In a green alga, Haematococcus pluvialis, a morphological change of vegetative cells into cyst cells was rapidly induced by the addition of acetate or acetate plus Fe2+ to the vegetative growth phase. Accompanied by cyst formation, algal astaxanthin formation was more enhanced by the addition of acetate plus Fe2+ than by the addition of acetate alone. Encystment and enhanced carotenoid biosynthesis were inhibited by either actinomycin D or cycloheximide. However, after cyst formation was induced by the addition of acetate alone, carotenoid formation could be enhanced with the subsequent addition of Fe2+ even in the presence of the inhibitors. The Fe2+-enhanced carotenogenesis was inhibited by potassium iodide, a scavenger for hydroxyl radical, suggesting that hydroxyl radical formed by an iron-catalyzed Fenton reaction may be required for enhanced carotenoid biosynthesis. Moreover, it was demonstrated that four active oxygen species, singlet oxygen, superoxide anion radical, hydrogen peroxide, and peroxy radical, were capable of replacing Fe2+ in its role in the enhanced carotenoid formation in the acetate-induced cyst. From these results, it was concluded that oxidative stress is involved in the posttranslational activation of carotenoid biosynthesis in acetate-induced cyst cells.
We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, beta-carotene ketolase (beta-carotene oxygenase), which converted beta-carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3'S), which was identified after purification by a variety of spectroscopic methods.
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