Symbiotic relationship between filamentous algae (Halomicronema sp.) and extracellular polymeric substance-producing algae (Chlamydomonas sp.) through biomimetic simulation of natural algal mats.

To lower the cost of biomass harvesting, the growth of natural biofilm is considered to be an optimal alternative to microalgae aggregation. This study investigated algal mats that naturally agglomerate into a lump and float on water surfaces. Halomicronema sp., a filamentous cyanobacterium with high cell aggregation and adhesion to substrates, and Chlamydomonas sp., which grows rapidly and produces high extracellular polymeric substances (EPS) in certain environments, are the main microalgae that make up selected mats through next-generation sequencing analysis. These two species play a major role in the formation of solid mats, and showed a symbiotic relationship as the medium and nutritional source, particularly owing to the large amount of EPS formed by the reaction between EPS and calcium ions through zeta potential and Fourier-transform infrared spectroscopy analysis. This led to the formation of an ecological biomimetic algal mat (BAM) that mimics the natural algal mat system, and this is a way to reduce costs in the biomass production process as there is no separate treatment process for harvesting.

Microbial biodegradation of recalcitrant synthetic dyes from textile-enriched wastewater by Fusarium oxysporum.

The present study reported the improvement of biological treatment for the removal of recalcitrant dyes including aniline blue, reactive black 5, orange II, and crystal violet in contaminated water. The biodegradation efficiency of Fusarium oxysporum was significantly enhanced by the addition of mediators and by adjusting the biomass density and nutrient composition. A supplementation of 1% glucose in culture medium improved the biodegradation efficiency of aniline blue, reactive black 5, orange II, and crystal violet by 2.24, 1.51, 4.46, and 2.1 folds, respectively. Meanwhile, the addition of mediators to culture medium significantly increased the percentages of total removal for aniline blue, reactive black 5, orange II, and crystal violet, reaching 86.07%, 68.29%, 76.35%, and 95.3%, respectively. Interestingly, the fungal culture supplemented with 1% remazol brilliant blue R boosted the biodegradation up to 97.06%, 89.86%, 91.38%, and 86.67% for aniline blue, reactive black 5, orange II, and crystal violet, respectively. Under optimal culture conditions, the fungal culture could degrade these synthetic dyes concentration up to 104 mg/L. The present study demonstrated that different recalcitrant dye types can be efficiently degraded using microorganism such as F. oxysporum.

Complete genome sequencing and comparative genomic analysis of Lactobacillus acidophilus C5 as a potential canine probiotics.

Lactobacillus acidophilus is a gram-positive, microaerophilic, and acidophilic bacterial species. L. acidophilus strains in the gastrointestinal tracts of humans and other animals have been profiled, but strains found in the canine gut have not been studied yet. Our study helps in understanding the genetic features of the L. acidophilus C5 strain found in the canine gut, determining its adaptive features evolved to survive in the canine gut environment, and in elucidating its probiotic functions. To examine the canine L. acidophilus C5 genome, we isolated the C5 strain from a Korean dog and sequenced it using PacBio SMRT sequencing technology. A comparative genomic approach was used to assess genetic relationships between C5 and six other strains and study the distinguishing features related to different hosts. We found that most genes in the C5 strain were related to carbohydrate transport and metabolism. The pan-genome of seven L. acidophilus strains contained 2,254 gene families, and the core genome contained 1,726 gene families. The phylogenetic tree of the core genes in the canine L. acidophilus C5 strain was very close to that of two strains (DSM20079 and NCFM) from humans. We identified 30 evolutionarily accelerated genes in the L. acidophilus C5 strain in the ratio of non-synonymous to synonymous substitutions (dN/dS) analysis. Five of these thirty genes were associated with carbohydrate transport and metabolism. This study provides insights into genetic features and adaptations of the L. acidophilus C5 strain to survive the canine intestinal environment. It also suggests that the evolution of the L. acidophilus genome is closely related to the host's evolutionary adaptation process.

Screening and Production of Manganese Peroxidase from Fusarium sp. on Residue Materials.

In this study, we report the manganese peroxidase production ability from a Fusarium sp. strain using an inexpensive medium of agriculture residues of either rice straw or wood chips as carbon source. The highest manganese peroxidase activity on rice straw medium and on wood chips was 1.76 U/mL by day 9 and 1.91 U/mL by day 12, respectively.

Enhancement of 2,3-butanediol production from Jerusalem artichoke tuber extract by a recombinant Bacillus sp. strain BRC1 with increased inulinase activity.

A Bacillus sp. strain named BRC1 is capable of producing 2,3-butanediol (2,3-BD) using hydrolysates of the Jerusalem artichoke tuber (JAT), a rich source of the fructose polymer inulin. To enhance 2,3-BD production, we undertook an extensive analysis of the Bacillus sp. BRC1 genome, identifying a putative gene (sacC) encoding a fructan hydrolysis enzyme and characterizing the activity of the resulting recombinant protein expressed in and purified from Escherichia coli. Introduction of the sacC gene into Bacillus sp. BRC1 using an expression vector increased enzymatic activity more than twofold. Consistent with this increased enzyme expression, 2,3-BD production from JAT was also increased from 3.98 to 8.10 g L-1. Fed-batch fermentation of the recombinant strain produced a maximal level of 2,3-BD production of 28.6 g L-1, showing a high theoretical yield of 92.3%.

A Mutant of the Bck1 Homolog from Cryphonectria parasitica Resulted in Sectorization with an Impaired Pathogenicity.

CpBck1, an ortholog of the cell-wall integrity mitogen-activated protein kinase kinase kinase of Saccharomyces cerevisiae, was cloned and characterized from the chestnut blight fungus Cryphonectria parasitica. The CpBck1-null mutant displayed cell wall integrity-related phenotypic changes such as abnormal cell morphology and wall formation and hypersensitivity to cell wall-disrupting agents. In addition, the mutant showed severely retarded growth without any sign of normal development, such as hyphal differentiation, conidiation, or pigmentation. As the culture proceeded, the mutant colony showed sporadic sectorization. Once sectored, the sectored phenotype of robust mycelial growth without differentiation was stably inherited. Compared with the wild type, both the parental CpBck1-null mutant and the sectored progeny exhibited marked impaired virulence. The present study revealed that a mutation in a signaling pathway component related to cell-wall integrity resulted in sporadic sectorization and these sectored phenotypes were stably inherited, suggesting that this signal transduction pathway is implicated in adaptive genetic changes for sectorization.

Characterization of a novel manganese dependent endoglucanase belongs in GH family 5 from Phanerochaete chrysosporium.

The cDNA encoding a putative glycoside hydrolase family 5, which has been predicted to be an endoglucanase (PcEg5A), was cloned from Phanerochaete chrysosporium and expressed in Pichia pastoris. PcEg5A contains a carbohydrate-binding domain and two important amino acids, E209 and E319, playing as proton donor and nucleophile in substrate catalytic domain. SDS-PAGE analysis indicated that the recombinant endoglucanase 5A (rPcEg5A) has a molecular size of 43 kDa which corresponds with the theoretical calculation. Optimum pH and temperature were found to be 4.5-6.0, and 50°C-60°C, respectively. Moreover, rPcEg5A exhibited maximal activity in the pH range of 3.0-8.0, whereas over 50% of activity still remained at 20°C and 80°C. rPcEg5A was stable at 60°C for 12 h incubation, indicating that rPcEg5A is a thermostable enzyme. Manganese ion enhanced the enzyme activity by 77%, indicating that rPcEg5A is a metal dependent enzyme. The addition of rPcEg5A to cellobiase (cellobiohydrolase and ß-glucosidase) resulted in a 53% increasing saccharification of NaOH-pretreated barley straw, whereas the glucose release was 47% higher than that cellobiase treatment alone. Our study suggested that rPcEg5A is an enzyme with great potential for biomass saccharification.

Identification of a Polyketide Synthase Gene in the Synthesis of Phleichrome of the Phytopathogenic Fungus Cladosporium phlei.

Phleichrome, a pigment produced by the phytopathogenic fungus Cladosporium phlei, is a fungal perylenequinone whose photodynamic activity has been studied intensively. To determine the biological function of phleichrome and to engineer a strain with enhanced production of phleichrome, we identified the gene responsible for the synthesis of phleichrome. Structural comparison of phleichrome with other fungal perylenequinones suggested that phleichrome is synthesized via polyketide pathway. We recently identified four different polyketide synthase (PKS) genes encompassing three major clades of fungal PKSs that differ with respect to reducing conditions for the polyketide product. Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis. Increased accumulation of Cppks1 transcript was observed in response to supplementation with the application of synthetic inducer cyclo-(l-Pro-l-Phe). In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome. These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.

Seminal CD38 is a pivotal regulator for fetomaternal tolerance.

A successful pregnancy depends on a complex process that establishes fetomaternal tolerance. Seminal plasma is known to induce maternal immune tolerance to paternal alloantigens, but the seminal factors that regulate maternal immunity have yet to be characterized. Here, we show that a soluble form of CD38 (sCD38) released from seminal vesicles to the seminal plasma plays a crucial role in inducing tolerogenic dendritic cells and CD4(+) forkhead box P3(+) (Foxp3(+)) regulatory T cells (Tregs), thereby enhancing maternal immune tolerance and protecting the semiallogeneic fetus from resorption. The abortion rate in BALB/c females mated with C57BL/6 Cd38(-/-) males was high compared with that in females mated with Cd38(+/+) males, and this was associated with a reduced proportion of Tregs within the CD4(+) T-cell pool. Direct intravaginal injection of sCD38 to CBA/J pregnant mice at preimplantation increased Tregs and pregnancy rates in mice under abortive sonic stress from 48 h after mating until euthanasia. Thus, sCD38 released from seminal vesicles to the seminal plasma acts as an immunoregulatory factor to protect semiallogeneic fetuses from maternal immune responses.

Putative endoglucanase PcGH5 from Phanerochaete chrysosporium is a ß-xylosidase that cleaves xylans in synergistic action with endo-xylanase.

A predicted endoglucanase gene (PcGH5) was cloned from Phanerochaete chysosporium, and expressed in Pichia pastoris. Although PcGH5 showed similarity with the conserved domains of a cellulase superfamily GH5, a ß-glucosidase/6-phospho-ß-glucosidase/ß-galactosidase superfamily, and an endoglucanase, recombinant PcGH5 exhibited a ß-xylosidase activity, rather than endoglucanase activity. Therefore, the predicted gene was named as PcXyl5. Further characterization of recombinant PcXyl5 showed not only catalysis of the hydrolysis of xylo-oligomers to xylose, but also displayed transglycosylation activity using alcohol as a receptor. Optimum pH of rPcXyl5 was found to be 5.5, whereas optimum temperature was 50°C. rPcXyl5 increased reducing sugar release of birchwood xylan, beechwood xylan, and arabinoxylan by 6.4%, 13%, 15.8%, respectively, in synergistic action with endo-xylanase. Interestingly, the late addition of rPcXyl5 into reaction with endo-xylanase resulted in a larger increase of reducing sugar release from pretreated barley straw that addition at the start or by treatment with endo-xylanases alone. The increases observed were 6.3% and 13.8%, respectively, showing a great potential application for hemicellulose saccharification.

Reduction of hexavalent chromium using Aerva lanata L.: elucidation of reduction mechanism and identification of active principles.

The effluent discharge treatment for controlling the environment from non biodegradable metal contaminants using plant extract is an efficient technique. The reduction of hexavalent chromium by abundantly available weed, Aerva lanata L. was investigated using batch equilibrium technique. The variables studied were Cr(VI) concentration, Aerva lanata L. dose, contact time, pH, temperature and agitation speed. Cyclic voltammetry and ICP-MS analysis confirmed the reduction of Cr(VI) to Cr(III). Electrochemical analysis proved that, the chromium has not been degraded and the valency of the chromium has only been changed. ICP-MS analysis shows that 100ng/L of hexavalent chromium was reduced to 97.01ng/L trivalent chromium. These results suggest that components present in the Aerva lanata L. are responsible for the reduction of Cr(VI) to Cr(III). The prime components ferulic acid, kaempherol and ß-carboline present in the Aerva lanata L. may be responsible for the reduction of Cr(VI) as evident from LC-MS analysis.

Manipulation of light wavelength at appropriate growth stage to enhance biomass productivity and fatty acid methyl ester yield using Chlorella vulgaris.

LEDs light offer several advantages over the conventional lamps, thereby being considered as the optimal light sources for microalgal cultivation. In this study, various light-emitting diodes (LEDs) especially red and blue color with different light wavelengths were employed to explore the effects of light source on phototrophic cultivation of Chlorella vulgaris. Blue light illumination led to significantly increased cell size, whereas red light resulted in small-sized cell with active divisions. Based on the discovery of the effect of light wavelengths on microalgal biology, we then applied appropriate wavelength at different growth stages; blue light was illuminated first and then shifted to red light. By doing so, biomass and lipid productivity of C. vulgaris could be significantly increased, compared to that in the control. These results will shed light on a novel approach using LED light for microalgal biotechnology.

Functional analysis of a tannic-acid-inducible and hypoviral-regulated small heat-shock protein Hsp24 from the chestnut blight fungus Cryphonectria parasitica.

A small heat-shock protein gene, CpHsp24, of Cryphonectria parasitica was selected based on its expression pattern, which showed that it was tannic acid inducible and that its induction was severely hampered by a hypovirus. The predicted protein sequence of CpHsp24 consisted of a hallmark α-crystalline domain flanked by a variable N-terminal and a short C-terminal region. Disruption of CpHsp24 resulted in a slow growth rate under standard growth conditions. The CpHsp24-null mutant showed enhanced sensitivity to heat shock, which was consistent with Northern and Western analyses displaying the heat-shock induction of the CpHsp24 gene and protein, respectively. Virulence tests on the excised bark revealed a severe decrease in the necrotic area of the CpHsp24-null mutant. When the hypovirus was transferred, virus-containing CpHsp24-null progeny displayed severely retarded growth patterns with hypovirulent characteristics of reduced pigmentation and sporulation. Because the tannic-acid-inducible and hypoviral-suppressible expression and the severely impaired virulence are also characteristics of the laccase3 gene (lac3), lac3 expression in the CpHsp24-null mutant was also examined. The resulting lac3 induction was severely affected in the CpHsp24-null mutant, suggesting that CpHsp24 is important for lac3 induction and that CpHsp24 may act as a molecular chaperone for the lac3 protein.

Manganese peroxidase h4 isozyme mediated degradation and detoxification of triarylmethane dye malachite green: optimization of decolorization by response surface methodology.

A cDNA encoding for manganese peroxidase isozyme H4 (MnPH4), isolated from Phanerochaete chrysosporium, was expressed in Pichia pastoris, under the control of alcohol oxidase I promoter. The recombinant MnPH4 was efficiently secreted onto media supplemented with hemin at a maximum concentration of 500 U/L, after which purified rMnPH4 was used to decolorize the triarylmethane dye malachite green (MG). Response surface methodology (RSM) was employed to optimize three different operational parameters for the decolorization of MG. RSM showed that the optimized variables of enzyme (0.662 U), MnSO4 (448 µM), and hydrogen peroxide (159 µM) decolorized 100 mg/L of MG completely at 3 h. Additionally, UV-VIS spectra, high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-electrospray ionization/mass spectrometry analysis confirmed the degradation of MG by the formation of main metabolites 4-dimethylamino-benzophenone hydrate, N, N-dimethylaniline (N,N-dimethyl-benzenamine), and methylbenzaldehyde. Interestingly, it was found that rMnPH4 mediates hydroxyl radical attack on the central carbon of MG. Finally, rMnPH4 degraded MG resulted in the complete removal of its toxicity, which was checked under in vitro conditions.

Characterization of a recombinant bifunctional xylosidase/arabinofuranosidase from Phanerochaete chrysosporium.

A bifunctional xylosidase/arabinofuranosidase gene (PcXyl) was cloned from the cDNA library of Phanerochaete chrysosporium and further expressed in Pichia pastoris. Enzymatic assay indicated that P. pastoris produced rPcXyl at a level of 26,141 U l⁻¹. The xylosidase and arabinofuranosidase activities of rPcXyl were maximized, respectively, at pHs of 5.0 and 5.5 and temperatures of 45°C and 50°C. SDS-PAGE revealed a single band of purified rPcXyl of 83 kDa. Cu²âº and Zn²âº completely inhibited the enzyme activity of rPcXyl. The enzyme activity of rPcXyl was increased 151%, 126% and 123%, respectively, in the presence of glucose, xylose and arabinose at concentrations of 5 mM. rPcXyl hydrolyzed xylobiose to xylose and xylotriose to xylose and xylobiose, indicating rPcXyl acts as an exo-type enzyme. Additionally, rPcXyl enhanced xylose release from xylan substrates in synergy with rPcXynC.

Expression and purification of an immunogenic dengue virus epitope using a synthetic consensus sequence of envelope domain III and Saccharomyces cerevisiae.

A synthetic consensus gene was designed based on residues of the amino acid sequences of dengue envelope domain III (scEDIII) from all four serotypes, and codon optimization for expression was conducted using baker's yeast, Saccharomyces cerevisiae. The synthetic gene was cloned into a yeast episomal expression vector, pYEGPD-TER, which was designed to direct cloned gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator. PCR and back-transformation into Escherichia coli confirmed the presence of the scEDIII gene-containing plasmid in the transformants. Northern blot analysis showed the presence of the scEDIII-specific transcript. Western blot analysis indicated that expressed scEDIII, with mobility similar to purified EDIII from E. coli, was successfully secreted into the culture media. Quantitative ELISA revealed that the recombinant scEDIII comprised approximately 0.1-0.6% of cell-free extract. In addition, 0.1-0.6 mg of scEDIII protein per liter of culture filtrate was detected on day 1 and peaked on day 3 after cultivation. The secreted scEDIII protein can be purified to ≥90% purity with 85% recovery using a simple ion-exchange FPLC followed by molecular weight cut-off. Upon administration of the purified protein to mice, mouse sera contained antibodies that were specific to all four serotypes of dengue virus. Moreover, a balanced immune response against all four serotypes was observed, suggesting that it may be possible to develop an effective tetravalent dengue vaccine using S. cerevisiae.

Cloning and characterization of a thermostable endo-arabinanase from Phanerochaete chrysosporium and its synergistic action with endo-xylanase.

Putative arabinanase (PcARA) was cloned from cDNA of Phanerochaete chrysosporium. The gene sequencing indicated that PcARA consisted of 939 nucleotides that encodes for 312 amino acid arabinanase-polypeptide chain, including a signal peptide of 19 amino acids. Three-dimensional homology indicated that this enzyme is a five-bladed ß-propeller, belonging to glycosidase family 43 and its secondary structure is consisted of 24 ß-sheets. The PcARA-cDNA was expressed in Pichia pastoris using pPICZαC. SDS-PAGE of purified arabinanase showed a single band of 33 kDa that is very close to theoretical molecular mass of 33.9 kDa calculated by its amino acid content. Recombinant arabinanase (rPcARA) exhibited maximum activity at pH and temperature of 5.0 and 60 °C, respectively. End-product analysis of debranched arabinan hydrolysis by thin-layer chromatography indicated that rPcARA acted as endo-type. The synergistic action of rPcARA with recombinant xylanase resulted in 72 and 9.3 % release of total soluble sugar of arabinoxylan and NaOH-pretreated barley straw, respectively.

Cloning and characterization of a novel bifunctional acetyl xylan esterase with carbohydrate binding module from Phanerochaete chrysosporium.

The cDNA of acetyl xylan esterase 2 (PcAxe2) gene containing a carbohydrate binding module (CBM) sequence from Phanerochaete chrysosporium was cloned and expressed in Pichia pastoris. The recombinant PcAxe2 protein (rPcAxe2) was efficiently produced, reaching a maximum of 1058 U l(-1) after 6 days of cultivation. Molecular mass of the rPcAxe2 on SDS-PAGE was approximately 63 kDa under hyperglycosylation. Optimal activity of the purified rPcAxe2 enzyme was observed at pH and temperature of 7.0 and 30-35°C, respectively. In addition to acetyl xylan esterase activity, rPcAxe2 also exhibited a xylanase activity at an optimum pH and temperature of 5.0 and 80°C, respectively. The synergistic action of rPcAxe2 with rPcXynC on birchwood xylan, beechwood xylan and wheat arabinoxylan enhanced the total reducing soluble sugar.

Biological function of a novel chrysovirus, CnV1-BS122, in the Korean Cryphonectria nitschkei BS122 strain.

Curing Cryphonectria nitschkei BS122 of a novel chrysovirus CnV1-BS122 infection was achieved by plating small hyphal fragments from an old plate and protoplasting followed by regeneration. Uneven distribution of mycoviruses within colonies was suggested. Comparing the CnV1-BS122-cured and -infected isogenic strains revealed that CnV1-BS122 infection resulted in reduced mycelial growth.

Occurrence of dsRNA Mycovirus (LeV-FMRI0339) in the Edible Mushroom Lentinula edodes and Meiotic Stability of LeV-FMRI0339 among Monokaryotic Progeny.

dsRNA was found in malformed cultures of Lentinula edodes strain FMRI0339, one of the three most popular sawdust cultivated commercial strains of shiitake, and was also found in healthy-looking fruiting bodies and actively growing mycelia. Cloning of the partial genome of the dsRNA revealed the presence of the RdRp sequence of a novel L. edodes mycovirus (LeV), and sequence comparison of the cloned amplicon showed identical sequences sequence to known RNA-dependent RNA polymerase genes of LeV found in strain HKA. The meiotic stability of dsRNA was examined by measuring the ratio of the presence of dsRNA among sexual monokaryotic progeny. More than 40% of the monokaryotic progeny still contained the dsRNA, indicating the persistence of dsRNA during sexual reproduction. Comparing the mycelia growth of monokaryotic progeny suggested that there appeared to be a tendency toward a lower frequency of virus incidence in actively growing progeny.