In vivoco-localization of enzymes on RNA scaffolds increases metabolic production in a geometrically dependent manner

Abstract

Co-localization of biochemical processes plays a key role in the directional control of metabolic fluxes toward specific products in cells. Here, we employ in vivo scaffolds made of RNA that can bind engineered proteins fused to specific RNA binding domains. This allows proteins to be co-localized on RNA scaffolds inside living Escherichia coli. We assembled a library of eight aptamers and corresponding RNA binding domains fused to partial fragments of fluorescent proteins. New scaffold designs could co-localize split green fluorescent protein fragments to produce activity as measured by cell-based fluorescence. The scaffolds consisted of either single bivalent RNAs or RNAs designed to polymerize in one or two dimensions. The new scaffolds were used to increase metabolic output from a two-enzyme pentadecane production pathway that contains a fatty aldehyde intermediate, as well as three and four enzymes in the succinate production pathway. Pentadecane synthesis depended on the geometry of enzymes on the scaffold, as determined through systematic reorientation of the acyl-ACP reductase fusion by rotation via addition of base pairs to its cognate RNA aptamer. Together, these data suggest that intra-cellular scaffolding of enzymatic reactions may enhance the direct channeling of a variety of substrates.

Self-assembly of large RNA structures: learning from DNA nanotechnology

“…AFM images provided in [3] indicate the assembly of structures with sizes in the range of 200-300 nm, however the structure of individual tiles or the yield of correct assemblies is unclear ( Figure 4D). Recently, an RNA DAO-O tile motif [11] was used to successfully co-localize enzymes in vivo [81], but features and yield of the assemblies are yet to be identified.…”

“…Artificial RNA scaffolds in cells could serve functional purposes and localize molecular components, as recently suggested [3,81]; it may be even possible to use cells as factories to produce and expel RNA scaffolds for other applications. In vivo assembled structures should form isothermally without a compromise in yield, and should not be toxic to cells or tissues, as well as having a functional role.…”

“…However, the complexity of an individual origami limits the scalability of its size relative to tile-based systems, which can assemble several microns in length. [45,32,89] 2D patterns [5] 3D patterns [12,38,43] Filaments, lattices [58,68,82] Rings, cubes, prisms, tubes [57,69,85] Lattices [78] Rings, cubes [77] Applications Algorithmic assembly [4,27,90] Functionalization with ligands [28,93] Scaffolding [91,92] Biomimetic assays [94] Drug delivery [56,60,61] Spatial organization of proteins [3,81] Recent work has tried to address this limitation with the development of origami tiling or connector systems. Individual origami tiles or blocks can be interfaced in two ways: the first approach is the use of sticky ends [39,40,41], where binding patterns are assigned by Watson-Crick base pairing.…”

Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

“…[141] These RNA-binding domains (aptamers) were fused with two modified enzymes (N-termini), the acyl-ACP reductase (AAR) and an aldehyde deformylation oxygenase (ADO), for the synthesis of pentadecane. This in vivo channeling systemi mprovedp roduction levels by 2.4-fold andi ntrinsic aldehyde reductase activity wasi nhibited.…”

Altering the orientation of a fused protein to the RNA-binding ribosomal protein L7Ae and its derivatives through circular permutation

“…This indicates that artificial RNP engineering is feasible and viable for exploring and modifying living systems. Recently, Sachdeva et al reported the in vivo expression of an RNP nanoobject displaying two enzymes that catalyze a cascade reaction of a pentadecane production pathway [4]. They found that the reaction efficiency depended on the geometry of the enzyme placement on the scaffold RNA, which can be changed by rotation via altering the length of the flanking RNA stem.…”