Abstract
This chapter discusses methods for the inducible expression of carotenoid biosynthesis genes in Rhodobacter capsulatus and Escherichia coli along with novel procedures for the purification of phytoene desaturases. Carotenoid desaturases are membrane-bound enzymes in both bacteria and plants. The functional expression of Neurospora crassa and Glycine max (soybean) cDNAs coding for carotenoid desaturases in mutants of the photosynthetic bacterium Rhodobacter capsulatus have been achieved. Thus, it is logical to expect functional expression in R. capsulatus of carotenoid desaturase cDNAs from other species. In addition, it is possible that cDNAs coding for carotenoid enzymes other than desaturases could be expressed in R. capsulatus carotenoid mutants. Phytoene desaturases from various organisms catalyze a different number of desaturations. Rhodobacter capsulatus phytoene desaturase converts phytoene to neurosporene, a three-step desaturation reaction. Neurospora crassa Al-1 catalyzes six desaturations. Soybean Pds1 converts phytoene to ζ-carotene, a two-step desaturation reaction. Expression of al-1 in a phytoene-accumulating strain of R. capsulatus leads to the accumulations of lycopene and 3,4 dehydrolycopene. Expression of pds in this bacterial strain results in the accumulation of ζ-carotene. Lycopene, 3,4-dehydrolycopene, and ζ-carotene are not normally accumulated in R. capsulatus .
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