A highly selective biosynthetic pathway to non-natural C 50 carotenoids assembled from moderately selective enzymes

Synthetic biology aspires to construct natural and non-natural pathways to useful compounds. However, pathways that rely on multiple promiscuous enzymes may branch, which might preclude selective production of the target compound. Here, we describe the assembly of a six-enzyme pathway in Escherichia coli for the synthesis of C 50-astaxanthin, a non-natural purple carotenoid. We show that by judicious matching of engineered size-selectivity variants of the first two enzymes in the pathway, farnesyl diphosphate synthase (FDS) and carotenoid synthase (CrtM), branching and the production of non-target compounds can be suppressed, enriching the proportion of C 50 backbones produced. We then further extend the C 50 pathway using evolved or wild-type downstream enzymes. Despite not containing any substrate-or product-specific enzymes, the resulting pathway detectably produces only C 50 carotenoids, including ∼90% C₅₀-astaxanthin. Using this approach, highly selective pathways can be engineered without developing absolutely specific enzymes.