, a transposon that induces sex would strongly favor its own spread. We demonstrate that a protein homologous to transposases, called α3, was essential for mating type switch in Kluyveromyces lactis. Mutational analysis showed that amino acids conserved among transposases were essential for its function. During switching, sequences in the 5′ and 3′ flanking regions of the α3 gene were joined, forming a DNA circle, showing that α3 mobilized from the genome. The sequences encompassing the α3 gene circle junctions in the mating type α (MATα) locus were essential for switching from MATα to MATa, suggesting that α3 mobilization was a coupled event. Switching also required a DNA-binding protein, Mating type switch 1 (Mts1), whose binding sites in MATα were important. Expression of Mts1 was repressed in MATa/MATα diploids and by nutrients, limiting switching to haploids in low-nutrient conditions. A hairpin-capped DNA double-strand break (DSB) was observed in the MATa locus in mre11 mutant strains, indicating that mating type switch was induced by MAT-specific DSBs. This study provides empirical evidence for selfish DNA promoting host sexual reproduction by mediating mating type switch. PMID:20008928

  14. Phenylpropanoid Glycoside Analogues: Enzymatic Synthesis, Antioxidant Activity and Theoretical Study of Their Free Radical Scavenger Mechanism

    PubMed Central

    López-Munguía, Agustín; Hernández-Romero, Yanet; Pedraza-Chaverri, José; Miranda-Molina, Alfonso; Regla, Ignacio; Martínez, Ana; Castillo, Edmundo

    2011-01-01

    Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols. PMID:21674039

  15. Exploring the yeast biodiversity of green table olive industrial fermentations for technological applications.

    PubMed

    Bautista-Gallego, J; Rodríguez-Gómez, F; Barrio, E; Querol, A; Garrido-Fernández, A; Arroyo-López, F N

    2011-05-27

    In recent years, there has been an increasing interest in identifying and characterizing the yeast populations associated with diverse types of table olive elaborations because of the many desirable technological properties of these microorganisms. In this work, a total of 199 yeast isolates were directly obtained from industrial green table olive fermentations and genetically identified by means of a RFLP analysis of the 5.8S-ITS region and sequencing of the D1/D2 domains of the 26S rDNA gene. Candida diddensiae, Saccharomyces cerevisiae and Pichia membranifaciens were the most abundant yeast species isolated from directly brined Aloreña olives, while for Gordal and Manzanilla cultivars they were Candida tropicalis, Pichia galeiformis and Wickerhamomyces anomalus. In the case of Gordal and Manzanilla green olives processed according to the Spanish style, the predominant yeasts were Debaryomyces etchellsii, C. tropicalis, P. galeiformis and Kluyveromyces lactis. Biochemical activities of technological interest were then qualitatively determined for isolates belonging to all yeast species. This preliminary screening identified two isolates of W. anomalus with interesting properties, such as a strong β-glucosidase and esterase activity, and a moderate catalase and lipolytic activity, which were also confirmed by quantitative assays. The results obtained in this survey show the potential use that some yeast species could have as starters, alone or in combination with lactic acid bacteria, during olive processing.

  16. Biodiversity of non-Saccharomyces yeasts in distilleries of the La Mancha region (Spain).

    PubMed

    Úbeda, Juan; Maldonado Gil, María; Chiva, Rosana; Guillamón, José M; Briones, Ana

    2014-06-01

    The aim of this pioneering study was to determine the biodiversity of non-Saccharomyces yeasts in ancient distilleries located in the La Mancha region, which is the principal area for the production of bioethanol and grape-based distillates in Spain. In this study, the yeast populations that were present during the process of extraction of alcohol and residual sugars from the byproducts of vinification, such as piquettes, pomace and grape skins, were studied. Non-Saccharomyces yeasts were identified by PCR-RFLP analysis of the 5.8S rRNA genes and, when necessary, by sequencing the D1/D2 domain of the 26S and/or 5.8S rRNA genes. Further, fermentation and the assimilation of carbon compounds were studied, to identify potential industrial applications. Phylogenetic trees and heat-maps were constructed for the genetic and phenotypic traits, respectively. Twenty yeast species belonging to eight genera were identified (Torulaspora, Candida, Zygosaccharomyces, Pichia, Hanseniaspora, Kluyveromyces, Ogataea and Saccharomycodes). Pichia galeiformis, Candida lactis-condensi, Hanseniaspora osmophila and Torulaspora delbrueckii were the most abundant species and were found principally in sweet and fermented piquettes.

  17. Utilization of concentrated cheese whey for the production of protein concentrate fuel alcohol and alcoholic beverages

    SciTech Connect

    Krishnamurti, R.

    1983-01-01

    The objective of this investigation was to recover the major components of whey and to develop food applications for their incorporation/conversion into acceptable products of commercial value. Reconstituted dried sweet whey with 36% solids was ultrafiltered to yield a protein concentrate (WPC) and a permeate containing 24% lactose and 3.7% ash. Orange juice fortified up to 2.07% and chocolate milks fortified up to 5.88% total protein levels with WPC containing 45% total protein were acceptable to about 90% of a panel of 24 individuals. Fermentation of demineralized permeate at 30/sup 0/C with Kluyveromyces fragilis NRRL Y 2415 adapted to 24% lactose levels, led to 13.7% (v/v) ethanol in the medium at the end of 34 hours. Batch productivity was 3.2 gms. ethanol per liter per hour and conversion efficiency was 84.26% of the theoretical maximum. Alcoholic fermentation of permeate and subsequent distillation produced compounds with desirable aroma characters in such products. This study suggests that there is potential for the production of protein fortified non-alcoholic products and alcoholic beverages of commercial value from whey, thus providing a cost effective solution to the whey utilization problem.

  18. Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: a strategy to enhance acidity and improve the overall quality of wine.

    PubMed

    Gobbi, Mirko; Comitini, Francesca; Domizio, Paola; Romani, Cristina; Lencioni, Livio; Mannazzu, Ilaria; Ciani, Maurizio

    2013-04-01

    In the last few years there is an increasing interest on the use of mixed fermentation of Saccharomyces and non-Saccharomyces wine yeasts for inoculation of wine fermentations to enhance the quality and improve complexity of wines. In the present work Lachancea (Kluyveromyces) thermotolerans and Saccharomyces cerevisiae were evaluated in simultaneous and sequential fermentation with the aim to enhance acidity and improve the quality of wine. In this specific pairing of yeast strains in mixed fermentations (S. cerevisiae EC1118 and L. thermotolerans 101), this non-Saccharomyces yeast showed a high level of competitiveness. Nevertheless the S. cerevisiae strain dominated the fermentation over the spontaneous S. cerevisiae strains also under the industrial fermentation conditions. The different condition tested (modalities of inoculum, temperature of fermentation, different grape juice) influenced the specific interactions and the fermentation behaviour of the co-culture of S. cerevisiae and L. thermotolerans. However, some metabolic behaviours such as pH reduction and enhancement of 2-phenylethanol and glycerol, were shown here under all of the conditions tested. The specific chemical profiles of these wines were confirmed by the sensory analysis test, which expressed these results at the tasting level as significant increases in the spicy notes and in terms of total acidity increases.

  19. Optimizing alcohol production from whey using computer technology.

    PubMed

    Zertuche, L; Zall, R R

    1985-04-01

    This study was undertaken with the major goal of optimizing the ethanol production from whey using computer technology. To reach this goal, a mathematical model that would describe the fermentation and that could be used for the optimization was developed. Kluyveromyces fragilis was the microorganism used to ferment the lactose in the whey into ethanol. Preliminary studies showed that K. fragilis produced about 90% of the theoretical ethanol yield when grown in whey-complemented media. However, when this yeast is grown in nonsupplemented whey media, it does not produce more than 32% of that yield. Comparative batch fermentations of lactose and whey-complemented media showed that whey possibly contains enhancing components for yeast growth and ethanol production. To obtain the mathematical model, the one-to-one effect of the process variables (lactose and yeast extract concentrations, air flowrate, pH, and dilution rate) on the ethanol production were first investigated. Experiments on the pH effect showed that a decrease in pH from 7 to 4 produced an increase in ethanol concentration from 16.5 to 26.5 g/L (50 g/L initial lactose). The results obtained from modeling of the continuous fermentation using the previously listed variables showed that air flowrate, pH, and dilution rate were the process variables that most influence the production of ethanol.

  20. A Conserved Motif within RAP1 Plays Diversified Roles in Telomere Protection and Regulation in Different Organisms

    PubMed Central

    Chen, Yong; Rai, Rekha; Zhou, Zi-Ren; Kanoh, Junko; Ribeyre, Cyril; Yang, Yuting; Zheng, Hong; Damay, Pascal; Wang, Feng; Tsujii, Hisayo; Hiraoka, Yasushi; Shore, David; Hu, Hong-Yu; Chang, Sandy; Lei, Ming

    2013-01-01

    Repressor activator protein 1 (RAP1) is the most highly conserved telomere protein. It is involved in protecting chromosome ends in fission yeast, promoting gene silencing in Saccharomyces cerevisiae while in Kluyveromyces lactis it is required to repress homology directed recombination (HDR) at telomeres. Since mammalian RAP1 requires TRF2 for stable expression, its role in telomere function has remained obscure. To understand how RAP1 plays such diverse functions at telomeres, we solved the crystal or solution structures of the C-terminal RCT domains of RAP1 from multiple organisms in complex with their respective protein-binding partners. Our comparative structural analysis establishes the RCT domain of RAP1 as an evolutionarily conserved protein-protein interaction module. In mammalian and fission yeast cells, this module interacts with TRF2 and Taz1, respectively, targeting RAP1 to chromosome ends for telomere end protection. While RAP1 repress NHEJ at fission yeast telomeres, at mammalian telomeres it is required to repress HDR. In contrast, S. cerevisiae RAP1 utilizes the RCT domain to recruit Sir3 to telomeres to mediate gene silencing. Together, our results reveal that depending on the organism, the evolutionarily conserved RAP1 RCT motif plays diverse functional roles at telomeres. PMID:21217703

  1. Dxo1 is a new type of eukaryotic enzyme with both decapping and 5'-3' exoribonuclease activity.

    PubMed

    Chang, Jeong Ho; Jiao, Xinfu; Chiba, Kunitoshi; Oh, ChanSeok; Martin, Charles E; Kiledjian, Megerditch; Tong, Liang

    2012-10-01

    Recent studies showed that Rai1 is a crucial component of the mRNA 5'-end-capping quality-control mechanism in yeast. The yeast genome encodes a weak homolog of Rai1, Ydr370C, but little is known about this protein. Here we report the crystal structures of Ydr370C from Kluyveromyces lactis and the first biochemical and functional studies on this protein. The overall structure of Ydr370C is similar to Rai1. Ydr370C has robust decapping activity on RNAs with unmethylated caps, but it has no detectable pyrophosphohydrolase activity. Unexpectedly, Ydr370C also possesses distributive, 5'-3' exoRNase activity, and we propose the name Dxo1 for this new eukaryotic enzyme with both decapping and exonuclease activities. Studies of yeast in which both Dxo1 and Rai1 are disrupted reveal that mRNAs with incomplete caps are produced even under normal growth conditions, in sharp contrast to current understanding of the capping process.

  2. A triple helix within a pseudoknot is a conserved and essential element of telomerase RNA.

    PubMed

    Shefer, Kinneret; Brown, Yogev; Gorkovoy, Valentin; Nussbaum, Tamar; Ulyanov, Nikolai B; Tzfati, Yehuda

    2007-03-01

    Telomerase copies a short template within its integral telomerase RNA onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Telomerase action extends the proliferative potential of cells, and thus it is implicated in cancer and aging. Nontemplate regions of telomerase RNA are also crucial for telomerase function. However, they are highly divergent in sequence among species, and their roles are largely unclear. Using in silico three-dimensional modeling, constrained by mutational analysis, we propose a three-dimensional model for a pseudoknot in telomerase RNA of the budding yeast Kluyveromyces lactis. Interestingly, this structure includes a U-A.U major-groove triple helix. We confirmed the triple-helix formation in vitro using oligoribonucleotides and showed that it is essential for telomerase function in vivo. While triplex-disrupting mutations abolished telomerase function, triple compensatory mutations that formed pH-dependent G-C.C(+) triples restored the pseudoknot structure in a pH-dependent manner and partly restored telomerase function in vivo. In addition, we identified a novel type of triple helix that is formed by G-C.U triples, which also partly restored the pseudoknot structure and function. We propose that this unusual structure, so far found only in telomerase RNA, provides an essential and conserved telomerase-specific function.

  3. Crystal structure analysis of c4763, a uropathogenic Escherichia coli-specific protein.

    PubMed

    Kim, Hun; Choi, Jongkeun; Kim, Doyoun; Kim, Kyeong Kyu

    2015-08-01

    Urinary-tract infections (UTIs), which are some of the most common infectious diseases in humans, can cause sepsis and death without proper treatment. Therefore, it is necessary to understand their pathogenicity for proper diagnosis and therapeutics. Uropathogenic Escherichia coli, the major causative agents of UTIs, contain several genes that are absent in nonpathogenic strains and are therefore considered to be relevant to UTI pathogenicity. c4763 is one of the uropathogenic E. coli-specific proteins, but its function is unknown. To investigate the function of c4763 and its possible role in UTI pathogenicity, its crystal structure was determined at a resolution of 1.45 Å by a multiple-wavelength anomalous diffraction method. c4763 is a homodimer with 129 residues in one subunit that contains a GGCT-like domain with five α-helices and seven β-strands. c4763 shows structural similarity to the C-terminal domain of allophanate hydrolase from Kluyveromyces lactis, which is involved in the degradation of urea. These results suggest that c4763 might be involved in the utilization of urea, which is necessary for bacterial survival in the urinary tract. Further biochemical and physiological investigation will elucidate its functional relevance in UTIs.

  4. DNA Damage Responses Are Induced by tRNA Anticodon Nucleases and Hygromycin B.

    PubMed

    Wemhoff, Sabrina; Klassen, Roland; Beetz, Anja; Meinhardt, Friedhelm

    2016-01-01

    Previous studies revealed DNA damage to occur during the toxic action of PaT, a fungal anticodon ribonuclease (ACNase) targeting the translation machinery via tRNA cleavage. Here, we demonstrate that other translational stressors induce DNA damage-like responses in yeast as well: not only zymocin, another ACNase from the dairy yeast Kluyveromyces lactis, but also translational antibiotics, most pronouncedly hygromycin B (HygB). Specifically, DNA repair mechanisms BER (base excision repair), HR (homologous recombination) and PRR (post replication repair) provided protection, whereas NHEJ (non-homologous end-joining) aggravated toxicity of all translational inhibitors. Analysis of specific BER mutants disclosed a strong HygB, zymocin and PaT protective effect of the endonucleases acting on apurinic sites. In cells defective in AP endonucleases, inactivation of the DNA glycosylase Ung1 increased tolerance to ACNases and HygB. In addition, Mag1 specifically contributes to the repair of DNA lesions caused by HygB. Consistent with DNA damage provoked by translation inhibitors, mutation frequencies were elevated upon exposure to both fungal ACNases and HygB. Since polymerase ζ contributed to toxicity in all instances, error-prone lesion-bypass probably accounts for the mutagenic effects. The finding that differently acting inhibitors of protein biosynthesis induce alike cellular responses in DNA repair mutants is novel and suggests the dependency of genome stability on translational fidelity.

  5. Yeast DNA plasmids.

    PubMed

    Gunge, N

    1983-01-01

    The study of yeast DNA plasmids has been initiated with the discovery of the 2-micron DNA in Saccharomyces cerevisiae. This multiple copy plasmid, organized into chromatin structure in vivo, probably exists in the nucleus and provides a good system to obtain information on eukaryotic DNA replication. Yeast transformation with the 2-micron DNA or artificially constructed chimeric plasmids had contributed significantly to the study of the molecular biology of yeast and eukaryotes, allowing the isolation and characterization of various genes, ars, centromeres, and telomeres, and also serving as a tool to study the expression of various heterologous genes. Encouraged by these fruitful results, new yeast plasmids have been screened among phylogenetically distant yeasts. The linear DNA plasmids (pGKl1 and pGKl2) from Kluyveromyces lactis are the first case of yeast plasmids associated with biological function (killer phenotype). This plasmid system would be ideal as a model to study the structure and function of eukaryotic linear chromosomes. The extracellular secretion of protein toxin suggests the plasmids to be an excellent candidate for a secretion vector. The importance of yeasts as suitable materials for the study of eukaryotic cell biology would be much enhanced by the advent of new transformation systems with diverse host yeasts of genetically and phylogenetically distinct properties.

  6. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase

    PubMed Central

    Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L.; Nie, Grace; Prescott, Nicholas A.; Zhang, Yan Jessie

    2016-01-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families. PMID:26933063

  7. Fermentation of deproteinized cheese whey powder solutions to ethanol by engineered Saccharomyces cerevisiae: effect of supplementation with corn steep liquor and repeated-batch operation with biomass recycling by flocculation.

    PubMed

    Silva, Ana Carina; Guimarães, Pedro M R; Teixeira, José A; Domingues, Lucília

    2010-09-01

    The lactose in cheese whey is an interesting substrate for the production of bulk commodities such as bio-ethanol, due to the large amounts of whey surplus generated globally. In this work, we studied the performance of a recombinant Saccharomyces cerevisiae strain expressing the lactose permease and intracellular beta-galactosidase from Kluyveromyces lactis in fermentations of deproteinized concentrated cheese whey powder solutions. Supplementation with 10 g/l of corn steep liquor significantly enhanced whey fermentation, resulting in the production of 7.4% (v/v) ethanol from 150 g/l initial lactose in shake-flask fermentations, with a corresponding productivity of 1.2 g/l/h. The flocculation capacity of the yeast strain enabled stable operation of a repeated-batch process in a 5.5-l air-lift bioreactor, with simple biomass recycling by sedimentation of the yeast flocs. During five consecutive batches, the average ethanol productivity was 0.65 g/l/h and ethanol accumulated up to 8% (v/v) with lactose-to-ethanol conversion yields over 80% of theoretical. Yeast viability (>97%) and plasmid retention (>84%) remained high throughout the operation, demonstrating the stability and robustness of the strain. In addition, the easy and inexpensive recycle of the yeast biomass for repeated utilization makes this process economically attractive for industrial implementation.

  8. Isolation of fungi from bats of the Amazon basin.

    PubMed Central

    Mok, W Y; Luizão, R C; Barreto da Silva, M do S

    1982-01-01

    A total of 2,886 bats captured in the Amazon Basin of Brazil were processed for the isolation of fungi. From the livers, spleens, and lungs of 155 bats (5.4%), 186 fungal isolates of the genera Candida (123 isolates), Trichosporon (26 isolates), Torulopsis (25 isolates), Kluyveromyces (11 isolates), and Geotrichum (1 isolate) were recovered. Seven known pathogenic species were present: Candida parapsilosis, C. guilliermondii, C. albicans, C. stellatoidea, C. pseudotropicalis, Trichosporon beigelii, and Torulopsis glabrata. Twenty-three culture-positive bats showed identical fungal colonization in multiple organs or mixed colonization in a single organ. The fungal isolation rates for individual bat species varied from 1 fungus per 87 bats to 3 fungi per 13 bats, and the mycoflora diversity for members of an individual fungus-bearing bat species varied from 16 fungi per 40 bats to 7 fungi per 6 bats. Of the 38 fungal species isolated, 36 had not been previously described as in vivo bat isolates. Of the 27 culture-positive bat species, 21 had not been previously described as mammalian hosts for medically or nonmedically important fungi. PMID:6890326

  9. Survival of cheese-ripening microorganisms in a dynamic simulator of the gastrointestinal tract.

    PubMed

    Adouard, Nadège; Magne, Laurent; Cattenoz, Thomas; Guillemin, Hervé; Foligné, Benoît; Picque, Daniel; Bonnarme, Pascal

    2016-02-01

    A mixture of nine microorganisms (six bacteria and three yeasts) from the microflora of surface-ripened cheeses were subjected to in vitro digestive stress in a three-compartment "dynamic gastrointestinal digester" (DIDGI). We studied the microorganisms (i) grown separately in culture medium only (ii) grown separately in culture medium and then mixed, (iii) grown separately in culture medium and then included in a rennet gel and (iv) grown together in smear-ripened cheese. The yeasts Geotrichum candidum, Kluyveromyces lactis and Debaryomyces hansenii, were strongly resistant to the whole DIDGI process (with a drop in viable cell counts of less than <1 log CFU mL(-1)) and there were no significant differences between lab cultures and cheese-grown cultures. Ripening bacteria such as Hafnia alvei survived gastric stress less well when grown in cheese (with no viable cells after 90 min of exposure of the cheese matrix, compared with 6 CFU mL(-1) in lab cultures). The ability of Corynebacterium casei and Staphylococcus equorum to withstand digestive stress was similar for cheese and pure culture conditions. When grow in a cheese matrix, Brevibacterium aurantiacum and Arthrobacter arilaitensis were clearly more sensitive to the overall digestive process than when grown in pure cultures. Lactococcus lactis displayed poorer survival in gastric and duodenal compartments when it had been grown in cheese. In vivo experiments in BALB/c mice agreed with the DIDGI experiments and confirmed the latter's reliability.

  10. A differential medium for the enumeration of the spoilage yeast Zygosaccharomyces bailii in wine.

    PubMed

    Schuller, D; Côrte-Real, M; Leão, C

    2000-11-01

    A collection of yeasts, isolated mostly from spoiled wines, was used in order to develop a differential medium for Zygosaccharomyces bailii. The 118 selected strains of 21 species differed in their origin and resistance to preservatives and belonged to the genera Pichia, Torulaspora, Dekkera, Debaryomyces, Saccharomycodes, Issatchenkia, Kluyveromyces, Kloeckera, Lodderomyces, Schizosaccharomyces, Rhodotorula, Saccharomyces, and Zygosaccharomyces. The design of the culture medium was based on the different ability of the various yeast species to grow in a mineral medium with glucose and formic acid (mixed-substrate medium) as the only carbon and energy sources and supplemented with an acid-base indicator. By manipulating the concentration of the acid and the sugar it was possible to select conditions where only Z. bailii strains gave rise to alkalinization, associated with a color change of the medium (positive response). The final composition of the mixed medium was adjusted as a compromise between the percentage of recovery and selectivity for Z. bailii. This was accomplished by the use of pure or mixed cultures of the yeast strains and applying the membrane filtration methodology. The microbiological analysis of two samples of contaminated Vinho Verde showed that the new medium can be considered as a differential medium to distinguish Z. bailii from other contaminating yeasts, having potential application in the microbiological control of wines and probably other beverages and foods.

  11. In vivo characterization of protein uptake by yeast cell envelope: single cell AFM imaging and μ-tip-enhanced Raman scattering study.

    PubMed

    Naumenko, Denys; Snitka, Valentinas; Serviene, Elena; Bruzaite, Ingrida; Snopok, Boris

    2013-09-21

    Direct detection of biological transformations of single living cells in vivo has been performed by the advanced combination of local topographic imaging by Atomic Force Microscopy (AFM) and label-free sub-surface chemical characterization using new μ-Tip-Enhanced Raman Spectroscopy (μ-TERS). The enhancing mechanism for μ-TERS tips with micrometre range radius differs significantly to that of the conventional tapered structures terminated by a sharp apex and conditioned by the effects of propagating instead of localizing surface plasmon resonance phenomena. Sub-wavelength light confinement in the form of a nonradiative evanescent wave near the tip surface with penetration depth in the sub-micrometre range opens the way for monitoring of subsurface processes near or within the cell wall, inaccessible by other methods. The efficiency of the approach has been demonstrated by the analysis of the cell envelope of genetically modified (by glucose dehydrogenase (GDH) gene bearing Kluyveromyces lactis toxin signal sequence) yeast cells enriched by GDH protein. The presence of trans-membrane fragments in GDH together with the tendency to form active dimers and tetramers causes the accumulation of the proteins within the periplasmic space. These results demonstrate that the advanced combination of AFM imaging and subsurface chemical characterization by the novel μ-TERS technique provides a new analytical tool for the investigation of single living cells in vivo.

  12. A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae.

    PubMed

    Piirainen, Mari A; Boer, Harry; de Ruijter, Jorg C; Frey, Alexander D

    2016-04-01

    N-glycosylation is an important feature of therapeutic and other industrially relevant proteins, and engineering of the N-glycosylation pathway provides opportunities for developing alternative, non-mammalian glycoprotein expression systems. Among yeasts, Saccharomyces cerevisiae is the most established host organism used in therapeutic protein production and therefore an interesting host for glycoengineering. In this work, we present further improvements in the humanization of the N-glycans in a recently developed S. cerevisiae strain. In this strain, a tailored trimannosyl lipid-linked oligosaccharide is formed and transferred to the protein, followed by complex-type glycan formation by Golgi apparatus-targeted human N-acetylglucosamine transferases. We improved the glycan pattern of the glycoengineered strain both in terms of glycoform homogeneity and the efficiency of complex-type glycosylation. Most of the interfering structures present in the glycoengineered strain were eliminated by deletion of the MNN1 gene. The relative abundance of the complex-type target glycan was increased by the expression of a UDP-N-acetylglucosamine transporter from Kluyveromyces lactis, indicating that the import of UDP-N-acetylglucosamine into the Golgi apparatus is a limiting factor for efficient complex-type N-glycosylation in S. cerevisiae. By a combination of the MNN1 deletion and the expression of a UDP-N-acetylglucosamine transporter, a strain forming complex-type glycans with a significantly improved homogeneity was obtained. Our results represent a further step towards obtaining humanized glycoproteins with a high homogeneity in S. cerevisiae.

  13. Improving ethanol productivity by modification of glycolytic redox factor generation in glycerol-3-phosphate dehydrogenase mutants of an industrial ethanol yeast.

    PubMed

    Guo, Zhong-peng; Zhang, Liang; Ding, Zhong-yang; Wang, Zheng-Xiang; Shi, Gui-Yang

    2011-08-01

    The GPD2 gene, encoding NAD(+)-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol-producing strain of Saccharomyces cerevisiae, was deleted. And then, either the non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus, or the NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Kluyveromyces lactis, was expressed in the obtained mutant AG2 deletion of GPD2, respectively. The resultant recombinant strain AG2A (gpd2Δ P (PGK)-gapN) exhibited a 48.70 ± 0.34% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.60 ± 0.12% (relative to the amount of substrate consumed) increase in ethanol yield, while recombinant AG2B (gpd2Δ P (PGK)-GAPDH) exhibited a 52.90 ± 0.45% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.34 ± 0.15% (relative to the amount of substrate consumed) increase in ethanol yield compared with the wild-type strain. More importantly, the maximum specific growth rates (μ (max)) of the recombinant AG2A and AG2B were higher than that of the mutant gpd2Δ and were indistinguishable compared with the wild-type strain in anaerobic batch fermentations. The results indicated that the redox imbalance of the mutant could be partially solved by expressing the heterologous genes.

  14. Interaction between transcriptional activator protein LAC9 and negative regulatory protein GAL80.

    PubMed Central

    Salmeron, J M; Langdon, S D; Johnston, S A

    1989-01-01

    In Saccharomyces cerevisiae, transcriptional activation mediated by the GAL4 regulatory protein is repressed in the absence of galactose by the binding of the GAL80 protein, an interaction that requires the carboxy-terminal 28 amino acids of GAL4. The homolog of GAL4 from Kluyveromyces lactis, LAC9, activates transcription in S. cerevisiae and is highly similar to GAL4 in its carboxyl terminus but is not repressed by wild-type levels of GAL80 protein. Here we show that GAL80 does repress LAC9-activated transcription in S. cerevisiae if overproduced. We sought to determine the molecular basis for the difference in the responses of the LAC9 and GAL4 proteins to GAL80. Our results indicate that this difference is due primarily to the fact that under wild-type conditions, the level of LAC9 protein in S. cerevisiae is much higher than that of GAL4, which suggests that LAC9 escapes GAL80-mediated repression by titration of GAL80 protein in vivo. The difference in response to GAL80 is not due to amino acid sequence differences between the LAC9 and GAL4 carboxyl termini. We discuss the implications of these results for the mechanism of galactose metabolism regulation in S. cerevisiae and K. lactis. Images PMID:2550790

  15. Occurrence and diversity of yeasts in the mangrove ecosystems in fujian, guangdong and hainan provinces of china.

    PubMed

    Chi, Zhen-Ming; Liu, Tian-Tian; Chi, Zhe; Liu, Guang-Lei; Wang, Zhi-Peng

    2012-09-01

    Mangrove wetland is a unique ecosystem and has rich bioresources. In this article, the roots, stems, branches, leaves, barks, fruits, and flowers from 12 species of the mangrove plants and six species of the accompanying mangrove plants, seawater and sediments in mangrove ecosystems in China were used as sources for isolation of yeasts. A total of 269 yeasts strains were obtained from the samples. The results of routine identification and phylogenetic analysis showed that they belonged to 22 genera and 45 species. Of all the 269 strains, Candida spp. was predominant with the proportion of 44.61%, followed by Kluyveromyces spp. (8.55%), Pichia spp. (7.44%), Kodamaea ohmeri (5.58%), Issatchenkia spp. (4.83%) and Debaryomyces hansenii (4.46%). We also found that strains N02-2.3 and ST3-1Y3 belonged to the undescribed species of Pichia sp. and Trichosporon sp. respectively while strain HN-12 was not related to any known yeast strains. This means that different yeast strains of Candida spp. especially C. tropicalis were widely distributed in the mangrove ecosystems and may have an important role in the mangrove ecosystems. The results also showed that some of them may have potential applications.

  16. Dynamics of telomeric DNA turnover in yeast.

    PubMed Central

    McEachern, Michael J; Underwood, Dana Hager; Blackburn, Elizabeth H

    2002-01-01

    Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover. PMID:11805045

  17. X-Ray Structure of the Amidase Domain of AtzF, the Allophanate Hydrolase from the Cyanuric Acid-Mineralizing Multienzyme Complex

    PubMed Central

    Balotra, Sahil; Newman, Janet; Cowieson, Nathan P.; French, Nigel G.; Campbell, Peter M.; Briggs, Lyndall J.; Warden, Andrew C.; Easton, Christopher J.; Peat, Thomas S.

    2014-01-01

    The activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s-triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis, when purified enzyme was tested in vitro. However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product. PMID:25362066

  18. The identification of antibacterial compounds for the development of enhanced media for the detection of foodborne fungi.

    PubMed

    Tournas, V H; Kohn, J S; Katsoudas, E J

    2007-08-15

    In an effort to identify a more suitable antibiotic for utilization in mycological media, 12 food borne fungal species from various genera including Alternaria alternata, Aspergillus flavus, A. niger, Eurotium chevalieri, Fusarium moniliforme, Penicillium sp., Rhizopus stolonifer, Saccharomyces cerevisiae, Candida tropicalis, Geotrichum candidum, Rhodotorula glutinis and Kluyveromyces thermotolerans along with 21 chloramphenicol-resistant bacterial isolates from fresh produce and ATCC cultures of Pseudomonas aeruginosa, P. fluorescens, E. coli, Pectobacterium carotovorum, Bacillus cereus and Staphylococcus spp. were tested for their abilities to grow on dichloran rose bengal agar containing various levels of gentamicin, chlortetracycline or chloramphenicol. Results indicated that all fungal isolates except for Rh. glutinis and R. stolonifer grew well on all media tested. Rh. glutinis did not grow on media containing gentamicin whereas R. stolonifer produced very restricted or no growth on these media. All bacterial isolates from fresh produce, P. aeruginosa (ATCC 27853) and P. fluorescens (ATCC BAA-477) grew well at 100, 125 and 150 mg chloramphenicol/liter medium, but they did not grow on media containing chlortetracycline (100, 125, or 150 mg/L) or gentamicin (15, 25, or 35 mg/L). P. aeruginosa (ATCC 10145) grew well on media containing chloramphenicol or gentamicin, but not in the presence of chlortetracycline. P. carotovorum, E. coli, B. cereus and Staphylococcus spp. did not grow on any of the selective media tested.

  19. Diversity of Saccharomyces and non-Saccharomyces yeasts in three red grape varieties cultured in the Serranía de Ronda (Spain) vine-growing region.

    PubMed

    Clavijo, Almudena; Calderón, Isabel L; Paneque, Patricia

    2010-10-15

    For the first time, an ecological survey of wine yeasts present in grapes growing in two vineyards located in the region of "Serranía de Ronda" (Málaga, southern Spain) has been carried out. During the 2006 and 2007 vintages, grapes from different varieties were aseptically collected and allowed to ferment spontaneously in the laboratory. From a total of 1586 colonies isolated from microvinifications, 1281 were identified according to ITS polymorphisms and their identity confirmed by sequencing of the D1/D2 region of 26S rDNA. Most of the isolates (84%) corresponded to thirteen different non-Saccharomyces species with Kluyveromyces thermotolerans, Hanseniaspora guilliermondii, Hanseniaspora uvarum and Issatchenkia orientalis accounting for 42.7% of the total. Mitochondrial DNA restriction analysis from the Saccharomyces cerevisiae isolates revealed a low diversity since only eleven different profiles were found, nine of them corresponding to local strains and two to commercial ones that had been used in different campaigns and that very likely were disseminated from the winery to the adjacent vineyard. A different distribution of strains was found in the three grape varieties studied.

  20. Recognition of the centromere-specific histone Cse4 by the chaperone Scm3

    SciTech Connect

    Cho, Uhn-Soo; Harrison, Stephen C.

    2011-09-20

    A specialized nucleosome is a component of all eukaryotic kinetochores. The core of this nucleosome contains a centromere-specific histone, CENP-A (the Cse4 gene product in budding yeast), instead of the usual H3. Assembly of a centromeric nucleosome depends on a specific chaperone, called Scm3 in yeast and HJURP in higher eukaryotes. We describe here the structure of a complex formed by an N-terminal fragment of Scm3 with the histone-fold domains of Cse4, and H4, all prepared as recombinant proteins derived from the budding yeast Kluyveromyces lactis. The contacts of Scm3 with Cse4 explain its selectivity for the centromere-specific histone; key residues at the interface are conserved in HJURP, indicating a common mechanism for centromeric-histone deposition. We also report the structure of a (Cse4 : H4)2 heterotetramer; comparison with the structure of the Scm3:Cse4:H4 complex shows that tetramer formation and DNA-binding require displacement of Scm3 from the nucleosome core. The two structures together suggest that specific contacts between the chaperone and Cse4, rather than an altered overall structure of the nucleosome core, determine the selective presence of Cse4 at centromeres.

  1. Design and analysis of an immobilized cell reactor with simultaneous product separation: ethanol from whey lactose

    SciTech Connect

    Dale, M.C.

    1983-01-01

    The simultaneous separation of volatile fermentation products from product inhibited fermentations can increase the productivity of a bioreactor by reducing the product concentration in the bioreactor. In this work, a simultaneous tubular reactor separator is developed in which the volatile product is removed from the reacting broth by an inert gas phase. The immobilized cell reactor separator (ICRS) consists of two column reactors: a cocurrent enriching column followed by an countercurrent stripping column. The application of the ICRS concept to the ethanol from whey lactose fermentation was investigated using the yeast Kluyveromyces fragilis 2415. An equilibrium stage model of the ICRS was developed including a surface renewal term for an adsorbed monolayer of reacting cells. This model demonstrated the effect of important operational parameters including temperature, pressure, and gas flow rates. Experimental results using yeast adsorbed to 1/4'' ceramic saddles were somewhat unsatisfactory but very high productivities, cell densities, and separation efficiency were obtained using an absorbant column packing in a gas continuous operating mode.

  2. Identification of base and backbone contacts used for DNA sequence recognition and high-affinity binding by LAC9, a transcription activator containing a C6 zinc finger

    SciTech Connect

    Halvorsen, Yuan-Di C.; Nandabalan, K.; Dickson, R.C. )

    1991-04-01

    The LAC9 protein of Kluyveromyces lactis is a transcriptional regulator of genes in the lactose-galactose regulon. To regulate transcription, LAC9 must bind to 17-bp upstream activator sequences (UASs) located in front of each target gene. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae, and the two proteins must bind DNA in a very similar manner. In this paper the authors show that high-affinity, sequence-specific binding by LAC9 dimers is mediated primarily by 3 bp at each end of the UAS. In addition, at least one half of the UAS must have a GC or CG base pair at position 1 for high-affinity binding; LAC9k binds preferentially to the half containing the GC base pair. Hydroxyl radical footprinting shows that a LAC9 dimer binds an unusually broad region on one face of the DNA helix. Because of the data, they suggest that LAC9 contacts positions 6, 7, and 8, both plus and minus, of the UAS, which are separated by more than one turn of the DNA helix, and twists part way around the DNA, thus protecting the broad region of the minor groove between the major-groove contacts.

  3. Construction of lactose-assimilating and high-ethanol-producing yeasts by protoplast fusion

    SciTech Connect

    Farahnak, F.; Seki, T.; Ryu, D.D.Y.; Ogrydziak, D.

    1986-02-01

    The availability of a yeast strain which is capable of fermenting lactose and at the same time is tolerant to high concentrations of ethanol would be useful for the production of ethanol from lactose. Kluyveromyces fragilis is capable of fermenting lactose, but it is not as tolerant as Saccharomyces cerevisiae to high concentrations of ethanol. In this study, the authors have used the protoplast fusion technique to construct hybrids between auxotrophic strains of S. cerevisiae having high ethanol tolerance and an auxotrophic strain of lactose-fermenting K. fragilis isolated by ethyl methanesulfonate mutagenesis. The fusants obtained were prototrophic and capable of assimilating lactose and producing ethanol in excess of 13% (vol/vol). The complementation frequency of fusion was about 0.7%. Formation of fusants was confirmed by the increased amount of chromosomal DNA per cell. Fusants contained 8 x 10/sup -9/ to 16 x 10/sup -8/ ..mu..g of DNA per cell as compared with about 4 x 10/sup -8/ ..mu..g of DNA per cell for the parental strains, suggesting that multiple fusions had taken place.

  4. Gene order in a 10 275 bp fragment of Yarrowia lipolytica, including adjacent YlURA5 and YlSEC65 genes conserved in four yeast species.

    PubMed

    Sánchez, M; Domínguez, A

    2001-06-30

    We have determined the sequence of a 10275 bp DNA segment of Yarrowia lipolytica located on chromosome VI. The sequence contains six complete open reading frames (ORFs) longer than 100 amino acids and two more partial ORFs at both ends. Two of the ORFs encode for the well-characterized genes YlURA5 (orotate phosphoribosyltransferase) and YlSEC65 (encoding a subunit of the signal recognition particle). These two genes show an identical organization-located on opposite strands and in opposite orientations-in four yeast species: Saccharomyces cerevisiae, Kluyveromyces lactis, Candida albicans and Y. lipolytica. One ORF and the two partial ORFs code for putative proteins showing significant homology with proteins from other organisms. YlVI-108w (partial) and YlVI-103w show 39% and 54% identity, respectively, with YDR430c and YHR088w from S. cerevisiae. YlVI-102c (partial) shows significant homology with a matrix protein, lustrin A from Haliotis rufescens, and with the PGRS subfamily (Gly-rich proteins) of Mycobacterium tuberculosis. The three remaining ORFs show weak or non-significant homology with previously sequenced genes. The nucleotide sequence has been submitted to the EMBL database under Accession No. AI006754.

  5. Structural and Biochemical Studies of the 5 - gt 3 Exoribonuclease Xrn1

    SciTech Connect

    J Chang; S Xiang; K Xiang; J Manley; L Tong

    2011-12-31

    The 5' {yields} 3' exoribonucleases (XRNs) have important functions in transcription, RNA metabolism and RNA interference. The structure of Rat1 (also known as Xrn2) showed that the two highly conserved regions of XRNs form a single, large domain that defines the active site of the enzyme. Xrn1 has a 510-residue segment after the conserved regions that is required for activity but is absent from Rat1/Xrn2. Here we report the crystal structures of Kluyveromyces lactis Xrn1 (residues 1-1,245, E178Q mutant), alone and in complex with a Mn{sup 2+} ion in the active site. The 510-residue segment contains four domains (D1-D4), located far from the active site. Our mutagenesis and biochemical studies show that their functional importance results from their ability to stabilize the conformation of the N-terminal segment of Xrn1. These domains might also constitute a platform that interacts with protein partners of Xrn1.

  6. Capsicum annuum L. trypsin inhibitor as a template scaffold for new drug development against pathogenic yeast.

    PubMed

    Ribeiro, Suzanna F F; Silva, Marciele S; Da Cunha, Maura; Carvalho, André O; Dias, Germana B; Rabelo, Guilherme; Mello, Erica O; Santa-Catarina, Claudete; Rodrigues, Rosana; Gomes, Valdirene M

    2012-03-01

    A 6,000 Da peptide, named CaTI, was isolated from Capsicum annuum L. seeds and showed potent inhibitory activity against trypsin and chymotrypsin. The aim of this study was to determine the effect of CaTI on Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Kluyveromyces marxiannus cells. We observed that CaTI inhibited the growth of S. cerevisiae, K. marxiannus as well as C. albicans and induced cellular agglomeration and the release of cytoplasmic content. No effect on growth was observed in C. tropicalis but morphological changes were noted. In the spot assay, different degrees of sensitivity were shown among the strains and concentrations tested. Scanning electron microscopy showed that S. cerevisiae, K. marxiannus and C. albicans, in the presence of CaTI, exhibited morphological alterations, such as the formation of pseudohyphae, cellular aggregates and elongated forms. We also show that CaTI induces the generation of nitric oxide and interferes in a dose-dependent manner with glucose-stimulated acidification of the medium mediated by H(+)-ATPase of S. cerevisiae cells.

  7. Synthesis, characterization and antimicrobial activity of Fe(II), Zn(II), Cd(II) and Hg(II) complexes with 2,6-bis(benzimidazol-2-yl) pyridine ligand.

    PubMed

    Aghatabay, Naz M; Neshat, A; Karabiyik, T; Somer, M; Haciu, D; Dülger, B

    2007-02-01

    2,6-Bis(benzimidazol-2-yl)pyridine (L) ligand and complexes [M(L)Cl(2)] and [Fe(L)(2)](ClO(4))(2) (M=Zn, Cd, Hg) have been synthesized. The geometries of the [M(L)Cl(2)] complexes were derived from theoretical calculation in DGauss/DFT level (DZVP basis set) on CACHE. The central M(II) ion is penta-coordinated and surrounded by N(3)Cl(2) environment, adopting a distorted trigonal bipyramidal geometry. The ligand is tridentate, via three nitrogen atoms to metal centre and two chloride ions lie on each side of the distorted benzimidazole ring. In the [Fe(L)(2)](ClO(4))(2) complex, the central Fe(II) ion is surrounded by two (3N) units, adopting a octahedral geometry. The elemental analysis, molecular conductivity, FT-Raman, FT-IR (mid-, far-IR), (1)H, and (13)C NMR were reported. The antimicrobial activities of the free ligand, its hydrochloride salt, and the complexes were evaluated using the disk diffusion method in dimethyl sulfoxide (DMSO) as well as the minimal inhibitory concentration (MIC) dilution method, against 10 bacteria and the results compared with that for gentamycin. Antifungal activities were reported for Candida albicans, Kluyveromyces fragilis, Rhodotorula rubra, Debaryomyces hansenii, Hanseniaspora guilliermondii, and the results were referenced against nystatin, ketaconazole, and clotrimazole antifungal agents. In most cases, the compounds tested showed broad-spectrum (Gram positive and Gram negative bacteria) activities that were either more effective than or as potent as the references. The binding of two most biologically effective compounds of zinc and mercury to calf thymus DNA has also been investigated by absorption spectra.

  8. Regulatory Genes Controlling Fatty Acid Catabolism and Peroxisomal Functions in the Filamentous Fungus Aspergillus nidulans†

    PubMed Central

    Hynes, Michael J.; Murray, Sandra L.; Duncan, Anna; Khew, Gillian S.; Davis, Meryl A.

    2006-01-01

    The catabolism of fatty acids is important in the lifestyle of many fungi, including plant and animal pathogens. This has been investigated in Aspergillus nidulans, which can grow on acetate and fatty acids as sources of carbon, resulting in the production of acetyl coenzyme A (CoA). Acetyl-CoA is metabolized via the glyoxalate bypass, located in peroxisomes, enabling gluconeogenesis. Acetate induction of enzymes specific for acetate utilization as well as glyoxalate bypass enzymes is via the Zn2-Cys6 binuclear cluster activator FacB. However, enzymes of the glyoxalate bypass as well as fatty acid beta-oxidation and peroxisomal proteins are also inducible by fatty acids. We have isolated mutants that cannot grow on fatty acids. Two of the corresponding genes, farA and farB, encode two highly conserved families of related Zn2-Cys6 binuclear proteins present in filamentous ascomycetes, including plant pathogens. A single ortholog is found in the yeasts Candida albicans, Debaryomyces hansenii, and Yarrowia lipolytica, but not in the Ashbya, Kluyveromyces, Saccharomyces lineage. Northern blot analysis has shown that deletion of the farA gene eliminates induction of a number of genes by both short- and long-chain fatty acids, while deletion of the farB gene eliminates short-chain induction. An identical core 6-bp in vitro binding site for each protein has been identified in genes encoding glyoxalate bypass, beta-oxidation, and peroxisomal functions. This sequence is overrepresented in the 5′ region of genes predicted to be fatty acid induced in other filamentous ascomycetes, C. albicans, D. hansenii, and Y. lipolytica, but not in the corresponding genes in Saccharomyces cerevisiae. PMID:16682457

  9. Yeast Community Structures and Dynamics in Healthy and Botrytis-Affected Grape Must Fermentations▿

    PubMed Central

    Nisiotou, Aspasia A.; Spiropoulos, Apostolos E.; Nychas, George-John E.

    2007-01-01

    Indigenous yeast population dynamics during the fermentation of healthy and Botrytis-affected grape juice samples from two regions in Greece, Attica and Arcadia, were surveyed. Species diversity was evaluated by using restriction fragment length polymorphism and sequence analyses of the 5.8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of cultivable yeasts. Community-level profiles were also obtained by direct analysis of fermenting samples through denaturing gradient gel electrophoresis of 26S rDNA amplicons. Both approaches revealed structural divergences in yeast communities between samples of different sanitary states or geographical origins. In all cases, Botrytis infection severely perturbed the bioprocess of fermentation by dramatically altering species heterogeneity and succession during the time course. At the beginning and middle of fermentations, Botrytis-affected samples possessed higher levels of biodiversity than their healthy counterparts, being enriched with fermentative and/or spoilage species, such as Zygosaccharomyces bailii and Issatchenkia spp. or Kluyveromyces dobzhanskii and Kazachstania sp. populations that have not been reported before for wine fermentations. Importantly, Botrytis-affected samples exposed discrete final species dominance. Selection was not species specific, and two different populations, i.e., Saccharomyces cerevisiae in samples from Arcadia and Z. bailii in samples from Attica, could be recovered at the end of Botrytis-affected fermentations. The governing of wine fermentations by Z. bailii is reported for the first time and could elucidate the origins and role of this particular spoilage microbe for the wine industry. This is the first survey to compare healthy and Botrytis-affected spontaneous fermentations by using both culture-based and -independent molecular methods in an attempt to further illuminate the complex yeast ecology of grape must fermentations. PMID:17766453

  10. The Composition of Camembert Cheese-Ripening Cultures Modulates both Mycelial Growth and Appearance

    PubMed Central

    Lessard, Marie-Hélène; Bélanger, Gaétan; St-Gelais, Daniel

    2012-01-01

    The fungal microbiota of bloomy-rind cheeses, such as Camembert, forms a complex ecosystem that has not been well studied, and its monitoring during the ripening period remains a challenge. One limitation of enumerating yeasts and molds on traditional agar media is that hyphae are multicellular structures, and colonies on a petri dish rarely develop from single cells. In addition, fungi tend to rapidly invade agar surfaces, covering small yeast colonies and resulting in an underestimation of their number. In this study, we developed a real-time quantitative PCR (qPCR) method using TaqMan probes to quantify a mixed fungal community containing the most common dairy yeasts and molds: Penicillium camemberti, Geotrichum candidum, Debaryomyces hansenii, and Kluyveromyces lactis on soft-cheese model curds (SCMC). The qPCR method was optimized and validated on pure cultures and used to evaluate the growth dynamics of a ripening culture containing P. camemberti, G. candidum, and K. lactis on the surface of the SCMC during a 31-day ripening period. The results showed that P. camemberti and G. candidum quickly dominated the ecosystem, while K. lactis remained less abundant. When added to this ecosystem, D. hansenii completely inhibited the growth of K. lactis in addition to reducing the growth of the other fungi. This result was confirmed by the decrease in the mycelium biomass on SCMC. This study compares culture-dependent and qPCR methods to successfully quantify complex fungal microbiota on a model curd simulating Camembert-type cheese. PMID:22247164

  11. Fungal Colonization and Biodeterioration of Plasticized Polyvinyl Chloride

    PubMed Central

    Webb, Jeremy S.; Nixon, Marianne; Eastwood, Ian M.; Greenhalgh, Malcolm; Robson, Geoffrey D.; Handley, Pauline S.

    2000-01-01

    Significant substratum damage can occur when plasticized PVC (pPVC) is colonized by microorganisms. We investigated microbial colonization of pPVC in an in situ, longitudinal study. Pieces of pPVC containing the plasticizers dioctyl phthalate and dioctyl adipate (DOA) were exposed to the atmosphere for up to 2 years. Fungal and bacterial populations were quantified, and colonizing fungi were identified by rRNA gene sequencing and morphological characteristics. Aureobasidium pullulans was the principal colonizing fungus, establishing itself on the pPVC between 25 and 40 weeks of exposure. A group of yeasts and yeast-like fungi, including Rhodotorula aurantiaca and Kluyveromyces spp., established themselves on the pPVC much later (after 80 weeks of exposure). Numerically, these organisms dominated A. pullulans after 95 weeks, with a mean viable count ± standard error of 1,000 ± 200 yeast CFU cm−2, compared to 390 ± 50 A. pullulans CFU cm−2. No bacterial colonization was observed. We also used in vitro tests to characterize the deteriogenic properties of fungi isolated from the pPVC. All strains of A. pullulans tested could grow with the intact pPVC formulation as the sole source of carbon, degrade the plasticizer DOA, produce extracellular esterase, and cause weight loss of the substratum during growth in vitro. In contrast, several yeast isolates could not grow on pPVC or degrade DOA. These results suggest that microbial succession may occur during the colonization of pPVC and that A. pullulans is critical to the establishment of a microbial community on pPVC. PMID:10919769

  12. Yeast community structures and dynamics in healthy and Botrytis-affected grape must fermentations.

    PubMed

    Nisiotou, Aspasia A; Spiropoulos, Apostolos E; Nychas, George-John E

    2007-11-01

    Indigenous yeast population dynamics during the fermentation of healthy and Botrytis-affected grape juice samples from two regions in Greece, Attica and Arcadia, were surveyed. Species diversity was evaluated by using restriction fragment length polymorphism and sequence analyses of the 5.8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of cultivable yeasts. Community-level profiles were also obtained by direct analysis of fermenting samples through denaturing gradient gel electrophoresis of 26S rDNA amplicons. Both approaches revealed structural divergences in yeast communities between samples of different sanitary states or geographical origins. In all cases, Botrytis infection severely perturbed the bioprocess of fermentation by dramatically altering species heterogeneity and succession during the time course. At the beginning and middle of fermentations, Botrytis-affected samples possessed higher levels of biodiversity than their healthy counterparts, being enriched with fermentative and/or spoilage species, such as Zygosaccharomyces bailii and Issatchenkia spp. or Kluyveromyces dobzhanskii and Kazachstania sp. populations that have not been reported before for wine fermentations. Importantly, Botrytis-affected samples exposed discrete final species dominance. Selection was not species specific, and two different populations, i.e., Saccharomyces cerevisiae in samples from Arcadia and Z. bailii in samples from Attica, could be recovered at the end of Botrytis-affected fermentations. The governing of wine fermentations by Z. bailii is reported for the first time and could elucidate the origins and role of this particular spoilage microbe for the wine industry. This is the first survey to compare healthy and Botrytis-affected spontaneous fermentations by using both culture-based and -independent molecular methods in an attempt to further illuminate the complex yeast ecology of grape must fermentations.

  13. Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation.

    PubMed

    Hector, Ronald E; Mertens, Jeffrey A; Bowman, Michael J; Nichols, Nancy N; Cotta, Michael A; Hughes, Stephen R

    2011-09-01

    Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited by metabolic imbalances, due to differing cofactor specificities of XR and XDH. The S. stipitis XR, which uses both NADH and NADPH, is hypothesized to reduce the cofactor imbalance, allowing xylose fermentation in this yeast. However, unadapted S. cerevisiae strains expressing this XR grow poorly on xylose, suggesting that metabolism is still imbalanced, even under aerobic conditions. In this study, we investigated the possible reasons for this imbalance by deleting genes required for NADPH production and gluconeogenesis in S. cerevisiae. S. cerevisiae cells expressing the XR-XDH, but not a xylose isomerase, pathway required the oxidative branch of the pentose phosphate pathway (PPP) and gluconeogenic production of glucose-6-P for xylose assimilation. The requirement for generating glucose-6-P from xylose was also shown for Kluyveromyces lactis. When grown in xylose medium, both K. lactis and S. stipitis showed increases in enzyme activity required for producing glucose-6-P. Thus, natural xylose-assimilating yeast respond to xylose, in part, by upregulating enzymes required for recycling xylose back to glucose-6-P for the production of NADPH via the oxidative branch of the PPP. Finally, we show that induction of these enzymes correlated with increased tolerance to the NADPH-depleting compound diamide and the fermentation inhibitors furfural and hydroxymethyl furfural; S. cerevisiae was not able to increase enzyme activity for glucose-6-P production when grown in xylose medium and was more sensitive to these inhibitors in xylose medium compared to glucose.

  14. Biological control as a strategy to reduce the impact of mycotoxins in peanuts, grapes and cereals in Argentina.

    PubMed

    Chulze, S N; Palazzini, J M; Torres, A M; Barros, G; Ponsone, M L; Geisen, R; Schmidt-Heydt, M; Köhl, J

    2015-01-01

    Mycotoxins including aflatoxins, deoxynivalenol, fumonisins and ochratoxin A are among the main fungal secondary metabolites detected as natural contaminants in South America in different commodities such as peanuts (aflatoxins), cereals (deoxynivalenol and fumonisins) or grapes (ochratoxin A). Different strategies including crop rotation, tillage practices, fungicide application and planting less susceptible cultivars are used in order to reduce the impact of these mycotoxins in both food and feed chains. The development of fungicide resistance in many fungal pathogens as well as rising of public concern on the risks associated with pesticide use led to the search for alternative environmentally friendly methods. Biological control of plant pathogens and toxigenic fungi offers an alternative that can complement chemical control in the frame of an integrated pest management to reduce the impact of mycotoxins in the food and feed chains. The advances made in Argentina on reducing the impact of toxigenic fungi and mycotoxins in peanut, grapes and cereals using the biocontrol strategy are summarised. Native bacteria, yeasts and filamentous fungi have been selected to evaluate them as potential biocontrol agents. Field trials showed that Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were effective at reducing deoxynivalenol accumulation in wheat. The application of Clonostachys rosea isolates on wheat stubble reduced Fusarium colonisation on the stubble. Bacillus amyloliquefaciens and Microbacterium oleovorans showed good activity to control both Fusarium verticillioides growth and the accumulation of fumonisins at pre-harvest stage in maize. Control of toxigenic Aspergillus flavus and aflatoxin accumulation in peanuts was achieved using a native atoxigenic Aspergillus flavus strain based on competitive exclusion of the toxigenic strains. Kluyveromyces thermotolerans strains were used as biocontrol agents to reduce the impact of Aspergillus section Nigri and

  15. Spectroscopic study, antimicrobial activity and crystal structures of N-(2-hydroxy-5-nitrobenzalidene)4-aminomorpholine and N-(2-hydroxy-1-naphthylidene)4-aminomorpholine

    NASA Astrophysics Data System (ADS)

    Yıldız, Mustafa; Ünver, Hüseyin; Dülger, Başaran; Erdener, Diğdem; Ocak, Nazan; Erdönmez, Ahmet; Durlu, Tahsin Nuri

    2005-03-01

    Schiff bases N-(2-hydroxy-3-nitrobenzalidene)4-aminomorpholine ( 1) and N-(2-hydroxy-1-naphthylidene)4-aminomorpholine ( 2) were synthesized from the reaction of 4-aminomorpholine with 2-hydroxy-5-nitrobenzaldehyde and 2-hydroxy-1-naphthaldehyde. Compounds 1 and 2 were characterized by elemental analysis, IR, 1H NMR, 13C NMR and UV-Visible techniques. The UV-Visible spectra of the Schiff bases with OH group in ortho position to the imino group were studied in polar and nonpolar solvents in acidic and basic media. The structures of compounds 1 and 2 have been examined cyrstallographically, for two compounds exist as dominant form of enol-imines in both the solutions and solid state. The title compounds 1 and 2 crystallize in the monoclinic space group P2 1/ c and P2 1/ n with unit cell parameters: a=8.410(1) and 11.911(3), b=6.350(9) and 4.860(9), c=21.728(3) and 22.381(6) Å, β=90.190(1) and 95.6(2)°, V=1160.6(3) and 1289.5(5) Å 3, Dx=1.438 and 1.320 g cm -3, respectively. The crystal structures were solved by direct methods and refined by full-matrix least squares. The antimicrobial activities of compounds 1 and 2 have also been studied. The antimicrobial activities of the ligands have been screened in vitro against the organisms Escherichia coli ATCC 11230, Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae UC57, Micrococcus luteus La 2971, Proteus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 27853, Mycobacterium smegmatis CCM 2067, Bacillus cereus ATCC 7064, Listeria monocytogenes ATCC 15313, Candida albicans ATCC 10231, Kluyveromyces fragilis NRRL 2415, Rhodotorula rubra DSM 70403, Debaryomyces hansenii DSM 70238 and Hanseniaspora guilliermondii DSM 3432.

  16. Engineering a cardosin B-derived rennet for sheep and goat cheese manufacture.

    PubMed

    Almeida, Carla Malaquias; Gomes, David; Faro, Carlos; Simões, Isaura

    2015-01-01

    Different sheep and goat cheeses with world-renowned excellence are produced using aqueous extracts of Cynara cardunculus flowers as coagulants. However, the use of this vegetable rennet is mostly limited to artisanal scale production, and no effective solutions to large-scale industrial applications have been reported so far. In this sense, the development of a synthetic rennet based on the most abundant cardoon milk-clotting enzymes (cardosins) would emerge as a solution for scalability of production and for application of these proteases as alternative rennets in dairy industry. In this work, we report the development of a new cardosin B-derived rennet produced in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. Using a stepwise optimization strategy-consisting of culture media screening, complemented with a protein engineering approach with removal of the plant-specific domain, and a codon optimization step-we successfully improved cardosin B production yield (35×) in K. lactis. We demonstrated that the secreted enzyme displays similar proteolytic properties, such as casein digestion profiles as well as optimum pH (pH 4.5) and temperature (40 °C), with those of native cardosin B. From this optimization process resulted the rennet preparation Vegetable Rennet (VRen), requiring no downstream protein purification steps. The effectiveness of VRen in cheese production was demonstrated by manufacturing sheep, goat, and cow cheeses. Interestingly, the use of VRen resulted in a higher cheese yield for all three types of cheese when compared with synthetic chymosin. Altogether, these results clearly position VRen as an alternative/innovative coagulant for the cheese-making industry.

  17. Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds.

    PubMed

    Games, Patrícia D; Dos Santos, Izabela S; Mello, Erica O; Diz, Mariângela S S; Carvalho, André O; de Souza-Filho, Gonçalo A; Da Cunha, Maura; Vasconcelos, Ilka M; Ferreira, Beatriz Dos S; Gomes, Valdirene M

    2008-12-01

    The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301-9], with some modifications. A DEAE-Sepharose, equilibrated with 20mM Tris-HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS-Tricine gel electrophoresis with a molecular mass of approximately 6kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.

  18. Characterization of yeasts involved in the ripening of Pecorino Crotonese cheese.

    PubMed

    Gardini, F; Tofalo, R; Belletti, N; Iucci, L; Suzzi, G; Torriani, S; Guerzoni, M E; Lanciotti, R

    2006-10-01

    The aims of this work were to identify and characterize for some important technological properties the yeast species present throughout the ripening process of Pecorino Crotonese, a traditional cheese produced in a well defined area of Southern Italy. In particular, the strain technological properties considered include fermentation/assimilation of galactose and lactose, assimilation of lactate and citrate in the presence of different NaCl concentrations, hydrolysis of butter fat, skim milk, gelatine and casein, production of brown pigments in cheese agar and ability to produce biogenic amines. High yeast levels were recorded in cheese samples already after 5 h of brining (about 5 log cfu/g) and these concentration remained constant during ripening. The yeast isolates belonged to restrict number of yeast species. While Kluyveromyces lactis and Saccharomyces cerevisiae were isolated prevalently in the first stages of Pecorino Crotonese production, Yarrowia lipolytica and Debaryomyces hansenii dominated during the later stages of maturation. Otherwise, the latter two were very NaCl resistant species. In fact, D. hansenii strains conserved the ability to assimilate lactose and galactose in the presence of 10% NaCl, while almost all the strains of Y. lipolytica isolated assimilated citrate and lactate up to 7.5% NaCl. Y. lipolytica isolates evidenced also the highest proteolytic and lipolytic activities and the capability to catabolize tyrosine producing brown pigment. In addition they resulted in the highest aminobiogenic potential decarboxylating ornithine, phenylalanine, tyrosine and lysine. However, they were not able to produce histamine, biogenic amine produced by three strains of D. hansenii.

  19. Fuel ethanol and high protein feed from corn and corn-whey mixtures in a farm-scale plant.

    PubMed

    Gibbons, W R; Westby, C A

    1983-09-01

    Distiller's wet grain (DWG) and 95% ethanol were produced from corn in a farm-scale process involving batch cooking-fermentation and continuous distillation-centrifugation. The energy balance was 2.26 and the cost was $1.86/gal (1981 cost). To improve the energy balance and reduce costs, various modifications were made in the plant. The first change, back-end (after liquefaction) serial recycling of stillage supernatant at 20 and 40% strengths, produced beers with 0.2 and 0.4% (v/v) more ethanol, respectively, than without recycling. This increased the energy balance by 0.22-0.43 units and reduced costs by $0.07-$0.10/gal. The DWGs from back-end recycling had increased fat. The second change, increasing the starch content from 17-19% to 27.5%, increased the ethanol in the beer from 10.5-14.9% at a cost saving of $0.41/gal. The energy balance increased by 1.08 units. No significant change was seen in DWG composition. The third change, using continuous cascade rather than batch fermentation, permitted batch-levels of ethanol (10%) in the beer but only at low dilution rates. Both the cost and energy balance were decreased slightly. The DWG composition remained constant. The last change, replacing part of the corn and all of the tap water in the mash with whole whey and using Kluyveromyces fragilis instead of Saccharomyces cerevisiae during fermentation, resulted in an energy balance increase of 0.16 units and a $0.27/gal cost reduction. Here, 10% ethanolic beers were produced and the DWGs showed increased protein and fat. Recommendations for farm-scale plants are provided.

  20. Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant.

    PubMed

    Guimarães, Pedro M R; François, Jean; Parrou, Jean Luc; Teixeira, José A; Domingues, Lucília

    2008-03-01

    The construction of Saccharomyces cerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (beta-galactosidase) genes of Kluyveromyces lactis was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the LAC construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between LAC4 and LAC12 and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of LAC4 and LAC12 by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous LAC genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for LAC4 and LAC12 resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media.

  1. Fuel ethanol and high protein feed from corn and corn-whey mixtures in a farm-scale plant

    SciTech Connect

    Gibbons, W.R.; Westby, C.A.

    1983-09-01

    Distiller's wet grain (DWG) and 95% ethanol were produced from corn in a farm-scale process involving batch cooking-fermentation and continuous distillation-centrifugation. The energy balance was 2.26 and the cost was $1.86/gal (1981 cost). To improve the energy balance and reduce costs, various modifications were made in the plant. The first change, back-end (after liquefaction) serial recycling of stillage supernatant at 20 and 40% strengths, produced beers with 0.2 and 0.4% (v/v) more ethanol, respectively, than without recycling. This increased the energy balance by 0.22-0.43 units and reduced costs by $0.07-$0.10/gal. The DWGs from back-end recycling had increased fat. The second change, increasing the starch content from 17-19% to 27.5%, increased the ethanol in the beer from 10.5-14.9% at a cost savings of $0.41/gal. The energy balance increased by 1.08 units. No significant change was seen in DWG composition. The third change, using continuous cascade rather than batch fermentation, permitted batch-levels of ethanol (10%) in the beer but only at low dilution rates. Both the cost and energy balance were decreased slightly. The DWG composition remained constant. The last change, replacing part of the corn and all of the tap water in the mash with whole whey and using Kluyveromyces fragilis instead of Saccharomyces cerevisiae during fermentation, resulted in an energy balance increase of 0.16 units and a $0.27/gal cost reduction. Here, 10% ethanolic beers were produced and the DWGs showed increased protein and fat. Recommendations for farm-scale plants are provided. (Refs. 46).

  2. Fuel ethanol and high protein feed from corn and corn-whey mixtures in a farm-scale plant

    SciTech Connect

    Gibbons, W.R.; Westby, C.A.

    1983-09-01

    Distiller's wet grain (DWG) and 95% ethanol were produced from corn in a farm-scale process involving batch cooking-fermentation and continuous distillation-centrifugation. The energy balance was 2.26 and the cost was $1.86/gal (1981 cost). To improve the energy balance and reduce costs, various modifications were made in the plant. The first change, back-end (after liquefaction) serial recycling of stillage supernatant at 20 and 40% strengths, produced beers with 0.2 and 0.4% (v/v) more ethanol, respectively, than without recycling. This increased the energy balance by 0.22-0.43 units and reduced costs by $0.07-$0.10/gal. The DWGs from back-end recycling had increased fat. The second change, increasing the starch content from 17-19% to 27.5%, increased the ethanol in the beer from 10.5-14.9% at a cost savings of $0.41/gal. The energy balance increased by 1.08 units. No significant change was seen in DWG composition. The third change, using continuous cascade rather than batch fermentation, permitted batch-levels of ethanol (10%) in the beer but only at low dilution rates. Both the cost and energy balance were decreased slightly. The DWG composition remained constant. The last change, replacing part of the corn and all of the tap water in the mash with whole whey and using Kluyveromyces fragilis instead of Saccharomyces cerevisiae during fermentation, resulted in an energy balance increase of 0.16 units and a $0.27/gal cost reduction. Here, 10% ethanolic beers were produced and the DWGs showed increased protein and fat. Recommendations for farm-scale plants are provided.

  3. Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica.

    PubMed

    Müller, S; Sandal, T; Kamp-Hansen, P; Dalbøge, H

    1998-10-01

    We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4.5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1 alpha and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters. Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning.

  4. Characterization of hexose transporters in Yarrowia lipolytica reveals new groups of Sugar Porters involved in yeast growth.

    PubMed

    Lazar, Zbigniew; Neuvéglise, Cécile; Rossignol, Tristan; Devillers, Hugo; Morin, Nicolas; Robak, Małgorzata; Nicaud, Jean-Marc; Crutz-Le Coq, Anne-Marie

    2017-03-01

    Sugar assimilation has been intensively studied in the model yeast S. cerevisiae, and for two decades, it has been clear that the homologous HXT genes, which encode a set of hexose transporters, play a central role in this process. However, in the yeast Yarrowia lipolytica, which is well-known for its biotechnological applications, sugar assimilation is only poorly understood, even though this yeast exhibits peculiar intra-strain differences in fructose uptake: some strains (e.g., W29) are known to be slow-growing in fructose while others (e.g., H222) grow rapidly under the same conditions. Here, we retrieved 24 proteins of the Sugar Porter family from these two strains, and determined that at least six of these proteins can function as hexose transporters in the heterologous host Saccharomyces cerevisiae EBY.VW4000. Transcriptional studies and deletion analysis in Y. lipolytica indicated that two genes, YHT1 and YHT4, are probably the main players in both strains, with a similar role in the uptake of glucose, fructose, and mannose at various concentrations. The other four genes appear to constitute a set of 'reservoir' hexose transporters with an as-yet unclear physiological role. Furthermore, through examining Sugar Porters of the entire Yarrowia clade, we show that they constitute a dynamic family, within which hexose transport genes have been duplicated and lost several times. Our phylogenetic analyses support the existence of at least three distinct evolutionary groups of transporters which allow yeasts to grow on hexoses. In addition to the well-known and widespread Hxt-type transporters (which are not essential in Y. lipolytica), we highlight a second group of transporters, represented by Yht1, which are phylogenetically related to sensors that play a regulatory role in S. cerevisiae, and a third group, represented by Yht4, previously thought to contain only high-affinity glucose transporters related to Hgt1of Kluyveromyces lactis.

  5. Characterization of yeasts isolated from artisanal short-ripened cows' cheeses produced in Galicia (NW Spain).

    PubMed

    Atanassova, M R; Fernández-Otero, C; Rodríguez-Alonso, P; Fernández-No, I C; Garabal, J I; Centeno, J A

    2016-02-01

    A total of 143 presumptive yeast isolates were obtained from the predominant microflora of 21 short-ripened starter-free raw cow's milk cheeses made in Galicia (NW Spain), and the following 68 isolates were identified by both genotyping and sequencing methods: Yarrowia lipolytica (21 isolates), Kluyveromyces lactis (18), Debaryomyces hansenii (11), Pichia guilliermondii (11), Pichia fermentans (4) and Saccharomyces cerevisiae (3). Of these, Y. lipolytica and K. lactis displayed the strongest extracellular proteolytic activity on skim milk agar, and none of the D. hansenii isolates showed any activity on this medium. Y. lipolytica also displayed the highest lipolytic activity on Tween 80 and on tributyrin. This species, which was characterized by production of butanoic acid, free fatty acid esters and sulfur compounds in pasteurized whole milk, was responsible for rancid and cheesy flavors. K. lactis mainly produced acetaldehyde, ethanol, branched chain aldehydes and alcohols, and acetic acid esters, which were responsible for alcoholic, fruity and acetic notes. The volatile profiles of D. hansenii were rather limited and characterized by high levels of methyl ketones. Most of the yeast isolates were described as tryptamine producers, although low concentrations of histamine were produced by five Y. lipolytica and two P. fermentans isolates. We conclude that selected Y. lipolytica strains could be used as adjunct cultures in the manufacture of Arzúa-Ulloa and Tetilla cheeses, and selected K. lactis strains could be used as co-starters in the manufacture of acid curd Cebreiro cheese, thus contributing to the sensory quality and typicality of the cheeses.

  6. Minireactor-based high-throughput temperature profiling for the optimization of microbial and enzymatic processes

    PubMed Central

    2014-01-01

    Background Bioprocesses depend on a number of different operating parameters and temperature is one of the most important ones. Unfortunately, systems for rapid determination of temperature dependent reaction kinetics are rare. Obviously, there is a need for a high-throughput screening procedure of temperature dependent process behavior. Even though, well equipped micro-bioreactors are a promising approach sufficient temperature control is quite challenging and rather complex. Results In this work a unique system is presented combining an optical on-line monitoring device with a customized temperature control unit for 96 well microtiter plates. By exposing microtiter plates to specific temperature profiles, high-throughput temperature optimization for microbial and enzymatic systems in a micro-scale of 200 μL is realized. For single well resolved temperature measurement fluorescence thermometry was used, combining the fluorescent dyes Rhodamin B and Rhodamin 110. The real time monitoring of the microbial and enzymatic reactions provides extensive data output. To evaluate this novel system the temperature optima for Escherichia coli and Kluyveromyces lactis regarding growth and recombinant protein production were determined. Furthermore, the commercial cellulase mixture Celluclast as a representative for enzymes was investigated applying a fluorescent activity assay. Conclusion Microtiter plate-based high-throughput temperature profiling is a convenient tool for characterizing temperature dependent reaction processes. It allows the evaluation of numerous conditions, e.g. microorganisms, enzymes, media, and others, in a short time. The simple temperature control combined with a commercial on-line monitoring device makes it a user friendly system. PMID:25126113

  7. L-methionine degradation potentialities of cheese-ripening microorganisms.

    PubMed

    Bonnarme, P; Lapadatescu, C; Yvon, M; Spinnler, H E

    2001-11-01

    Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses. These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms. The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized. The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus lutens and Staphylococcus equorum of technological interest for cheese-ripening. The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared. The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain. Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G. candidum that produced S-methyl thioacetate. Bacteria, especially Arth. sp. and Brevi. linens, produced the highest amounts and the greatest variety of volatile sulphur compounds includling methanethiol, sulfides and S-methylthioesters, e.g. S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate. Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e. L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase. They all possessed L-methionine demethiolase activity, while some (K. lactis, Deb. hansenii, Arth. sp., Staph. equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase

  8. Controlled production of Camembert-type cheeses. Part II. Changes in the concentration of the more volatile compounds.

    PubMed

    Leclercq-Perlat, Marie-Noëlle; Latrille, Eric; Corrieu, Georges; Spinnler, Henry-Eric

    2004-08-01

    Flavour generation in cheese is a major aspect of ripening. In order to enhance aromatic qualities it is necessary to better understand the chemical and microbiological changes. Experimental Camembert-type cheeses were prepared in duplicate from pasteurized milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti and Brevibacterium linens under aseptic conditions. Two replicates performed under controlled conditions of temperature (12 degrees C), relative humidity (95 +/- 2%), and atmosphere showed similar ripening characteristics. The evolutions of metabolite concentrations were studied during ripening. The volatile components were extracted by dynamic headspace extraction, separated and quantified by gas chromatography and identified by mass spectrometry. For each cheese the volatile concentrations varied with the part considered (rind or core). Except for ethyl acetate and 2-pentanone, the volatile quantities observed were higher than their perception thresholds. The flavour component production was best correlated with the starter strains. During the first 10 days the ester formations (ethyl, butyl and isoamyl acetates) were associated with the concentrations of K. lactis and G. candidum. The rind quantity of esters was lower than that observed in core probably due to (1) a diffusion from the core to the surface and (2) evaporation from the surface to the chamber atmosphere. G. candidum and Brev. linens association produced 3 methyl butanol and methyl 3-butanal from leucine, respectively. DMDS came from the methionine catabolism due to Brev. linens. Styrene production was attributed to Pen. camemberti. 2-Pentanone evolution was associated with Pen. camemberti spores and G. candidum. 2-Heptanone changes were not directly related to flora activities while 2-octanone production was essentially due to G. candidum. This study also demonstrates the determining role of volatile component diffusion.

  9. Isolation of an acetyl-CoA synthetase gene (ZbACS2) from Zygosaccharomyces bailii.

    PubMed

    Rodrigues, Fernando; Zeeman, Anne-Marie; Cardoso, Helena; Sousa, Maria João; Steensma, H Yde; Côrte-Real, Manuela; Leão, Cecília

    2004-03-01

    A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl-CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches. A probe obtained by PCR amplification from Z. bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210-219) and RVDDVVNVSG (ScAcs1p; 574-583), was used for screening a Z. bailii genomic library. Nine clones with partially overlapping inserts were isolated. The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S. cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively). Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins. Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S. cerevisiae, K. lactis, Candida albicans, C. glabrata and Debaryomyces hansenii lineages. Additionally, the cloned gene allowed growth of S. cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl-CoA synthetase. Also, S. cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1-0.35%, v/v). No differences in cell response to acetic acid stress were detected both by specific growth and death rates. The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid-grown cells.

  10. An extranuclear expression system for analysis of cytoplasmic promoters of yeast linear killer plasmids.

    PubMed

    Schründer, J; Meinhardt, F

    1995-03-01

    Based on the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis two new linear hybrid plasmids were constructed which consist of pGKL1, into which in addition to the previously developed cytoplasmically expressible LEU2* selectable marker a glucose dehydrogenase-encoding bacterial gene (gdh A) has been integrated. One of the hybrid plasmids carries the bacterial gene preceded by an arbitrarily placed cytoplasmic promoter (upstream conserved sequence) in front of the coding region (pRKL121). The other plasmid was constructed in such a way that the ATG start codon of the gdh A gene was fused in frame to the ATG start codon of the killer plasmid's open reading frame 5 (pRKL122). The structures of both linear hybrid plasmids were confirmed by restriction analysis, Southern hybridization, and sequencing of the junction sites. Yeast strains carrying either of the plasmids expressed the glucose dehydrogenase gene; however, expression of the in phase fused gene was 40-fold higher compared to the arbitrarily placed cytoplasmic promoter. In general, an in phase fusion was not required for expression, but efficiency is dramatically enhanced when the 5' noncoding sequences in front of the heterologous genes are the same as those found on the native killer plasmids. The developed system can serve as a reporter for determining the efficiency of the different cytoplasmic promoters present on both linear plasmids. Hybrid plasmids were stably maintained without selective pressure in K. lactis and they were transferred and expressed also in Saccharomyces cerevisiae.

  11. Protein kinase Ymr291w/Tda1 is essential for glucose signaling in saccharomyces cerevisiae on the level of hexokinase isoenzyme ScHxk2 phosphorylation*.

    PubMed

    Kaps, Sonja; Kettner, Karina; Migotti, Rebekka; Kanashova, Tamara; Krause, Udo; Rödel, Gerhard; Dittmar, Gunnar; Kriegel, Thomas M

    2015-03-06

    The enzyme ScHxk2 of Saccharomyces cerevisiae is a dual-function hexokinase that besides its catalytic role in glycolysis is involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by the phosphorylation of the nuclear fraction of ScHxk2 at serine 15 and the translocation of the phosphoenzyme into the cytosol. Different studies suggest different serine/threonine protein kinases, Ymr291w/Tda1 or Snf1, to accomplish ScHxk2-S15 phosphorylation. The current paper provides evidence that Ymr291w/Tda1 is essential for that modification, whereas protein kinases Ydr477w/Snf1, Ynl307c/Mck1, Yfr014c/Cmk1, and Ykl126w/Ypk1, which are co-purified during Ymr291w/Tda1 tandem affinity purification, as well as protein kinase PKA and PKB homolog Sch9 are dispensable. Taking into account the detection of a significantly higher amount of the Ymr291w/Tda1 protein in cells grown in low-glucose media as compared with a high-glucose environment, Ymr291w/Tda1 is likely to contribute to glucose signaling in S. cerevisiae on the level of ScHxk2-S15 phosphorylation in a situation of limited external glucose availability. The evolutionary conservation of amino acid residue serine 15 in yeast hexokinases and its phosphorylation is illustrated by the finding that YMR291W/TDA1 of S. cerevisiae and the homologous KLLA0A09713 gene of Kluyveromyces lactis allow for cross-complementation of the respective protein kinase single-gene deletion strains.

  12. Identification of base and backbone contacts used for DNA sequence recognition and high-affinity binding by LAC9, a transcription activator containing a C6 zinc finger.

    PubMed Central

    Halvorsen, Y D; Nandabalan, K; Dickson, R C

    1991-01-01

    The LAC9 protein of Kluyveromyces lactis is a transcriptional regulator of genes in the lactose-galactose regulon. To regulate transcription, LAC9 must bind to 17-bp upstream activator sequences (UASs) located in front of each target gene. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae, and the two proteins must bind DNA in a very similar manner. In this paper we show that high-affinity, sequence-specific binding by LAC9 dimers is mediated primarily by 3 bp at each end of the UAS: [Formula: see text]. In addition, at least one half of the UAS must have a GC or CG base pair at position 1 for high-affinity binding; LAC9 binds preferentially to the half containing the GC base pair. Bases at positions 2, 3, and 4 in each half of the UAS make little if any contribution to binding. The center base pair is not essential for high-affinity LAC9 binding when DNA-binding activity measured in vitro. However, the center base pair must play an essential role in vivo, since all natural UASs have 17, not 16, bp. Hydroxyl radical footprinting shows that a LAC9 dimer binds an unusually broad region on one face of the DNA helix. Because of the data, we suggest that LAC9 contacts positions 6, 7, and 8, both plus and minus, of the UAS, which are separated by more than one turn of the DNA helix, and twists part way around the DNA, thus protecting the broad region of the minor groove between the major-groove contacts. Images PMID:2005880

  13. Sequencing-Based Analysis of the Bacterial and Fungal Composition of Kefir Grains and Milks from Multiple Sources

    PubMed Central

    Marsh, Alan J.; O’Sullivan, Orla; Hill, Colin; Ross, R. Paul; Cotter, Paul D.

    2013-01-01

    Kefir is a fermented milk-based beverage to which a number of health-promoting properties have been attributed. The microbes responsible for the fermentation of milk to produce kefir consist of a complex association of bacteria and yeasts, bound within a polysaccharide matrix, known as the kefir grain. The consistency of this microbial population, and that present in the resultant beverage, has been the subject of a number of previous, almost exclusively culture-based, studies which have indicated differences depending on geographical location and culture conditions. However, culture-based identification studies are limited by virtue of only detecting species with the ability to grow on the specific medium used and thus culture-independent, molecular-based techniques offer the potential for a more comprehensive analysis of such communities. Here we describe a detailed investigation of the microbial population, both bacterial and fungal, of kefir, using high-throughput sequencing to analyse 25 kefir milks and associated grains sourced from 8 geographically distinct regions. This is the first occasion that this technology has been employed to investigate the fungal component of these populations or to reveal the microbial composition of such an extensive number of kefir grains or milks. As a result several genera and species not previously identified in kefir were revealed. Our analysis shows that the bacterial populations in kefir are dominated by 2 phyla, the Firmicutes and the Proteobacteria. It was also established that the fungal populations of kefir were dominated by the genera Kazachstania, Kluyveromyces and Naumovozyma, but that a variable sub-dominant population also exists. PMID:23894461

  14. A genetic overhaul of Saccharomyces cerevisiae 424A(LNH-ST) to improve xylose fermentation.

    PubMed

    Bera, Aloke K; Ho, Nancy W Y; Khan, Aftab; Sedlak, Miroslav

    2011-05-01

    Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD(+)-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.

  15. Carbohydrate utilization in Streptococcus thermophilus: characterization of the genes for aldose 1-epimerase (mutarotase) and UDPglucose 4-epimerase.

    PubMed Central

    Poolman, B; Royer, T J; Mainzer, S E; Schmidt, B F

    1990-01-01

    The complete nucleotide sequences of the genes encoding aldose 1-epimerase (mutarotase) (galM) and UDPglucose 4-epimerase (galE) and flanking regions of Streptococcus thermophilus have been determined. Both genes are located immediately upstream of the S. thermophilus lac operon. To facilitate the isolation of galE, a special polymerase chain reaction-based technique was used to amplify the region upstream of galM prior to cloning. The galM protein was homologous to the mutarotase of Acinetobacter calcoaceticus, whereas the galE protein was homologous to UDPglucose 4-epimerase of Escherichia coli and Streptomyces lividans. The amino acid sequences of galM and galE proteins also showed significant similarity with the carboxy-terminal and amino-terminal domains, respectively, of UDPglucose 4-epimerase from Kluyveromyces lactis and Saccharomyces cerevisiae, suggesting that the yeast enzymes contain an additional, yet unidentified (mutarotase) activity. In accordance with the open reading frames of the structural genes, galM and galE were expressed as polypeptides with apparent molecular masses of 39 and 37 kilodaltons, respectively. Significant activities of mutarotase and UDPglucose 4-epimerase were detected in lysates of E. coli cells containing plasmids encoding galM and galE. Expression of galE in E. coli was increased 300-fold when the gene was placed downstream of the tac promoter. The gene order for the gal-lac gene cluster of S. thermophilus is galE-galM-lacS-lacZ. The flanking regions of these genes were searched for consensus promoter sequences and further characterized by primer extension analysis. Analysis of mRNA levels for the gal and lac genes in S. thermophilus showed a strong reduction upon growth in medium containing glucose instead of lactose. The activities of the lac (lactose transport and beta-galactosidase) and gal (UDPglucose 4-epimerase) proteins of lactose- and glucose-grown S. thermophilus cells matched the mRNA levels. Images PMID:1694527

  16. Separable Crossover-Promoting and Crossover-Constraining Aspects of Zip1 Activity during Budding Yeast Meiosis

    PubMed Central

    Voelkel-Meiman, Karen; Johnston, Cassandra; Thappeta, Yashna; Subramanian, Vijayalakshmi V.; Hochwagen, Andreas; MacQueen, Amy J.

    2015-01-01

    Accurate chromosome segregation during meiosis relies on the presence of crossover events distributed among all chromosomes. MutSγ and MutLγ homologs (Msh4/5 and Mlh1/3) facilitate the formation of a prominent group of meiotic crossovers that mature within the context of an elaborate chromosomal structure called the synaptonemal complex (SC). SC proteins are required for intermediate steps in the formation of MutSγ-MutLγ crossovers, but whether the assembled SC structure per se is required for MutSγ-MutLγ-dependent crossover recombination events is unknown. Here we describe an interspecies complementation experiment that reveals that the mature SC is dispensable for the formation of Mlh3-dependent crossovers in budding yeast. Zip1 forms a major structural component of the budding yeast SC, and is also required for MutSγ and MutLγ-dependent crossover formation. Kluyveromyces lactis ZIP1 expressed in place of Saccharomyces cerevisiae ZIP1 in S. cerevisiae cells fails to support SC assembly (synapsis) but promotes wild-type crossover levels in those nuclei that progress to form spores. While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery. Moreover, K. lactis Zip1-mediated crossovers rely on S. cerevisiae synapsis initiation proteins Zip3, Zip4, Spo16, as well as the Mlh3 protein, as do the crossovers mediated by S. cerevisiae Zip1. Surprisingly, however, K. lactis Zip1-mediated crossovers are largely Msh4/Msh5 (MutSγ)-independent. This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast. Our data

  17. Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts

    PubMed Central

    Mehlgarten, Constance; Krijger, Jorrit-Jan; Lemnian, Ioana; Gohr, André; Kasper, Lydia; Diesing, Anne-Kathrin; Grosse, Ivo; Breunig, Karin D.

    2015-01-01

    Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK) functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of Saccharomyces cerevisiae to that of Kluyveromyces lactis. In high glucose, S. cerevisiae displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive) while K. lactis has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative), which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the Saccharomyces lineage. We find that in K. lactis, but not in S. cerevisiae, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of KlSIP4 gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in K. lactis. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and KlCat8, to

  18. Yeasts from Canastra cheese production process: Isolation and evaluation of their potential for cheese whey fermentation.

    PubMed

    Andrade, Rafaela Pereira; Melo, Carolina Naves; Genisheva, Zlatina; Schwan, Rosane Freitas; Duarte, Whasley Ferreira

    2017-01-01

    Canastra cheese is a cheese with geographical indication recognized by the Brazilian National Institute of Industrial Protection under number IG201002. It is produced in seven municipalities in the state of Minas Gerais in a region called Serra da Canastra. In this work, samples of milk, "pingo" (natural starter), whey and Canastra cheese were collected on a farm in Medeiros-MG/Brazil to evaluate the yeast microbiota and select yeasts for whey fermentation to produce ethanol and volatile aromatic compounds of relevance in the production of cheese. Thirty-nine isolates capable of fermenting lactose in a synthetic medium were identified by MALDI-TOF as Kluyveromyces lactis (29), Torulaspora delbrueckii (7) and Candida intermedia (3). Eleven isolates of K. lactis and three of T. delbrueckii efficiently fermented lactose until 4th day, and due to this reason were selected for cheese whey fermentation with Brix 12, 14 and 18. Generally, the isolates T. delbrueckii B14, B35, and B20 and K. lactis B10 were the most effective regardless of the initial Brix value. The identification of these four isolates by MALDI TOF was confirmed by sequencing of the ITS region. In the fermentation of cheese whey 14 Brix, T. delbrueckii B14 and B35, respectively yielded 24.06g/L and 16.45g/L of ethanol, while K. lactis B10 was more efficient in the consumption of lactose. In sequential culture with K. lactis B10 inoculated 48h after T. delbrueckii B14, 97.82% of the total sugars were consumed resulting in the production of 19.81g/L ethanol and 39 aromatic volatile compounds. The most abundant compounds were 3-methyl-1-butanol, octanoic acid and ethyl decanoate, which are reported as important for the aroma and flavor of cheeses. Based in our results, B10 isolate inoculated 48h after B14 isolate is a promising yeast inoculum to be used for fermentation of dairy substrates.

  19. Yeast Biodiversity from DOQ Priorat Uninoculated Fermentations

    PubMed Central

    Padilla, Beatriz; García-Fernández, David; González, Beatriz; Izidoro, Iara; Esteve-Zarzoso, Braulio; Beltran, Gemma; Mas, Albert

    2016-01-01

    Climate, soil, and grape varieties are the primary characteristics of terroir and lead to the definition of various appellations of origin. However, the microbiota associated with grapes are also affected by these conditions and can leave a footprint in a wine that will be part of the characteristics of terroir. Thus, a description of the yeast microbiota within a vineyard is of interest not only to provide a better understanding of the winemaking process, but also to understand the source of microorganisms that maintain a microbial footprint in wine from the examined vineyard. In this study, two typical grape varieties, Grenache and Carignan, have been sampled from four different vineyards in the DOQ Priorat winegrowing region. Afterward, eight spontaneous alcoholic fermentations containing only grapes from one sampling point and of one variety were conducted at laboratory scale. The fermentation kinetics and yeast population dynamics within each fermentation experiment were evaluated. Yeast identification was performed by RFLP-PCR of the 5.8S-ITS region and by sequencing D1/D2 of the 26S rRNA gene of the isolates. The fermentation kinetics did not indicate clear differences between the two varieties of grapes or among vineyards. Approximately 1,400 isolates were identified, exhibiting high species richness in some fermentations. Of all the isolates studied, approximately 60% belong to the genus Hanseniaspora, 16% to Saccharomyces, and 11% to Candida. Other minor genera, such as Hansenula, Issatchenkia, Kluyveromyces, Saccharomycodes, and Zygosaccharomyces, were also found. The distribution of the identified yeast throughout the fermentation process was studied, and Saccharomyces cerevisiae was found to be present mainly at the end of the fermentation process, while Aureobasidium pullulans was isolated primarily during the first days of fermentation in three of the eight spontaneous fermentations. This work highlights the complexity and diversity of the vineyard

  20. Yarrowia lipolytica vesicle-mediated protein transport pathways

    PubMed Central

    Swennen, Dominique; Beckerich, Jean-Marie

    2007-01-01

    Background Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. Results We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular transport shows that 40% of Y

  1. Controlled production of Camembert-type cheeses. Part I: Microbiological and physicochemical evolutions.

    PubMed

    Leclercq-Perlat, Marie-Noëlle; Buono, Frédéric; Lambert, Denis; Latrille, Eric; Spinnler, Henry-Eric; Corrieu, Georges

    2004-08-01

    A holistic approach of a mould cheese ripening is presented. The objective was to establish relationships between the different microbiological and biochemical changes during cheese ripening. Model cheeses were prepared from pasteurized milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti and Brevibacterium linens under aseptic conditions. Two cheese-making trials with efficient control of environmental parameters were carried out and showed similar ripening characteristics. K. lactis grew rapidly between days 1 and 6 (generation time around 48 h). G. candidum grew exponentially between days 4 and 10 (generation time around 4.6 d). Brevi. linens also grew exponentially but after day 6 when Pen. camemberti mycelium began developing and the pH of the rind was close to 7. Its exponential growth presented 3 phases in relation to carbon and nitrogen substrate availability. Concentrations of Pen. camemberti mycelium were not followed by viable cell count but they were evaluated visually. The viable microorganism concentrations were well correlated with the carbon substrate concentrations in the core and in the rind. The lactose concentrations were negligible after 10 d ripening, and changes in lactate quantities were correlated with fungi flora. The pH of the inner part depended on NH3. Surface pH was significantly related to NH3 concentration and to fungi growth. The acid-soluble nitrogen (ASN) and non-protein nitrogen (NPN) indexes and NH3 concentrations of the rind were low until day 6, and then increased rapidly to follow the fungi concentrations until day 45. The ASN and NPN indexes and NH3 concentrations in the core were lower than in the rind and they showed the same evolution. G. candidum and Pen. camemberti populations have a major effect on proteolysis; nevertheless, K. lactis and Brevi. linens cell lysis also had an impact on proteolysis. Viable cell counts of K. lactis, G. candidum, Pen. camemberti and Brevi. linens were

  2. From yeast genetics to biotechnology.

    PubMed

    Maráz, Anna

    2002-01-01

    Roots of classical yeast genetics go back to the early work of Lindegreen in the 1930s, who studied thallism, sporulation and inheritance of wine yeast strains belonging to S. cerevisiae. Consequent mutation and hybridization of heterothallic S. cerevisae strains resulted in the discovery of life cycle and mating type system, as well as construction of the genetic map. Elaboration of induced mutation and controlled hybridization of yeast strains opened up new possibilities for the genetic analysis of technologically important properties and for the production of improved industrial strains, but a big drawback was the widely different genetic properties of laboratory and industrial yeast strains. Genetic analysis and mapping of industrial strains were generally hindered because of homothallism, poor sporulation and/or low spore viability of brewing and wine yeast strains [1, 2]. In spite of this, there are a few examples of the application of sexual hybridization in the study of genetic control of important technological properties, e.g. sugar utilization, flocculation and flavor production in brewing yeast strains [3] or in the improvement of ethanol producing S. cerevisiae strains [4]. Rare mating and application of karyogamy deficient (kar-) mutants also proved useful in strain improvement [5]. Importance of yeasts in biotechnology is enormous. This includes food and beverage fermentation processes where a wide range of yeast species are playing role, but S. cerevisiae is undoubtedly the most important species among them. New biotechnology is aiming to improve these technologies, but besides this, a completely new area of yeast utilization has been emerged, especially in the pharmaceutical and medical areas. Without decreasing the importance of S. cerevisiae, numerous other yeast species, e.g. Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica have gained increasing potentialities in the modern

  3. Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts.

    PubMed

    Mehlgarten, Constance; Krijger, Jorrit-Jan; Lemnian, Ioana; Gohr, André; Kasper, Lydia; Diesing, Anne-Kathrin; Grosse, Ivo; Breunig, Karin D

    2015-01-01

    Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK) functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of Saccharomyces cerevisiae to that of Kluyveromyces lactis. In high glucose, S. cerevisiae displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive) while K. lactis has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative), which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the Saccharomyces lineage. We find that in K. lactis, but not in S. cerevisiae, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of KlSIP4 gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in K. lactis. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and KlCat8, to

  4. The transfer of aflatoxin M1 in milk of ewes fed diet naturally contaminated by aflatoxins and effect of inclusion of dried yeast culture in the diet.

    PubMed

    Battacone, G; Nudda, A; Palomba, M; Mazzette, A; Pulina, G

    2009-10-01

    An experiment was carried out to investigate 1) the transfer of aflatoxin M1 (AFM1) into the milk of dairy ewes fed diets naturally contaminated with aflatoxin B1 (AFB1); 2) the effect of the addition of dried yeast culture in the diet on this transfer; and 3) the alteration of enzymatic activities in the liver of ewes fed diets contaminated with AFB1. Twenty-four Sarda dairy ewes were divided in 4 groups and fed a concentrate mix containing 4 amounts of wheat meal naturally contaminated with aflatoxins. The diet of the control group had no wheat meal, whereas that of treated groups had low, medium, or high amounts of contaminated wheat, which corresponded to 1.13, 2.30, and 5.03 microg of AFB1/kg of feed, respectively. The experiment lasted 14 d. On d 8 to 14 from the beginning of the trial, 12 g/d of a commercial dried yeast product (DYP) of Kluyveromyces lactis was added to the diet of each ewe. The AFM1 concentration in individual milk samples and the blood serum metabolites were measured periodically. The presence of AFM1 was first detected in milk on d 1 of administration, and then its concentration increased and approached a steady-state condition on d 3 simultaneously in all treated groups. The AFM1 in milk at the steady-state condition, which was linearly related to the AFB1 intake, was 39.72, 50.38, and 79.29 ng/L in the low-aflatoxin, medium-aflatoxin, and high-aflatoxin groups, respectively. The AFM1 concentration in milk of the high-aflatoxin group was approximately 1.5-fold greater than the European Commission maximum tolerance level (50 ng/kg). The addition of DYP to the diet did not affect the AFM1 concentration in milk. After the withdrawal of the contaminated concentrate mix, the AFM1 mean concentrations decreased quickly and were no longer detected after 3 d in all treated groups. Daily milk yield and composition did not differ because of aflatoxin treatment. Blood serum parameters (creatinine, glutamic oxalacetic transaminase, glutamic pyruvic

  5. Biochemical conversions of lignocellulosic biomass for sustainable fuel-ethanol production in the upper Midwest

    NASA Astrophysics Data System (ADS)

    Brodeur-Campbell, Michael J.

    species results. Chapter 4 is an evaluation of the potential for producing Trichoderma reesei cellulose hydrolases in the Kluyveromyces lactis yeast expression system. The exoglucanases Cel6A and Cel7A, and the endoglucanase Cel7B were inserted separately into the K. lactis and the enzymes were analyzed for activity on various substrates. Recombinant Cel7B was found to be active on carboxymethyl cellulose and Avicel powdered cellulose substrates. Recombinant Cel6A was also found to be active on Avicel. Recombinant Cel7A was produced, but no enzymatic activity was detected on any substrate. Chapter 5 presents a new method for enzyme improvement studies using enzyme co-expression and yeast growth rate measurements as a potential high-throughput expression and screening system in K. lactis yeast. Two different K. lactis strains were evaluated for their usefulness in growth screening studies, one wild-type strain and one strain which has had the main galactose metabolic pathway disabled. Sequential transformation and co-expression of the exoglucanase Cel6A and endoglucanase Cel7B was performed, and improved hydrolysis rates on Avicel were detectable in the cell culture supernatant. Future work should focus on hydrolysis of natural substrates, developing the growth screening method, and utilizing the K. lactis expression system for directed evolution of enzymes.

  6. DNA typing methods for differentiation of Debaryomyces hansenii strains and other yeasts related to surface ripened cheeses.

    PubMed

    Petersen, K M; Møller, P L; Jespersen, L

    2001-09-19

    The discriminative power of ITS-PCR, ITS-PCR RFLP and mitochondrial (mt)-DNA RFLP were evaluated for differentiation of yeasts of importance for surface ripened cheeses. In total 60 isolates were included. Of these, 40 strains of the following species, Debaryomyces hansenii var. hansenii, D. hansenii var. fabryi, Saccharomyces cerevisiae, Candida zeylanoides, Kluyveromyces lactis and Yarrowia lipolytica, were obtained from culture collections and 20 isolates of D. hansenii representing six different phenotypes were collected from seven Danish producers of surface ripened cheeses. ITS-PCR was evaluated for differentiation at species level on the 40 strains obtained from culture collections. Ten strains of each variety of D. hansenii and five strains of each of the above mentioned species were analysed. For each of the investigated species, a specific ITS1-5.8S rDNA-ITS2 region size was observed. Accordingly ITS-PCR was found valuable for differentiation at species level of yeasts of importance for surface ripened cheeses. ITS-PCR RFLP was investigated for the purpose of strain typing of D. hansenii. Ten CBS strains of each variety of D. hansenii were analysed. Only one enzyme (TaqI) out of several investigated (BamHI, DpnI, Fnu4HI, HaeIII, HindIII, HpaII, NlaII, Sau3AI, TaqI) demonstrated genetic diversity within the strains. This enzyme divided the 20 strains in three groups. Sequence analysis of the ITS1-5.8S rDNA-ITS2 region for the type strains of each variety of D. hansenii showed an identity of 99.84%, corresponding to a difference in one basepair. Based on these results, ITS-PCR RFLP was found ineffective for strain typing of D. hansenii. MtDNA RFLP using HaeIII and HpaII was evaluated for strain typing of D. hansenii on the 20 CBS strains of D. hansenii. The CBS strains were divided into 16 groups according to their restriction profiles, which proved the method useful for typing of D. hansenii at subspecies level. The 20 dairy isolates showed a lower genetic