Project description: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.
Project description:A fungus, Thelonectria discophora SANK 18292 (JCM 30947), produces nectrisine that has a nitrogen-containing heterocyclic 5-membered ring acting as a glycosidase inhibitor. Our previous study showed the possibility that 4-amino-4-deoxyarabinitol was enzymatically converted to nectrisine but the enzyme was not known. In order to characterize the enzyme, which is designated as NecC, it was purified from the fungus using ammonium sulfate precipitation and anion exchange chromatography. Liquid chromatography-tandem mass spectrometry analysis of NecC tryptic digests revealed partial NecC protein sequences. Subsequently, the partial DNA fragments were amplified by polymerase chain reaction with degenerate oligonucleotide primers and cloned. Then, necC complete genomic DNA was cloned by screening a genomic library of the fungus. Recombinant NecC also had NecC enzymatic activity, thus providing verification for the necC gene. NecC presumably belonged to the family of glucose methanol choline oxidoreductases, forming oligomers ranging approximately from 8 mer to 16 mer based on the results of native PAGE, and was also found to have a melting temperature of 57 °C, an optimal reaction condition of pH 7 at 30 °C, an activity inhibited by Cu(2+) or ethylenediaminetetraacetic acid, and 4-amino-4-deoxyarabinitol as its preferred substrate. It was also indicated that not nectrisine but 4-amino-4-deoxyarabinitol was mainly extracted from the mycelium, and then was converted to nectrisine by the enzyme NecC in vitro. We believe that these findings are helpful to establish a nectrisine manufacturing process at large scale with the fungus.
Project description:The distribution of microbial species, including fungi, has long been considered cosmopolitan. Recently, this perception has been challenged by molecular studies in historical biogeography, phylogeny and population genetics. Here we explore this issue using the fungal morphological species Thelonectria discophora, one of the most common species of fungi in the family Nectriaceae, encountered in almost all geographic regions and considered as a cosmopolitan taxon. In order to determine if T. discophora is a single cosmopolitan species or an assemblage of sibling species, we conducted various phylogenetic analyses, including standard gene concatenation, Bayesian concordance methods, and coalescent-based species tree reconstruction on isolates collected from a wide geographic range. Results show that diversity among isolates referred as T. discophora is greatly underestimated and that it represents a species complex. Within this complex, sixteen distinct highly supported lineages were recovered, each of which has a restricted geographic distribution and ecology. The taxonomic status of isolates regarded as T. discophora is reconsidered, and the assumed cosmopolitan distribution of this species is rejected. We discuss how assumptions about geographically widespread species have implications regarding their taxonomy, true diversity, biological diversity conservation, and ecological functions.
Project description:Our project provides long and short read genome sequences, assembly and functional predictions for Penicillium sclerotiorum SNB-CN111 and Neonectria discophora SNB-CN63. Penicillium sclerotiorum was isolated from Nasutitermes similis from French Guiana. Neonectria discophora was isolated from Nasutitermes corniger from French Guiana.