, 2007). Cloning and the heterogeneous expression of crtI from Rba. azotoformans were performed to understand the product pattern of CrtI. A 1557 bp crtI gene was amplified via PCR from the Rba. azotoformans CGMCC 6086 genome with primers Ra-If and Ra-Ir (Table 1). A 518-amino acid protein was encoded with a predicted molecular

mass of 57.28 kDa. The crtI gene was inserted into pET22b and transformed into E. coli BL21 (DE3). The ratio of CrtI to total Ku-0059436 price E. coli protein was approximately 7–10% after induction with IPTG. The subunit molecular mass of 57 kDa determined via SDS-PAGE (Fig. 3) was consistent with the predicted molecular mass. The product pattern of CrtI from

Rba. azotoformans was examined in vivo by co-transforming plasmid pET22b-I with plasmid pACYCDuet-EB into the E. coli BL21 (DE3). The transformant acquired a red color in LB culture after induction with IPTG. After cultivation for 5 h in LB medium with 0.5 mM IPTG, the recombinant E. coli produced three carotenoids (Fig. 4a) identified by molecular mass and absorption spectra as neurosporene, lycopene, and 3,4-didehydrolycopene Hormones antagonist (Fig. S3). Neurosporene has a relative molecular mass of 538.4 and three absorption maxima at 416, 440, and 469 nm. Lycopene has a relative molecular mass of 536.4 and three absorption maxima at 444, 472, and 504 nm. Meanwhile, 3,4-didehydrolycopene has a relative molecular mass of 534.4 and three absorption maxima at 469, 496, and 528 nm. After cultivation for 24 h, the relative 5-Fluoracil ic50 contents of neurosporene and lycopene in recombinant E. coli were approximately 23% and 75%, respectively, whereas 3,4-didehydrolycopene almost disappeared (Fig. 4b). This in vivo result showed that CrtI from Rba. azotoformans

CGMCC 6086 could produce three-step desaturated neurosporene and four-step desaturated lycopene as major products, together with small amounts of five-step desaturated 3,4-didehydrolycopene. The present study is the first to report that 3,4-didehydrolycopene could be produced by CrtI from Rhodobacter. CrtI would be a three-step phytoene desaturase in situ because carotenoids of the spheroidene series were synthesized in Rba. azotoformans CGMCC 6086. Therefore, the formation of lycopene and 3,4-didehydrolycopene in recombinant E. coli were probably due to neurosporene accumulation caused by the lack of hydroxyneurosporene synthase (CrtC) and CrtI kinetics. In a crtC deletion mutant of Rba. azotoformans CGMCC 6086 obtained via EMS and LiCl mutagenesis, carotenoid products contained approximately 90% neurosporene and 10% lycopene (data not shown). The kinetics could also affect product patterns of CrtI. CrtI from Rvi. gelatinosus and P.