Carotenoid biosynthesis by a cell-free preparation from a Flavobacterium species.

Previous work with Flavobacterium strain R1519 has involved the use of inhibitors, especially nicotine, to demonstrate the conversions, in vioo, of lycopene (#,$-carotene) into P-carotene (P,P-carotene) and of lycopene, 8-carotene and rubixanthin (PAcaroten-3-01) into P-cryptoxanthin (D,P-caroten-3-01) and zeaxanthin (D,P-carotene3,3'-diol) (McDermott et al., 1974). The next aim was to develop a cell-free system that could be used to study in detail the individual reactions of carotenoid biosynthesis in bacteria. The only systematic work reported on carotenoid biosynthesis in cell-free systems has been that of Porter (1971) with a tomato plastid preparation, and Davies (1 973) with crude extracts of the fungus Phycomyces blakesleeanus. In the present work, a high-zeaxanthin-producing Flavobacterium strain, R1560, was used, and, in the method finally adopted, bacterial cells, in 0.1 M-Tris-HCl buffer, pH7.0, were subjected to ultrasonic disruption for a total of 2min, with intermittent periods of cooling. This gave an orange cell-free supernatant (100OOOg) capable of efficient incorporation of [2-14C]mevalonate into the carotenoid precursor phytoene (7,8,11,12,7',8',11',12'-octahydro-yl,yl-carotene) (up to 70% incorporation in 3 h in a O S m l incubation containing approx. 30mg of protein). With crude lower-speed but substantially cell-free supernatants (3000-5000g), [2-'4C]mevalonate was also incorporated into other carotenoids, e.g. phytofluene (7,8,11,12,7',8'-hexahydro-yl,yl-carotene) (-carotene (7,8,7',8'-tetrahydro-p,ylv-carotene) and its unsymmetrical isomer (7,8,11,12-tetrahydro-yl,y/-carotene), neurosporene (7,8-dihydro-yl,yl-carotene), lycopene, P-carotene and zeaxanthin. The most active preparations were obtained when the cells underwent ultrasonic treatment in the presence of the detergent Tween 80 (2%, v/v). ATP (5mM), Mgz+ (15m~) and Mn2+ ( 3 m ~ ) were essential requirements, and the formation of phytoene was considerably enhanced by the presence of a range of cofactors (NAD+, NADH, NADP+, NADPH, FAD, all at 1 mM) similar to that used by Davies (1973) with the Phycomyces system. In contrast with the Phycomyces system and the tomato plastid system of Porter (1971), the use of anaerobic conditions and thiol reagents (glutathione, dithiothreitol, mercaptoethanol) did not improve the rate of phytoene production by the Flavobacterium preparation. The phytoene produced was almost entirely (99%) the 15-cis isomer (the isomer normally predominating in whole cells of Flavobacteriurn R1560), whereas it was mainly the all-trans isomers of the other carotenoids that were labelled from [2-14C]mevalonate. The incorporation of [2'*C]mevalonate into other carotenoids was improved by increasing the concentrations of added cofactors (up to 6 m ) but never reached the same rates as the incorporation into phytoene. Apart from phytoene, the best incorporation of mevalonate was into phytofluene and jl-carotene (approx. 5 %). Incorporation into zeaxanthin, the normal main carotenoid of Flavobacterium R1560 was relatively low and required the presence of 0 2 . With the same preparation, the conversion of ['4C]phytoene (15-cis isomer) into other carotenoids, notably phytofluene and P-carotene (approx. 5 % incorporation) and zeaxanthin has also been demonstrated. These conversions were enhanced by the presence of the range of cofactors, as before, and did not require anaerobic conditions or illumination. Substantial isomerization of 1 5-cis-phytoene to the all-trans isomer was