Fatty acid profiles of oomycetes (Halophytophthora and Phytopythium spp.) from Philippines mangroves.

Mangroves create an ecological environment for a diverse assemblage of organisms, including marine and mangrove oomycetes. Halophytophthora spp., in particular, are isolated from fallen senescent mangrove leaves. Studies reported on Philippines oomycetes are mostly on their distribution and taxonomy, while fatty acid studies have not yet been fully explored. Recently, oomycetes were reported as efficient producers of various fatty acids; therefore, bioprospecting efforts are aimed at obtaining more industrially important fatty acid compounds for aquaculture, biodiesel production, and human consumption. In this study, 21 isolated oomycetes, identified as Halophytophthora spp., and two type species of Phytopythium, were grown in a broth medium containing peptone, yeast extract, glucose, and 50% seawater and incubated at room temperature for 3 weeks for fatty acid production and identification. Results revealed the presence of various fatty acids, mainly palmitic acid (C16:0), linoleic acid (C18:2n6c), oleic acid (C18:1n9c), cis-11,14,17-eicosatrienoic acid (ETA, C20:3n3), and stearic acid (C18:0), from Halophytophthora and Phytopythium isolates ranging from 2% to 30% total fatty acids. An omega-6 fatty acid, Ƴ-linolenic acid (GLA, C18:3n6), was found in Phytopythium isolates with considerably higher concentrations compared to Halophytophthoras. Further, omega-3 polyunsaturated fatty acid, cis-11,14,17-eicosatrienoic acid (ETA, C20:3n3), was detected on most oomycete isolates.

Characterization of Biogenic PbS Quantum Dots.

Heavy metals in a polluted environment are toxic to life. However, some microorganisms can remove or immobilize heavy metals through biomineralization. These bacteria also form minerals with compositions similar to those of semiconductors. Here, this bioprocess was used to fabricate semiconductors with low energy consumption and cost. Bacteria that form lead sulfide (PbS) nanoparticles were screened, and the crystallinity and semiconductor properties of the resulting nanoparticles were characterized. Bacterial consortia that formed PbS nanoparticles were obtained. Extracellular particle size ranged from 3.9 to 5.5 nm, and lattice fringes were observed. The lattice fringes and electron diffraction spectra corresponded to crystalline PbS. The X-ray diffraction (XRD) patterns of bacterial PbS exhibited clear diffraction peaks. The experimental and theoretical data of the diffraction angles on each crystal plane of polycrystalline PbS were in good agreement. Synchrotron XRD measurements showed no crystalline impurity-derived peaks. Thus, bacterial biomineralization can form ultrafine crystalline PbS nanoparticles. Optical absorption and current-voltage measurements of PbS were obtained to characterize the semiconductor properties; the results showed semiconductor quantum dot behavior. Moreover, the current increased under light irradiation when PbS nanoparticles were used. These results suggest that biogenic PbS has band gaps and exhibits the general fundamental characteristics of a semiconductor.

Der f 34, a Novel Major House Dust Mite Allergen Belonging to a Highly Conserved Rid/YjgF/YER057c/UK114 Family of Imine Deaminases.

The high prevalence of house dust mite (HDM) allergy is a growing health problem worldwide, and the characterization of clinically important HDM allergens is a prerequisite for the development of diagnostic and therapeutic strategies. Here, we report a novel HDM allergen that belongs structurally to the highly conserved Rid/YjgF/YER057c/UK114 family (Rid family) with imine deaminase activity. Isolated HDM cDNA, named der f 34, encodes 128 amino acids homologous to Rid-like proteins. This new protein belongs to the Rid family and has seven conserved residues involved in enamine/imine deaminase activity. Indeed, we demonstrated that purified Der f 34 had imine deaminase activity that preferentially acted on leucine and methionine. Native Der f 34 showed a high IgE binding frequency as revealed by two-dimensional immunoblotting (62.5%) or ELISA (68%), which was comparable with those of a major HDM allergen Der f 2 (77.5 and 79%, respectively). We also found that Der f 34 showed cross-reactivity with another prominent indoor allergen source, Aspergillus fumigatus This is the first report showing that the Rid family imine deaminase represents an additional important pan-allergen that is conserved across organisms.

Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases.

Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat Δ6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of Δ5 desaturase (D5d), which converts dihomo-γ-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-γ-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish Δ5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (Δ9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids.

Dysgonomonas alginatilytica sp. nov., an alginate-degrading bacterium isolated from a microbial consortium.

Gram-stain-negative, facultatively anaerobic, non-motile, non-spore-forming, rod-shaped bacterium, designated strain HUA-2T, was isolated from an alginate-degrading microbial consortium. Strain HUA-2T was related to Dysgonomonas capnocytophagoides JCM 16697T, Dysgonomonas macrotermitis JCM 19375T and Dysgonomonas mossii CCUG 43457T with 95.1 %, 94.1 % and 92.1 % 16S rRNA gene sequence similarity, respectively. The optimal growth temperature and pH for strain HUA-2T were 35 °C and pH 8.0, respectively. Enzyme production, major fermentation products from glucose, and the major cellular fatty acids were different from those of D. capnocytophagoides CCUG 17966T or other members of the genus Dysgonomonas. Therefore, strain HUA-2T is proposed to represent a novel species of the genus Dysgonomonas, for which we propose the name Dysgonomonas alginatilytica sp. nov. The type strain is HUA-2T ( = DSM 100214T = HUT 8134T).

Value-added lipid production from brown seaweed biomass by two-stage fermentation using acetic acid bacterium and thraustochytrid.

Thraustochytrid production of polyunsaturated fatty acids and xanthophylls have been generally sourced from crop-derived substrates, making the exploration of alternative feedstocks attractive since they promise increased sustainability and lower production costs. In this study, a distinct two-stage fermentation system was conceptualized for the first time, using the brown seaweed sugar mannitol as substrate for the intermediary biocatalyst Gluconobacter oxydans, an acetic acid bacterium, along with the marine thraustochytrid Aurantiochytrium sp. to produce the value-added lipids and xanthophylls. Jar fermenter culture resulted in seaweed mannitol conversion to fructose with an efficiency of 83 % by G. oxydans and, after bacteriostasis with sea salts, production of astaxanthin and docosahexaenoic acid by Aurantiochytrium sp. KH105. Astaxanthin productivity was high at 3.60 mg/L/day. This new system, therefore, widens possibilities of obtaining more varieties of industrially valuable products including foods, cosmetics, pharmaceuticals, and biofuel precursor lipids from seaweed fermentation upon the use of suitable thraustochytrid strains.

Unexpected T cell regulatory activity of anti-histone H1 autoantibody: its mode of action in regulatory T cell-dependent and -independent manners.

Induction of anti-nuclear antibodies against DNA or histones is a hallmark of autoimmune disorders, but their actual contribution to disease predisposition remains to be clarified. We have previously reported that autoantibodies against histone H1 work as a critical graft survival factor in a rat model of tolerogeneic liver transplantation. Here we show that an immunosuppressive anti-histone H1 monoclonal antibody (anti-H1 mAb) acts directly on T cells to inhibit their activation in response to T cell receptor (TCR) ligation. Intriguingly, the T cell activation inhibitory activity of anti-H1 mAb under suboptimal dosages required regulatory T (Treg) cells, while high dose stimulation with anti-H1 mAb triggered a Treg cell-independent, direct negative regulation of T cell activation upon TCR cross-linking. In the Treg cell-dependent mode of immunosuppressive action, anti-H1 mAb did not induce the expansion of CD4(+-)Foxp3(+) Treg cells, but rather potentiated their regulatory capacity. These results reveal a previously unappreciated T cell regulatory role of anti-H1 autoantibody, whose overproduction is generally thought to be pathogenic in the autoimmune settings.

Development of a Transformation System for Nitratireductor sp.

Nitratireductor sp. OM-1 can accumulate butenoic acid, which is a short-chain unsaturated carboxylic acid utilized for chemical products. So far, we have predicted the thioesterase gene, te, as a candidate gene for butenoic acid biosynthesis, based on comparative transcriptome analysis. To confirm the function of te, the gene transfer system in Nitratireductor sp. OM-1 was required. Thus, in this study, we used electroporation as a transformation system and pRK415, a broad host range plasmid, and optimized the conditions. As a result, a maximum transformation efficiency of 7.9 × 104 colonies/µg DNA was obtained at 22.5 kV/cm. Moreover, an expression vector, pRK415-te, was constructed by insertion of te, which was successfully transferred into strain OM-1, using electroporation. The recombinant OM-1 strain produced butenoic acid at 26.7 mg/g of dried cell weight, which was a 254% increase compared to transformants harboring an empty vector. This is the first report of a gene transfer system for Nitratireductor sp., which showed that the te gene was responsible for butenoic acid production.

Preparation of egg-koji for developing a novel food.

While chicken eggs contain many nutrients necessary for humans and there are various cooking methods, the nutritional components are used as they are, and there are no traditional foods that utilize microorganisms. Koji-mold, containing Aspergillus oryzae, A. sojae, and A. luchuensis, which has been used in various fermented foods since ancient times, grows on raw grain materials such as rice and barley to become koji. This can give flavors not found in the raw materials that can decompose and convert the nutritional components of the raw materials. Here, we succeeded for the first time in developing egg-koji that uses only eggs and koji-mold by selecting and combining cooked egg powder (CEP) and A. oryzae AO101 as the most suitable combination. To suppress the explosive growth of harmful bacteria, we improved the sterilization method, watering method, and amount of water. In addition, it was found that egg-koji has a characteristic enzyme activity balance, in which amylase is extremely low and protease at pH 6 was high compared to grain koji, such as rice and barley. Egg-koji might produce enzymes suitable for taking in nutrients when growing into CEP and would be expected to give a flavor that could not be achieved by cooking or additives.

Utilization of Sugarcane Bagasse as a Substrate for Lipid Production by Aurantiochytrium sp.

Thraustochytrid, Aurantiochytrium sp., produces various lipids such as polyunsaturated and saturated fatty acids, carotenoids, and other hydrocarbons, which are useful in the fields of health foods, cosmetics, fine chemicals, and biofuels. Lignocellulosic biomass, which is abundant and cheap, is a promising feedstock for producing cheaper bulk and high-value-added products using Aurantiochytrium sp. However, the steam explosion of lignocellulosic biomass for efficient enzymatic saccharification generates substances that inhibit the growth of microorganisms. In this study, the inhibitory activities of these by-products on the growth and lipid production of Aurantiochytrium sp. were investigated. Aurantiochytrium sp. was found to be highly sensitive to furfural and vanillin and moderately sensitive to 5-hydroxymethylfurfural and syringaldehyde. Washing steam-exploded bagasse with water, followed by activated charcoal treatment, significantly reduced furfural, which was a major inhibitory component in the saccharified solution.

Sake lees fermented with lactic acid bacteria prevents allergic rhinitis-like symptoms and IgE-mediated basophil degranulation.

We tested the effect of oral administration of fermented sake lees with lactic acid bacteria (FESLAB) on a murine model of allergic rhinitis upon immunization and nasal sensitization with ovalbumin (OVA). We used Lactobacillus paracasei NPSRIk-4 (isolated from sake lees), and L. brevis NPSRIv-8 (from fermented milk) as starter strains to produce the FESLAB. Oral FESLAB administration resulted in the development of significantly fewer sneezing symptoms than those seen in sham control animals given sterile water. We also found that FESLAB suppressed the allergen-induced degranulation of RBL2H3 rat basophilic leukemia cells.

Visualization of Gene Reciprocity among Lactic Acid Bacteria in Yogurt by RNase H-Assisted Rolling Circle Amplification-Fluorescence In Situ Hybridization.

Recently, we developed an in situ mRNA detection method termed RNase H-assisted rolling circle amplification-fluorescence in situ hybridization (RHa-RCA-FISH), which can detect even short mRNA in a bacterial cell. However, because this FISH method is sensitive to the sample condition, it is necessary to find a suitable cell permeabilization and collection protocol. Here, we demonstrate its further applicability for detecting intrinsic mRNA expression using lactic acid bacteria (LAB) as a model consortium. Our results show that this method can visualize functional gene expression in LAB cells and can be used for monitoring the temporal transition of gene expression. In addition, we also confirmed that data obtained from bulk analyses such as RNA-seq or microarray do not always correspond to gene expression in individual cells. RHa-RCA-FISH will be a powerful tool to compensate for insufficient data from metatranscriptome analyses while clarifying the carriers of function in microbial consortia. By extending this technique to capture spatiotemporal microbial gene expression at the single-cell level, it will be able to characterize microbial interactions in phytoplankton-bacteria interactions.

Improvement of fatty acid productivity of thraustochytrid, Aurantiochytrium sp. by genome editing.

Thraustochytrid strains belonging to the genus Aurantiochytrium accumulate significant amounts of lipids including polyunsaturated fatty acids and carotenoids and, therefore, are expected to be used for industrial production of various valuable materials. Although various efforts such as chemical mutagenesis and homologous gene recombination have been made to improve lipid productivity of Aurantiochytrium species, low specificity and efficiency in the conventional methods hinder the research progress. Here, we attempted to apply a genome editing technology, the CRISPR-Cas9 system as an alternative molecular breeding technique for Aurantiochytrium species to accelerate the metabolic engineering. The efficiency of specific gene knock-in by the homologous recombination increased more than 10-folds by combining the CRISPR-Cas9 system. As a result of disrupting the genes associated with ß-oxidation of fatty acids by the improved method, the genome edited strains with higher fatty acid productivity were isolated, demonstrating for the first time that the CRISPR-Cas9 system was effective for molecular breeding of the strains in the genus Aurantiochytrium to improve lipid productivity.

Molecular cloning and immunochemical characterization of a novel major Japanese cedar pollen allergen belonging to the aspartic protease family.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis in Japan. Protease activity in the pollen grains may trigger pro-allergic responses but no such proteases have yet been identified as pollen allergens. OBJECTIVES: We report the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to the aspartic protease family. METHODS: We focused on the C. japonica pollen allergen spot No. 63 (CPA63, 47.5% IgE binding frequency) on our 2-dimensional IgE immunoblot map. The internal amino acid sequences were determined using time-of-flight mass spectrometry. Full-length cpa63 cDNA was cloned by rapid amplification of cDNA ends (RACE)-PCR. Recombinant CPA63 (r-CPA63) was expressed using the baculovirus-insect cell culture system and its IgE binding capacity was analyzed by enzyme-linked immunosorbent assay (ELISA). The proteolytic activity of r-CPA63 was also assessed using a putative mature enzyme produced upon autolysis. RESULTS: cpa63 cDNA encoded a 472 amino acid polypeptide showing about 40% sequence identity to members of the plant atypical aspartic protease family. ELISA showed that r-CPA63 was recognized by IgE antibodies in the serum of 58% (18/31) of Japanese cedar pollinosis patients. We also demonstrated an aspartic protease-like enzyme activity of the putative mature r-CPA63. CONCLUSIONS: We have identified the first plant aspartic protease allergen from Japanese cedar pollen. The availability of the CPA63 sequence and its recombinant allergen production system are useful not only for pharmaceutical applications but also for further examination of the role of protease activity in the pathogenesis of cedar pollinosis.

A new lipid transfer protein homolog identified as an IgE-binding antigen from Japanese cedar pollen.

Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal rhinitis and conjunctivitis in Japan, and an understanding of its full allergen repertoire is prerequisite for the development of future molecular diagnostics and immunotherapeutic strategies. Here we report the identification of a new C. japonica pollen IgE-binding antigen (CJP-8) homologous to lipid transfer proteins (LTPs), a class of plant cross-reactive allergens found in foods, latex, and pollen grains. The cjp-8 cDNA encodes a 165-amino acid polypeptide possessing the conserved eight cysteines characteristic of plant LTP family members. Escherichia coli-expressed recombinant CJP-8 (r-CJP-8) reacted with IgE antibody from Japanese cedar pollinosis patients at a 37.5% frequency (6/16).

Direct detection of mRNA expression in microbial cells by fluorescence in situ hybridization using RNase H-assisted rolling circle amplification.

Meta-analyses using next generation sequencing is a powerful strategy for studying microbiota; however, it cannot clarify the role of individual microbes within microbiota. To know which cell expresses what gene is important for elucidation of the individual cell's function in microbiota. In this report, we developed novel fluorescence in situ hybridization (FISH) procedure using RNase-H-assisted rolling circle amplification to visualize mRNA of interest in microbial cells without reverse transcription. Our results show that this method is applicable to both Gram-negative and Gram-positive microbes without any noise from DNA, and it is possible to visualize the target mRNA expression directly at the single-cell level. Therefore, our procedure, when combined with data of meta-analyses, can help to understand the role of individual microbes in the microbiota.

Improvement of Stearidonic acid production in Oleaginous Saccharomyces cerevisiae.

Rat Delta6 desaturase was introduced in "oleaginous" Saccharomyces cerevisiae genetically modified by the snf2 disruption, DGA1 overexpression, and LEU2 expression to improve stearidonic acid (18:4n-3 (Delta6, 9, 12, 15)) production. When this transformed yeast was cultured in the presence of 0.7 g/l alpha-linolenic acid for 7 d, 0.4 g of alpha-linolenic acid was accumulated and 44 mg of stearidonic acid was produced, mainly as a triacylglycerol in the 1 liter broth.

Metabolite Profile Analysis of Aurantiochytrium limacinum SR21 Grown on Acetate-based Medium for Lipid Fermentation.

Thraustochytrids, a group of marine protists, are continuously gaining attention due to their capability in producing lipids for various biotechnological applications towards foods, medicines, chemicals, and biofuels. Although various substrates, predominantly glucose, have been used as carbon source for this microalga, it is desirable to adopt cheaper and more diversified substrate to expand their application range. In this study, we aimed to examine the ability of acetate, which can be easily generated from various resources by acetogenic microorganisms, as a substrate of Aurantiochytrium limacinum SR21. As a result of flask-scale analysis, specific growth rates (µ) of the strain SR21 grown in 3% acetate- or glucose-based medium were 0.55 and 0.98 h-1, respectively. The maximum yield of total fatty acid in acetate medium was 4.8 g/L at 48 h while that in glucose medium was 6.8 g/L at 30 h, indicating that acetate has potential as substrate. Metabolome analysis was performed to comprehensively elucidate characteristic metabolic fluctuations caused by acetate assimilation and identify targets to improve the fatty acid productivity from acetate. It was found that the use of glyoxylate cycle, which bypasses release of energy molecules such as NADH and GTP, and the inhibition of utilization of compounds from TCA cycle for anabolic reactions, may cause the slow growth in acetate which has an effect also in lipid productivity. The activity of the pentose phosphate pathway was found to be weak in acetate cultivation, thus NADPH was mainly produced in malate-pyruvate cycle. Lastly, mevalonate pathway was found to be activated in acetate cultivation which additionally competes with acetyl-CoA as starting material of fatty acid synthesis.

Marine Oomycetes (Halophytophthora and Salispina): A Potential Source of Fatty Acids with Cytotoxic Activity Against Breast Adenocarcinoma Cells (MCF7).

Marine oomycetes are ubiquitous, fungus-like eukaryotes known to produce fatty acids with potential anticancer activity. The long chain omega-3 and omega-6 fatty acids are currently popular and considered as safe when used as nutraceuticals in cancer treatment. In this study, crude fatty acids from three marine oomycetes, Halophytophthora spp. (T12GP1 and T12YBP2) and Salispina hoi (USTCMS 1611), were explored for their cytotoxic and apoptotic potentials against human breast adenocarcinoma cells (MCF7) and normal human dermal fibroblasts (HDFn). Extracts from mycelia mats consisted of diverse saturated, monounsaturated, and polyunsaturated fatty acids such as linoleic, α-linolenic, γ-linolenic, eicosatrienoic and eicosapentaenoic acids. The crude fatty acids from all three oomycetes in in vitro assays for cytotoxicity showed no toxicity (30% toxicity values) on HDFn cells. On MCF7 cells, however, IC50 values of 23.44, 15.63, and 26.15 µg/mL were obtained with extracts from Halophytophthora T12GP1 and T12YBP2 and S. hoi, respectively. Treated MCF7 cells exhibited deformed cell membrane in MTT assay and also aggregation of DNA and disruption of nuclear membrane aggregation in nuclear staining; further, green signals indicative of apoptosis was recorded in caspase 3/7 assay.

Complete genome sequence of Nitratireductor sp. strain OM-1: A lipid-producing bacterium with potential use in wastewater treatment.

Reducing CO2 emissions is necessary to alleviate rising global temperature. Renewable sources of energy are becoming an increasingly important substitute for fossil fuels. An important step in this direction is the isolation of novel, technologically relevant microorganisms. Nitratireductor sp. strain OM-1 can convert volatile short-chain fatty acids in wastewater into 2-butenoic acid and its ester and can accumulate intracellularly esterified compounds up to 50% of its dried cell weight under nitrogen-depleted conditions. It is believed that a novel fatty acid biosynthesis pathway including an esterifying enzyme is encoded in its genome. In this study, we report the whole-genome sequence (4.8 Mb) of OM-1, which comprises a chromosome (3,977,827 bp) and a megaplasmid (857,937 bp). This sequence information provides insight into the genome organization and biochemical pathways of OM-1. In addition, we identified lipid biosynthesis pathways in OM-1, paving the way to a better understanding of its biochemical characterization.