Design
Glyphosate is first converted to glyoxylate by GOX and then to glycolic acid while NADPH is consumed by GRHPR. Then, the amount of NADPH is determined by iNap.
Fig 1. Mechanism of GOX, GRHPR and iNap.
For the sake of
avoiding the interference of intracellular NADPH, the surface display system was employed (Fig 2) to anchor the enzymes mentioned above on the surface of engineered bacteria. Notably, this strategy
subtly solves the difficulty that there are
few natural regulators which could sense glyphosate or its derivatives in the glyphosate metabolic pathways, as scarcely reported.
Fig 2. Gene circuits of detection system.
Results
Every enzyme (GOX, GRHPR and iNap) and its fusion protein forms were characterized separately. The value of optical density at 340 nm (OD
340) was used to represented the amount of NADPH, except for the experiments related to iNap.
Fig 3. Results of GOX and GRHPR. OD340 was monitored along time in order to a, test the catalytic capability of GOX with GRHPR; b, test the enzymatic activity of anchored GOX; c, test the enzymatic activity of GRHPR; d, test the enzymatic activity of anchored GRHPR.
The single GOX could slightly catalyze the conversion of glyphosate (data not shown), while the second reaction catalyzed by GRHPR has a
faster kinetic behavior (Fig 3c). When tested with GRHPR, the GOX showed moderate enzymatic activity to catalyze the conversion of glyphosate to the substrate of GRHPR, which indicates the conjugation of the second reaction could actually
accelerate the conversion of glyphosate (Fig 3a). INPNC-GOX and INPNC-GRHPR retain the enzymatic activity in a large part (Fig 3b and 3d), and the latter (BBa_K3332057) is our
FAVORITE.
Fig 4. Results of iNap. a, iNap fluorescence intensity with excitation at 420 nm in the presence of different concentrations of NADPH. The fluorescence intensity of b, INPNC-iNap and c, iNap-AIDA were monitored along time in the presence of different concentrations of NADPH.
iNap manifests excellent SENSITIVITY and a large dynamic range to NADPH (Fig 4a). Compared to iNap-AIDA, INPNC-iNap has a larger dynamic range since the fluorescence intensities distinguished more significantly among different groups (concentrations of NADPH) (Fig 4b and 4c). Regretfully, BrkA-iNap has not been obtained due to the limit of time.
Proof of Concept
Fig 5. Fluorescence intensity was monitored along time to test the function of reaction mixture.
Three types of E. coli BL21 (DE3) respectively carrying INPNC-GOX (BBa_K3332052), INPNC-GRHPR (BBa_K3332057), and INPNC-iNap (BBa_K3332047) were mixed to react with glyphosate and NADPH. The decrease of fluorescence intensity suggested that the detection system could work well.