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New Insights into the Biosynthesis of Fosfazinomycin.

ABSTRACT: The biosynthetic origin of a unique hydrazide moiety in the phosphonate natural product fosfazinomycin is unknown. This study presents the activities of five proteins encoded in its gene cluster. The flavin dependent oxygenase FzmM catalyses the oxidation of L-Asp to N-hydroxy-Asp. When FzmL is added, fumarate is produced in addition to nitrous acid. The adenylosuccinate lyase homolog FzmR eliminates acetylhydrazine from N-acetylhydrazinosuccinate, which in turn is the product of FzmQ-catalysed acetylation of hydrazinosuccinate. Collectively, these findings suggest a path to N-acetylhydrazine from L-Asp. The incorporation of nitrogen from L-Asp into fosfazinomycin was confirmed by isotope labelling studies. Installation of the N-terminal Val of fosfazinomycin is catalysed by FzmI in a Val-tRNA dependent process.

SUBMITTER: Huang Z

PROVIDER: S-EPMC5215806 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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New Insights into the Biosynthesis of Fosfazinomycin.

Huang Zedu Z Wang Kwo-Kwang Abraham KA van der Donk Wilfred A WA

Chemical science 20160506 8

The biosynthetic origin of a unique hydrazide moiety in the phosphonate natural product fosfazinomycin is unknown. This study presents the activities of five proteins encoded in its gene cluster. The flavin dependent oxygenase FzmM catalyses the oxidation of L-Asp to <i>N</i>-hydroxy-Asp. When FzmL is added, fumarate is produced in addition to nitrous acid. The adenylosuccinate lyase homolog FzmR eliminates acetylhydrazine from <i>N</i>-acetylhydrazinosuccinate, which in turn is the product of F ...[more]

PMID: 28070267

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Project description:BackgroundStreptomyces albus J1074 produces glycosylated antibiotics paulomycin A, B and E that derive from chorismate and contain an isothiocyanate residue in form of paulic acid. Paulomycins biosynthesis pathway involves two glycosyltransferases, three acyltransferases, enzymes required for paulic acid biosynthesis (in particular an aminotransferase and a sulfotransferase), and enzymes involved in the biosynthesis of two deoxysugar moieties: D-allose and L-paulomycose.ResultsInactivation of genes encoding enzymes involved in deoxysugar biosynthesis, paulic acid biosynthesis, deoxysugar transfer, and acyl moieties transfer has allowed the identification of several biosynthetic intermediates and shunt products, derived from paulomycin intermediates, and to propose a refined version of the paulomycin biosynthesis pathway. Furthermore, several novel bioactive derivatives of paulomycins carrying modifications in the L-paulomycose moiety have been generated by combinatorial biosynthesis using different plasmids that direct the biosynthesis of alternative deoxyhexoses.ConclusionsThe paulomycins biosynthesis pathway has been defined by inactivation of genes encoding glycosyltransferases, acyltransferases and enzymes involved in paulic acid and L-paulomycose biosynthesis. These experiments have allowed the assignment of each of these genes to specific paulomycin biosynthesis steps based on characterization of products accumulated by the corresponding mutant strains. In addition, novel derivatives of paulomycin A and B containing L-paulomycose modified moieties were generated by combinatorial biosynthesis. The production of such derivatives shows that L-paulomycosyl glycosyltransferase Plm12 possesses a certain degree of flexibility for the transfer of different deoxysugars. In addition, the pyruvate dehydrogenase system form by Plm8 and Plm9 is also flexible to catalyze the attachment of a two-carbon side chain, derived from pyruvate, into both 2,6-dideoxyhexoses and 2,3,6-trideoxyhexoses. The activity of the novel paulomycin derivatives carrying modifications in the L-paulomycose moiety is lower than the original compounds pointing to some interesting structure-activity relationships.

Project description:BackgroundGypenosides are a group of triterpene saponins from Gynostemma pentaphyllum that are the same as or very similar to ginsenosides from the Panax species. Several enzymes involved in ginsenoside biosynthesis have been characterized, which provide important clues for elucidating the gypenoside biosynthetic pathway. We suppose that gypenosides and ginsenosides may have a similar biosynthetic mechanism and that the corresponding enzymes in the two pathways may have considerable similarity in their sequences. To further understand gypenoside biosynthesis, we sequenced the G. pentaphyllum transcriptome with a hybrid sequencing-based strategy and then determined the candidate genes involved in this pathway using phylogenetic tree construction and gene expression analysis.ResultsFollowing the PacBio standard analysis pipeline, 66,046 polished consensus sequences were obtained, while Illumina data were assembled into 140,601 unigenes with Trinity software. Then, these output sequences from the two analytical routes were merged. After removing redundant data with CD-HIT software, a total of 140,157 final unigenes were obtained. After functional annotation, five 2,3-oxidosqualene cyclase genes, 145 cytochrome P450 genes and 254 UDP-glycosyltransferase genes were selected for the screening of genes involved in gypenoside biosynthesis. Using phylogenetic analysis, several genes were divided into the same subfamilies or closely related evolutionary branches with characterized enzymes involved in ginsenoside biosynthesis. Using real-time PCR technology, their expression patterns were investigated in different tissues and at different times after methyl jasmonate induction. Since the genes in the same biosynthetic pathway are generally coexpressed, we speculated that GpOSC1, GpCYP89, and GpUGT35 were the leading candidates for gypenoside biosynthesis. In addition, six GpWRKYs and one GpbHLH might play a possible role in regulating gypenoside biosynthesis.ConclusionsWe developed a hybrid sequencing strategy to obtain longer length transcriptomes with increased accuracy, which will greatly contribute to downstream gene screening and characterization, thus improving our ability to elucidate secondary metabolite biosynthetic pathways. With this strategy, we found several candidate genes that may be involved in gypenoside biosynthesis, which laid an important foundation for the elucidation of this biosynthetic pathway, thus greatly contributing to further research in metabolic regulation, synthetic biology and molecular breeding in this species.

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New Insights into the Biosynthesis of Fosfazinomycin.

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New Insights into the Biosynthesis of Fosfazinomycin.

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