In vivo co-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.

Figure 1.

Three dimensionalities of RNA scaffolds. Our designs allow formation of three different types of RNA scaffolds (14). (A) In the 0D case, a single RNA strand folds into a discrete unit presenting two aptamers. (B) The 1D polymerizing strands have ‘sticky’ ends that allow the individual units to form linear chains of aptamer sites through complementary base pairing. (C) In the case of 2D scaffolds, two different RNA oligonucleotides, A and B, come together to form unit tiles A₂B₂, which then polymerize in two dimensions through sticky end base pairs. Each corner of the unit-tile interactions brings together two different aptamers in close proximity. Red and blue represent different aptamers and dimmed shades show aptamers in a pointing downwards from the scaffold plane.

Figure 2.

A library of aptamer-RNA binding domain pairs can be functionally expressed invivo. (A) Schematic for split GFP complementation on an RNA scaffold (B) List of four different pairs—with eight unique aptamer-RNA binding sets tested. Values of dissociation constants reported previously (24,27,28) are tabulated with domain and aptamer sizes. (C) FITC images showing enhanced split GFP fluorescence invivo in the presence of RNA scaffolds for all sets tested. Scale bars: 1 μm.

Figure 3.

Aptamers incorporated into 0D, 1D and 2D RNA scaffolds bind proteins invivo (A) FITC images showing GFP fluorescence observed in cells on expression of split GFP fragments (GFPA-PP7 and GFPB-BIV-TAT) with either no scaffold, 0D, 1D or 2D scaffolds containing PP7 and BIV-TAT binding aptamers (B) Corresponding FITC quantification (histograms) for 0, 1 and 2D scaffold designs (P < 10⁻³, one-tailed t-test).

Figure 4.

Localizing enzymes on in vivo RNA scaffolds leads to increased metabolic flux (A) The alkane synthesis pathway with the aldehyde reductase side reaction (19). (B) Scaffolding designs implemented to channel the hexadecanal intermediate toward alkane production. (C) Synthesis of alkanes from E. coli expressing PP7-ADO, BIV-Tat-AAR, an empty vector or RNA scaffolds of zero, one or two dimensions (with aptamers for BIV-TAT and PP7), and a 2D scaffold with mismatched aptamers (containing anti-RevR11Q and MS2). (D) One of the pentadecane pathway side-products, hexadecanol, production measured on expression of pathway enzymes with the same empty vector, correct 2D scaffold and mismatched 2D scaffold. (E) Direct evidence of enzyme–RNA interaction from experimental flow for detecting RNA associated invivo with 6xHis tagged BIV-TAT-AAR enzymes. Differently shaded boxes indicate whether amplification was detected by SYBR green or RNA species were below detection threshold. (F) Pathway enzyme production levels measured by quantitative western blotting using 6xHis (AAR) and Strep (ADO) tags and normalized to GAPDH. Levels are shown for co-expression with an empty vector or scaffolds of different dimensionalities (n = 3, error bars = SEM) (* indicates P < 0.05, one-tailed t-test).

Figure 5.

Length and orientation of aptamers affect metabolic flux increase on scaffolds. (A) Schematic showing design details of a pair of aptamers interacting on the 2D scaffold using PDB IDs: 2QUX (34), 2A9X (35) and 4KVQ (36). Different base pair lengths (7–18bp) were used for the hairpin presenting aptamer bound by BIV-Tat-AAR so that rotation of the complex may bring AAR in closer proximity to ADO on the scaffold. (B) Pentadecane yields are shown for 2D scaffolds with the different BIV-Tat binding aptamer stem lengths. Concentrations were normalized to yield from enzyme expression without scaffolds and plotted as relative production levels. (C) RNA scaffold production levels, measured by qRT-PCR and normalized to endogenous gapA mRNA, for the strains showing greatest variation in yields. (Log base value 2r ∼1.84—see ‘Materials and Methods’ section). (D) Pathway enzyme production levels measured by quantitative western blotting using 6xHis (AAR) and Strep (ADO) tags. Levels are shown for co-expression of pathway enzymes with RNA scaffolds of varying BIV-TAT-aptamer lengths and are normalized to GAPDH levels. (E) Proposed model for two maximal configurations of intermediate flux channeling. On varying the anti-BIV-TAT aptamer stem loop length, different rotational conformations of the BIV-Tat-AAR moiety are possible, relative to the PP7-ADO dimer (n = 3, error bars = SEM) (* indicates P < 0.05, one-tailed t-test).

Figure 6.

Enhanced succinate production on scaffolds. (A) Enzymes expressed to increase succinate production in E. coli with the intermediate molecules shown in boxes. (B) Schematics for two scaffolding approaches on 2D scaffolds to increase pathway flux toward succinate. PYC and FDH are scaffolded with MDH in the first approach to prevent loss of oxaloacetate and NADH respectively. In the scaffold with four aptamers, eCA is also scaffolded to provide HCO₃⁻ for PYC. (C) Succinate production from expressing three enzyme fusions (PP7-PYC, BIV-TAT-MDH, lambaN-FDH) or four enzymes (also RevR11Q-eCA) with and without the corresponding 2D scaffolds compared to wild-type (WT) levels (n = 3, error bars = SEM) (* indicates P < 0.05, one-tailed t-test).