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

• Published in: Journal of biotechnology 2015-02, Vol.196-197, p.33-41
• Main Authors: Kathiresan, S., Chandrashekar, Arun, Ravishankar, G.A., Sarada, R.
• Format: Article
• Language: English
• Subjects: Agrobacterium; Agrobacterium - genetics; Algal Proteins - genetics; Astaxanthin; Beta carotene ketolase; Biotechnology; Canthaxanthin - biosynthesis; Carotenoids - biosynthesis; Chlorophyta - enzymology; Chlorophyta - genetics; Cloning; Cloning, Molecular; Genes; Genetic Vectors - genetics; Haematococcus pluvialis; Mathematical analysis; Oxygenases - genetics; Transformation; Transformation, Genetic; Transformations; Vectors (mathematics); Xanthophylls - biosynthesis
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2015.01.006

•Beta carotene ketolase (bkt) was cloned from genomic DNA of Haematococcus pluvialis.•Agrobacterium-mediated transformation of bkt to H. pluvialis for over-expression.•Transformed H. pluvialis showed higher content of total carotenoids and astaxanthin.•Echinenone and canthaxanthin were 8–10-fold higher in transformants cells.•Expression level of carotenogenic genes were found higher in transformed cells. 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, β-carotene ketolase (BKT) on β-carotene through intermediates echinenone and canthaxanthin. In this study, the β-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), phytoene desaturase (pds), lycopene cyclase (lcy), bkt, and β-carotene hydroxylase (bkh) were found to be higher in transformed cells compared to the non-transformed (NT) H. pluvialis.