# Biosensors-Based In Vivo Quantification of 2-Oxoglutate in Cyanobacteria and Proteobacteria
## Results
### Evaluation of Single-Protein Based FRET Sensors for 2-OG Measurements  
We first tested whether the biosensors respond to 2-OG in vitro. While the YFP/CFP F-NtAc_1, _3, _4, and _7 to _10 biosensors were not produced or aggregated as inclusion bodies, the F-PII, F-NtAc_2, F-NtAc_5 and F-NtAc_6 were successfully purified (Figure S1). Their FRET ratio (YFPm/CFPem) was then measured in presence (1 mM) or absence of 2-OG. In agreement with the 2-OG-induced conformational change observed in the crystal structures, the FRET ratio of F_PII in presence of 2-OG increased by \(\sim\)40% compared to the control without 2-OG. For the F_NtAcA biosensors, only F-NtAc_2 presented significant increased FRET ratio upon addition of 2-OG compared to the control (Figure 1E). While F-NtAc_5 contains the shortest fragment of NtAcA and F-NtAc_6 the longest, they both weakly responded to 2-OG. One possible explanation is that the two fluorophores are already at close proximity and the distance between the two is not significantly modified upon 2-OG binding. Based on these data, F_NtAc_2 was chosen for further studies and renamed F_NtAcA. To engineer 2-OG biosensors usable for in vivo 2-OG measurements, less sensitive to photobleaching, and exploitable for corroborative intensity-based FRET experiments, we replaced CFP by mTurquoise. The two new biosensors named F-mT_PII and F-mT_NtAc were purified and tested in vitro. Measurements of the FRET ratio showed results comparable to that measured with CFP, with an increase of 20-30% upon addition of 2-OG compared to control conditions without 2-OG (Figure 1F,G). The increase of the FRET ratio for F-mT_PII and F-mT_NtcA was dependent on the 2-OG binding since PII and NtcA variants previously shown to be affected for 2-OG binding (K58M substitution in PII [12]; R88E substitution in NtcA [44]) did not show any increase in the FRET ratio compared to the controls (Figure 1F,G). To gain further insights into the responsiveness of the two biosensors, a titration curve representing the FRET ratio as a function of 2-OG concentration was determined for different concentrations of the two mono-protein biosensors as previously described [35]. Varying the biosensor concentrations between 0.25 and 1 uM did not cause significant changes on the response of the two biosensors to 2-OG (Figure 2A,B). However, we noted that F-mT_PII and F-mT_NtcA responded to  
Figure 1: 2-oxoglutarate (2-OG) fluorescence resonance energy transfer (FRET) biosensors based on mono-proteins. (**A,D**) Schematic (**A**) and linear (**C**) representations of the FRET biosensor F_PII. The CFP is fused at the C-terminal end of PII whereas the YFP is inserted within the flexible, 2-OG-responsive T-loop; (**B,D**) schematic (**B**) and linear (**D**) representations of the FRET biosensor F_NtcA. The DNA binding domain was removed and the biosensors have conserved parts of the effector binding domain (EBD) and of the C-helix. The YFP is fused at the N-terminus of the EBD fragments whereas the CFP is fused to the C-terminus of the C-helix fragment. The yellow and blue spheres correspond to YFP and CFP, respectively. The red ball indicates the 2-OG binding site. The orange arrow indicates the conformational changes triggered by 2-OG binding. The green arrows indicate the substituted residues that prevent 2-OG binding and used as controls (K58M for PII, R88E for NtcA). The 10 different NtcA biosensor length variants, highlighting the first and last residues of each construct, are shown in panel (**D**); (**E**) FRET ratio change (%) of the biosensors purified after the addition of 1 mM of 2-OG; (**F,G**) _In vitro_ relative FRET ratio of F-mT_PII and its K58M mutant (**F**) and of F-mT_NtcA_2 and its R88E mutant (**G**) after addition of 10 mM of 2-OG (FRET ratio without 2-OG was set as 1). different ranges of 2-OG concentrations. F-mT_PII is \(\sim\)10 times more sensitive to 2-OG than F-mT_NtcA with a K\({}_{\rm d}\) of 120 uM and a K\({}_{\rm d}\) of 1.9 mM, respectively.