## 2 Results
### CrtR from C. glutamicum Binds to Heterologous crtR Promoter DNA Sequences
glutamicum_ in the absence of endogenous chromosomally encoded CrtR, but in the presence of CrtR orthologs from other actinobacteria. The CrtR orthologs were at different phylogenetic distances compared to CrtR from _C. glutamicum_, and therefore different protein identities: _C. callunae_ (62% identity), _A. jensenii_ (57% identity), _P. nicotinovorans_ (53% identity), _M. luteus_ (35% identity) and _P. chlorophenolicus_ (35% identity). To this end, the _crtR_ orthologs from _C. callunae_, _A. jensenii_, _P. nicotinovorans_, _M. luteus_ and _P. chlorophenolicus_, as well as the _crtR_ from _C. glutamicum_ as reference, were expressed from the strong, constitutive promoter _Pgap_ in divergent orientation to the _PcrtE_gfpuv_ transcriptional fusion. The respective vectors were named pTEST_CrtR\({}_{\rm Cg}\), pTEST_CrtR\({}_{\rm Cg}\), pTEST_CrtR\({}_{\rm Aj}\), pTEST_CrtR\({}_{\rm Pn}\), pTEST_CrtR\({}_{\rm Ml}\) and pTEST_CrtR\({}_{\rm Pc}\) (Figure S1). First, maximal repression using the vector pTEST-_crtR\({}_{\rm Cg}\)_ was determined and compared to a two-vector system, in which the expression of _crtR_ and the reporter gene fusion P_crtE_gfpuv_ were decoupled; _crtR_ was expressed by IPTG-inducible pEC-XT_crtR\({}_{\rm Cg}\), while pEPR1_PcrtE_ contained the reporter gene fusion P_crtE_gfpuv_ (Figure S1). In the strains carrying the P_crtE_gfpuv_ fusion, but lacking _crtR_ (WT\(\Delta\)_crtR_(pTEST) and WT\(\Delta\)_crtR_(pEPR1_PcrtE), pigmentation due to decaprenoxanthin accumulation (3.2-3.3 mg/g CDW) and GFPnuv fluorescence (1.3 normalized MFI) were high (Figure S2). The IPTG-inducible expression of _crtR_ in the two-vector system reduced decaprenoxanthin accumulation and GFPnuv fluorescence 38- and 32-fold, respectively (Figure S2). Reduction of decaprenoxanthin accumulation and GFPnuv fluorescence using pTEST_crtR\({}_{\rm Cg}\) (200- and 55-fold, respectively; Figure S2) was even higher (_crtR\({}_{\rm Cg}\)_ is transcribed from the strong and constitutive _Pgap_, shown above). Next, the in vivo effects of the CrtR orthologs were tested. Constitutive overexpression of the _crtR_ orthologs from the _gap_ promoter revealed differential effects on carotenogenesis and expression of the P_crtE_gfpuv_ transcriptional fusion (Figure 4). WT\(\Delta\)_crtR_ carrying the empty vector pTEST showed the expected intense yellow pigmentation due to derepression of the chromosomal carotenoid biosynthesis genes (Figure 4A), as well as the derepressed expression of the P_crtE_gfpuv_ transcriptional fusion (Figure 4B). Upon plasmid-borne expression of CrtR repressor genes from _C. glutamicum_, _C. callunae_, _A. jensenii_ and _P. nicotinovorans_, pigmentation was strongly reduced to less than 1 mg/g CDW (Figure 4A), which corresponded to the strongly reduced expression of the P_crtE_gfpuv_ transcriptional fusion (Figure 4B). Repression by CrtR from _C. callunae_ was nearly as tight as that by endogenous CrtR, leading to a relative GFPnuv signal of less than 0.1 (Figure 4B). CrtR orthologs from _A. jensenii_ and _P. nicotinovorans_ also repressed the _crtE_ promoter fusion very efficiently, resulting in a GFPnuv signal of less than 0.2 (Figure 4B). Upon expression of _M. luteus crtR_, carotenoid biosynthesis was reduced to a much lesser extent (Figure 4A), while the expression of the P_crtE_gfpuv_ transcriptional fusion was as high as in the empty vector control (Figure 4B). CrtR from _P. chlorophenolicus_ reduced pigmentation, but expression of the P_crtE_gfpuv_ transcriptional fusion was unaffected (Figure 4A,B). Taken together, the repression of carotenogenesis, and of the expression of _crtE_ transcriptional fusion, in _C. glutamicum_ in vivo by CrtR orthologs from other actinobacteria was possible. The efficacy was highest for closely related species, and it decreased with phylogenetic distance.