• ALGAE
• /
• v.32 no.4
• /
• pp.359-377
• /
• 2017

Pyropia haitanensis (T. J. Chang et B. F. Zheng) N. Kikuchi et M. Miyata is one of the most commercially useful macroalgae cultivated in southeastern China. In red algae, the biosynthesis of terpenoids through 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway can produce a direct influence on the synthesis of many biologically important metabolites. In this study, two genes of cDNAs, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductase (DXR), which encoding the first two rate-limiting enzymes among MEP pathway were cloned from P. haitanensis. The cDNAs of P. haitanensis DXS (PhDXS) and DXR (PhDXR) both contained complete open reading frames encoding polypeptides of 764 and 426 amino acids residues, separately. The expression analysis showed that PhDXS was significant differently expressed between leafy thallus and conchocelis as PhDXR been non-significant. Additionally, expression of PhDXR and its downstream gene geranylgeranyl diphosphate synthase were both inhibited by fosmidomycin significantly. Meanwhile, we constructed types of phylogenetic trees through different algae and higher plants DXS and DXR encoding amino acid sequences, as a result we found tree clustering consequences basically in line with the "Cavalier-Smith endosymbiotic theory." Whereupon, we speculated that in red algae, there existed only complete MEP pathway to meet needs of terpenoids synthesis for themselves; Terpenoids synthesis of red algae derivatives through mevalonate pathway came from two or more times endosymbiosis of heterotrophic eukaryotic parasitifer. This study demonstrated that PhDXS and PhDXR could play significant roles in terpenoids biosynthesis at molecular levels. Meanwhile, as nuclear genes among MEP pathway, PhDXS and PhDXR could provide a new way of thinking to research the problem of chromalveolata biological evolution.

• Korean Journal of Veterinary Research
• /
• v.46 no.4
• /
• pp.295-304
• /
• 2006

Residual materials such as veterinary drugs, environmental contaminants, and pesticides are affecting food safety. High resolution techniques and quality controls are needed to analyze these materials from part per million to part per trillion quantities in food. In order to achieve quality results, standardized methods and techniques are required. Our laboratories were prepared to obtain a certificate of accreditation for ISO/IEC 17025 in the analytical criteria of animal drugs, dioxins, pesticides, and heavy metals. ISO together with IEC has built a strategic partnership with the World Trade Organization with the common goal of promoting a free and fair global trading system. ISO collaborates with the United Nations Organization and its specialized agencies and commissions, particularly those involved in the harmonization of regulations and public policies including the World Health Organization and CODEX Alimentarius for food safety measurement, management and traceability. Our goal was to have high quality analysts, proper analytical methods, good laboratory facilities, and safety systems within guidelines of ISO/IEC 17025. All staff members took requirement exams. We applied proficiency tests in the analysis of veterinary drugs (nitrofuran metabolites, sulfonamide and tetracyclines), dioxins, organophosphorus pesticides, and heavy metals (Cd, Pb, As) to the Food Analysis Performance Assessment Scheme (FAPAS) at Central Science Laboratory, Department for Environment Food and Rural Affairs (DEFRA), England. The results were very satisfactory. All documents were prepared, including system management, laboratory management, standard operational procedures for testing, reporting, and more. The criteria encompassed the requirements of ISO/IEC 17025:1999. Finally, the Korea Laboratory Accreditation Scheme (KOLAS) accredited our testing laboratories in accordance with the provisions of Article 23 of the National Standards Act. The accreditation will give us the benefit of becoming a regional reference laboratory in Asia.

• Journal of Life Science
• /
• v.17 no.2 s.82
• /
• pp.266-271
• /
• 2007

Bisphenol A (BPA) has been widely used as a monomer for production of epoxy resins and polycarbonate plastics. The annual production of BPA exceeds 640,000 metric tons in worldwide. BPA, a suspected phenolic endocrine disrupter, is moderately soluble and frequently detected in industrial wastewater. To date, HPLC and GC has been used for BPA analysis. However, HPLC and GC-analysis need high operation lost, experts, and an elaborate pre-treatment of samples, and is difficult to apply on-time and mass analysis. Therefore, simple, mass and rapid detection of BPA in environments is necessary. In the present study, spectrophotometric method of BPA quantification was developed. Based on blue-color product formation with BPA and ferric chloride/ferricyanide under the optimized conditions, the standard curve was acquired $({\lambda}_{750}=0.061\;BPA\;[{\mu}M]+0.07155,\;R^2=0.992)$. Using an established method, the BPA contents in the soil extract, and different water samples and living products, including disposable syringe, cup and plastic tube, were analyzed. The results suggested that the method is useful for BPA determination from different massive samples. Since the BPA metabolites, nontoxic 4-hydroxyacetophenone or 4-hydroxybenzaldehyde, did not form blue-color product, this method is also useful to screen a microorganism for BPA bioremediation.

• Microbiology and Biotechnology Letters
• /
• v.49 no.1
• /
• pp.75-87
• /
• 2021

Type I Interferons (IFNα) are known for their role as biological anticancer agents owing to their cell-apoptosis inducing properties. Development of an appropriate, cost-effective host expression system is crucial for meeting the increasing demand for proteins. Therefore, this study aims to develop codon-optimized IFNα-2b in L. lactis NZ3900. These cells express extracellular protein using the NICE system and Usp₄₅ signal peptide. To validate the mature form of the expressed protein, the recombinant IFNα-2b was screened in a human colorectal cancer cell line using the cytotoxicity assay. The IFNα-2b was successfully cloned into the pNZ8148 vector, thereby generating recombinant L. lactis pNZ8148-SP_Usp45-IFNα-2b. The computational analysis of codon-optimized IFNα-2b revealed no mutation and amino acid changes; additionally, the codon-optimized IFNα-2b showed 100% similarity with native human IFNα-2b, in the BLAST analysis. The partial size exclusion chromatography (SEC) of extracellular protein yielded a 19 kDa protein, which was further confirmed by its positive binding to anti-IFNα-2b in the western blot analysis. The crude protein and SEC-purified partial fraction showed IC₅₀ values of 33.22 ㎍/ml and 127.2 ㎍/ml, respectively, which indicated better activity than the metabolites of L. lactis NZ3900 (231.8 ㎍/ml). These values were also comparable with those of the regular anticancer drug tamoxifen (105.5 ㎍/ml). These results demonstrated L. lactis as a promising host system that functions by utilizing the pNZ8148 NICE system. Meanwhile, codon-optimized usage of the inserted gene increased the optimal protein expression levels, which could be beneficial for its large-scale production. Taken together, the recombinant L. lactis IFNα-2b is a potential alternative treatment for colorectal cancer. Furthermore, its activity was analyzed in the WiDr cell line, to assess its colorectal anticancer activities in vivo.

• Journal of Applied Biological Chemistry
• /
• v.65 no.4
• /
• pp.439-446
• /
• 2022

Soybean is a crop with high-quality of protein and oil, and it is one of the most widely used genetically modified (GM) crops in the world today. In South Korea, Kwangan is the most utilized variety as a parental line for GM soybean development. In this study, untargeted LC-MS metabolomic approaches were used to compare metabolite profiles of Kwangan and three other commercial varieties cultivated in Gunwi and Jeonju in 2020 year. Metabolomic studies revealed that the 4 soybean varieties were distinct based on the partial least squares-discriminant analysis (PLS-DA) score plots; 18 metabolites contributed to variety distinction, including phenylalanine, isoflavones, and fatty acids. All varieties were clearly differentiated by location on the PLS-DA score plot, indicating that the growing environment is also attributable to metabolite variability. In particular, isoflavones and linolenic acid levels in Kwangan were significantly lower and higher, respectively compared to those of the three varieties. It was discussed that it might need to include more diverse conventional varieties as comparators in regard to metabolic characteristics of Kwangan for the assessment of substantial equivalence biogenetically engineered soybeans in a Kwangan-variety background.

• Mycobiology
• /
• v.51 no.1
• /
• pp.36-48
• /
• 2023

Xanthoria elegans is a lichen symbiosis, that inhabits extreme environments and can absorb UV-B. We reported the de novo sequencing and assembly of X. elegans genome. The whole genome was approximately 44.63 Mb, with a GC content of 40.69%. Genome assembly generated 207 scaffolds with an N50 length of 563,100 bp, N90 length of 122,672 bp. The genome comprised 9,581 genes, some encoded enzymes involved in the secondary metabolism such as terpene, polyketides. To further understand the UV-B absorbing and adaptability to extreme environments mechanisms of X. elegans, we searched the secondary metabolites genes and gene-cluster from the genome using genome-mining and bioinformatics analysis. The results revealed that 7 NR-PKSs, 12 HR-PKSs and 2 hybrid PKS-PKSs from X. elegans were isolated, they belong to Type I PKS (T1PKS) according to the domain architecture; phylogenetic analysis and BGCs comparison linked the putative products to two NR-PKSs and three HR-PKSs, the putative products of two NR-PKSs were emodin xanthrone (most likely parietin) and mycophelonic acid, the putative products of three HR-PKSs were soppilines, (+)-asperlin and macrolactone brefeldin A, respectively. 5 PKSs from X. elegans build a correlation between the SMs carbon skeleton and PKS genes based on the domain architecture, phylogenetic and BGC comparison. Although the function of 16 PKSs remains unclear, the findings emphasize that the genes from X. elegans represent an unexploited source of novel polyketide and utilization of lichen gene resources.

• The Korean Journal of Pesticide Science
• /
• v.19 no.3
• /
• pp.174-184
• /
• 2015

In the present study, the metabolism of [$^{14}C$]butachlor was investigated in rice plant according to the OECD test guideline No. 501. [$^{14}C$]Butachlor was treated as granule to paddy water by application of 1.5 kg ingredient (a.i.)/ha at the 3~4 leave stage of rice plant. At 85 days after treatment (DAT), samples of panicle, foliage, and roots were taken for radioactivity analysis. Upon harvest at 126 DAT, rice plants were separated into brown rice, husk, straw, and root parts. Amounts of total radioactivity absorbed by rice plant ranged from 8.6 to 9.8% of applied radioactivity (AR). Total radioactive residues (TRRs) of rice plant at 126 DAT was the highest as 4.0421 mg/kg (7.3% AR) in the straw followed by 1.4595 mg/kg (2.4% AR) in the root, 0.7257 mg/kg (0.1% AR) in the husk. The lowest level recording 0.1020 mg/kg (0.1% AR) was found in brown rice. Each part was extracted with various solvents and solvent/water mixtures. Greater than 70% of TRRs was readily extractable from foliage, panicle, husk and straw. Only 34.0% of the brown rice and 43% of root based on TRRs were extractable showing that the residues were completely assimilated in the plant tissue. The level of non-extractable radioactivity was ranged from 26.2 to 66.0% of TRRs. From this study, five tentative major metabolites (M1, M2, M3, M4 and M5) were observed in rice extracts. Among the metabolites, 2,6-diethylaniline assigned as M4 was identified in rice plant by comparing to retention time of reference standard. Un-metabolized butachlor was not detected in any fractions. In soil extracts, N-(butoxymethyl)-N-(2,6-diethyl phenyl)acetamide, 2,6-diethylaniline, M2, M3 and M5 were observed. And the concentration of butachlor was low level (ca. 0.03 mg/kg).

• 한국균학회소식:학술대회논문집
• /
• 2014.05a
• /
• pp.27-28
• /
• 2014

Beauveria bassiana (Cordycipitaceae, Hypocreales, Ascomycota) is an anamorphic fungus having a potential to be used as a biological control agent because it parasitizes a wide range of arthropod hosts including termites, aphids, beetles and many other insects. A number of bioactive secondary metabolites (SMs) have been isolated from B. bassiana and functionally verified. Among them, beauvericin and bassianolide are cyclic depsipeptides with antibiotic and insecticidal effects belonging to the enniatin family. Non-ribosomal peptide synthetases (NRPSs) play a crucial role in the synthesis of these secondary metabolites. NRPSs are modularly organized multienzyme complexes in which each module is responsible for the elongation of proteinogenic and non-protein amino acids, as well as carboxyl and hydroxyacids. A minimum of three domains are necessary for one NRPS elongation module: an adenylation (A) domain for substrate recognition and activation; a tholation (T) domain that tethers the growing peptide chain and the incoming aminoacyl unit; and a condensation (C) domain to catalyze peptide bond formation. Some of the optional domains include epimerization (E), heterocyclization (Cy) and oxidation (Ox) domains, which may modify the enzyme-bound precursors or intermediates. In the present study, we analyzed genomes of B. bassiana and its allied species in Hypocreales to verify the distribution of NRPS-encoding genes involving biosynthesis of beauvericin and bassianolide, and to unveil the evolutionary processes of the gene clusters. Initially, we retrieved completely or partially assembled genomic sequences of fungal species belonging to Hypocreales from public databases. SM biosynthesizing genes were predicted from the selected genomes using antiSMASH program. Adenylation (A) domains were extracted from the predicted NRPS, NRPS-like and NRPS-PKS hybrid genes, and used them to construct a phylogenetic tree. Based on the preliminary results of SM biosynthetic gene prediction in B. bassiana, we analyzed the conserved gene orders of beauvericin and bassianolide biosynthetic gene clusters among the hypocrealean fungi. Reciprocal best blast hit (RBH) approach was performed to identify the regions orthologous to the biosynthetic gene cluster in the selected fungal genomes. A clear recombination pattern was recognized in the inferred A-domain tree in which A-domains in the 1st and 2nd modules of beauvericin and bassianolide synthetases were grouped in CYCLO and EAS clades, respectively, suggesting that two modules of each synthetase have evolved independently. In addition, inferred topologies were congruent with the species phylogeny of Cordycipitaceae, indicating that the gene fusion event have occurred before the species divergence. Beauvericin and bassianolide synthetases turned out to possess identical domain organization as C-A-T-C-A-NM-T-T-C. We also predicted precursors of beauvericin and bassianolide synthetases based on the extracted signature residues in A-domain core motifs. The result showed that the A-domains in the 1st module of both synthetases select D-2-hydroxyisovalerate (D-Hiv), while A-domains in the 2nd modules specifically activate L-phenylalanine (Phe) in beauvericin synthetase and leucine (Leu) in bassianolide synthetase. antiSMASH ver. 2.0 predicted 15 genes in the beauvericin biosynthetic gene cluster of the B. bassiana genome dispersed across a total length of approximately 50kb. The beauvericin biosynthetic gene cluster contains beauvericin synthetase as well as kivr gene encoding NADPH-dependent ketoisovalerate reductase which is necessary to convert 2-ketoisovalarate to D-Hiv and a gene encoding a putative Gal4-like transcriptional regulator. Our syntenic comparison showed that species in Cordycipitaceae have almost conserved beauvericin biosynthetic gene cluster although the gene order and direction were sometimes variable. It is intriguing that there is no region orthologous to beauvericin synthetase gene in Cordyceps militaris genome. It is likely that beauvericin synthetase was present in common ancestor of Cordycipitaceae but selective gene loss has occurred in several species including C. militaris. Putative bassianolide biosynthetic gene cluster consisted of 16 genes including bassianolide synthetase, cytochrome P450 monooxygenase, and putative Gal4-like transcriptional regulator genes. Our synteny analysis found that only B. bassiana possessed a bassianolide synthetase gene among the studied fungi. This result is consistent with the groupings in A-domain tree in which bassianolide synthetase gene found in B. bassiana was not grouped with NRPS genes predicted in other species. We hypothesized that bassianolide biosynthesizing cluster genes in B. bassiana are possibly acquired by horizontal gene transfer (HGT) from distantly related fungi. The present study showed that B. bassiana is the only species capable of producing both beauvericin and bassianolide. This property led to B. bassiana infect multiple hosts and to be a potential biological control agent against agricultural pests.

• Korean Journal of Environmental Agriculture
• /
• v.21 no.4
• /
• pp.261-268
• /
• 2002

Biodegradation of monochlorophenols by wood rot fungi such as Daldina concentrica, Trametes versicolor and Pleurotus ostreatus was evaluated by determining their resistance or toxic test and biodegradability. The metabolites of monochlorophenols were also analyzed. Among the three fungi, T. versicolor was the most resistant to 200 ppm of 2-, 3- and 4-chlorophenols, and did not show any inhibitory mycellium growth. But D. concentrica had a little inhibition effect at more than 100 ppm of 3- or 4-chlorophenol. Control cultures of P. ostreatus took even 14 days far the completion of mycellium growth, but the hyphal growth was improved when 2- or 3-chlorophenol were added to the culture. In biodegradation analysis, P. ostreatus showed the highest degradation of 2- or 3-chlorophenol, while T. versicolor was the most effective in 4-chlorophenol. D. concentrica and P. ostreatus slowly degraded 4-chlorophenol. However, T. versicolor had similar degradation capability in the three monochlorophenols, suggesting that the biode- gradation nude is dependent on the fungi as well as the type of monochlorophenol. Several metabolites such as 1,3,5-trihydroxyl benzene, 1-ethyl-1-hydroxyl pentane, 2-propenoicacid, methylmalonic acid and 2-methyl-4-keto-pentan-2-ol were found as products of primary oxidation of 2-, 3- and 4-chlorophenols by intact fungal cultures. fatty acids including tetradecanoic, heptadecanoic and octadecanoic acids were also detected The order of increase of mycellium weight during incubation were P. ostreatus > T. versicolor > D. concentrica. The pH in the culture was not constantly changed depending on incubation days, but the mycellium weight was slightly increased, indicating that the biodegradation of monochlorophenol might have low relationship with the mycellium growth Laccase activities of T. versicolor and P. ostreatus were continuously increased depending on the incubation days, suggesting that the ligninolytic enzyme activity play an important role in the biodegradation of monochlorophenol.

• Investigative Magnetic Resonance Imaging
• /
• v.12 no.2
• /
• pp.100-106
• /
• 2008

The purpose of this study was to confirm the exactitude of in vitro nuclear magnetic resonance spectroscopy(NMRS) and to complement the defect of in vivo NMRS. It has been difficult to understand the metabolism of a cerebellum using in vivo NMRS owing to the generated inhomogeneity of magnetic fields (B0 and B1 field) by the complexity of the cerebellum structure. Thus, this study tried to more exactly analyze the metabolism of a canine cerebellum using the cell extraction and high resolution NMRS. In order to conduct the absolute metabolic quantification in a canine cerebellum, the spectrum of our phantom included in various brain metabolites (i.e., NAA, Cr, Cho, Ins, Lac, GABA, Glu, Gln, Tau and Ala) was obtained. The canine cerebellum tissue was extracted using the methanol-chloroform water extraction (M/C extraction) and one group was filtered and the other group was not under extract processing. Finally, NMRS of a phantom solution and two extract solution (90% D2O) was progressed using a 500MHz (11.4 T) NMR machine. Filtering a solution of the tissue extract increased the signal to noise ratio (SNR). The metabolic concentrations of a canine cerebellum were more close to rat’s metabolic concentration than human’s metabolic concentration. The present study demonstrates the absolute quantification technique in vitro high resolution NMRS with tissue extraction as the method to accurately measure metabolite concentration.