P96a: Enhanced b-carotene hydroxylase genes: A simple high throughput screen, and their impact on astaxanthin biosynthesis

Carotenoids are natural products with significant commercial value due to their capacity to function as potent antioxidants. This results in imparted health benefits and commercial appeal as nutraceutical compounds. Carotenoid biosynthesis is divided into two parts. Carotene synthesis, proceeds in a linear highly conserved fashion, increasing both carbon number and the level of desaturation of the compound, and culminates with the production of b-carotene. Xanthophyll biosynthesis proceeds from b-carotene via the addition of functional groups to the two b-ionone rings. This proceeds in a less well conserved fashion. Astaxanthin is the ultimate product of carotenoid biosynthesis, based on commercial value and antioxidant capacity. The biosynthesis of astaxanthin requires the co-operative activity of two proteins, b-carotene hydroxylase and b-carotene ketolase. Several metabolic engineering projects have attempted to elucidate the specific route of astaxanthin biosynthesis, progressing from b-carotene, and develop systems capable of efficient astaxanthin biosynthesis. However, most efforts have been plagued by low final product purities, while also producing conflicting results with respect to the predicted route of biosynthesis. We previously demonstrated that efficient astaxanthin biosynthesis (>90% of total carotenoids) can be achieved through tight regulation of the two genes involved. Our current hypothesis states that increasing the catalytic activity of a b-carotene hydroxylase will increase astaxanthin biosynthesis within an expression system. In pursuit of this, we present a simple high throughput screen for the selection of enhanced b-carotene hydroxylase genes, a feat not possible via a simple visual screen, and subsequently demonstrate their impact on the biosynthesis of astaxanthin.