A One-Pot Chemoenzymatic Synthesis for the Universal Precursor of Antidiabetes and Antiviral Bis-Indolylquinones

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.

Section: Discussionsupporting

confidence: 72%

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

Miyauchi

Ono

Ohnuki

et al. 2016

“…Mycelia were filtered under vacuum, washed with distilled H 2 O (dH 2 O), and ground by mortar and pestle under liquid nitrogen before total RNA extraction using the SV Total RNA Isolation System (Promega) by following the manufacturer's spin protocol. First-strand synthesis of armH1 and armH2 was accomplished with the oligonucleotide primer cHalR2 (Table 1) and ImProm-II reverse transcriptase, using previously described parameters (24). Using 1 l of the first-strand synthesis reaction mixture as the template, the armH1 cDNA was amplified by PCR using Pfu polymerase (94°C for 2 min; 40 cycles of 94°C for 40 s, 58°C for 30 s, and 72°C for 4 min; final extension at 72°C for 10 min) and oligonucleotide primers cHalF1 and cHalR1 ( Table 1).…”

Section: Methodsmentioning

confidence: 99%

A Fivefold Parallelized Biosynthetic Process Secures Chlorination of Armillaria mellea (Honey Mushroom) Toxins

Wick

Heine

Lackner

et al. 2016

Self Cite

The basidiomycetous tree pathogen Armillaria mellea (honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemical in vitro characterization of five flavin-dependent A. mellea enzymes (ArmH1 to ArmH5) that were heterologously produced in Escherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A 5-fold, secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier-protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share an ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.

“…The reactions (100 l) were incubated at 37°C (varied between 4 and 45°C to determine the temperature optimum) and consisted of Tris buffer (pH 7.5 [varied from pH 5.0 to 8.0 to determine the optimum pH]), 5 mM MgCl 2 , 125 nM EDTA, 5 mM ATP, 100 nM purified protein, 0.1 M [ 32 P]pyrophosphate, and 1 mM substrate (fumaric acid, L-phenylalanine, L-tyrosine). The reactions proceeded for 1 h before they were stopped and further processed as described previously (14). Exchange of pyrophosphate was quantified using a Perkin-Elmer TriCarb 2910TR scintillation counter.…”

Section: Methodsmentioning

confidence: 99%

Genetic Engineering Activates Biosynthesis of Aromatic Fumaric Acid Amides in the Human Pathogen Aspergillus fumigatus

Kalb

Heinekamp

Lackner

et al. 2015

Self Cite

The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro. Fumaric acid was identified as preferred substrate of the first and both L-tyrosine and L-phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR, encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl-L-tyrosine and fumaryl-L-phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds.