The strains and plasmids used in this work are listed in Table 1. C. glutamicum ATCC 13032 was used as wild type (WT) and the prophage cured C. glutamicum MB001 (Baumgart et al., 2013) as basal strain for genetic engineering. C. glutamicum cultivations were performed in CGXII medium with 100 mM glucose as carbon and energy source (Eggeling and Reyes, 2005) after inoculation to an initial OD₆₀₀ of 1 at 30°C in either a volume of 50 ml in 500 ml flasks with two baffles shaking at 120 rpm or in 1 ml volume in microtiterplates at 1100 rpm at 30°C using Biolector® micro fermentation system (m2p-labs GmbH, Baesweiler, Germany). The growth was followed by measuring the OD₆₀₀ using a Shimadzu UV-1202 spectrophotometer (Duisburg, Germany). Cloning was conducted with E. coli DH5α as host, cultivated in LB medium at 37°C. When appropriate, kanamycin (25 μg/ml), spectinomycin (100 μg/ml), or ampicillin (100 μg/ml) was added to the culture medium. If not stated otherwise 1 mM of IPTG was added for induction of gene expression at inoculation of the main culture.

Plasmids were constructed in E. coli DH5α from PCR-generated fragments (KOD, Novagen, Darmstadt, Germany) and isolated with the NucleoSpin kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). Oligonucleotides used in this study were obtained from Eurofins MWG Operon (Ebersberg, Germany) and Metabion (Planegg/Steinkirchen, Germany) and are listed in Table 1. Plasmid construction was executed by standard PCR, restriction and ligation reactions as described previously (Sambrook and Russell, 2001) as well as Gibson assembly (Gibson et al., 2009). The RbCl method was used for transformation of E. coli (Hanahan, 1983) and C. glutamicum was transformed via electroporation at 2.5 kV, 200 Ω, and 25 μF (Eggeling and Reyes, 2005). The correctness of the cloned DNA fragments was verified by sequencing.

Plasmids for inducible gene expression were constructed on the basis of pEKEx3 (Stansen et al., 2005) or pVWEx1 (Peters-Wendisch et al., 2001). The gene crtR (cg0725) was amplified from genomic DNA of C. glutamicum WT, which was prepared as described (Eikmanns et al., 1995). Amplification was carried out by PCR using the respective crtR-fw/rv primers (Table 1).

Deletion of the gene crtR (cg0725) was carried out using the suicide vector pK19mobsacB (Schäfer et al., 1994). The construction of pK19mobsacB-crtR has been described (Henke et al., 2016). The genomic regions flanking the gene to be deleted by homologous recombination were amplified from genomic DNA of C. glutamicum WT using the primer pairs crtR_A/B and crtR_C/D (Table 1). The PCR products were purified and assembled and simultaneously cloned into SmaI restricted pK19mobsacB by Gibson assembly, which resulted in the deletion vector pK19mobsacB-crtR (Table 1). Targeted gene deletion proceeds via two-step homologous recombination as described previously using the before mentioned deletion vector (Eggeling and Bott, 2005). The first recombination event, the integration of the vector into one of the gene flanking regions, was selected via kanamycin resistance. Integration of the deletion vector into the genome triggers sucrose sensitivity due to the expression of sacB, encoding a levansucrase. In a second recombination event the deletion vector is excised and clones can be selected upon sucrose-resistance. By PCR analysis of three selected mutants using the primer pair crtR_E/F, deletion of the respective gene was verified (Table 1). The PCR products were then sequenced and in-frame deletion of crtR was confirmed.

The extraction of carotenoids from C. glutamicum was performed as described previously (Heider et al., 2014b) using 0.8 or 1 ml aliquots of the cell cultures. The pigments were extracted from the cell pellets with a methanol:acetone mixture (7:3) at 60°C for 30 min with thorough vortexing every 10 min. When necessary, extraction was repeated to remove all visible colors from the cell pellet. Subsequently, extraction mixtures were centrifuged for 5 min at 13,000 × g and the clear supernatant was used for analysis.

The carotenoid content of cell extracts was determined through absorbance at 470 nm by high performance liquid chromatography (HPLC) analysis as described previously (Heider et al., 2014b). HPLC analyses were performed on an Agilent 1200 series HPLC system (Agilent Technologies GmbH & Co. KG, Böblingen, Germany), including a diode array detector (DAD) for UV/visible (Vis) spectrum recording for detection. Separation of the carotenoids was accomplished by application of a column system (all columns from CS Chromatographie Service GmbH, Langerwehe, Germany) consisting of a pre-column (10 × 4 mm MultoHigh 100 RP18-5) and a main column (ProntoSIL 200-5 C30, 250 × 4 mm). Alternatively, 50 μL of the sample was separated with a column system consisting of a precolumn (LiChrospher 100 RP18 EC-5, 40 × 4 mm) and a main column (LiChrospher 100 RP18 EC-5, 125 × 4 mm) with methanol (A) and methanol/water (9:1) (B) as mobile phase. The following gradient was used at a flow rate of 1.5 mL/min; 0 min B: 0%, 10 min B: 100%, 32.5 min B: 100%. Quantification of lycopene and β-carotene was performed running a standard curve with HPLC grade standards (Sigma-Aldrich, Taufkirchen, Germany). Due to the lack of appropriate standards for decaprenoxanthin, sarcinaxanthin and C.p. 450 the quantification was calculated based on a β-carotene standard and reported as β-carotene equivalents (y = 79.7x; R² = 0.96). The standards were dissolved in chloroform according to its solubility and diluted in methanol:acetone (7:3) for generating a standard curve.

For transcriptome analysis triplicate cultures were grown in LB or CgXII medium. At an optical density of OD₆₀₀ between 4 and 5 cells were harvested and total RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. DNA was degraded in two reactions, in solution as well as on-column, using the RNase-Free DNase set (Qiagen, Hilden, Germany). Fluorescently labeled cDNA from 20 μg of total RNA was generated by indirect labeling as described by Hüser et al. (2003). cDNA probes labeled with Cy3 and Cy5 were combined for hybridization. Hybridization and scanning was performed as described earlier (Hüser et al., 2003). Signal-background segmentation, spot finding and intensity quantification were executed by ImaGene 6.0 software (BioDiscovery, Hawthorne, United States). Normalization (lowess) and t-test statistics were accomplished with EMMA 2.2, a software platform for consistent storage and efficient analysis of microarray data (Dondrup et al., 2003, 2009). Genes were regarded as differentially expressed when transcript abundance changed 2-fold or more (M ≥ 1 or M ≤ −1) with a statistical significance of p ≤ 0.05 in a student's t-test was determined.

In vivo expression levels of crtR and crtE were assayed by transcriptional fusion of the intergenic region of crtR and crtE to the promoter-less GFP_UV gene using the pEPR1 plasmid system (Knoppova et al., 2007). Therefore, the corresponding promoter sequences were amplified from genomic DNA of C. glutamicum WT with primer pairs PcrtE-pEPR-fw/PcrtE-pEPR-rv and PcrtR-pEPR-fw/PcrtR-pEPR-rv, respectively and cloned into the BamHI restricted pEPR1 vector upstream of the promoter-less gfp_UV gene. Site-directed mutagenesis of putative motifs was performed on both constructed vectors using the primer pairs SDM-fw-nat/ SDM-rv-mut, SDM-fw-mut/ SDM-rv-nat and SDM-fw-mut/SDM-rv-mut. The GFP_UV shows characteristic emission at 509 nm with an excitation wavelength of 385 nm. Measurements of fluorescence were performed on a FACS Gallios™ (Beckmann Coulter GmbH, Krefeld, Germany) with 405 nm excitation from a blue solid-state laser. Forward-scatter characteristics (FSC) and side-scatter characteristics (SSC) were detected as small- and large-angle scatters of the 405 nm laser. GFP_UV fluorescence was detected using a 500/50 nm band-pass filter. C. glutamicum MB001ΔcrtR and MB001 harboring the plasmids pEPR1-PcrtR and pEPR1-PcrtE or the plasmid with the mutated promoter sequences, were harvested in mid-exponential growth washed once in phosphate-buffered saline and the optical density was adjusted to OD₆₀₀ <1. Cells harboring the empty vector pEPR1 were used to determine background fluorescence.

The vector pET16b-crtR was constructed using PCR-amplified crtR using oligonucleotides crtR-NdeI-fw and crtR-BamHI-rv and ligating it into pET16b restricted with BamHI and NdeI. E. coli BL21(DE3) cells carrying the plasmid pET16b-crtR were grown at 37°C in 500 ml LB medium with 100 μg/ml ampicillin to an OD₆₀₀ of 0.5 before adding IPTG for induction of the gene expression to a final concentration of 0.5 mM. After induction, cells were cultivated at 21°C for additional 5 h. Cells were harvested by centrifugation and stored at −20°C. For cell extract preparation, thawed cells were re-suspended in TNI buffer (20 mM Tris-HCl, pH 7.9, 300 mM NaCl, 5% (v/v) glycerol) with 5 mM imidazole containing a proteinase inhibitor cocktail tablet (Complete Mini, Roche, Basel, Switzerland). Cells were disrupted by ultrasonification using Hielscher UP200S2 (Teltow, Germany) with an amplitude of 60% and a pulsing cycle of 0.5 (power discharge 0.5 s, pause 0.5 s) for 2 min. After ultracentrifugation (1.5 h, 45,000 × g, 4°C) the supernatant was filtered through a 0.2 μm filter and purified by nickel affinity chromatography using nickel-activated nitrilotriacetic acid-agarose (NTA) (Novagen, San Diego, USA). TNI buffer containing 5 mM or 100 mM imidazole was used for sequentially washing of the column. The regulator protein was eluted with TNI buffer containing 400 mM imidazole and the fractions of highest protein concentrations were pooled. The elution buffer was exchanged against band shift (BS) buffer (50 mM Tris–HCl, 10% (v/v) glycerol, 50 mM KCl, 10 mM MgCl₂, 0.5 mM EDTA, pH 7.5) using PD10 columns (GE Healthcare, Chalfont St. Giles, Great Britain). Protein concentrations were determined with the Bradford assay kit (Bio-Rad Laboratories, Hercules, Canada) using bovine serum albumin as reference. Protein purification was ascertained by 12% SDS-PAGE (polyacrylamide gel electrophoresis). The purified protein was applied for band shift assay without removing the N-terminal His-tag.

For verification of a physical Protein-DNA interaction between the regulator CrtR and the promoter region of crtE and crtR, band shift assay was performed according to Krause et al. (2012). His-tagged CrtR in varying molar excess was mixed with 90 ng of purified promoter-fragments of the target genes in band shift (BS) buffer (50 mM Tris–HCl, 10% (v/v) glycerol, 50 mM KCl, 10 mM MgCl₂, 0.5 mM EDTA, pH 7.5) in a total volume of 20 μL. The 5′UTR of crtE was PCR-amplified with the oligonucleotides SFT8 and SFT3 (fragment A) listed in Table 1 and purified with NucleoSpin kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). Truncated and mutated promoter fragments were amplified using oligonucleotide pairs SFT8/SFT10 (fragment B), SFT8/SFT12 (fragment C), SFT4/SFT27 (fragment D), SFT28/SFT27 (fragment E), SFT29/SFT27 (fragment F), SFT30/SFT27 (fragment G), SFT8/SFT23 (fragment H), SFT8/SFT26 (fragment I), SFT8/SFT25 (fragment K). A 78 bp-fragment of the upstream region of cg2228 was added in every sample as a negative control using oligonucleotides cg2228_fw and cg2228_rv. After 30 min of incubation at room temperature, gel shift samples were separated on a 6% DNA Retardation gel (Life Technologies GmbH, Darmstadt, Germany) at 100 V buffered in 44.5 mM Tris, 44.5 mM boric acid, and 1 mM EDTA at pH 8.3. Additionally, the binding affinity in presence of selected effectors was analyzed by incubation of the protein with the effector substance under buffered conditions for 15 min at room temperature prior to the addition of the promoter. Subsequently, the gel shift samples were separated on a 6% DNA Retardation gel (Life Technologies GmbH, Darmstadt, Germany) at 100 V buffered in 44.5 mM Tris, 44.5 mM boric acid, and 1 mM EDTA at pH 8.3. Staining of the DNA was achieved with ethidium bromide.

In the genome of C. glutamicum the carotenogenic genes responsible for conversion of the building blocks IPP and DMAPP to decaprenoxanthin are organized in the crt operon (crtE-cg0722-crtBIY_eY_fEb) (Heider et al., 2012). The major GGPP synthase IdsA (Heider et al., 2014a) and a second phytoene synthase are encoded elsewhere on the chromosome (Heider et al., 2012). Divergent to the crt operon and 200 bp upstream of its first gene, crtE, one of nine MarR-type transcriptional regulator genes in the genome of C. glutamicum (Brune et al., 2005) is found. This gene cg0725 was named crtR based on the results described in this study. Previously, a transposon mutagenesis screen showed that insertion of a transposon into crtR enhanced carotenoid production (Krubasik et al., 2001a). A model of the protein secondary structure of the 195 aa containing crtR product revealed a pfam12802 domain with a centrally located winged HTH motif located from aa position 73 to 134 as is typical for MarR-type regulator proteins of C. glutamicum (Brune et al., 2005).

A sequence comparison by BLAST showed that C. glutamicum CrtR is distinct from MarR-type regulators of Bacillus subtilis, Escherichia coli, Mycobacterium, and Streptomyces species (data not shown), but showed highest similarities to MarR-type proteins of the high GC content family of Actinobacteria, primarily of species of the genera Corynebacterium, Microbacterium, Leucobacter, Arthrobacter, Propionibacterium, and Gulosibacter (Figure S1). A sequence alignment with the closest relatives of CrtR (CAF19334) from C. vitaeruminis, C. efficiens, C. callunae, Leucobacter sp. Ag1, M. mangrovi, Brevibacterium sp. VCM10, and L. chironomi shows high similarity especially in the central region of the proteins where the proposed HTH motif is located (Figure S2). Since crtR is localized in proximity to the crt operon in C. glutamicum, it was tested if this pattern is conserved among bacteria containing a CrtR ortholog. First, sequence comparisons of C. glutamicum CrtR to completely sequenced genomes identified bacteria possessing a CrtR homolog showing at least 25% amino acid identity and an e-value <10⁻¹⁰. Subsequently, the completely sequenced genomes of these bacteria were analysed for the presence of homologs (25% amino acid identity and e-value <10⁻¹⁰) of the C. glutamicum proteins encoded by crtEb (cg0717), crtY_f (cg0718), crtY_e (cg0719), crtI (cg0720), crtB (cg0721), cg0722, crtE (cg0723), crtX (cg0730), and idsA (cg2384). Although the low identity cut-off of 25% may have led to more false-positives than searches with higher identity cut-offs (Rost, 1999), this was compensated for by the high significance value of e⁻¹⁰ and the combinatory search for CrtR homologs and other carotenogenesis genes. If at least one homolog of the proteins in this list were encoded in a completely sequenced genome in addition to a homolog of C. glutamicum CrtR, the genome was considered further and the results depicted as heatmap (Figure S3). Opposite and in divergent orientation to the crtR homologous genes, individual genes or crt operons were found. For the most part, the individual genes or the first genes of the crt operons encoded GGPP synthase (crtE), IPP isomerase (idi), and putative multidrug efflux protein (mmpl), respectively (Figure 1).

With respect to the genes encoding homologs of C. glutamicum CrtR and genes involved in carotenogenesis four distinct patterns emerged (Figure 1). First, a group of bacteria possessing a full complement of carotenogenesis genes including genes for carotenoid elongase (crtEb), carotenoid cyclase (crtY, crtY_e/Y_f) and a carotenoid glycosyltransferase (crtX). This group comprised bacteria known to produce glycosylated, cyclic C50 carotenoids (C. glutamicum, C. callunae, C. marinum, Micrococcus luteus), but also bacteria of which it is unknown which carotenoids they synthesize (Arthrobacter arilaitensis, Microbacterium barkeri, and Microbacterium hydrocarbonoxydans; Figure 1). A second group with Agromyces subbeticus and Leucobacter chironomi lacks crtX homologs likely synthesizing non-glucosylated carotenoids and a third group with Propionibacterium jensenii and Corynebacterium lubricantis lacks crtX and crtY homologs likely synthesizing noncyclic nonglycosylated carotenoids. Finally, a group with Brevibacterium linens, Gulosibacter molinativorax and Timonella senegalensis lacks crtX, crtY and crtE/ubiA homologs and these are likely unable to synthesize glycosylated or cyclic or elongated carotenoids. Taken together, homologs of C. glutamicum CrtR are conserved in various bacteria (mostly actinobacteria) that were shown to synthesize various carotenoids (cyclic and noncyclic, elongated or not, glycosylated or not) or do have the potential to synthesize these according to their gene repertoire. Moreover, the crtR homologs are found in diverse genomic locations, but mostly adjacent to crt operons or genes involved in carotenogenesis. These findings supported the hypothesis that CrtR homologs may have a conserved role in regulation of carotenogenesis.

To test if C. glutamicum CrtR plays a role in transcriptional regulation of genes of carotenogenesis a crtR deletion mutant was constructed and characterized with respect to global gene expression. Comparative transcriptome analyses of C. glutamicum MB001ΔcrtR and C. glutamicum MB001 were performed to identify genes directly or indirectly regulated by CrtR. The transcriptomes of cells cultured in triplicates either in glucose minimal medium or LB complex medium revealed only eight differentially expressed genes (Table 2). All of the differentially expressed genes showed 8 to 69-fold increased mRNA levels and they belonged to the crt operon or, as in the case of cg0726 that codes for a putative secreted lipoprotein, are localized adjacent to it. Deletion of crtR led to considerably higher mRNA ratios for cg0717 and cg0719 during growth in LB than in minimal medium, however, this does not pertain to the other genes of the crt operon. Currently, it remains unclear if these differences may be caused by technical issues or have a biological cause, however, it has to be noted that RNAseq analysis revealed expression of cg0717 and cg0718 independent of the other crt genes, thus, sub-operons may exist (Pfeifer-Sancar et al., 2013). As expected from the operon structure all transcripts were decreased from the first to the last genes in the crt operon. Taken together, the crt operon of C. glutamicum is directly or indirectly regulated by CrtR.

The pEPR1-promoter probe vector system for transcriptional fusion analysis (Knoppova et al., 2007) was used to determine if CrtR regulates transcription of its own gene and of the crt operon in vivo. The intergenic region between crtR and crtE, the first gene of the crt operon, was cloned upstream of the promoter-less gfp_UV gene in both orientations (named PcrtE and PcrtR, respectively). Reporter gene expression in C. glutamicum MB001(pEPR1-PcrtE), MB001(pEPR1-PcrtR), MB001ΔcrtR(pEPR1-PcrtR), and MB001ΔcrtR(pEPR1-PcrtE) was determined by fluorescence cell scanning in LB medium in the early exponential growth phase (Table 3). About 2-fold higher expression of the PcrtR fusion in the absence of CrtR revealed that CrtR is subject to negative autoregulation (Table 3). Expression of the PcrtE fusion was about 5-fold higher when crtR was deleted indicating that CrtR represses transcription of the crt operon (Table 3).

As CrtR was shown to negatively regulate transcription of the crt operon and of its own gene, gel mobility shift assays were performed to determine if CrtR directly interacts with the intergenic region between crtR and crtE. Therefore, CrtR protein was produced in recombinant E. coli BL21 (DE3) as N-terminal His-tagged protein and purified to apparent homogeneity by affinity chromatography (Figure S4). Gel mobility shift analysis revealed that His-tagged CrtR bound to the intergenic region between crtR and crtE (fragment A; Figures 2a,c), whereas BSA as a control did not bind to this DNA fragment (data not shown). At a 30 to 40-fold molar excess of CrtR a complete shift of the promoter fragment C was observed, whereas a negative control fragment comprising the sequence upstream of the unrelated gene cg2228 was not bound by CrtR (Figure 2b). To localize the CrtR binding site, truncated fragments of the intergenic region were used for further gel shift assays. In line with the observation that MarR-type regulators typically bind to palindromic sites of 16–20 bp which often overlap the −10 and −35 regions for steric inhibition of RNA polymerase (Wilkinson and Grove, 2006), two inverted repeats were found in the intergenic region between crtR and crtE (Figure 2a). CrtR bound to fragments A, C, D, E, and F that contained a TTAA sequence motif 25–28 bases upstream of the transcription start site of crtE (Figures 2c,d). Fragments B and G that lack the complete TTAA motif were not bound by CrtR (Figures 2c,d) indicating that this sequence motif might be essential for CrtR binding. Thus, this motif and a similar motif (AAATTT overlapping with the −10 hexamer of the crtE promoter; Figure 2a) were mutated. Mutating the latter motif (fragment H) did not influence binding of CrtR, whereas CrtR did not bind to fragments with a mutated TTAA motif (fragments I and K; Figure 2d). Thus, the TTAA motif was shown to be required for binding of CrtR in vitro.

In order to determine the role of the TTAA motif for regulation by CrtR in vivo, transcriptional fusions of the promoters PcrtR and PcrtE containing mutations in the TTAA and/or the AAATTT motifs were constructed. Mutation of the AAATTT motif changed the −10 hexamer of the PcrtE promoter from TATAAA to TATGGG (Figure 2a). Consequently, only very low reporter gene activities were measured (Table 3) and thus, the relevance of the AAATTT motif for regulation of the PcrtE promoter could not be determined. Reporter gene activities of a transcriptional fusion of PcrtR containing the mutated AAATTT motif were comparable to those obtained with the non-mutated PcrtR promoter and revealed that this motif is dispensable for negative autoregulation by CrtR (Table 3). Mutation of the TTAA motif led to very low reporter gene activity with PcrtR, which precluded deducing its involvement in crtR autoregulation. By contrast, mutation of the TTAA motif led to high reporter gene activities of PcrtE regardless of the absence or presence of CrtR (Table 3) which supports the notion that this motif is required for function of CrtR as transcriptional repressor of the crt operon in vivo.

Regulation of carotenogenesis in response to low-molecular-weight compounds has not yet been described. However, since CrtR belongs to the MarR family of transcriptional regulators and representatives of this family show regulation in response to low-molecular-weight compounds, such as hydrogen peroxide in the case of RosR of C. glutamicum (Bussmann et al., 2010), low-molecular-weight compounds were tested as effectors of CrtR in gel mobility shift assays. Intermediates of carotenogenesis (isoprenoid pyrophosphates), precursors of the MEP pathway (GAP and pyruvate), but also phosphorylated intermediates of glycolysis (DHAP, phosphoenolpyruvate and D-glucosamine 6-phosphate), organic acids (acetate, propionate) and compounds affecting other MarR-type regulators [vitamin B12, salicylic acid, urea, protocatechuic acid ‘(data not shown)’] were tested as effectors of CrtR with concentrations in the mM range. Neither DHAP (Figure 3) nor pyruvate (data not shown) had an effect on CrtR binding to its DNA target. In the contrary, in the presence of GAP CrtR binding was prevented (Figure 3). In addition, the isoprenoid pyrophosphates IPP, DMAPP, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and GGPP perturbed binding of His-tagged CrtR to the intergenic region between crtR and crtE (Figure 3). Among these isoprenoid pyrophosphates, GGPP was effective at the lowest concentration (50 μM), whereas FPP, GPP, IPP and DMAPP exerted similar effects on CrtR binding in vitro at a 5-fold higher concentration (Figure 3). Thus, GGPP, shorter isoprenoid pyrophosphates and the MEP-pathway precursor GAP interfere with the in vitro binding of CrtR to the intergenic region between crtR and crtE and the strength of interference follows the order GGPP >> FPP, GPP, IPP, DMAPP >> GAP (Figure S5).

Since CrtR represses the crt operon the effect of deletion and overexpression of crtR on decaprenoxanthin accumulation in cells of C. glutamicum wild type and the phage-cured strain MB001 was determined. While growth was not significantly affected by the deletion or overexpression of crtR, neither in complex medium (data not shown) nor in minimal medium, the crtR deletion mutant showed more intense yellow pigmentation, while pigmentation of the overexpressing strain was slightly decreased (Figure S6). HPLC analyses of extracts from cells grown in CGXII medium revealed that the crtR deletion mutants accumulated decaprenoxanthin to 15 to 30-fold higher levels than the corresponding parental strains (Table 4). By contrast, as consequence of plasmid-borne overexpression of crtR decaprenoxanthin levels were significantly decreased (Table 4).

Since lycopene is a central intermediate of C40 and C50 carotenoid biosynthesis, the effect of deletion of crtR was elucidated in a metabolically engineered lycopene producing LYC5. Lycopene concentration increased about 5-fold (Figure 4). To test if this type of regulatory engineering can also be applied to the production of non-native carotenoids, crtR was deleted in the β-carotene producing strain BETA3, the sarcinaxanthin producing strain LYC5 (pEKEx3-crtE2Y), and the C.p.450 (2,2′-bis-(4-hydroxy-3-methybut-2-enyl)-β,β-carotene) producing strain LYC5 (pEKEx3-lbtABC). Indeed, all strains lacking CrtR produced 1.5 to 2 fold more carotenoid than the parental strains (Figure 4). Thus, deletion of crtR is a general strategy to boost production of native and non-native C40 and C50 carotenoids by C. glutamicum.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer BL and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.