Continuous artificial synthesis of glucose precursor using enzyme-immobilized microfluidic reactors

Abstract

Food production in green crops is severely limited by low activity and poor specificity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in natural photosynthesis (NPS). This work presents a scientific solution to overcome this problem by immobilizing RuBisCO into a microfluidic reactor, which demonstrates a continuous production of glucose precursor at 13.8 μmol g^-1 RuBisCO min^-1 from CO₂ and ribulose-1,5-bisphosphate. Experiments show that the RuBisCO immobilization significantly enhances enzyme stabilities (7.2 folds in storage stability, 6.7 folds in thermal stability), and also improves the reusability (90.4% activity retained after 5 cycles of reuse and 78.5% after 10 cycles). This work mimics the NPS pathway with scalable microreactors for continuous synthesis of glucose precursor using very small amount of RuBisCO. Although still far from industrial production, this work demonstrates artificial synthesis of basic food materials by replicating the light-independent reactions of NPS, which may hold the key to food crisis relief and future space colonization.

Fig. 1

Design and characterization of RuBisCO immobilized microfluidic reactors (RIMRs). a Scheme of light-independent reactions of NPS: Phase 1: Carbon fixation starts with ribulose 1,5-bisphosphate (RuBP) and uses the enzyme RuBisCO to fix CO₂ into 3-phosphoglycerate (3-PGA); Phase 2: Reduction reaction uses adenosine triphosphate (ATP), nicotinamide adenine dinucleotide phosphate (NADPH) and the enzyme phosphoglycerate kinase (PGK) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) to reduce 3-PGA into glyceraldehyde 3-phosphate (G3P), two of which can form the end product glucose; Phase 3: RuBP regeneration from G3P using up to 9 steps of enzymatic reactions. b Three-dimensional diagram and the photograph (inset) of the RIMRs, the scale bar of the inset is 1 cm. c SEM image of the inner surfaces of RIMRs. Flat and smooth PDMS becomes rough and is covered by PDA nanoparticles after PDA modification (brownish color). Large blocks are the immobilized RuBisCO (green blocks). The scale bar is 1 μm. d Protein-loading amount and protein-loading efficiency as a function of the concentrations of injected RuBisCO to find the optimal RuBisCO concentration for further experiments. Error bars represent the standard deviations from three independent experiments. Source data are provided as a Source Data file

Fig. 2

Feasibility of RIMRs. a Production amount of 3-PGA as a function of the reaction time for the RIMRs (solid red circles) and the BIMRs (open dark squares). The red dash-dotted line and dark dashed line are the liner fitting regressions. The slope of liner fitting regression of 3-PGA production in RIMRs is 13.8 μmol g⁻¹ RuBisCO min⁻¹ and that in BIMRs is almost 0. The initially injected RuBisCO and BSA concentrations for immobilization are both 6.25 μg μL⁻¹. The collected production solutions I from RIMRs and BIMRs are 21 μL. RuBP concentration is 0.5 mM. ${\mathrm{HCO}}_3^{-}$ in the reaction buffer is 66 mM. Reaction temperature is 30 °C. b HPLC–MS/MS chromatography of RuBP and 3-PGA in the production solutions I obtained from RIMRs and BIMRs at the reaction time of 5 min. Error bars represent the standard deviations from three independent experiments. Source data are provided as a Source Data file

Fig. 3

Stability and reusability of RIMRs. a Storage stability (incubation at 4 °C) of immobilized (red line) and free RuBisCO (dark line). b Thermal stability of immobilized (red line) and free RuBisCO (dark line). All samples are incubated for 10 min before the activity assay. c Reusability of the RIMRs when RuBP is injected at 1.4 μL min⁻¹ (the reaction time is 5 min). Red dash-dotted line is the third-order polynomial fitting, representing that the activity drop trend tends to slow down with the increase of cycles of reuse. d Relative activity as a function of the flow rate of RuBP injection (from 7 to 0.7 μL min⁻¹) for repeated uses. The amount of RuBisCO is 21.875 μg for both the immobilized and free ones in all the experiments. The volume of the collected production solutions for the storage and thermal stability tests are 100 μL. RuBP concentration is 0.5 mM and ${\mathrm{HCO}}_3^{-}$ in the reaction buffer is 66 mM. Error bars represent the standard deviations from three independent experiments. Source data are provided as a Source Data file

Fig. 4

Continuous production of 3-PGA as a function of the volume of collected production solution. The dark open squares represent the 3-PGA production in the bulk reaction with free RuBisCO and the red solid circles represent the 3-PGA production in the RIMRs with the immobilized RuBisCO. The amount of RuBisCO used is 21.875 μg for both the immobilized and free ones. RuBP concentration is 0.5 mM and ${\mathrm{HCO}}_3^{-}$ in the reaction buffer is 66 mM. Reaction temperature is 30 °C. Reaction time is 1 min. Error bars represent the standard deviations from three independent experiments. Source data are provided as a Source Data file