PTS
Introduction
It codes the key enzyme which catalyst FPP (farnesyl pyrophosphate) transform into patchoulol[1].
Characterization
In order to test the function of PTS (Patchoulol synthase), we construct "Promoter-PTS-terminator" (Figure 1). If PTS is functional, we can test the product of patchoulol after fermentation of recombinant P.pastoris not only in the static system but also dynamic regulation.
Figure 1 Gene circuit of PTS (static system)
Figure 2 Gene circuit of light-control system of PTS (dynamic system)
Analysis of Terpenoids
The quantification of patchoulol was performed on a GC system (Agilent 7820 a, USA) equipped with HP-5 column (30 m × 0.25 mm, 0.25μM U.M film thickness) and a flame ionization detector (FID). The oven temperature shall be kept at 90℃ for 0.5 min, then gradually increased to 120℃ at the speed of 20℃/ min, then increased to 160℃ at the speed of 3℃/ min for 5 min, and finally increased to 250℃ at the speed of 20℃/ min for 10 min. Patchoulol was quantified by external standard method (figure 3,4,5).
Figure 3 The blue bars and the vertical coordinates on the right show the yield of patchoulol for different promoters.
Figure 4 The blue bars and the vertical coordinates on the right show the yield of patchoulol for different light condition.
Figure 5 Gas Chromatogram of Patchoulol
Weigh 0.5mg of patchoulol standard with an analytical balance and dissolve it in 5ml of n-dodecane. Volume it to 5ml with a volumetric flask. The concentration of the standard mother liquor is 1g/L; Dilute the standard mother liquor to 0.75g/l, 0.75g/l, 0.5g/l, 0.25g/l and 0.1g/l, and then determine the relationship between the absorption peak of the standard and the concentration of the standard according to the GC method. Fit the linear regression equation y=3×107 - 556362, R2 = 0.9995, Y represents the peak area, X represents the concentration of the standard patchoulol (figure 6).
Figure 6 The standard curve of patchoulol
Reference
[1] Xu, H.; Goldfuss, B.; Schnakenburg, G.; Dickschat, J. S. Beilstein J. Org. Chem. 2022, 18, 13-24. doi:10.3762/bjoc.18.2