Light-powered CO2 fixation in a chloroplast mimic with natural and synthetic parts

Abstract

Nature integrates complex biosynthetic and energy-converting tasks within compartments such as chloroplasts and mitochondria. Chloroplasts convert light into chemical energy, driving carbon dioxide fixation. We used microfluidics to develop a chloroplast mimic by encapsulating and operating photosynthetic membranes in cell-sized droplets. These droplets can be energized by light to power enzymes or enzyme cascades and analyzed for their catalytic properties in multiplex and real time. We demonstrate how these microdroplets can be programmed and controlled by adjusting internal compositions and by using light as an external trigger. We showcase the capability of our platform by integrating the crotonyl-coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a synthetic network for carbon dioxide conversion, to create an artificial photosynthetic system that interfaces the natural and the synthetic biological worlds.

Figure 1. Light-driven cofactor regeneration by thylakoid membrane-based energy modules (TEM)

(a) NADPH production is dependent on light and externally added ferredoxin with an optimum concentration between 2.5 and 10 μM ferredoxin (Supplementary Fig. 1a) (b) ATP production is dependent on light, with some background reaction in the dark due to membrane bound adenylate kinase (Supplementary Fig. 1b). (c) Scheme of TEM-driven carboxylation reactions of propionyl-CoA carboxylase (Pcc) and crotonyl-CoA carboxylase/reductase (Ccr) utilizing light-produced ATP and NADPH, respectively. (d) Pcc reaction (blue lines, carboxylation product) and Ccr reaction (orange lines, combined reduction and carboxylation product) coupled to TEM (2.5 or 5 μg Chl and 60 μmol photons m^-2 s^-1) in the light (solid lines) and dark (dashed lines) (N=6).

Figure 2. Light-driven, continuous fixation of CO₂ into organic acids

(a) Scheme of CETCH version 6.0 for the conversion of CO₂ into glycolate coupled to TEM. The CETCH cycle is started by addition of 80 μM of the intermediate propionyl-CoA (or crotonyl-CoA). Operation of the CETCH cycle was assessed by following the 13C-labeling patterns and total levels (shown as integrated peak area of the extracted ion chromatogram) of the intermediates methylmalonyl-CoA (blue) and ethylmalonyl-CoA (orange). (b) CETCH cycle version 6.0 directly operated by 125 μg Chl mL^-1 TEM under constant illumination (60 μmol photons m^-2 s^-1). Shown is the extracted ion peak area and the fractional labeling of ethylmalonyl-CoA and methylmalonyl-CoA (shown in shades of orange and blue, respectively, see Supplementary Fig. 4 for explanation of the labeling pattern) (c) same as in (b) but in the dark, showing that light is required to operate the cycle. Ethylmalonyl-CoA is not produced in the dark when starting from propionyl-CoA (n.d., not detected). (d) Glycolate production in the light and the dark by CETCH version 6.0.

Figure 3. Encapsulation of a functional TEM in micro-droplets and light-driven enzymatic reaction coupled to TEM in micro-droplets.

(a) Scheme of the TEM system encapsulated in micro-droplets. Light triggers TEM activity to produce NADPH and ATP. NADPH production is monitored by NADPH fluorescence (365 nm) of individual droplets. Populations of droplets can be distinguished from one another through the addition of a barcoding dye. (b) Microscopic pictures of a representative 4-bit emulsion of droplets containing four different TEM concentrations. First row, left to right: barcode fluorescence, bright field. Second row, left to right: NADPH fluorescence at time point 0, NADPH fluorescence after ten minutes. The scale bar represents 100 μm. A time-lapse video of the increasing NADPH fluorescence is available as Supplementary movie 1. (c) NADPH concentration versus time of micro-droplets with varying TEM (40, 80, 160 μg/mL Chl content). (d) Scheme of the TEM-powered Ghr reaction encapsulated in micro-droplets. Light-driven NADP⁺ photoreduction allows Ghr catalyzed reduction of glyoxylate into glycolate. (e) TEM-driven conversion (64 μg Chl mL^-1) of glyoxylate (5 mM) by 50 and 1,000 nM Ghr in micro-droplets (N = 50). The concentration of Ghr changes the steady state level of NADPH at 50 μmol photons m^-2 s^-1 (f) TEM-driven conversion of glyoxylate (120 μg Chl mL^-1 and 5 mM respectively) in micro-droplets under programmed light-dark cycles. Photoreduction of NADP⁺ in droplets (N=50) with 53.5 nM Ghr (teal line) under alternating cycles of illumination (50 μmol photons m^-2 s^-1) and darkness. Control: Droplets containing no Ghr (green line) and droplets containing 1 mM NADPH (black line). A time-lapse video of oscillations is available as Supplementary movie 2. In c, e, and f, bold lines indicate the mean of the droplet population. Shaded areas indicate the ± standard deviation (N=50). (g) Time and space control of metabolic activity in droplets. TEM is coupled to the NADPH-dependent reduction of glyoxylate in droplets. As described in (d), energy levels are controlled in time by varying the internal content and externally regulating the droplets by illumination. This experiment demonstrates how metabolic activity can be controlled both in time and space in droplets. To do this, a binary emulsion of droplets are programmed with different TEM and Ghr concentrations and these droplets are filled into an observation chamber. Two different droplet populations (200 μg Chl + 40 nM Ghr, green circle; and 40 μg Chl + 2.5 nM Ghr, grey circle) were patterned in space, each population filling approximately half of the chamber. Bright-field and 550 nm (barcoding) fluorescence images of a binary emulsion composed of two droplets populations are shown (scale bar represents 250 μm). The plot shows relative NADPH fluorescence over time under fluctuating light conditions (dark, 50 μmol photons m^-2 s^-1, dark) for both droplet populations, with the same color coding. NADPH fluorescence images are displayed below the plot and a time-lapse of the images is available (see Supplementary movie 3).

Figure 4. Light-driven, continuous fixation of CO₂ into organic acids by CETCH version 7.0 coupled to TEM in micro-droplets.

(a) Scheme of the CETCH version 7.0 coupled to TEM operating inside micro-droplets. (b) Dynamic equilibrium states of NADPH fluorescence offour populations of droplets: Droplets containing TEM (82 μg Chl mL^-1), 1 mM NADP⁺ and CETCH version 7.0 (teal line), droplets containing TEM, 1 mM NADP⁺ and an additional ATP regeneration system (Ppk and polyphosphate, coral line), as well as control droplets containing 1 mM NADPH and all CETCH version 7.0 components except for Ccr (black line), and control droplets containing TEM, 1 mM NADP⁺ and all CETCH version 7.0 components except for Ccr (green line). (c) Images of the droplets from (b) using the same color coding; first row, left to the right: bright field, thylakoid fluorescence with overlap from the coding dye, coding dye; second row, left to the right: NADPH fluorescence before illumination, after 15 minutes, and after 30 minutes illumination (scale bar corresponds to 100 μM). A time-lapse video is available as Supplementary movie 4. (d) Glycolate formed per acceptor molecule (sum of crotonyl-CoA and 3- hydroxybutyryl-CoA) over time in droplets and in bulk solution. The light and dark curves represent droplets maintained in the light and in the dark. The bulk curve shows an experiment with the same reaction mixture but on the micro-tube scale, kept in the dark for the duration of droplet manufacture. The bulk solution and the droplets were simultaneously exposed to light for parallel comparison.