Whole-Genome Transformation of Yeast Promotes Rare Host Mutations with a Single Causative SNP Enhancing Acetic Acid Tolerance.

Whole-genome (WG) transformation (WGT) with DNA from the same or another species has been used to obtain strains with superior traits. Very few examples have been reported in eukaryotes-most apparently involving integration of large fragments of foreign DNA into the host genome. We show that WGT of a haploid acetic acid-sensitive Saccharomyces cerevisiae strain with DNA from a tolerant strain, but not from nontolerant strains, generated many tolerant transformants, some of which were stable upon subculturing under nonselective conditions. The most tolerant stable transformant contained no foreign DNA but only seven nonsynonymous single nucleotide polymorphisms (SNPs), of which none was present in the donor genome. The SNF4 mutation c.[805G→T], generating Snf4E269*, was the main causative SNP. Allele exchange of SNF4E269* or snf4Δ in industrial strains with unrelated genetic backgrounds enhanced acetic acid tolerance during fermentation under industrially relevant conditions. Our work reveals a surprisingly small number of mutations introduced by WGT, which do not bear any sequence relatedness to the genomic DNA (gDNA) of the donor organism, including the causative mutation. Spontaneous mutagenesis under protection of a transient donor gDNA fragment, maintained as extrachromosomal circular DNA (eccDNA), might provide an explanation. Support for this mechanism was obtained by transformation with genomic DNA of a yeast strain containing NatMX and selection on medium with nourseothricin. Seven transformants were obtained that gradually lost their nourseothricin resistance upon subculturing in nonselective medium. Our work shows that WGT is an efficient strategy for rapidly generating and identifying superior alleles capable of improving selectable traits of interest in industrial yeast strains.

Polygenic analysis of very high acetic acid tolerance in the yeast Saccharomyces cerevisiae reveals a complex genetic background and several new causative alleles.

BACKGROUND: High acetic acid tolerance is of major importance in industrial yeast strains used for second-generation bioethanol production, because of the high acetic acid content of lignocellulose hydrolysates. It is also important in first-generation starch hydrolysates and in sourdoughs containing significant acetic acid levels. We have previously identified snf4 E269* as a causative allele in strain MS164 obtained after whole-genome (WG) transformation and selection for improved acetic acid tolerance. RESULTS: We have now performed polygenic analysis with the same WG transformant MS164 to identify novel causative alleles interacting with snf4 E269* to further enhance acetic acid tolerance, from a range of 0.8-1.2% acetic acid at pH 4.7, to previously unmatched levels for Saccharomyces cerevisiae. For that purpose, we crossed the WG transformant with strain 16D, a previously identified strain displaying very high acetic acid tolerance. Quantitative trait locus (QTL) mapping with pooled-segregant whole-genome sequence analysis identified four major and two minor QTLs. In addition to confirmation of snf4 E269* in QTL1, we identified six other genes linked to very high acetic acid tolerance, TRT2, MET4, IRA2 and RTG1 and a combination of MSH2 and HAL9, some of which have never been connected previously to acetic acid tolerance. Several of these genes appear to be wild-type alleles that complement defective alleles present in the other parent strain. CONCLUSIONS: The presence of several novel causative genes highlights the distinct genetic basis and the strong genetic background dependency of very high acetic acid tolerance. Our results suggest that elimination of inferior mutant alleles might be equally important for reaching very high acetic acid tolerance as introduction of rare superior alleles. The superior alleles of MET4 and RTG1 might be useful for further improvement of acetic acid tolerance in specific industrial yeast strains.

Polygenic analysis and targeted improvement of the complex trait of high acetic acid tolerance in the yeast Saccharomyces cerevisiae.

BACKGROUND: Acetic acid is one of the major inhibitors in lignocellulose hydrolysates used for the production of second-generation bioethanol. Although several genes have been identified in laboratory yeast strains that are required for tolerance to acetic acid, the genetic basis of the high acetic acid tolerance naturally present in some Saccharomyces cerevisiae strains is unknown. Identification of its polygenic basis may allow improvement of acetic acid tolerance in yeast strains used for second-generation bioethanol production by precise genome editing, minimizing the risk of negatively affecting other industrially important properties of the yeast. RESULTS: Haploid segregants of a strain with unusually high acetic acid tolerance and a reference industrial strain were used as superior and inferior parent strain, respectively. After crossing of the parent strains, QTL mapping using the SNP variant frequency determined by pooled-segregant whole-genome sequence analysis revealed two major QTLs. All F1 segregants were then submitted to multiple rounds of random inbreeding and the superior F7 segregants were submitted to the same analysis, further refined by sequencing of individual segregants and bioinformatics analysis taking into account the relative acetic acid tolerance of the segregants. This resulted in disappearance in the QTL mapping with the F7 segregants of a major F1 QTL, in which we identified HAA1, a known regulator of high acetic acid tolerance, as a true causative allele. Novel genes determining high acetic acid tolerance, GLO1, DOT5, CUP2, and a previously identified component, VMA7, were identified as causative alleles in the second major F1 QTL and in three newly appearing F7 QTLs, respectively. The superior HAA1 allele contained a unique single point mutation that significantly improved acetic acid tolerance under industrially relevant conditions when inserted into an industrial yeast strain for second-generation bioethanol production. CONCLUSIONS: This work reveals the polygenic basis of high acetic acid tolerance in S. cerevisiae in unprecedented detail. It also shows for the first time that a single strain can harbor different sets of causative genes able to establish the same polygenic trait. The superior alleles identified can be used successfully for improvement of acetic acid tolerance in industrial yeast strains.

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

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).

[Knowledge, attitudes and use of dietary supplement among students of the University of Nis (Serbia)].

INTRODUCTION: Dietary supplements, being concentrated sources of vitamins, minerals and other substances with nutritional and physiological effects, are products that supplement the normal diet. The aim of this study was to determine the use, attitudes and knowledge on dietary supplements among students of the University of Nis, paying special attention to differences in responses between male and female students. MATERIAL AND METHODS: The study used the original questionnaire which was distributed to 330 students from the University of Nis. The sample was stratified by school, year of study and sex. Out of 330 questionnaires, 301 were classified as correct and they were further analyzed. RESULTS: Dietary supplements were used by 68.10% of the respondents. Females used dietary supplements more than males. Two or more products were most commonly used. Multivitamins were the most widely used products. The majority of respondents adhered to the declaration (77.07%). A small number had health damage (2.93%). More than half of the students stated that they were not properly informed about dietary supplements. The most common source of information was the media and friends. CONCLUSION: Health professionals and users of dietary supplements should be educated better on the use of these products in order to make the supplement use safe, controlled and rational.

Comparative analysis of autodermal graft and polypropylene mesh use in large incisional hernia defects reconstruction.

BACKGROUND: Large defects of the abdominal wall caused by incisional hernia still represent a challenging problem in plastic, reconstructive, and abdominal surgery. For their successful tension-free repair a proper selection of reconstructive material is essential. In the last decades, the use of synthetic meshes was dominant while biological autodermal grafts were rarely used. The aim of the study was to comparatively analyse efficacy and safety of autodermal graft and polypropylene mesh in surgical treatment of large abdominal wall defects. METHODS: This prospective comparative clinical study enroled 40 patients surgically treated for large incisional hernia repair in a 10-year period. The patients were divided into two equal groups consisting of 20 subjects and treated either by biological autodermal graft or by synthetic polypropylene mesh. The surgical techniques of reconstruction, duration of surgery, the occurrence of early, minor, and major (severe) and delyed complications and hospital stay were analysed. The average follow-up took 2 years. RESULTS: Statistically significant differences in demographic characteristics of patients and in size of defects were not found. The surgical technique of reconstruction with an autodermal graft was more complicated. The duration of surgery in patients treated with autodermal grafts was significantly longer. There was no statistically significant difference regarding occurrence of early, minor postoperative complications and hospital stay in our study. Two severe complications were registered in the synthetic mesh group: intestinal obstruction and enterocutaneous fistula. The recurrence rate was 10% in the autodermal graft group and 15% in the group with a synthetic mesh. CONCLUSION: Tension-free repair of large incisional hernia with autodermal grafts was unjustly neglected despite the fact that it is safe and effective. It can be applied in all cases where synthetic mesh are not indicated (presence of infection, immunodeficient patients, after radiotherapy). They are especially important in war surgery and in lack of funds when commercial grafts cannot be purchased.

Tobacco use among youth: findings from the global youth tobacco survey in Montenegro.

Smoking is a burning healthcare and economy issue, especially in underdeveloped countries. The aim of this study was to determine the number of smokers among elementary school students in Montenegro and to assess the correlates of tobacco use. The study was done in 2003 using the World Health Organization Global Youth Tobacco Survey. Our data showed that children as young as ten years smoked. There were 3.6% permanent smokers and one in three students (30.6%) experimented with smoking. More than two thirds who smoked agreed that they should quit smoking, and three fourths tried to quit. This study has also shown that children talk too little about smoking in schools and are exposed to passive smoking at home and elsewhere. Activities to solve the elementary school smoking problem should include preventive programs to be introduced into regular school curricula because this is the only way to address the issue properly. In addition, legislation prohibiting indoor tobacco smoking should be implemented rigorously to protect children from passive smoking in public places.