Saccharopolyspora Species: Laboratory Maintenance and Enhanced Production of Secondary Metabolites

Dipesh Dhakal1, Anaya Raj Pokhrel1, Amit Kumar Jha1, Nguyen Huy Thuan2, Jae Kyung Sohng3

1 Department of Life Science and Biochemical Engineering, Sun Moon University, Chungnam, 2 Center for Molecular Biology, Institute of Research and Development, Duy Tan University, Danang, 3 Department of BT‐Convergent Pharmaceutical Engineering, Sun Moon University, Chungnam
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10H.1
DOI:  10.1002/cpmc.21
Online Posting Date:  February, 2017
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Abstract

Saccharopolyspora spp. are aerobic, Gram‐positive, non‐acid‐fast, and non‐motile actinomycetes. Various species of the genus Saccharopolyspora have been reported with an ability to produce various bioactive compounds for pharmaceutical and agricultural uses. This unit includes general protocols for the laboratory maintenance of Saccharopolyspora species, including growth in liquid medium, growth on solid agar, long‐term storage, and generation of a higher producer strain by mutagenesis. Saccharopolyspora spinosa ATCC 49460 is used as a prototype for explaining the considerations for efficient laboratory maintenance of Saccharopolyspora spp. Saccharopolyspora spinosa is a producer of spinosad, a prominent insecticide with selective activity against various insects. © 2017 by John Wiley & Sons, Inc.

Keywords: Saccharopolyspora ; medium; spinosad; storage; UV mutagenesis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Growth of Saccharopolyspora on Solid Medium
  • Basic Protocol 2: Growth of Saccharopolyspora in Liquid Medium
  • Basic Protocol 3: Long‐Term Storage
  • Basic Protocol 4: Production of Secondary Metabolites (Spinosads) from Saccharopolyspora Spinosa
  • Basic Protocol 5: Generation of a Higher Producer Strain of Secondary Metabolite (Spinosad) by UV Mutagenesis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Growth of Saccharopolyspora on Solid Medium

  Materials
  • Tryptic soy broth (TSB) solid medium (see recipe)
  • Saccharopolyspora spinosa ATCC 49460 glycerol stock
  • 100 15‐mm petri dishes
  • Sterile inoculation needle or wooden stick
  • 28°C incubator

Basic Protocol 2: Growth of Saccharopolyspora in Liquid Medium

  Materials
  • Tryptic soy broth (TSB) liquid medium (see recipe)
  • Saccharopolyspora spinosa ATCC 49460 on solid medium ( protocol 1) or glycerol stock ( protocol 3)
  • 250‐ml baffled culture flask, sterile (glass or plastic with vented caps)
  • Toothpicks, sterile
  • Sterile cotton
  • Incubator or shaker apparatus
  • Light microscope

Basic Protocol 3: Long‐Term Storage

  Materials
  • Saccharopolyspora spinosa ATCC 49460 liquid culture
  • Sterile glycerol
  • 1.5‐ml microcentrifuge tube
  • 1‐ml pipet
  • Pipettors
  • −80°C freezer

Basic Protocol 4: Production of Secondary Metabolites (Spinosads) from Saccharopolyspora Spinosa

  Materials
  • GYSC medium (see recipe)
  • Different Saccharopolyspora spinosa liquid culture in tryptic soy broth (TSB) medium
  • C18‐reversed phase HPLC column (4.6 × 250 mm, 50 μm; KANTO Reagents)
  • Acetonitrile
  • Methanol
  • 2% ammonium acetate buffer
  • 500‐ml baffled culture flask, sterile (glass or plastic with vented caps)
  • Sterilized 1‐ml pipettor tips
  • Pipettors
  • Sterile cotton
  • Incubator or shaker apparatus
  • 0.22‐μm syringe filter

Basic Protocol 5: Generation of a Higher Producer Strain of Secondary Metabolite (Spinosad) by UV Mutagenesis

  Materials
  • GYSC medium (see recipe)
  • Saccharopolyspora spinosa ATCC 49460 liquid culture in tryptic soy broth (TSB) medium ( protocol 2)
  • 500‐ml baffled culture flask, sterile (glass or plastic with vented caps)
  • Sterilized 1‐ml pipettor tips
  • Pipettors
  • Sterile cotton
  • Incubator or shaker apparatus
  • Sterile inoculation loop
  • Aluminum foil
  • UV lamp (λ max 254 nm and 40 W)
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Figures

Videos

Literature Cited

    
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Internet Resources
   http://www.bacterio.net/index.html
  Website is a valuable resource for anyone with an interest in prokaryotic nomenclature.
   http://www.bacterio.net/saccharopolyspora.html
  Website provides comprehensive information about different Saccharopolyspora species.
   http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
  Website provides guidelines about the critical considerations for biosafety in microbiological and biomedical laboratories.
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