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. 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.

Streptomyces lydicus M01 Regulates Soil Microbial Community and Alleviates Foliar Disease Caused by Alternaria alternata on Cucumbers

“…Since this fungus is widely distributed in soil and most species are harmless, plant pathogenic species were not considered in the present study. Some species of Ascobolus were found on rotting Brassica stems (Buczacki and Wilkinson, 1992) and Thelonectria was reported to produce glycosidase inhibitors (Miyauchi et al, 2016), both genera were decreased in the presence of S. lydicus M01. Plants are tightly associated with complex microbial communities, modification of the plant microbiome play an important role to reduce the occurrence of plant disease, increase agricultural production, and reduce chemical inputs.…”

Metal-mediated synthesis of pyrrolines

“…2). Examples include the iminosugar nectrisine (1), 60 discovered as an immunomodulator; the iminosaccharide 2 that has glycosidase inhibitory activity; 15,61 and the 1-pyrroline 3 that has antihypertensive properties. 14 b-Triuoromethylated 1-pyrrolines (4-6) 62 are nitric oxide synthase inhibitors 63 and as such possess anti-infective, [64][65][66] antitumor, 67 and anti-inammatory activities.…”

“…Wang et al 75 constructed novel 1-fulleropyrrolines 24 using a cuprous bromide-catalyzed heteroannulation reaction of [60] fullerene with ketoxime acetates 23 (Scheme 8). The proposed mechanism begins with ketoxime N-O cleavage by Cu(I) to give an imino radical with formation of the C-C and C-N bonds (Scheme 9).…”

“…Synthesis of 1-pyrrolines by silver catalysis. Bai et al 22 reported a one-pot asymmetric Michael-type conjugate Zang and coworkers 108 used a novel three-component annulation reaction between [60]fullerene, sulfonylhydrazones 160 and nitriles 161 mediated by Ag(I) as a general entry to disubstituted [60]fullerene-fused pyrrolines 162 (Scheme 50). The reaction exhibits a broad substrate scope and excellent functional-group tolerance.…”

“…Coordination of the alkene 265 to A followed by carbopalladation would produce intermediate B. Aminopalladation and subsequent ring opening of A could generate intermediate C, which would furnish the bicyclic product 267 via a nal b-hydride elimination. Reoxidation of palladium by the Cu(II) completes the catalytic cycle.Peng et al reported an efficient Pd-catalyzed synthesis of the scarce N-unsubstituted 2-fulleropyrrolines 269 employing[60] fullerene and benzoyl hydrazone esters 268 (Scheme 89) 139. The reaction involves the use of 20 mol% of Pd(OAc) 2 and 3 equivalent of Cu(OAc) 2 as oxidant in a solvent mixture of chlorobenzene/TFA (10 : 1) at 120 C. The presence of TFA generates a highly electrophilic cationic Pd(II) species and provides better results.…”