Production of Insect Toxin Beauvericin From Entomopathogenic Fungi Cordyceps Militaris by Heterologous Expression of Global Regulator

Cordyceps militaris is one of entomopathogenic fungi species that is well known to be a traditional medicine in China for decades. Although the pharmaceutical and/or toxic properties of C. militaris has attracted attention as a promising resource for finding bioactive compounds, only a few substances including cordycepin have been reported so far. In the previous report heterologous expression of LaeA, a global regulator for secondary metabolites production in fungi, has been succeeded in C. militaris. The LaeA-engineered transformants are proved to produce new and/or elevated production of secondary metabolites, as detected by HPLC analysis. In order to further characterize the secondary metabolites that were being significantly produced by LaeA transformant, HPLC profiling and structure elucidation by proton NMR were conducted in two target compounds, designated as compound 1 and compound 2. Compound 1 possessed the highly similar characters to insect toxin beauvericin in UV spectrum, molecular weight, and retention time in HPLC analysis. Proton NMR analysis revealed that compound 1 had the same proton signals as beauvericin

ISSUES IN BIOTECHNOLOGY TEACHING - International experience in human resource development in biotechnology: achievements and lessons learnt

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Identification of actinomycetes from plant rhizospheric soils with inhibitory activity against Colletotrichum spp., the causative agent of anthracnose disease

<p>Abstract</p> <p>Background</p> <p><it>Colletotrichum </it>is one of the most widespread and important genus of plant pathogenic fungi worldwide. Various species of <it>Colletotrichum </it>are the causative agents of anthracnose disease in plants, which is a severe problem to agricultural crops particularly in Thailand. These phytopathogens are usually controlled using chemicals; however, the use of these agents can lead to environmental pollution. Potential non-chemical control strategies for anthracnose disease include the use of bacteria capable of producing anti-fungal compounds such as actinomycetes spp., that comprise a large group of filamentous, Gram positive bacteria from soil. The aim of this study was to isolate actinomycetes capable of inhibiting the growth of <it>Colletotrichum </it>spp, and to analyze the diversity of actinomycetes from plant rhizospheric soil.</p> <p>Results</p> <p>A total of 304 actinomycetes were isolated and tested for their inhibitory activity against <it>Colletotrichum gloeosporioides </it>strains DoA d0762 and DoA c1060 and <it>Colletotrichum capsici </it>strain DoA c1511 which cause anthracnose disease as well as the non-pathogenic <it>Saccharomyces cerevisiae </it>strain IFO 10217. Most isolates (222 out of 304, 73.0%) were active against at least one indicator fungus or yeast. Fifty four (17.8%) were active against three anthracnose fungi and 17 (5.6%) could inhibit the growth of all three fungi and <it>S. cerevisiae </it>used in the test. Detailed analysis on 30 selected isolates from an orchard at Chanthaburi using the comparison of 16S rRNA gene sequences revealed that most of the isolates (87%) belong to the genus <it>Streptomyces </it>sp., while one each belongs to <it>Saccharopolyspora </it>(strain SB-2) and <it>Nocardiopsis </it>(strain CM-2) and two to <it>Nocardia </it>(strains BP-3 and LK-1). Strains LC-1, LC-4, JF-1, SC-1 and MG-1 exerted high inhibitory activity against all three anthracnose fungi and yeast. In addition, the organic solvent extracts prepared from these five strains inhibited conidial growth of the three indicator fungi. Preliminary analysis of crude extracts by high performance liquid chromatography (HPLC) indicated that the sample from strain JF-1 may contain a novel compound. Phylogenetic analysis revealed that this strain is closely related to <it>Streptomyces cavurensis </it>NRRL 2740 with 99.8% DNA homology of 16S rRNA gene (500 bp).</p> <p>Conclusion</p> <p>The present study suggests that rhizospheric soil is an attractive source for the discovery of a large number of actinomycetes with activity against <it>Colletotrichum </it>spp. An interesting strain (JF-1) with high inhibitory activity has the potential to produce a new compound that may be useful in the control of <it>Colletotrichum </it>spp.</p

トリプトファナーゼオヨビベータ - チロシナーゼノカッセイチュウシンノキノウテキ コウゾウテキソクメン

京都大学0048新制・課程博士工学博士甲第2370号工博第654号新制||工||468(附属図書館)6204UT51-55-E46京都大学大学院工学研究科工業化学専攻(主査)教授 福井 三郎, 教授 田伏 岩夫, 教授 伊勢 典夫学位規則第5条第1項該当Kyoto UniversityDA

Effect of chondroitin sulfate and hyaluronic acid on gene expression in a three-dimensional culture of chondrocytes

The effect of glycosaminoglycan addition on a three-dimensional (3D) culture of porcine chondrocyte cells was investigated with a view to use in cartilage regenerative medicine. Chondroitin sulfate C increased the mRNA expression of type 2 collagen, while chondroitin sulfate A did not. Hyaluronic acid of high molecular weight markedly decreased the mRNA expression of both aggrecan and type 2 collagen, although hyaluronic acid of low molecular weight showed no apparent effect

barS1, a Gene for Biosynthesis of a γ-Butyrolactone Autoregulator, a Microbial Signaling Molecule Eliciting Antibiotic Production in Streptomyces Species

From Streptomyces virginiae, in which production of streptogramin antibiotic virginiamycin M(1) and S is tightly regulated by a low-molecular-weight Streptomyces hormone called virginiae butanolide (VB), which is a member of the γ-butyrolactone autoregulators, the hormone biosynthetic gene (barS1) was cloned and characterized by heterologous expression in Escherichia coli and by gene disruption in S. virginiae. The barS1 gene (a 774-bp open reading frame encoding a 257-amino-acid protein [M(r), 27,095]) is situated in the 10-kb regulator island surrounding the VB-specific receptor gene, barA. The deduced BarS1 protein is weakly homologous to β-ketoacyl-acyl carrier protein/coenzyme A reductase and belongs to the superfamily of short-chain alcohol dehydrogenase. The function of the BarS1 protein in VB biosynthesis was confirmed by BarS1-dependent in vitro conversion of 6-dehydro-VB-A to VB-A, the last catalytic step in VB biosynthesis. Of the four possible enantiomeric products from racemic 6-dehydro-VB-A as a substrate, only the natural enantiomer of (2R,3R,6S)-VB-A was produced by the purified recombinant BarS1 (rBarS1), indicating that rBarS1 is the stereospecific reductase recognizing (3R)-isomer as a substrate and reducing it stereospecifically to the (6S) product. In the ΔbarS1 mutant created by homologous recombination, the production of VB as well as the production of virginiamycin was lost. The production of virginiamycin by the ΔbarS1 mutant was fully recovered by the external addition of VB to the culture, which indicates that the barS1 gene is essential in the biosynthesis of the autoregulator VBs in S. virginiae and that the failure of virginiamycin production was a result of the loss of VB production

Production of Insect Toxin Beauvericin from Entomopathogenic Fungi Cordyceps militaris by Heterologous Expression of Global Regulator

Cordyceps militaris is one of entomopathogenic fungi species that is well known to be a traditional medicine in China for decades. Although the pharmaceutical and/or toxic properties of C. militaris has attracted attention as a promising resource for finding bioactive compounds, only a few substances including cordycepin have been reported so far. In the previous report heterologous expression of LaeA, a global regulator for secondary metabolites production in fungi, has been succeeded in C. militaris. The LaeA-engineered transformants are proved to produce new and/or elevated production of secondary metabolites, as detected by HPLC analysis. In order to further characterize the secondary metabolites that were being significantly produced by LaeA transformant, HPLC profiling and structure elucidation by proton NMR were conducted in two target compounds, designated as compound 1 and compound 2. Compound 1 possessed the highly similar characters to insect toxin beauvericin in UV spectrum, molecular weight, and retention time in HPLC analysis. Proton NMR analysis revealed that compound 1 had the same proton signals as beauvericin