Isolation

1
Isolation Characterization of Polyketide
Synthase Thioestherase DomainsEvelyn Walenta
Christopher N. BoddyDepartment of Chemistry,
Syracuse UniversitySyracuse, NY 13244
Polyketides are important clinical agents
Amphotericin TE Protein Expression
Project Goal Characterize multiple TE Domains

• Amphotericine, Nystatin, Avermectin, Oleandomycin
  TE domains were selected

• BL21 (CDE3) were used for protein expression)
• Time course for protein expression at 30C shows
  10h to be optimal
• Amphotericin TE was purified by Ni-NTA
  chromatography
• 4 ml of 22 µM high purity Amphotericin TE was
  obtained from 0.5 l of culture

Amphotericin
Nystatin

• TE domains were sequenced
• bacterial strains were commercially available
• Polyketide products were commercially available

Why?
Avermctin
Oleandomycin
6.5
5.5

• large family of structurally diverse and natural
  products produced by bacteria and fungi

Generation of TE expression vector
Activity of Amphotericin TE
Polyketides are made in an assembly-line fashion

• Amphotericin TE does not catalyze lactone
  hydrolosys

producing organisms
isolate genomic DNA
grow culture
TE domain
PCR
LCMS analysis shows no hydrolysis activity
clone into expression vecoor
950

• Thioester Domain can hydrolyze simple Thioesters

TE domain
PCR products for all 4 TE domains were generated
Expression vector for Amphotericin TE was
generated

• Enormous multifunktional enzymes
• Growing polyketide is covalently attached to
  enzyme
• Complex mulecules are made from simple starting
  materials
• Different organisation of modules gives different
  polyketides

LCMS analysis shows hydrolysis activity

Acknowledgements
Release of Polyketide from enzyme in crucial
Ben Lundgren (Syracuse University) Mike Henry
(Syracuse University) The Boddy Lab (Syracuse
University) Syracuse University University of
Technology (Graz)
cyclorelease
- HSR
R ACP or N-Acetulcysteamine

• Thioester domain catalyzes the release of the
  polyketide via cyclization