title: Metabolomics-guided analysis of isocoumarin production byStreptomycesspecies MBT76 and biotransformation of flavonoids and phenylpropanoids.
abstract: BACKGROUND
  Actinomycetes produce the majority of the antibiotics currently in clinical use. The efficiency of antibiotic production is affected by multiple factors such as nutrients, pH, temperature and growth phase. Finding the optimal harvesting time is crucial for successful isolation of the desired bioactive metabolites from actinomycetes, but for this conventional chemical analysis has limitations due to the metabolic complexity.
  
  
  OBJECTIVE
  This study explores the utility of NMR-based metabolomics for (1) optimizing fermentation time for the production of known and/or unknown bioactive compounds produced by actinomycetes; (2) elucidating the biosynthetic pathway for microbial natural products; and (3) facilitating the biotransformation of nature-abundant chemicals.
  
  
  METHODS
  The aqueous culture broth of actinomyceteStreptomycessp. MBT76 was harvested every 24 h for 5 days and each broth was extracted by ethyl acetate. The extracts were analyzed by1H NMR spectroscopy and the data were compared with principal component analysis (PCA) and orthogonal projection to latent structures (OPLS) analysis. Antimicrobial test were performed by agar diffusion assay.
  
  
  RESULTS
  The secondary metabolites production byStreptomycessp. MBT76 was growth phase-dependent. Isocoumarins (1-9), undecylprodiginine (10), streptorubin B (11), 1H-pyrrole-2-carboxamide (12), acetyltryptamine (13), and fervenulin (14) were identified, and their optimal production time was determined in crude extracts without tedious chromatographic fractionation. Of these compounds, 5,6,7,8-tetramethoxyl-3-methyl-isocoumarin (9) is as a novel compound, which was most likely synthesized by a type I iterative polyketide synthase (PKS) encoded by theicmgene cluster. Multivariate data analysis of the1H NMR spectra showed that acetyltryptamine (13) andtri-methoxylated isocoumarins (7and8) were the major determinants of antibiotic activity during later time points. The methoxylation was exploited to allow bioconversion of exogenously added genistein into a suite of methoxylated isoflavones (15-18). Methoxylation increased the antimicrobial efficacy of isocoumarins, but decreased that of the isoflavones.
  
  
  CONCLUSIONS
  Our results show the applicability of NMR-based metabolic profiling to streamline microbial biotransformation and to determine the optimal harvesting time of actinomycetes for antibiotic production.
authors.affiliation: Molecular Biotechnology, Institute of Biology, Leiden University, Sylviusweg, 72, 2333 BE Leiden, The Netherlands.|Natural Products Laboratory, Institute of Biology, Leiden University, Sylviusweg, 72, 2333 BE Leiden, The Netherlands.
authors.forename: Changsheng
authors.initials: C
authors.lastname: Wu
authors.affiliation: Molecular Biotechnology, Institute of Biology, Leiden University, Sylviusweg, 72, 2333 BE Leiden, The Netherlands.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Molecular Biotechnology, Institute of Biology, Leiden University, Sylviusweg, 72, 2333 BE Leiden, The Netherlands.
authors.forename: Gilles P
authors.initials: GP
authors.lastname: van Wezel
authors.affiliation: Natural Products Laboratory, Institute of Biology, Leiden University, Sylviusweg, 72, 2333 BE Leiden, The Netherlands.
authors.forename: Young Hae
authors.initials: YH
authors.lastname: Choi
keywords: Antibiotics; Biotransformation; Growth phase-dependence; NMR-based metabolomics; Streptomyces