Assembly of Metabolons in Yeast Using Cas6-Mediated RNA Scaffolding

Cells often localize pathway enzymes in close proximity to reduce substrate loss via diffusion and to ensure that carbon flux is directed toward the desired product. To emulate this strategy for the biosynthesis of heterologous products in yeast, we have taken advantage of the highly specific Cas6-RNA interaction and the predictability of RNA hybridizations to demonstrate Cas6-mediated RNA-guided protein assembly within the yeast cytosol. The feasibility of this synthetic scaffolding technique for protein localization was first demonstrated using a split luciferase reporter system with each part fused to a different Cas6 protein. In Saccharomyces cerevisiae, the luminescence signal increased 3.6- to 20-fold when the functional RNA scaffold was also expressed. Expression of a trigger RNA, designed to prevent the formation of a functional scaffold by strand displacement, decreased the luminescence signal by nearly 2.3-fold. Temporal control was also possible, with induction of scaffold expression resulting in an up to 11.6-fold increase in luminescence after 23 h. Cas6-mediated assembly was applied to create a two-enzyme metabolon to redirect a branch of the violacein biosynthesis pathway. Localizing VioC and VioE together increased the amount of deoxyviolacein (desired) relative to prodeoxyviolacein (undesired) by 2-fold. To assess the generality of this colocalization method in other yeast systems, the split luciferase reporter system was evaluated in Kluyveromyces marxianus; RNA scaffold expression resulted in an increase in the luminescence signal of up to 1.9-fold. The simplicity and flexibility of the design suggest that this strategy can be used to create metabolons in a wide range of recombinant hosts of interest.