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

Abstract

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.

Importance: Iminosugars 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.

FIG 1

Structures of nectrisine (compound 1) and 4-amino-4-deoxyarabinitol (compound 2).

FIG 2

Nectrisine biosynthetic gene cluster. The restriction endonuclease map, inserted positions of cosmid RB185, cosmid RB246, and plasmid RB237, and locations of genes are shown. The arrows indicate the putative direction of transcription, based on sequence analysis and homology searches. Black arrows indicate the biosynthesis genes. B, BamHI.

FIG 3

Disruption and complementation of necC. (A) Predicted disruption and complementation events caused by homologous recombination with the XhoI-digested plasmids pNEC001disnecC and pNEC002compnecC. The deduced restriction patterns for genomic DNA from the parent strain (top), a necC disruptant (ΔnecC) (middle), and a necC-complemented mutant (CompnecC) (bottom) are shown. Bars and white arrows indicate necC genes. necC fragments from a plasmid (pNEC001disnecC) are shown as black bars. Bg, BglII; Sp, SphI; Ba, BamHI. (B) Genomic Southern hybridization patterns with DIG-labeled necC for genomic DNA from the parent strain, the necC disruptant, and the necC-complemented mutant, digested with the indicated enzymes. (C) HPLC chromatograms (left) and ESI mass spectra (right) of reduced and NBD-labeled authentic nectrisine, authentic 4-amino-4-deoxyarabinitol, and metabolites produced by the parent strain, the necC disruptant, and the necC-complemented mutant.

FIG 4

Disruption and complementation of necA. (A) Predicted disruption and complementation events caused by homologous recombination with the XhoI-digested plasmids pNEC001disnecA and pNEC002compnecA. The deduced restriction patterns for genomic DNA from the parent strain (top), a necA disruptant (ΔnecA) (middle), and a necA-complemented mutant (CompnecA) (bottom) are shown. Bars and white arrows indicate necA genes. necA fragments from a plasmid (pNEC001disnecA) are shown as black bars. Sc, ScaI; H, HindIII; Ba, BamHI. (B) Genomic Southern hybridization patterns with DIG-labeled necA for genomic DNA from the parent strain, the necA disruptant, and the necA-complemented mutant, digested with the indicated enzymes. (C) HPLC chromatograms (left) and ESI mass spectrum (right) of reduced and NBD-labeled metabolites produced by the parent strain, the necA disruptant, and the necA-complemented mutant.

FIG 5

Disruption of necB. (A) Predicted disruption events caused by homologous recombination with the XhoI-digested plasmid pNEC001disnecB. The deduced restriction patterns for genomic DNA from the parent strain (top) and a necB disruptant (ΔnecB) (bottom) are shown. Bars and white arrows indicate necB genes. necB fragments from a plasmid (pNEC001disnecB) are shown as black bars. H, HindIII; Sc, ScaI; Ba, BamHI. (B) Genomic Southern hybridization patterns with DIG-labeled necB for genomic DNA from the parent strain and the necB disruptant, digested with the indicated enzymes. (C) HPLC chromatograms (left) and ESI mass spectrum (right) of reduced and NBD-labeled metabolites produced by the parent strain and the necB disruptant.

FIG 6

Coexpression of necA, necB, and necC by recombinant E. coli. (A) Western blot of soluble fractions probed with anti-His antibodies. Lane 1, molecular weight markers; lane 2, E. coli BL21(DE3) harboring the plasmid pETDuetnecCnecA; lane 3, E. coli BL21(DE3) harboring the plasmids pETDuetnecCnecA and pACYCDuetnecB. (B) HPLC chromatograms (left) and ESI mass spectra (right) of reduced and NBD-labeled supernatants of the E. coli cultures. (i) Reduced and NBD-labeled authentic nectrisine, for which the calculated molecular mass ([M+H]) is 297.08. (ii) E. coli BL21(DE3) harboring the plasmid pETDuetnecCnecA. (iii) E. coli BL21(DE3) harboring the plasmids pETDuetnecCnecA and pACYCDuetnecB.

FIG 7

Proposed pathway for nectrisine biosynthesis in T. discophora.