High-Level Production of the Industrial Product Lycopene by the Photosynthetic Bacterium Rhodospirillum rubrum

ABSTRACTThe biosynthesis of the major carotenoid spirilloxanthin by the purple nonsulfur bacteriumRhodospirillum rubrumis thought to occur via a linear pathway proceeding through phytoene and, later, lycopene as intermediates. This assumption is based solely on early chemical evidence (B. H. Davies, Biochem. J. 116:93–99, 1970). In most purple bacteria, the desaturation of phytoene, catalyzed by the enzyme phytoene desaturase (CrtI), leads to neurosporene, involving only three dehydrogenation steps and not four as in the case of lycopene. We show here that the chromosomal insertion of a kanamycin resistance cassette into thecrtC-crtDregion of the partial carotenoid gene cluster, whose gene products are responsible for the downstream processing of lycopene, leads to the accumulation of the latter as the major carotenoid. We provide spectroscopic and biochemical evidence thatin vivo, lycopene is incorporated into the light-harvesting complex 1 as efficiently as the methoxylated carotenoids spirilloxanthin (in the wild type) and 3,4,3′,4′-tetrahydrospirilloxanthin (in acrtDmutant), both under semiaerobic, chemoheterotrophic, and photosynthetic, anaerobic conditions. Quantitative growth experiments conducted in dark, semiaerobic conditions, using a growth medium for high cell density and high intracellular membrane levels, which are suitable for the conventional industrial production in the absence of light, yielded lycopene at up to 2 mg/g (dry weight) of cells or up to 15 mg/liter of culture. These values are comparable to those of many previously describedEscherichia colistrains engineered for lycopene production. This study provides the first genetic proof that theR. rubrumCrtI produces lycopene exclusively as an end product.