amplified and ligated. These ligated manXZ and yjfP fragments had been then cloned into the

amplified and ligated. These ligated manXZ and yjfP fragments had been then cloned into the pACYC184 involving the EcoRI and NcoI web sites. The resulting plasmids had been named pAC-manXYZ and pAC-yjfP. The KDPT fragment was digested with EcoRV and SacI and inserted in to the SmaI and SacI web-sites of pAC-manXYZ and pAC-yjfP, resulting within the plasmids pAC-manXYZ-KDPT and pAC-yjfP-KDPT, respectively. The IDI gene fragment was amplified by PCR and inserted in to the XhoI and KpnI sites between Ptac and TrrnB with the pAC-manXYZ-KDPT, yielding the plasmid pAC-manXYZKDPT-HI. The Aacl-pnbA fragment was amplified by PCR with pAC-Mev/Scidi/Aacl/pnbA as a template and inserted in to the plasmid pAC-yjfP-KDPT, resulting in the plasmid pAC-yjfP-KDPTAacl-pnbA. The DNA fragments for the recombination had been also obtained by PCR applying the primers shown in Supplementary Table S2. Either E. coli JM101(DE3) or JM101 (DE3) (manXYZ)[IDI] carrying the Red helper plasmid pKD46 (25) was grown in SOB medium with ampicillin and 1 mM L-arabinose. The electroporationcompetent cells had been LPAR1 Antagonist site prepared as described previously (26). The cells had been mixed with PCR fragments in an ice-cold 0.1cm cuvette and electroporated at 1.eight kV (25 , 200 ; Gene Pulser Xcell, Bio-Rad, USA). Right after choice with kanamycin, the transformants had been cultured overnight at 42 C and tested for ampicillin sensitivity to verify for loss on the helper plasmid. Colony PCR was then performed to confirm the genome recombination. The FLP helper plasmid pCP20 (27) was introduced in to the transformants to remove the NPT gene involving FRT sequences at 30 C. Just after choice with ampicillin resistance, the transformants were cultured at 42 C overnight and tested for kanamycin and ampicillin sensitivity to check for the loss with the NPT gene and helper plasmid, respectively.two.four Fermentation conditionsCells were cultured overnight in liquid Luria Broth (LB) medium at 30 C, after which, 10 ml of the cell culture was inoculated into 1 l of modified Terrific Broth (TB) medium (per liter: 12 g Bacto Tryptone; Gibco), 24 g Bacto yeast extract, 9.4 g K2 HPO4 , two.two g KH2 PO4 and appropriate antibiotics (100 mg spectinomycin, 10 mg tetracycline and 30 mg chloramphenicol). Cultures have been grown at 25 C inside a 3-l jar fermenter (BMJ-03P, In a position). The pH was maintained at 7.0 by automatic addition of 28 NH4 OH and 25 H3 PO4 . The agitation speed was one hundred rpm. In the time of inoculation, CB2 Antagonist list dissolved oxygen levels had been permitted to fall to ten of O2 saturation with a continuous air provide of 1 volume per minute. The glucose concentration was maintained at 0.four g/l by the addition of 15 (w/v) glucose option. 0.1 mM IPTG and 0.1 (v/v) ethyl 3-oxobutanate had been then added for the culture when Optical Density at 600 nm (OD600 ) reached ten.2.five Detection and quantification of chemical compoundsGlucose in the culture medium was analyzed by the mutarotaseglucose method working with a Glucose CII Test Wako (Wako, Japan). To analyze carotenoid compounds within the culture medium, cultures have been collected every single 12 h by an autosampler (LA-11, In a position). Cells have been corrected by centrifugation at 5000 g for 5 min and stored at -20 C. Cells from 0.two ml culture medium have been homogenized with 0.5 ml acetone. About 1 ml hexane/diethyl ether (1:1) was added to acetone extract and vortexed nicely. Also, 1 ml water was added andFigure two. Effect in the -monocyclase around the -carotene production. HPLC chromatograms from the extracts from E. coli obtaining the plasmids pAC-HIEBIYm (A), pAC-HIEBIA (B),