A Novel Strategy to Construct Yeast Saccharomyces cerevisiae Strains for Very High Gravity Fermentation

Very high gravity (VHG) fermentation is aimed to considerably increase both the fermentation rate and the ethanol concentration, thereby reducing capital costs and the risk of bacterial contamination. This process results in critical issues, such as adverse stress factors (ie., osmotic pressure and ethanol inhibition) and high concentrations of metabolic byproducts which are difficult to overcome by a single breeding method. In the present paper, a novel strategy that combines metabolic engineering and genome shuffling to circumvent these limitations and improve the bioethanol production performance of Saccharomyces cerevisiae strains under VHG conditions was developed. First, in strain Z5, which performed better than other widely used industrial strains, the gene GPD2 encoding glycerol 3-phosphate dehydrogenase was deleted, resulting in a mutant (Z5ΔGPD2) with a lower glycerol yield and poor ethanol productivity. Second, strain Z5ΔGPD2 was subjected to three rounds of genome shuffling to improve its VHG fermentation performance, and the best performing strain SZ3-1 was obtained. Results showed that strain SZ3-1 not only produced less glycerol, but also increased the ethanol yield by up to 8% compared with the parent strain Z5. Further analysis suggested that the improved ethanol yield in strain SZ3-1 was mainly contributed by the enhanced ethanol tolerance of the strain. The differences in ethanol tolerance between strains Z5 and SZ3-1 were closely associated with the cell membrane fatty acid compositions and intracellular trehalose concentrations. Finally, genome rearrangements in the optimized strain were confirmed by karyotype analysis. Hence, a combination of genome shuffling and metabolic engineering is an efficient approach for the rapid improvement of yeast strains for desirable industrial phenotypes

The evolutionary rate variation among genes of HOG-signaling pathway in yeast genomes

Abstract Background Responses to extracellular stress are required for microbes to survive in changing environments. Although the stress response mechanisms have been characterized extensively, the evolution of stress response pathway remains poorly understood. Here, we studied the evolution of High Osmolarity Glycerol (HOG) pathway, one of the important osmotic stress response pathways, across 10 yeast species and underpinned the evolutionary forces acting on the pathway evolution. Results Although the HOG pathway is well conserved across the surveyed yeast species, the evolutionary rate of the genes in this pathway varied substantially among or within different lineages. The fast divergence of MSB2 gene indicates that this gene is subjected to positive selection. Moreover, transcription factors in HOG pathway tend to evolve more rapidly, but the genes in conserved MAPK cascade underwent stronger functional selection. Remarkably, the dN/dS values are negatively correlated with pathway position along HOG pathway from Sln1 (Sho1) to Hog1 for transmitting external signal into nuclear. The increased gradient of selective constraints from upstream to downstream genes suggested that the downstream genes are more pleiotropic, being required for a wider range of pathways. In addition, protein length, codon usage, gene expression, and protein interaction appear to be important factors to determine the evolution of genes in HOG pathway. Conclusions Taken together, our results suggest that functional constraints play a large role in the evolutionary rate variation in HOG pathway, but the genetic variation was influenced by quite complicated factors, such as pathway position, protein length and so on. These findings provide some insights into how HOG pathway genes evolved rapidly for responding to environmental osmotic stress changes. Reviewers This article was reviewed by Han Liang (nominated by Laura Landweber), Georgy Bazykin (nominated by Mikhail Gelfand) and Zhenguo Lin (nominated by John Logsdon).</p

New Polyketides from the Marine-Derived Fungus Letendraea Sp. 5XNZ4-2

Marine-derived fungi have been reported to have great potential to produce structurally unique metabolites. Our investigation on secondary metabolites from marine-derived fungi resulted in the isolation of seven new polyketides (phomopsiketones D&ndash;G (1&ndash;4) and letendronols A&ndash;C (5&ndash;7)) as well as one known xylarinol (8) in the cultural broth of Letendraea sp. Their structures and absolute configurations were elucidated using a set of spectroscopic and chemical methods, including HRESIMS, NMR, single-crystal X-ray diffraction, ECD calculation, and a modified version of Mosher&rsquo;s method. Compound 2 showed weak inhibition against nitric oxide production in lipopolysaccaride-activated macrophages with an IC50 value of 86 &mu;M

Trehalose concentrations and related enzymatic activity of stains Z5 and SZ3-1.

<p>Yeast strains were harvested at the stationary phase and exposed to ethanol stress. Trehalose concentration (A) of strains Z5 (light gray) and SZ3-1 (gray) subjected to different ethanol stress (0%, 5%, 10%, and 15% (v/v) ethanol) was measured. Finally, we determined related enzymatic activities, namely, trehalose-P-synthase (B), acid trehalase (C), and neutral trehalase (D) under 0% ethanol and 10% ethanol conditions.</p

Fatty acid compositions in plasma membrane and ergosterol content of strains Z5 and SZ3-1 cultivated in different conditions.

<p>Data are the mean values and standard deviation of three dependent experiments.</p>a<p>Fatty acids are denoted by the number of carbon atoms: number of unsaturated linkeages.</p>b<p>Unsaturation Index(Δ/mol) was calculated as: Δ/mol = [1×(% monoene)+2×(% diene)+3×(% triene)]/100.</p

The fermentation performance of strains Z5, Z5Δ<i>GPD2</i> and SZ3-1.

<p>The ethanol yield (A), glucose consumption (B), glycerol (filled symbols) and acetate (open symbols) production (C), and cell survival rate (D) of control strains Z5 (squares), Z5Δ<i>GPD2</i> (circles), and shuffled strain SZ3-1 (triangles) during fermentation were monitored and compared.</p

Karyotype profiles of strains Z5 and SZ3-1 obtained by PFGE.

<p>Lanes M, Z5 and SZ3-1 respectively represent the chromosomal profiles of strains BY4743, Z5 and SZ3-1. Numbers corresponding to each band are designated in the left.</p

Relative expression level of six genes related to trehalose metabolism.

<p>Strains Z5 and SZ3-1 were grown in the absence (0%) or presence (10%) of ethanol. Gene expression of four cases were compared: strain Z5 grown in the presence and absence of ethanol (Z5 (10%)/Z5 (0%)); strain SZ3-1 grown in the presence and absence of ethanol (SZ3-1 (10%)/SZ3-1 (0%)); strain SZ3-1 and Z5 grown in absence of ethanol (SZ3-1(0%)/Z5(0%)); and strain SZ3-1 and Z5 grown in the presence of ethanol (SZ3-1 (10%)/Z5 (10%)).</p