Hexokinase PII: structural analysis and glucose signalling in the yeast Saccharomyces cerevisiae.

Hexokinase PII (Hxk2) is a yeast glucose phosphorylating enzyme that, besides its role in glycolysis, seems to have an additional role in glucose signalling. To study the domains in Hxk2 that may participate in this latter process, we have constructed 11 mutant alleles using site-directed mutagenesis. Six of them were clustered charged-to-alanine mutants in which clusters of charged residues were changed to alanine residues. Two of them contained substitutions in Ser15 to either alanine or glutamic acid and three of them had deletions at either the N-terminus or the C-terminus of the protein. In most of them, the catalytic activity correlated directly with their functionality in glucose signalling. However, we found two mutants (Delta1-15 and Delta476-486) that, having low catalytic activity, were still fully functional in glucose signalling. This may indicate that other factors and not just the catalytic activity of the enzyme may be important for the functionality of the protein in glucose signalling.     

Characterization of a gene similar to BIK1 in the yeast Kluyveromyces lactis

In Saccharomyces cerevisiae, Bik1p is a microtubule plus-end-tracking protein that plays several roles in mitosis and ploidy. KlBik1p (from Kluyveromyces lactis) maintains the same structural-domain organization as does S. cerevisiae Bik1p. As part of its characterization, we constructed a stable klbik1 mutant which is sensitive to benomyl only at 14 degrees C and has a higher frequency of crescent-shaped nuclei than S. cerevisiae bik1 mutants. This phenotype is partially rescued by S. cerevisiae BIK1. Other phenotypes associated with bik1 are not present in the K. lactis mutant. By fusion to GFP we were able to show the functionality of the KlBik1p CAP-Gly domain and found that the fusion protein changes its cellular location during the cell cycle.     

Isolation of a lager yeast with an increased copy number of the YCK1 gene and high fermentation performance

The selection of yeast with good fermentation characteristics is critical for producing beer with desirable qualities. A yeast population was selected with an enhanced fermentation rate, referred to as high‐fermentation yeast (HFY), which was derived from the wild‐type Sacchromyces pastorianus yeast population (WTY). To identify genes that contribute to the fermentation performance, we compared the genetic profiles of the WTY and HFY populations by next‐generation sequencing. Several chromosomal regions were found to exhibit markedly different sequence coverage, suggesting chromosomal duplications and deletions, which might have occurred during selection of the HFY population. Among the genes with altered coverage, the copy number of the Saccharomyces eubayanus‐type YCK1 (SeYCK1) gene was almost two times higher in the HFY population than in the WTY population. The gene which is involved in glucose sensing in Saccharomyces cerevisiae was at a higher level in the HFY population throughout fermentation. These findings suggest that the chromosomal duplication of a region including the SeYCK1 gene locus of the HFY population is at least partially responsible for the differences in the fermentation properties between the WTY and HFY populations. © 2018 The Institute of Brewing & Distilling     

Heat shock transiently enhances the synthesis rate of Sis1p,a ribosome-associated DnaJ protein in the oleagenous yeast Apiotrichum curvatum

DnaJ proteins have been localized in different intracellular compartments of eukaryotes. In Apiotrichum curvatum, a fat-storing yeast, we found a DnaJ homolog associated with ribosomes and large cytosolic complexes as well. Using a plant DnaJ probe and a cDNA library constructed from poly(A)⁺ -RNA of A. curvatum grown on oleate we isolated a SIS1 cDNA coding for a 39·5 kDa protein. The putative protein contains neither a zinc finger motif nor a CAAX motif but is characterized by a J-domain at the N-terminal region and a large G-rich region in the middle part of the molecule. Heat shock applied for 1 h resulted in a pronounced but transient increase of the SIS1 mRNA. An antiserum was raised against the bacterially expressed protein. Cell fractions from A. curvatum were further separated by sedimentation centrifugation on sucrose gradients. Analysing the sub-fractions, we detected Sis1p mainly associated with ribosomes, and with particles sedimenting at approximately 200S. Hsp70 was found to be associated with the 200S fraction. The respective cytosolic A. curvatum Hsp70 cDNA was cloned and sequenced. High salt conditions caused the removal of Hsp70 and Sis1p from the 200S complexes. Mild RNase treatment of the 200S fraction afforded monosomes and 200S complexes unaffected by RNase. Heat shock led to a pronounced increase in the rate of de novo synthesis. However, due to the large pools of Sis1p on ribosomes and large cytosolic complexes, the increase in gene activation did not lead to a significant change of the total amount of Sis1p. Accession numbers are: Y12079 for ACHSP70 and Y12080 for ACSIS1. © 1998 John Wiley & Sons, Ltd.     

Use of a YAP1 overexpression cassette conferring specific resistance to cerulenin and cycloheximide as an efficient selectable marker in the yeast Saccharomyces cerevisiae.

Drug-resistance markers for yeast transformation are useful because they can be applied to strains without auxotrophic mutations. However, they are susceptible to technical difficulties, namely lower transformation efficiency and the appearance of drug-resistant mutants without the marker. To avoid these problems, we have constructed a phosphoglycerate kinase (PGK) promoter-driven YAP1 expression cassette, called PGKp-YAP1. Yeast cells containing PGKp-YAP1 were resistant to cycloheximide, a protein synthesis inhibitor, and also to cerulenin, a fatty acid synthesis inhibitor, but not to other drugs tested. The transformation efficiency of PGKp-YAP1 using cerulenin selection was comparable to that using a URA3 auxotrophic marker when low concentrations of cerulenin were used. Non-transformed drug-resistant colonies did appear on the low-concentration cerulenin plates. However, these non-transformed colonies could easily be identified, based on their cycloheximide sensitivity and/or their resistance to aureobasidin A to which the transformants were sensitive. Therefore, the dual drug resistance of PGKp-YAP1 could be used as an effective selection for PGKp-YAP1 recipient cells. The PGKp-YAP1 marker was used to disrupt the LYS2 gene and to transform an industrial yeast strain, indicating that this marker can be used for efficient and reliable gene manipulations in any Saccharomyces cerevisiae strain.     

发酵促进剂的应用研究——复方发酵促进剂1#应用于酒精发酵的研究报告

对复方发酵促进剂 1#在酒精发酵中的使用浓度及使用效果进行了研究 ,结果表明该促进剂对酒精发酵的多个环节具有促进作用。促进剂添加浓度为 1×10 -6g/mL醪液时 ,可使发酵液中酵母出芽率提高10 %以上 ,酵母细胞数提高 2 0 %左右 ,发酵速率加快 ,出酒率提高 ,醪液中酒精含量可提高 6%左右。文中还探讨了促进剂的添加条件和应用领域 ,为进一步推广提供了必要的依据     

高产乙酸乙酯酵母菌筛选及固态发酵应用研究

为提高清香型麸曲白酒中乙酸乙酯的含量,从已有菌株中筛选一株高产乙酸乙酯的酵母菌,将其应用于白酒固态发酵实验,并对其发酵条件进行研究。结果表明,筛选得到一株高产乙酸乙酯的酵母菌J-4,乙酸乙酯产量为1.38 g/L,经初步发酵实验表明,其最适固态发酵工艺条件为高粱粉与酒糟质量比1.0∶4.5,发酵时间7 d,酒醅入发酵容器后,以正常压力进行压醅（醅料密度为331.43 kg/m3）,所得原酒中乙酸乙酯含量为1.31 g/L,出酒率为46.3%,均达到较高水平。进一步将酵母菌J-4应用于麸曲白酒酿造生产中,以不添加产酯酵母发酵为对照,采用相同生产工艺酿造白酒。结果表明,添加酵母菌J-4发酵生产原酒酒样中乙酸乙酯含量达1.07 g/L,比对照组增长50.7%,出酒率为45.3%,比对照组降低0.87%,原酒口感品质有明显改善。     

First aspects on acetate metabolism in the yeast Dekkera bruxellensis: a few keys for improving ethanol fermentation

Dekkera bruxellensis is continuously changing its status in fermentation processes, ranging from a contaminant or spoiling yeast to a microorganism with potential to produce metabolites of biotechnological interest. In spite of that, several major aspects of its physiology are still poorly understood. As an acetogenic yeast, minimal oxygen concentrations are able to drive glucose assimilation to oxidative metabolism, in order to produce biomass and acetate, with consequent low yield in ethanol. In the present study, we used disulfiram to inhibit acetaldehyde dehydrogenase activity to evaluate the influence of cytosolic acetate on cell metabolism. D. bruxellensis was more tolerant to disulfiram than Saccharomyces cerevisiae and the use of different carbon sources revealed that the former yeast might be able to export acetate (or acetyl‐CoA) from mitochondria to cytoplasm. Fermentation assays showed that acetaldehyde dehydrogenase inhibition re‐oriented yeast central metabolism to increase ethanol production and decrease biomass formation. However, glucose uptake was reduced, which ultimately represents economical loss to the fermentation process. This might be the major challenge for future metabolic engineering enterprises on this yeast.     

1H NMR‐based metabolomics approach for understanding the fermentation behaviour of Bacillus licheniformis

Bacillus licheniformis has been found to be one of the persistent dominant microorganisms in Daqu, which is a traditional fermentation starter, and it has been used to intensify certain strains. To understand the impact of B. licheniformis on Daqu, the fermentation behaviour of B. licheniformis was studied using ¹H NMR‐based non‐targeted analysis and principal component analysis. During the fermentation, 53 compounds were identified. Among them, seven compounds were largely consumed and 17 metabolites were largely accumulated. The macromolecular starch and cellulose were degraded by B. licheniformis, and then utilized to produce acetic acid, lactic acid, propionic acid, succinate acid, etc. Principal component analysis was carried out to study the variations of analytes during the fermentation. Samples collected at each time point could be clearly discriminated and the biomarkers of each time point were defined. A variety of biochemical compounds (such as acetate, ethanol, lactate, pyruvate, malate, maltose and sucrose) were changed during the fermentation of B. licheniformis. The results are useful to reveal how and why B. licheniformis becomes dominant in Daqu, and to reveal its impact on the aroma formation of Daqu and its derived products. Copyright © 2015 The Institute of Brewing & Distilling     

山药多糖和燕麦多糖发酵产酸及发酵产物对结肠癌细胞的增殖抑制作用

采用水提取、醇沉淀法制备山药多糖和燕麦多糖,利用健康成人粪便提取物中的肠道微生物体外发酵多糖,确定发酵产酸情况（乙酸,丙酸,丁酸,乳酸）以及发酵产物对结肠癌细胞HCT-116的增殖抑制作用。山药多糖和燕麦多糖的糖含量分别为82.63%和80.32%。发酵24 h,山药多糖和燕麦多糖产生23.28、25.14 mmol/L乙酸、2.49、1.97 mmol/L丙酸、11.04、7.99 mmol/L丁酸和0.19、0.46 mmol/L乳酸;发酵48 h,它们产生四种酸为28.14、42.53,6.48、2.45,13.76、9.64,0.08、0.09 mmol/L。发酵时间从24 h延长至48 h,产物中乙酸、丙酸和丁酸含量显著提高（p<0.05）,而乳酸含量明显降低（p<0.05）。两种多糖对细胞的抑制作用小于24.5%,而两种多糖发酵产物有更强的抑制作用,分别达到46.2%⁶9.1%、44.6%⁶7.3%;48 h发酵产物抑制作用更强,处理细胞48 h产生的抑制作用更大。结果表明,两种多糖经过肠道微生物发酵后,其产物对结肠癌细胞具有更强的增殖抑制活性。      

Effects of supplementing a Saccharomyces cerevisiae fermentation product during the transition period on rumen fermentation of dairy cows fed fresh diets differing in starch content

The objective of this study was to evaluate the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) during the transition period (d −28 ± 3 to 23 ± 3 relative to calving) on rumen fermentation and mRNA abundance of genes in the rumen epithelium of fresh cows (d 1 to 23 ± 3 after calving) fed diets differing in starch content. Eighteen ruminally cannulated multiparous Holstein cows were fed diets with SCFP (n = 9) or without (CON; n = 9) throughout the experiment. All cows were fed a common basal controlled-energy close-up diet (1.43 Mcal/kg, net energy for lactation; 13.8% starch) before calving. Cows within each treatment (CON or SCFP) were fed either a low-starch (LS; 22.1% starch) or high-starch (HS; 28.3% starch) diet during the fresh period. Cows were assigned to treatment after balancing for parity, body condition score, and expected calving date. Rumen pH was measured continuously for 72 h starting on d −10, −3, 1, 7, and 21 relative to calving date. Rumen papillae were collected on d −10 and 21 relative to calving. Supplementation of SCFP had no effect on rumen pH during d −10 to −8, but mean rumen pH tended to be higher (6.64 vs. 6.49) for SCFP cows than for CON cows during d −3 to −1. Feeding SCFP decreased the range of rumen pH variation compared with CON within the HS group during both d 7 to 9 (1.08 vs. 1.38) and d 21 to 23 (1.03 vs. 1.30) after calving. In addition, nadir rumen pH tended to be higher (5.64 vs. 5.44) and duration of pH below 5.8 tended to be shorter (116 vs. 323 min/d) for the SCFP group than for the CON group during d 21 to 23 after calving. Supplementation of SCFP increased the mRNA abundance of insulin-like growth factor-6 (1.10 vs. 0.69) before calving and decreased the mRNA abundance of putative anion transporter isoform 1 (1.12 vs. 2.27) after calving. Nadir rumen pH tended to be higher during d 1 to 3 (5.63 vs. 5.41) for LS cows than for HS cows, but rumen pH was not affected by dietary starch content during other time periods. Dietary starch content had no effect on mRNA abundance of genes in the rumen epithelium after calving. These results suggest that supplementation of SCFP may reduce the range of variation in rumen pH in fresh cows fed HS diets and the duration of subacute ruminal acidosis by the end of the fresh period regardless of dietary starch content and that decreasing dietary starch content during the fresh period may reduce the decrease in rumen pH immediately after parturition.     

一株耐盐产香鲁氏接合酵母FA-1的鉴定及其在酱油酿造中的应用

为提高酱油风味与品质,从酱油醪液中筛选耐盐产香酵母菌株并应用于酱油酿造。对筛选菌株进行鉴定,并采用顶空固相微萃取-气质联用法（HS-SPME-GC-MS）分别检测其在不同NaCl浓度的发酵液及酱油成品中的挥发性香气成分。结果表明,筛选出1株耐盐产香菌株FA-1,经鉴定为鲁氏接合酵母（Zygosaccharomyces rouxii）,可耐受22%的食盐浓度。在不同NaCl浓度的酱油发酵液中共检出56种挥发性风味物质,且当NaCl质量分数为18%时,更有利于醇类化合物的富集。在酱油生产试验中,添加鲁氏接合酵母FA-1的实验组中醇类化合物占比最大（72.03%）,其中苯乙醇、3-甲基-1-丁醇、乙醇的含量较高,且醇类和酯类化合物的含量分别较添加酵母As 1.039的对照组提高131.05%和189.65%。因此,菌株FA-1具有良好的耐盐产香性能,可作为潜在的酱油发酵菌株。     

Production of α‐amylase by Aspergillus oryzae As 3951 in solid state fermentation using spent brewing grains as substrate

BACKGROUND: The optimisation of nutrient levels for the production of α‐amylase by Aspergillus oryzae As 3951 in solid state fermentation (SSF) with spent brewing grains (SBG), an inexpensive substrate and solid support, was carried out using response surface methodology (RSM) based on Plackett–Burman design (PBD) and Box–Behnken design (BBD). RESULTS: In the first optimisation step a PBD was used to evaluate the influences of related factors. Corn steep liquor, CaCl₂ and MgSO₄ were found to be the most compatible supplements to the substrate of SBG and influenced α‐amylase activity positively. In the second step the concentrations of these three nutrients were optimised using a BBD. The final concentrations (g/g dry substrate basis) in the medium optimised with RSM were 1.8% corn steep liquor, 0.22% CaCl₂ and 0.2% MgSO₄ · 7H₂O using SBG as the solid substrate. The average α‐amylase activity reached 6186 U g^?1 dry substrate under the optimised conditions at 30 °C after 96 h. Under the optimised conditions of SSF an approximately 17.5% increase in enzyme yield was observed. CONCLUSION: SBG was found to be a good substrate for the production of α‐amylase by A. oryzae As 3951 under SSF. Copyright © 2007 Society of Chemical Industry     

Functional inactivation of the conserved Sem1p in yeast by intrabodies

Intrabody technology was applied to characterize the function and intracellular localization of a highly conserved Saccharomyces cerevisiae Sem1 protein. DSS1, the mammalian homologue of Sem1p, is functionally conserved between yeast and mammalian cells, and in mammalian cells physically interacts with the strong tumour supressor BRCA2. Yeast and the generated intrabodies are thus expected to offer a useful system for studies on Sem1p/DSS1 function. Sem1p-specific antibody isolated from a phage display library was expressed intracellularily and targeted to either the cytosol or the nucleus of yeast cells. Analysis of the applicability of different antibody fragments as intrabodies showed that the Fab intrabody was expressed most efficiently. Expression of nuclear-targeted anti-Sem1p Fab intrabodies inhibited the growth of the sigma1278b yeast strain in a manner similar to deletion of the SEM1 gene. This indicates that the Fab intrabodies interact in vivo with Sem1p and result in inactivation of Sem1p. Localization of the Fab intrabody with or without the nuclear localization signal to the nucleus in Sem1p-dependent manner suggests that Sem1p mediates the nuclear transport of the intrabody without any targeting signal. Our results suggest that Sem1p function in yeast cells is in part manifested in the nucleus.     

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

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.     

A 37·5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes,a TCP-1-related gene,an open reading frame similar to the DAL80 gene,and a tRNAArg

The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromsome X was determined from an ordered set of subclones. The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons. Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor-like protein, D678 is identical to the yeast GSH1 gene encoding γ-glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation. The deduced amino acid sequence of A550 is 63% identical to the Ccη subunit of a murine TCP-1-containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP-1 family. Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein. In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI. Finally, the sequence contained a tRNA^Arg3 (AGC) gene. The nucleotide sequence data reported in this paper have been deposited in the EMBL and GenBank databases under the accession number X85021.