Abstract The integration of first‐ (1G) and second‐generation (2G) ethanol production by adding sugarcane juice or molasses to lignocellulosic hydrolysates offers the possibility to overcome the problem of inhibitors (acetic acid, furfural, hydroxymethylfurfural and phenolic compounds), and add nutrients (such as salts, sugars and nitrogen sources) to the fermentation medium, allowing the production of higher ethanol titers. In this work, an 1G2G production process was developed with hemicellulosic hydrolysate (HH) from a diluted sulfuric acid pretreatment of sugarcane bagasse and sugarcane molasses. The industrial Saccharomyces cerevisiae CAT‐1 was genetically modified for xylose consumption and used for co‐fermentation of sucrose, fructose, glucose, and xylose. The fed‐batch fermentation with high cell density that mimics an industrial fermentation was performed at bench scale fermenter, achieved high volumetric ethanol productivity of 1.59 g L⁻¹ h⁻¹, 0.39 g g⁻¹ of ethanol yield, and 44.5 g L⁻¹ ethanol titer, and shown that the yeast was able to consume all the sugars present in must simultaneously. With the results, it was possible to establish a mass balance for the global process: from pretreatment to the co‐fermentation of molasses and HH, and it was possible to establish an effective integrated process (1G2G) with sugarcane molasses and HH co‐fermentation employing a recombinant yeast.

Acetogens grow autotrophically and use hydrogen (H2) as the energy source to fix carbon dioxide (CO2). This feature can be applied to gas fermentation, contributing to a circular economy. A challenge is the gain of cellular energy from H2 oxidation, which is substantially low, especially when acetate formation coupled with ATP production is diverted to other chemicals in engineered strains. Indeed, an engineered strain of the thermophilic acetogen Moorella thermoacetica that produces acetone lost autotrophic growth on H2 and CO2. We aimed to recover autotrophic growth and enhance acetone production, in which ATP production was assumed to be a limiting factor, by supplementing with electron acceptors. Among the four selected electron acceptors, thiosulfate and dimethyl sulfoxide (DMSO) enhanced both bacterial growth and acetone titers. DMSO was the most effective and was further analyzed. We showed that DMSO supplementation enhanced intracellular ATP levels, leading to increased acetone production. Although DMSO is an organic compound, it functions as an electron acceptor, not a carbon source. Thus, supplying electron acceptors is a potential strategy to complement the low ATP production caused by metabolic engineering and to improve chemical production from H2 and CO2.

Simultaneous saccharification and fermentation (SSF) has been investigated for the efficient production of ethanol because it has several advantages such as simplifying the manufacturing process, operating easily, and reducing energy input. Previously, using lignocellulosic biomass as source materials, we succeeded in producing ethanol by SSF with Pichia kudriavzevii NBRC1279 and NBRC1664. However, various acids that fermentation inhibitors are also produced by the hydrolysis of lignocellulosic biomass, and the extent to which these acids affect the growth and ethanol productivity of the two strains has not yet been investigated. In this study, to better understand the acid tolerance mechanism of the two strains, a spot assay, growth experiment, and transcriptome analysis were carried out using Saccharomyces cerevisiae BY4742 as a control. When the three strains were cultured in SCD medium containing 15 mM formic acid, 35 mM sulfuric acid, 60 mM hydrochloric acid, 100 mM acetic acid, or 550 mM lactic acid, only P. kudriavzevii NBRC1664 could grow well under all conditions, and it showed the fastest growth rates. The transcriptome analysis showed that “MAPK signaling pathway-yeast” was significantly enriched in P. kudriavzevii NBRC1664 cultured with 60 mM hydrochloric acid, and most genes involved in the high osmolarity glycerol (HOG) pathway were up-regulated. Therefore, the up-regulation of the HOG pathway may be important for adapting to acid stress in P. kudriavzevii. Moreover, the log2-transformed fold change value in the expression level of Gpd1 was 1.3-fold higher in P. kudriavzevii NBRC1664 than in P. kudriavzevii NBRC1279, indicating that high Gpd1 expression may be accountable for the higher acid tolerance of P. kudriavzevii NBRC1664. The transcriptome analysis performed in this study provides preliminary knowledge of the molecular mechanism of acid stress tolerance in P. kudriavzevii. Our data may be useful for future studies on methods to improve the tolerance of P. kudriavzevii to acids produced from lignocellulose hydrolysis.

Hydrogen (H2) converted to reducing equivalents is used by acetogens to fix and metabolize carbon dioxide (CO2) to acetate. The utilization of H2 enables not only autotrophic growth, but also mixotrophic metabolism in acetogens, enhancing carbon utilization. This feature seems useful, especially when the carbon utilization efficiency of organic carbon sources is lowered by metabolic engineering to produce reduced chemicals, such as ethanol. The potential advantage was tested using engineered strains of Moorella thermoacetica that produce ethanol. By adding H2 to the fructose-supplied culture, the engineered strains produced increased levels of acetate, and a slight increase in ethanol was observed. The utilization of a knockout strain of the major acetate production pathway, aimed at increasing the carbon flux to ethanol, was unexpectedly hindered by H2-mediated growth inhibition in a dose-dependent manner. Metabolomic analysis showed a significant increase in intracellular NADH levels due to H2 in the ethanol-producing strain. Higher NADH level was shown to be the cause of growth inhibition because the decrease in NADH level by dimethyl sulfoxide (DMSO) reduction recovered the growth. When H2 was not supplemented, the intracellular NADH level was balanced by the reversible electron transfer from NADH oxidation to H2 production in the ethanol-producing strain. Therefore, reversible hydrogenase activity confers the ability and flexibility to balance the intracellular redox state of M. thermoacetica. Tuning of the redox balance is required in order to benefit from H2-supplemented mixotrophy, which was confirmed by engineering to produce acetone.

Gas fermentation is a promising biological process for the conversion of CO2 or syngas into valuable chemicals. Homoacetogens are microorganisms growing autotrophically using CO2 and H2 or CO and metabolizing them to form acetate coupled with energy conservation. The challenge in the metabolic engineering of the homoacetogens is divergence of the acetate formation, whose intermediate is acetyl-CoA, to a targeted chemical with sufficient production of adenosine triphosphate (ATP). In this study, we report that an engineered strain of the thermophilic homoacetogen Moorella thermoacetica, in which a pool of acetyl-CoA is diverted to ethanol without ATP production, can maintain autotrophic growth on syngas. We estimated the ATP production in the engineered strains under different gaseous compositions by considering redox-balanced metabolism for ethanol and acetate formation. The culture test showed that the combination of retaining a level of acetate production and supplying the energy-rich CO allowed maintenance of the autotrophic growth during ethanol production. In contrast, autotrophy was collapsed by complete elimination of the acetate pathway or supplementation of H2-CO2. We showed that the intracellular level of ATP was significantly lowered on H2-CO2 in consistent with the incompetence. In the meantime, the complete disruption of the acetate pathway resulted in the redox imbalance to produce ethanol from CO, albeit a small loss in the ATP production. Thus, preservation of a fraction of acetate formation is required to maintain sufficient ATP and balanced redox in CO-containing gases for ethanol production.

First-generation ethanol (E1G) is based on the fermentation of sugars released from saccharine or starch sources, while second-generation ethanol (E2G) is focused on the fermentation of sugars released from lignocellulosic feedstocks. During the fractionation process to release sugars from hemicelluloses (mainly xylose), some inhibitor compounds are released hindering fermentation. Thus, the biggest challenge of using hemicellulosic hydrolysate is selecting strains and processes able to efficiently ferment xylose and tolerate inhibitors. With the aim of diluting inhibitors, sugarcane molasses (80% of sucrose content) can be mixed to hemicellulosic hydrolysate in an integrated E1G-E2G process. Cofermentations of xylose and sucrose were evaluated for the native xylose consumer Spathaspora passalidarum and a recombinant Saccharomyces cerevisiae strain. The industrial S. cerevisiae strain CAT-1 was modified to overexpress the XYL1, XYL2, XKS1 genes and a mutant ([4-59Δ]HXT1) version of the low-affinity HXT1 permease, generating strain MP-C5H1. Although S. passalidarum showed better results for xylose fermentation, this yeast showed intracellular sucrose hydrolysis and low sucrose consumption in microaerobic conditions. Recombinant S. cerevisiae showed the best performance for cofermentation, and a batch strategy at high cell density in bioreactor achieved unprecedented results of ethanol yield, titer and volumetric productivity in E1G-E2G production process.

Klebsiella pneumoniae subsp. pneumoniae CCI2 was isolated from leaf soil collected in Hiroshima Prefecture, Japan. The draft genome sequence comprises 78 contigs and contains 5,075,115 bp with a G+C content of 57.7%.

The filamentous fungus Talaromyces cellulolyticus is a well-characterized cellulolytic and hemicellulolytic enzyme producer. In this study, the function of the tclB2 gene, which is a homolog of the manR/clrB/clr-2 gene in other filamentous fungi, in mannanolytic enzyme production by T. cellulolyticus was investigated. When a tclB2-disrupted strain (YDTclB) was grown in the presence of glucomannan, the production of β-mannanase, β-mannosidase, and α-galactosidase was decreased at the protein and transcriptional levels when compared to the control strain. In addition, a tclB2-overexpressing strain (YHTclB) showed higher β-mannanase and β-mannosidase production. When cellulose was used as a carbon source, the expression of genes encoding mannanolytic enzymes also decreased in YDTclB. These results suggested that TclB2 contributes to mannanolytic enzyme production in T. cellulolyticus. This work is the first study to identify a transcriptional regulator of mannanolytic enzyme genes in T. cellulolyticus.

Simultaneous saccharification and fermentation (SSF) is capable of performing enzymatic saccharification and fermentation for biofuel production in a single vessel. Thus, SSF has several advantages such as simplifying the manufacturing process, operating easily, and reducing energy input. Here, we describe the application of Pichia kudriavzevii NBRC1279 and NBRC1664 to SSF for bioethanol production. When each strain was incubated for 144 h at 35 °C with Japanese cedar particles, the highest ethanol concentrations were reached 21.9 ± 0.50 g/L and 23.8 ± 3.9 g/L, respectively. In addition, 21.6 ± 0.29 g/L and 21.3 ± 0.21 g/L of bioethanol were produced from Japanese eucalyptus particles when each strain was incubated for 144 h at 30 °C. Although previous methods require pretreatment of the source material, our method does not require pretreatment, which is an advantage for industrial use. To elucidate the different characteristics of the strains, we performed genome sequencing and genome comparison. Based on the results of the eggNOG categories and the resulting Venn diagram, the functional abilities of both strains were similar. However, strain NBRC1279 showed five retrotransposon protein genes in the draft genome sequence, which indicated that the stress tolerance of both strains is slightly different.

Strain CCI5, an oligotrophic bacterium, was isolated from leaf soil collected in Japan. Strain CCI5 grew at temperatures between 25 °C and 43 °C (optimum temperature, 40 °C) and at pHs between 6.0 and 10.0 (optimum pH, 9.0). Its major fatty acids were anteiso-C15:0 and iso-C16:0, and menaquinone 7 was the only detected quinone system. In a phylogenetic analysis based on 16S rRNA gene sequences, strain CCI5 presented as a member of the genus Paenibacillus. Moreover, multilocus sequence analysis based on partial sequences of the atpD, dnaA, gmk, and infB genes showed that strain CCI5 tightly clustered with P. glycanilyticus DS-1T. The draft genome of strain CCI5 consisted of 6,864,972 bp with a G+C content of 50.7% and comprised 6,189 predicted coding sequences. The genome average nucleotide identity value (97.8%) between strain CCI5 and P. glycanilyticus DS-1T was below the cut-off value for prokaryotic subspecies delineation. Based on its phenotypic, chemotaxonomic, and phylogenetic features, strain CCI5 (= HUT-8145T = KCTC 43270T) can be considered as a novel subspecies within the genus Paenibacillus with the proposed name Paenibacillus glycanilyticus subsp. hiroshimensis subsp. nov.

Gas fermentation is one of the promising bioprocesses to convert CO2 or syngas to important chemicals. Thermophilic gas fermentation of volatile chemicals has the potential for the development of consolidated bioprocesses that can simultaneously separate products during fermentation. This study reports the production of acetone from CO2 and H2, CO, or syngas by introducing the acetone production pathway using acetyl-coenzyme A (Ac-CoA) and acetate produced via the Wood-Ljungdahl pathway in Moorella thermoacetica. Reducing the carbon flux from Ac-CoA to acetate through genetic engineering successfully enhanced acetone productivity, which varied on the basis of the gas composition. The highest acetone productivity was obtained with CO-H2, while autotrophic growth collapsed with CO2-H2. By adding H2 to CO, the acetone productivity from the same amount of carbon source increased compared to CO gas only, and the maximum specific acetone production rate also increased from 0.04 to 0.09 g-acetone/g-dry cell/h. Our development of the engineered thermophilic acetogen M. thermoacetica, which grows at a temperature higher than the boiling point of acetone (58 °C), would pave the way for developing a consolidated process with simplified and cost-effective recovery via condensation following gas fermentation.

Enterobacter oligotrophicus CCA3 was isolated from leaf soil collected in Hiroshima Prefecture, Japan. Here, we report the draft genome sequence of E. oligotrophicus CCA3. The draft genome sequence of E. oligotrophicus CCA3 consists of 29 contigs of 4,425,100 bp, with a GC content of 54.2%.

Strain CCI9, which was isolated from leaf soil collected in Japan, was capable of growth on poor-nutrient medium, at temperatures of 10°C to 45°C, at pHs of 4.5 to 10, and in the presence of 7.0% NaCl. We determined a draft genome sequence of strain CCI9, which consists of a total of 28 contigs containing 4,644,734 bp with a GC content of 56.1%. This assembly yielded 4,154 predicted coding sequences. Multilocus sequence analysis (MLSA) based on atpD, gyrB, infB, and rpoB gene sequences were performed to further identify strain CCI9. The MLSA revealed that strain CCI9 clustered tightly with Enterobacter roggenkampii EN-117T. Moreover, the average nucleotide identity value (98.6%) between genome sequences of strain CCI9 and E. roggenkampii EN-117T exceeds the cutoff value for prokaryotic subspecies delineation. Therefore, strain CCI9 was identified as E. roggenkampii CCI9. To clarify differences between E. roggenkampii EN-117T and CCI9, the coding proteins were compared against the eggNOG database.

meso-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible NADP+-dependent oxidative deamination of meso-2,6-diaminopimelate (meso-DAP) to produce l-2-amino-6-oxopimelate. meso-DAPDH is divided into two major clusters, types I and II, based on substrate specificity and structural characteristic. Here, we describe a novel type II meso-DAPDH from Thermosyntropha lipolytica (TlDAPDH). The gene encoding a putative TlDAPDH was expressed in Escherichia coli cells, and then the enzyme was purified 7.3-fold to homogeneity from the crude cell extract. The molecule of TlDAPDH seemed to form a hexamer, which is the typical structural characteristic of type II meso-DAPDHs. The purified enzyme exhibited oxidative deamination activity toward meso-DAP with both NADP+ and NAD+ as coenzymes. TlDAPDH exhibited reductive amination activity of corresponding 2-oxo acid to produce d-amino acid. In particular, the productivities for d-aspartate and d-glutamate have not been reported in the type II enzymes. The optimum pH and temperature for oxidative deamination of meso-DAP were 10.5 and 55°C, respectively. TlDAPDH retained more than 80% of its activity after incubation for 30 min at temperatures between 50°C and 65°C and in the pH range of 4.5-9.5. Moreover, the coenzyme and substrate recognition mechanisms of TlDAPDH were elucidated based on a multiple sequence alignment and the homology model. The results of these analyses suggested that the molecular mechanisms for coenzyme and substrate recognition of TlDAPDH were similar to those of meso-DAPDH from S. thermophilum, which is the representative type II enzyme. Based on the kinetic characteristics and structural comparison, TlDAPDH was considered to be a novel type II meso-DAPDH.

The discharge of industrial dyes and their breakdown products are often environmentally harmful. Here, we describe a biodegradation method using Burkholderia multivorans CCA53, which exhibits a capacity to degrade azo dyes, particularly ethyl red. Under the optimized culture conditions, 100 μM ethyl red was degraded more than 99% after incubation for 8 h. Real-time PCR analysis of azoR1 and azoR2, encoding two azoreductases, revealed that transcription level of these genes is enhanced at early phase under the optimized conditions. For a more practical approach, hydrolysates were prepared from eucalyptus or Japanese cedar chips or rice straw, and rice straw hydrolysate was used as the best medium for ethyl red biodegradation. Under those conditions, ethyl red was also degraded with high efficiency (>91%). We have thus constructed a potentially economical method for the biodegradation of ethyl red.

GH30-7 endoxylanase C from the cellulolytic fungus Talaromyces cellulolyticus (TcXyn30C) belongs to glycoside hydrolase family 30 subfamily 7, and specifically releases 22-(4-O-methyl-α-D-glucuronosyl)-xylobiose from glucuronoxylan, as well as various arabino-xylooligosaccharides from arabinoxylan. TcXyn30C has a modular structure consisting of a catalytic domain and a C-terminal cellulose-binding module 1 (CBM1). In this study, the crystal structure of a TcXyn30C mutant which lacks the CBM1 domain was determined at 1.65 Å resolution. The structure of the active site of TcXyn30C was compared with that of the bifunctional GH30-7 xylanase B from T. cellulolyticus (TcXyn30B), which exhibits glucuronoxylanase and xylobiohydrolase activities. The results revealed that TcXyn30C has a conserved structural feature for recognizing the 4-O-methyl-α-D-glucuronic acid (MeGlcA) substituent in subsite -2b. Additionally, the results demonstrated that Phe47 contributes significantly to catalysis by TcXyn30C. Phe47 is located in subsite -2b and also near the C-3 hydroxyl group of a xylose residue in subsite -2a. Substitution of Phe47 with an arginine residue caused a remarkable decrease in the catalytic efficiency towards arabinoxylan, suggesting the importance of Phe47 in arabinoxylan hydrolysis. These findings indicate that subsite -2b of TcXyn30C has unique structural features that interact with arabinofuranose and MeGlcA substituents.

Pyruvate, a potential precursor of various chemicals, is one of the fundamental chemicals produced by the fermentation process. We previously reported a pyruvate-producing Escherichia coli strain LAFCPCPt-accBC-aceE (PYR) that has the potential to be applied to the industrial production of pyruvate. In this study, the availability of the PYR strain for the production of pyruvate-derivative chemicals was evaluated using a d-lactate-producing strain (LAC) based on the PYR strain. The LAC strain expresses a d-lactate dehydrogenase-encoding gene from Lactobacillus bulgaricus under the control of a T7 expression system. The d-lactate productivity of the LAC strain was further improved by limiting aeration and changing the induction period for the expression of d-lactate dehydrogenase-encoding gene expression. Under combined conditions, the LAC strain produced d-lactate at 21.7 ± 1.4 g·L−1, which was compatible with the pyruvate production by the PYR strain (26.1 ± 0.9 g·L−1). These results suggest that we have succeeded in the effective conversion of pyruvate to d-lactate in the LAC strain, demonstrating the wide versatility of the parental PYR strain as basal strain for various chemicals production.

Xylanase B, a member of subfamily 7 of the GH30 (glycoside hydrolase family 30) from Talaromyces cellulolyticus (TcXyn30B), is a bifunctional enzyme with glucuronoxylanase and xylobiohydrolase activities. In the present study, crystal structures of the native enzyme and the enzyme-product complex of TcXyn30B expressed in Pichia pastoris were determined at resolutions of 1.60 and 1.65 Å, respectively. The enzyme complexed with 22 -(4-O-methyl-α-d-glucuronyl)-xylobiose (U4m2 X) revealed that TcXyn30B strictly recognizes both the C-6 carboxyl group and the 4-O-methyl group of the 4-O-methyl-α-d-glucuronyl side chain by the conserved residues in GH30-7 endoxylanases. The crystal structure and site-directed mutagenesis indicated that Asn-93 on the β2-α2-loop interacts with the non-reducing end of the xylose residue at subsite-2 and is likely to be involved in xylobiohydrolase activity. These findings provide structural insight into the mechanisms of substrate recognition of GH30-7 glucuronoxylanase and xylobiohydrolase.

Talaromyces cellulolyticus is a promising strain for industrial cellulase production. In this study, the thaB gene, which is a homologue of the hap2/B gene in other filamentous fungi, was isolated and characterized. When grown in the presence of cellulose, culture supernatants of a thaB-disrupted strain (YDTha) exhibited decreased cellulase and xylanase enzymatic activities compared to the control strain. Furthermore, YDTha exhibited lower expression of the genes encoding cellulases and xylanases compared to the control strain. When cellobiose and lactose (soluble carbon sources) were used as carbon sources, the expression of the genes encoding cellulases and xylanases was decreased in both the YDTha and the control strains, though the expression levels in YDTha remained lower than those in the control strain. These results suggested that thaB has a positive role in cellulase and xylanase production in T. cellulolyticus.

Glycoside hydrolase family 30 subfamily 7 (GH30-7) enzymes include various types of xylanases, such as glucuronoxylanase, endoxylanase, xylobiohydrolase, and reducing-end xylose-releasing exoxylanase. Here, we characterized the mode of action and gene expression of the GH30-7 endoxylanase from the cellulolytic fungus Talaromyces cellulolyticus (TcXyn30C). TcXyn30C has a modular structure consisting of a GH30-7 catalytic domain and a C-terminal cellulose binding module 1, whose cellulose-binding ability has been confirmed. Sequence alignment of GH30-7 xylanases exhibited that TcXyn30C has a conserved Phe residue at the position corresponding to a conserved Arg residue in GH30-7 glucuronoxylanases, which is required for the recognition of the 4-O-methyl-α-d-glucuronic acid (MeGlcA) substituent. TcXyn30C degraded both glucuronoxylan and arabinoxylan with similar kinetic constants and mainly produced linear xylooligosaccharides (XOSs) with 2 to 3 degrees of polymerization, in an endo manner. Notably, the hydrolysis of glucuronoxylan caused an accumulation of 22-(MeGlcA)-xylobiose (U4m2X). The production of this acidic XOS is likely to proceed via multistep reactions by putative glucuronoxylanase activity that produces 22-(MeGlcA)-XOSs (X n U4m2X, n ≥ 0) in the initial stages of the hydrolysis and by specific release of U4m2X from a mixture containing X n U4m2X. Our results suggest that the unique endoxylanase activity of TcXyn30C may be applicable to the production of linear and acidic XOSs. The gene xyn30C was located adjacent to the putative GH62 arabinofuranosidase gene (abf62C) in the T. cellulolyticus genome. The expression of both genes was induced by cellulose. The results suggest that TcXyn30C may be involved in xylan removal in the hydrolysis of lignocellulose by the T. cellulolyticus cellulolytic system.IMPORTANCE Xylooligosaccharides (XOSs), which are composed of xylose units with a β-1,4 linkage, have recently gained interest as prebiotics in the food and feed industry. Apart from linear XOSs, branched XOSs decorated with a substituent such as methyl glucuronic acid and arabinose also have potential applications. Endoxylanase is a promising tool in producing XOSs from xylan. The structural variety of XOSs generated depends on the substrate specificity of the enzyme as well as the distribution of the substituents in xylan. Thus, the exploration of endoxylanases with novel specificities is expected to be useful in the provision of a series of XOSs. In this study, the endoxylanase TcXyn30C from Talaromyces cellulolyticus was characterized as a unique glycoside hydrolase belonging to the family GH30-7, which specifically releases 22-(4-O-methyl-α-d-glucuronosyl)-xylobiose from hardwood xylan. This study provides new insights into the production of linear and branched XOSs by GH30-7 endoxylanase.

Construction of acid-tolerant strains of Saccharomyces cerevisiae is required for various bioproduction processes. We previously isolated the gene IoGAS1 from multiple stress-tolerant Issatchenkia orientalis as a gene conferring sulfuric acid resistance in S. cerevisiae, but its acid tolerance was only investigated using sulfuric acid. Here, we evaluated the growth and ethanol fermentation ability of the IoGAS1-expressing S. cerevisiae strain, B4-IoGAS1, by using various acidic reagents. B4-IoGAS1 exhibited faster growth than the control strain, B4-CON, when cultured aerobically with sulfuric, hydrochloric, formic, acetic, and lactic acids at pH below 2.4. However, the growth of B4-IoGAS1 was suppressed at pH above 2.48, irrespective of the type of acid reagents. Furthermore, B4-IoGAS1 exhibited higher performance of ethanol fermentation than B4-CON under 250 mM lactic acid condition at pH 2.37. These results demonstrate that IoGAS1 could facilitate the aerobic growth and anaerobic ethanol production under different acidic stressed conditions.

In this study, we characterized the mode of action of reducing-end xylose-releasing exoxylanase (Rex), which belongs to the glycoside hydrolase family 30-7 (GH30-7). GH30-7 Rex, isolated from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), exists as a dimer. The purified Xyn30A released xylose from linear xylooligosaccharides (XOSs) 3 to 6 xylose units in length with similar kinetic constants. Hydrolysis of branched, borohydride-reduced, and p-nitrophenyl XOSs clarified that Xyn30A possesses a Rex activity. 1H nuclear magnetic resonance (1H NMR) analysis of xylotriose hydrolysate indicated that Xyn30A degraded XOSs via a retaining mechanism and without recognizing an anomeric structure at the reducing end. Hydrolysis of xylan by Xyn30A revealed that the enzyme continuously liberated both xylose and two types of acidic XOSs: 22-(4-O-methyl-α-d-glucuronyl)-xylotriose (MeGlcA2Xyl3) and 22-(MeGlcA)-xylobiose (MeGlcA2Xyl2). These acidic products were also detected during hydrolysis using a mixture of MeGlcA2Xyl n (n = 2 to 14) as the substrate. This indicates that Xyn30A can release MeGlcA2Xyl n (n = 2 and 3) in an exo manner. Comparison of subsites in Xyn30A and GH30-7 glucuronoxylanase using homology modeling suggested that the binding of the reducing-end residue at subsite +2 was partially prevented by a Gln residue conserved in GH30-7 Rex; additionally, the Arg residue at subsite -2b, which is conserved in glucuronoxylanase, was not found in Xyn30A. Our results lead us to propose that GH30-7 Rex plays a complementary role in hydrolysis of xylan by fungal cellulolytic systems.IMPORTANCE Endo- and exo-type xylanases depolymerize xylan and play crucial roles in the assimilation of xylan in bacteria and fungi. Exoxylanases release xylose from the reducing or nonreducing ends of xylooligosaccharides; this is generated by the activity of endoxylanases. β-Xylosidase, which hydrolyzes xylose residues on the nonreducing end of a substrate, is well studied. However, the function of reducing-end xylose-releasing exoxylanases (Rex), especially in fungal cellulolytic systems, remains unclear. This study revealed the mode of xylan hydrolysis by Rex from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), which belongs to the glycoside hydrolase family 30-7 (GH30-7). A conserved residue related to Rex activity is found in the substrate-binding site of Xyn30A. These findings will enhance our understanding of the function of GH30-7 Rex in the cooperative hydrolysis of xylan by fungal enzymes.

ABSTRACT Ureibacillus thermosphaericus A1 was isolated from compost collected in Munakata City, Fukuoka Prefecture, Japan. Here, we report the first complete genome sequence of U. thermosphaericus . The complete genome of this strain consists of 3,488,104 bp with a GC content of 36.3% and comprises 3,362 predicted coding sequences.

We previously showed that overexpression of IoGAS1, which was isolated from the multiple stress-tolerant yeast Issatchenkia orientalis, endows Saccharomyces cerevisiae cells with the ability to grow and ferment under acidic and high-salt conditions. The deduced amino acid sequence of the IoGAS1 gene product exhibits 60% identity with the S. cerevisiae Gas1 protein, a glycosylphosphatidylinositol-anchored protein essential for maintaining cell wall integrity. However, the functional roles of ScGAS1 in stress tolerance and pH regulation remain unclear. In the present study, we characterized ScGAS1 regarding its roles in tolerance to low pH and high salt concentrations. Transcriptional analysis indicated that, as for the IoGAS1 gene, ScGAS1 expression was pH dependent, with maximum expression at pH 3.0; the presence of salt increased endogenous expression of both GAS1 genes at almost all pH levels. These results suggested that ScGAS1, like IoGAS1, is involved in a novel acid- and salt-stress adaptation mechanism in S. cerevisiae. Overexpression of ScGAS1 in S. cerevisiae improved growth and ethanol production from glucose under acid stress without added salt, although the stress tolerance of the ScGAS1-overexpressing strain was inferior to that of the IoGAS1-overexpressing strain. However, overexpression of ScGAS1 did not result in increased tolerance of S. cerevisiae to combined acid and salt stress, even though ScGAS1 appears to be a salt-responsive gene. Thus, ScGAS1 is directly implicated in tolerance to low pH but does not confer salinity tolerance, supporting the view that ScGAS1 and IoGAS1 have overlapping yet distinct roles in stress tolerance in yeast. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.

The pentose phosphate pathway (PPP) plays an important role in the efficiency of xylose fermentation during cellulosic ethanol production. In simultaneous saccharification and co-fermentation (SSCF), the optimal temperature for cellulase hydrolysis of lignocellulose is much higher than that of fermentation. Successful use of SSCF requires optimization of the expression of PPP genes at elevated temperatures. This study examined the combinatorial expression of PPP genes at high temperature. The results revealed that over-expression of TAL1 and TKL1 in Saccharomyces cerevisiae (S. cerevisiae) at 30 A degrees C and over-expression of all PPP genes at 36 A degrees C resulted in the highest ethanol productivities. Furthermore, combinatorial over-expression of PPP genes derived from S. cerevisiae and a thermostable yeast Kluyveromyces marxianus allowed the strain to ferment xylose with ethanol productivity of 0.51 g/L/h, even at 38 A degrees C. These results clearly demonstrate that xylose metabolism can be improved by the utilization of appropriate combinations of thermostable PPP genes in high-temperature production of ethanol.

The use of yeasts tolerant to acid (low pH) and salt stress is of industrial importance for several bioproduction processes. To identify new candidate genes having potential roles in low-pH tolerance, we screened an expression genomic DNA library of a multiple-stress-tolerant yeast, Issatchenkia orientalis (Pichia kudriavzevii), for clones that allowed Saccharomyces cerevisiae cells to grow under highly acidic conditions (pH 2.0). A genomic DNA clone containing two putative open reading frames was obtained, of which the putative protein-coding gene comprising 1629 bp was retransformed into the host. This transformant grew significantly at pH 2.0, and at pH 2.5 in the presence of 7.5% Na2SO4. The predicted amino acid sequence of this new gene, named I. orientalis GAS1 (IoGAS1), was 60% identical to the S. cerevisiae Gas1 protein, a glycosylphosphatidylinositol-anchored protein essential for maintaining cell wall integrity, and 58-59% identical to Candida albicans Phr1 and Phr2, pH-responsive proteins implicated in cell wall assembly and virulence. Northern hybridization analyses indicated that, as for the C. albicans homologs, IoGAS1 expression was pH-dependent, with expression increasing with decreasing pH (from 4.0 to 2.0) of the medium. These results suggest that IoGAS1 represents a novel pH-regulated system required for the adaptation of I. orientalis to environments of diverse pH. Heterologous expression of IoGAS1 complemented the growth and morphological defects of a S. cerevisiae gas1 Delta mutant, demonstrating that IoGAS1 and the corresponding S. cerevisiae gene play similar roles in cell wall biosynthesis. Site-directed mutagenesis experiments revealed that two conserved glutamate residues (E161 and E262) in the IoGas1 protein play a crucial role in yeast morphogenesis and tolerance to low pH and salt stress. Furthermore, overexpression of IoGAS1 in S. cerevisiae remarkably improved the ethanol fermentation ability at pH 2.5, and at pH 2.0 in the presence of salt (5% Na2SO4), compared to that of a reference strain. Our results strongly suggest that constitutive expression of the IoGAS1 gene in S. cerevisiae could be advantageous for several fermentation processes under these stress conditions.

The Saccharomyces cerevisiae HAP4 gene encodes a transcription activator that plays a key role in controlling the expression of genes involved in mitochondrial respiration and reductive pathways. This work examines the effect of knockout of the HAP4 gene on aerobic ethanol production in a xylose-utilizing S. cerevisiae strain. A hap4-deleted recombinant yeast strain (B42-DHAP4) showed increased maximum concentration, production rate, and yield of ethanol compared with the reference strain MA-B42, irrespective of cultivation medium (glucose, xylose, or glucose/xylose mixtures). Notably, B42-DHAP4 was capable of producing ethanol from xylose as the sole carbon source under aerobic conditions, whereas no ethanol was produced by MA-B42. Moreover, the rate of ethanol production and ethanol yield (0.44 g/g) from the detoxified hydrolysate of wood chips was markedly improved in B42-DHAP4 compared to MA-B42. Thus, the results of this study support the view that deleting HAP4 in xylose-utilizing S. cerevisiae strains represents a useful strategy in ethanol production processes.

An hxt-null S. cerevisiae strain, lacking the major hexose transporters (hxt1 Delta-hxt7 Delta and gal2 Delta) but having high xylose reductase, xylitol dehydrogenase and xylulokinase activities, was transformed with a genomic DNA library from S. stipitis. Four plasmids allowing growth on xylose contained three genes encoding sugar transporters: the previously characterized XUT1 permease, and two new genes (HXT2.6 and QUP2) not previously identified as xylose transporters. High cell density fermentations with the recombinant strains showed that the XUT1 gene allowed ethanol production from xylose or xylose plus glucose as carbon sources, while the HXT2.6 permease produced both ethanol and xylitol, and the strain expressing the QUP2 gene produced mainly xylitol during xylose consumption. Cloning novel sugar transporters not previously identified in the S. stipitis genome using an hxt-null S. cerevisiae strain with a high xylose-utilizing pathway provides novel promising target genes for improved lignocellulosic ethanol production by yeasts.

The industrial Saccharomyces cerevisiae IR-2 is a promising host strain to genetically engineer xylose-utilizing yeasts for ethanol fermentation from lignocellulosic hydrolysates. Two IR-2-based haploid strains were selected based upon the rate of xylulose fermentation, and hybrids were obtained by mating recombinant haploid strains harboring heterogeneous xylose dehydrogenase (XDH) (wild-type NAD(+)-dependent XDH or engineered NADP(+)-dependent XDH, ARSdR), xylose reductase (XR) and xylulose kinase (XK) genes. ARSdR in the hybrids selected for growth rates on yeast extract-peptone-dextrose (YPD) agar and YP-xylose agar plates typically had a higher activity than NAD(+)-dependent XDH. Furthermore, the xylose-fermenting performance of the hybrid strain SE12 with the same level of heterogeneous XDH activity was similar to that of a recombinant strain of IR-2 harboring a single set of genes, XR/ARSdR/XK. These results suggest not only that the recombinant haploid strains retain the appropriate genetic background of IR-2 for ethanol production from xylose but also that ARSdR is preferable for xylose fermentation.

Since the uptake of xylose is believed to be one of the rate-limiting steps for xylose ethanol fermentation by recombinant Saccharomyces cerevisiae strains, we transformed a hxt-null strain lacking the major hexose transporters (hxt1 Delta-hxt7 Delta, and gal2 Delta) with an integrative plasmid to overexpress the genes for xylose reductase (XYL1), xylitol dehydrogenase (XYL2) and xylulokinase (XKS1), and analyzed the impact that overexpression of the HXT1, HXT2, HXT5 or HXT7 permeases have in anaerobic batch fermentations using xylose, glucose, or xylose plus glucose as carbon sources. Our results revealed that the low-affinity HXT1 permease allowed the maximal consumption of sugars and ethanol production rates during xylose/glucose co-fermentations, but was incapable to allow xylose uptake when this sugar was the only carbon source. The moderately high-affinity HXT5 permease was a poor glucose transporter, and it also did not allow significant xylose uptake by the cells. The moderately high-affinity HXT2 permease allowed xylose uptake with the same rates as those observed during glucose consumption, even under co-fermentation conditions, but had the drawback of producing incomplete fermentations. Finally, the high-affinity HXT7 permease allowed efficient xylose fermentation, but during xylose/glucose co-fermentations this permease showed a clear preference for glucose. Thus, our results indicate that approaches to engineer S. cerevisiae HXT transporters to improve second generation bioethanol production need to consider the composition of the biomass sugar syrup, whereby the HXT1 transporter seems more suitable for hydrolysates containing xylose/glucose blends, whereas the HXT7 permease would be a better choice for xylose-enriched sugar streams. (C) 2014 Elsevier Inc. All rights reserved.

Flocculation is a desirable property in industrial yeasts and is particularly important in the fuel ethanol industry because it provides a simple and cost-free way to separate yeast cells from fermentation products. In the present study, the effect of pH and lignocellulose-derived sugars on yeast flocculation was investigated using a flocculent Saccharomyces cerevisiae, MA-R4, which has been recombinantly engineered to simultaneously co-ferment glucose and xylose to ethanol with high productivity. The flocculation level of MA-R4 dramatically decreased at pH values below 3.0 during co-fermentation of glucose and xylose. Sedimentation and microscopic observation revealed that flocculation was induced in MA-R4 when it fermented glucose, a glucose/xylose mixture, or mannose, whereas attempts to ferment xylose, galactose, and arabinose led to the loss of flocculation. MA-R4 fermented xylose and galactose more slowly than glucose and mannose. Therefore, the various flocculation behaviors shown by MA-R4 should be useful in the control of ethanol fermentation processes.

In the bioethanol production process, high solid saccharification and glucose/xylose co-fermentation are important technologies for obtaining increased ethanol concentrations; however, bench-scale studies using combinations of these methods are limited. In this study, we hydrolyzed high solid concentration of milled eucalyptus using commercial enzymes and obtained 138.4 g/L total monomeric sugar concentration. These sugars were fermented to 53.5 g/L of ethanol by a xylose-utilizing recombinant Saccharomyces cerevisiae strain, MA-R4. These experiments were performed in bench scale (using 50 L scale solid mixer and 70 L scale fermenter). The results obtained in this study were comparable to our previous results in laboratory scale, indicating that we successfully achieved an efficient high solid saccharification and glucose/xylose co-fermentation system in bench scale.

Background: There has been much research on the bioconversion of xylose found in lignocellulosic biomass to ethanol by genetically engineered Saccharomyces cerevisiae. However, the rate of ethanol production from xylose in these xylose-utilizing yeast strains is quite low compared to their glucose fermentation. In this study, two diploid xylose-utilizing S. cerevisiae strains, the industrial strain MA-R4 and the laboratory strain MA-B4, were employed to investigate the differences between anaerobic fermentation of xylose and glucose, and general differences between recombinant yeast strains, through genome-wide transcription analysis. Results: In MA-R4, many genes related to ergosterol biosynthesis were expressed more highly with glucose than with xylose. Additionally, these ergosterol-related genes had higher transcript levels in MA-R4 than in MA-B4 during glucose fermentation. During xylose fermentation, several genes related to central metabolic pathways that typically increase during growth on non-fermentable carbon sources were expressed at higher levels in both strains. Xylose did not fully repress the genes encoding enzymes of the tricarboxylic acid and respiratory pathways, even under anaerobic conditions. In addition, several genes involved in spore wall metabolism and the uptake of ammonium, which are closely related to the starvation response, and many stress-responsive genes mediated by Msn2/4p, as well as trehalose synthase genes, increased in expression when fermenting with xylose, irrespective of the yeast strain. We further observed that transcript levels of genes involved in xylose metabolism, membrane transport functions, and ATP synthesis were higher in MA-R4 than in MA-B4 when strains were fermented with glucose or xylose. Conclusions: Our transcriptomic approach revealed the molecular events underlying the response to xylose or glucose and differences between MA-R4 and MA-B4. Xylose-utilizing S. cerevisiae strains may recognize xylose as a non-fermentable carbon source, which induces a starvation response and adaptation to oxidative stress, resulting in the increased expression of stress-response genes.

We constructed a xylose-fermenting recombinant strain of thermotolerant yeast Kluyveromyces marxianus, DMB3-7. Both xylose consumption and ethanol production were remarkably increased in DMB3-7 compared to the control strain at 30°C. Furthermore, DMB3-7 produced ethanol from xylose at both 42°C and 45°C, above which xylose metabolic activity decreased. © 2013 The Society for Biotechnology, Japan.

Antarctic basidiomycetous yeast Mrakia blollopis SK-4 has unique fermentability for various sugars under a low temperature condition. Hence, this yeast was used for ethanol fermentation from glucose and also for direct ethanol fermentation (DEF) from cellulosic biomass without/with Tween 80 at 10 degrees C. Maximally, 48.2 g/l ethanol was formed from 12% (w/v) glucose. DEF converted filter paper, Japanese cedar and Eucalyptus to 12.2 g/l, 12.5 g/l and 7.2 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80, ethanol concentration increased by about 1.1-1.6-fold compared to that without Tween 80. This is the first report on DEF using cryophilic fungi under a low temperature condition. We consider that M. blollopis SK-4 has a good potential for ethanol fermentation in told environments. (C) 2013 Elsevier Inc. All rights reserved.

In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7-phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4. These results provide a metabolic explanation for the low ethanol productivity on xylose compared to glucose.

We prepared eight recombinant Saccharomyces cerevisae strains, including three strains generated in this study that were produced by chromosomal integration of xylose utilization pathway enzymes genes. Among these strains, MA-R4 was the most efficient at producing ethanol from rice straw enzymatic hydrolysate, indicating that it is a superior strain for bioethanol production.

The yeast Kluyveromyces marxianus is considered as a potential alternative to Saccharomyces cerevisiae in producing ethanol as a biofuel. In this study, we investigated the ethanol fermentation properties of novel K marxianus strain DMB1, isolated from bagasse hydrolysates. This strain utilized sorbitol as well as various pentoses and hexoses as single carbon sources under aerobic conditions and produced ethanol from glucose in hydrolysates of the Japanese cedar at 42 degrees C. Reference strains K marxianus NBRC1777 and S. cerevisiae BY4743 did not assimilate sorbitol or ferment lignocellulosic hydrolysates to ethanol at this temperature. Thus strain DMB1 appears to be optimal for producing bioethanol at high temperatures, and might provide a valuable means of increasing the efficiency of ethanol fermentation.

Glucose/xylose mixtures (90 g/L total sugar) were evaluated for their effect on ethanol fermentation by a recombinant flocculent Saccharomyces cerevisiae, MA-R4. Glucose was utilized faster than xylose at any ratio of glucose/xylose, although MA-R4 can simultaneously co-ferment both sugars. A high percentage of glucose can increase cell biomass production and therefore increase the rate of glucose utilization (1.224 g glucose/g biomass/h maximum) and ethanol formation (0.493 g ethanol/g biomass/h maximum). However, the best ratio of glucose/xylose for the highest xylose consumption rate (0.209 g xylose/g biomass/h) was 2:3. Ethanol concentration and yield increased and by-product (xylitol, glycerol, and acetic acid) concentration decreased as the proportion of glucose increased. The maximum ethanol concentration was 41.6 and 21.9 g/L after 72 h of fermentation with 90 g/L glucose and 90 g/L xylose, respectively, while the ethanol yield was 0.454 and 0.335 g/g in 90 g/L glucose and 90 g/L xylose media, respectively. High ethanol yield when a high percentage of glucose is available is likely due to decreased production of by-products, such as glycerol and acetic acid. These results suggest that ethanol selectivity is increased when a higher proportion of glucose is available and reduced when a higher proportion of xylose is available.

The inhibitory effects of pH and acetic acid on the co-fermentation of glucose and xylose in complex medium by recombinant flocculent Saccharomyces cerevisiae MA-R4 were evaluated. In the absence of acetic acid, the fermentation performance of strain MA-R4 was similar between pH 4.0-6.0, but was negatively affected at pH 2.5. The addition of acetic acid to batch cultures resulted in negligible inhibition of several fermentation parameters at pH 6.0, whereas the interactive inhibition of pH and acetic acid on the maximum cell and ethanol concentrations, and rates of sugar consumption and ethanol production were observed at pH levels below 5.4. The inhibitory effect of acetic acid was particularly marked for the consumption rate of xylose, as compared with that of glucose. With increasing initial acetic acid concentration, the ethanol yield slightly increased at pH 5.4 and 6.0, but decreased at pH values lower than 4.7. Notably, ethanol production was nearly completely inhibited under low pH (4.0) and high acetic acid (150-200 mM) conditions. Together, these results indicate that the inhibitory effects of acetic acid and pH on ethanol fermentation by MA-R4 are highly synergistic, although the inhibition can be reduced by increasing the medium pH.

The activity of transaldolase and transketolase, key enzymes in the non-oxidative pentose phosphate pathway, is rate-limiting for xylose utilization in recombinant Saccharomyces cerevisiae. Overexpression of TAL1 and TKL1, the major transaldolase and transketolase genes, increases the flux from the pentose phosphate pathway into the glycolytic pathway. However, the functional roles of NQM1 and TKL2, the secondary transaldolase and transketolase genes, especially in xylose utilization, remain unclear. This study focused on characterization of NQM1 and TKL2, together with TAL1 and TKL1, regarding their roles in xylose utilization and fermentation. Knockout or overexpression of these four genes on the phenotype of xylose-utilizing S. cerevisiae strains was also examined. Transcriptional analysis indicated that the expression of TAL1, NQM1, and TKL1 was up-regulated in the presence of xylose. A significant decrease in both growth on xylose and xylose-fermenting ability in tal1 Delta and tkl1 Delta mutants confirmed that TALI and TKL1 are essential for xylose assimilation and fermentation. Gene disruption analysis using a tkl1 Delta mutant revealed that TKL1 is also required for utilization of glucose. Growth on xylose and xylose-fermenting ability were slightly influenced by deletion of NQM1 or TKL2 when xylose was used as the sole carbon source. Moreover, the rate of xylose consumption and ethanol production was slightly impaired in TKL1- and TKL2-overexpressing strains. NQM1 and TKL2 may thus play a physiological role via an effect on the non-oxidative pentose phosphate pathway in the xylose metabolic pathway, although their roles in xylose utilization and fermentation are less important than those of TAL1 and TKL1. (C) 2012 Elsevier Inc. All rights reserved.

The potential of spent mushroom substrate after cultivation of Pleurotus eryngii as a biomass resource for bioethanol production was investigated. Materials were pretreated by ball milling for 1 h, and enzymatic hydrolysis was then carried out. Glucose, xylose, arabinose and galactose were detected in enzymatic hydrolysates, and a > 59.0% yield of total sugars was obtained, even at a substrate concentration of 30% (w/v). Enzymatic hydrolysates were fermented using Pichia stipitis. When enzymatic hydrolysates obtained from a 20% (w/v) substrate concentration were fermented, the maximum ethanol concentration was 17.7 g and ethanol yield was 67.0%. These results indicate that corncob-based spent mushroom substrate can be used as a raw material for bioethanol production.

Construction of xylose- and xylo-oligosaccharide-fermenting Saccharomyces cerevisiae strains is important, because hydrolysates derived from lignocellulosic biomass contain significant amounts of these sugars. We have obtained recombinant S. cerevisiae strain MA-D4 (D-XKXDHXR), expressing xylose reductase, xylitol dehydrogenase and xylulokinase. In the present study, we generated recombinant strain D-XSD/XKXDHXR by transforming MA-D4 with a beta-xylosidase gene cloned from the filamentous fungus Trichoderma reesei. The intracellular beta-xylosidase-specific activity of D-XSD/XKXDHXR was high, while that of the control strain was under the limit of detection. D-XSD/XKXDHXR produced ethanol, and xylose accumulated in the culture supernatant under fermentation in a medium containing xylo-oligosaccharides as sole carbon source. beta-Xylosidase-specific activity in D-XSD/XKXDHXR declined due to xylose both in vivo and in vitro. D-XSD/XKXDHXR converted xylo-oligosaccharides in an enzymatic hydrolysate of eucalyptus to ethanol. These results indicate that D-XSD/XKXDHXR efficiently converted xylo-oligosaccharides to xylose and subsequently to ethanol.

Ethanol production from xylose is important for the utilization of lignocellulosic biomass as raw materials. Recently, we reported the development of an industrial xylose-fermenting Saccharomyces cerevisiae strain. MA-R4, which was engineered by chromosomal integration to express the genes encoding xylose reductase and xylitol dehydrogenase from Pichia sripitis along with S. cerevisiae xylulokinase gene constitutively using the alcohol-fermenting flocculent yeast strain, IR-2. IR-2 has the highest xylulose-fermenting ability of the industrial diploid strains, making it a useful host strain for genetically engineering xylose-utilizing S. cerevisiae. To optimize the activities of xylose metabolizing enzymes in the metabolic engineering of IR-2 for further improvement of ethanol production from xylose, we constructed a set of recombinant isogenic strains harboring different combinations of genetic modifications present in MA-R4, and investigated the effect of constitutive expression of xylulokinase and of different levels of xylulokinase and xylose reductase activity on xylose fermentation. This strain comparison showed that constitutive expression of xylulokinase increased ethanol production from xylose at the expense of xylitol excretion, and that high activity of xylose reductase resulted in an increased rate of xylose consumption and an increased glycerol yield. Moreover, strain MA-R6, which has moderate xylulokinase activity, grew slightly better but accumulated more xylitol than strain MA-R4. These results suggest that fine-tuning of introduced enzyme activity in S. cerevisiae is important for improving xylose fermentation to ethanol. (C) 2011 Elsevier Inc. All rights reserved.

Different initial cell concentrations of a recombinant flocculent Saccharomyces cerevisiae MA-R4 were evaluated for their effects on xylose fermentation and glucose-xylose cofermentation. A high initial cell concentration greatly increased both the substrate utilization and ethanol production rates. During xylose fermentation, the highest rates of xylose consumption (2.58 g/L h) and ethanol production (0.83 g/L h) were obtained at an initial cell concentration of 13.1 g/L. During cofermentation, the highest rates of glucose consumption (14.4 g/L h), xylose consumption (2.79 g/L h), and ethanol production (6.68 g/L h) were obtained at an initial cell concentration of 12.7 g/L. However, a high initial cell density had no positive effect on the maximum ethanol concentration and ethanol yield mainly due to the increased amount of by-products including xylitol. The ethanol yield remained almost constant (0.34 g/g) throughout xylose fermentation (initial cell concentration range, 1.81-13.1 g/L), while it was slightly lower at high initial cell concentrations (9.87 and 12.7 g/L) during cofermentation. The determination of the appropriate initial cell concentration is necessary for the improvement of substrate utilization and ethanol yield.

We examined the evolutionary adaptation of recombinant shochu yeast by serial anaerobic cultivation in xylose-based minimal medium. Compared with the parental strain, the adapted strain MA-S4-M1 (M1) markedly improved the growth on xylose and the anaerobic xylose consumption rate. M1 gained improved xylose utilization properties by optimizing the metabolic pathway enzymes and enhancing the uptake of xylose. (C) 2010, The Society for Biotechnology, japan. All rights reserved.

Bioethanol production from xylose is important for utilization of lignocellulosic biomass as raw materials. The research on yeast conversion of xylose to ethanol has been intensively studied especially for genetically engineered Saccharomyces cerevisiae during the last 20 years. S. cerevisiae, which is a very safe microorganism that plays a traditional and major role in industrial bioethanol production, has several advantages due to its high ethanol productivity, as well as its high ethanol and inhibitor tolerance. However, this yeast cannot ferment xylose, which is the dominant pentose sugar in hydrolysates of lignocellulosic biomass. A number of different strategies have been applied to engineer yeasts capable of efficiently producing ethanol from xylose, including the introduction of initial xylose metabolism and xylose transport, changing the intracellular redox balance, and overexpression of xylulokinase and pentose phosphate pathways. In this review, recent progress with regard to these studies is discussed, focusing particularly on xylose-fermenting strains of S. cerevisiae. Recent studies using several promising approaches such as host strain selection and adaptation to obtain further improved xylose-utilizing S. cerevisiae are also addressed.

The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP(+)-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.

In this study, five recombinant Saccharomyces cerevisiae strains were compared for their xylose-fermenting ability. The most efficient xylose-to-ethanol fermentation was found by using the industrial strain MA-R4, in which the genes for xylose reductase and xylitol dehydrogenase from Pichia stipitis along with an endogenous xylulokinase gene were expressed by chromosomal integration of the flocculent yeast strain IR-2. The MA-R4 strain rapidly converted xylose to ethanol with a low xylitol yield. Furthermore, the MA-R4 strain had the highest ethanol production when fermenting not only a mixture of glucose and xylose, but also mixed sugars in the detoxified hydrolysate of wood chips. These results collectively suggest that MA-R4 may be a suitable recombinant strain for further study into large-scale ethanol production from mixed sugars present in lignocellulosic hydrolysates. (c) 2008 Elsevier Ltd. All rights reserved.

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD+-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP+. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP+-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP+-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.

We varied the promoter strength of xylose reductase (XR) gene and the copy number of xylulokinase (XK) gene to determine how XR and XK activities affect the xylose-fermenting abilities of recombinant Saccharomyces cerevisiae expressing xylitol dehydrogenase (XDH). The most enhanced ethanol yield and lowered xylitol yield occurred in strain I-PGK/AUR, which has high activity of both XR and XDH and moderate XK activity.

We constructed a set of recombinant Saccharomyces cerevisiae strains with xylose-fermenting ability. A recombinant S. cerevisiae strain D-XR/ARSdR/XK, in which protein engineered NADP(+)-dependent XDH was expressed, showed 40% increased ethanol production and 23% decrease in xylitol excretion as compared with the reference strain D-XR/XDH/XK expressing the wild-type XDH.

His-Asp phosphorelays are widespread signal transduction mechanisms in bacteria, fungi, and higher plants. In order to investigate a His-Asp phosphorelay network in filamentous fungi, which has been genetically characterized in part, we attempted to construct an in vitro phosphotransfer network in Aspergillus nidulans comprising all the necessary components. As a first step, we established an in vitro phosphotransfer system with a histidine-containing phosphotransmitter YpdA, a response regulator SrrA, and a bacterial histidine kinase ArcB as a phosphate donor. We demonstrated the phosphotransfer from ArcB to A. nidulans YpdA and the subsequent transfer from YpdA to SrrA. This is the first direct biochemical evidence for the presence of the phosphotransfer system in filamentous fungi. Furthermore, a retrograde phosphorylation from YpdA to FphA, a histidine kinase similar to bacterial phytochrome, was found. The overall picture of the His-Asp phosphorelays in A. nidulans is discussed based on the results of the in vitro study.

Together with PRR1/TOC1, PRR5 belongs to the small family of PSEUDO-RESPONSE REGULATORs (PRRs), which function as clock components of Arabidopsis thaliana. We employed a set of transgenic lines, each of which was designed to misexpress a truncated form of the PRR5 molecule, together with the original transgenic line (named PRR5-ox) that misexpresses the entire PRR5 polypeptide. The results of genetic analysis suggested that PRR5-ox seedlings showed a phenotype of hypersensitivity to red light during early photomorphogenesis in a manner dependent on red light photoreceptors (PhyA and PhyB), but independent of PRR1/TOC1. The set of newly constructed transgenic lines (named PRR5-N-ox and PRR5-C-ox) were also characterized in terms of cireadian-associated phenotypes. The results suggest that the N-terminal pseudoreceiver domain of the PRR5 molecule seems to be dispensable for the misexpressed PRR5 molecule to bring about the phenotype of red light sensitivity. However, PRR5-N-ox plants, misexpressing only the pseudo-receiver domain, showed a phenotype of long period of free-running circadian rhythms of certain clock-controlled genes. Considering these and other results, we discuss the structure and function of PRR5 in the context of current views of the circadian clock in higher plants.

Every member of a small family of PSEUDO-RESPONSE REGULATOR (PRR) genes, including TIMING OF CAB EXPRESSION 1 (TOC1 [or PRR1]), are believed to play roles close to the circadian clock in the model higher plant Arabidopsis thaliana. In this study we established a transgenic line that misexpresses (or overexpresses) the PRR7 gene. As compared with wildtype plants, the resulting PRR7-misexpressing plants (designated PRR7-ox) showed characteristic phenotypes as to hallmarked circadian-associated biological events: (i) early flowering in a manner independent of photoperiodicity, (ii) hypersensitive response to red light during early photomorphogenesis, and (iii) altered free-running rhythms with long period of clock-associated genes. Finally, a series of all transgenic lines (PRR1-ox, PRR3-ox, PRR5-ox, PRR7-ox, and PRR9-ox) were characterized comparatively with regard to their clock-associated roles. The results suggested that the five homologous PRR factors play coordinate roles, distinctively from one another, and closely to the circadian clock in higher plants.

In the model higher plant Arabidopsis thaliana, a number of circadian clock-associated protein components have recently been identified. Among them, a small family of ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9) is interesting because the most probable clock component TIMING OF CAB EXPRESSION I (TOC1) belongs to this family. Several lines of evidence have already been accumulated to support the view that not only APRR1/TOC1 but also other APRR family members are crucial for a better understanding of the molecular link between circadian rhythm and light-signal transduction. Among the APRR1/TOC1 family members, the circadian-controlled APRR9 gene is unique in that its expression is rapidly induced by light at the level of transcription. In this study we dissected the regulatory cis-elements of the light-induced and/or circadian-controlled APRR9 promoter by employing not only a mutant plant carrying a T-DNA insertion in the APRR9 promoter, but also a series of APRR9-promoter::LUC (luciferase) reporters that were introduced into an Arabidopsis cultured cell line (T87 cells). Taking the results of these approaches together, we provide several lines of evidence that the APRR9 promoter contains at least two distinctive and separable regulatory cis-elements: an "L element" responsible for the light-induced expression, followed by an "R element" necessary for the fundamental rhythmic expression of APRR9. Furthermore, APRR1/ TOC1 was implicated in the L-element-mediated light response of APRR9, directly or indirectly.

A small family of plant proteins, designated PSEUDO RESPONSE REGULATORS (PRRs), is crucial for a better understanding of the molecular link between circadian rhythm and photoperiodic control of flowering time in the dicotyledonous model plant Arabidopsis thaliana. Recently, we showed that the monocotyledonous model plant Oryza sativa also has homologous members of the OsPRR family (Oryza sativa PRR). In the previous experiments with rice, we mainly characterized a japonica variety (Nipponbare). By employing an indica variety (Kasalath), in this study we further characterized OsPRRs with reference to the photoperiod sensitivity Hd (Heading date) QTL (quantitative trait loci) implicated in the control of flowering time in rice. The circadian-controlled and sequential expression profiles of the five OsPRR genes were observed not only for Nipponbare but also for Kasalath. Then each of these OsPRR genes was mapped on the rice chromosomes. Among these OsPRR genes, OsPRR37 was mapped very closely to Hd2-QTL, which was identified as the major locus that enhances the photoperiod sensitivity of flowering in Nipponbare. Furthermore, we found that Kasalath has a severe mutational lesion in the OsPRR37 coding sequence.

In Arabidopsis thaliana, a number of cireadian-associated factors have been identified, including TOC1 (TIMING OF CAB EXPRESSION1) that is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). As demonstrated previously, these APRR1/TOC1 quintet members are crucial for a better understanding of the molecular links between circadian rhythms and photosensory signal transduction. Here we focused on the light-induced quintet member, APRR9, and three critical issues with regard to this member were simultaneously addressed: (i) clarification of the mechanism underlying the light-dependent acute response of APRR9, (ii) clarification of the phenotype of a null mutant of APRR9, (iii) identification of protein(s) that interacts with APRR9. In this study, we present the results that support the following views. (i) A phytochrome-mediated signaling pathway(s) activates the transcription of APRR9, leading to the acute light response of APRR9. (ii) The severe mutational lesion of APRR9 singly, if not directly, affects the period (and/or phase) of free-running rhythms, in continuous light, of every circadian-controlled gene tested, including the clock genes, APRR1/TOC1, CCA1, and LHY. (iii) The APRR9 protein is capable of interacting with APRR1/TOC1, suggesting a hetrodimer formation between these cognate family members. These results are discussed within the context of a current consistent model of the Arabidopsis circadian oscillator.

APRR1 (ARABIDPSIS PSUEDO-RESPONSE REGULATOR 1) (or TOC1, TIMING OF CAB EXPRESSION 1) is believed to be a crucial component of biological clocks of Arabidopsis thaliana. Nevertheless, its molecular function remains to be fully elucidated. Based on the results of yeast two-hybrid and in vitro binding assays, we previously showed that APRR1/TOC1 interacts with certain bHLH factors (i.e. P1F3 and PIL1, which are PHYTOCHROME INTERACTING FACTOR 3 and its homolog (PIF3-LIKE 1), respectively). To critically examine the relevance of PILI with reference to the function of APRR1/TOC1, T-DNA insertion mutants were isolated for PILL No phenotype was observed for such homozygous pill mutants, in terms of circadian-associated events in plants. We then examined more extensively a certain set of bHLH factors, which are considerably similar to PILI in their structural designs. The results of extensive analyses of such bHLH factors (namely, HFR1, PIL2, PIF4, PIL5 and PIL6) in wild-type and APRR1-overexressing (APRR1-ox) transgenic lines provided us with several new insights into a link between APRR1/TOC1 and these bHLH factors. In yeast two-hybrid assays, APRR1/TOC1 showed the ability to interact with these proteins (except for HFR1), as well as PIL1 and PIF3. Among them, it was found that the expressions of PIF4 and PIL6 were regulated in a circadian-dependent manner, exhibiting free-running robust rhythms. The expressions of PIF4 and PIL6 were regulated also by light in a manner that their transcripts were rapidly accumulated upon exposure of etiolated seedlings to light. The light-induced expressions of PIF4 and PIL6 were severely impaired in APRR1-ox transgenic lines. Taken together, here we propose the novel view that these bHLH factors (PIF4 and PIL6) might play roles, in concert with APRR1/TOC1, in the integration of light-signals to control both circadian and photomorphogenic processes.

A small family of genes, named ARABIDOPSIS PSEUDO RESPONSE REGULATOR (APRR), are intriguing with special reference to circadian rhythms in plants, based on the fact that one of the members (APRR1) is identical to TOC1 (TIMING OF CAB EXPRESSION 1) that is believed to encode a clock component. In Arabidopsis plants, each transcript of the APRR1/TOC1 quintet genes starts accumulating after dawn rhythmically and one after another at intervals in the order of APRR9 --> APRR7 --> APRR5 --> APRR3 --> APRR1/TOC1. To characterize such intriguing circadian-associated events, we employed an established Arabidopsis cell line (named T87). When T87 cells were grown in an appropriate light and dark cycle, cell autonomous diurnal oscillations of the APRR1/TOC1 quintet genes were observed at the level of transcription, as seen in intact plants. After transfer to the conditions without any environmental time cues, particularly in constant dark, we further showed that free-running circadian rhythms persisted in the cultured cells, not only for the APRR1/TOC1 quintet genes, but also other typical circadian-controlled genes including CCA1 (CIRCADIAN CLOCK ASSOCIATED 1), LHY (LATE ELONGATED HYPOCOTYL) and CCR2 (COLD CIRCADIAN RHYTHM RNA BINDING 2). To our knowledge, this is the first indication of cell autonomous circadian rhythms in cultured cells in Arabidopsis thaliana, which will provide us with an alternative and advantageous means to characterize the plant biological clock.

Several Arabidopsis genes have been proposed to encode potential clock-associated components, including the Myb-related CCA1 and LHY transcription factors and a member (APRR1/TOC1) of the family of pseudo-response regulators. We previously showed that transcripts of the APRR1/TOC1 family genes each start accumulating after dawn rhythmically and sequentially at intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1/TOC1, under the conditions of continuous light. Nevertheless, no evidence has been provided that each member of the APRR1/TOC1 quintet, except for APRR1/TOC1, is indeed relevant to the mechanisms underlying circadian rhythms. Here we attempt to provide such evidence by characterizing transgenic plants that aberrantly (or constitutively) express the APRR9 gene in a manner independent of circadian rhythms. The resulting APRR9-ox plants showed intriguing phenotypes with regard to circadian rhythms, in two aspects. First, the aberrant expression of APRR9 resulted in a characteristic phenotype with regard to transcriptional events, in which short-period rhythms were commonly observed for certain circadian-regulated genes, including CCA1, LHY, APRR1/TOC1, other APRR1/TOC1 members, ELF3, and CAB2. With regard to biological consequences, such APRR9-ox plants flowered much earlier than wildtype plants, in a manner independent of photoperiodicity (or under short-day conditions). These results suggest that APRR9 (and perhaps other members of the APRR1/TOC1 quintet) must also be taken into consideration for a better understanding of the molecular mechanisms underlying circadian rhythms, and also underlying control of the flowering time through the photoperiodic long-day pathway.

We previously identified a novel class of proteins, named pseudo-response regulators (APRRs) in Arabidopsis thaliana, each of which (APRR1, APRR3, APRR5, APRR7, and APRR9) has an intriguing structural design containing an N-terminal pseudo receiver domain and a C-terminal CONSTANS motif. Among them, APRR1 is identical to TOC1, previously proposed to be a candidate component of an Arabidopsis circadian clock. Intriguingly, expressions of the APRR1/TOC1 family of genes are under control of coordinate circadian rhythms at the level of transcription, in the manner that each APRR-transcript starts accumulating sequentially after dawn with 2 to 3 It intervals in the order: APRR9-->4PRR7-->4PRR5-->APRR3-->APRR1/TOC1. Here we examined this circadian-related event, "circadian waves of the APRR1/TOC1 quintet", by employing CCA1-overexpression (CCA1-ox) transgenic plants, based on the fact that CCA1 is a well-characterized and the most plausible oscillator component. It was found that aberrant overexpression of the CCA1 gene severely perturbed free-running and sequential rhythms of the APRR1/TOC1 family of genes. In the accompanying paper, it was shown that overexpression of APRR1 also results in a marked alteration of the CCA1 circadian rhythm, and vice versa. Taken together, it was suggested that there are intimate and mutual links between these two types of circadian-associated components (APRRs and CCA1).

Several Arabidopsis genes have been proposed to encode potential clock-associated components, including the Myb-related CCA1 and LHY transcription factors and a member of the novel family of pseudo response regulators (APRR1/TOC1). We previously showed that mRNAs of the APRR1/TOC1 family of genes start accumulating after dawn rhythmically and sequentially at approximately 2 h intervals in the order: APRR9-->APRR7-->APRR5-->APRR3 -->APRR1/TOC1. Here we constructed APRR1-overexpressing (APRR1-ox) plants, and examined certain circadian profiles for APRRs, CCA1, LHY, GI, CCR2, and CAB2. The free-running circadian rhythms of the APRR1/TOC1 family of genes, including APRR1, were dampened in APRR1-ox plants. In particular, the light-inducible expression of APRR9 was severely repressed in APRR1-ox plants, suggesting that there is a negative APRR1-->APRR9 regulation. The free-running robust rhythm of CAB2 was also dampened in APRR1-ox. The circadian profiles of potential clock-associated genes, CCA1, LHY, GI, and CCR2 were all markedly altered in APRR1-ox, each in characteristic fashion. To gain further insight into the molecular function of APRR1, we then identified a novel Myc-related bHLH transcription factor, which physically associated with APRR1. This protein (named PIL1) is similar in its amino acid sequence to PIF3, which has been identified as a phytochrome-interacting transcription factor. These results are discussed in relation to the current idea that APRR1 (TOC1) plays a role within, or close to, the Arabidopsis central oscillator.

We previously identified a novel class of proteins, named Arabidopsis pseudo-response regulators (APRRs), each of which (APRR1/TOC1, APRR3, APRR5, APRR7, APRR9) has an intriguing structural design containing an N-terminal pseudo-receiver domain and a C-terminal CONSTANS motif, Expression of these APRR1/TOC1 family members is under the control of a coordinate circadian rhythm at the level of transcription such that the APRR-mRNAs start accumulating sequentially after dawn with 2 to 3h intervals in the order of APRR9 --> APRR7 --> APRR5 --> APRR3 --> APRR1/TOC1 in a given 24 h photo-period. Based on these data, we previously proposed that these sequential and rhythmic events of transcription, termed 'circadian waves of APRR1/TOC1 quintet', may be a basis of a presumed Arabidopsis biological clock (named 'bar code clock') [Matsushika et al, (2000) Plant and Cell Physiol. 41: 1002], Here we further characterized the event of circadian waves, by demonstrating that certain light stimuli ape crucial determinants to induce the robust circadian waves, and accordingly, the first-boosted and light-induced APRR9 appears to be primarily responsible for this light response of the circadian waves. Also, as such a light stimulus, a red Light pulse that is presumably perceived by phytochromes appears to be sufficient to induce (or synchronize) the APRR1/TOC1 circadian waves.

The Arabidopsis pseudo-response regulator, APRR1, has a unique structural design containing a pseudo-receiver domain and a C-terminal CONSTANS motif. This protein was originally characterized as a presumed component of the His-to-Asp phosphorelay systems in Arabidopsis thaliana, Recently, it was reported that APRR1 is identical to the TOC1 gene product, a mutational lesion of which affects the periods of many circadian rhythms in Arabidopsis plants. TOC1 is believed to be a component of the presumed circadian clock (or central oscillator). Based on these facts, in this study four more genes, each encoding a member of the APRR1/TOC1 family of pseudo-response regulators were identified and characterized with special reference to circadian rhythms. It was found that all these members of the APRR1/TOC1 family (APRR1, APRR3, APRR5, APRR7, and APRR9) are subjected to a circadian rhythm at the level of transcription. Furthermore, in a given 24 h period, the APRR-mRNAs started accumulating sequentially after dawn with 2-3 h intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1. These sequential events of transcription, termed 'circadian waves of APRR1/TOC1', were not significantly affected by the photoperiod conditions, if any (e.g. both long and short days), and the expression of APRR9 was first boosted always after dawn. Among these APRRs, in fact, only the expression of APRR9 was rapidly and transiently induced also by white light, whereas such light responses of others were very dull, if any, These results collectively support the view that these members of the APRR1/TOC1 family are together all involved in an as yet unknown mechanism underlying the Arabidopsis circadian clock, Here we propose that the circadian waves of the APRR1/TOC1 family members are most likely a molecular basis of such a biological clock in higher plants.

The ArcB sensor plays a crucial role in the histidine to aspartate (His-to-Asp) phosphorelay signal transduction, which is involved in the transcriptional regulatory network that allows Escherichia coli cells to sense various respiratory growth conditions. ArcB is one of the best-studied hybrid His-kinases involved in the multi-step His-to-Asp phosphorelay. However, a major question that remains to be elucidated is how does ArcB sense an anoxic signal? The N-terminal region of ArcB is considered to be! a signal-input domain, which probably plays a role in such signal-perception. In this study, this N-terminal region of ArcB was dissected into three putative sub-domains, a "transmembrane domain," a "leucine-zipper-like domain," and a "PAS-like domain." The importance of these structural domains was assessed in vivo and in vitro by systematically analyzing a number of arcB mutants, each of which encodes a mutant ArcB protein having an amino acid substitution or a deletion within one of these sub-domains. The results are discussed with special reference to the nature of the ArcB anaerobic sensor.

The Escherichia coli sdhCDAB operon encodes succinate dehydrogenase, an enzyme complex involved in the tricarboxylic acid (TCA) cycle. Expression of this operon is under complex transcriptional regulation in response to growth conditions, such as anaerobiosis and carbon sources. Typically, the expression of sdhCDAB is known to be subjected to "an aerobic repression" and "a glucose repression." The molecular mechanism underlying the anaerobic repression has been well documented, involving both the ArcB-ArcA two-component system and the Fnr global anaerobic regulator. However, the mechanism underlying the glucose repression is not yet clear, because the involvment of the general catabolite regulators such as CRP and CRA has been dismissed. In this study, we conducted a series of genetic analyses to identify the regulator gene(s) involved in the glucose repression of sdh. The results demonstrate that the EIICBGlc protein (the ptsG gene product), a component of the major glucose transporter, acts as a crucial mediator in glucose repression. These results support the view that the EIICBGlc protein functions not only as a glucose transporter, but also as a glucose-sensing signal transducer that modulates the glucose repression of the sdhCDAB operon.

The Escherichia coli ArcB sensor is involved in anaerobic phosphotransfer signal transduction. ArcB is a hybrid sensor that contains three types of phosphotransfer signaling domains in its primary amino acid sequence, namely, transmitter (or His-kinase), receiver, and histidine-containing phosphotransfer (HPt) domains. However, examination of the function of the newly-discovered HPt domain (named ArcB(c)) is still at a very early stage. To gain a general insight into the structure and function of the widespread HPt domains, on the basis of ifs three-dimensional crystal structure, in this study we constructed a certain set of mutants each having a single amino acid substitution in the HPt domain of ArcB. These ArcB(c) mutants were characterized and evaluated, based on the in vivo ability to signal the OmpR receiver via trans-phosphorylation.

The Escherichia coli ArcB sensor is involved in anaerobic signal transduction. ArcB is an unorthodox His-kinase, in that it contains three types of phosphotransfer signaling domains in its primary amino acid sequence, namely, transmitter (or His-kinase), receiver, and histidine-containing phosphotransfer (HPt) domains. In this study, we first conducted an in. vivo experiment to determine whether or not the phosphorylation of the HPt domain is crucial for ArcB/ArcA-mediated anaerobic signal transduction. The results are best interpreted as meaning that the HPt domain of ArcB is important for the anaerobic signal transduction, as far as the expression of the succinate dehydrogenase (sdh) operon is concerned. We then isolated a set of ArcB mutant each with a single amino acid substitution in the HPt domain, which has lost the ability to function as a phospho-transmitter. The results of such mutational analyses, together with the three-dimensional crystal structure of the HPt domain, provided an insight into the structure and function of the HPt domain of ArcB.

The two components ArcB and ArcA play a crucial role in the signal transduction implicated in the complex transcriptional regulatory network that allows Escherichia coli to sense various respiratory growth conditions. ArcB is a hybrid sensor kinase having multiple phosphorylation sites in its primary amino acid sequence, including a transmitter, a receiver, and a histidine-containing phosphotransfer (HPt) domain. ArcA is a DNA-binding transcriptional regulator with a receiver domain. Results of recent in vitro studies revealed multistep His-to-Asp phosphotransfer circuitry in the ArcB-ArcA signaling system. For this report we conducted a series of in vivo experiments using a set of crucial ArcB mutants to evaluate the regulation of the sdh operon, The results suggested that the phosphorylated His-717 site in the HPt domain of ArcB is essential for anaerobic repression of sdh, Nonetheless, the ArcB mutant lacking this crucial His-717 site does not necessarily exhibit a null phenotype,vith respect to ArcB-ArcA signaling, The HPt mutant appears to maintain an ability to signal ArcA, particularly under aerobic conditions, which results in a significant repression of sdh. Based on these and other in vivo results, we propose a model in which ArcB functions in its own right as a dual-signaling sensor that is capable of propagating two types of stimuli through two distinct phosphotransfer pathways.

The electrofermentation process is a biomanufacturing process that utilizes bacteria that eat electricity (electrode oxidizing bacteria; EUB) and bacteria that generates electricity (electrode reducing bacteria).In this study, we aimed to improve the electro-fermentation process by isolating two types of bacteria that contribute to the enhancement of electron flux in the electroreduction tank (4.1-4-2 in the main text of the report), cultivating an accumulation of aerobic EUB that contribute to the enhancement of electron flux in the cathode chamber (4-3), the development of solid phase culture system to expand the bioresources of EUB (4.4), and the establishment of a genome editing systems for EUB to accelerate the genetic modification of EUB (4.5).