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Ammonia is an essential substance for agriculture and the chemical industry. The intracellular production of ammonia in yeast (Saccharomyces cerevisiae) by metabolic engineering is difficult because yeast strongly assimilates ammonia, and the knockout of genes enabling this assimilation is lethal. Therefore, we attempted to produce ammonia outside the yeast cells by displaying a glutaminase (YbaS) from Escherichia coli on the yeast cell surface. YbaS-displaying yeast successfully produced 3.34 g/L ammonia from 32.6 g/L glutamine (83.2% conversion rate), providing it at a higher yield than in previous studies. Next, using YbaS-displaying yeast, we also succeeded in producing ammonia from glutamine in soybean residues (okara) produced as food waste from tofu production. Therefore, ammonia production outside cells by displaying ammonia-lyase on the cell surface is a promising strategy for producing ammonia from food waste as a novel energy resource, thereby preventing food loss. [ABSTRACT FROM AUTHOR]

Brown macroalgae are characterized by a large size and high productivity without requiring arable land, fresh water, and fertilizer. Furthermore, since brown macroalgae contain little or no lignin, simple biorefinery processing can efficiently produce sugars from this material. Therefore, brown macroalgae have attracted attention as an alternative feedstock for bioethanol production. However, the utilization of biotechnologies previously developed for terrestrial biomass processing results in difficulties in the bioconversion of brown macroalgae. Recently, several studies have developed biotechnologies for using major carbohydrates of brown macroalgae, such as laminarin, mannitol, and alginate. This review focuses on these fermentation biotechnologies using natural or engineered microorganisms. [ABSTRACT FROM AUTHOR]

Biological technologies for recycling rare metals, which are essential for high-tech products, have attracted much attention because they could prove to be more environmentally friendly and energy-saving than other methods. We have developed biological recycling technologies by cell surface engineering for the selective recovery of toxic heavy metal ions and rare metal ions from aqueous wastes. In this study, we aimed to construct a unique biological technique to recover rare metals 'in solid' form by reducing rare metal ions, leading to a practical next-generation recovery system. Sulfate-reducing bacteria (SRB) can reduce Pt(II) to Pt(0), and hydrogenases of SRB contribute to the reduction. Therefore, we constructed yeasts displaying their hydrogenases on the 'cell membrane', and reduction experiments were performed under anaerobic conditions without any electron donors. As a result, hydrogenase-displaying yeasts produced black precipitates in PtCl solution. Based on X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM) observations, the constructed yeasts were found to successfully produce the precipitates of Pt(0) through the reduction of Pt(II). Interestingly, the precipitates of Pt(0) were formed as nanoparticles, suitable for industrial usage. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide-1 (GLP1) is a 30-amino acid peptide hormone activating the GLP1 receptor (GLP1R), a class B G-protein coupled receptor (GPCR), and is considered to be effective for treating diabetes and other metabolic diseases. Phage display is the first innovative technology in order to prepare and screen a large polypeptide library including GLP1R agonists, but this methodology is not as effective in discovering functional peptides such as activators for GPCRs. Here, we report a novel functional screening system for GPCR-acting peptides, which integrates a yeast peptide secretion system into a biological detection system with GPCR-producing mammalian cells. Using this screening system, we found attractive GLP1R agonists with several substitutions from a random mutant GLP1 library which was secreted by yeast, Saccharomyces cerevisiae . This system established here not only enables peptides to be analyzed in the soluble form but also needs no chemical synthesis, purification, and condensation of peptides of interests, and therefore, can be widely applied to the discovery of novel bioactive peptides acting on GPCRs. [ABSTRACT FROM AUTHOR]

Rhizopus oryzae lipase (ROL) has a propeptide at its N-terminus that functions as an intramolecular chaperone and facilitates the folding of mature ROL (mROL). In this study, we successfully generated a functionally distinct imprinted mROL (mROLimp) through protein folding memory using a mutated propeptide. The mutated propeptide left its structural memory on mROL and produced mROLimp that exhibited different substrate specificities compared with mROLWT (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROLimp showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate. In addition, ROLimp was more stable than mROLWT. These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity. [ABSTRACT FROM AUTHOR]

We report the demonstration of ultrathin Ge-on-insulator (GOI) inversion-type nMOSFETs, fabricated by the optimized Ge condensation technique and solid-phase diffusion of Sb. The GOI structures with low hole concentration of \(10^{\mathrm {\mathbf {17}}}\) cm \(^{\mathrm {\mathbf {-3}}}\) or less are realized by optimizing oxidation temperature and inserting annealing process for enhancement of intermixing in the Ge condensation process recipe. The hole concentration in the GOI body has been systematically analyzed with different oxidation temperature and insertion of annealing process. Highly doped n+ source/drain regions are formed in 16-nm-thick GOI layers by Sb solid-phase diffusion doping from spin-on-glass at 650 °C. The high I \(_{\mathbf {\mathrm{{\scriptstyle ON}}}}\) /I \(_{\mathbf {\mathrm{{\scriptstyle OFF}}}}\) ratio of \(10^{\mathrm {\mathbf {4}}}\) is observed in the fabricated nMOSFETs. The peak electron mobility of 107 cm \(^{\mathrm {\mathbf {2}}}\) /Vs is obtained for the inversion-type GOI nMOSFETs. [ABSTRACT FROM PUBLISHER]

Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to the surface-displayed proteins/peptides. A part of various cell wall or plasma membrane proteins can be genetically fused to the proteins/peptides of interest to be displayed. This technology, leading to the generation of so-called "arming technology", can be employed for basic and applied research purposes. In this article, we describe various strategies for the construction of arming yeasts, and outline the diverse applications of this technology to industrial processes such as biofuel and chemical productions, pollutant removal, and health-related processes, including oral vaccines. In addition, arming technology is suitable for protein engineering and directed evolution through high-throughput screening that is made possible by the feature that proteins/peptides displayed on cell surface can be directly analyzed using intact cells without concentration and purification. Actually, novel proteins/peptides with improved or developed functions have been created, and development of diagnostic/therapeutic antibodies are likely to benefit from this powerful approach. [ABSTRACT FROM AUTHOR]

By cell surface display of ModE protein that is a transcriptional regulator of operons involved in the molybdenum metabolism in Escherichia coli, we have constructed a molybdate-binding yeast (Nishitani et al., Appl Microbiol Biotechnol 86:641-648, ). In this study, the binding specificity of the molybdate-binding domain of the ModE protein displayed on yeast cell surface was improved by substituting the amino acids involved in oxyanion binding with other amino acids. Although the displayed S126T, R128E, and T163S mutant proteins adsorbed neither molybdate nor tungstate, the displayed ModE mutant protein (T163Y) abolished only molybdate adsorption, exhibiting the specific adsorption of tungstate. The specificity of the displayed ModE mutant protein (T163Y) for tungstate was increased by approximately 9.31-fold compared to the displayed wild-type ModE protein at pH 5.4. Therefore, the strategy of protein design and its cell surface display is effective for the molecular breeding of bioadsorbents with metal-specific adsorption ability based on a single species of microorganism without isolation from nature. [ABSTRACT FROM AUTHOR]

Antibiotics have significantly improved our living environments. However, overuse of antibiotics has led to the emergence of multi-drug resistant microorganisms, and the subsequent constant demand for the exploration of novel antibiotics. To this end, antimicrobial peptides (AMPs) have attracted much attention as a novel class of antibiotics. AMPs have strong antimicrobial activity against a wide-range of species, including gram-positive and gram-negative bacteria, fungi, and viruses. In addition, they are also effective against pathogenic organisms that are resistant to conventional drugs. Despite their great potential, the hemolytic activity and a highly broad spectrum of activity of AMPs dictate the need for amendments to develop safe pharmaceuticals. The human body contains commensal microflora as an integral part of complex mucosal surfaces that offers protection against pathogenic organisms. Administration of antibiotics with broad spectra of activity disrupts the indigenous microflora and increases the risks of diarrhea and other fatal infections. Therefore, it is difficult, but vital, to develop treatments capable of rapidly eliminating pathogenic organisms while maintaining the commensal microbiota. As such, novel pharmaceuticals, safe designer AMPs have been heavily researched. In this article, we review recent attempts to spatially and temporally regulate AMPs to enhance the quality-of-life of patients. [ABSTRACT FROM AUTHOR]

Abstract: Saccharomyces cerevisiae cannot utilize cellulose as a carbon source. In nature, cellulose is mainly degraded into glucose by 3 cellulases: endoglucanase II (EG), cellobiohydrolase II (CBH), and β-glucosidase I (BG). A yeast codisplaying these 3 enzymes was constructed by cell surface engineering to directly ferment cellulose into ethanol. The constructed yeast was used to directly ferment newspaper—a carbon source. The newspaper was first pretreated with Trametes sp. Ha1 laccase, and the laccase-treated newspaper was used for investigating the fermentation by the constructed yeast. The results indicated that the laccase-pretreated waste paper could serve as a resource for the production of ethanol by direct fermentation mediated by yeast codisplaying EG, CBH, and BG. [Copyright &y& Elsevier]

The article presents the findings of a research conducted on the identification of interaction site of propeptide toward mature carboxypeptidase Y (mCPY), based on the similarity between propeptide and CPY inhibitor (IC). Under the research, sequence alignment between IC and proCPY indicated that a sequence similar to the N-terminal region of IC was present in proCPY. It concludes that C-terminal region of the propeptide might bind to the substrate-binding site of mCPY.

The article discusses a study which investigated the production of reactive oxygen species (ROS), as well as its apoptosis induction through Gts1p-mediated protein aggregate formation. The study used agarose gel electrophoresis to examine the degrees of Gts1p-mediated protein aggregation. Results suggested that protein aggregates mediated by Gts1p influence the mitochondrial regulation of ROS apoptosis and production.

The genetic engineering of microorganisms to adsorb metal ions is an attractive method to facilitate the environmental cleanup of metal pollution and to enrich the recovery of metal ions such as rare metal ions. For the recovery of metal ions by microorganisms, cell surface design is an effective strategy for the molecular breeding of bioadsorbents as an alternative to intracellular accumulation. The cell surface display of known metal-binding proteins/peptides and the molecular design of novel metal-binding proteins/peptides have been performed using a cell surface engineering approach. The adsorption of specific metal ions is the important challenge for the practical recovery of metal ions. In this paper, we discuss the recent progress in surface-engineered bioadsorbents for the recovery of metal ions. [Copyright &y& Elsevier]

The article presents a study on how GTS1, a pleiotropic gene, affects a variety of biological phenomena in a gene-dose-dependent manner. It notes that a genome-wide transcriptional analysis determined genes in response to GTS1 induction which said to have caused derepression of Tup1-Cyc8-repressing genes and chromatin remodeling through the interaction of Gts1p with Cyc8p. Moreover, the study finds that inhibition of Tup1-Cyc8 results in Gts1p-mediated gene depression.

The article discusses a study which determines the interaction partners of Saccharomyces cerevisiae gene GTSI. It states that tandem affinity, purification and immunoprecipitation were made to know the factors which are important on the inhibition process of GTSI. Findings of the study reveal Ssa1p and Ssa2p, members of heat-shock protein family, as the valuable factors.

Large amounts of fossil fuels are consumed every day in spite of increasing environmental problems. To preserve the environment and construct a sustainable society, the use of biofuels derived from different kinds of biomass is being practiced worldwide. Although bioethanol has been largely produced, it commonly requires food crops such as corn and sugar cane as substrates. To develop a sustainable energy supply, cellulosic biomass should be used for bioethanol production instead of grain biomass. For this purpose, cell surface engineering technology is a very promising method. In biobutanol and biodiesel production, engineered host fermentation has attracted much attention; however, this method has many limitations such as low productivity and low solvent tolerance of microorganisms. Despite these problems, biofuels such as bioethanol, biobutanol, and biodiesel are potential energy sources that can help establish a sustainable society. [ABSTRACT FROM AUTHOR]

The bioadsorption of metal ions using microorganisms is an attractive technology for the recovery of rare metal ions as well as removal of toxic heavy metal ions from aqueous solution. In initial attempts, microorganisms with the ability to accumulate metal ions were isolated from nature and intracellular accumulation was enhanced by the overproduction of metal-binding proteins in the cytoplasm. As an alternative, the cell surface design of microorganisms by cell surface engineering is an emerging strategy for bioadsorption and recovery of metal ions. Cell surface engineering was firstly applied to the construction of a bioadsorbent to adsorb heavy metal ions for bioremediation. Cell surface adsorption of metal ions is rapid and reversible. Therefore, adsorbed metal ions can be easily recovered without cell breakage, and the bioadsorbent can be reused or regenerated. These advantages are suitable for the recovery of rare metal ions. Actually, the cell surface display of a molybdate-binding protein on yeast led to the enhanced adsorption of molybdate, one of the rare metal ions. An additional advantage is that the cell surface display system allows high-throughput screening of protein/peptide libraries owing to the direct evaluation of the displayed protein/peptide without purification and concentration. Therefore, the creation of novel metal-binding protein/peptide and engineering of microorganisms towards the recovery of rare metal ions could be simultaneously achieved. [ABSTRACT FROM AUTHOR]

Vector engineering and gene disruption in host cells were attempted for the enhancement of α-agglutinin-based display of proteins on the cell surface in yeast. To evaluate the display efficiency by flow cytometric analysis, DsRed-monomer fused with FLAG-tag was displayed and immunostained as a model protein. The use of leu2- d in the expression vector resulted in the enhanced efficiency and ratio of the accessible display of proteins. Moreover, the amount of displayed proteins in SED1-disrupted cells increased particularly during the stationary growth phase. The combination of these improvements resulted in the quantitatively enhanced accessible display of DsRed-monomer on the yeast cell surface. The improved yeast display system would be useful in a wider range of its applications in biotechnology. [ABSTRACT FROM AUTHOR]

The cell wall of Saccharomyces cerevisiae plays an essential role in the biophysical characteristics of the cell surface. The modification of the cell wall property is an important factor for cellular adaptation to a stressful environment. In this study, we randomly modified the cell wall by displaying combinatorial random peptides on the yeast cell surface, and by screening, we successfully obtained a novel peptide, Scr35, that endowed yeasts with acid tolerance. The yeast, surface-modified by Scr35, was able to grow well under acidic condition and low glucose condition and showed high glucose uptake activity. However, the growth of the modified yeast became inferior as extracellular pH became higher. This inferiority was rescued by decreasing glucose concentration in a medium. Our results suggest that the optimum pH of a medium becomes low when the newly created Scr35 affects glucose uptake activity through cell-surface modification. Therefore, such artificial modification of the cell surface has a great potential as a useful tool for breeding acid-tolerant yeasts for industrial applications of S. cerevisiae as a biocatalyst. [ABSTRACT FROM AUTHOR]

The analysis of protein–protein interactions is important for developing a better understanding of the functional annotations of proteins that are involved in various biochemical reactions in vivo. The discovery that a protein with an unknown function binds to a protein with a known function could provide a significant clue to the cellular pathway concerning the unknown protein. Therefore, information on protein–protein interactions obtained by the comprehensive analysis of all gene products is available for the construction of interactive networks consisting of individual protein–protein interactions, which, in turn, permit elaborate biological phenomena to be understood. Systems for detecting protein–protein interactions in vitro and in vivo have been developed, and have been modified to compensate for limitations. Using these novel approaches, comprehensive and reliable information on protein–protein interactions can be determined. Systems that permit this to be achieved are described in this review. [ABSTRACT FROM AUTHOR]

A novel protein molecular targeting system was created using a cytoplasmic face of a plasma membrane-targeting system in Saccharomyces cerevisiae. The technique involves a molecular display for the creation of a novel reaction site and interaction sites in the field of biotechnology. In a model system, a fluorescent protein was targeted as a reporter to the cytoplasmic face of the plasma membrane. The C-terminal transmembrane domain (CTM) of Ras2p and Snc2p was examined as the portions with anchoring ability to the cytoplasmic face of the plasma membrane. We found that the CTM of Snc2p targeted the enhanced cyan fluorescent protein (ECFP)–protein A fusion protein on the cytoplasmic face of the plasma membrane more strongly than that of Ras2p. To develop it for use as a detection system for protein–protein interactions, the Fc fragment of IgG (Fc) was genetically fused with the enhanced yellow fluorescent protein (EYFP) and expressed in the cytoplasm of the ECFP–protein A-anchored cell. A microscopic analysis showed that fluorescence resonance energy transfer (FRET) between ECFP–protein A and EYFP–Fc occurred, and the change in fluorescence was observed on the cytoplasmic face of the plasma membrane. The detection of protein–protein interactions at the cytoplasmic face of a plasma membrane using FRET combined with a cytoplasmic face-targeting system for proteins provides a novel method for examining the molecular interactions of cytoplasmic proteins, in addition to conventional methods, such as the two-hybrid method in the nuclei. [ABSTRACT FROM AUTHOR]

To increase the level of adsorption of heavy metal ions in surface-engineered yeasts, a yeast metallothionein (YMT) was tandemly fused and displayed by means of an α-agglutinin-based display system. The display of the YMT and its tandem repeats was examined by immunofluorescent labeling. The adsorption and recovery of Cd2+ on the cell surface was increasingly enhanced with increasing number of tandem repeats. All Cd2+-binding sites in the YMT tandem repeats were suggested to be completely occupied. To investigate the relationship between cell-surface adsorption and protection against heavy metal ion toxicity, the tolerance of these surface-engineered yeasts to Cd2+ was examined by growing in Cd2+-containing liquid medium. The rate of growth was found to be dependent on the number of displayed tandem repeats of YMT. These results suggest that the characteristics of surface-engineered yeasts as a bioadsorbent were dependent on the ability of the displayed proteins to bind metal ions, and the adsorption of heavy metal ions on the cell surface plays a major role in the ability of the cells to resist the toxic effects of metal ions. [ABSTRACT FROM AUTHOR]

Examines the applicability of a set of grooves of multi-roll wire mills for different materials having different stress-strain curves. Evaluation of stability of geometry among different materials when rolled under the same mill setting conditions; Influence of inter-stand tension upstream side four-stand tandem rolling.

Ammonia is used as a fertilizer for agriculture, chemical raw material, and carrier for transporting hydrogen, and with economic development, the demand for ammonia has increased. The Haber–Bosch process, which is the main method for producing ammonia, can produce ammonia with high efficiency. However, since it consumes a large amount of fossil energy, it is necessary to develop an alternative method for producing ammonia with less environmental impact. Ammonia production from food by-products is an appealing production process owing to unused resource usage, including waste, and mild reaction conditions. However, when food by-products and biomass are used as feedstocks, impurities often reduce productivity. Using metabolic profiling, glucose was identified as a potential inhibitor of ammonia production from impure food by-products. We constructed the recombinant Escherichia coli, in which glucose uptake was reduced by ptsG gene disruption and amino acid catabolism was promoted by glnA gene disruption. Ammonia production efficiency from okara, a food by-product, was improved in this strain; 35.4 mM ammonia was produced (47% yield). This study might provide a strategy for efficient ammonia production from food by-products. [ABSTRACT FROM AUTHOR]

Spatial reorganization of metabolic enzymes to form the "metabolic enzymes transiently assembling (META) body" is increasingly recognized as a mechanism contributing to regulation of cellular metabolism in response to environmental changes. A number of META body-forming enzymes, including enolase (Eno2p) and phosphofructokinase, have been shown to contain condensate-forming regions. However, whether all META body-forming enzymes have condensate-forming regions or whether enzymes have multiple condensate-forming regions remains unknown. The condensate-forming regions of META body-forming enzymes have potential utility in the creation of artificial intracellular enzyme assemblies. In the present study, the whole sequence of yeast pyruvate kinase (Cdc19p) was searched for condensate-forming regions. Four peptide fragments comprising 27–42 amino acids were found to form condensates. Together with the fragment previously identified from Eno2p, these peptide regions were collectively termed "META body-forming sequences (METAfos)." METAfos-tagged yeast alcohol dehydrogenase (Adh1p) was found to co-localize with META bodies formed by endogenous Cdc19p under hypoxic conditions. The effect of Adh1p co-localization with META bodies on cell metabolism was further evaluated. Expression of Adh1p fused with a METAfos-tag increased production of ethanol compared to acetic acid, indicating that spatial reorganization of metabolic enzymes affects cell metabolism. These results contribute to understanding of the mechanisms and biological roles of META body formation. [ABSTRACT FROM AUTHOR]

In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we created a donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid) that was composed of sequence-extended gRNA and single-stranded oligodeoxynucleotide (ssODN) combined with complementary bases without chemical modifications to increase the concentration of donor DNA at the cleavage site. The efficiency of genome editing using DGybrid was evaluated in Saccharomyces cerevisiae. The results show a 1.8-fold (from 35% to 62%) improvement in HDR-mediated editing efficiency compared to genome editing in which gRNA and donor DNA were introduced separately. In addition, analysis of the nucleic acid introduction efficiency using flow cytometry indicated that both RNA and ssODNs are efficiently incorporated into cells together by using the DNA/RNA hybrid. Our technique would be preferred as a universal and concise tool for improving the efficiency of HDR-mediated genome editing. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, loosening of chromatin causes changes in transcription and DNA replication. The artificial conversion of tightly packed chromatin (heterochromatin) to loosely packed chromatin (euchromatin) enables gene expression and regulates cell differentiation. Although some chemicals convert chromatin structures through histone modifications, they lack sequence specificity. This study attempted to establish a novel technology for inducing chromatin loosening in target regions of Saccharomyces cerevisiae. We focused on histone acetylation, which is one of the mechanisms of euchromatin induction. The sequence-recognizing ability of the dead Cas9 (dCas9) and guide RNA (gRNA) complex was used to promote histone acetylation at a targeted genomic locus. We constructed a plasmid to produce a fusion protein consisting of dCas9 and histone acetyltransferase Gcn5 and a plasmid to express gRNA recognizing the upstream region of heterochromatic URA3. Confocal microscopy revealed that the fusion proteins were localized in the nucleus. The yeast strain producing the fusion protein and gRNA grew well in the uracil-deficient medium, while the strain harboring empty plasmids or the strain containing the mutations that cause loss of nucleosomal histone acetylation activity of Gcn5 did not. This suggests that the heterochromatin was loosened as much as euchromatin through nucleosomal histone acetylation. The amount of euchromatic DNA at the target locus increased, indicating that chromatin loosening was induced by our system. Nucleosomal histone acetylation in heterochromatic loci by our developed system is a promising method for inducing euchromatic state in a target locus. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae, we have demonstrated that organic solvent stress activated the pleiotropic drug resistance (PDR) pathway, which involves the transcription factors Pdr1p and Pdr3p. Pdr1p and Pdr3p are functionally homologous in multidrug resistance, although Pdr3p has been reported to have some distinct functions. Here, we analyzed the functions of Pdr1p and Pdr3p during the cellular response to isooctane, as a representative of organic solvents, and observed the differential functions of Pdr1p and Pdr3p. In response to organic solvent stress, only Pdr3p contributed to the regulation of downstream genes of the PDR pathway, while Pdr1p had a rather inhibitory role on transcriptional induction through competition with Pdr3p for binding to their recognition sequence, pleiotropic drug response element. Our results demonstrated that organic solvent stress was likely to damage mitochondria, causing generation of reactive oxygen species and mitochondrial fragmentation, and to activate retrograde signaling pathway via Pdr3p to upregulate PDR5 expression. Therefore, the unique function of Pdr3p in organic solvent stress distinguishes this pathway from the multidrug response. [ABSTRACT FROM AUTHOR]

Organic solvents are toxic to living cells. In eukaryotes, cells with organic solvent tolerance have only been found in Saccharomyces cerevisiae. Although several factors contributing to organic solvent tolerance have been identified in previous studies, the mechanism of how yeast cells naturally respond to organic solvent stress is not known. We demonstrated that the pleiotropic drug resistance (PDR) pathway contributed to response to organic solvent stress. Activation of the PDR pathway by mutations in the transcription factors Pdr1p and Pdr3p led to organic solvent tolerance. Exposure to organic solvents also induced transcription levels of PDR5, which encodes a major drug efflux pump. Overproduction of Pdr5p improved organic solvent tolerance, presumably by exporting organic solvents out of the cell. In addition, we showed that the cell wall integrity (CWI) pathway was induced in response to organic solvents to upregulate genes encoding the cell wall-related proteins Wsc3p and Ynl190wp. WSC3 and YNL190W were upregulated independently of the PDR pathway. Among the components of the CWI pathway, the cell surface sensors (Wsc3p and Mid2p) and the transcription factors (Swi4p and Swi6p) appeared to be particularly involved in the response to organic solvents. Our findings indicate that S. cerevisiae activates two different signaling pathways, the PDR pathway and the CWI pathway, to cope with stresses from organic solvents. [ABSTRACT FROM AUTHOR]

Since nitrogenase is irreversibly inactivated within a few minutes after exposure to oxygen, current studies on the heterologous expression of nitrogenase are limited to anaerobic conditions. This study comprehensively identified genes showing oxygen-concentration-dependent expression only under nitrogen-fixing conditions in Azotobacter vinelandii, an aerobic diazotroph. Among the identified genes, nafU, with an unknown function, was greatly upregulated under aerobic nitrogen-fixing conditions. Through replacement and overexpressing experiments, we suggested that nafU is involved in the maintenance of nitrogenase activity under aerobic nitrogenase activity. Furthermore, heterologous expression of nafU in nitrogenase-producing Escherichia coli increased nitrogenase activity under aerobic conditions by 9.7 times. Further analysis of NafU protein strongly suggested its localization in the inner membrane and raised the possibility that this protein may lower the oxygen concentration inside the cells. These findings provide new insights into the mechanisms for maintaining stable nitrogenase activity under aerobic conditions in A. vinelandii and provide a platform to advance the use of nitrogenase under aerobic conditions. [ABSTRACT FROM AUTHOR]

Cellulose, a primary component of lignocellulosic biomass, is the most abundant carbohydrate polymer in nature. Only a limited number of microorganisms are known to degrade cellulose, which is highly recalcitrant due to its crystal structure. Anaerobic bacteria efficiently degrade cellulose by producing cellulosomes, which are complexes of cellulases bound to scaffoldins. The underlying mechanisms that are responsible for the assembly and efficiency of cellulosomes are not yet fully understood. The cohesin-dockerin specificity has been extensively studied to understand cellulosome assembly. Moreover, the recent progress in proteomics has enabled integral analyses of the growth-substrate-dependent variations in cellulosomal systems. Furthermore, the proximity and targeting effects of cellulosomal synergistic actions have been investigated using designed minicellulosomes. The recent findings about cellulosome assembly, strategies for optimal cellulosome production, and beneficial features of cellulosomes as an arming microcompartment on the microbial cell surface are summarized here. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The budding yeast, Saccharomyces cerevisiae, is an attractive host for studying G protein-coupled receptors (GPCRs). We developed a system in which a peptide ligand specific for GPCR is displayed on yeast plasma membrane. The model system described here is based on yeast plasma membrane display of an analogue of α-factor, which is a peptide ligand for Ste2p, the GPCR that activates the yeast pheromone response pathway. α-Factor analogues, containing linkers of varying lengths and produced in yeast cells, became attached to the cell plasma membrane by linking to the glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein Yps1p. We were able to demonstrate that an optimized α-factor analogue activated the pheromone response pathway in S. cerevisiae, as assessed by a fluorescent reporter assay. Furthermore, it was shown that linker length strongly influenced signalling pathway activation. To our knowledge, this is the first report documenting functional signalling by a plasma membrane-displayed ligand in S. cerevisiae. [ABSTRACT FROM PUBLISHER]

The propeptide of carboxypeptidase Y precursor (proCPY) acts as an intramolecular chaperone that ensures the correct folding of the mature CPY (mCPY). Here, to further characterize the folding mechanism mediated by the propeptide, folding analysis was performed using a yeast molecular display system. CPYs with mutated propeptides were successfully displayed on yeast cell surface, and the mature enzymes were purified by the selective cleavage of mutated propeptides. Measurement of the activity and kinetics of the displayed CPYs indicated that the propeptide mutation altered the catalytic efficiency of mCPY. Although the mature region of the wild-type and mutant CPYs had identical amino acid sequences, the mCPYs from the mutant proCPYs had higher catalytic efficiency than the wild-type. These results indicate that proteins with identical amino acid sequence can fold into isomeric proteins with conformational microchanges through mutated intramolecular chaperones. [ABSTRACT FROM AUTHOR]

This study is the first to demonstrate the activity of putative cellulosomal protease/peptidase inhibitors (named cyspins) of Clostridium cellulovorans, using the Saccharomyces cerevisiae display system. Cyspins exhibited inhibitory activities against several representative plant proteases. This suggests that these inhibitors protect their microbe and cellulosome from external attack by plant proteases. [ABSTRACT FROM AUTHOR]

Summary Clostridium cellulovorans, an anaerobic and mesophilic bacterium, degrades native substrates in soft biomass such as corn fibre and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Recently, we have reported the whole-genome sequence of C. cellulovorans comprising 4220 predicted genes in 5.10 Mbp [Y. Tamaru et al., (2010) J. Bacteriol., 192: 901-902]. As a result, the genome size of C. cellulovorans was about 1 Mbp larger than that of other cellulosome-producing clostridia, mesophilic C. cellulolyticum and thermophilic C. thermocellum. A total of 57 cellulosomal genes were found in the C. cellulovorans genome, and they coded for not only carbohydrate-degrading enzymes but also a lipase, peptidases and proteinase inhibitors. Interestingly, two novel genes encoding scaffolding proteins were found in the genome. According to KEGG metabolic pathways and their comparison with 11 Clostridial genomes, gene expansion in the C. cellulovorans genome indicated mainly non-cellulosomal genes encoding hemicellulases and pectin-degrading enzymes. Thus, by examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way natural selection moulds functional DNA sequence evolution. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced cellulosome-producing Clostridium strains for industrial applications such as biofuel production. [ABSTRACT FROM AUTHOR]

Abstract: We determined the genetic background that would result in a more optimal display of heterologously expressed β-glucosidase (BGL) on the cell surface of yeast Saccharomyces cerevisiae. Amongst a collection of 28 strains carrying deletions in genes for glycosylphosphatidyl inositol (GPI)-anchored proteins, the Δsed1 and Δtos6 strains had significantly higher BGL-activity whilst maintaining wild type growth. Absence of Sed1p, which might facilitate incorporation of anchored BGL on the cell-surface, could also influence the activity of BGL on the cell surface with the heterologous gene being placed under the control of the SED1 promoter. For the evaluation of its industrial applicability we tested this system in heterologous and homogenous SED1-disruptants of sake yeast, a diploid S. cerevisiae strain, in which either the SED1 ORF or the complete gene including the promoter was deleted by use of the high-efficiency loss of heterozygosity method. Evaluation of disruptants displaying BGL showed that deletion of the SED1 ORF enhanced BGL activity on the cell surface, while additional deletion of the SED1 promoter increased further BGL activity on the cell surface. Compared to heterozygous disruption, homozygous disruption resulted generally in a higher BGL activity. Thus, homozygous deletion of both SED1 gene and promoter resulted in the most efficient display of BGL reaching a 1.6-fold increase of BGL-activity compared to wild type. [Copyright &y& Elsevier]

In modern industrial society, molybdenum is one of the important metals for development of the industry of rare metals. It is important to recycle the rare metals from wastes because they are technically and economically difficult to be dug and be purified, and they exist in only a few regions in the world. In this study, ModE protein derived from Escherichia coli, which is a molybdate-dependent transcriptional regulator with the ability to bind molybdate as a form of soluble molybdenum, was displayed on the yeast cell surface by α-agglutinin-based cell surface display system for the adsorption and recovery of molybdate. Displayed ModE, confirmed by immunofluorescence labeling, caught molybdate more preferably at pH 3.0 than at basic pH. Yeast cells displaying C-terminal domain of ModE, which lacks N-terminal DNA binding domain, more effectively adsorbed molybdate than those displaying full-length ModE, suggesting that the deletion of the domain unrelated to metal binding enhanced the binding ability. Our results indicated that the adsorption system on cell surface of yeast cells displaying ModE is effective not only for adsorption of molybdate as a rare metal bioadsorbent but also for the easy recovery of molybdate located on the cell surface. [ABSTRACT FROM AUTHOR]

Chitosanase from Paenibacillus fukuinensis D2 is an attractive enzyme, and it exhibits both chitosanase and β-1, 4 glucanase activities. In our previous study, we generated P. fukuinensis chitosanase-displaying yeast cells using a yeast cell surface-displaying system. Chitosanase-displaying yeast can be utilized as a chitosanase cluster without many time-consuming purification steps. In this study, using the system, we have investigated whether Glu302, which is supposed as a putative proton acceptor, is an essential amino acid residue for exhibiting chitosanase activity and analyzed the contribution of mutual interaction between Glu302 and Asn312 to the activity. A mutant library in which Glu302 and Asn312 were comprehensively substituted by the other amino acid residues was constructed on the yeast cell surface. From the results of chitosanase and β-1, 4 glucanase activity assays, we demonstrated that Glu302 was a proton acceptor for chitosanase activity, and Asn312 also participated in the hydrolysis of chitosan and cellulose. [ABSTRACT FROM AUTHOR]

To utilize glucoamylase-displaying yeast cells for enzymatic desizing of starched cotton cloth, we constructed yeast strains that codisplayed Rhizopus oryzae glucoamylase and two kinds of Trichoderma reesei cellulose-binding domains (CBD1, CBD of cellobiohydrolase I (CBHI); and CBD2, CBD of cellobiohydrolase II (CBHII)). In this study, we aimed to obtain a high efficiency of enzymatic desizing of starched cotton cloth. Yeast cells that codisplayed glucoamylase and CBD had higher activity on starched cotton cloth than yeast cells that displayed only glucoamylase. Glucoamylase and double CBDs (CBD1 and CBD2) codisplaying yeast cells exhibited the highest activity ratio (4.36-fold), and glucoamylase and single CBD (CBD1 or CBD2) codisplaying yeast cells had higher relative activity ratios (2.78- and 2.99-fold, respectively) than glucoamylase single-displaying cells. These results indicate that the glucoamylase activity of glucoamylase-displaying cells would be affected by the binding ability of CBD codisplayed on the cell surface to starched cotton cloth. These novel strains might play useful roles in the enzymatic desizing of starched cotton cloth in the textile industry. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae mutant strain, KK-211, isolated from serial culture in medium containing isooctane showed an extremely higher tolerance to the hydrophobic organic-solvents, which are toxic to yeast cells compared to the wild-type parent strain, DY-1. To detect genes that are related to this tolerance, a DNA microarray analysis was performed using mRNAs isolated from strains DY-1 and KK-211. Fourteen genes were identified as being related to the tolerance. The expression of 12 genes including ICT1, YNL190W, and PRY3, was induced while the expression of two genes including PHO84 was repressed in strain KK-211. Two genes, ICT1 and YNL190W showed the same profile in the DNA microarray analysis and a differential display-polymerase chain reaction analysis. But, there is no detectable difference in the expression profile of KK-211 cells cultured with or without isooctane. The results suggest that change in expression levels of multiple genes that confer the modification function of the cell surface, not by a single gene, might be required for yeast cell tolerance to organic solvents. [ABSTRACT FROM AUTHOR]

Biological nitrogen fixation by nitrogenase has attracted attention as an alternative method to chemical nitrogen fixation, which requires large amounts of fossil fuels. Azotobacter vinelandii , which produces an oxygen-sensitive nitrogenase, can fix nitrogen even under aerobic conditions; therefore, the heterologous expression of nif-related genes from A. vinelandii is a promising strategy for developing a biological nitrogen fixation method. We assembled 17 nif-related genes, which are scattered throughout the genome of A. vinelandii , into synthetic gene clusters by overlap-extension-PCR and seamless cloning and expressed them in Escherichia coli. The transcription and translation of the 17 nif-related genes were evaluated by RT-qPCR and LC-MS/MS, respectively. The constructed E. coli showed nitrogenase activity under anaerobic and microaerobic conditions. This strain would be a useful model for examining the effect of other genes from A. vinelandii on nitrogen fixation by expressing them in addition to the minimal set of nif-related genes. [ABSTRACT FROM AUTHOR]

At normal oxygen concentration, glycolytic enzymes are scattered in the cytoplasm of Saccharomyces cerevisiae. Under hypoxia, however, most of these enzymes, including enolase, pyruvate kinase, and phosphoglycerate mutase, spatially reorganize to form cytoplasmic foci. We tested various small‐scale hypoxic culture systems and showed that enolase foci formation occurs in all the systems tested, including in liquid and on solid media. Notably, a small‐scale hypoxic culture in a bench‐top multi‐gas incubator enabled the regulation of oxygen concentration in the media and faster foci formation. Here, we demonstrate that the foci formation of enolase starts within few hours after changing the oxygen concentration to 1% in a small‐scale cultivation system. The order of foci formation by each enzyme is tightly regulated, and of the three enzymes, enolase was the fastest to respond to hypoxia. We further tested the use of the small‐scale cultivation method to screen reagents that can control the spatial reorganization of enzymes under hypoxia. An AMPK inhibitor, dorsomorphin, was found to delay formation of the foci in all three glycolytic enzymes tested. These methods and results provide efficient ways to investigate the spatial reorganization of proteins under hypoxia to form a multienzyme assembly, the META body, thereby contributing to understanding and utilizing natural systems to control cellular metabolism via the spatial reorganization of enzymes. [ABSTRACT FROM AUTHOR]