Astaxanthin (3,3'-dihydroxy-4,4'-diketo-β-carotene) (1) 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 1 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 1 is well studied; however, questions remain that prevent optimized production in heterologous systems. Presented is a direct comparison of 12 β-carotene (2) hydroxylases derived from archaea, bacteria, cyanobacteria, and plants. Expression in Escherichia coli enables a comparison of catalytic activity with respect to zeaxanthin (3) and 1 biosynthesis. The most suitable β-carotene hydroxylases were subsequently expressed from an efficient dual expression vector, enabling 1 biosynthesis at levels up to 84% of total carotenoids. This supports efficient 1 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.