Characterization of a novel oxidase from Thelonectria discophora SANK 18292 involved in nectrisine biosynthesis

Miyauchi, Ryuki; Sakurai, Hidetaka; Shiba, Yoichiro

doi:10.1186/s13568-016-0176-1

Cited by 2 publications

(8 citation statements)

References 35 publications

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“…Since NecC is an alcohol oxidase, 4-amino-4-deoxyarabinitol is considered to be first converted to an aldehyde (compound 3) with intramolecular addition of the amino group to the carbonyl group, resulting in a cyclic compound (compound 4) that could be spontaneously dehydrated (without an enzymatic reaction) to afford nectrisine (20). Our results also indicated that necA, encoding a putative aminotransferase, is involved in 4-amino-4-deoxyarabinitol biosynthesis.…”

Section: Discussionmentioning

confidence: 52%

“…A cosmid library of T. discophora genomic DNA was constructed and necCpositive cells were screened as described previously (20). Briefly, the cosmid library was constructed using SuperCos1 cosmid vector (Agilent Technologies), with E. coli XL1-Blue MR as the host strain.…”

Section: Methodsmentioning

confidence: 99%

“…To detect and to efficiently separate nectrisine and 4-amino-4-deoxyarabinitol by high-performance liquid chromatography (HPLC), samples were reduced with NaBH 4 and derivatized with 4-fluoro-7-nitrobenzofurazan (NBD-F) (Dojindo Laboratories, Kumamoto, Japan) (26,27); liquid chromatography (LC)-photodiode array (PDA)-positive electrospray ionization (ESI) mass spectrometry was performed as described previously (20). The peak areas of the PDA signal at 500 nm for the samples and the nectrisine standard were compared to quantify the concentrations.…”

Section: Construction Of Complementation Vectorsmentioning

confidence: 99%

“…Total RNA was extracted from ground cells of T. discophora with RNeasy Plant kits (Qiagen) and was reverse transcribed with ReverTraAce-␣-reverse transcriptase and oligo(dT) 20 primer (Toyobo, Osaka, Japan), according to the supplier's instructions. Then, necB cDNA was amplified by PCR with the forward and reverse primers BamHI-AK-f and AK-HindIII-r, respectively.…”

Section: Construction Of Complementation Vectorsmentioning

confidence: 99%

“…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. Furthermore, the other genes required for nectrisine biosynthesis remain unclear.…”

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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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Section: Discussionmentioning

confidence: 52%

Section: Methodsmentioning

confidence: 99%

Section: Construction Of Complementation Vectorsmentioning

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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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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Characterization of a novel oxidase from Thelonectria discophora SANK 18292 involved in nectrisine biosynthesis

Cited by 2 publications

References 35 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

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

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