Part:BBa_K3733006

ScGS: Streptomyces coelicolor geosmin synthase

Gesomin synthase from Streptomyces coelicolor A3(2) (ScGS) is a single 726-amino acid protein which catalyze the Mg²⁺ dependent conversion of farnesyl diphosphate to a mixture including geosmin. ScGS is a bifunctional enzyme in which the N-terminal domain catalyze the cyclization of FPP to form germacradienol, while the C-terminal domain then convert this sesquiterpenoid product to geosmin.

Usage and Biology

The ScGS is a bifunctional sesquiterpene cyclase, with the presence of Mg²⁺, the N-terminal half of this protein catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate(PP_i). Then the C-terminal domain, highly homologous with the former, catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

To obtain ScGS, pET-28a(+)-ScGS(with His-tag) was transferred into E.coli BL21(DE3), and the cells were inoculated in 25 mL cultures of LB medium with 10 μg/mL kanamycin. These cultures were grown at 37℃ with 250 rpm shaking until the OD₆₀₀ reached 0.5-0.8, then 0.3 mM isopropyl β-D-1-thiogalactopyranoside(IPTG) were added, following by an overnight cultivation at 16℃ with 250 rpm shaking to induce protein expression. The washed and harvested cells were resuspended with a Binding Buffer, and then the cells were lysed by ultrasonication. Purification was performed according to the protocol of Ni-NTA Sefinose^TM Resin (Sangon Biotech, Shanghai, China). As it shows in the following figure(figure 1.), the existence of ScGS in our chasis is clearly proved by SDS-PAGE analysis.