초록
<▼1><P>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 <I>Escherichia coli</I> for the synthesis of C<SUB>50</SUB>-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<SUB>50</SUB> backbones produced. We then further extend the C<SUB>50</SUB> pathway using evolved or wild-type downstream enzymes. Despite not containing any substrate- or product-specific enzymes, the resulting pathway detectably produces only C<SUB>50</SUB> carotenoids, including ∼90% C<SUB>50</SUB>-astaxanthin. Using this approach, highly selective pathways can be engineered without developing absolutely specific enzymes.</P></▼1><▼2><P>[GRAPHIC OMISSION]Synthetic engineering of complex pathways is often hindered by pathway branching and generation of non-target compounds. Here, the authors show that by judicious combination of moderately selective enzyme variants, a non-natural C50 carotenoid can be generated in bacteria with minimal production of unwanted compounds.</P></▼2>