Identification of a carotenoid oxygenase synthesizing acyclic xanthophylls: Combinatorial biosynthesis and directed evolution

A carotenoid desaturase homolog from Staphylococcus aureus (CrtOx) was identified. When expressed in engineered E. coli cells synthesizing linear C-30 carotenoids, polar carotenoid products were generated, identified as aldehyde and carboxylic acid C-30 carotenoid derivatives. The major product in this engineered pathway is the fully desaturated C-30 dialdehyde carotenoid 4,4'-diapolycopen-4,4'-dial. Very low carotenoid yields were observed when CrtOx was complemented with the C-40 carotenoid lycopene pathway. But extension of an in vitro evolved pathway of the fully desaturated 2,4,2',4'-tetradehydrolycopene produced the structurally novel fully desaturated C-40 dialdehyde carotenoid 2,4,2',4'-tetradehydrolycopendial. Directed evolution of CrtOx by error-prone PCR resulted in a number of variants with higher activity on C-40 carotenoid substrates and improved product profiles. These findings may provide new biosynthetic routes to highly polar carotenoids with unique spectral properties desirable for a number of industrial and pharmaceutical applications.