Biosynthesis of nectrisine in Thelonectria discophora SANK 18292

Miyauchi, Ryuki; Takatsu, Toshio; Suzuki, Tetsuya; Ono, Yasunori; Shiba, Yoichiro

doi:10.1016/j.phytochem.2015.03.011

Cited by 4 publications

(14 citation statements)

References 33 publications

(43 reference statements)

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“…1), also known as FR-900483 (10), is an iminosugar that has a five-membered nitrogen-containing ring and shows inhibitory activity against ␣-and ␤-glucosidases, ␣-and ␤-mannosidases, ␤-N-acetylglucosaminidase, and other glycosidases (11). Nectrisine can be biologically produced by Nectria lucida F-4490 (12), which is synonymous with Thelonectria lucida, as described by Chaverri et al (13), and Thelonectria discophora SANK 18292 (JCM 30947), as determined in our screening program for nectrisine-producing organisms (14). Nectrisine, having three stereogenic centers, can also be chemically synthesized from chiral starting materials, such as D-arabinose or D-serine (15)(16)(17)(18)(19).…”

supporting

confidence: 62%

“…ORF3 showed homology to choline/ethanol amine kinases. Our previous study suggested that, besides oxidation by NecC, amination and dephosphorylation should be required for nectrisine biosynthesis (14). Thus, in addition to ORF5 (necC), ORF2 and ORF3, designated necA and necB, respectively, were expected to be genes potentially required for nectrisine biosynthesis.…”

Section: Resultsmentioning

confidence: 99%

“…T. discophora SANK 18292, deposited in the Japan Collection of Microorganisms (Tsukuba, Japan) under accession number JCM 30947, was used in this study. The spore suspension was inoculated into A1 medium (14) and incubated for 5 days at 23°C on a rotary shaker. Then glucose-potato-yeast extract-calcium carbonate no.…”

Section: Methodsmentioning

confidence: 99%

“…His-tagged proteins were detected with Penta-His-horseradish peroxidase (HRP) conjugate (Qiagen) and visualized with ECL Prime Western blotting detection reagent (GE Healthcare). A supernatant of the culture was reduced and labeled with NBD-F, and then nectrisine in the solution was quantified by HPLC as described previously (14).…”

Section: Construction Of Complementation Vectorsmentioning

confidence: 99%

“…In order to begin to identify enzymes or genes required for nectrisine biosynthesis, the biosynthetic pathway in T. discophora was studied using stable isotope labeling techniques, with 4-amino-4-deoxyarabinitol (compound 2) ( Fig. 1) being identified as a potential late intermediate (14). Moreover, a potential enzyme, designated NecC, that catalyzes the conversion of 4-amino-4-deoxyarabinitol to nectrisine was purified from the fungus and showed characteristics of glucose-methanol-choline oxidoreductase (20); however, it was not verified that the necC gene was involved in nectrisine biosynthesis.…”

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confidence: 99%

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Nectrisine Biosynthesis Genes in Thelonectria discophora SANK 18292: Identification and Functional Analysis

Miyauchi

Ono

Ohnuki

et al. 2016

Appl Environ Microbiol

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The fungus Thelonectria discophora SANK 18292 produces the iminosugar nectrisine, which has a nitrogen-containing heterocyclic 5-membered ring and acts as a glycosidase inhibitor. In our previous study, an oxidase (designated NecC) that converts 4-amino-4-deoxyarabinitol to nectrisine was purified from T. discophora cultures. However, the genes required for nectrisine biosynthesis remained unclear. In this study, the nectrisine biosynthetic gene cluster in T. discophora was identified from the contiguous genome sequence around the necC gene. Gene disruption and complementation studies and heterologous expression of the gene showed that necA, necB, and necC could be involved in nectrisine biosynthesis, during which amination, dephosphorylation, and oxidation occur. It was also demonstrated that nectrisine could be produced by recombinant Escherichia coli coexpressing the necA, necB, and necC genes. These findings provide the foundation to develop a bacterial production system for nectrisine or its intermediates through genetic engineering. IMPORTANCEIminosugars might have great therapeutic potential for treatment of many diseases. However, information on the genes for their biosynthesis is limited. In this study, we report the identification of genes required for biosynthesis of the iminosugar nectrisine in Thelonectria discophora SANK 18292, which was verified by disruption, complementation, and heterologous expression of the genes involved. We also demonstrate heterologous production of nectrisine by recombinant E. coli, toward developing an efficient production system for nectrisine or its intermediates through genetic engineering.

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supporting

confidence: 62%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Construction Of Complementation Vectorsmentioning

confidence: 99%

mentioning

confidence: 99%

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Nectrisine Biosynthesis Genes in Thelonectria discophora SANK 18292: Identification and Functional Analysis

Miyauchi

Ono

Ohnuki

et al. 2016

Appl Environ Microbiol

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The genus Thelonectria (Nectriaceae, Hypocreales, Ascomycota) and closely related species with cylindrocarpon-like asexual states

2016

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Functional Analysis of a Gene Cluster from Chitinophaga pinensis Involved in Biosynthesis of the Pyrrolidine Azasugar DAB-1

Nuñez

Horenstein

2019

J. Nat. Prod.

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Azasugars, "nitrogen in the ring" analogues of monosaccharides, are known to be distributed in select plant, fungal. and bacterial species. We identify Chitinophaga pinensis DSM 2588 as the first bacterial source of the plant pyrrolidine azasugar 1,4-dideoxy-1,4-aminoarabinitol (DAB-1). Comparative sequence analyses identified C. pinensis as a putative azasugar producer, via observation of a three-gene cluster coding for putative aminotransferase, alcohol dehydrogenase, and sugar phosphatase enzymes, similar to the previously reported azasugar biosynthetic signature identified in Bacillus amyloliquefaciens FZB42. Multistep fractionation of C. pinensis culture media guided by a maltase inhibition assay yielded a component with a mass consistent with the structure of DAB-1. Heterologous expression of the three-gene cluster in E. coli, a non-azasugar producer, led to the isolation of nectrisine, a biosynthetic precursor to DAB-1, which displayed potent slow tight binding inhibition of maltase. Reduction of nectrisine with NaBH 4 removed the slow tight binding inhibition kinetics, and MS analysis provided evidence for the production of a compound matching that of the isolated DAB-1 from C. pinensis. 1 H NMR analysis of the nectrisine produced in E. coli after NaBD 4 reduction produced a spectrum consistent with DAB-1 deuterated at C-1, primarily at the pro-S position. These results support the idea that the azasugar three-gene cluster represents a general biosynthetic path leading to several different compounds, which may prove useful for the identification of other azasugarproducing organisms.

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Biosynthesis of nectrisine in Thelonectria discophora SANK 18292

Cited by 4 publications

References 33 publications

Nectrisine Biosynthesis Genes in Thelonectria discophora SANK 18292: Identification and Functional Analysis

Nectrisine Biosynthesis Genes in Thelonectria discophora SANK 18292: Identification and Functional Analysis

The genus Thelonectria (Nectriaceae, Hypocreales, Ascomycota) and closely related species with cylindrocarpon-like asexual states

Functional Analysis of a Gene Cluster from Chitinophaga pinensis Involved in Biosynthesis of the Pyrrolidine Azasugar DAB-1

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