D90087). These primer pairs were used in amplification of the farnesyl-diphosphate Ganetespib molecular weight synthase gene (crtE) and phytoene synthase gene (crtB). The genomic DNA of P. ananatis ATCC 19321 was used as the

template. The crtE and crtB genes were inserted into the co-expressing vector pACYCDuet-1 (Novagen, Germany) at BamHI and SacI, NdeI and KpnI restriction sites to construct the plasmid pACYCDuet-EB. pACYCDuet-EB was transformed into E. coli BL21 (DE3). After induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25 °C for 15 h, recombinant E. coli cells were harvested in preparation for phytoene extraction. The crtI gene of Rba. azotoformans CGMCC 6086 was amplified through PCR from its genome with primers Ra-If and Ra-Ir (Table 1). The crtI fragment was digested AG-014699 order with NdeI and HindIII restriction enzymes and inserted into the expression vector pET22b (Novagen, Germany) to form pET22b-I. The plasmid pET22b-I was transformed into E. coli BL21 (DE3) to obtain the product of the crtI gene. After induction with IPTG as described previously, recombinant E. coli cells harboring plasmid pET22b-I were harvested and resuspended in 100 mM Tris–HCl buffer (pH 7.9). The suspension cells were disintegrated via ultrasonication,

and the supernatant was used as the crude enzyme in the in vitro reaction. The purification of CrtI was performed via nickel affinity chromatography (Qiagen, Switzerland). The total protein content of the supernatant was determined using the Bradford method (Bradford, 1976). The relative content of CrtI in the supernatant was calculated by comparing the scanning density of the CrtI band with the lane from SDS-PAGE. The plasmid pET22b-I was co-transformed

Branched chain aminotransferase with pACYCDuet-EB into E. coli BL21 (DE3) to examine the product pattern of CrtI in vivo. The transformant showing a red color was selected and cultured in LB medium containing 50 μg mL−1 of ampicillin and 25 μg mL−1 of chloramphenicol and induced with IPTG as described previously. The cells were then harvested in preparation for carotenoid extraction. The supernatant obtained from the lysate of the E. coli BL21 (DE3) transformant harboring the plasmid pET22b-I was used as the crude enzyme for the in vitro reaction. The reaction mixture (0.5 mL) contained 65 μg CrtI in 400 μL supernatant (final concentration 130 μg mL−1), 400 μg emulsified soybean l-α-phosphatidylcholine, and phytoene with final concentrations of 0.13, 0.26, 0.65, 1.3, and 2.6 μM. After mixing by ultrasonication and incubating in the dark at 30 °C for 5 h with shaking at 200 r.p.m., the reaction was terminated with the successive addition of 15 μL NaOH (2 M), 15 μL SDS (10%), and 300 μL CH3COONa (3 M, pH 4.8) solutions. The mixture was centrifuged, and the precipitate was prepared for carotenoid extraction. Carotenoids in Rba. azotoformans CGMCC 6086 cells, recombinant E. coli cells, and the precipitate in vitro were extracted.