Colocalization of enzymes in Saccharomyces cerevisiae via dynamic assembly of Cas6-mediated RNA scaffold

Date
March 22, 2022

Slow diffusion of intracellular molecules has been a critical challenge to maintaining efficient cellular functions. Cells have naturally evolved to compartmentalize many of their metabolic pathways within intracellular organelles or to cluster their enzymes into functional metabolons for production of specific natural products. To overcome this diffusion-limited bottleneck in the biosynthesis of heterologous products in S. cerevisiae, we have devised a novel strategy to dynamically control the formation of synthetic metabolon complexes by utilizing a Cas6-mediated RNA scaffold and strand displacement mechanism. Using split luciferase as a reporter system, we have demonstrated the feasibility of utilizing these scaffolds to dynamically colocalize two enzymes in close proximity. The luminescence in the strains containing functional scaffold constructs was four times higher than in the control strains. We then assessed the system for the localization of pathway enzymes to form metabolons. Using the highly branched Violacein pathway, the Cas6-mediated RNA scaffold allowed redirection of carbon flux away from prodeoxyviolacein and toward either deoxyviolacein or proviolacein. We have also adapted this novel synthetic biology tool for the dynamic assembly of a metabolon for the synthesis of the polyketide triacetic acid lactone (TAL) in S. cerevisiae. The approach and our results demonstrating the benefits of this dynamic colocalization method will be presented.

Related Products

Thumbnail for Novel carbon source-repressible promoter for stationary phase production of polyketides in the non-conventional yeast Kluyveromyces marxianus | Poster Board #3607
Novel carbon source-repressible promoter for stationary phase production of polyketides in the non-conventional yeast Kluyveromyces marxianus | Poster Board #3607
The non-conventional yeast _Kluyveromyces marxianus_ has garnered significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. K…
Thumbnail for Controlling chemoenzymatic chemistry for the construction of coiled-coil nanostructures
Controlling chemoenzymatic chemistry for the construction of coiled-coil nanostructures
The foundation of nature’s most impressive materials are proteins, macromolecules composed of amino acid sequences that can fold and assemble into unique structures. The modularity and precision of proteinaceous building-blocks affords a bottom-up design of complex bioinspired structures…
Thumbnail for Enhanced production of acetyl-CoA-based products via yeast peroxisomal surface display
Enhanced production of acetyl-CoA-based products via yeast peroxisomal surface display
Colocalization of enzymes is a proven approach to increase pathway flux and the synthesis of non-native products. Here, we develop a method for enzyme colocalization using the yeast peroxisomal membrane as an anchor point…
Thumbnail for Developing a highly specific, modular platform for conditional protein degradation
Developing a highly specific, modular platform for conditional protein degradation
Current approaches to balancing dysregulated protein levels focus on either RNA or protein level control schemes. Protein level control mechanisms can deplete both pre-existing targets and those that will continue to be produced…