## 2 Results
### Construction and Analysis of a GGPP Biosensor  
The expression control of P_crtE_gfpuv_ transcriptional fusion by CrtR from _C. glutamicum_ has been described previously [19], and the results from above prompted us to consider its use in combination with chromosomally encoded _crtR_ as a GGPP biosensor, i.e., using _C. glutamicum_ WT(pEPR1_PcrtE) as a biosensor strain. Targeted metabolic engineering typically addresses four modules: carotenoid biosynthesis, precursor supply, central carbon metabolism and redox cofactor regeneration. A CrtR-based biosensor may allow us to simultaneously optimize the latter three modules via fluorescence reporter output. While GGPP inhibits the DNA binding of CrtR [23], no feeding regimen is known to predictably alter the intracellular GGPP concentration. Therefore, a genetic approach was chosen, and two _C. glutamicum_ strains, expected to accumulate GGPP to intracellular concentrations different from the wild type, were constructed (Figure 5). Figure 4: In vivo characterization of CrtR orthologs in _C. glutamicum_ WT\(\Delta\)_crtR_. (**A**) Phenotypes on LB plates after incubation at 30 \({}^{\circ}\)C for 24 h and carotenoid concentration in mg/g CDW (\(\beta\)-carotene equivalents) of WT\(\Delta\)_crtR_ strains harboring pTEST derivatives expressing _crtR_ genes from the indicated bacteria. (**B**) Flow cytometry analysis of the strains depicted in (A) during exponential growth in LB. Mean fluorescence intensities (MFI) of GFPuv signals were normalized to autofluorescence and shown for at least two biological replicates. First, the conversion of GGPP by the endogenous phytoene synthases CrtB [24] was prevented through the deletion of _crtB_ and _crtB2I'I2_ from the chromosome of the _C. glutamicum_ WT yielding strain WT_\(\Delta\)crtB\(\Delta\)crtB2I'I2_ (Table 1). Second, the supply of the precursor molecules DMAPP and IPP was increased by overexpression of the genes encoding the MEP pathway enzymes 1-deoxy-D-xylulose 5-phosphate synthase (Dxs) and Idi. It is known for _C. glutamicum_[28] and other organisms that the first enzymatic step in the MEP pathway strongly limits the flux [38; 39; 40]. Dxs is supposed to be feedback-regulated by isoprenoid pyrophosphates [38], and the overexpression of _dxs_ was shown to increase the flux towards terpenoid biosynthesis [28; 30; 33]. In addition, the ratio of DMAPP to IPP was shown to be important for optimized isoprenoid production, and _idi_ overexpression equilibrates intracellular concentrations of DMAPP and IPP [28; 33; 39]. Third, GGPP synthesis was improved by plasmid-driven overexpression of the major GGPP synthase gene _idsA_ from _C. glutamicum_[26] (pEC-XT_idsA_; Table 1). Combining the three strategies resulted in strain GGPPA (WT_\(\Delta\)crtB\(\Delta\)crtB2I'I2_ (pEKEx3-_dxs_idi_) (pECXT-_idsA_)) (Table 1) (Figure 5). Since engineering of the RNA polymerase sigma factor A improved isoprenoid carotenoid production [41], _sigA_ from _C. glutamicum_ was overexpressed in a synthetic operon with _idsA_ (pEC-XT_idsA_sigA_) (Table 1). The resulting strain GGPPB (WT_\(\Delta\)crtB\(\Delta\)crtB2I'I2_ (pEKEx3-_dxs_idi_) (pECXT-_idsA_sigA_)) differs from strain GGPPA only by the additional overexpression of _sigA_ (Table 1) (Figure 5). The intracellular GGPP concentrations of the _C. glutamicum_ strains WT, GGPPA and GGPPB were expected to differ. After transformation with the biosensor plasmid pEPR1_P_crtE_, the strains were grown in CGXII minimal medium with glucose. Samples were taken during exponential growth 12 h after inoculation and analyzed by LC-MS (Figure 6). As expected, WT (pEPR1_P_crtE_) accumulated the lowest GGPP concentration, with less than 0.1 mM GGPP (Figure 6). In comparison, GGPPA (pEPR1_P_crtE_) accumulated about 23-fold more GGPP (2.3 \(\pm\) 0.5 mM; Figure 6). With 4.0 \(\pm\) 0.4 mM, strain GGPPB (pEPR1_P_crtE_) exhibited the highest concentration of GGPP, i.e., about 1.7-fold higher than GGPPA (pEPR1_P_crtE_).