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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]

Modification of the genome of the yeast Saccharomyces cerevisiae has great potential for application in biological research and biotechnological advancements, and the CRISPR-Cas9 system has been increasingly employed for these purposes. The CRISPR-Cas9 system enables the precise and simultaneous modification of any genomic region of the yeast to a desired sequence by altering only a 20-nucleotide sequence within the guide RNA expression constructs. However, the conventional CRISPR-Cas9 system has several limitations. In this review, we describe the methods that were developed to overcome these limitations using yeast cells. We focus on three types of developments: reducing the frequency of unintended editing to both non-target and target sequences in the genome, inducing desired changes in the epigenetic state of the target region, and challenging the expansion of the CRISPR-Cas9 system to edit genomes within intracellular organelles such as mitochondria. These developments using yeast cells to overcome the limitations of the CRISPR-Cas9 system are a key factor driving the advancement of the field of genome editing. [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]

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]

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]

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 a sustainable and promising source for bioethanol production because they are abundant in ocean ecosystems and contain negligible quantities of lignin. Brown macroalgae contain cellulose, hemicellulose, mannitol, laminarin, and alginate as major carbohydrates. Among these carbohydrates, brown macroalgae are characterized by high levels of alginate and mannitol. The direct bioconversion of alginate and mannitol into ethanol requires extensive bioengineering of assimilation processes in the standard industrial microbe Saccharomyces cerevisiae. Here, we constructed an alginate-assimilating S. cerevisiae recombinant strain by genome integration and overexpression of the genes encoding endo- and exo-type alginate lyases, DEH (4-deoxy- l-erythro-5-hexoseulose uronic acid) transporter, and components of the DEH metabolic pathway . Furthermore, the mannitol-metabolizing capacity of S. cerevisiae was enhanced by prolonged culture in a medium containing mannitol as the sole carbon source. When the constructed strain AM1 was anaerobically cultivated in a fermentation medium containing 6% ( w/ v) total sugars (approximately 1:2 ratio of alginate/mannitol), it directly produced ethanol from alginate and mannitol, giving 8.8 g/L ethanol and yields of up to 32% of the maximum theoretical yield from consumed sugars. These results indicate that all major carbohydrates of brown macroalgae can be directly converted into bioethanol by S. cerevisiae. This strain and system could provide a platform for the complete utilization of brown macroalgae. [ABSTRACT FROM AUTHOR]

Xylose isomerase (XylC) from Clostridium cellulovorans can simultaneously perform isomerization and fermentation of d-xylose, the main component of lignocellulosic biomass, and is an attractive candidate enzyme. In this study, we optimized a specified metal cation in a previously established Saccharomyces cerevisiae strain displaying XylC. We investigated the effect of each metal cation on the catalytic function of the XylC-displaying S. cerevisiae. Results showed that the divalent cobalt cations (Co2+) especially enhanced the activity by 46-fold. Co2+ also contributed to d-xylose fermentation, which resulted in improving ethanol yields and xylose consumption rates by 6.0- and 2.7-fold, respectively. Utility of the extracellular xylose isomerization system was exhibited in the presence of mixed sugar. XylC-displaying yeast showed the faster d-xylose uptake than the yeast producing XI intracellularly. Furthermore, direct xylan saccharification and fermentation was performed by unique yeast co-culture system. A xylan-degrading yeast strain was established by displaying two kinds of xylanases; endo-1,4-β-xylanase (Xyn11B) from Saccharophagus degradans, and β-xylosidase (XlnD) from Aspergillus niger. The yeast co-culture system enabled fine-tuning of the initial ratios of the displayed enzymes (Xyn11B:XlnD:XylC) by adjusting the inoculation ratios of Xylanases (Xyn11B and XlnD)-displaying yeast and XylC-displaying yeast. When the enzymes were inoculated at the ratio of 1:1:2 (1.39 × 1013: 1.39 × 1013: 2.78 × 1013 molecules), 6.0 g/L ethanol was produced from xylan. Thus, the cofactor optimization and the yeast co-culture system developed in this study could expand the prospect of biofuels production from lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1068-1076, 2017 [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]

Clostridium cellulovorans can effectively assimilate not only cellulose but also hemicellulose by producing cellulosomal and non-cellulosomal enzymes. However, little is known about how C. cellulovorans assimilates various saccharides in media containing polysaccharides and oligosaccharides. In this research, we investigated the property of saccharide incorporation and assimilation by C. cellulovorans. Faster growth in media containing xylan and cellulose was achieved by switching polysaccharides, in which xylan was first assimilated, followed by cellulose. Furthermore, the presence of polysaccharides that can be easily degraded might increase the assimilation rate of lignocellulose by promoting growth. These properties of C. cellulovorans could be suitable for the effective utilization of lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Alginate-degrading bacteria play an important role in alginate degradation by harboring highly efficient and unique alginolytic genes. Although the general mechanism for alginate degradation by these bacteria is fairly understood, much is still required to fully exploit them. Here, we report the isolation of a novel strain, Falsirhodobacter sp. alg1, the first report for an alginate-degrading bacterium from the family Rhodobacteraceae. Genome sequencing reveals that strain alg1 harbors a primary alginate degradation pathway with only single homologs of an endo- and exo-type alginate lyase, AlyFRA and AlyFRB, which is uncommon among such bacteria. Subsequent functional analysis showed that both enzymes were extremely efficient to depolymerize alginate suggesting evolutionary interests in the acquirement of these enzymes. The exo-type alginate lyase, AlyFRB in particular could depolymerize alginate without producing intermediate products making it a highly efficient enzyme for the production of 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Based on our findings, we believe that the discovery of Falsirhodobacter sp. alg1 and its alginolytic genes hints at the potentiality of a more diverse and unique population of alginate-degrading bacteria. [ABSTRACT FROM AUTHOR]

Alginate is a major component of brown macroalgae. In macroalgae, an endolytic alginate lyase first degrades alginate into oligosaccharides. These oligosaccharides are further broken down into monosaccharides by an exolytic alginate lyase. In this study, genes encoding various alginate lyases derived from alginate-assimilating marine bacterium Saccharophagus degradans were isolated, and their enzymes were displayed using the yeast cell surface display system. Alg7A-, Alg7D-, and Alg18J-displaying yeasts showed endolytic alginate lyase activity. On the other hand, Alg7K-displaying yeast showed exolytic alginate lyase activity. Alg7A, Alg7D, Alg7K, and Alg18J, when displayed on yeast cell surface, demonstrated both polyguluronate lyase and polymannuronate lyase activities. Additionally, polyguluronic acid could be much easily degraded by Alg7A, Alg7K, and Alg7D than polymannuronic acid. In contrast, polymannuronic acid could be much easily degraded by Alg18J than polyguluronic acid. We further constructed yeasts co-displaying endolytic and exolytic alginate lyases. Degradation efficiency by the co-displaying yeasts were significantly higher than single alginate lyase-displaying yeasts. Alg7A/Alg7K co-displaying yeast had maximum alginate-degrading activity, with production of 1.98 g/L of reducing sugars in a 60-min reaction. This system developed, along with our findings, will contribute to the efficient utilization and production of useful and non-commercialized monosaccharides from alginate by Saccharomyces cerevisiae. [ABSTRACT FROM AUTHOR]

Quantitative proteomic analysis was conducted to assess the assimilation processes of Saccharophagus degradans cultured with glucose, pectin, and alginate as carbon sources. A liquid chromatography-tandem mass spectrometry approach was used, employing our unique, long monolithic silica capillary column. In an attempt to select candidate proteins that correlated to alginate assimilation, the production of 23 alginate-specific proteins was identified by statistical analyses of the quantitative proteomic data. Based on the analysis, we propose that S. degradans has an alginate-specific gene cluster for efficient alginate utilization. The alginate-specific proteins of S. degradans were comprised of alginate lyases, enzymes related to carbohydrate metabolism, membrane transporters, and transcription factors. Among them, the short-chain dehydrogenase/reductase Sde_3281 annotated in the alginate-specific cluster showed 4-deoxy- l-erythro-5-hexoseulose uronic acid reductase (DehR) activity. Furthermore, we found two different genes ( Sde_3280 and Sde_0939) encoding 2-keto-3-deoxy- d-gluconic acid (KDG) kinases (KdgK) that metabolize the KDG derived from alginate and pectin in S. degradans. S. degradans used Sde_3280 to phosphorylate the KDG derived from alginate and Sde_0939 to phosphorylate the KDG derived from pectin. The distinct selection of KdgKs provides an important clue toward the elucidation of how S. degradans recognizes and processes polysaccharides. [ABSTRACT FROM AUTHOR]

Influenza viruses have periodically caused pandemic due to frequent mutation of viral proteins. Influenza viruses have two major membrane glycoproteins: hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin plays a crucial role in viral entry, while NA is involved in the process of a viral escape. In terms of developing antiviral drugs, HA is a more important target than NA in the prevention of pandemic, since HA is likely to change the host specificity of a virus by acquiring mutations, thereby to increase the risk of pandemic. To characterize mutated HA functions, current approaches require immobilization of purified HA on plastic wells and carriers. These troublesome methods make it difficult to respond to emerging mutations. In order to address this problem, a yeast cell surface engineering approach was investigated. Using this technology, human HAs derived from various H1N1 subtypes were successfully and rapidly displayed on the yeast cell surface. The yeast-displayed HAs exhibited similar abilities to native influenza virus HAs. Using this system, human HAs with 190E and 225G mutations were shown to exhibit altered recognition specificities from human to avian erythrocytes. This system furthermore allowed direct measurement of HA binding abilities without protein purification and immobilization. Coupled with the ease of genetic manipulation, this system allows the simple and comprehensive construction of mutant protein libraries on yeast cell surface, thereby contributing to influenza virus pandemic prevention. [ABSTRACT FROM AUTHOR]

Clostridium cellulovorans produces multi‐enzyme complexes called cellulosomes capable of efficiently degrading cellulosic biomass. There are three xylanase genes containing a sequence corresponding to a dockerin domain that are necessary for constructing cellulosomes in the genome. Among the xylanases encoded by these genes, xylanase B (XynB) contains a catalytic domain belonging to glycoside hydrolase family 10 and a carbohydrate‐binding module (CBM) at the N‐terminus, making it a member of CBM family 22. In this study, XynB was cloned, overexpressed, purified and crystallized. XynB was crystallized using the hanging‐drop vapour‐diffusion method in the presence of 0.2 M sodium acetate trihydrate, 0.1 M Tris–HCl pH 8.5, 32%(w/v) PEG 4000 at 293 K. X‐ray diffraction analysis revealed that the crystal diffracted to 1.95 Å resolution and belonged to space group P212121, with unit‐cell parameters a = 74.28, b = 77.55, c = 88.20 Å, α = β = γ = 90°. The data‐evaluation statistics revealed high quality of the collected data, thereby establishing a solid basis for determination of the structure of cellulosomal xylanase from C. cellulovorans. [ABSTRACT FROM AUTHOR]

Clostridium cellulovorans is an anaerobic, cellulolytic bacterium, capable of effectively degrading and metabolizing various types of substrates, including cellulose, hemicellulose (xylan and galactomannan), and pectin. Among Clostridia, this ability to degrade and metabolize a wide range of hemicellulose and pectin substrates is a unique feature; however, the mechanisms are currently unknown. To clarify the mechanisms of hemicelluloses and pectin recognition and metabolism, we carried out a quantitative proteome analysis of C. cellulovorans cultured with these substrates. C. cellulovorans was cultured in the medium of glucose (control), xylan, galactomannan (Locus bean gum, LBG), or pectin for 36 h. Xylan and galactomannan were used to search for the common recognition mechanisms of hemicellulose, and pectin was used to search for unique recognition systems in C. cellulovorans. Using an isobaric tag method and liquid chromatograph/mass spectrometer equipped with a long monolithic silica capillary column, we identified 734 intracellular proteins from all substrates. We performed KEGG analyses and cluster analyses of the resulting proteins. In the KEGG analyses, we found common degradation mechanisms for hemicellulose and pectin. In the cluster analysis corresponding to the genome analysis, we detected substrate-specific clusters that include genes involved in substrate recognition, substrate degradation, and metabolism. Combining the results of the KEGG analyses and cluster analyses, we propose the mechanisms involved in the recognition and metabolism of hemicellulose and pectin in C. cellulovorans. [ABSTRACT FROM AUTHOR]

Clostridium cellulovorans is an anaerobic, cellulolytic bacterium, capable of effectively degrading various types of soft biomass. Its excellent capacity for degradation results from optimization of the composition of the protein complex (cellulosome) and production of non-cellulosomal proteins according to the type of substrates. In this study, we performed a quantitative proteome analysis to determine changes in the extracellular proteins produced by C. cellulovorans for degradation of several types of natural soft biomass. C. cellulovorans was cultured in media containing bagasse, corn germ, rice straw (natural soft biomass), or cellobiose (control). Using an isobaric tag method and a liquid chromatograph equipped with a long monolithic silica capillary column/mass spectrometer, we identified 372 proteins in the culture supernatant. Of these, we focused on 77 saccharification-related proteins of both cellulosomal and non-cellulosomal origins. Statistical analysis showed that 18 of the proteins were specifically produced during degradation of types of natural soft biomass. Interestingly, the protein Clocel_3197 was found and commonly involved in the degradation of every natural soft biomass studied. This protein may perform functions, in addition to its known metabolic functions, that contribute to effective degradation of natural soft biomass. [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]

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]

Phenotypic and genomic heterogeneity among single cells in a cell population leads to inaccuracy and obscuration in research about mammalian cell differentiation. In order to address the problems regarding bulk analysis on heterogeneous cell populations, it is necessary to accurately regulate and analyze changes in differentiating cells at the single-cell level. To investigate the single-cell changes in PC12 neuronal differentiation that occur when inhibited by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), we directly injected the chemical into individual target cells and analyzed the outcomes (neurite outgrowth) at the single-cell level. As a result, we could accurately regulate the quantity of U0126 being introduced into each target cell, which was previously not possible using the common method of simply adding the inhibitor to the culture medium. It was possible to analyze the inhibitive effect of U0126 even when the injected quantity was lower than the lower limit for inhibition when added to culture medium (0.1 μM, identical to 1.2 × 108 molecules per cell on dish). In particular, injection of 1.5 × 107 molecules into each cell resulted in a 59% decrease of the mean total neurite length. Time-course analysis of neurite outgrowth at the single-cell level using fluorescence staining method showed that the changes in neurite length of differentiating PC12 cells were not homogeneous, but were largely variable across individual target cells. [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]

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]

Abstract: Neuraminidase (NA) is a surface glycoprotein produced by the influenza virus. Specific NA mutations that confer resistance to anti-viral drugs have been reported. The aim of this study was to demonstrate quick preparation of the mutated NAs using the yeast surface display and its applicability for screening inhibitors. Plasmids encoding the head domain of wild-type and drug-resistant NAs were constructed and introduced into yeast, and these were successfully displayed on the yeast surface, with biochemical properties similar to the native virus NAs. This system using mutated NAs-displaying yeast provides an efficient and convenient tool for screening novel inhibitors against the drug-resistant influenza virus. [Copyright &y& Elsevier]

A thermotolerant Saccharomyces cerevisiae yeast strain, YK60-1, was bred from a parental strain, MT8-1, via stepwise adaptation. YK60-1 grew at 40°C, a temperature at which MT8-1 could not grow at all. YK60-1 exhibited faster growth than MT8-1 at 30°C. To investigate the mechanisms how MT8-1 acquired thermotolerance, DNA microarray analysis was performed. The analysis revealed the induction of stress-responsive genes such as those encoding heat shock proteins and trehalose biosynthetic enzymes in YK60-1. Furthermore, nontargeting metabolome analysis showed that YK60-1 accumulated more trehalose, a metabolite that contributes to stress tolerance in yeast, than MT8-1. In conclusion, S. cerevisiae MT8-1 acquired thermotolerance by induction of specific stress-responsive genes and enhanced intracellular trehalose levels. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1116-1123, 2013 [ABSTRACT FROM AUTHOR]

The development of simple, portable, inexpensive, and rapid analytical methods for detecting and monitoring toxic heavy metals are important for the safety and security of humans and their environment. Herein, we describe the application of phytochelatin (PC) synthase, which plays a critical role in heavy metal responses in higher plants and green algae, in a novel fluorescent sensing platform for cadmium (Cd). We first created surface-engineered yeast cells on which the PC synthase from Arabidopsis (AtPCS1) was displayed with retention of enzymatic activity. The general concept for the sensor is based on the Cd level-dependent synthesis of PC2 from glutathiones by AtPCS1-displaying yeast cells, followed by simple discriminative detection of PC2 via sensing of excimer fluorescence of thiol-labeling pyrene probes. The intensity of excimer fluorescence increased in the presence of Cd up to 1.0 μM in an approximately dose-dependent manner. This novel biosensor achieved a detection limit of as low as 0.2 μM (22.5 μg/L) for Cd. Although its use may be limited by the fact that Cu and Pb can induce cross-reaction, the proposed simple biosensor holds promise as a method useful for cost-effective screening of Cd contamination in environmental and food samples. The AtPCS1-displaying yeast cells also might be attractive tools for dissection of the catalytic mechanisms of PCS. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1197-1202, 2013 [ABSTRACT FROM AUTHOR]

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]

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 main sterol of the human cell membrane is cholesterol, whereas in yeast it is ergosterol. In this study, we constructed a cholesterol-producing yeast strain by disrupting the genes related to ergosterol synthesis and inserting the genes related to cholesterol synthesis. The total sterols of the mutant yeast were extracted and the sterol composition was analyzed by GC-MS. We confirmed that cholesterol was produced instead of ergosterol in yeast and subsequently examined the activity of the yeast G-protein-coupled receptor (GPCR) Ste2p. Ste2p signaling was assessed in wild type (WT) with ergosterol and the cholesterol-producing yeast instead of ergosterol to determine whether sterol composition affects the activity of the yeast GPCR. Our results demonstrated that Ste2p could transduce a signal even in the cholesterol-rich membrane, but the maximum signal intensity was weaker than that transduced in the ergosterol-rich original (WT) membrane. This result indicates that sterol composition affects the activity of yeast GPCRs, and thus, this provides new insight into GPCR-mediated transduction using yeast for future fundamental and applied studies on GPCRs from yeast to other organisms. [ABSTRACT FROM AUTHOR]

Pyrosequencing system utilizing luciferase is one of the next-generation DNA sequencing systems. However, there is a crucial problem with the current pyrosequencing system: luciferase cannot discriminate between ATP and dATP completely, and dATPαS must be used as the dATP analogue. dATPαS is expensive and has low activity for the enzyme. If luciferase can clearly recognize the difference between ATP and dATP, dATP could be used instead of the expensive dATPαS in the pyrosequencing system. We attempted to prepare a novel luciferase with improved specific activity and dATP discrimination with the molecular display method. First, we selected two amino acid residues, Ser440 and Ser456, as target residues for mutation from the whole sequence of Photinus pyralis luciferase; we comprehensively mutated these two amino acids. A mutant luciferase library was constructed using yeast cell surface engineering. Through three step-wide screenings with individual conditions, we easily and speedily isolated three candidate mutants from 1,152 candidates and analyzed the properties of these mutants. Consequently, we succeeded in obtaining interesting mutant luciferases with improved specific activity and dATP discrimination more conveniently than with other methods. [ABSTRACT FROM AUTHOR]

Xylose isomerase (XI) is a key enzyme in the conversion of d-xylose, which is a major component of lignocellulosic biomass, to d-xylulose. Genomic analysis of the bacterium Clostridium cellulovorans revealed the presence of XI-related genes. In this study, XI derived from C. cellulovorans was produced and displayed using the yeast cell-surface display system, and the xylose assimilation and fermentation properties of this XI-displaying yeast were examined. XI-displaying yeast grew well in medium containing xylose as the sole carbon source and directly produced ethanol from xylose under anaerobic conditions. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 346-351, 2013 [ABSTRACT FROM AUTHOR]

Neuraminidase (NA) is a surface glycoprotein produced by the influenza virus. Specific NA mutations that confer resistance to anti‐viral drugs have been reported. The aim of this study was to demonstrate quick preparation of the mutated NAs using the yeast surface display and its applicability for screening inhibitors. Plasmids encoding the head domain of wild‐type and drug‐resistant NAs were constructed and introduced into yeast, and these were successfully displayed on the yeast surface, with biochemical properties similar to the native virus NAs. This system using mutated NAs‐displaying yeast provides an efficient and convenient tool for screening novel inhibitors against the drug‐resistant influenza virus. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides are promising antibiotics as they possess strong antimicrobial activity and very broad spectra of activity. However, administration of an antibiotic with a very broad spectrum of activity disrupts normal microflora and increases the risks of other fatal infections. To solve the problem, we designed a novel antimicrobial peptide that is activated by virulent proteases of pathogenic organisms. We constructed a peptide composed of three domains, namely an antimicrobial peptide (lactoferricin) as the active center, a protective peptide (magainin intervening sequence) that suppresses antimicrobial activity, and a specific linker that joins these two components and is efficiently cleaved by virulent proteases. We utilized Candida albicans as a model organism that produces secreted aspartic proteases as a virulence attribute. We screened for a peptide sequence efficiently cleaved by secreted aspartic proteases isozymes and identified a GFIKAFPK peptide as the most favorable substrate. Subsequently, we chemically synthesized a peptide containing the GFIKAFPK sequence. The designed peptide possessed no antimicrobial activity until it was activated by secreted aspartic proteases isozymes. Furthermore, it demonstrated selective antimicrobial activity against C. albicans, but not against Saccharomyces cerevisiae. A designed peptide like the one described in this study may protect normal microflora, resulting in enhanced safety as a therapeutic. [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]

Candida albicans is normally present in nearly all humans but can cause fatal diseases in immunocompromised patients. The agglutinin-like sequence ( ALS) gene family of C. albicans has been suggested to be important for biofilm formation on medical devices. Here, we cloned all ALS genes and determined the binding properties of their gene products by cell surface engineering of Saccharomyces cerevisiae. Most of the ALS homologues had the ability to bind polypropylene, borosilicate glass and polyvinyl chloride, which are often used as materials in medical devices. However, ALS homologues were not able to bind polyurethane, polymethyl methacrylate, polytetrafluoroethylene or titanium. These findings will aid in the development of biofilm-resistant medical devices. [ABSTRACT FROM AUTHOR]

A gene encoding laccase I was identified and cloned from the white-rot fungus Trametes sp. Ha1. Laccase I contained 10 introns and an original secretion signal sequence. After laccase I without introns was prepared by overlapping polymerase chain reaction, it was inserted into expression vector pULD1 for yeast cell surface display. The oxidation activity of a laccase-I-displaying yeast as a whole-cell biocatalyst was examined with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and the constructed yeast showed a high oxidation activity. After the pretreatment of hydrothermally processed rice straw (HPRS) with laccase-I-displaying yeast with ABTS, fermentation was conducted with yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase with HPRS. Fermentation of HPRS treated with laccase-I-displaying yeast was performed with 1.21-fold higher activities than those of HPRS treated with control yeast. The results indicated that pretreatment with laccase-I-displaying yeast with ABTS was effective for direct fermentation of cellulosic materials by yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase. [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 article discusses a study which determines functionality of various c Gα proteins in yeasts G-protein-coupled receptor (GPCR). It mentions that Gα protein located at 37 gustducin in yeasts maintains functionality in yeast while its common variant found in mammalian experimental systems is not functional. It also reports saccharomyces cerevisiae yeast an effective experimental model for the characterization of GPCR-Gα interactions.

Background: Candida albicans, a commensal organism, is a part of the normal flora of healthy individuals. However, once the host immunity is compromised, C. albicans opportunistically causes recurrent superficial or fatal systemic candidiasis. Secreted aspartic proteases (Sap), encoded by 10 types of SAP genes, have been suggested to contribute to various virulence processes. Thus, it is important to elucidate their biochemical properties for better understanding of the molecular mechanisms that how Sap isozymes damage host tissues. Methodology/Principal Findings: The SAP7 gene was cloned from C. albicans SC5314 and heterogeneously produced by Pichia pastoris. Measurement of Sap7 proteolytic activity using the FRETS-25Ala library showed that Sap7 was a pepstatin Ainsensitive protease. To understand why Sap7 was insensitive to pepstatin A, alanine substitution mutants of Sap7 were constructed. We found that M242A and T467A mutants had normal proteolytic activity and sensitivity to pepstatin A. M242 and T467 were located in close proximity to the entrance to an active site, and alanine substitution at these positions widened the entrance. Our results suggest that this alteration might allow increased accessibility of pepstatin A to the active site. This inference was supported by the observation that the T467A mutant has stronger proteolytic activity than the wild type. Conclusions/Significance: We found that Sap7 was a pepstatin A-insensitive protease, and that M242 and T467 restricted the accessibility of pepstatin A to the active site. This finding will lead to the development of a novel protease inhibitor beyond pepstatin A. Such a novel inhibitor will be an important research tool as well as pharmaceutical agent for patients suffering from candidiasis. [ABSTRACT FROM AUTHOR]

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.

Candida albicans is a commensal organism, but causes life-threatening infections in immunocompromised patients. Certain factors such as yeast-hyphae transition and hydrolytic enzymes are suggested as virulence attributes of C. albicans. Among them, 10 types of secreted aspartic protease (SAP) genes have received particular attention as a major virulence gene family. However, their full functional repertoire, including its biochemical properties, remains to be elucidated. Hence, we purified all Sap isozymes using Pichia pastoris and comprehensively determined and compared their biochemical properties. While optimum pH of Sap7 was 6.5 and that of Sap8 was 2.5, presence of other Sap isozymes functioning within a broad range of optimum pH could allow C. albicans to survive and cause infections in various tissues. The substrate specificities of Sap isozymes were analysed by using FRETS-25Xaa libraries. Sap7 and Sap10 showed high substrate specificity, while other Sap isozymes had broad substrate specificities. Principal component analysis revealed that the 10 Sap isozymes were clustered into 3 distinct groups in terms of their substrate specificities. Interestingly, Sap4–6, which are coproduced in the hyphal form, were clustered as the same group, indicating that they may target similar host proteins. These results will lead to further understanding of C. albicans pathogenicity. [ABSTRACT FROM PUBLISHER]

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 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.