## 2 Results
### Construction and Analysis of a GGPP Biosensor
Thus, it was confirmed that the genetic approach altered the intracellular GGPP concentrations as anticipated. Figure 5: GGPP and decaprenoxanthin biosynthesis pathway in _C. glutamicum_. GAP: glyceraldehyde 3-phosphate, DXP: 1-deoxy-1-xylulose-5-phosphate; MEP: methylerythnitol phosphate; IPP: isopentenyl pyrophosphate; DMAPP: dimethylallyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate; RNAP: RNA-Polymerase core enzyme; SigA: housekeeping primary sigma factor A; Dxs: 1-deoxy-1-xylulose5-phosphate synthase; Idi: isopentenyl pyrophosphate isomerase; IdsA/CrtE: GGPP synthase; CrtB/CrtB2: phytoene synthase; CrtI/I2: phytoene desantarse; CrtEb: lycopene elongase; CrtY\(\mathrm{{}_{ef}}\): C50 \(\varepsilon\)-cyclase; genes overexpressed in strains GGPPA and GGPPB are shown in green, genes deleted on both strains are indicated by red crosses; blue shows genes overexpressed only in strain GGPPB. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Strain, gDNA or Plasmid** & **Relevant Characteristics of Sequence** & **Reference** \\ \hline \multicolumn{3}{c}{_E. coli_ strains} \\ \hline _E.coli_ DH5\(\alpha\) & F-_thi-1 endAI Inst17(\%, m)- supE4A IntC1169 (@@@@@duczAM15) recAI gyrA96_ & [42] \\ SI7-1 & _rcA9_ batch RR4 -2-Te-X-Mn-T20F integrated into the chromosome & [43] \\ _E.coli_ BL21 (DE3) & F-_omp7 gal dom lon hsd8(@-pn-)-JD5(JDS) lac lacI LCI/V-T20F/ _int1 sumT mics1_) [_hall8_]-IK-12(_3S_) & [44] \\ _E.coli_ BL21 (DE3) (@@LysS) & F-_omp7 gal dom lon hsd8(@-pn-)-JD5(JDS lacI lacI/V-T20F) \({}^{\prime}\) int1 sumT mics1_) [_hall8_+I-12(_15_) _t1 playsSI(TIP20 orpi15A)(CmR)_ & Promega \\ \hline \multicolumn{3}{c}{_G. glutamic strains_} \\ \hline _C. glutamicium_ WT & ATC 13032, will type & [45] \\ WTAC\(r\)R\(r\)R & ATC 13,032 with deletion of _crtR_ (eq0725) & [23] \\ WTAC\(r\)R\(r\)R\(r\)\(2\)T & ATCC 13,032 with deletion of _crtR_ (eq0721) and _crtR2I_ (2P \(\_\)cg2672) & this work \\ GGPPA & WTAC\(r\)R\(r\)R\(2\)T/12 derivative with plasmid-driven IPTG-inducible expression of MEF pathway genes _dss_ (eg2083) and _idi_ & this work \\ GGPPB & WTAC\(r\)R\(r\)\(2\)T derivative with plasmid-driven IPTG-inducible expression of MEF pathway genes _dss_ (eg2380) and _idi_ & this work \\ GGPPB & \(\overline{\text{W}}\)TA\(r\)R\(r\)\(2\)T derivative with plasmid-driven IPTG-inducible expression of MEF pathway genes _dss_ (eg2380) and _idi_ & this work \\  & (eg2531) from pEKEs3 and the GGPP synthase gene _idsA_ (2884) and primary sigma factor gene _s_g_  
\begin{table}
\begin{tabular}{c c c c} \hline
**Strain, gDNA or Plasmid** & **Relevant Characteristics or Sequence** & **Reference** \\ \hline \multicolumn{3}{c}{**Plasmids**} \\ \hline pET160\({}_{c}\)_crtR\({}_{\text{Pr}}\)_ & pET160 derivate for expression of the _crtR_ from _P. inc  
Since the strains harbored plasmid pEPR1_P_CrtE, the sensing of intracellular GGPP by chromosomally encoded CrtR could be tested. The normalized mean fluorescence intensity (MFI) of the _P_CrtE_gfpuv fusion observed after 12 h growth in glucose minimal medium was lowest for WT (pEPR1_P_CrtE) (MFI of 4.0 \(\pm\) 0.3). The biosensor signal of GGPPB (pEPR1_P_CrtE), of 14.0 \(\pm\) 1.8, was about two-fold higher than that of GGPPA (pEPR1_P_CrtE) (MFI of 7.0 \(\pm\) 0.7; Figure 6). Thus, biosensor signal output correlated with intracellular GGPP concentration. In order to determine if the _P_CrtE_gfpuv fusion can be used to monitor variations in the GGPP concentration during growth, strain GGPPB (pEPR1_P_CrtE) was analyzed in a time-course experiment in CGXII minimal medium supplemented with 100 mM glucose and 100 uM IPTG (Figure 7). Samples were taken every 1.5 h for determination of the intracellular GGPP concentration and flow cytometry analysis (Figure 7). Both the intracellular GGPP concentration and the GFPuv signal strongly increased in the first 7.5-10.5 h after inoculation. An offset between the GGPP concentration and the GFPuv signal was observed, with the increase in the latter being delayed by about 4 h (Figure 7).