• Title/Summary/Keyword: nitrilase

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Transcriptome analyses of the ginseng root rot pathogens Cylindrocarpon destructans and Fusarium solani to identify radicicol resistance mechanisms

  • Li, Taiying;Kim, Jin-Hyun;Jung, Boknam;Ji, Sungyeon;Seo, Mun Won;Han, You Kyoung;Lee, Sung Woo;Bae, Yeoung Seuk;Choi, Hong-Gyu;Lee, Seung-Ho;Lee, Jungkwan
    • Journal of Ginseng Research
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    • v.44 no.1
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    • pp.161-167
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    • 2020
  • Background: The ascomycete fungi Cylindrocarpon destructans (Cd) and Fusarium solani (Fs) cause ginseng root rot and significantly reduce the quality and yield of ginseng. Cd produces the secondary metabolite radicicol, which targets the molecular chaperone Hsp90. Fs is resistant to radicicol, whereas other fungal genera associated with ginseng disease are sensitive to it. Radicicol resistance mechanisms have not yet been elucidated. Methods: Transcriptome analyses of Fs and Cd mycelia treated with or without radicicol were conducted using RNA-seq. All of the differentially expressed genes (DEGs) were functionally annotated using the Fusarium graminearum transcript database. In addition, deletions of two transporter genes identified by RNA-seq were created to confirm their contributions to radicicol resistance. Results: Treatment with radicicol resulted in upregulation of chitin synthase and cell wall integrity genes in Fs and upregulation of nicotinamide adenine dinucleotide dehydrogenase and sugar transporter genes in Cd. Genes encoding an ATP-binding cassette transporter, an aflatoxin efflux pump, ammonium permease 1 (mep1), and nitrilase were differentially expressed in both Fs and Cd. Among these four genes, only the ABC transporter was upregulated in both Fs and Cd. The aflatoxin efflux pump and mep1 were upregulated in Cd, but downregulated in Fs, whereas nitrilase was downregulated in both Fs and Cd. Conclusion: The transcriptome analyses suggested radicicol resistance pathways, and deletions of the transporter genes indicated that they contribute to radicicol resistance.

Analysis of the Melithiazol Biosynthetic Gene Cluster in Myxococcus stipitatus DSM 14675 (Myxococcus stipitatus DSM 14675의 melithiazol 생합성 유전자 분석)

  • Hyun, Hyesook;Park, Soohyun;Cho, Kyungyun
    • Microbiology and Biotechnology Letters
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    • v.44 no.3
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    • pp.391-399
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    • 2016
  • Melithiazols are antifungal substances produced by the myxobacteria Melitangium lichenicola, Archangium gephyra, and Myxococcus stipitatus. Melithiazol biosynthetic genes have been identified in M. lichenicola, but not in A. gephyra and M. stipitatus until now. We identified a 37.3-kb melithiazol biosynthetic gene cluster from M. stipitatus DSM 14675 using genome sequence analysis and mutational analysis. The cluster is comprised of 9 genes (MYSTI_04973 to MYSTI_04965) that encode 4 polyketide synthase modules, 3 non-ribosomal peptide synthase modules, a putative fumarylacetoacetate hydrolase, a putative S-adenosylmethionine-dependent methyltransferase, and a putative nitrilase. Disruption of the MYSTI_04972 or MYSTI_04973 gene by plasmid insertion resulted in defective melithiazol production. The organization of the melithiazol biosynthetic modules encoded by 8 genes from MYSTI_04972 to MYSTI_04965 was similar to that in M. lichenicola Me l46. However, the loading module encoded by the first gene (MYSTI_04973) was different from that of M. lichenicola Me l46, explaining the difference in the production of melithiazol derivatives between the M. lichenicola Me l46 and M. stipitatus strains.

Cyanide Degradation by Two Recombinant Cyanide Hydratases (Recombinant Cyanide Hydratases에 의한 시안화물 분해)

  • Kwon, Sung-Hyun;Cho, Dae-Chul
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.10 no.6
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    • pp.1287-1291
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    • 2009
  • The genes of cyanide hydratase(CHT), a kind of nitrilases whichhydrolyze cyanide to formamide were extracted from N. crassa and A. nidulans, the two fungal strains. The recombinant forms of the CHT originated from N. crassa and A. nidulans were prepared with N-terminal hexahistidine purificationtags or no tags, and expressed in E. coli. The enzymes were purified using immobilized metal affinity chromatography. They were compared according to their pH activity profiles, and kinetic parameters. The N. crassa CHT has the wider pH range of activity above 50% and three-fold higher turnover rate (6.6 ${\times}$ $10^8$ $min^{-1}$) than the A. nidulans, meanwhile the CHT of A. nidulans has the higher $K_m$ value. Expression of CHT in both N. crassa and A. nidulans were induced by the presence of KCN, regardless of any presence of nitrogen sources. Max. 82% of KCN was degraded in 60 min for biological degradation tests.

Characterization of Nitrile-hydrolyzing Enzymes Produced from Rhodococcus erythropolis (니트릴 분해효소 생산균인 Rhodococcus erythropolis의 발굴 및 효소 특성 연구)

  • Park Hyo-Jung;Park Ha-Joo;Uhm Ki-Nam;Kim Hyung-Kwoun
    • Microbiology and Biotechnology Letters
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    • v.34 no.3
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    • pp.204-210
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    • 2006
  • Ethyl (S)-4-chloro-3-hydroxybutyrate is a useful intermediate for the synthesis of Atorvastatin, a chiral drug to hypercholesterolemia. In this research, two 4-chloro-3-hydroxybutyro-nitrile-degrading strains were isolated from soil sample. They were identified as Rhodococcus erythropolis strains by 16S rRNA analysis. The nitrile-degrading enzyme(s) were suggested to be nitrile hydratase and amidase rather than nitrilase from the result of thin layer chromatography analysis. The corresponding genes were obtained by PCR cloning method. The predicted protein sequences had identities more than 96% with nitrile hydratase ${\alpha}-subunit$, nitrile hydratase ${\beta}-subunit$, and amidase of R. erythropolis. The 4-chloro-3-hydroxybutyronitrile-hydrolyzing activities in both strains were increased dramatically by ${\varepsilon}-caprolactam$ which was known as good inducer for nitrile hydratase. Both intact cells and cell-free extract could hydrolyze the nitrile compound. So, the intact cell and the enzymes could be used as potential biocatalyst for the production of 4-chloro-3-hydroxybutyric acid.

The Effect of Sodium Tungstate on the Aldehyde Oxidase and the Growth in the Primary Root of Maize (Zea mays) (옥수수 (Zea mays) 뿌리의 알데히드 산화효소와 생장에 미치는 텅스텐산 나트륨의 영향)

  • Oh, Young-Joo;Cho, Young-Jun;Park, Woong-June
    • Journal of Life Science
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    • v.17 no.7 s.87
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    • pp.990-995
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    • 2007
  • We tested the effect of sodium tungstate, which disturbs the molybdenum cofactor formation, on the activities of aldehyde oxidase(AO) and the growth of maize(Zea mays) primary roots. As reported in other plants, sodium tungstate inhibited AO also in the maize root concentration-dependently. The inhibitory effect of sodium tungstate was observed only when the inhibitor was applied to the living plants. Application of tungstate to the extracted protein did not show any effect. Western analysis revealed slightly decreased level of AO protein in the presence of tungstate, indicating a positive feedback of gene regulation by the product. We also tested the effects of tungstate on the root growth. The elongation of primary root and the development of lateral roots, which are sensitive to the absolute level of auxin, were decreased in the presence of sodium tungstate. However, the gravitropic curvature of the primary root, which is dependent on the relative amount of auxin at both sides, was unaffected. These data suggested the decrease of auxin biosynthesis by the application of tungstate. However, the level of free IAA was unaffected by tungstate application. We discuss the possible explanations for the observed results.