Life Cycle Plasticity in Typhula and Pistillaria in the Arctic and the Temperate Zone.

Typhulaceae Jülich is one of the cold-adapted fungal families in basidiomycetes. The representative genera, Typhula (Pers.) Fr. and Pistillaria Fr., are distinguished by the discontinuity between stems and hymenia in the former and the continuity in the latter (Fries 1821). This taxonomic criterion is ambiguous, and consequently, the view of Karsten (1882) has been widely accepted: Typhula develops basidiomata from sclerotia, while basidiomata develop directly from substrata in Pistillaris. However, Corner (1970) observed basidiomata of Pistillaria petasitis S. Imai developing from sclerotia in Hokkaido, Japan. We later recognized that P. petasitis basidiomata also emerged directly from substrates on the ground in Hokkaido. An aberrant form of Typhula hyperborea H. Ekstr. was found in Upernavik, West Greenland. This specimen had a stem-like structure on a Poaceae plant, and sclerotia developed on its tip. Similar phenomena were found in other Typhula species in Japan. In this study, we aimed to elucidate the life cycle plasticity in the genera Typhula and Pistillaria through the interactions between their ecophysiological potential and environmental conditions in their localities. We collected and prepared strains of the above fungi from sclerotia or basidiomata, and we elucidated the taxonomical relationship and determined the physiological characteristics of our strains. Our findings imply that both Typhula and Pistillaria have the potential to produce sclerotia as well as the capacity for mycelial growth at ambient air temperatures in each locality where samples were collected. These findings suggest that Typhula spp. develope basidiomata not only from the sclerotia dispersed by the basidiospores but also from mycelia generated by the spore germination, which formed basidiomata multiple times, depending on their growth environments.

Taxonomic revision of the Typhula ishikariensis complex.

Typhula ishikariensis and the related fungi were separated into three biological species by morphological and physiological characteristics, as well as DNA sequences and mating reactions. We propose that the T. ishikariensis complex should be divided into three species (T. ishikariensis, T. canadensis and T. hyperborea) and two varieties (T. ishikariensis var. ishikariensis and var. idahoensis). Typhula hyperborea was reappraised to be recognized also as a separate species of the T. ishikariensis complex.

Identification and Isolation Pattern of Globisporangium spp. from a Sanionia Moss Colony in Ny-Ålesund, Spitsbergen Is., Norway from 2006 to 2018.

Globisporangium spp. are soil-inhabiting oomycetes distributed worldwide, including in polar regions. Some species of the genus are known as important plant pathogens. This study aimed to clarify the species construction of Globisporangium spp. and their long-term isolation pattern in Sanionia moss in Ny-Ålesund, Spitsbergen Is., Norway. Globisporangium spp. were isolated at two-year intervals between 2006 and 2018 at a Sanionia moss colony, Ny-Ålesund, Spitsbergen Is., Norway. The isolates were obtained by using three agar media and were identified based on sequences of the rDNA-ITS region and cultural characteristics. Most of the Globisporangium isolates obtained during the survey were identified into six species. All six species were grown at 0 °C on an agar plate and used to infect Sanionia moss at 4 and/or 10 °C under an in vitro inoculation test. The total isolation frequency of Globisporangium gradually decreased throughout the survey period. The isolation frequency varied among the six species, and four of the species that showed a high frequency in 2006 were rarely isolated after 2016. The results suggested that Globisporangium inhabiting Sanionia moss in Ny-Ålesund has a unique composition of species and that most of the species reduced their population over the recent decade.

Basidiomycetous Yeast, Glaciozyma antarctica, Forming Frost-Columnar Colonies on Frozen Medium.

The basidiomycetous yeast, Glaciozyma antarctica, was isolated from various terrestrial materials collected from the Sôya coast, East Antarctica, and formed frost-columnar colonies on agar plates frozen at -1 °C. Thawed colonies were highly viscous, indicating that the yeast produced a large number of extracellular polysaccharides (EPS). G. antarctica was then cultured on frozen media containing red food coloring to observe the dynamics of solutes in unfrozen water; pigments accumulated in frozen yeast colonies, indicating that solutes were concentrated in unfrozen water of yeast colonies. Moreover, the yeast produced a small quantity of ice-binding proteins (IBPs) which inhibited ice crystal growth. Solutes in unfrozen water were considered to accumulate in the pore of frozen colonies. The extracellular IBPs may have held an unfrozen state of medium water after accumulation in the frost-columnar colony.

High-temperature ethanol production by a series of recombinant xylose-fermenting Kluyveromyces marxianus strains.

Thermotolerant yeast Kluyveromyces marxianus can assimilate xylose but cannot produce ethanol from xylose under anaerobic conditions. Here, we constructed two recombinant K. marxianus strains, DMB5 and DMB13, that express xylose reductase (XR), NAD+- or protein-engineered NADP+-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK) from K. marxianus. These strains, together with previously reported strain DMB3-7, which expresses Scheffersomyces stipitis XR and NAD+-dependent XDH and Saccharomyces cerevisiae XK, were compared to evaluate enzymatic activities and ethanol productivities at 30 °C and 40 °C. Unlike the activities of xylose metabolic enzymes in DMB3-7, enzymatic activities of XR, XDH, and XK in both DMB5 and DMB13 hardly decreased even at 40 °C, suggesting that these enzymes from K. marxianus are highly thermostable. The most efficient glucose/xylose co-fermentation at 40 °C was found in DMB13; namely, DMB13 rapidly converted xylose to ethanol, especially after glucose depletion, and showed the highest ethanol yield (0.402 g/g). These findings support the view that alteration of coenzyme specificity of XDH expressed in K. marxianus will be efficacious for high-temperature ethanol production from mixed sugars containing xylose.

Pseudomonas humi sp. nov., isolated from leaf soil.

An aerobic, Gram-negative, non-sporulating, motile, rod-shaped and lignin-degrading bacterial strain, Pseudomonas sp. CCA1, was isolated from leaf soil collected in Japan. This strain grew at 20-45 °C (optimum 20 °C), at pH 5.0-10.0 (optimum pH 5.0), and in the presence of 2% NaCl. Its major cellular fatty acids were C16:0 and summed feature 8 (C18:1ω6c and/or C18:1ω7c). The predominant quinone system was ubiquinone-9. Comparative 16S rRNA gene sequence analysis showed that Pseudomonas sp. CCA1 was related most closely to P. citronellolis NBRC 103043T (98.9%), but multilocus sequence analysis based on fragments of the atpD, gyrA, gyrB and rpoB gene sequences showed strain CCA1 to branch separately from its most closely related Pseudomonas type strains. DNA-DNA hybridization values between Pseudomonas sp. CCA1 and type strains of closely related Pseudomonas species were less than 53%. Based on its phenotypic, chemotaxonomic and phylogenetic features, we propose that Pseudomonas sp. CCA1 represents a novel species within the genus Pseudomonas, for which the name Pseudomonas humi sp. nov. is proposed. The type strain is CCA1 (= HUT 8136T = TBRC 8616T).

Ice-binding proteins from the fungus Antarctomyces psychrotrophicus possibly originate from two different bacteria through horizontal gene transfer.

Various microbes, including fungi and bacteria, that live in cold environments produce ice-binding proteins (IBPs) that protect them from freezing. Ascomycota and Basidiomycota are two major phyla of fungi, and Antarctomyces psychrotrophicus is currently designated as the sole ascomycete that produces IBP (AnpIBP). However, its complete amino acid sequence, ice-binding property, and evolutionary history have not yet been clarified. Here, we determined the peptide sequences of three new AnpIBP isoforms by total cDNA analysis and compared them with those of other microbial IBPs. The AnpIBP isoforms and ascomycete-putative IBPs were found to be phylogenetically close to the bacterial ones but far from the basidiomycete ones, which is supported by the higher sequence identities to bacterial IBPs than basidiomycete IBPs, although ascomycetes are phylogenetically distant from bacteria. In addition, two of the isoforms of AnpIBP share low sequence identity and are not close in the phylogenetic tree. It is hence presumable that these two AnpIBP isoforms were independently acquired from different bacteria through horizontal gene transfer (HGT), which implies that ascomycetes and bacteria frequently exchange their IBP genes. The non-colligative freezing-point depression ability of AnpIBP was not very high, whereas it exhibited significant abilities of ice recrystallization inhibition, ice shaping, and cryo-protection against freeze-thaw cycles even at submicromolar concentrations. These results suggest that HGT is crucial for the cold-adaptive evolution of ascomycetes, and their IBPs offer freeze resistance to organisms to enable them to inhabit the icy environments of Antarctica. DATABASES: Nucleotide sequence data are available in the DDBJ database under the accession numbers LC378707, LC378707, LC378707 for AnpIBP1a, AnpIBP1b, AnpIBP2, respectively.

A novel non-segmented double-stranded RNA virus from an Arctic isolate of Pythium polare.

We investigated virus infection in the oomycete Pythium polare from the Arctic. From 39 isolates investigated, 14 contained virus-like double-stranded RNA (dsRNA). Next generation sequencing revealed that the P. polare isolate OPU1176 contained three different virus-like sequences. We determined the full-length genome sequence of one of them. The 5397 nt-length genome had two overlapped open reading frames (ORFs) consistent with a toti and toti-like viruses, that we named Pythium polare RNA virus 1 (PpRV1). The ORF2 encoded an RNA-dependent RNA polymerase (RdRp). The shifty heptamer motif and RNA pseudoknot were predicted near the stop codon of ORF1, implying that the RdRp could be translated as a fusion protein with the ORF1 protein. Phylogenetic analysis with deduced RdRp amino acid sequences indicated that oomycete virus PpRV1 was closely related to the unclassified arthropod toti-like viruses. The comparison of PpRV1-free and -infected lines suggested that PpRV1 infected in a symptomless manner.

Development of tailor-made synergistic cellulolytic enzyme system for saccharification of steam exploded sugarcane bagasse.

Designing a tailor-made synergistic system is a promising strategy for developing an effective enzyme for saccharification of lignocellulosic materials. In this study, a cellulolytic enzyme mixture comprising selected core recombinant enzymes for hydrolysis of sugarcane bagasse pretreated by alkaline-catalyzed steam explosion was optimized using a mixture design approach. The optimized enzyme system comprised a cellobiohydrolase (Cel7A) from Talaromyces cellulolyticus, an endo-glucanase (Cel7B) from Thielavia terrestris, a ß-glucosidase (BGL) and an endo-ß1,4-xylanase (XYN) from Aspergillus aculeatus at the ratio of 0.34:0.27:0.14:0.25. The maximum reducing sugar yield of 797 mg/g biomass, comprising 543 and 96.8 mg/g glucose and xylose, respectively were achieved, equivalent to 92.44% and 47.50% recoveries, respectively from the pretreated substrate at the enzyme dosage of 20 mg/g biomass. The sugar yield from the quaternary enzyme mixture was 17.37% higher than that obtained with Accellerase 1500.

Production of acetoin from hydrothermally pretreated oil mesocarp fiber using metabolically engineered Escherichia coli in a bioreactor system.

Acetoin is used in the biochemical, chemical and pharmaceutical industries. Several effective methods for acetoin production from petroleum-based substrates have been developed, but they all have an environmental impact and do not meet sustainability criteria. Here we describe a simple and efficient method for acetoin production from oil palm mesocarp fiber hydrolysate using engineered Escherichia coli. An optimization of culture conditions for acetoin production was carried out using reagent-grade chemicals. The final concentration reached 29.9gL-1 with a theoretical yield of 79%. The optimal pretreatment conditions for preparing hydrolysate with higher sugar yields were then determined. When acetoin was produced using hydrolysate fortified with yeast extract, the theoretical yield reached 97% with an acetoin concentration of 15.5gL-1. The acetoin productivity was 10-fold higher than that obtained using reagent-grade sugars. This approach makes use of a compromise strategy for effective utilization of oil palm biomass towards industrial application.

Identification and characterization of Burkholderia multivorans CCA53.

OBJECTIVE: A lignin-degrading bacterium, Burkholderia sp. CCA53, was previously isolated from leaf soil. The purpose of this study was to determine phenotypic and biochemical features of Burkholderia sp. CCA53. RESULTS: Multilocus sequence typing (MLST) analysis based on fragments of the atpD, gltD, gyrB, lepA, recA and trpB gene sequences was performed to identify Burkholderia sp. CCA53. The MLST analysis revealed that Burkholderia sp. CCA53 was tightly clustered with B. multivorans ATCC BAA-247T. The quinone and cellular fatty acid profiles, carbon source utilization, growth temperature and pH were consistent with the characteristics of B. multivorans species. Burkholderia sp. CCA53 was therefore identified as B. multivorans CCA53.

Deletion Analysis of GH7 Endoglucanase Gene (cel7B) Promoter Region in a Talaromyces cellulolyticus ligD-Disrupted Strain.

Talaromyces cellulolyticus is expected to become an industrial cellulase producer. In this study, we performed deletion analysis of the promoter region of the GH7 endoglucanase gene (cel7B), which encodes one of the major cellulases, using a ß-glucuronidase reporter system. To obtain strains that harbor each gene cassette at the same locus, we had to improve the homologous recombination frequency. Hence, the ligD gene, encoding DNA ligase IV, was disrupted by homologous recombination. After that, the introduced pyrF marker gene, encoding orotate phosphoribosyl transferase, was deleted by a marker recycling system. The resultant strain, YDLP, exhibits high homologous recombination frequency. These data suggest that this approach will drastically improve the genetic modification tools of T. cellulolyticus. We obtained 7 strains for reporter analysis using YDLP as the host strain. Reporter analysis revealed that the promoter region between -812 and -612 is important for expression of cel7B. These results imply a relationship between this region and novel transcriptional factors.

Thermophilic ethanol fermentation from lignocellulose hydrolysate by genetically engineered Moorella thermoacetica.

A transformant of Moorella thermoacetica was constructed for thermophilic ethanol production from lignocellulosic biomass by deleting two phosphotransacetylase genes, pdul1 and pdul2, and introducing the native aldehyde dehydrogenase gene (aldh) controlled by the promoter from glyceraldehyde-3-phosphate dehydrogenase. The transformant showed tolerance to 540mM and fermented sugars including fructose, glucose, galactose and xylose to mainly ethanol. In a mixed-sugar medium of glucose and xylose, all of the sugars were consumed to produce ethanol at the yield of 1.9mol/mol-sugar. The transformant successfully fermented sugars in hydrolysate prepared through the acid hydrolysis of lignocellulose to ethanol, suggesting that this transformant can be used to ferment the sugars in lignocellulosic biomass for ethanol production.

Evaluation of Saccharomyces cerevisiae GAS1 with respect to its involvement in tolerance to low pH and salt stress.

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.

Production of d-lactate using a pyruvate-producing Escherichia coli strain.

To generate an organism capable of producing d-lactate, NAD+-dependent d-lactate dehydrogenase was expressed in our pyruvate-producing strain, Escherichia coli strain LAFCPCPt-accBC-aceE. After determining the optimal culture conditions for d-lactate production, 18.4 mM d-lactate was produced from biomass-based medium without supplemental mineral or nitrogen sources. Our results show that d-lactate can be produced in simple batch fermentation processes.

Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase.

A stable NADP+-dependent d-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericusmeso-diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 µmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 µmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP+ and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains.IMPORTANCE In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d-amino acids, but all include tedious steps. The use of NAD(P)+-dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs.

Genetic improvement of xylose metabolism by enhancing the expression of pentose phosphate pathway genes in Saccharomyces cerevisiae IR-2 for high-temperature ethanol production.

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 °C and over-expression of all PPP genes at 36 °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 °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.

Typhula cf. subvariabilis, new snow mould in Antarctica.

We collected snow blight of moss, Polytrichum juniperinum on King George Island, maritime Antarctica. Host died in a circle of about 10-30 cm after snow melts. Clamp connected hyphae and no sclerotia were observed on tip of host leaves. DNA sequence of ITS region from moss symptoms were perfectly matched with fruit bodies of Typhula sp. on Macquarie Island in the maritime Antarctica and high homology with Typhula cf. subvariabilis from Iran. Therefore, we suggested that T. cf. subvariabilis caused snow blight on moss in Antarctica, and this is first record of Typhula snow blight in Southern Hemisphere. These results also suggested that fungi in same genera gained similar ecological niche in both Polar Regions.

Draft Genome Sequence of Pseudomonas sp. Strain CCA1, Isolated from Leaf Soil.

Pseudomonas sp. strain CCA1 was isolated from leaf soil collected in Higashi-Hiroshima City in Hiroshima Prefecture, Japan. Here, we present a draft genome sequence of this strain. The genome consists of 24 contigs for a total of 6,993,992 bp, 8,917 predicted coding sequences, and a GC content of 67.2%.