Comparative Analysis of β-Carotene Hydroxylase Genes for Astaxanthin Biosynthesis

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.