ISOLATION AND FUNCTIONAL IDENTIFICATION OF A NOVEL CDNA FOR ASTAXANTHIN BIOSYNTHESIS FROM HAEMATOCOCCUS-PLUVIALIS, AND ASTAXANTHIN SYNTHESIS IN ESCHERICHIA-COLI

We succeeded in isolating a novel cDNA involved in astaxanthin biosynt hesis from the green alga Haematococcus pluvialis, by an expression cl oning method using an Escherichia coli transformant as a host that syn thesizes beta-carotene due to the Erwinia uredovora carotenoid biosynt hesis genes. The cloned cDNA was shown to encode a novel enzyme, beta- carotene ketolase (beta-carotene oxygenase), which converted beta-caro tene to canthaxanthin via echinenone, through chromatographic and spec troscopic analysis of the pigments accumulated in an E, coli transform ant. This indicates that the encoded enzyme is responsible for the dir ect conversion of methylene to keto groups, a mechanism that usually r equires two different enzymatic reactions proceeding via a hydroxy int ermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvi alis cDNA and the E. uredovora genes required for zeaxanthin biosynthe sis was also found to synthesize astaxanthin (3S, 3'S), which was iden tified after purification by a variety of spectroscopic methods.