Comparative Analysis of β-Carotene Hydroxylase Genes for Astaxanthin Biosynthesis

Astaxanthin (3,3′-dihydroxy-4,4′-diketo-β-carotene) (<b>1</b>) is a carotenoid of significant commercial value due to its superior antioxidant potential, application as a component of animal feeds, and ongoing research that links its application to the treatment and prevention of human pathologies. The high commercial cost of <b>1</b> is also based upon its complex synthesis. Chemical synthesis has been demonstrated, but produces a mixture of stereoisomers with limited applications. Production from biological sources is limited to natural producers with complex culture requirements. The biosynthetic pathway for <b>1</b> is well studied; however, questions remain that prevent optimized production in heterologous systems. Presented is a direct comparison of 12 β-carotene (<b>2</b>) hydroxylases derived from archaea, bacteria, cyanobacteria, and plants. Expression in <i>Escherichia coli</i> enables a comparison of catalytic activity with respect to zeaxanthin (<b>3</b>) and <b>1</b> biosynthesis. The most suitable β-carotene hydroxylases were subsequently expressed from an efficient dual expression vector, enabling <b>1</b> biosynthesis at levels up to 84% of total carotenoids. This supports efficient <b>1</b> biosynthesis by balanced expression of β-carotene ketolase and β-carotene hydroxylase genes. Moreover, our work suggests that the most efficient route for astaxanthin biosynthesis proceeds by hydroxylation of β-carotene to zeaxanthin, followed by ketolation