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1.  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
2.  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
3.  Characterization of Antimicrobial Peptides toward the Development of Novel Antibiotics 
Pharmaceuticals  2013;6(8):1055-1081.
Antimicrobial agents have eradicated many infectious diseases and significantly improved our living environment. However, abuse of antimicrobial agents has accelerated the emergence of multidrug-resistant microorganisms, and there is an urgent need for novel antibiotics. Antimicrobial peptides (AMPs) have attracted attention as a novel class of antimicrobial agents because AMPs efficiently kill a wide range of species, including bacteria, fungi, and viruses, via a novel mechanism of action. In addition, they are effective against pathogens that are resistant to almost all conventional antibiotics. AMPs have promising properties; they directly disrupt the functions of cellular membranes and nucleic acids, and the rate of appearance of AMP-resistant strains is very low. However, as pharmaceuticals, AMPs exhibit unfavorable properties, such as instability, hemolytic activity, high cost of production, salt sensitivity, and a broad spectrum of activity. Therefore, it is vital to improve these properties to develop novel AMP treatments. Here, we have reviewed the basic biochemical properties of AMPs and the recent strategies used to modulate these properties of AMPs to enhance their safety.
doi:10.3390/ph6081055
PMCID: PMC3817730  PMID: 24276381
antibiotic; antimicrobial peptide; drug delivery; activity regulation
4.  Disclosure of the differences of Mesorhizobium loti under the free-living and symbiotic conditions by comparative proteome analysis without bacteroid isolation 
BMC Microbiology  2013;13:180.
Background
Rhizobia are symbiotic nitrogen-fixing soil bacteria that show a symbiotic relationship with their host legume. Rhizobia have 2 different physiological conditions: a free-living condition in soil, and a symbiotic nitrogen-fixing condition in the nodule. The lifestyle of rhizobia remains largely unknown, although genome and transcriptome analyses have been carried out. To clarify the lifestyle of bacteria, proteome analysis is necessary because the protein profile directly reflects in vivo reactions of the organisms. In proteome analysis, high separation performance is required to analyze complex biological samples. Therefore, we used a liquid chromatography-tandem mass spectrometry system, equipped with a long monolithic silica capillary column, which is superior to conventional columns. In this study, we compared the protein profile of Mesorhizobium loti MAFF303099 under free-living condition to that of symbiotic conditions by using small amounts of crude extracts.
Result
We identified 1,533 and 847 proteins for M. loti under free-living and symbiotic conditions, respectively. Pathway analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that many of the enzymes involved in the central carbon metabolic pathway were commonly detected under both conditions. The proteins encoded in the symbiosis island, the transmissible chromosomal region that includes the genes that are highly upregulated under the symbiotic condition, were uniquely detected under the symbiotic condition. The features of the symbiotic condition that have been reported by transcriptome analysis were confirmed at the protein level by proteome analysis. In addition, the genes of the proteins involved in cell surface structure were repressed under the symbiotic nitrogen-fixing condition. Furthermore, farnesyl pyrophosphate (FPP) was found to be biosynthesized only in rhizobia under the symbiotic condition.
Conclusion
The obtained protein profile appeared to reflect the difference in phenotypes under the free-living and symbiotic conditions. In addition, KEGG pathway analysis revealed that the cell surface structure of rhizobia was largely different under each condition, and surprisingly, rhizobia might provided FPP to the host as a source of secondary metabolism. M. loti changed its metabolism and cell surface structure in accordance with the surrounding conditions.
doi:10.1186/1471-2180-13-180
PMCID: PMC3750425  PMID: 23898917
Mesorhizobium loti; Lotus japonicus; Symbiosis; Proteome analysis; Plant-microbe interaction; Monolithic column; Nitrogen fixation; Rhizobase; KEGG
5.  Secretome Analysis of the Pine Wood Nematode Bursaphelenchus xylophilus Reveals the Tangled Roots of Parasitism and Its Potential for Molecular Mimicry 
PLoS ONE  2013;8(6):e67377.
Since it was first introduced into Asia from North America in the early 20th century, the pine wood nematode Bursaphelenchus xylophilus has caused the devastating forest disease called pine wilt. The emerging pathogen spread to parts of Europe and has since been found as the causal agent of pine wilt disease in Portugal and Spain. In 2011, the entire genome sequence of B. xylophilus was determined, and it allowed us to perform a more detailed analysis of B. xylophilus parasitism. Here, we identified 1,515 proteins secreted by B. xylophilus using a highly sensitive proteomics method combined with the available genomic sequence. The catalogue of secreted proteins contained proteins involved in nutrient uptake, migration, and evasion from host defenses. A comparative functional analysis of the secretome profiles among parasitic nematodes revealed a marked expansion of secreted peptidases and peptidase inhibitors in B. xylophilus via gene duplication and horizontal gene transfer from fungi and bacteria. Furthermore, we showed that B. xylophilus secreted the potential host mimicry proteins that closely resemble the host pine’s proteins. These proteins could have been acquired by host–parasite co-evolution and might mimic the host defense systems in susceptible pine trees during infection. This study contributes to an understanding of their unique parasitism and its tangled roots, and provides new perspectives on the evolution of plant parasitism among nematodes.
doi:10.1371/journal.pone.0067377
PMCID: PMC3689755  PMID: 23805310
6.  Tracing Putative Trafficking of the Glycolytic Enzyme Enolase via SNARE-Driven Unconventional Secretion 
Eukaryotic Cell  2012;11(8):1075-1082.
Glycolytic enzymes are cytosolic proteins, but they also play important extracellular roles in cell-cell communication and infection. We used Saccharomyces cerevisiae to analyze the secretory pathway of some of these enzymes, including enolase, phosphoglucose isomerase, triose phosphate isomerase, and fructose 1,6-bisphosphate aldolase. Enolase, phosphoglucose isomerase, and an N-terminal 28-amino-acid-long fragment of enolase were secreted in a sec23-independent manner. The enhanced green fluorescent protein (EGFP)-conjugated enolase fragment formed cellular foci, some of which were found at the cell periphery. Therefore, we speculated that an overview of the secretory pathway could be gained by investigating the colocalization of the enolase fragment with intracellular proteins. The DsRed-conjugated enolase fragment colocalized with membrane proteins at the cis-Golgi complex, nucleus, endosome, and plasma membrane, but not the mitochondria. In addition, the secretion of full-length enolase was inhibited in a knockout mutant of the intracellular SNARE protein-coding gene TLG2. Our results suggest that enolase is secreted via a SNARE-dependent secretory pathway in S. cerevisiae.
doi:10.1128/EC.00075-12
PMCID: PMC3416056  PMID: 22753847
7.  Membrane-displayed somatostatin activates somatostatin receptor subtype-2 heterologously produced in Saccharomyces cerevisiae 
AMB Express  2012;2:63.
The G-protein-coupled receptor (GPCR) superfamily, which includes somatostatin receptors (SSTRs), is one of the most important drug targets in the pharmaceutical industry. The yeast Saccharomyces cerevisiae is an attractive host for the ligand screening of human GPCRs. Here, we demonstrate the utility of the technology that was developed for displaying peptide ligands on yeast plasma membrane, termed “PepDisplay”, which triggers signal transduction upon GPCR activation. A yeast strain that heterologously produced human somatostatin receptor subtype-2 (SSTR2) and chimeric Gα protein was constructed along with membrane-displayed somatostatin; somatostatin was displayed on the yeast plasma membrane by linking it to the anchoring domain of the glycosylphosphatidylinositol anchored plasma membrane protein Yps1p. We demonstrate that the somatostatin displayed on the plasma membrane successfully activated human SSTR2 in S. cerevisiae. The methodology presented here provides a new platform for identifying novel peptide ligands for both liganded and orphan mammalian GPCRs.
doi:10.1186/2191-0855-2-63
PMCID: PMC3558460  PMID: 23193953
Membrane-displayed ligand; PepDisplay; Yeast GPCR assay; Cyclic peptide; Somatostatin receptor subtype-2; Chimeric Gα protein
8.  Construction of a novel selection system for endoglucanases exhibiting carbohydrate-binding modules optimized for biomass using yeast cell-surface engineering 
AMB Express  2012;2:56.
To permit direct cellulose degradation and ethanol fermentation, Saccharomyces cerevisiae BY4741 (Δsed1) codisplaying 3 cellulases (Trichoderma reesei endoglucanase II [EG], T. reesei cellobiohydrolase II [CBH], and Aspergillus aculeatus β-glucosidase I [BG]) was constructed by yeast cell-surface engineering. The EG used in this study consists of a family 1 carbohydrate-binding module (CBM) and a catalytic module. A comparison with family 1 CBMs revealed conserved amino acid residues and flexible amino acid residues. The flexible amino acid residues were at positions 18, 23, 26, and 27, through which the degrading activity for various cellulose structures in each biomass may have been optimized. To select the optimal combination of CBMs of EGs, a yeast mixture with comprehensively mutated CBM was constructed. The mixture consisted of yeasts codisplaying EG with mutated CBMs, in which 4 flexible residues were comprehensively mutated, CBH, and BG. The yeast mixture was inoculated in selection medium with newspaper as the sole carbon source. The surviving yeast consisted of RTSH yeast (the mutant sequence of CBM: N18R, S23T, S26S, and T27H) and wild-type yeast (CBM was the original) in a ratio of 1:46. The mixture (1 RTSH yeast and 46 wild-type yeasts) had a fermentation activity that was 1.5-fold higher than that of wild-type yeast alone in the early phase of saccharification and fermentation, which indicates that the yeast mixture with comprehensively mutated CBM could be used to select the optimal combination of CBMs suitable for the cellulose of each biomass.
doi:10.1186/2191-0855-2-56
PMCID: PMC3534607  PMID: 23092441
Biorefinery; Carbohydrate-binding module (CBM); Cellulase; Yeast cell-surface engineering
9.  Profile of native cellulosomal proteins of Clostridium cellulovorans adapted to various carbon sources 
AMB Express  2012;2:37.
We performed a focused proteome analysis of cellulosomal proteins predicted by a genome analysis of Clostridium cellulovorans [Tamaru, Y., et al.. 2010. J. Bacteriol. 192:901–902]. Our system employed a long monolithic column (300 cm), which provides better performance and higher resolution than conventional systems. Twenty-three cellulosomal proteins were, without purification, identified by direct analysis of the culture medium. Proteome analysis of the C. cellulovorans cellulosome after culture in various carbon sources demonstrated the production of carbon source-adapted cellulosome components.
doi:10.1186/2191-0855-2-37
PMCID: PMC3444338  PMID: 22839966
Clostridium cellulovorans; Cellulosome; Focused proteome analysis; Monolithic column
10.  Putative Role of Cellulosomal Protease Inhibitors in Clostridium cellulovorans Based on Gene Expression and Measurement of Activities▿ 
Journal of Bacteriology  2011;193(19):5527-5530.
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.
doi:10.1128/JB.05022-11
PMCID: PMC3187468  PMID: 21784939
11.  Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease 
PLoS ONE  2012;7(2):e32513.
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 A-insensitive 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.
doi:10.1371/journal.pone.0032513
PMCID: PMC3287985  PMID: 22384266
12.  Molecular Breeding of Advanced Microorganisms for Biofuel Production 
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.
doi:10.1155/2011/416931
PMCID: PMC3035169  PMID: 21318120
13.  Comparison of the mesophilic cellulosome‐producing Clostridium cellulovorans genome with other cellulosome‐related clostridial genomes 
Microbial biotechnology  2010;4(1):64-73.
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.
doi:10.1111/j.1751-7915.2010.00210.x
PMCID: PMC3815796  PMID: 21255373
14.  Genome Sequence of the Cellulosome-Producing Mesophilic Organism Clostridium cellulovorans 743B▿  
Journal of Bacteriology  2009;192(3):901-902.
Clostridium cellulovorans 743B was isolated from a wood chip pile and is an anaerobic and mesophilic spore-forming bacterium. This organism degrades native substrates in soft biomass such as corn fiber and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Here we report the genome sequence of C. cellulovorans 743B.
doi:10.1128/JB.01450-09
PMCID: PMC2812471  PMID: 19948806
15.  Improvement in organophosphorus hydrolase activity of cell surface-engineered yeast strain using Flo1p anchor system 
Biotechnology Letters  2010;32(5):655-659.
Organophosphorus hydrolase (OPH) hydrolyzes organophosphorus esters. We constructed the yeast-displayed OPH using Flo1p anchor system. In this system, the N-terminal region of the protein was fused to Flo1p and the fusion protein was displayed on the cell surface. Hydrolytic reactions with paraoxon were carried out during 24 h of incubation of OPH-displaying cells at 30°C. p-Nitrophenol produced in the reaction mixture was detected by HPLC. The strain with highest activity showed 8-fold greater OPH activity compared with cells engineered using glycosylphosphatidylinositol anchor system, and showed 20-fold greater activity than Escherichia coli using the ice nucleation protein anchor system. These results indicate that Flo1p anchor system is suitable for display of OPH in the cell surface-expression systems.
doi:10.1007/s10529-010-0204-1
PMCID: PMC2852028  PMID: 20111980
Cell surface engineering; Flo1p anchor system; GPI anchor system; Organophosphorus hydrolase
16.  Regulation of the Display Ratio of Enzymes on the Saccharomyces cerevisiae Cell Surface by the Immunoglobulin G and Cellulosomal Enzyme Binding Domains▿  
Applied and Environmental Microbiology  2009;75(12):4149-4154.
We constructed a novel cell surface display system to control the ratio of target proteins on the Saccharomyces cerevisiae cell surface, using two pairs of protein-protein interactions. One protein pair is the Z domain of protein A derived from Staphylococcus aureus and the Fc domain of human immunoglobulin G. The other is the cohesin (Coh) and dockerin (Dock) from the cellulosome of Clostridium cellulovorans. In this proposed displaying system, the scaffolding proteins (fusion proteins of Z and Coh) were displayed on the cell surface by fusing with the 3′ half of α-agglutinin, and the target proteins fused with Fc or Dock were secreted. As a target protein, a recombinant Trichoderma reesei endoglucanase II (EGII) was secreted into the medium and immediately displayed on the yeast cell surface via the Z and Fc domains. Display of EGII on the cell surface was confirmed by hydrolysis of β-glucan as a substrate, and EGII activity was detected in the cell pellet fraction. Finally, two enzymes, EGII and Aspergillus aculeatus β-glucosidase 1, were codisplayed on the cell surface via Z-Fc and Dock-Coh interactions, respectively. As a result, the yeast displaying two enzymes hydrolyzed β-glucan to glucose very well. These results strongly indicated that the proposed strategy, the simultaneous display of two enzymes on the yeast cell surface, was accomplished by quantitatively controlling the display system using affinity binding.
doi:10.1128/AEM.00318-09
PMCID: PMC2698344  PMID: 19411409
17.  Discovery of a Modified Transcription Factor Endowing Yeasts with Organic-Solvent Tolerance and Reconstruction of an Organic-Solvent-Tolerant Saccharomyces cerevisiae Strain▿  
Applied and Environmental Microbiology  2008;74(13):4222-4225.
Organic-solvent tolerance in Saccharomyces cerevisiae strain KK-211, which was first isolated as an organic-solvent-tolerant strain, depends on point mutation (R821S) of the transcription factor Pdr1p. The integration of the PDR1 R821S mutation into wild-type yeast results in organic-solvent tolerance, and the PDR1 R821S mutant can reduce carbonyl compounds in organic solvents.
doi:10.1128/AEM.02874-07
PMCID: PMC2446499  PMID: 18469127
18.  Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents†  
Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with α-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 × 104-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 × 104-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H2O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.
doi:10.1128/AEM.71.8.4335-4338.2005
PMCID: PMC1183351  PMID: 16085821
19.  Direct Production of Ethanol from Raw Corn Starch via Fermentation by Use of a Novel Surface-Engineered Yeast Strain Codisplaying Glucoamylase and α-Amylase 
Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis α-amylase by using the C-terminal-half region of α-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.
doi:10.1128/AEM.70.8.5037-5040.2004
PMCID: PMC492352  PMID: 15294847
20.  Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme 
A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on α-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus β-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of β-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying β-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.
doi:10.1128/AEM.70.2.1207-1212.2004
PMCID: PMC348929  PMID: 14766607
21.  Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes 
Applied and Environmental Microbiology  2002;68(10):5136-5141.
For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on α-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His6) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-α-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley β-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and β-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing β-glucan as the sole carbon source and could directly ferment 45 g of β-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.
doi:10.1128/AEM.68.10.5136-5141.2002
PMCID: PMC126432  PMID: 12324364
22.  Construction of Yeast Strains with High Cell Surface Lipase Activity by Using Novel Display Systems Based on the Flo1p Flocculation Functional Domain 
We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3′ region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5′ upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.
doi:10.1128/AEM.68.9.4517-4522.2002
PMCID: PMC124111  PMID: 12200308
23.  Genetically Controlled Self-Aggregation of Cell-Surface-Engineered Yeast Responding to Glucose Concentration 
We constructed an arming (cell-surface-engineered) yeast displaying two types of agglutinin (modified a-agglutinin and α-agglutinin) on the cell surface, with agglutination being independent of both mating type and pheromones. The modified a-agglutinin was artificially prepared by the fusion of the genes encoding Aga1p and Aga2p. The modified a-agglutinin could induce agglutination of cells displaying Agα1p (α-agglutinin). The upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL), active at a low glucose concentration, was used as the promoter to express the modified a-agglutinin- and α-agglutinin-encoding genes. The arming yeast displaying both agglutinins agglutinated and sedimented in response to decreased glucose concentration. When the glucose concentration was high, the arming yeast grew normally. In the late log phase, when the glucose concentration became very low, agglutination occurred suddenly and drastically and yeast cells sedimented completely. Sedimentation was confirmed by weighing the aggregated cells after filtration of the broth. Strains in which aggregation can be genetically controlled can be used in industrial processes in which the separation of yeast cells from the supernatant is necessary.
doi:10.1128/AEM.67.5.2083-2087.2001
PMCID: PMC92840  PMID: 11319085
24.  Screening of Genes Involved in Isooctane Tolerance in Saccharomyces cerevisiae by Using mRNA Differential Display 
Applied and Environmental Microbiology  2000;66(11):4883-4889.
A Saccharomyces cerevisiae strain, KK-211, isolated by the long-term bioprocess of stereoselective reduction in isooctane, showed extremely high tolerance to the solvent, which is toxic to yeast cells, but, in comparison with its wild-type parent, DY-1, showed low tolerance to hydrophilic organic solvents, such as dimethyl sulfoxide and ethanol. In order to detect the isooctane tolerance-associated genes, mRNA differential display (DD) was employed using mRNAs isolated from strains DY-1 and KK-211 cultivated without isooctane, and from strain KK-211 cultivated with isooctane. Thirty genes were identified as being differentially expressed in these three types of cells and were classified into three groups according to their expression patterns. These patterns were further confirmed and quantified by Northern blot analysis. On the DD fingerprints, the expression of 14 genes, including MUQ1, PRY2, HAC1, AGT1, GAC1, and ICT1 (YLR099c) was induced, while the expression of the remaining 16 genes, including JEN1, PRY1, PRY3, and KRE1, was decreased, in strain KK-211 cultivated with isooctane. The genes represented by HAC1, PRY1, and ICT1 have been reported to be associated with cell stress, and AGT1 and GAC1 have been reported to be involved in the uptake of trehalose and the production of glycogen, respectively. MUQ1 and KRE1, encoding proteins associated with cell surface maintenance, were also detected. Based on these results, we concluded that alteration of expression levels of multiple genes, not of a single gene, might be the critical determinant for isooctane tolerance in strain KK-211.
PMCID: PMC92395  PMID: 11055939
25.  An n-Alkane-Responsive Promoter Element Found in the Gene Encoding the Peroxisomal Protein of Candida tropicalis Does Not Contain a C6 Zinc Cluster DNA-Binding Motif 
Journal of Bacteriology  2000;182(9):2492-2497.
When an asporogenic diploid yeast, Candida tropicalis, is cultivated on n-alkane, the expression of the genes encoding enzymes of the peroxisomal β-oxidation pathway is highly induced. An upstream activation sequence (UAS) which can induce transcription in response to n-alkane (UASALK) was identified on the promoter region of the peroxisomal 3-ketoacyl coenzyme A (CoA) thiolase gene of C. tropicalis (CT-T3A). The 29-bp region (from −289 to −261) present upstream of the TATA sequence was sufficient to induce n-alkane-dependent expression of a reporter gene. Besides n-alkane, UASALK-dependent gene expression also occurred in the cells grown on oleic acid. Several kinds of mutant UASALK were constructed and tested for their UAS activity. It was clarified that the important nucleotides for UASALK activity were located within 10-bp region from −273 to −264 (5′-TCCTGCACAC-3′). This region did not contain a CGG triplet and therefore differed from the sequence of the oleate-response element (ORE), which is a UAS found on the promoter region of 3-ketoacyl-CoA thiolase gene of Saccharomyces cerevisiae. Similar sequences to UASALK were also found on several peroxisomal enzyme-encoding genes of C. tropicalis.
PMCID: PMC111312  PMID: 10762250

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