Helicovermis profundi gen. nov., sp. nov., a novel mesophilic, asporogenous bacterium within the Clostridia isolated from a deep-sea hydrothermal vent chimney.

A novel mesophilic bacterial strain, designated S502T, was isolated from a deep-sea hydrothermal vent at Suiyo Seamount, Japan. Cells were Gram-positive, asporogenous, motile, and curved rods, measuring 1.6-5.6 µm in length. The strain was an obligate anaerobe that grew fermentatively on complex substrates such as yeast extract and Bacto peptone. Elemental sulfur stimulated the growth of the strain, and was reduced to hydrogen sulfide. The strain grew within a temperature range of 10-23 °C (optimum at 20 °C), pH range of 4.8-8.3 (optimum at 7.4), and a NaCl concentration range of 1.0-4.0% (w/v) (optimum at 3.0%, w/v). Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolate was a member of the class Clostridia, with Fusibacter paucivorans strain SEBR 4211T (91.1% sequence identity) being its closest relative. The total size of the genome of the strain was 3.12 Mbp, and a G + C content was 28.2 mol%. The highest values for average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) value of strain S502T with relatives were 67.5% (with Marinisporobacter balticus strain 59.4MT), 51.5% (with M. balticus strain 59.4MT), and 40.9% (with Alkaliphilus serpentinus strain LacTT), respectively. Based on a combination of phylogenetic, genomic, and phenotypic characteristics, we propose strain S502T to represent a novel genus and species, Helicovermis profundi gen. nov., sp. nov., with the type strain S502T (= DSM 112048T = JCM 39167T).

A Simple and Effective Method for Solid Medium Cultivation of Strictly Hydrogen- and Sulfur-oxidizing Chemolithoautotrophs Predominant in Deep-|sea Hydrothermal Fields.

Strictly hydrogen- and sulfur-oxidizing chemolithoautotrophic bacteria, particularly members of the phyla Campylobacterota and Aquificota, have a cosmopolitan distribution in deep-sea hydrothermal fields. The successful cultivation of these microorganisms in liquid media has provided insights into their physiological, evolutionary, and ecological characteristics. Notably, recent population genetic studies on Sulfurimonas (Campylobacterota) and Persephonella (Aquificota) revealed geographic separation in their populations. Advances in this field of research are largely dependent on the availability of pure cultures, which demand labor-intensive liquid cultivation procedures, such as dilution-to-extinction, given the longstanding assumption that many strictly or facultatively anaerobic chemolithoautotrophs cannot easily form colonies on solid media. We herein describe a simple and cost-effective approach for cultivating these chemolithoautotrophs on solid media. The results obtained suggest that not only the choice of gelling agent, but also the gas phase composition significantly affect the colony-forming ratio of diverse laboratory strains. The use of gellan gum as a gelling agent combined with high concentrations of H2 and CO2 in a pouch bag promoted the formation of colonies. This contrasted with the absence of colony formation on an agar-solidified medium, in which thiosulfate served as an electron donor, nitrate as an electron acceptor, and bicarbonate as a carbon source, placed in anaerobic jars under an N2 atmosphere. Our method efficiently isolated chemolithoautotrophs from a deep-sea vent sample, underscoring its potential value in research requiring pure cultures of hydrogen- and sulfur-oxidizing chemolithoautotrophs.

Pyrofollis japonicus gen. nov. sp. nov., a novel member of the family Pyrodictiaceae isolated from the Iheya North hydrothermal field.

A novel hyperthermophilic, heterotrophic archaeon, strain YC29T, was isolated from a deep-sea hydrothermal vent in the Mid-Okinawa Trough, Japan. Cells of strain YC29T were non-motile, irregular cocci with diameters of 1.2-3.0 µm. The strain was an obligatory fermentative anaerobe capable of growth on complex proteinaceous substrates. Growth was observed between 85 and 100 °C (optimum 90-95 °C), pH 4.9-6.4 (optimum 5.1), and in the presence of 1.4-4.0% (w/v) NaCl (optimum 3.0%). Inorganic carbon was required as a carbon source. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolate was a member of the family Pyrodictiaceae. The genome size was 2.02 Mbp with a G+C content of 49.4%. The maximum values for average nucleotide identity (ANI), average amino acid identity (AAI), and in silico DNA-DNA hybridization (dDDH) value of strain YC29T with relatives were 67.9% (with Pyrodictium abyssi strain AV2T), 61.1% (with Pyrodictium occultum strain PL-19T), and 33.8% (with Pyrolobus fumarii strain 1AT), respectively. Based on the phylogenetic, genomic, and phenotypic characteristics, we propose that strain YC29T represents a novel genus and species, Pyrofollis japonicus gen. nov., sp. (type strain YC29T = DSM 113394T = JCM 39171T).

Haliovirga abyssi gen. nov., sp. nov., a mesophilic fermentative bacterium isolated from the Iheya North hydrothermal field, and proposal of Haliovirgaceae fam. nov.

A novel mesophilic, obligately anaerobic, facultatively sulphur-reducing bacterium, designated strain IC12T, was isolated from a deep-sea hydrothermal field in the Mid-Okinawa Trough, Japan. The cells were Gram-negative, motile, short rods with a single polar flagellum. The ranges and optima of the growth temperature, NaCl concentration and pH of strain IC12T were 15-40 °C (optimum, 30-35 °C), 10-60 g l-1 (optimum, 20-30 g l-1) and pH 4.9-6.7 (optimum, pH 5.8), respectively. Yeast extract was utilized as a sole carbon and energy source for fermentative growth. Major fatty acids of strain IC12T were C14 : 0, C16 : 0 and C16 : 1 ω7. Results of phylogenetic analysis based on 16S rRNA gene sequences indicated that strain IC12T was affiliated to the phylum Fusobacteriota and was most closely related to Ilyobacter insuetus VenChi2T (86.5 % sequence similarity). Strain IC12T contained a chromosome of 2.43 Mbp and a large plasmid of 0.30 Mbp. The G+C content of the genomic DNA was 26.4 mol%. The maximum values for average nucleotide identity and in silico DNA-DNA hybridization between strain IC12T and related strains of the phylum Fusobacteriota were 71.4 and 26.4 %, respectively. Phylogenomic, physiological and chemotaxonomic analyses indicate that strain IC12T represents a novel genus and species within the phylum Fusobacteriota, for which the name Haliovirga abyssi gen. nov., sp. nov. is proposed, with strain IC12T (= DSM 112164T=JCM 39166T) as the type strain. We also propose the family Haliovirgaceae fam. nov. to accommodate this novel genus.

Transcriptome analysis of Aurantiochytrium limacinum under low salt conditions.

Aurantiochytrium limacinum can accumulate high amounts of omega-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA). Although salinity affects the DHA content, its impact on the metabolic pathway responsible for DHA production in A. limacinum is not completely understood. To address this issue, we investigated the transcriptional profile of A. limacinum under hypoosmotic stress. We first cultured A. limacinum under typical and low salinity for RNA sequencing, respectively. Transcriptome analyses revealed that 933 genes exhibited significant changes in expression under hypoosmotic conditions, of which 81.4% were downregulated. Strikingly, A. limacinum downregulated genes related to polyketide synthesis and fatty acid synthase pathways, while upregulating ß-oxidation-related genes. In accordance with this, DHA production significantly decreased under hypoosmotic conditions, while antioxidant-related genes were significantly upregulated. Considering that ß-oxidation of fatty acids generates energy and reactive oxygen species (ROS), our results suggest that A. limacinum utilizes fatty acids for energy to survive under hypoosmotic conditions and detoxifies ROS using antioxidant systems.

Improvement of Astaxanthin Production in Aurantiochytrium limacinum by Overexpression of the Beta-Carotene Hydroxylase Gene.

Aurantiochytrium limacinum is a heterotrophic eukaryotic microorganism that can accumulate high levels of commercial products such as astaxanthin and docosahexaenoic acid. Due to its rapid growth and relatively simple extraction method, A. limacinum is considered a promising astaxanthin resource to replace the conventional microalgal production. However, the astaxanthin biosynthetic process in A. limacinum remains incompletely understood, especially in those catalysed by ß-carotene hydroxylase (CrtZ) and ketolase. In this study, we overexpressed a crtZ candidate gene to increase astaxanthin production and expand our understanding of the conversion from beta-carotene to astaxanthin. The resultant transformant AlcrtZ#10 cultivated for 5 days showed a significant increase in astaxanthin production per culture (2.8-fold) and per cell (4.5-fold) compared with that of the wild-type strain. Strikingly, longer light exposure increased astaxanthin production and decreased the beta-carotene content in the wild-type strain, suggesting that light exposure duration is important for astaxanthin production in A. limacinum. Among several predicted intermediates, furthermore, the cantaxanthin produced from ß-carotene by ketolase activity were enhanced in the transformant AlcrtZ#10. Although the further investigation is needed, this result suggested that the main route of astaxanthin was via cantaxanthin. Thus, our findings will be valuable not only for its application, but also for understanding the astaxanthin biosynthetic process in A. limacinum.

Physiological and comparative proteomic characterization of Desulfolithobacter dissulfuricans gen. nov., sp. nov., a novel mesophilic, sulfur-disproportionating chemolithoautotroph from a deep-sea hydrothermal vent.

In deep-sea hydrothermal environments, inorganic sulfur compounds are important energy substrates for sulfur-oxidizing, -reducing, and -disproportionating microorganisms. Among these, sulfur-disproportionating bacteria have been poorly understood in terms of ecophysiology and phylogenetic diversity. Here, we isolated and characterized a novel mesophilic, strictly chemolithoautotrophic, diazotrophic sulfur-disproportionating bacterium, designated strain GF1T, from a deep-sea hydrothermal vent chimney at the Suiyo Seamount in the Izu-Bonin Arc, Japan. Strain GF1T disproportionated elemental sulfur, thiosulfate, and tetrathionate in the presence of ferrihydrite. The isolate also grew by respiratory hydrogen oxidation coupled to sulfate reduction. Phylogenetic and physiological analyses support that strain GF1T represents the type strain of a new genus and species in the family Desulfobulbaceae, for which the name Desulfolithobacter dissulfuricans gen. nov. sp. nov. is proposed. Proteomic analysis revealed that proteins related to tetrathionate reductase were specifically and abundantly produced when grown via thiosulfate disproportionation. In addition, several proteins possibly involved in thiosulfate disproportionation, including those encoded by the YTD gene cluster, were also found. The overall findings pointed to a possible diversity of sulfur-disproportionating bacteria in hydrothermal systems and provided a refined picture of microbial sulfur disproportionation.

Transcriptional responses of Aurantiochytrium limacinum under light conditions.

AIMS: Astaxanthin-producing protist Aurantiochytrium limacinum can accumulate higher amounts of astaxanthin under light conditions; however, little is known about the impact of light exposure on its metabolism. Here, we investigated the transcriptional profile of A. limacinum under light conditions. METHODS AND RESULTS: Transcriptomic analyses revealed that 962 genes of A. limacinum showed a significant change in expression under light conditions, most of which (94.5%) were downregulated. Furthermore, gene ontology enrichment analysis indicated that A. limacinum mainly downregulated genes associated with cell motility, proliferation and gene expression processes, whose activities depend on ATP as an energy source. Additionally, the quantification of carotenoid and its transcripts suggested that ß-carotene and astaxanthin biosynthesis pathways were rate-limiting and tightly regulated steps, respectively. In comparison, these processes were enhanced under light conditions. CONCLUSIONS: Considering that astaxanthin accumulation was highly correlated with reactive oxygen species (ROS) levels in microalgae, our results suggest that A. limacinum reduces ATP consumption to decrease the occurrence of ROS in mitochondria while accumulating astaxanthin to prevent ROS damage. SIGNIFICANCE AND IMPACT OF STUDY: This study provides novel insights into the impact of light exposure on A. limacinum metabolism, thereby facilitating a complete understanding of this protist for efficient astaxanthin production.

Desulfomarina profundi gen. nov., sp. nov., a novel mesophilic, hydrogen-oxidizing, sulphate-reducing chemolithoautotroph isolated from a deep-sea hydrothermal vent chimney.

A novel mesophilic, strictly anaerobic, chemolithoautotrophic sulphate-reducing bacterium, designated strain KT2T, was isolated from a deep-sea hydrothermal vent chimney at the Suiyo Seamount in the Izu-Bonin Arc. Strain KT2T grew at 25-40 °C (optimum 35 °C) and pH 5.5-7.0 (optimum 6.6) in the presence of 25-45 g l-1 NaCl (optimum 30 g l-1). Growth occurred with molecular hydrogen as the electron donor and sulphate, thiosulphate, and sulphite as the electron acceptors. The isolate utilized CO2 as the sole carbon source for chemolithoautotrophic growth on H2. Glycerol, succinate, fumarate, malate, glutamate, or casamino acids could serve as an alternative electron donor in the presence of CO2. Malate, citrate, glutamate, and casamino acids were used as fermentative substrates for weak growth. The G+C content of genomic DNA was 46.1 %. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain KT2T is a member of the family Desulfobulbaceae, showing a sequence similarity of 94.3 % with Desulforhopalus singaporensis. Phylogenomic analysis based on concatenated 156 single-copy marker genes confirmed the same topology as the 16S rRNA gene phylogeny. The ANI and AAI values between strain KT2T and related genera of the family Desulfobulbaceae were 65.6-68.6 % and 53.1-62.9 %. Based on the genomic, molecular, and physiological characteristics, strain KT2T represents a novel genus and species within the family Desulfobulbaceae, for which the name Desulfomarina profundi gen. nov., sp. nov. is proposed, with KT2T (=JCM 34118T = DSM 111364T) as the type strain.

Enhanced Lutein Production in Chlamydomonas reinhardtii by Overexpression of the Lycopene Epsilon Cyclase Gene.

Chlamydomonas reinhardtii is a well-established microalgal model species with a shorter doubling time, which is a promising natural source for the efficient production of high-value carotenoids. In the microalgal carotenoid biosynthetic pathway, lycopene is converted either into ß-carotene by lycopene ß-cyclase or into α-carotene by lycopene ε-cyclase (LCYE) and lycopene ß-cyclase. In this study, we overexpressed the LCYE gene in C. reinhardtii to estimate its effect on lycopene metabolism and lutein production. Chlamydomonas transformants (CrLCYE#L1, #L5, and #L6) produced significantly increased amounts of lutein per culture (up to 2.6-fold) without a decrease in cell yields. Likewise, the expression levels of LCYE gene in transformants showed a significant increase compared with that of the wild-type strain. These results suggest that LCYE overexpression enhances the conversion of lycopene to α-carotene, which in turn improves lutein productivity. Interestingly, their ß-carotene productivity appeared to increase slightly rather than decrease. Considering that the inhibition of the lycopene cyclization steps often induces higher expression in genes upstream of metabolic branches, this result implies that the redirection from ß-carotene to α-carotene by LCYE overexpression might also enhance upstream gene expression, thereby leading to auxiliary ß-carotene production.

Enhanced Production of Astaxanthin without Decrease of DHA Content in Aurantiochytrium limacinum by Overexpressing Multifunctional Carotenoid Synthase Gene.

Aurantiochytrium limacinum produces both docosahexaenoic acid (DHA) and astaxanthin, respectively. Organisms that produce these industrially important materials more efficiently than microalgae are currently needed. In this study, we overexpressed a putative homolog of CarS, which is involved in synthesizing the astaxanthin precursor, ß-carotene, in A. limacinum to increase carotenoid synthesis with the goal of obtaining strains that produce large amounts of both DHA and carotenoids. AlCarS transformants #1 and #18 produced significantly increased amounts of astaxanthin as assessed according to culture (up to 5.8-fold) and optical density (up to 9.3-fold). The improved astaxanthin production of these strains did not affect their DHA productivity. Additionally, their CarS expression levels were higher than those of the wild-type strain, suggesting that CarS overexpression enhanced ß-carotene production, which in turn improved astaxanthin productivity. Although cell yields were slightly decreased, these features will be valuable in health food, medical care, and animal feed fields.

Draft Genome Sequence of the Astaxanthin-Producing Microalga Haematococcus lacustris Strain NIES-144.

Haematococcus lacustris is an industrially important eukaryotic microalga that is thought to be a great source of natural astaxanthin with strong antioxidant activity. Here, we report the draft assembly and annotation results of the genome of H. lacustris NIES-144. These data will expand our knowledge of the molecular biological features of this microalga.

Overexpression of the DOF-Type Transcription Factor Enhances Lipid Synthesis in Chlorella vulgaris.

In the present study, DNA binding with one finger (DOF)-type transcription factors were screened from the Chlorella vulgaris genome database. The candidate DOF transcription factor was endogenously overexpressed in C. vulgaris to improve neutral lipid production. The protein expression vector contains the heat shock protein 70 and ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit promoters and self-cleaving 2A peptide to reduce the transgene-silencing effect of C. vulgaris. A total of 74 phleomycin-resistant transformants were obtained. Under nitrogen-deficient conditions, the transformant CvDOF#3 showed approximately 1.5-fold higher neutral lipid content per cell compared to the original strain and also showed a His-tagged DOF candidate protein expression of 0.6%. Microscopic observations revealed that CvDOF#3 cells were larger in size. However, the observed differences in average cell diameter between CvDOF#3 and control cells were not statistically significant. These results indicated that the protein expression vector harboring the dual promoters and the 2A peptide, when used in combination with enzymatic cell wall degradation and glass bead transformation, could be useful for transgene and protein expression in C. vulgaris. Further experiment is necessary to confirm the expression efficiency of the HSP70 and RBCS dual promoter and 2A peptide strategy after construction of homologous recombination system in C. vulgaris. Our findings suggested that the overexpression of the endogenous DOF-type transcription factor can be used for improving the lipid content in C. vulgaris.

Seasonal distribution of Gambierdiscus spp. in Wakasa Bay, the Sea of Japan, and antagonistic relationships with epiphytic pennate diatoms.

The occurrence of the ciguatera fish poisoning (CFP) causative Gambierdiscus spp. was confirmed in the Sea of Japan for the first time in 2009. This paper reports seasonal distribution of Gambierdiscus spp. and epiphytic diatoms in the Sea of Japan. Monitoring results suggested an antagonistic interaction in abundances between epiphytic diatoms and the dinoflagellate Gambierdiscus spp. Allelopathic effects of diatoms were considered to be involved in the competitive phenomenon. Therefore it is hypothesized that cell densities of epiphytic pennate diatoms on macroalgae are a novel determinant affecting the abundance of Gambierdiscus spp. other than sea water temperature, salinity and nutrients. Monitorings of the abundance of epiphytic diatoms would lead us to predict the occurrences of Gambierdiscus spp. blooms in the CFP area, and thereby the CFP risk assessments would be developed. Phylogenetic analyses indicated that Gambierdiscus spp. in the Sea of Japan belonged to Gambierdiscus sp. type 2 which was reported to be non-toxic. Nevertheless, based on morphological characteristics, at least two types of Gambierdiscus spp. were found in the Sea of Japan. It is needed to test the toxicity of the both types of Gambierdiscus recognized in the present study for evaluation of the probability of CFP outbreak risks in the Sea of Japan in the future.

Overexpression of DnaJ-Like Chaperone Enhances Carotenoid Synthesis in Chlamydomonas reinhardtii.

Production of functional carotenoids using microalgae may facilitate the commercialization of anti-aging nutritional supplements. The green alga Chlamydomonas reinhardtii uses a non-mevalonate (MEP) pathway for isopentenyl diphosphate (IPP) synthesis. Two enzymes thought to play important roles in this MEP pathway to IPP synthesis are 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and reductase (DXR). DnaJ-like chaperone (Orange protein) is thought to support phytoene synthase, a key enzyme in plant carotenoid synthesis. Genes for Orange (OR), DXS, and DXR were overexpressed via nuclear transformation into C. reinhardtii. CDS of OR, DXS, and DXR were amplified and connected with dual promoters of heat-shock protein 70A and ribulose bisphosphate carboxylase small chain 2. Compared with the parental strain, transformant CrOR#2 produced increased lutein and ß-carotene (1.9-fold and 1.7-fold per cell, respectively). Transformant CrDXS#1 produced lutein and ß-carotene at lower per-cell abundances than those for the parental strain. CrDXR#2 transformant produced lutein and ß-carotene at higher per-cell abundances than their parental counterpart; however, these transformants produced lutein and ß-carotene at lower per-medium abundances than their parental counterparts. These results suggest that OR protein supports phytoene synthase in C. reinhardtii and that the phytoene synthesis step is rate-limiting in carotenoid synthesis.

A Simple and Efficient RNA Extraction Method from Deep-Sea Hydrothermal Vent Chimney Structures.

RNA-based microbiological analyses, e.g., transcriptome and reverse transcription-quantitative PCR, require a relatively large amount of high quality RNA. RNA-based analyses on microbial communities in deep-sea hydrothermal environments often encounter methodological difficulties with RNA extraction due to the presence of unique minerals in and the low biomass of samples. In the present study, we assessed RNA extraction methods for deep-sea vent chimneys that had complex mineral compositions. Mineral-RNA adsorption experiments were conducted using mock chimney minerals and Escherichia coli total RNA solution, and showed that detectable RNA significantly decreased possibly due to adsorption onto minerals. This decrease in RNA was prevented by the addition of sodium tripolyphosphate (STPP), deoxynucleotide triphosphates (dNTPs), salmon sperm DNA, and NaOH. The addition of STPP was also effective for RNA extraction from the mixture of E. coli cells and mock chimney minerals when TRIzol reagent and the RNeasy column were used, but not when the RNeasy PowerSoil total RNA kit was used. A combination of STPP, TRIzol reagent, the RNeasy column, and sonication resulted in the highest RNA yield from a natural chimney. This indirect extraction procedure is simple, rapid, inexpensive, and may be used for large-scale RNA extraction.

Microbiota in the coelomic fluid of two common coastal starfish species and characterization of an abundant Helicobacter-related taxon.

Marine invertebrates associate with diverse microorganisms. Microorganisms even inhabit coelomic fluid (CF), namely, the fluid filling the main body cavity of echinoderms. The CF microbiota potentially impacts host health and disease. Here, we analysed the CF microbiota in two common coastal starfish species, Patiria pectinifera and Asterias amurensis. Although microbial community structures were highly variable among individual starfish, those of P. pectinifera were compositionally similar to those in the surrounding seawater. By contrast, many A. amurensis individuals harboured unique microbes in the CF, which was dominated by the unclassified Thiotrichales or previously unknown Helicobacter-related taxon. In some individuals, the Helicobacter-related taxon was the most abundant genus-level taxon, accounting for up to 97.3% of reads obtained from the CF microbial community. Fluorescence in situ hybridization using a Helicobacter-related-taxon-specific probe suggested that probe-reactive cells in A. amurensis were spiral-shaped, morphologically similar to known Helicobacter species. Electron microscopy revealed that the spiral cells had a prosthecate-like polar appendage that has never been reported in Helicobacter species. Although culture of Helicobacter-related taxon was unsuccessful, this is the first report of the dominance of a Helicobacter-related taxon in invertebrates and non-digestive organs, reshaping our knowledge of the phylogeography of Helicobacter-related taxa.

Lebetimonas natsushimae sp. nov., a novel strictly anaerobic, moderately thermophilic chemoautotroph isolated from a deep-sea hydrothermal vent polychaete nest in the Mid-Okinawa Trough.

A moderately thermophilic, strictly anaerobic, chemoautotrophic bacterium, designated strain HS1857T, was isolated from a deep-sea hydrothermal vent at the Noho site in the Mid-Okinawa Trough. Strain HS1857T grew between 35 and 63°C (optimum 55°C), in the presence of 10-55gl-1 NaCl (optimum 25gl-1), and pH 5.5-7.1 (optimum 6.4). Growth occurred with molecular hydrogen as the electron donor and elemental sulfur, nitrate, or selenate as the electron acceptors. Formate could serve as an alternative electron donor with nitrate as an electron acceptor. During growth with nitrate as the electron acceptor, strain HS1857T produced ammonium and formed a biofilm. CO2 was utilized as the sole carbon source. The G+C content of the genomic DNA was 33.2mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain HS1857T is a member of the order Nautiliales, showing a sequence similarity of 95.0% with Lebetimonas acidiphila Pd55T. The fatty acid composition was similar to that of L. acidiphila, which was dominated by C18:0 (47.0%) and C18:1 (23.7%). Based on the genomic, chemotaxonomic, phenotypic characteristics, the name Lebetimonas natsushimae sp. nov., is proposed. The type strain is HS1857T (=NBRC 112478T=DSM 104102T).

Production of ß-xylosidase from Trichoderma asperellum KIF125 and its application in efficient hydrolysis of pretreated rice straw with fungal cellulase.

On-site cellulase and hemicellulase production is a promising way to reduce enzyme cost in the commercialization of the lignocellulose-to-ethanol process. A hemicellulase-producing fungal strain suitable for on-site enzyme production was selected from cultures prepared using wet disc-milling rice straw (WDM-RS) and identified as Trichoderma asperellum KIF125. KIF125 hemicellulase showed uniquely high abundance of ß-xylosidase in the xylanolytic enzyme system compared to other fungal hemicellulase preparations. Supplementation of Talaromyces cellulolyticus cellulase with KIF125 hemicellulase was more effective than that with the hemicellulases from other fungal sources in reducing the total enzyme loading for the improvement of xylose yield in the hydrolysis of ball-milling RS, due to its high ß-xylosidase dominance. ß-Xylosidase in KIF125 hemicellulase was purified and classified as a glycosyl hydrolase family 3 enzyme with relatively high specificity for xylobiose. The production of KIF125 ß-xylosidase in the fermentor was estimated as 118 U/g-WDM-RS (2350 U/L culture) at 48 h. These results demonstrate that KIF125 is promising as a practical hemicellulase source to combine with on-site cellulase production using T. cellulolyticus.

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis.

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The kcat/Km value with d-arabinose (1.27 min-1 mM-1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.