Regulation of astaxanthin and its intermediates through cloning and genetic transformation of Beta-carotene ketolase in Haematococcus pluvialis

Astaxanthin, a high-value ketocarotenoid used in the pharmaceutical and nutraceutical industries is
mainly produced from green alga, Haematococcus pluvialis. It is biosynthesized by the action of key
enzyme, ␤-carotene ketolase (BKT) on ␤-carotene through intermediates echinenone and canthaxan-
thin. In this study, the ␤-carotene ketolase (bkt) gene was isolated from H. pluvialis and cloned in a vector
pRT100 and further mobilized to a binary vector pCAMBIA 1304. The T-DNA of pCAMBIA 1304, which
consists of cloned bkt, was successfully transformed to H. pluvialis through Agrobacterium mediation. The
cloning and transformation of bkt in H. pluvialis was confirmed by Southern blotting and also by PCR
analysis. Total carotenoids and astaxanthin content in the transformed cells were found to be 2–3-fold
higher, while the intermediates like echinenone and canthaxanthin were found to be 8–10-fold higher
than in the control cells. The expression level of carotenogenic genes like phytoene synthase (psy), phy-
toene desaturase (pds), lycopene cyclase (lcy), bkt, and ␤-carotene hydroxylase (bkh) were found to be
higher in transformed cells compared to the non-transformed (NT) H. pluvialis.