New Method For Identification and Expression of gene involved in Polyketide Biosynthesis

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New Method For Identification and Expression of
gene involved in Polyketide Biosynthesis
Michael R. Henry and Christopher N. Boddy
Department of Chemistry Syracuse
University Syracuse, NY 13244
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Modular Polyketide Synthases

• Assembly line production of complex molecules
• Similar mechanism to Fatty Acid Synthesis- not
  fixed order sequence
• Each protein contains many catalytically active
  Domains

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What are Polyketides?

• Secondary Metabolites produced by microorganisms
• Natural products with remarkable diversity in
  structure and function
• Potential for Anticancer, Antibiotic and
  Immunosuppressant agents

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Myxococcus Xanthus

• Under environmental stress forms fruiting bodies
• Can express polyketide synthase pathways from
  other myxobacteria
• High levels of homologous recombination and
  polyketide production

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Homologous Recombination

• Replacement of single gene in Myxococcus Xanthus
  with constructed Asg E Locus gene
• Asg E Locus composed of polyketide synthase genes
  for different antibiotic compounds
• Incorporate large amounts of DNA into genome

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Polymerase Chain Reaction

• Using PCR able to extract Asg E locus from M.
  Xanthus genomic DNA
• Digestion with restriction enzymes isolated the
  Asg E locus for ligation

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Transformation and Ligation

• Transformation of Asg E into E.coli strain
  creates stockpile of cosmid DNA
• Ligation of Asg E gene with vector creates cosmid
  system for insertion into M. xanthus and
  identifying PKS

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Oxytetracycline Trial Run

• Antibiotic produced by Streptomyces via
  Polyketide synthase
• Insertion of oxytetracycline gene cluster into
  M.xanthus to see if production occurs

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Future of Polyketide Synthesis

• Endless possibilities of products that can be
  cost efficient

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Acknowledgements

• Dr. Anthony Garza Syracuse University
• Dr. Yi Tang UCLA
• Dr. Roy Welch Syracuse University
• Ben Lundgren
• Steve Houghton