Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli

• Published in: Plant molecular biology Vol. 29; no. 2; pp. 343 - 352
• Main Authors: Kaiiwara, S, Kakizono, T, Saito, T, Kondo, K, Ohtani, T, Nishio, N, Nagai, S, Misawa, N
• Format: Journal Article
• Language: English
• Published: Netherlands 01.10.1995
• Subjects: Amino Acid Sequence; amino acid sequences; astaxanthin; Bacterial Proteins - genetics; Base Sequence; beta Carotene; beta-carotene ketolase; beta-carotene oxygenase; Binding Sites - genetics; biosynthesis; Blotting, Northern; canthaxanthin; carotenoids; Carotenoids - analysis; Carotenoids - biosynthesis; Carotenoids - metabolism; Chlorophycota; Chlorophyta - enzymology; Chlorophyta - genetics; Chromatography, High Pressure Liquid; Circular Dichroism; complementary DNA; Conserved Sequence; DNA Mutational Analysis; DNA, Complementary - genetics; enzyme activity; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; genbank/d45881; Gene Library; gene transfer; genetic transformation; Molecular Sequence Data; nucleotide sequences; oxygenases; Oxygenases - genetics; Pantoea ananatis; Sequence Analysis, DNA; Sequence Deletion; Sequence Homology, Amino Acid; Xanthophylls
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1007/bf00043657

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.