Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression

<p>Abstract</p> <p>Background</p> <p>Hydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and β-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation. In this study, using our previously developed method to optimize cellulase-expression levels in yeast, we constructed a diploid <it>Saccharomyces cerevisiae </it>strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw, one of the most abundant sources of lignocellulosic biomass.</p> <p>Results</p> <p>The engineered diploid strain, which contained multiple copies of three cellulase genes integrated into its genome, was precultured in molasses medium (381.4 mU/g wet cell), and displayed approximately six-fold higher phosphoric acid swollen cellulose (PASC) degradation activity than the parent haploid strain (63.5 mU/g wet cell). When used to ferment PASC, the diploid strain produced 7.6 g/l ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/l ethanol from pretreated rice straw in 72 hours.</p> <p>Conclusions</p> <p>We have developed diploid yeast strain optimized for expression of cellulolytic enzymes, which is capable of directly fermenting from cellulosic materials. Although this is a proof-of-concept study, it is to our knowledge, the first report of ethanol production from agricultural waste biomass using cellulolytic enzyme-expressing yeast without the addition of exogenous enzymes. Our results suggest that combining multigene expression optimization and diploidization in yeast is a promising approach for enhancing ethanol production from various types of lignocellulosic biomass.</p

Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus <it>T. reesei </it>effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant <it>Saccharomyces cerevisiae</it>, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration.</p> <p>Results</p> <p>In cocktail δ-integration, several kinds of cellulase expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (i.e., expressing an optimum ratio of cellulases) are easily obtained. Although the total integrated gene copy numbers of cocktail δ-integrant strain was about half that of a conventional δ-integrant strain, the phosphoric acid swollen cellulose (PASC) degradation activity (64.9 mU/g-wet cell) was higher than that of a conventional strain (57.6 mU/g-wet cell). This suggests that optimization of the cellulase expression ratio improves PASC degradation activity more so than overexpression.</p> <p>Conclusions</p> <p>To our knowledge, this is the first report on the expression of cellulase genes by δ-integration and optimization of various foreign genes by δ-integration in yeast. This method should be very effective and easily applied for other multi-enzymatic systems using recombinant yeast.</p

Loss of Parp-1 affects gene expression profile in a genome-wide manner in ES cells and liver cells

BACKGROUND: Many lines of evidence suggest that poly(ADP-ribose) polymerase-1 (Parp-1) is involved in transcriptional regulation of various genes as a coactivator or a corepressor by modulating chromatin structure. However, the impact of Parp-1-deficiency on the regulation of genome-wide gene expression has not been fully studied yet. RESULTS: We employed a microarray analysis covering 12,488 genes and ESTs using mouse Parp-1-deficient (Parp-1(-/-)) embryonic stem (ES) cell lines and the livers of Parp-1(-/- )mice and their wild-type (Parp-1(+/+)) counterparts. Here, we demonstrate that of the 9,907 genes analyzed, in Parp-1(-/- )ES cells, 9.6% showed altered gene expression. Of these, 6.3% and 3.3% of the genes were down- or up-regulated by 2-fold or greater, respectively, compared with Parp-1(+/+ )ES cells (p < 0.05). In the livers of Parp-1(-/- )mice, of the 12,353 genes that were analyzed, 2.0% or 1.3% were down- and up-regulated, respectively (p < 0.05). Notably, the number of down-regulated genes was higher in both ES cells and livers, than that of the up-regulated genes. The genes that showed altered expression in ES cells or in the livers are ascribed to various cellular processes, including metabolism, signal transduction, cell cycle control and transcription. We also observed expression of the genes involved in the pathway of extraembryonic tissue development is augmented in Parp-1(-/- )ES cells, including H19. After withdrawal of leukemia inhibitory factor, expression of H19 as well as other trophoblast marker genes were further up-regulated in Parp-1(-/- )ES cells compared to Parp-1(+/+ )ES cells. CONCLUSION: These results suggest that Parp-1 is required to maintain transcriptional regulation of a wide variety of genes on a genome-wide scale. The gene expression profiles in Parp-1-deficient cells may be useful to delineate the functional role of Parp-1 in epigenetic regulation of the genomes involved in various biological phenomena

Erythropoietin Receptor Signaling Mitigates Renal Dysfunction-Associated Heart Failure by Mechanisms Unrelated to Relief of Anemia

ObjectivesWe examined the effect of asialoerythropoietin (asialoEPO), a nonerythrogenic derivative of erythropoietin (EPO), on renal dysfunction-associated heart failure.BackgroundAlthough EPO is known to exert beneficial effects on cardiac function, the clinical benefits in patients with chronic kidney disease are controversial. It remains to be addressed whether previously reported outcomes were the result of relief of the anemia, adverse effects of EPO, or direct cardiovascular effects.MethodsMice underwent 5/6 nephrectomy to cause renal dysfunction. Eight weeks later, when renal dysfunction was established, anemia and cardiac dysfunction and remodeling were apparent. Mice were then assigned to receive saline (control), recombinant human erythropoietin (rhEPO) at 5,000 IU (714 pmol)/kg, or asialoEPO at 714 pmol/kg, twice/week for 4 weeks.ResultsAlthough only rhEPO relieved the nephrectomy-induced anemia, both rhEPO and asialoEPO significantly and similarly mitigated left ventricular dilation and dysfunction. The hearts of rhEPO- or asialoEPO-treated mice showed less hypertrophy, reflecting decreases in cardiomyocyte hypertrophy and degenerative subcellular changes, as well as significant attenuation of fibrosis, leukocyte infiltration, and oxidative deoxyribonucleic acid damage. These phenotypes were accompanied by restored expression of GATA-4, sarcomeric proteins, and vascular endothelial growth factor and decreased inflammatory cytokines and lipid peroxidation. Finally, myocardial activation was observed of extracellular signal-regulated protein kinase and signal transducer and activator of transcription pathways in the treated mice.ConclusionsEPO receptor signaling exerts direct cardioprotection in an animal model of renal dysfunction-associated heart failure, probably by mitigating degenerative, pro-fibrosis, inflammatory, and oxidative processes but not through relief of anemia

Disposition of protein-bound 3-nitrotyrosine in rat plasma analysed by a novel protocol for HPLC-ECD

金沢大学医薬保健研究域医学系3-Nitrotyrosine (NTyr) is considered as a biomarker of the generation of reactive nitrogen species (RNS). However, it is still difficult to determine its concentration in biological samples. To develop a reliable and high-throughput method, we optimized the conditions for high performance liquid chromatography and electrochemical detection (HPLC-ECD). The best separation of NTyr was achieved using a highly acidic mobile phase (pH 2.5). The concentration of protein-bound NTyr in plasma protein was 593.6 ± 53.8 fmol/mg in rats treated with lipopolysaccharide (LPS) and 114.4 ± 27.6 fmol/mg in control. After intravenous administration of in vitro-nitrated plasma protein, NTyr concentration decreased; the half-life was 63.4 ± 16.8 h. Consistently, protein-bound NTyr concentration in plasma after LPS treatment declined gradually, but was detectable for 1 week. Our protocol is reproducible and suitable for analysing multiple clinical samples to study RNS production in vivo. © 2007 The Japanese Biochemical Society

Existence of a Neuropathic Pain Component in Patients with Osteoarthritis of the Knee

∙ The authors have no financial conflicts of interest. © Copyright: Yonsei University College of Medicine 2012 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens

Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface

G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP) strategy). In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs