Random mutagenesis and overexpression of rhodopin-3,4-desaturase allows the production of highly conjugated carotenoids in Rhodospirillum rubrum

The Methoxylated, Highly Conjugated C40 Carotenoids, Spirilloxanthin and Anhydrorhodovibrin, Can Be Separated Using High Performance Liquid Chromatography with Safe and Environmentally Friendly Solvents

“…It has been shown previously that Spx can be easily separated from its biosynthetic precursors (such as lycopene and anhydrorhodovibrin (Anhdrvbr) (Figure 1)) by using a simple isocratic HPLC method (on a BioRad ® system) with an ODS-2 (C 18 ) column and MeOH/THF (98/2 ( v/v )) as running solvent [4,10]. In the present study, we initially wanted to reproduce this data by using a modern UHPLC-system (Dionex ® Ultimate 3000) and a new C 18 column (for details, see Materials and Methods section).…”

“…Comparison of HPLC runs with ( A ) the BioRad ® HPLC system [4], and ( B ) the Dionex ® UHPLC system. In both cases, a sample containing Spx as main component, and with minor components Anhdrvbr and lycopene, was injected.…”

“…Whereas those plant-derived carotenoids contain only 11 conjugated double bonds, which, depending on configuration and concentration, leads to colors from deep yellow to red, several bacterial species can produce deep-purple colored carotenoids with up to 13 conjugated double bonds, such as spirilloxanthin (Spx), a linear C 40 carotenoid with a methoxy group at both ends of the molecule (Figure 1), which is the wild-type carotenoid found in the photosynthetic membranes of the purple, Gram-negative, non-sulphur bacterium, Rhodospirillum rubrum . Even higher conjugation lengths (14–15 double bonds) can be achieved by using in vitro mutagenesis strategies [3,4]. Carotenoids with higher conjugation lengths have been suggested to be more effective anti-oxidants in cancer prevention and treatment than the commonly used carotenoids, such as lycopene [5].…”

“…However, the use of Ac obstructs UV-detection during the HPLC run, and the relatively high water content (nearly 30% at the start of the gradient) led to solubility problems with samples containing lipids. Therefore, in this study, we re-focused on the use of MeOH as the main solvent for the HPLC-method (MeOH/THF (98/2 ( v/v ))), which, in previous studies [4,10] had been shown to yield an excellent separation of Spx and all of the precursors starting from phytoene. The method had to be adapted for its use in an UHPLC system.…”