Cloning and expression inEscherichia coli of the gene encoding β‐C‐4‐oxygenase, that converts β‐carotene to the ketocarotenoid canthaxanthin inHaematococcus pluvialis

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Abstract
In the green algaHaematococcus pluvialis the ketocarotenoid astaxanthin accumulates under stress conditions. Astaxanthin is a red carotenoid pigment which possess antioxidative activity. We have cloned the gene for β-C-4 oxygenase (β-carotene ketolase) from the green algaeH. pluvialis. The cloning method took advantage of a strain ofE. coli which was genetically engineered to produce β-carotene. An expression cDNA library ofH. pluvialis was transfected to cells of this strain and visually screened for brown-red pigmented colonies. One colony out of 100,000 transformants showed color change due to accumulation of canthaxanthin. The cDNA clone in this transformant colony encodes the enzyme β-C-4 oxygenase that catalyzes the conversion of β carotene to canthaxanthin via echinenone. This enzyme does not convert zeaxanthin to astaxanthin. It is concluded that inH. pluvialis astaxanthin is synthesized via canthaxanthin and therefore an additional enzyme is predicted, which converts canthaxanthin to astaxanthin.