Heterologous biosynthesis of lutein in S. cerevisiae enabled by temporospatial pathway control

The market-expanding lutein is currently mainly supplied by plant extraction, with microbial fermentation using engineered cell factory emerging as a promising substitution. During construction of lutein-producing yeast, α-carotene formation through asymmetric ε- and β-cyclization of lycopene was found as the main limiting step, attributed to intra-pathway competition of the cyclases for lycopene, forming β-carotene instead. To solve this problem, temperature-responsive expression of β-cyclase was coupled to constitutive expression of ε-cyclase for flux redirection to α-carotene by allowing ε-cyclization to occur first. Meanwhile, the ε-cyclase was engineered and re-localized to the plasma membrane for further flux reinforcement towards α-carotene. Finally, pathway extension with proper combination of carotenoid hydroxylases enabled lutein (438 μg/g dry cells) biosynthesis in S. cerevisiae. The success of heterologous lutein biosynthesis in yeast suggested temporospatial pathway control as a potential strategy in solving intra-pathway competitions, and may also be applicable for promoting the biosynthesis of other natural products.