Recipient Organization
NORTHERN REGIONAL RES CENTER
(N/A)
PEORIA,IL 61604
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
Task 1. To investigate the biosynthetic pathway for apramycin by the producing strain(s) (S. tenebrarius and S. hindustanus) using isotopic labeling coupled with mass spectrometry and nuclear magnetic resonance (NMR) techniques. The aim is to identify biosynthetic intermediates as lead compounds for chemical synthesis and for antibiotic/ototoxicity testing. Task 2. To produce apramycin biosynthetic intermediates by microbial fermentation for antibiotic and ototoxicity testing by the collaborator.
Project Methods
The scientific approach requires: 1. The chemical synthesis of modified apramycins and apramycin analogs; 2. The identification of apramycin biosynthetic intermediates produced by apramycin-producing bacterial strains; and 3. Ototoxicity and antibiotic testing of chemically modified apramycins and apramycin biosynthetic intermediates. ARS part will be small-scale culturing (50 ml scale) of apramycin-producing Streptomyces strains with isotopically-enriched (non-radioactive stable isotope 13-carbon label) precursor molecules apramycins will be recovered from the spent culture supernatants using ion exchange resin, and the positions of isotopic incorporation will be assessed by mass spectrometry and nuclear magnetic resonance (NMR) using techniques developed previously. Briefly, the total number of isotopic incorporations into apramycin(s) will be assessed by matrix-assisted laser desorption/ionazation time-of-flight (MALDI-TOF) mass spectrometry. The positional incorporation of these isotopes is then determined in two ways. First, by acid-hydrolysis of the labeled apramycin(s) to simpler degradation products. These products are then analyzed (after suitable derivatization) by gas chromatography-electron impact ionization mass spectrometry (GC-EI-MS). The EI-MS technique fragments the labeled products giving small mass ions, which can then be mapped backed to the parent compound to reveal which atoms are 13C (or 15N) enriched. Next we will use heteronuclear single quantum correlation 2D-NMR (HSQC-NMR) to localize and quantify 13C-enrichment into labeled apramycin, using the technique as described previously. A 500 MHz NMR, MALDI-TOF mass spectrometer, and GC-MS equipment are all already available at National Center for Agricultural Utilization Research (NCAUR). The second part to be undertaken at the ARS lab will be to scale-up the production of apramycin biosynthetic intermediates by fermentation of identified Streptomyces strains. The aim is to provide antibacterial-active intermediates from the pathway to collaborator¿s laboratory for ototoxicity testing. How the structural-activity of these intermediates relates to ototoxicity and antibiotic activity a major aim of the project.