Synthetic CO2-fixation enzyme cascades immobilized on self-assembled nanostructures that enhance CO2/O2 selectivity of RubisCO

Abstract

Background: With increasing concerns over global warming and depletion of fossil-fuel reserves, it is attractive to develop innovative strategies to assimilate CO₂, a greenhouse gas, into usable organic carbon. Cell-free systems can be designed to operate as catalytic platforms with enzymes that offer exceptional selectivity and efficiency, without the need to support ancillary reactions of metabolic pathways operating in intact cells. Such systems are yet to be exploited for applications involving CO₂ utilization and subsequent conversion to valuable products, including biofuels. The Calvin-Benson-Bassham (CBB) cycle and the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) play a pivotal role in global CO₂ fixation.

Results: We hereby demonstrate the co-assembly of two RubisCO-associated multienzyme cascades with self-assembled synthetic amphiphilic peptide nanostructures. The immobilized enzyme cascades sequentially convert either ribose-5-phosphate (R-5-P) or glucose, a simpler substrate, to ribulose 1,5-bisphosphate (RuBP), the acceptor for incoming CO₂ in the carboxylation reaction catalyzed by RubisCO. Protection from proteolytic degradation was observed in nanostructures associated with the small dimeric form of RubisCO and ancillary enzymes. Furthermore, nanostructures associated with a larger variant of RubisCO resulted in a significant enhancement of the enzyme's selectivity towards CO₂, without adversely affecting the catalytic activity.

Conclusions: The ability to assemble a cascade of enzymes for CO₂ capture using self-assembling nanostructure scaffolds with functional enhancements show promise for potentially engineering entire pathways (with RubisCO or other CO₂-fixing enzymes) to redirect carbon from industrial effluents into useful bioproducts.

Keywords: Bioconversion; CO2 fixation; Cell-free systems; Nanostructures; Pathway engineering; RubisCO.

Fig. 1

Illustration of the assembly of nanostructures with RubisCO driven by electrostatic interactions. a Structures of compounds that self-assemble into either nanotubes (A/B) or nanofibers (C). b Either the dimeric R. rubrum form II RubisCO (PDB id—5RUB) or the hexadecameric R. eutropha form I RubisCO (PDB id—1BXN) were used in these studies

Fig. 2

RubisCO loading with fixed amounts of pre-assembled nanotube A (a 0.75 mg/mL) or nanofiber C (b 0.9 mg/mL). Plots show activities measured from nanostructure-RubisCO complexes that had been loaded with varying amounts of either form I (blue) or form II (orange) RubisCO enzymes. Recovery percentages were calculated relative to the enzyme activities in the corresponding samples with unbound RubisCO. Activities were also measured from identical form I (gray) or form II (yellow) nanofiber C preparations (b) that had been supplemented with 1 mg/mL bovine serum albumin (BSA) during RubisCO loading

Fig. 3

TEM images of nanostructures. a Nanotubes and nanofibers formed from compounds A (left) and C (right), respectively, stained with uranyl acetate. b TEM images of Ni–NTA Nanogold^® particles bound to the hexa-histidine tagged form I RubisCO and associated with either nanotube A (left) or nanofiber C (right). Images are representative of multiple samples imaged from independent preparations. For better clarity, a close-up view of a single nanostructure is shown to the left of each image

Fig. 4

Schematic of CO₂-fixation pathways assembled in nanostructures. Cascade of enzymatic steps employed to convert either ribose-5-phosphate (R-5-P) (pathway 1 a) or glucose (pathway 2 b) to 3-PGA

Fig. 5

Proteolytic sensitivities of RubisCO and PRK enzymes in nanostructure complexes. Unbound or nanostructure-associated RubisCO (a, b) or PRK (c) were treated with subtilisin for various times and residual enzymatic activities were measured for each time point. Data shown here is representative of two independent preparations that gave similar results. Nanotubes A or B provided identical levels of protection to all enzymes and hence the data is shown for only one of them (i.e., nanotube B), along with the data for nanofiber C

Fig. 6

Reaction velocity measurements of R. eutropha form I RubisCO as a function of CO₂ concentration in the presence (open circles) or absence (closed circles) of saturating levels of oxygen (i.e., 1230 μM). a Michaelis–Menten curves for carboxylation activities measured with unbound R. eutropha form I RubisCO. b Michaelis–Menten curves for carboxylation activities measured with nanotube B-form I RubisCO complexes. The extent of oxygen inhibition for each enzyme preparation is indicated (double-headed arrows). The K _O (K _I) for O₂ was enhanced about 1.6-fold for the nanotube B-form I enzyme complex compared to the unbound enzyme (see Table 3 for kinetic constants). The average value and error bar for each data point was calculated with values obtained from two independent assays (with different preparations) performed with identical CO₂ concentrations