Phenotypic characterisation of Saccharomyces spp. yeast for tolerance to stresses encountered during fermentation of lignocellulosic residues to produce bioethanol.

BACKGROUND: During industrial fermentation of lignocellulose residues to produce bioethanol, microorganisms are exposed to a number of factors that influence productivity. These include inhibitory compounds produced by the pre-treatment processes required to release constituent carbohydrates from biomass feed-stocks and during fermentation, exposure of the organisms to stressful conditions. In addition, for lignocellulosic bioethanol production, conversion of both pentose and hexose sugars is a pre-requisite for fermentative organisms for efficient and complete conversion. All these factors are important to maximise industrial efficiency, productivity and profit margins in order to make second-generation bioethanol an economically viable alternative to fossil fuels for future transport needs. RESULTS: The aim of the current study was to assess Saccharomyces yeasts for their capacity to tolerate osmotic, temperature and ethanol stresses and inhibitors that might typically be released during steam explosion of wheat straw. Phenotypic microarray analysis was used to measure tolerance as a function of growth and metabolic activity. Saccharomyces strains analysed in this study displayed natural variation to each stress condition common in bioethanol fermentations. In addition, many strains displayed tolerance to more than one stress, such as inhibitor tolerance combined with fermentation stresses. CONCLUSIONS: Our results suggest that this study could identify a potential candidate strain or strains for efficient second generation bioethanol production. Knowledge of the Saccharomyces spp. strains grown in these conditions will aid the development of breeding programmes in order to generate more efficient strains for industrial fermentations

The genetic basis of variation in clean lineages of Saccharomyces cerevisiae in response to stresses encountered during bioethanol fermentations.

Saccharomyces cerevisiae is the micro-organism of choice for the conversion of monomeric sugars into bioethanol. Industrial bioethanol fermentations are intrinsically stressful environments for yeast and the adaptive protective response varies between strain backgrounds. With the aim of identifying quantitative trait loci (QTL's) that regulate phenotypic variation, linkage analysis on six F1 crosses from four highly divergent clean lineages of S. cerevisiae was performed. Segregants from each cross were assessed for tolerance to a range of stresses encountered during industrial bioethanol fermentations. Tolerance levels within populations of F1 segregants to stress conditions differed and displayed transgressive variation. Linkage analysis resulted in the identification of QTL's for tolerance to weak acid and osmotic stress. We tested candidate genes within loci identified by QTL using reciprocal hemizygosity analysis to ascertain their contribution to the observed phenotypic variation; this approach validated a gene (COX20) for weak acid stress and a gene (RCK2) for osmotic stress. Hemizygous transformants with a sensitive phenotype carried a COX20 allele from a weak acid sensitive parent with an alteration in its protein coding compared with other S. cerevisiae strains. RCK2 alleles reveal peptide differences between parental strains and the importance of these changes is currently being ascertained

The genetic basis of variation in clean lineages of Saccharomyces cerevisiae in response to stresses encountered during bioethanol fermentations.

Saccharomyces cerevisiae is the micro-organism of choice for the conversion of monomeric sugars into bioethanol. Industrial bioethanol fermentations are intrinsically stressful environments for yeast and the adaptive protective response varies between strain backgrounds. With the aim of identifying quantitative trait loci (QTL's) that regulate phenotypic variation, linkage analysis on six F1 crosses from four highly divergent clean lineages of S. cerevisiae was performed. Segregants from each cross were assessed for tolerance to a range of stresses encountered during industrial bioethanol fermentations. Tolerance levels within populations of F1 segregants to stress conditions differed and displayed transgressive variation. Linkage analysis resulted in the identification of QTL's for tolerance to weak acid and osmotic stress. We tested candidate genes within loci identified by QTL using reciprocal hemizygosity analysis to ascertain their contribution to the observed phenotypic variation; this approach validated a gene (COX20) for weak acid stress and a gene (RCK2) for osmotic stress. Hemizygous transformants with a sensitive phenotype carried a COX20 allele from a weak acid sensitive parent with an alteration in its protein coding compared with other S. cerevisiae strains. RCK2 alleles reveal peptide differences between parental strains and the importance of these changes is currently being ascertained

PCR Applications to Brewing: Differentiation of Brewing Yeast Strains by PCR Fingerprinting

The increased use of pure starter cultures in the wine industry has made it necessary to develop a rapid and simple identification system for yeast strains. A method based upon the PCR using oligonucleotide primers that are complementary to intron splice sites has been developed. Since most introns are not essential for gene function, introns have evolved with minimal constraint. By targeting these highly variable sequences, the PCR has proved to be very effective in uncovering polymorphisms in commercial yeast strains. The speed of the method and the ability to analyze many samples in a single day permit the monitoring of specific yeast strains during fermentations. Furthermore, the simplicity of the technique, which does not require the isolation of DNA, makes it accessible to industrial laboratories that have limited molecular expertise and resources

Phenotypic microarray screening of (A) heterozygous diploid transformants (transformants labelled T1–T6) harbouring reciprocal deletions of COX20 alleles to acetic acid stress.

<p>DNA sequence comparisons (B) and protein sequence comparisons (C) of T5 and T6 transformants are shown along with their parental strains DBVPG6044 and Y12.</p

Expression data for genes using qPCR under (A) acetic acid stress present in loci identified on chromosome IV (region 921–1021) in the isogenic diploid parental strain Y12×DBVPG6044, (B) Expression data for genes present in loci identified on chromosome XII (region 599–699 kb) under osmotic stress in the isogenic diploid parental YPS128×DBVPH6765 and (C) Expression data for genes present in loci identified on chromosome XII (region 389–489 kb) under osmotic stress in the isogenic diploid parental YPS128×Y12.

<p>Expression data for genes using qPCR under (A) acetic acid stress present in loci identified on chromosome IV (region 921–1021) in the isogenic diploid parental strain Y12×DBVPG6044, (B) Expression data for genes present in loci identified on chromosome XII (region 599–699 kb) under osmotic stress in the isogenic diploid parental YPS128×DBVPH6765 and (C) Expression data for genes present in loci identified on chromosome XII (region 389–489 kb) under osmotic stress in the isogenic diploid parental YPS128×Y12.</p

Statistical comparison (using R) of F1 haploid segregants.

<p>F1 haploid segregants were grown under stress conditions from crosses (A) Y12×YPS128, (B) YPS128×DBVPG6765, (C) DBVPG6044×DBVPG6765, (D) Y12×DBVPG6044, (E) DBVPG6765×Y12 and (F) YPS128×DBVPG6044 for shared phenotypic response to acetic acid, formic acid, furfural, HMF, vanillin, osmotic (sorbitol), ethanol and temperature (35°C and 40°C).</p

Phenotypic microarray screening of (A) heterozygous diploid transformants (transformants labelled T1–T10) to osmotic stress harbouring reciprocal deletions of <i>RCK2</i> alleles (B) DNA and (C) Protein sequence comparison of <i>RCK2</i>-<i>::rck2</i> T2 and T10 transformants and their parental strains DBVPG6567 and YPS128.

<p>Phenotypic microarray screening of (A) heterozygous diploid transformants (transformants labelled T1–T10) to osmotic stress harbouring reciprocal deletions of <i>RCK2</i> alleles (B) DNA and (C) Protein sequence comparison of <i>RCK2</i>-<i>::rck2</i> T2 and T10 transformants and their parental strains DBVPG6567 and YPS128.</p

Assessment of variation of yeast populations to stresses encountered during bioethanol fermentations.

<p>F1 segregants from six pairwise crosses of four parental <i>S. cerevisiae</i> clean lineages were tested for (A) acetic acid, (B) formic acid, (C) HMF, (D) furfural (E) vanillin (F) sorbitol, (G) ethanol and (H) thermal (35°C) stress. Each population exhibited a range of tolerance and sensitivity beyond the parameters set by the phenotypic response of either parent.</p

Phenotypic microarray analysis (redox signal intensity) of F1 haploid segregants from a Y12×DBVPG6044 cross.

<p>Tolerance to (A) 10% and 15% sorbitol, (B) 10% and 15% ethanol, (C) 35°C and 40°C, (D) 25 mM acetic acid, (E) 10 mM formic acid, (F) 10 mM levulinic acid, (G) 5 mM HMF, (H) 5 mM furfural and (I) 5 mM vanillin are shown. The Y axis represents the % of RSI (redox signal intensity) where wells containing the listed stresses are compared to unstressed conditions. All yeast cells were grown in minimal medium with 6% glucose added at 30°C with the final data shown at the 25 hr time point. The values shown are an average of triplicate experiments including standard deviations.</p