, 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 screening assay 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 Opaganib (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 (-)-p-Bromotetramisole Oxalate 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.