Constitutive expression of heterologous genes for astaxanthin production in Synechocystis sp. PCC 6803

Astaxanthin (Asx) is a keto-carotenoid which possesses a unique antioxidant activity compared to other carotenoids, more than 10 times stronger than ?-carotene and hundred times stronger than vitamin E. Moreover, it plays a key-role in aquaculture, being responsible of the characteristic pinkish-red colour of salmon, trout, shrimp, and crustaceans. Due to its properties, Asx is largely employed in a wide spectrum of industrial applications in the food, feed, nutraceutical, cosmetic and pharmaceutical sectors. To better meet the increasing market demand, the need to find an alternative natural source beside that of Haematococcus pluvialis, hampered by slow growth times and low cell density, is emerging. The model cyanobacterium Synechocystis sp. PCC 6803 has gained significant attention as sustainable source for biomass, biofuels and added-value compounds. Synechocystis is naturally able to accumulate ?-carotene (?-car) and zeaxanthin (Zea), the metabolic precursors of Asx. Thus, the addition of a ?-carotene ketolase (CrtW) and a ?-carotene hydroxylase (CrtZ) is expected to allow the synthesis of the desired compound. The aim of our study is to obtain an optimized system for the production of Asx and other valuable keto-carotenoids, such as canthaxanthin (Can). To this end, we successfully transformed Synechocystis with an integrative plasmid vector, carrying the CrtW and CrtZ genes from Brevundimonas. The engineered strains constitutively express the two genes singularly (W and Z) or in tandem (WZ and ZW). The obtained results, whole cells absorption spectra and carotenoid profiles, demonstrate the potential of Synechocystis as useful platform for non-endogenous keto-carotenoids biosynthesis. The W strain almost exclusively accumulates Can, although showing some production instability depending on the age of the culture, while both the double-mutant strains accumulate substantial amounts of Can and Asx, with the ZW strain being the most efficient Asx producer.