Cloning and expression in Escherichia coli of the gene encoding beta-C-4-oxygenase, that converts beta-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis.

In the green alga Haematococcus pluvialis the ketocarotenoid astaxanthin accumulates under stress conditions. Astaxanthin is a red carotenoid pigment which possess antioxidative activity. We have cloned the gene for beta-C-4 oxygenase (beta-carotene ketolase) from the green algae H. pluvialis. The cloning method took advantage of a strain of E. coli which was genetically engineered to produce beta-carotene. An expression cDNA library of H. 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 beta-C-4 oxygenase that catalyzes the conversion of beta carotene to canthaxanthin via echinenone. This enzyme does not convert zeaxanthin to astaxanthin. It is concluded that in H. pluvialis astaxanthin is synthesized via canthaxanthin and therefore an additional enzyme is predicted, which converts canthaxanthin to astaxanthin.