Metabolic engineering of ketocarotenoids biosynthetic pathway in Chlamydomonas reinhardtii strain CC-4102

In Chlamydomonas reinhardtii, ketocarotenoid biosynthesis is limited to the diploid zygospore stage. In this study, we attempted to engineer the ketocarotenoid pathway into Chlamydomonas haploid vegetative green cells by overexpressing the key enzyme beta-carotene ketolase (CrBKT). We chose strain CC-4102 for the approach; competitive pathways, alpha-carotene biosynthesis and xanthophyll cycle are silenced in this strain. Driven by the strong constitutive HSP70/RBCS2 promoter CrBKT overexpression resulted in the production of canthaxanthin, the ketolation product from beta-carotene as well as a drastic reduction in the chlorophyll concentration. Intriguingly, these phenotypes could only be detected from lines transformed and grown heterotrophically in the dark. Once exposed to light, these transformants lost the aforementioned phenotypes as well as their antibiotic resistance. This phenomenon is in agreement with the fact that we were unable to recover any canthaxanthin-producing line among light-selected transformants.