YdfH identified as a repressor of rspA by the use of reduced genome Escherichia coli MGF-01.

We have reported the construction of 1 Mb reduced genome Escherichia coli MGF-01 by a 28-step operation. This time, transcriptome analysis of MGF-01 was performed. Although the transcriptome profiles of the exponential phase in parental strain W3110red were well-conserved in MGF-01, the rspAB operon was highly expressed. A LacZ reporter assay of a series of stepwise deletion strains prepared in the course of MGF-01 construction indicated that rspA was highly expressed after the 5th step. Further analysis indicated that Δ29, one of the deleted regions at the 5th step, relates to an increase in rspA expression, and that transcriptional regulator ydfH, in the Δ29 region, is responsible for the expression of rspA, gel shift assay indicated that YdfH bound directly to the upstream region of rspA. Based on these results, it was concluded that YdfH is a transcriptional repressor of the rspAB operon.

Development of Targeted Protein-Displaying Technology with a Novel Carbon Material.

This study reports a new carbon material and its specific display of targeted protein. The properties of the carbon materials fabricated with carbon black MOGUL® were analyzed. The carbon materials were spherical structures with 55.421 µm as a median value. The specific surface area, pore volume, average pore diameter, and total of the acidic functional group were 130 m2·g-1, 0.55 cm3·g-1, 17.2 nm, and 0.29 mEq·g-1, respectively. The adsorption-desorption isoform of the carbon materials showed type IV of the hysteresis loop as defined by IUPAC, indicating non-uniform mesoporous structures (2-50 nm). The distribution of the log differential pore volume also indicated non-uniform porous structures because (i) the difference between the average pore size and the most frequent pore size was significant and (ii) the σ value was larger than the average value regarding the pore sizes. However, 10-90% of the integrated values of the log differential pore volume were 57.4% of the total integrated values, and the distribution was similar to the Gauss distribution model. Although the value of the total of the acidic functional group was 2.5-5.4 times lower than the values of the HPLC columns, the carbon materials require good scaffold quality rather than good HPLC quality. Therefore, the amounts could be enough for the scaffold of biotin hydrazide. To demonstrate the property of displaying the targeted proteins, carbon materials displaying biotin hydrazide by covalent bonding were prepared and avidin-labeled horse radish peroxidase (HRP) was bound to the biotin region. The carbon materials were porous structures, so the unspecific adsorption of HRP was estimated. Then, the maintenance ratios of HRP activities were analyzed in the repeated-use-with-wash processes after each evaluation, resulting in the activities of HRP on the carbon materials being treated with biotin hydrazide being significantly maintained compared to that of the ones without biotin hydrazide. The study revealed the properties of the carbon materials and indicated the display of HRP, suggesting that the carbon materials could be a new material for displaying targeted proteins.

Pretreatment of extruded Napier grass byhydrothermal process with dilute sulfuric acid and fermentation using a cellulose-hydrolyzing and xylose-assimilating yeast for ethanol production.

One of the potential bioresources for bioethanol production is Napier grass, considering its high cellulose and hemicellulose content. However, the cost of pretreatment hinders the bioethanol produced from being economical. This study examines the effect of hydrothermal process with dilute acid on extruded Napier grass, followed by enzymatic saccharification prior to simultaneous saccharification and co-fermentation (SScF). Extrusion facilitated lignin removal by 30.2 % prior to dilute acid steam explosion. Optimum pretreatment condition was obtained by using 3% sulfuric acid, and 30-min retention time of steam explosion at 190 °C. Ethanol yield of 0.26 g ethanol/g biomass (60.5% fermentation efficiency) was attained by short-term liquefaction and fermentation using a cellulose-hydrolyzing and xylose-assimilating Saccharomyces cerevisiae NBRC1440/B-EC3-X ΔPHO13, despite the presence of inhibitors. This proposed method not only reduced over-degradation of cellulose and hemicellulose, but also eliminated detoxification process and reduced cellulase loading.

Substrate-induced differential degradation and partitioning of the two tryptophan permeases Tat1 and Tat2 into eisosomes in Saccharomyces cerevisiae.

Tryptophan is a relatively rare amino acid whose influx is strictly controlled to meet cellular demands. The yeast Saccharomyces cerevisiae has two tryptophan permeases, namely Tat1 (low-affinity type) and Tat2 (high-affinity type). These permeases are differentially regulated through ubiquitination based on inducible conditions and dependence on arrestin-related trafficking adaptors, although the physiological significance of their degradation remain unclear. Here, we demonstrated that Tat2 was rapidly degraded in an Rsp5-Bul1-dependent manner upon the addition of tryptophan, phenylalanine, or tyrosine, whereas Tat1 was unaffected. The expression of the ubiquitination-deficient variant Tat25K>R led to a reduction in cell yield at 4 µg/mL tryptophan, suggesting the occurrence of an uncontrolled, excessive consumption of tryptophan at low tryptophan concentrations. Eisosomes are membrane furrows that are thought to be storage compartments for some nutrient permeases. Tryptophan addition caused rapid Tat2 dissociation from eisosomes, whereas Tat1 distribution was unaffected. The 5 K > R mutation had no marked effect on Tat2 dissociation, suggesting that dissociation is independent of ubiquitination. Interestingly, the D74R mutation, which was created within the N-terminal acidic patch, stabilized Tat2 while reducing the degree of partitioning into eisosomes. Moreover, the hyperactive I285V mutation in Tat2, which increases Vmax/Km for tryptophan import by 2-fold, reduced the degree of segregation into eisosomes. Our findings illustrate the coordinated activity of Tat1 and Tat2 in the regulation of tryptophan transport at various tryptophan concentrations and suggest the positive role of substrates in inducing a conformational transition in Tat2, resulting in its dissociation from eisosomes and subsequent ubiquitination-dependent degradation.

Construction of cell-plastics as neo-plastics consisted of cell-layer provided green alga Chlamydomonas reinhardtii covered by two-dimensional polymer.

Green alga Chlamydomonas reinhardtii has gained interest as a sustainable resource because it can be easily grown using CO2 as a carbon source owing to its high CO2 assimilating activity. Although the robustness of the cell wall of C. reinhardtii makes it difficult to extract its intracellular products, such property is beneficial when using the cell as an ingredient to fabricate "cell-plastic" in this study. The cell layer, which is a component of the cell-plastic, was prepared with an intercellular filler to connect each cell because C. reinhardtii is a single-cell strain. The cell layers were then repeatedly piled to increase the strength of the cell-plastic. To avoid slippage between the cell layers, they were covered with a small amount of a two-dimensional polymer to maintain the flat surface structure of the cell-plastic. Based on the evaluation, the cell-plastic has the potential to be a novel, sustainable plastic using ubiquitous green algal cells in nature.

Increased flux in acetyl-CoA synthetic pathway and TCA cycle of Kluyveromyces marxianus under respiratory conditions.

Yeasts are extremely useful, not only for fermentation but also for a wide spectrum of fuel and chemical productions. We analyzed the overall metabolic turnover and transcript dynamics in glycolysis and the TCA cycle, revealing the difference in adaptive pyruvate metabolic response between a Crabtree-negative species, Kluyveromyces marxianus, and a Crabtree-positive species, Saccharomyces cerevisiae, during aerobic growth. Pyruvate metabolism was inclined toward ethanol production under aerobic conditions in S. cerevisiae, while increased transcript abundances of the genes involved in ethanol metabolism and those encoding pyruvate dehydrogenase were seen in K. marxianus, indicating the augmentation of acetyl-CoA synthesis. Furthermore, different metabolic turnover in the TCA cycle was observed in the two species: malate and fumarate production in S. cerevisiae was higher than in K. marxianus, irrespective of aeration; however, fluxes of both the reductive and oxidative TCA cycles were enhanced in K. marxianus by aeration, implying both the cycles contribute to efficient electron flux without producing ethanol. Additionally, decreased hexokinase activity under aerobic conditions is expected to be important for maintenance of suitable carbon flux. These findings demonstrate differences in the key metabolic trait of yeasts employing respiration or fermentation, and provide important insight into the metabolic engineering of yeasts.

A novel sensitive immunoassay method based on the Invader technique.

A novel and sensitive immunoassay method has been developed in which the conventional sandwich immunoassay and the highly sensitive DNA detection method, the Invader method, are combined. The signal amplification function of the latter method has been successfully used to enhance the sensitivity of the sandwich immunoassay. The new assay method may be called the Immuno-Invader assay. The assay format involves three important steps: (1) a target antigen is captured and flagged with a biotin-conjugated detection antibody by the sandwich method, (2) streptavidin and a biotin-conjugated oligonucleotide are added to form a complex with the detection antibody, and (3) the oligonucleotide in the complex is detected using the Invader method. The method was applied to the assay of human tumor necrosis factor-alpha (hTNF-alpha). Detection limits obtained were 0.1 pg/ml hTNF-alpha when a luminescent europium chelate was used with a time-resolved measurement mode, and 0.8 pg/ml when fluorescein was used with a normal prompt fluorescence measurement mode. On the other hand, the detection limit of a commercially available hTNF-alpha enzyme-linked immunosorbent assay that uses horseradish peroxidase was 3.5 pg/ml. These results demonstrate the feasibility and potential of the new assay method for highly sensitive immunoassay.

Selection of yeast Saccharomyces cerevisiae promoters available for xylose cultivation and fermentation.

To efficiently utilize xylose, a major sugar component of hemicelluloses, in Saccharomyces cerevisiae requires the proper expression of varied exogenous and endogenous genes. To expand the repertoire of promoters in engineered xylose-utilizing yeast strains, we selected promoters in S. cerevisiae during cultivation and fermentation using xylose as a carbon source. To select candidate promoters that function in the presence of xylose, we performed comprehensive gene expression analyses using xylose-utilizing yeast strains both during xylose and glucose fermentation. Based on microarray data, we chose 29 genes that showed strong, moderate, and weak expression in xylose rather than glucose fermentation. The activities of these promoters in a xylose-utilizing yeast strain were measured by lacZ reporter gene assays over time during aerobic cultivation and microaerobic fermentation, both in xylose and glucose media. In xylose media, PTDH3, PFBA1, and PTDH1 were favorable for high expression, and PSED1, PHXT7, PPDC1, PTEF1, PTPI1, and PPGK1 were acceptable for medium-high expression in aerobic cultivation, and moderate expression in microaerobic fermentation. PTEF2 allowed moderate expression in aerobic culture and weak expression in microaerobic fermentation, although it showed medium-high expression in glucose media. PZWF1 and PSOL4 allowed moderate expression in aerobic cultivation, while showing weak but clear expression in microaerobic fermentation. PALD3 and PTKL2 showed moderate promoter activity in aerobic cultivation, but showed almost no activity in microaerobic fermentation. The knowledge of promoter activities in xylose cultivation obtained in this study will permit the control of gene expression in engineered xylose-utilizing yeast strains that are used for hemicellulose fermentation.

Crystal structure of family GH-8 chitosanase with subclass II specificity from Bacillus sp. K17.

Crystal structures of chitosanase from Bacillus sp. K17 (ChoK) have been determined at 1.5 A resolution in the active form and at 2.0 A resolution in the inactive form. This enzyme belongs to the family GH-8, out of 93 glycoside hydrolase families, and exhibits the substrate specificity of subclass II chitosanase. The catalytic site is constructed on the scaffold of a double-alpha(6)/alpha(6)-barrel, which is formed by six repeating helix-loop-helix motifs. This structure is quite different from those of the GH-46 chitosanases and of GH-5. Structural comparison with CelA (a cellulase belonging to the same family GH-8) suggests that the proton donor Glu122 is conserved, but the proton acceptor is the inserted Glu309 residue, and that the corresponding Asp278 residue in CelA is inactivated in ChoK. The four acidic residues, Asp179, Glu309, Asp183 and Glu107, can be involved in substrate recognition through interactions with the amino groups of the glucosamine residues bound in the -3, -2, -1 and +1 sites, respectively. The hydrophobic Trp235, Trp166, Phe413 and Tyr318 residues are highly conserved for binding of the hexose rings at the -3, -2, +1 and +2 sites, respectively. These structural features indicate that enzymes in GH-8 can be further divided into three subfamilies. Different types of chitosanases are discussed in terms of convergent evolution from different structural ancestors.

Improved ethanol production from xylose in the presence of acetic acid by the overexpression of the HAA1 gene in Saccharomyces cerevisiae.

The hydrolysis of lignocellulosic biomass liberates sugars, primarily glucose and xylose, which are subsequently converted to ethanol by microbial fermentation. The rapid and efficient fermentation of xylose by recombinant Saccharomyces cerevisiae strains is limited by weak acids generated during biomass pretreatment processes. In particular, acetic acid negatively affects cell growth, xylose fermentation rate, and ethanol production. The ability of S. cerevisiae to efficiently utilize xylose in the presence of acetic acid is an essential requirement for the cost-effective production of ethanol from lignocellulosic hydrolysates. Here, an acetic acid-responsive transcriptional activator, HAA1, was overexpressed in a recombinant xylose-fermenting S. cerevisiae strain to yield BY4741X/HAA1. This strain exhibited improved cell growth and ethanol production from xylose under aerobic and oxygen limited conditions, respectively, in the presence of acetic acid. The HAA1p regulon enhanced transcript levels in BY4741X/HAA1. The disruption of PHO13, a p-nitrophenylphosphatase gene, in BY4741X/HAA1 led to further improvement in both yeast growth and the ability to ferment xylose, indicating that HAA1 overexpression and PHO13 deletion act by different mechanisms to enhance ethanol production.

Optimized membrane process to increase hemicellulosic ethanol production from pretreated rice straw by recombinant xylose-fermenting Saccharomyces cerevisiae.

UNLABELLED: Oligomeric sugars in the liquid fraction of hot water-pretreated rice straw are more amenable to membrane process than monomeric sugars, as lower pressure is required. Following membrane process was employed: nanofiltration (NF) concentration → (dilution → NF concentration) × 2 times → enzymatic hydrolysis (EH) → ultrafiltration (UF) permeation [ IMPLICATION: NF for recovery of oligomeric sugars, dilution and NF for removal of low molecular weight fermentation inhibitors, UF for removal of high molecular weight fermentation inhibitors and recovery of monomeric sugars after EH]. This process provided the liquid fraction containing 111.4 g L(-1) of sugars, corresponding to 681.0mM as monomeric sugars, from the original liquid fraction (181.1mM monomeric sugars). Concentrations of low molecular weight fermentation inhibitors, acetic and formic acids, were decreased to 24% and 48%, respectively. Xylose-fermenting recombinant Saccharomyces cerevisiae produced 34.5 ± 2.2 g L(-1) ethanol from the 0.8 times liquid fraction (76% of theoretical yield).

Ethanol fermentation by xylose-assimilating Saccharomyces cerevisiae using sugars in a rice straw liquid hydrolysate concentrated by nanofiltration.

Concentrating sugars using membrane separation, followed by ethanol fermentation by recombinant xylose-assimilating Saccharomyces cerevisiae, is an attractive technology. Three nanofiltration membranes (NTR-729HF, NTR-7250, and ESNA3) were effective in concentrating glucose, fructose, and sucrose from dilute molasses solution and no permeation of sucrose. The separation factors of acetate, formate, furfural, and 5-hydroxymethyl furfural, which were produced by dilute acid pretreatment of rice straw, over glucose after passage through these three membranes were 3.37-11.22, 4.71-20.27, 4.32-16.45, and 4.05-16.84, respectively, at pH 5.0, an applied pressure of 1.5 or 2.0 MPa, and 25 °C. The separation factors of these fermentation inhibitors over xylose were infinite, as there was no permeation of xylose. Ethanol production from approximately two-times concentrated liquid hydrolysate using recombinant S. cerevisiae was double (5.34-6.44 g L(-1)) that compared with fermentation of liquid hydrolysate before membrane separation (2.75 g L(-1)).

The N-terminal region is important for the nuclease activity and thermostability of the flap endonuclease-1 from Sulfolobus tokodaii.

This paper reports the biochemical properties of two types of recombinant flap endonuclease-1 (FEN-1) proteins obtained from the thermophilic crenarchaeon, Sulfolobus tokodaii strain 7. One of the two FEN-1 proteins is a product of the gene with AUG as the translational start codon (StoS-FEN-1), which is originally assigned in the database. The other is a product of the gene with a new AUG start codon (StoL-FEN-1), which is inserted at 153 bases upstream of the original AUG codon. Although StoL-FEN-1 showed activity and thermostability, StoS-FEN-1 showed neither activity nor thermostability. The N-terminal region in StoL-FEN-1 was also conserved in all of the FEN-1 homologs deduced from genes from newly isolated Sulfolobus spp. These results strongly suggest that the actual start codon of the fen-1 gene from S. tokodaii is not the originally assigned AUG, but rather is located at about 100 bases upstream of this codon.

Isolation and metabolic characteristics of previously uncultured members of the order aquificales in a subsurface gold mine.

Culture-dependent and -independent techniques were combined to characterize the physiological properties and the ecological impacts of culture-resistant phylotypes of thermophiles within the order Aquificales from a subsurface hot aquifer of a Japanese gold mine. Thermophilic bacteria phylogenetically associated with previously uncultured phylotypes of Aquificales were successfully isolated. 16S ribosomal DNA clone analysis of the entire microbial DNA assemblage and fluorescence in situ whole-cell hybridization analysis indicated that the isolates dominated the microbial population in the subsurface aquifer. The isolates were facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotrophs utilizing molecular oxygen, nitrate, ferric iron, arsenate, selenate, and selenite as electron acceptors. Their versatile energy-generating systems may reflect the geochemical conditions of their habitat in the geothermally active subsurface gold mine.

Molecular phylogenetic analyses of reverse-transcribed bacterial rRNA obtained from deep-sea cold seep sediments.

A depth profile of naturally occurring bacterial community structures associated with the deep-sea cold seep push-core sediment in the Japan Trench at a depth of 5343 m were evaluated using molecular phylogenetic analyses of RNA reverse transcription-PCR (RT-PCR) amplified 16S crDNA fragments. A total of 137 clones of bacterial crDNA (complimentary rDNA) phylotypes (phylogenetic types) obtained at three different depths (2-4, 8-10 and 14-16 cm) were identified in partial crDNA sequencings. crDNA phylotypes from the cold seep sediment were dominantly composed of delta- and epsilon-Proteobacteria (36% and 42% respectively). Phylotype analysis of crDNA clone libraries and terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the majority of bacterial components shifted from delta- Proteobacteria to epsilon-Proteobacteria with increasing depth. Among the delta-proteobacterial crDNA clones, the sequences related to the genus Desulfosarcina were dominant. Almost all sequences of crDNA belonging to epsilon-Proteobacteria were affiliated with the same cluster (epsilon-CSG: epsilon-proteobacterial cold seep group), and were closely related with rDNA sequences from deep-sea hydrothermal vent environments.

Crystallization and preliminary X-ray analyses of the active and the inactive forms of family GH-8 chitosanase with subclass II specificity from Bacillus sp. strain K17.

Chitosanase from Bacillus sp. strain K17 (ChoK) belongs to glycoside hydrolase family 8 and exhibits subclass II specificity. The purified protein is structurally stable over a wide pH range (3-10), but is active in a much narrower pH range (4.5-7.5), with optimal activity around pH 6.0. The protein has been successfully crystallized at two different pH values corresponding to the active and inactive states. The crystals diffract to 1.5 and 2.0 A resolution, respectively.

A novel chitosanase from Bacillus sp. strain K17: gene cloning and expression in Escherichia coli.

The gene encoding a novel chitosanase from Bacillus sp. strain K17 was cloned and sequenced. The nucleotide sequence of the gene contained an open reading frame corresponded to a protein of 453 amino acids. The deduced amino acid sequence of the K17 chitosanase exhibited the highest homology to those of family 8 glycanases, suggesting that the enzyme belonged to family 8.