Comparative analysis of ß-carotene hydroxylase genes for astaxanthin biosynthesis.

Astaxanthin (3,3'-dihydroxy-4,4'-diketo-ß-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 ß-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 ß-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 ß-carotene ketolase and ß-carotene hydroxylase genes. Moreover, our work suggests that the most efficient route for astaxanthin biosynthesis proceeds by hydroxylation of ß-carotene to zeaxanthin, followed by ketolation.

A high-throughput screen for the identification of improved catalytic activity: ß-carotene hydroxylase.

Astaxanthin is a natural product of immense value. Its biosynthesis has been investigated extensively and typically requires the independent activity of two proteins, a ß-carotene ketolase and ß-carotene hydroxylase. Rational engineering of this pathway has produced limited success with respect to the biological production of astaxanthin. Random mutagenesis of the ß-carotene ketolase has also been pursued. However, to date, no suitable method has been developed for the investigation of the ß-carotene hydroxylase because ß-carotene and zeaxanthin cannot be differentiated visually, unlike ß-carotene and canthaxanthin. Thus, random mutagenesis and efficient selection of improved ß-carotene hydroxylase clones is not feasible. Presented here are the steps required for the efficient generation of a ß-carotene hydroxylase random mutagenesis library in Escherichia coli. Subsequently presented is a novel high-throughput screening method for the rapid identification of clones with enhanced ß-carotene hydroxylase activity. The validity of the presented method is confirmed by functional expression of the mutated proteins, combined with accurate quantification of produced carotenoids. The developed method has potential applications in the development of biological systems for improved carotenoid biosynthesis, as well as robust astaxanthin production.

Efficient extraction of canthaxanthin from Escherichia coli by a 2-step process with organic solvents.

Canthaxanthin has a substantial commercial market in aquaculture, poultry production, and cosmetic and nutraceutical industries. Commercial production is dominated by chemical synthesis; however, changing consumer demands fuel research into the development of biotechnology processes. Highly productive microbial systems to produce carotenoids can be limited by the efficiency of extraction methods. Extraction with hexane, acetone, methanol, 2-propanol, ethanol, 1-butanol, tetrahydrofuran and ethyl acetate was carried out with each solvent separately, and subsequently the most efficient solvents were tested in combination, both as mixtures and sequentially. Sequential application of methanol followed by acetone proved most efficient. Extraction efficiency remained stable over a solvent to biomass range of 100:1 to 55:1, but declined significantly at a ratio of 25:1. Application of this method to a canthaxanthin-producing Escherichia coli production system enabled efficient canthaxanthin extraction of up to 8.5 mg g(-1) dry biomass.