Central carbon metabolism of characterized yeast species in this study.

<p>Different yeast species were studied for their carbon metabolism and the results illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-g003" target="_blank">figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-g004" target="_blank">4</a> are summarized in this table. Yield of products (biomass and ethanol) relative to consumed substrate (glucose) is presented in the unit g/g (gram product per gram substrate) and is calculated by dividing the amount of product (at the maximum of ethanol concentration for Crabtree positive yeasts, or when glucose was depleted for Crabtree negative yeasts) with the amount of substrate consumed. The consumption rates of substrate and production rate of products is presented in the unit g/g,h (gram substrate or product per gram biomass per hour), and is calculated during the exponential phase of growth by dividing the amount of consumed glucose or produced product with the amount of produced biomass and multiplied with the corresponding specific growth rate. Characterized species names and different collections (Y, CBS and other) that provide them are mentioned in separate columns.</p

Yeast glucose consumption, ethanol production and growth rates.

<p>Different yeast species were studied for their carbon metabolism and the results are shown as: specific glucose consumption rate (g/g h^-1 amount of consumed glucose by 1 g of biomass and multiplied with the corresponding specific growth rate, shown in blue), specific ethanol production rate (g/g h^-1 ethanol produced by 1 g biomass and multiplied with the corresponding specific growth rate, shown in red), and specific growth rate (per hour, shown in green). Detailed results and biological replicates are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-t001" target="_blank">table 1</a> but hereby either a single measurement or an average of two replicates are presented. The yeast species are presented starting with the <i>Saccharomyces</i> genus at the top and then following a decreasing phylogenetic relationship, according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-g001" target="_blank">figure 1</a>. The species related the least to <i>S. cerevisiae</i> are at the bottom, and the gap divides the <i>Saccharomycotina</i> and non-<i>Saccharomycotina</i> yeasts. The four groups of yeasts (1, 2, 3 and 4) used in the statistical analysis are shown (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-g003" target="_blank">figure 3</a>). Specific glucose consumption rate decreases with the phylogenetic distance from the <i>Saccharomyces</i> genus, indicating that Crabtree negative yeasts have only a moderate rate, while Crabtree positive have faster glucose consumption rate. On the other hand, the growth rate (in green) is very similar among all <i>Saccharomycotina</i> yeasts (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-g005" target="_blank">figure 5</a> C and D).</p

Statistical analysis of the fermentation parameters.

<p>Yeasts have been grouped into the following groups: Group 1 included all tested species belonging to <i>Eremothecium</i> and <i>Kluyveromyces</i>, group 2 all strains of the <i>Lachancea</i>, <i>Torulaspora</i> and <i>Zygotorulaspora</i> genera, group 3 contain the <i>Vandervaltozyma</i> and <i>Tetrapisispora</i>, and group 4 all species belonging to the <i>Saccharomyces</i>, <i>Kazachstania</i>, <i>Naumovozyma</i> and <i>Nakaseomyces</i> genera. The group mean values on four parameters: ethanol yield (A), biomass yield (B), glucose consumption rate (C) and growth rate (D) are illustrated. All error bars in the figure cover a 95% confidence interval for each group. Group 3 appears to be an intermediate between group 2 and 4 for all parameters except growth rate. Hence, no significant difference in growth rates among groups can be observed. These results are also supported by statistical analysis of variance and pairwise t-test (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-t002" target="_blank">table 2</a>).</p

Yeast ethanol and biomass yield.

<p>Different yeast species were studied for their carbon metabolism: ethanol yield as g of ethanol per g of glucose (red), and biomass yield as g of biomass per g of glucose (blue). Detailed results and biological replicates are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-t001" target="_blank">table 1</a> but hereby either a single measurement or an average of two replicates are presented. The yeast species are presented starting with the <i>Saccharomyces</i> genus at the top and then following a decreasing phylogenetic relationship, following <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-g001" target="_blank">figure 1</a>. The species related the least to <i>S. cerevisiae</i> are at the bottom, and the gap divides the <i>Saccharomycotina</i> and non-<i>Saccharomycotina</i> yeasts. The four groups of yeasts (1, 2, 3 and 4) used in the statistical analysis are shown. In general, the ethanol yield gradually drops and the biomass yield gradually increases with the genetic distance from the <i>Saccharomyces</i> yeasts (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-g005" target="_blank">figure 5 A and B</a>).</p

Statistical test and correlation analysis.

<p>This table summarizes the results from the statistical analysis conducted on our results. Tests between two parameters (under x- and y-column) are considered to be significant at a significance level (alpha = 5%) or p-value lower than 0.05. ANOVA and Kruskal-Wallis test amongst the four groups reveal significant differences in all parameters (except for growth rate). Hence, there is no significant difference in growth rate among groups 1, 2, 3 and 4. The groups were defined in the main text as well as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-g005" target="_blank">figure 5</a>. Pairwise t-tests performed separately on all combinations of groups, on all variables (except growth rate) reveal significant differences between all group combinations (except between groups 2–3, and groups 3–4). Once more, t-test failed to detect any significant differences in growth rates between groups 1, 2, 3 and 4, and the results indicate that group 3 can be seen as an intermediate between group 2 and 4 (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-g005" target="_blank">figure 5</a>). A highly significant correlation between glucose consumption rate and biomass yield is seen on both parametric and non-parametric tests. Thus, our data support a linear model that explains 49% of the variation (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone.0068734.s007" target="_blank">table S3</a> for comparison within groups). Furthermore a significant correlation between glucose consumption rate and growth rate, and no significant correlation between growth rate and ethanol yield can be seen with both parametric and non-parametric tests.</p

Phylogenetic relationship among yeast.

<p>A schematic phylogenetic relationship, based on the phylogenetic tree from Kurtzman and Robnett (2003) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone.0068734-Kurtzman2" target="_blank">[16]</a>, covering twelve genera of <i>Saccharomycetaceae</i> and all employed species. Note that alternative models to explain the phylogenetic relationship between the <i>Lachancea</i>, <i>Kluyveromyces</i> and <i>Eremothecium</i> genera have been proposed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone.0068734-Jeffroy1" target="_blank">[23]</a> but here we follow the tree in ref. 15. Several evolutionary events, which are relevant for the modern traits, are shown. Note that the relative timing of some events, especially those which left a clear finger-print in the modern genomes (green arrows) is relatively precise, such as WGD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone.0068734-Wolfe1" target="_blank">[10]</a>, the horizontal transfer of a bacterial »anaerobic« DHODase (encoded by <i>URA1</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone.0068734-Gojkovic1" target="_blank">[19]</a>, complete rewiring of the respiration related promoters (RGE stands for Rapid Growth Elements) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone.0068734-Pronk1" target="_blank">[3]</a>, and the loss of respiratory Complex I <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone.0068734-Dujon1" target="_blank">[9]</a>, while the timing of more complex traits (red arrows), such as the capability for anaerobic growth <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone.0068734-Moller1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone.0068734-Merico1" target="_blank">[13]</a> and petite positivity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone.0068734-Merico1" target="_blank">[13]</a>, might be less precise.</p

Yeast growth profiles.

<p>A few examples of a representative batch culture experiment showing different capacity to produce ethanol and biomass in the presence of excess glucose and oxygen: <i>Sac. eubayanus</i> (A), <i>Kaz. exiguus</i> (B), <i>Zto. mrakii</i> (C), <i>Lac. waltii</i> (D), <i>Klu. wickerhamii</i> (E) and <i>Ere. sinecaudum</i> (F). The graphs show time dependence of yeast glucose consumption, and appearance of the fermentation products and biomass. The ethanol and biomass yields vary enormously among the six shown species, as well as among other studied yeasts (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-t001" target="_blank">table 1</a>), and are related to the phylogenetic position of each studied yeast species (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068734#pone-0068734-g001" target="_blank">figure 1</a>).</p

Location of the breakpoint sequences of the deletion in the <i>KLK15</i> gene.

<p>The deletion in the <i>KLK15</i> gene is located between nucleotide 56 022 311 in intron 2 and nucleotide 56 025 704 in intron 1 of the <i>KLK15</i> gene, which results in a 3394-bp deletion eliminating exon 2.</p

<i>PARS2</i> gene region with exons and analyzed SNP markers.

<p>The linkage disequilibrium pattern (LD) are reported as R² and estimated using the EUR population of the 1000Genomes project.</p

Comparison of variants found in 310 CRS patients and 379 EUR background population controls from the 1000Genomes project.

<p>Circles denote synonymous mutations and stars denote missense mutations. Intron 1 has been shortened by 3.5 kb to increase resolution.</p