Difference between revisions of "Part:BBa K3733006"

Revision as of 15:13, 21 October 2021

ScGS: Streptomyces coelicolor geosmin synthase

Geosmin synthase from Streptomyces coelicolor A3(2) (ScGS) is a single 726-amino acid protein which catalyzes the Mg²⁺ dependent conversion of farnesyl diphosphate to a mixture including geosmin. ScGS is a bifunctional enzyme which the N-terminal domain catalyzes the cyclization of FPP to form germacradienol, while the C-terminal domain then converts 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 was proved by SDS-PAGE analysis.

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Figure 1. SDS-PAGE analysis of ScGS with His-tag expression

In order to identify the synthesis of geosmin, engineered bacteria in TB medium containing 5% glycerol were first induced ScGS expression with 0.7mM IPTG when OD₆₀₀ reached about 0.7, following by an overnight culture at 18℃ and continuing cultivation for next 72h at 25℃. From this way we could smell a strong and unusual odor from the culture comparing to the control.

For further demonstration, we prepared the sample via headspace liguid-phase microextraction(HS-LPME) and a gas chromatography-mass spectrometry(GC-MS) test was conducted. The results given by GC-MS fairly shows the existence of geosmin in our culture(Figure 2.), thus proves the feasibility of the part.

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Figure 2. Identification of geosmin by GC-MS. A. Total ion current chromatogram of geosmin standard and extracted product. B. Mass spectrum of geosmin standard. C. Mass spectrum of the extracted product.

References

[1] Harris G G, Lombardi P M, Pemberton T A, et al. Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization–Fragmentation Reaction Sequence[J]. Biochemistry, 2015, 54(48): 7142-7155.

[2] Xie Y, He J, Huang J, et al. Determination of 2-methylisoborneol and geosmin produced by Streptomyces sp. and Anabaena PCC7120[J]. Journal of agricultural and food chemistry, 2007, 55(17): 6823-6828.

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 <center><img src="https://static.igem.org/mediawiki/parts/a/ae/T--HZAU-China-gcms.png" style="width:869px;height:433px"></center>
-<b>Figure 2. </b>Identification of geosmin by GC-MS. <b>A.</b> Total ion current chromatogram of geosmin standard(the peak of it is marked with an arrow <b>Peak 1</b>) and the extracted product(the peak of it is marked with an arrow <b>Peak 2</b>), which the retention time is similar to each other. <b>B.</b> Mass spectrum of geosmin standard. <b>C.</b> Mass spectrum of the extracted product.
+<b>Figure 2. </b>Identification of geosmin by GC-MS. <b>A.</b> Total ion current chromatogram of geosmin standard and extracted product. <b>B.</b> Mass spectrum of geosmin standard. <b>C.</b> Mass spectrum of the extracted product.
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