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.

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.

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.

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.

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.

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.

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.

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.

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.

RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection.

RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3'-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes. When padlock probes are hybridized to the target RNA molecule, they are converted to the circular form by SplintR ligase. Subsequently, RNase H creates nick sites only in the hybridized RNA sequence, and single-stranded DNA is finally synthesized from the nick site by phi29 DNA polymerase. This method could specifically detect at least 10 fmol of the target RNA molecule without reverse transcription. Moreover, this method detected GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without background DNA amplification. Therefore, this method can potentially detect almost all types of RNA molecules without reverse transcription and reveal full-length sequence information.

Isolation of High Carotenoid-producing Aurantiochytrium sp. Mutants and Improvement of Astaxanthin Productivity Using Metabolic Information.

The marine eukaryotic microheterotroph thraustochytrid genus Aurantiochytrium is a known producer of polyunsaturated fatty acids, carotenoids, and squalene. We previously constructed a lipid fermentation system for Aurantiochytrium sp. strains using underutilized biomass, such as canned syrup and brown macroalgae. To improve the productivity, in this study, Aurantiochytrium sp. RH-7A and RH-7A-7 that produced high levels of carotenoids, such as astaxanthin and canthaxanthin, were isolated through chemical mutagenesis. Moreover, metabolomic analysis of the strain RH-7A revealed that oxidative stress impacts carotenoid accumulation. Accordingly, the addition of ferrous ion (Fe2+), as an oxidative stress compound, to the culture medium significantly enhanced the production of astaxanthin by the mutants. These approaches improved the productivity of astaxanthin up to 9.5 mg/L/day at the flask scale using not only glucose but also fructose which is the main carbon source in fermentation systems with syrup and brown algae as the raw materials.

A Possible Trifunctional ß-Carotene Synthase Gene Identified in the Draft Genome of Aurantiochytrium sp. Strain KH105.

Labyrinthulomycetes have been regarded as a promising industrial source of xanthophylls, including astaxanthin and canthaxanthin, polyunsaturated fatty acids such as docosahexaenoic acid and docosapentaenoic acid, ω-3 oils, and terpenic hydrocarbons, such as sterols and squalene. A Thraustochytrid, Aurantiochytrium sp. KH105 produces carotenoids, including astaxanthin, with strong antioxidant activity. To gain genomic insights into this capacity, we decoded its 97-Mbp genome and characterized genes for enzymes involved in carotenoid biosynthesis. Interestingly, all carotenogenic genes, as well as other eukaryotic genes, appeared duplicated, suggesting that this strain is diploid. In addition, among the five genes involved in the pathway from geranylgeranyl pyrophosphate to astaxanthin, geranylgeranyl phytoene synthase (crtB), phytoene desaturase (crtI) and lycopene cyclase (crtY) were fused into single gene (crtIBY) with no internal stop codons. Functionality of the trifunctional enzyme, CrtIBY, to catalyze the reaction from geranylgeranyl diphosphate to ß-carotene was confirmed using a yeast assay system and mass spectrometry. Furthermore, analyses of differential gene expression showed characteristic up-regulation of carotenoid biosynthetic genes during stationary and starvation phases under these culture conditions. This suggests genetic engineering events to promote more efficient production of carotenoids. We also showed an occurrence of crtIBY in other Thraustochytrid species.

Efficient conversion of mannitol derived from brown seaweed to fructose for fermentation with a thraustochytrid.

Macroalgae are a promising biomass feedstock for energy and valuable chemicals. Mannitol and alginate are the major carbohydrates found in the microalga Laminaria japonica (Konbu). To convert mannitol to fructose for its utilization as a carbon source in mannitol non-assimilating bacteria, a psychrophile-based simple biocatalyst (PSCat) was constructed using a psychrophile as a host by expressing mesophilic enzymes, including mannitol 2-dehydrogenase for mannitol oxidation, and NADH oxidase and alkyl hydroxyperoxide reductase for NAD+ regeneration. PSCat was treated at 40 °C to inactivate the psychrophilic enzymes responsible for byproduct formation and to increase the membrane permeability of the substrate. PSCat efficiently converted mannitol to fructose with high conversion yield without additional input of NAD+. Konbu extract containing mannitol was converted to fructose with hydroperoxide scavenging, inhibiting the mannitol dehydrogenase activity. Auranthiochytrium sp. could grow well in the presence of fructose converted by PSCat. Thus, PSCat is a potential carbohydrate converter for mannitol non-assimilating microorganism.

Prominent IgE-binding and cytokine-inducing capacities of a newly cloned N-terminal region of Der f 14, an apolipophorin-like house dust mite allergen.

We previously characterized a 177-kDa allergen, M-177, from Dermatophagoides farinae. Thereafter, a counterpart to M-177 for Euroglyphus maynei was cloned as Eur m 14, and its sequence revealed that two environmental allergens, Mag 1 and Mag 3, are digested fragments of M-177. The aims of this study were to clone the cDNA of Der f 14 corresponding to M-177 and to elucidate the allergenic capacities of the N-terminal fragment of Der f 14 (Der f 14-N). Recombinant allergens were produced as trigger-factor-fused proteins in Escherichia coli. Der f 14-N showed the highest IgE-binding frequency among Der f 14-derived fragments in patients allergic to house dust mite by enzyme-linked immunosorbent assay. Der f 14-N showed the highest capacity to induce cell proliferation in murine lymphocyte and human peripheral mononuclear cells among Der f 14-derived fragments. Der f 14-N induced IL-13, IFN-γ and IL-17 production more than Der f 1 and Der f 2 in mouse, and induced IL-5 and IFN-γ production at levels comparable to those of Der f 1 and Der f 2 in some patients. The high prevalence of IgE binding to the Der f 14-N indicates that it could be an important mite allergen.

Improved methanization and microbial diversity during batch mode cultivation with repetition of substrate addition using defined organic matter and marine sediment inoculum at seawater salinity.

The activation of microbes, which are needed to initiate continuous methane production, can be accomplished by fed-batch methanization. In the present study, marine sediment inoculum was activated by batch mode methanization with repetition of substrate addition using defined organic matter from sugar, protein, or fat at seawater salinity to investigate the potential for application of the activation method to various types of saline waste and microbial community compositions. All substrates had methane potentials close to the theoretical value except for bovine serum albumin (BSA) whose methane potential was lower, but the maximum methane potential reached the value during repeated methanization. Beta diversity analysis revealed that substrate (especially BSA)-fed and non-fed cultures had distinct microbial community compositions. Bacterial members depended on substrate. Thus, marine sediment inocula activated via the methanization method can be used to effectively treat various types of saline waste.

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.

Detection of Acyl-CoA Derivatized with Butylamide for in vitro Fatty Acid Desaturase Assay.

Membrane-bound fatty acid desaturases acting on acyl-CoA contribute to the biosynthesis of unsaturated fatty acids, such as arachidonic acid and docosahexaenoic acid in higher organisms. We propose a simplified method for measuring the desaturase activity that combines the in vitro reaction by desaturase-expressing yeast cell homogenate and the detection of acyl-CoA product as butylamide derivatives by gas chromatography. To set up the in vitro reaction, we traced the in vivo activity of rat liver ∆6 fatty acid desaturase (D6d) expressed in the yeast, Saccharomyces cerevisiae, and determined the time taken for the D6d activity to reach its maximum level. The cell homogenate of yeast expressing the maximum D6d activity was made to react in vitro with linoleoyl-CoA to generate the D6d product, γlinolenoyl-CoA. This product was successfully detected as a peak corresponding to γ-linolenoyl butylamide on gas chromatography. This procedure, with low background expression, using non-labeled acyl-CoA as substrate, will contribute toward developing a simple in vitro desaturase assay. It will also help in elucidating the functions of membrane-bound fatty acid desaturases with various substrate specificities and regioselectivities.

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.