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1.  Genome Sequence of Formosa haliotis Strain MA1, a Brown Alga-Degrading Bacterium Isolated from the Gut of Abalone Haliotis gigantea 
Genome Announcements  2016;4(6):e01312-16.
Formosa haliotis is a brown alga-degrading bacterium isolated from the gut of abalone Haliotis gigantea. Here, we report the draft genome sequence of this bacterium and pointed out possible important features related to alginate degradation.
doi:10.1128/genomeA.01312-16
PMCID: PMC5114390  PMID: 27856598
2.  Recovery of platinum(0) through the reduction of platinum ions by hydrogenase-displaying yeast 
AMB Express  2016;6:88.
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 PtCl4 2− 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.
doi:10.1186/s13568-016-0262-4
PMCID: PMC5050174  PMID: 27704470
Platinum; Metal reduction; Metal recovery; Hydrogenase; Cell membrane display; Saccharomyces cerevisiae
3.  Characteristic strategy of assimilation of various saccharides by Clostridium cellulovorans 
AMB Express  2016;6(1):64.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-016-0237-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s13568-016-0237-5
PMCID: PMC5009059  PMID: 27586595
Clostridium cellulovorans; Polysaccharide assimilation; Cellulose; Hemicellulose
4.  Rhizobial gibberellin negatively regulates host nodule number 
Scientific Reports  2016;6:27998.
In legume–rhizobia symbiosis, the nodule number is controlled to ensure optimal growth of the host. In Lotus japonicus, the nodule number has been considered to be tightly regulated by host-derived phytohormones and glycopeptides. However, we have discovered a symbiont-derived phytohormonal regulation of nodule number in Mesorhizobium loti. In this study, we found that M. loti synthesized gibberellic acid (GA) under symbiosis. Hosts inoculated with a GA-synthesis-deficient M. loti mutant formed more nodules than those inoculated with the wild-type form at four weeks post inoculation, indicating that GA from already-incorporated rhizobia prevents new nodule formation. Interestingly, the genes for GA synthesis are only found in rhizobial species that inhabit determinate nodules. Our findings suggest that the already-incorporated rhizobia perform GA-associated negative regulation of nodule number to prevent delayed infection by other rhizobia.
doi:10.1038/srep27998
PMCID: PMC4910070  PMID: 27307029
5.  Falsirhodobacter sp. alg1 Harbors Single Homologs of Endo and Exo-Type Alginate Lyases Efficient for Alginate Depolymerization 
PLoS ONE  2016;11(5):e0155537.
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.
doi:10.1371/journal.pone.0155537
PMCID: PMC4866713  PMID: 27176711
6.  A comparative proteomics study of a synovial cell line stimulated with TNF‐α 
FEBS Open Bio  2016;6(5):418-424.
To elucidate the pathogenesis of rheumatoid arthritis (RA), we used proteomic analysis to determine the protein profile in a synovial cell line, MH7A, established from patients with RA. Proteins were extracted from MH7A cells that were or were not stimulated with tumor necrosis factor‐α (TNF‐α), and then analyzed on a liquid chromatography/mass spectrometry system equipped with a unique long monolithic silica capillary. On the basis of the results of this proteomic analysis, we identified 2650 proteins from untreated MH7A cells and 2688 proteins from MH7A cells stimulated with TNF‐α. Next, we selected 269 differentially produced proteins that were detected only under TNF‐α stimulation, and classified these proteins by performing gene ontology analysis by using DAVID as a functional annotation tool. In TNF‐α‐stimulated MH7A cells, we observed substantial production of plasminogen‐activator inhibitor 2 and apoptosis‐regulating proteins such as BH3‐interacting domain death agonist, autophagy protein 5, apolipoprotein E, and caspase‐3. These results indicate that the upregulation of plasminogen‐activator inhibitor 2 and apoptosis‐regulating proteins in synovial cells in response to TNF‐α stimulation might represent a predominant factor that contributes to the pathogenesis of RA.
doi:10.1002/2211-5463.12049
PMCID: PMC4856420  PMID: 27419047
apoptosis; comparative proteomics; gene ontology analysis; rheumatoid arthritis; synovial cell line; TNF‐α
7.  Reconstruction of thermotolerant yeast by one-point mutation identified through whole-genome analyses of adaptively-evolved strains 
Scientific Reports  2016;6:23157.
Saccharomyces cerevisiae is used as a host strain in bioproduction, because of its rapid growth, ease of genetic manipulation, and high reducing capacity. However, the heat produced during the fermentation processes inhibits the biological activities and growth of the yeast cells. We performed whole-genome sequencing of 19 intermediate strains previously obtained during adaptation experiments under heat stress; 49 mutations were found in the adaptation steps. Phylogenetic tree revealed at least five events in which these strains had acquired mutations in the CDC25 gene. Reconstructed CDC25 point mutants based on a parental strain had acquired thermotolerance without any growth defects. These mutations led to the downregulation of the cAMP-dependent protein kinase (PKA) signaling pathway, which controls a variety of processes such as cell-cycle progression and stress tolerance. The one-point mutations in CDC25 were involved in the global transcriptional regulation through the cAMP/PKA pathway. Additionally, the mutations enabled efficient ethanol fermentation at 39 °C, suggesting that the one-point mutations in CDC25 may contribute to bioproduction.
doi:10.1038/srep23157
PMCID: PMC4794720  PMID: 26984760
8.  Rapid preparation of mutated influenza hemagglutinins for influenza virus pandemic prevention 
AMB Express  2016;6:8.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-016-0179-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s13568-016-0179-y
PMCID: PMC4722048  PMID: 26797882
Influenza; Hemagglutinin; Yeast display; Hemagglutination assay
9.  Inactivation of the Antifungal and Immunomodulatory Properties of Human Cathelicidin LL-37 by Aspartic Proteases Produced by the Pathogenic Yeast Candida albicans 
Infection and Immunity  2015;83(6):2518-2530.
Constant cross talk between Candida albicans yeast cells and their human host determines the outcome of fungal colonization and, eventually, the progress of infectious disease (candidiasis). An effective weapon used by C. albicans to cope with the host defense system is the release of 10 distinct secreted aspartic proteases (SAPs). Here, we validate a hypothesis that neutrophils and epithelial cells use the antimicrobial peptide LL-37 to inactivate C. albicans at sites of candidal infection and that C. albicans uses SAPs to effectively degrade LL-37. LL-37 is cleaved into multiple products by SAP1 to -4, SAP8, and SAP9, and this proteolytic processing is correlated with the gradual decrease in the antifungal activity of LL-37. Moreover, a major intermediate of LL-37 cleavage—the LL-25 peptide—is antifungal but devoid of the immunomodulatory properties of LL-37. In contrast to LL-37, LL-25 did not affect the generation of reactive oxygen species by neutrophils upon treatment with phorbol esters. Stimulating neutrophils with LL-25 (rather than LL-37) significantly decreased calcium flux and interleukin-8 production, resulting in lower chemotactic activity of the peptide against neutrophils, which may decrease the recruitment of neutrophils to infection foci. LL-25 also lost the function of LL-37 as an inhibitor of neutrophil apoptosis, thereby reducing the life span of these defense cells. This study indicates that C. albicans can effectively use aspartic proteases to destroy the antimicrobial and immunomodulatory properties of LL-37, thus enabling the pathogen to survive and propagate.
doi:10.1128/IAI.00023-15
PMCID: PMC4432748  PMID: 25847962
10.  Description of the interaction between Candida albicans and macrophages by mixed and quantitative proteome analysis without isolation 
AMB Express  2015;5:41.
Candida albicans is an opportunistic pathogen that causes fatal diseases in immunocompromised hosts. Host resistance against C. albicans relies on ingestion of the pathogen by macrophages. Analysis of the escaping behavior of C. albicans from macrophages is required to understand the onset of systemic candidiasis. In this study, native interactions of C. albicans with macrophages were investigated by proteome analysis using high efficiency of long monolithic silica capillary column. Using this system, we developed a method of “mixed and quantitative proteome analysis” in which C. albicans and macrophages were simultaneously analyzed by nanoLC–MS/MS without the need to isolate the two individual living cells. Two hundred twenty-seven proteins from C. albicans and five proteins from macrophages were identified as candidate interaction-specific molecules. C. albicans seemed to produce glucose through a β-oxidation pathway, a glyoxylate cycle, and gluconeogenesis for escape from macrophages. Up-regulation of stress-related and candidate pathogenic proteins in C. albicans indicated how C. albicans endured the harsh environment inside the macrophages. Down-regulation of apoptosis-associated protein NOA1- and chaperone HSPA1A-syntheses in macrophage indicated that C. albicans was able to escape from macrophages in part by suppressing the production of these macrophage proteins.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-015-0127-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13568-015-0127-2
PMCID: PMC4503712  PMID: 26179440
Candida albicans; Macrophage; Mixed proteome analysis; Quantitative proteome analysis; Apoptosis; Chaperone
11.  Proximity Effect among Cellulose-Degrading Enzymes Displayed on the Saccharomyces cerevisiae Cell Surface 
Proximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields.
doi:10.1128/AEM.02864-14
PMCID: PMC4272747  PMID: 25304511
12.  Elucidation of the recognition mechanisms for hemicellulose and pectin in Clostridium cellulovorans using intracellular quantitative proteome analysis 
AMB Express  2015;5:29.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-015-0115-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13568-015-0115-6
PMCID: PMC4441647  PMID: 26020016
Clostridium cellulovorans; Proteome analysis; Monolithic column; Substrate recognition; Hemicellulose; Pectin; Metabolism
13.  Functional screening system for yeast-secreted peptides acting on G-protein coupled receptors 
AMB Express  2015;5:26.
We established a novel functional screening system for peptides acting on G-protein coupled receptors (GPCRs). Peptides are a promising drug scaffold because of their intermediate molecular size between that of therapeutic small molecules and antibodies. They also offer potential advantages of targeting not only membrane proteins but also intracellular protein–protein interactions. Phage display technology has been used for exploring novel peptides acting on GPCRs, but it is unclear whether the identified peptides functionally modulate targets because the technology selects peptides based on binding ability but not functional activity to targets. In a novel screening system that we established, yeast cells were utilized as a peptide producer while mammalian cells stably producing the receptor for glucagon-like peptide 1 (GLP1R) were used as a biosensor for receptor activation. Three kinds of GLP1R agonists secreted by yeasts were successfully detected for their functional activities without any purification and condensation of those peptides. By applying the functional screening system, we were able to identify GLP1R agonist-secreting yeasts based on GLP1R activation from the cell mixture containing a number of background yeasts that produced non-active control peptides. Further applications of this system would include not only activity evaluation of bioactive peptides without chemical synthesis but also discovery of novel peptides activating druggable GPCRs.
doi:10.1186/s13568-015-0113-8
PMCID: PMC4427575  PMID: 25992304
Functional screening system; G-protein coupled receptors; Peptides; Yeast; Glucagon-like peptide-1
14.  Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory 
PLoS ONE  2015;10(5):e0124545.
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.
doi:10.1371/journal.pone.0124545
PMCID: PMC4430139  PMID: 25970342
15.  Kinin release from human kininogen by 10 aspartic proteases produced by pathogenic yeast Candida albicans 
BMC Microbiology  2015;15:60.
Background
Candida albicans yeast produces 10 distinct secreted aspartic proteases (Saps), which are some of the most important virulence factors of this pathogenic fungus. One of the suggested roles of Saps is their deregulating effect on various proteolytic cascades that constitute the major homeostatic systems in human hosts, including blood coagulation, fibrinolysis, and kallikrein-kinin systems. This study compared the characteristics of the action of all 10 Saps on human kininogens, which results in generating proinflammatory bradykinin-related peptides (kinins).
Results
Recombinant forms of Saps, heterologously overexpressed in Pichia pastoris were applied. Except for Sap7 and Sap10, all Saps effectively cleaved the kininogens, with the highest hydrolytic activity toward the low-molecular-mass form (LK). Sap1–6 and 8 produced a biologically active kinin—Met-Lys-bradykinin—and Sap3 was exceptional in terms of the kinin-releasing yield (>60% LK at pH 5.0 after 24 hours). Des-Arg1-bradykinin was released from LK by Sap9 at a comparably high yield, but this peptide was assumed to be biologically inactive because it was unable to interact with cellular B2-type kinin receptors. However, the collaborative actions of Sap9 and Sap1, −2, −4–6, and −8 on LK rerouted kininogen cleavage toward the high-yield release of the biologically active Met-Lys-bradykinin.
Conclusions
Our present results, together with the available data on the expression of individual SAP genes in candidal infection models, suggest a biological potential of Saps to produce kinins at the infection foci. The kinin release during candidiasis can involve predominant and complementary contributions of two different Sap3- and Sap9-dependent mechanisms.
doi:10.1186/s12866-015-0394-8
PMCID: PMC4357070  PMID: 25879450
Candidiasis; Human kininogen; Met-Lys-bradykinin; Des-Arg-kinins; Bradykinin B2-subtype receptors; Pichia pastoris
16.  Exoproteome analysis of Clostridium cellulovorans in natural soft-biomass degradation 
AMB Express  2015;5(1):2.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-014-0089-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s13568-014-0089-9
PMCID: PMC4305082  PMID: 25642399
Clostridium cellulovorans; Cellulosome; Soft-biomass degradation; Proteome analysis; Monolithic column
17.  Exoproteome analysis of Clostridium cellulovorans in natural soft-biomass degradation 
AMB Express  2015;5:2.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-014-0089-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s13568-014-0089-9
PMCID: PMC4305082  PMID: 25642399
Clostridium cellulovorans; Cellulosome; Soft-biomass degradation; Proteome analysis; Monolithic column
18.  Oral Immunization Against Candidiasis Using Lactobacillus casei Displaying Enolase 1 from Candida albicans 
Scientia Pharmaceutica  2014;82(3):697-708.
Abstract
Candidiasis is a common fungal infection that is prevalent in immunocompromised individuals. In this study, an oral vaccine against Candida albicans was developed by using the molecular display approach. Enolase 1 protein (Eno1p) of C. albicans was expressed on the Lactobacillus casei cell surface by using poly-gamma-glutamic acid synthetase complex A from Bacillus subtilis as an anchoring protein. The Eno1p-displaying L. casei cells were used to immunize mice, which were later challenged with a lethal dose of C. albicans. The data indicated that the vaccine elicited a strong IgG response and increased the survival rate of the vaccinated mice. Furthermore, L. casei acted as a potent adjuvant and induced high antibody titers that were comparable to those induced by strong adjuvants such as the cholera toxin. Overall, the molecular display method can be used to rapidly develop vaccines that can be conveniently administered and require minimal processing.
doi:10.3797/scipharm.1404-07
PMCID: PMC4318230  PMID: 25853077
Eno1p; Candida albicans; Lactococcus casei; Molecular display technology; Candidiasis
19.  Draft Genome Sequence of Falsirhodobacter sp. Strain alg1, an Alginate-Degrading Bacterium Isolated from Fermented Brown Algae 
Genome Announcements  2014;2(4):e00826-14.
Falsirhodobacter sp. alg1 is an alginate-degrading bacterium, the second species from the nonphototrophic bacterial genus Falsirhodobacter. We report the first draft genome of a bacterium from this genus and point out possible important features related to alginate assimilation and its evolutionary aspects.
doi:10.1128/genomeA.00826-14
PMCID: PMC4153483  PMID: 25146138
20.  Enhanced Adsorption and Recovery of Uranyl Ions by NikR Mutant-Displaying Yeast 
Biomolecules  2014;4(2):390-401.
Uranium is one of the most important metal resources, and the technology for the recovery of uranyl ions (UO22+) from aqueous solutions is required to ensure a semi-permanent supply of uranium. The NikR protein is a Ni2+-dependent transcriptional repressor of the nickel-ion uptake system in Escherichia coli, but its mutant protein (NikRm) is able to selectively bind uranyl ions in the interface of the two monomers. In this study, NikRm protein with ability to adsorb uranyl ions was displayed on the cell surface of Saccharomyces cerevisiae. To perform the binding of metal ions in the interface of the two monomers, two metal-binding domains (MBDs) of NikRm were tandemly fused via linker peptides and displayed on the yeast cell surface by fusion with the cell wall-anchoring domain of yeast α-agglutinin. The NikRm-MBD-displaying yeast cells with particular linker lengths showed the enhanced adsorption of uranyl ions in comparison to the control strain. By treating cells with citrate buffer (pH 4.3), the uranyl ions adsorbed on the cell surface were recovered. Our results indicate that the adsorption system by yeast cells displaying tandemly fused MBDs of NikRm is effective for simple and concentrated recovery of uranyl ions, as well as adsorption of uranyl ions.
doi:10.3390/biom4020390
PMCID: PMC4101488  PMID: 24970221
cell surface engineering; arming yeast; bioadsorption; uranyl ions; NikR
21.  Exoproteome Profiles of Clostridium cellulovorans Grown on Various Carbon Sources 
Applied and Environmental Microbiology  2013;79(21):6576-6584.
The cellulosome is a complex of cellulosomal proteins bound to scaffolding proteins. This complex is considered the most efficient system for cellulose degradation. Clostridium cellulovorans, which is known to produce cellulosomes, changes the composition of its cellulosomes depending on the growth substrates. However, studies have investigated only cellulosomal proteins; profile changes in noncellulosomal proteins have rarely been examined. In this study, we performed a quantitative proteome analysis of the whole exoproteome of C. cellulovorans, including cellulosomal and noncellulosomal proteins, to illustrate how various substrates are efficiently degraded. C. cellulovorans was cultured with cellobiose, xylan, pectin, or phosphoric acid-swollen cellulose (PASC) as the sole carbon source. PASC was used as a cellulose substrate for more accurate quantitative analysis. Using an isobaric tag method and a liquid chromatography mass spectrometer equipped with a long monolithic silica capillary column, 639 proteins were identified and quantified in all 4 samples. Among these, 79 proteins were involved in saccharification, including 35 cellulosomal and 44 noncellulosomal proteins. We compared protein abundance by spectral count and found that cellulosomal proteins were more abundant than noncellulosomal proteins. Next, we focused on the fold change of the proteins depending on the growth substrates. Drastic changes were observed mainly among the noncellulosomal proteins. These results indicate that cellulosomal proteins were primarily produced to efficiently degrade any substrate and that noncellulosomal proteins were specifically produced to optimize the degradation of a particular substrate. This study highlights the importance of noncellulosomal proteins as well as cellulosomes for the efficient degradation of various substrates.
doi:10.1128/AEM.02137-13
PMCID: PMC3811513  PMID: 23956399
22.  Spatial Reorganization of Saccharomyces cerevisiae Enolase To Alter Carbon Metabolism under Hypoxia 
Eukaryotic Cell  2013;12(8):1106-1119.
Hypoxia has critical effects on the physiology of organisms. In the yeast Saccharomyces cerevisiae, glycolytic enzymes, including enolase (Eno2p), formed cellular foci under hypoxia. Here, we investigated the regulation and biological functions of these foci. Focus formation by Eno2p was inhibited temperature independently by the addition of cycloheximide or rapamycin or by the single substitution of alanine for the Val22 residue. Using mitochondrial inhibitors and an antioxidant, mitochondrial reactive oxygen species (ROS) production was shown to participate in focus formation. Focus formation was also inhibited temperature dependently by an SNF1 knockout mutation. Interestingly, the foci were observed in the cell even after reoxygenation. The metabolic turnover analysis revealed that [U-13C]glucose conversion to pyruvate and oxaloacetate was accelerated in focus-forming cells. These results suggest that under hypoxia, S. cerevisiae cells sense mitochondrial ROS and, by the involvement of SNF1/AMPK, spatially reorganize metabolic enzymes in the cytosol via de novo protein synthesis, which subsequently increases carbon metabolism. The mechanism may be important for yeast cells under hypoxia, to quickly provide both energy and substrates for the biosynthesis of lipids and proteins independently of the tricarboxylic acid (TCA) cycle and also to fit changing environments.
doi:10.1128/EC.00093-13
PMCID: PMC3754543  PMID: 23748432
23.  Construction of a convenient system for easily screening inhibitors of mutated influenza virus neuraminidases☆ 
FEBS Open Bio  2013;3:484-489.
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.
Highlights
•Neuraminidase (NA) is a surface glycoprotein produced by the influenza virus.•Yeasts displaying wild-type and mutated NAs were constructed.•Biochemical properties of the displayed NAs were similar to those on the native virus.•Direct and rapid assays of NA enzyme activity were carried out.•This system can be developed for screening chemical libraries for novel inhibitors.
doi:10.1016/j.fob.2013.10.007
PMCID: PMC3836197  PMID: 24265981
Yeast surface display; Influenza A virus neuraminidase; Avian influenza virus H5N1; NA, neuraminidase; HNA, head domain of neuraminidase
24.  Fixation of CO2 in Clostridium cellulovorans analyzed by 13C-isotopomer-based target metabolomics 
AMB Express  2013;3:61.
Clostridium cellulovorans has been one of promising microorganisms to use biomass efficiently; however the basic metabolic pathways have not been completely known. We carried out 13C-isotopomer-based target metabolome analysis, or carbohydrate conversion process analysis, for more profound understanding of metabolic pathways of the bacterium. Our findings that pyruvate + oxaloacetate, fumarate, and malate inside and outside cells exhibited 13C incorporation suggest that C. cellulovorans exactly fixed CO2 and partly operated the TCA cycle in a reductive manner. Accompanied with CO2 fixation, the microorganism was also found to produce and secrete lactate. Overall, our study demonstrates that a part of C. cellulovorans metabolic pathways related to glycolysis and the TCA cycle are involved in CO2 fixation.
doi:10.1186/2191-0855-3-61
PMCID: PMC4124662  PMID: 24103325
CO2 fixation; Clostridium cellulovorans; Target metabolomics
25.  Arming Technology in Yeast—Novel Strategy for Whole-cell Biocatalyst and Protein Engineering 
Biomolecules  2013;3(3):632-650.
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.
doi:10.3390/biom3030632
PMCID: PMC4030959  PMID: 24970185
cell surface engineering; arming yeast; whole-cell biocatalyst; molecular display; arming technology; cell surface display; protein engineering; directed evolution; combinatorial bioengineering; single cell analysis

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