Genome shuffling to improve thermotolerance,ethanol tolerance and ethanol productivity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Genome shuffling is a powerful strategy for rapid engineering of microbial strains for desirable industrial phenotypes. Here we improved the thermotolerance and ethanol tolerance of an industrial yeast strain SM-3 by genome shuffling while simultaneously enhancing the ethanol productivity. The starting population was generated by protoplast ultraviolet irradiation and then subjected for the recursive protoplast fusion. The positive colonies from the library, created by fusing the inactivated protoplasts were screened for growth at 35, 40, 45, 50 and 55°C on YPD-agar plates containing different concentrations of ethanol. Characterization of all mutants and wild-type strain in the shake-flask indicated the compatibility of three phenotypes of thermotolerance, ethanol tolerance and ethanol yields enhancement. After three rounds of genome shuffling, the best performing strain, F34, which could grow on plate cultures up to 55°C, was obtained. It was found capable of completely utilizing 20% (w/v) glucose at 45–48°C, producing 9.95% (w/v) ethanol, and tolerating 25% (v/v) ethanol stress.     

Thermotolerance behavior in sugar cane syrup fermentations of wild type yeast strains selected under pressures of temperature,high sugar and added ethanol

Summary The selected yeast strains were examined for their ability to grow, to retain cell viability and to ferment diluted sugar cane juice (15 % total sugar, w/v) to ethanol at 40°C. The degree of agitation (aeration) affects the thermotolerance while the method used for isolation of the strains appears to have no significant effect. The yeast isolated are aerobically fermentative with increased levels of fermentation and growth resulting from agitation (aeration), the exact level of these increases being dependent on the strain used.     

从老抽酱醪中分离耐盐性酵母的研究

以老抽酱醪为实验材料进行耐盐性酵母菌种分离,并做菌种鉴定。分析了在不同盐度条件下耐盐性酵母菌的生长情况和生长过程中培养基总糖的消耗,可以发现实验得到的酵母在22％（质量与体积}E）盐度下依然能够良好生长。结果表明,实验分离出的No．2菌在同级盐度的条件下的生长量要明显高于No．1菌,但在乙醇产率方面,两株菌在相同的含盐量为16％（质量与体积比）的麦芽汁培养基中发酵8d,No．1菌的乙醇产率为3．1％（体积比）,No．2菌的乙醇产率2．9％（体积比）。     

Characterization of yeast starter cultures used in household alcoholic beverage preparation by a few ethnic communities of Northeast India

In this study, we report the characterization of traditional household starter cultures from a few ethnic groups of northeast India. Pure cultures obtained from the study have been deposited in the Microbial Type Culture Collection (MTCC), India. These isolates have been analyzed for their growth characteristics, sensitivity to temperature and pH, alcohol tolerance, alcohol production, and alcohol dehydrogenase (ADH) content. The pure cultures obtained from different starter cultures revealed the presence of Debaryomyces, Wickerhamomyces, and Candida along with the fermenting yeast Saccharomyces. The growth behavior at different temperatures, pH and alcohol tolerance revealed numerous facts and behavior of the yeast strains associated with traditional alcoholic fermentation. All the isolates were found to be thermotolerant up to 37 °C, fairly pH-resistant, good in ADH secretion, and with appreciable alcohol production. For all the strains studied, the Saccharomyces cerevisiae MTCC 3976 strain from the Tea Tribes of Assam and the Wickerhamomyces anomalus MTCC 3979 from the Apatani Tribe of Arunachal Pradesh were found to be exceptional in terms of thermotolerance, alcohol tolerance, alcohol production, and ADH activity, and hence may be identified as potential strains for industrial fermentation.     

Ethanol tolerance, sugar tolerance and invertase activities of some yeast strains isolated from steep water of fermenting cassava tubers

E kunsanmi , T.J. & O dunfa , S.A. 1990. Ethanol tolerance, sugar tolerance and invertase activities of some yeast strains isolated from steep water of fermenting cassava tubers. Journal of Applied Bacteriology 69 , 672–675.
Thirteen yeasts isolated from the steep water of fermenting cassava tubers were screened for ethanol tolerance. Three strains which showed measurable growth in medium containing 10% (v/v) ethanol were also sugar-tolerant and grew well in medium containing 25% (w/v) glucose. One of the strains, YC3, was found to possess much higher invertase activity than the other two and could be of value in ethanol production from molasses. Further search for industrially useful yeasts in African fermented foods is suggested.     

Characterization of yakju brewed from glutinous rice and wild-type yeast strains isolated from nuruks

Korean traditional rice wines yakju and takju are generally brewed with nuruk as the source of the saccharogenic enzymes by natural fermentation. To improve the quality of Korean rice wine, the microorganisms in the nuruk need to be studied. The objective of this research was to improve the quality of Korean wine with the wild-type yeast strains isolated from the fermentation starter, nuruk. Only strain YA-6 showed high activity in 20% ethanol. Precipitation of Y89-5-3 was similar to that of very flocculent yeast (?80%) at 75.95%. Using 18S rRNA sequencing, all 10 strains were identified as Saccharomyces cerevisiae. Volatile compounds present in yakju were analyzed by gas chromatography-mass selective detector. The principal component analysis (PCA) of the volatile compounds grouped long-chain esters on the right side of the first principal component, PC1; these compounds were found in yakju that was made with strains YA-6, Y89-5-3, Y89-5- 2, Y90-9, and Y89-1-1. On the other side of PC1 were short-chain esters; these compounds were found in wines that were brewed with strains Y183-2, Y268-3, Y54-3, Y98-4, and Y88-4. Overall, the results indicated that using different wild-type yeast strains in the fermentation process significantly affects the chemical characteristics of the glutinous rice wine.     

Modelling the freezing response of baker's yeast prestressed cells: a statistical approach

Aims: To study the effect of prestress conditions on the freezing and thawing (FT) response of two baker’s yeast strains and the use of statistical analysis to optimize resistance to freezing. Methods and Results: Tolerance to FT of industrial strains of Saccharomyces cerevisiae was associated to their osmosensitivity and growth phase. Pretreatments with sublethal stresses [40°C, 0·5 mol l^?1 NaCl, 1·0 mol l^?1 sorbitol or 5% (v/v) ethanol] increased freeze tolerance. Temperature or hyperosmotic prestresses increased trehalose contents, nevertheless no clear correlation was found with improved FT tolerance. Plackett–Burman design and response surface methodology were applied to improve freeze tolerance of the more osmotolerant strain. Optimal prestress conditions found were: 0·779 mol l^?1 NaCl, 0·693% (v/v) ethanol and 32·15°C. Conclusions: Ethanol, saline, osmotic or heat prestresses increased freezing tolerance of two phenotypically distinct baker’s yeast strains. A relationship among prestresses, survival and trehalose content was not clear. It was possible to statistically find optimal combined prestress conditions to increase FT tolerance of the osmotolerant strain. Significance and Impact of the Study: Statistically designed combination of prestress conditions that can be applied during the production of baker’s yeast could represent a useful tool to increase baker’s yeast FT resistance.     

Isolation and utilization of indigenous yeast strain for brewing of millet beer (OYOKPO)

Three yeast strains were isolated from a spontaneously fermented native millet (Pennisetum typhoideum) malt beer (Oyokpo). One of the yeast isolates found to have the most highly fermenting capacity was characterised and identified as a strain of Saccharomyces cerevisiae. The yeast was then utilised as the pitching yeast in a subsequent controlled fermentation of millet wort at 20°C for 120 hours. Bitter leaf (Vernonia amagdalina) extract was used as the bittering and flavouring agent. The Oyokpo beer sample produced under these conditions was found to possess both chemical and organoleptic qualities comparable to some extent, to the conventional barley malt beer. At the end of fermentation, the pH, specific gravity, alcohol content, reducing sugar content and protein content of the beer were 4.11, 1.0308, 2.81% (v/v), 4.00 (mg/ml) and 0.84 (mg/ml) respectively.     

The application of PCR fingerprinting to the differentiation of Yersinia enterocolitica strains isolated from humans and pigs

The distribution of different genotypes of Yersinia enterocolitica strains recovered from humans and from healthy pigs was investigated using PCR fingerprinting. The thirty six strains of Y. enterocolitica from humans, thirty five strains from pigs and Y. enterocolitica ATCC 9610 strain were included in this study. The tested strains of Y. enterocolitica belonged to O3 and O9 serogroups. The PCR fingerprinting using EAE5 primer (5' CTT AAT CTC AGT AAT GCT GGC CTT GG) made it possible to form five groups among the tested Y. enterocolitica strains. Two groups were very numerously represented by the tested strains. The thirty of Y. enterocolitica O3 strains from humans (thirty one of tested) and eighteen of Y. enterocolitica O3 strains from pigs (twenty of tested) belonged to one group. This group also included Y. enterocolitica ATCC9610 strain and four Y. enterocolitica O9 strains from pigs. All investigated Y. enterocolitica O9 strains from humans and the majority of Y. enterocolitica O9 strains isolated from pigs created a second, numerous group. The third genotype was created by two strains O9 from pigs, and the remaining two strains, isolated from pigs, belonging to O3 and O9 serogroups showed different binding patterns revealed by gel electrophoresis and created two other genotypes. The tested Y. enterocolitica strains which were isolated from humans formed only two groups but Y. enterocolitica strains isolated from pigs were found in five groups but such as the Y. enterocolitica strains from humans, the majority of strains from pigs were in first and second group. The Y. enterocolitica O3 strains regardless of their origin mostly represented the same PCR fingerprinting profile. The tested Y. enterocolitica O9 strains were more genetically diverse and represented four PCR fingerprinting profiles.     

Intergeneric yeast fusants with efficient ethanol production from cheese whey powder solution: Construction of a Kluyveromyces marxianus and Saccharomyces cerevisiae AY‐5 hybrid

Due to its high content of lactose and abundant availability, cheese whey powder (CWP) has received much attention for ethanol production in fermentation processes. However, lactose‐fermenting yeast strains including Kluyveromyces marxianus can only produce alcohol at a relatively low level, while the most commonly used distiller yeast strain Saccharomyces cerevisiae cannot ferment lactose since it lacks both β‐galactosidase and the lactose permease system. To combine the unique aspects of these two yeast strains, hybrids of K. marxianus TY‐22 and S. cerevisiae AY‐5 were constructed by protoplast fusion. The fusants were screened and characterized by DNA content, β‐galactosidase activity, ethanol tolerance, and ethanol productivity. Among the genetically stable fusants, the DNA content of strain R‐1 was 6.94%, close to the sum of the DNA contents of TY‐22 (3.99%) and AY‐5 (3.51%). The results obtained by random‐amplified polymorphic DNA analysis suggested that R‐1 was a fusant between AY‐5 and TY‐22. During the fermentation process with CWP, the hybrid strain R‐1 produced 3.8% v/v ethanol in 72 h, while the parental strain TY‐22 only produced 3.1% v/v ethanol in 84 h under the same conditions.     

Genome-wide identification of genes involved in tolerance to various environmental stresses inSaccharomyces cerevisiae

During fermentation, yeast cells are exposed to a number of stresses — such as high alcohol concentration, high osmotic pressure, and temperature fluctuation — so some overlap of mechanisms involved in the response to these stresses has been suggested. To identify the genes required for tolerance to alcohol (ethanol, methanol, and 1-propanol), heat, osmotic stress, and oxidative stress, we performed genome-wide screening by using 4828 yeast deletion mutants. Our screens identified 95, 54, 125, 178, 42, and 30 deletion mutants sensitive to ethanol, methanol, 1-propanol, heat, NaCl, and H₂O₂, respectively. These deleted genes were then classified based on their cellular functions, and cross-sensitivities between stresses were determined. A large number of genes involved in vacuolar H⁺-ATPase (V-ATPase) function, cytoskeleton biogenesis, and cell wall integrity, were required for tolerance to alcohol, suggesting their protective role against alcohol stress. Our results revealed a partial overlap between genes required for alcohol tolerance and those required for thermotolerance. Genes involved in cell wall integrity and the actin cytoskeleton are required for both alcohol tolerance and thermotolerance, whereas the RNA polymerase II mediator complex seems to be specific to heat tolerance. However, no significant overlap of genes required for osmotic stress and oxidative stress with those required for other stresses was observed. Interestingly, although mitochondrial function is likely involved in tolerance to several stresses, it was found to be less important for thermotolerance. The genes identified in this study should be helpful for future research into the molecular mechanisms of stress response.     

Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis

During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.     

Effect of UV radiation on thermotolerance,ethanol tolerance and osmotolerance of Saccharomyces cerevisiae VS1 and VS3 strains

After a previous mass screening and enrichment programme for the isolation of thermotolerant yeasts, VS1, VS2, VS3 and VS4 strains isolated from soil samples, collected within the hot regions of Kothagudem Thermal Power Plant, AP, India, had a better thermotolerance, osmotolerance and ethanol tolerance than the other isolates. Among these isolates VS1 and VS3 were best performers. Efforts were made to further improve their osmotolerance, thermotolerance and ethanol tolerance by treating them with UV radiation. Mutants of VS1 and VS3 produced more biomass and ethanol than the parent strains at high temperature and glucose concentrations. The amount of biomass produced by VS1 and VS3 mutants was 0.25 and 0.20 g l(-1) more than the parent strains at 42 degrees C using 2% glucose. At high glucose concentrations VS1 and VS3 mutants produced biomass which was 0.70 and 0.30 g l(-1) at 30 degrees C and 0.10 and 0.20 g l(-1) at 40 degrees C more than the parent strains. The amount of ethanol produced by the mutants (VS1 and VS3) was 8.20 and 1.20 g l(-1) more than the parent strains at 42 degrees C using 150 g l(-1) glucose. More ethanol was produced by mutants (VS1 and VS3) than the parents at high glucose concentrations of 5.0 and 6.0 g l(-1) at 30 degrees C and 13.0 and 3.0 g l(-1) at 42 degrees C, respectively. These results indicated that UV mutagenesis can be used for improving thermotolerance, ethanol tolerance and osmotolerance in VS1 and VS3 yeast strains.     

Screening and characterization of butanol‐tolerant micro‐organisms

Aims: Poor butanol tolerance of solventogenic stains directly limits their butanol production during industrial‐scale fermentation process. This study was performed to search for micro‐organisms possessing elevated tolerance to butanol. Methods and Results: Two strains, which displayed higher butanol tolerance compared to commonly used solventogenic Clostridium acetobutylicum, were isolated by evolution and screening strategies. Both strains were identified as lactic acid bacteria (LAB). On this basis, a LAB culture collection was tested for butanol tolerance, and 60% of the strains could grow at a butanol concentration of 2·5% (v/v). In addition, an isolated strain with superior butanol tolerance was transformed using a certain plasmid. Conclusions: The results indicate that many strains of LAB possessed inherent tolerance of butanol. Significance and Impact of the Study: This study suggests that LAB strains may be capable of producing butanol to elevated levels following suitable genetic manipulation.     

Survival characteristics of environmental and clinically derived strains of Cronobacter sakazakii in infant milk formula (IMF) and ingredients

Aims: The study aimed to compare survival of Cronobacter sakazakii strains in plant‐derived infant milk formula (IMF) ingredients and their thermotolerance in reconstituted IMF. Methods and Results: Inulin and lecithin were inoculated with isolates of C. sakazakii including the typed clinical strains, NCTC 11467^T and BAA 894; a mutant strain in which the wcaD gene had been disrupted; and two environmental strains isolated from IMF processing facilities. Samples were stored and examined for C. sakazakii. All strains were still detectable in both matrices after 338 days storage, except for the mutant strain that was no longer detectable at that time. Higher numbers of the environmental strains were recoverable after 338 days than the clinical strains. The thermotolerance of the five strains was investigated in reconstituted IMF at 55, 60 and 65°C. The clinically derived type strain, NCTC 11467^T, and the mutant strain were shown to be significantly more thermotolerant than other strains tested. Conclusions: Environmental strains were more persistent than the clinical strains in inulin and lecithin, indicating that patho‐adaptation may have contributed to a reduction in the desiccation tolerance phenotype. However, the thermotolerance results could indicate that the ability to produce extracellular polysaccharide decreases thermotolerance. Significance and Impact of the Study: These results indicate that desiccation resistance may play a role in survival of C. sakazakii in dry IMF ingredients and processing plants; however, this trait may be of less importance in clinical environs.     

GroESL overexpression imparts Escherichia coli tolerance to i-, n-, and 2-butanol, 1,2,4-butanetriol and ethanol with complex and unpredictable patterns

Strain tolerance to toxic metabolites remains a limiting issue in the production of chemicals and biofuels using biological processes. Here we examined the impact of overexpressing the autologous GroESL chaperone system with its natural promoter on the tolerance of Escherichia coli to several toxic alcohols. Strain tolerance was examined using both a growth assay as well as viable cell counts employing a CFU (colony-forming unit) assay. GroESL over expression enhanced cell growth to all alcohols tested, including a 12-fold increase in total growth in 48-h cultures under 4% (v/v) ethanol, a 2.8-fold increase under 0.75% (v/v) n-butanol, a 3-fold increase under 1.25% (v/v) 2-butanol, and a 4-fold increase under 20% (v/v) 1,2,4-butanetriol. GroESL overexpression resulted in a 9-fold increase in CFU numbers compared to a plasmid control strain after 24 h of culture under 6% (v/v) ethanol, and a 3.5-fold and 9-fold increase for culture under 1% (v/v) n-butanol and i-butanol, respectively. The toxicity of the alcohols was examined against their octanol–water partition coefficient, a measure commonly used to predict solvent toxicity. For both the control and the GroESL overexpressing strains, the calculated membrane concentration of each alcohol based on the octanol–water partition coefficient could be correlated, but with different patterns, to the impact of the various alcohols on cell growth, but not on cell viability (CFUs). Our data suggest a complex pattern of growth inhibition and differential protection by GroESL overexpression depending on the specific alcohol molecule. Overall, however, GroESL overexpression appears to provide molecule-agnostic tolerance to toxic chemicals.     

Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases

We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41°C, tolerate to 1.5% acetic acid or 4% ethanol at 39°C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37°C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4–5% ethanol at the same temperature. The fermentation ability at 37°C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37°C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.     

Isolation and characterization of ethanol tolerant yeast strains

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains.     

Respiratory chain analysis of Zymomonas mobilis mutants producing high levels of ethanol

We previously isolated respiratory-deficient mutant (RDM) strains of Zymomonas mobilis, which exhibited greater growth and enhanced ethanol production under aerobic conditions. These RDM strains also acquired thermotolerance. Morphologically, the cells of all RDM strains were shorter compared to the wild-type strain. We investigated the respiratory chains of these RDM strains and found that some RDM strains lost NADH dehydrogenase activity, whereas others exhibited reduced cytochrome bd-type ubiquinol oxidase or ubiquinol peroxidase activities. Complementation experiments restored the wild-type phenotype. Some RDM strains seem to have certain mutations other than the corresponding respiratory chain components. RDM strains with deficient NADH dehydrogenase activity displayed the greatest amount of aerobic growth, enhanced ethanol production, and thermotolerance. Nucleotide sequence analysis revealed that all NADH dehydrogenase-deficient strains were mutated within the ndh gene, which includes insertion, deletion, or frameshift. These results suggested that the loss of NADH dehydrogenase activity permits the acquisition of higher aerobic growth, enhanced ethanol production, and thermotolerance in this industrially important strain.     

几株啤酒酵母的筛选及发酵性能比较*

啤酒酵母是啤酒酿造的灵魂,可以直接影响啤酒品质。在啤酒酿造过程中,由于啤酒酵母被多次传代和保藏,造成优良菌种发酵性能衰退等问题,导致发酵不彻底,影响最后啤酒的风味质量。为此以8株Lager型啤酒酵母为出发菌株,通过平板分离纯化获得80株分离菌株,再经过三角瓶发酵初筛和复筛、发酵罐中试发酵实验最终获得了8株发酵性能优良的啤酒酵母。其中,6株酵母可应用于酿造双乙酰含量低于0.1 mg/L的啤酒;3株酵母发酵度高于70%,适合酿造干啤酒;1株酵母发酵度低于50%,适合酿造低醇啤酒。在风味方面:1株酵母酿造的啤酒醇酯比为3.3,啤酒酯香味较突出;另1株酵母酿造的啤酒醇酯比为4.5,啤酒高级醇含量较高。8株经过选育的啤酒酵母发酵特征明显,便于精酿啤酒厂实际应用。