title: [Functional characterization of gouC and gouD in gougerotin biosynthesis].
abstract: OBJECTIVE
  To determine the functions of gouC and gouD in gougerotin biosynthesis, disruption of these two genes was performed. As gougerotin producing strain Streptomyces graminearus lacks efficient genetic manipulation system, the gene cluster for gougerotin biosynthesis was heterologously expressed in Streptomyces coelicolor M1146 to facilitate genetic manipulations of gouC and gouD.
  
  
  METHODS
  By using fosmid D6-4H containing the complete gougerotin biosynthetic gene cluster, gouC and gouD were disrupted by PCR-targeting method to generate pGOUe-ΔC and pGOUe-ΔD. Both pGOUe-ΔC and pGOUe-ΔD were introduced into Streptomyces coelicolor M1146 by intergeneric conjugation, thus gouC and gouD disrpution mutants (Ml146-GOUe-AC and M1146-GOUe-ΔD) were obtained. The gougerotin production of M1146-GOUe-ΔC and M1146-GOUe-ΔD were assayed by HPLC analysis. The intermediates accumulated in these mutants were purified and subjected to MS and NMR analyses for structure determinations. Bioassay of these intermediates against tumor cell line were also carried out.
  
  
  RESULTS
  Disruption mutants of gouC and gouD failed to produce gougerotin and the mutants accumulated different gougerotin intermediates, which lost their ability to inhibit cancer cell proliferation.
  
  
  CONCLUSIONS
  gouC and gouD are key structual genes in the biosynthesis of gougerotin peptidyl moieties. This study will pave the way for the elucidation of gougerotin biosynthetic pathway.
authors.forename: Junhong
authors.initials: J
authors.lastname: Wei
authors.forename: Jihui
authors.initials: J
authors.lastname: Zhang
authors.forename: Lingjuan
authors.initials: L
authors.lastname: Jiang
authors.forename: Huarong
authors.initials: H
authors.lastname: Tan
authors.forename: Guoqing
authors.initials: G
authors.lastname: Niu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D053898:Biosynthetic Pathways; D015394:Molecular Structure; D005810:Multigene Family; D011741:Pyrimidine Nucleosides / Q000096:biosynthesis / Q000737:chemistry; D013302:Streptomyces / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048372:Streptomyces coelicolor / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011741:Pyrimidine Nucleosides; C004942:gougerotin