Recombinant DNA work Plasmids were constructed in E. coli DH5α from PCR-generated fragments (KOD, Novagen, Darmstadt, Germany) and isolated with the QIAprep spin miniprep kit (QIAGEN, Hilden, Germany). Oligonucleotides used in this study were obtained from Eurofins MWG Operon (Ebersberg, Germany) and are listed in Additional file 3: Table S1. Standard reactions like restriction, ligation and PCR were performed as described previously [37]. If applicable, PCR products were purified using the PCR purification kit or MinElute PCR purification

kit (QIAGEN, Hilden, Germany). For transformation of E. coli the RbCl method was used [38] and C. glutamicum was transformed via electroporation [39] at 2.5 kV, 200 Ω and 25 μF. All cloned DNA fragments were shown to be correct by sequencing. Determination of the transcriptional start point of crtE and crtB2 Total RNA was isolated from an exponentially growing culture of C. glutamicum WT as described GSK461364 mw previously [40]. Purified RNA was analyzed by UV-spectrometry in regard to quantity and quality and was stored at −20°C until use. 2 μg of total RNA were used to GSK126 supplier perform 5’ rapid amplification of cDNA ends-PCR (5’ RACE_PCR) basically CH5424802 concentration as described previously [41] with use of crtE-rv and crtB2-rv primers, respectively, for

reverse transcription. Both, individual C tailing and A tailing were performed and analyzed. RACE_PCR was performed with primers crtE-RACE and crtB2-RACE and either OligoT or OligoG. Sequencing of the generated PCR fragments was accomplished

using the suitable RACE primers and gave identical results for C tailing and A tailing reactions. Reverse transcription (RT) for the analysis of transcription units Total RNA was isolated from an exponentially growing culture of C. glutamicum WT as described previously [40]. Purified RNA was analyzed by UV-spectrometry in regard to quantity Fluorometholone Acetate and quality and was stored at −20°C until use. 2 μg of total RNA were used to perform reverse transcription to generate cDNA that was subsequently used as template for PCRs applying primer that bind at adjacent genes. The reverse transcription reactions were performed using SuperScript™ II reverse transcriptase (Invitrogen, Karlsruhe, Germany), and the remaining RNA was removed by the use of RNase H (MBI Fermentas GmbH, St. Leon-Rot). Overexpression of carotenogenic genes from C. glutamicum Plasmids harboring a carotenogenic gene allowed its IPTG-inducible overexpression and were based on pEKEx3 [42] or pVWEx1 [43], respectively. Amplification of a carotenogenic gene by polymerase chain reaction (PCR) from genomic DNA of C. glutamicum WT, which was prepared as described [44], was carried out using the respective primers (Additional file 3: Table S1). The amplified products were cloned into the appropriately restricted pEKEx3 or pVWEx1 plasmid DNA. Deletion of carotenogenic genes in C. glutamicum WT For deletion of a carotenogenic gene, the suicide vector pK19mobsacB was used [36].