Engineering artificial photosynthesis based on rhodopsin for CO₂ fixation

The study addresses whether a simple rhodopsin-based phototrophic module, when combined with an engineered extracellular electron uptake pathway, can drive full CO₂ fixation in a non-photosynthetic bacterium. Microbial CO₂ fixation underpins the global carbon cycle, and while chlorophyll-based systems split water and generate both ATP and reductant, rhodopsin systems primarily pump protons to establish proton motive force (pmf) but lack an intrinsic electron source. Recent evidence suggests rhodopsin-generated pmf can drive reverse electron transport to synthesize NAD(P)H, implying potential for CO₂ reduction if electrons are supplied. The authors propose engineering Ralstonia eutropha H16 to couple an inward extracellular electron transfer conduit (MtrCAB from Shewanella oneidensis) with Gloeobacter rhodopsin (GR), thereby creating a synthetic photosynthetic electron transport chain that enables photoelectroautotrophic CO₂ fixation using light as the energy source and an electrode as the electron source.

Prior work has shown rhodopsin expression in bacteria (e.g., E. coli, R. eutropha, S. oneidensis) can convert light into intracellular chemical energy and enhance bioproduction in light. In electroactive S. oneidensis, rhodopsin can power electrosynthesis under cathodic conditions. Earlier, a GR-expressing R. eutropha system used soluble flavin mediators to shuttle electrons from an electrode, achieving CO₂ incorporation but with low electron transfer rates and efficiencies. Natural electroactive bacteria use outer-membrane Mtr complexes to exchange electrons with extracellular substrates, and phototrophs like Rhodopseudomonas palustris have outer-membrane cytochromes enabling extracellular electron uptake. Increasing rhodopsin proton-pumping capacity (e.g., via antennas) can enhance ATP generation and reverse electron transfer through NADH dehydrogenase to regenerate reducing equivalents for carbon fixation. These insights motivate combining a robust extracellular electron uptake interface (MtrCAB) with rhodopsin-driven pmf in R. eutropha to emulate photosynthetic electron flow.

• Strain engineering: Constructed plasmids under arabinose-inducible PBAD promoter to express MtrCAB (outer-membrane electron conduit from S. oneidensis MR-1) and Gloeobacter violaceus rhodopsin (GR) in R. eutropha H16. Created variants including R. eutropha Δpha (RHM5) to divert carbon from PHA to biomass, and overexpressed a native β-carbonic anhydrase (can) to enhance CO₂ hydration. GR induction included exogenous trans-retinal; canthaxanthin was added as an antenna to enhance GR proton pumping; FMN was supplied as an electron mediator cofactor for MtrC.
• Verification of expression: Single-cell Raman spectroscopy identified increased c-type cytochrome bands (748, 1128, 1312, 1584 cm⁻¹) upon Mtr induction and heme-stained MtrC/MtrA bands from SDS-PAGE. GR expression was confirmed by a characteristic ~1530 cm⁻¹ Raman band and pigmentation changes.
• Electrochemical setup: Dual-chamber photoelectrochemical reactors (Nafion PEM) in three-electrode configuration; cathode: carbon paper (2.5 × 2.5 cm²), reference: Ag/AgCl; anode: stainless steel mesh. A solar panel with a custom voltage regulator provided cathodic bias (−1.7 V applied) to avoid hydrogen evolution. N₂/CO₂ gas maintained anaerobic cathode conditions; anodic oxygen was isolated by PEM.
• Functional tests: Assessed inward electron transfer by monitoring current consumption during nitrate respiration in potentiostatic bioreactors (−500 mVAg/AgCl) after nitrate addition. Quantified intracellular NADH/NAD⁺ and NADPH/NADP⁺ ratios under light versus dark. Measured GR proton pumping via extracellular pH changes in unbuffered suspensions with and without canthaxanthin. Used single-cell Raman to detect Mtr-bound FMN (~1340 cm⁻¹) and canthaxanthin bands (1005, 1155, 1517 cm⁻¹). Tracked photoelectroautotrophic growth on CO₂ (continuous supply) under light/dark for 5 days; monitored OD600, doubling time, total charge transfer, and faradaic efficiency. Stable isotope probing with ¹³C-bicarbonate and phenylalanine band shifts (1003→987/975/961 cm⁻¹) quantified CO₂ incorporation. Statistical analyses used two-sided Student’s t-tests (or Welch’s t-test for SCRS quantification).

• MtrCAB expression: Induced R. eutropha-Mtr showed elevated c-type cytochrome Raman bands (single-cell n=234), consistent with MtrC/MtrA synthesis; visible red pigmentation appeared upon induction.
• Inward electron transfer: Upon addition of 20 mM nitrate under cathodic conditions (−500 mVAg/AgCl), induced R. eutropha-Mtr consumed current markedly over 6 h, whereas uninduced controls showed little change, indicating Mtr-mediated electron flow to native nitrate reductases.
• GR expression and reductant regeneration: GR-positive cells were identified by a ~1530 cm⁻¹ Raman band; the fraction of GR-expressing cells increased with arabinose induction (p=0.0057). Under light, engineered cells exhibited higher NADH/NAD⁺ and NADPH/NADP⁺ ratios than in dark (p=0.0017 and p=0.00798, respectively), consistent with rhodopsin-driven reverse electron transport via NADH dehydrogenase.
• Photoelectroautotrophic growth: In solar-driven reactors with −1.7 V applied and CO₂ as the sole carbon source, RHM5-GR-Mtr grew only in light (OD600 increased to 0.237 over 5 days); all dark controls did not grow, ruling out H₂-mediated growth at this potential.
• Electron transfer enhancement by Mtr: Compared to RHM5-GR using flavin shuttle alone, RHM5-GR-Mtr achieved nearly half the doubling time and increased faradaic efficiency from 23.9% to 35.4%. Total charge transfer increased by 54.4% (105 C vs 68 C), indicating a more efficient electron pathway when MtrCAB bridges the outer membrane.
• Antenna-enhanced proton pumping: Adding canthaxanthin nearly doubled ΔpH over 1 min and produced characteristic Raman bands; cell suspensions shifted from pink to red. Canthaxanthin reduced generation time by 22% to 70 h and raised faradaic efficiency by 21% to 42.9% relative to GR alone.
• Carbonic anhydrase overexpression: Raman isotope probing showed greater ¹³C incorporation from bicarbonate in can-overexpressing strains. In reactors, overexpressing can further reduced generation time to 67.3 h and increased faradaic efficiency to 45.0% (with canthaxanthin), demonstrating improvements in the CO₂ fixation module.

The results demonstrate a synthetic photosynthetic electron transport chain in R. eutropha by coupling MtrCAB-mediated extracellular electron uptake with GR-driven proton motive force. Electrons from a cathode reduce the quinone pool via the Mtr conduit, while rhodopsin-generated pmf drives reverse electron flow through proton-translocating NADH dehydrogenase to regenerate NADH/NADPH, thereby supplying energy and reducing power for the Calvin-Benson-Bassham cycle and enabling growth on CO₂ under illumination. The system mimics natural photosynthesis: a light-powered anode splits water (analogous to PSII), and pmf facilitates reductant regeneration (analogous to PSI), but without intermediates such as H₂ or formate. Performance enhancements via MtrCAB, FMN binding, canthaxanthin antennas, and carbonic anhydrase underscore the modularity of the approach. Comparisons with Rhodopseudomonas palustris highlight shared capacity for electrode-assisted CO₂ fixation under light, but distinct mechanisms (engineered rhodopsin pmf versus native P870 photochemistry). Discussion of electron transfer components suggests endogenous inner-membrane proteins (e.g., nuo complex) are likely critical for reverse electron transfer, corroborated by inhibition with the protonophore CCCP. Potential ROS generation was mitigated by a dual-chamber reactor and anaerobic cathode operation. The work suggests broader ecological and biotechnological relevance: rhodopsin–Mtr couplings may occur in nature, enabling autotrophic CO₂ fixation powered by diverse extracellular electron sources in sunlit environments.

This study establishes a modular artificial photosynthesis platform in a non-phototrophic bacterium, R. eutropha, by integrating an extracellular electron conduit (MtrCAB) with a rhodopsin-based proton pump to drive reverse electron transport and CO₂ fixation. The system supports photoelectroautotrophic growth on CO₂ under light, with significant gains in electron transfer efficiency, reductant regeneration, and growth when augmented by FMN, canthaxanthin, and carbonic anhydrase. The approach offers a blueprint to convert other heterotrophs into photoelectrotrophs and suggests ecological scenarios wherein rhodopsin phototrophy and extracellular electron uptake jointly support carbon fixation. Future work should optimize Mtr expression and periplasmic relays (e.g., STC), refine codon/RBS and heme biosynthesis, engineer electrodes and reactor conditions, expand rhodopsin spectral properties, manage ROS while leveraging controlled oxygen for energy yields, and quantify overall solar-to-biomass efficiencies under standardized conditions.

• MtrCAB expression in R. eutropha appears relatively low; electron transfer enhancement is modest compared to some heterologous systems (e.g., E. coli). Precise functional attribution to MtrCAB requires further validation; increased membrane permeability or endogenous mediators could contribute to observed effects.
• The system operates at low cathodic potentials (−1.7 V) to avoid H₂ evolution; while no H₂ was detected, generalization to other potentials or long-term stability remains to be assessed.
• ROS risks necessitated anaerobic cathode operation and PEM separation, potentially trading off energy from aerobic respiration; scalable strategies to balance ROS control and energy yield are needed.
• Overall photosynthetic efficiency could not be meaningfully calculated due to multiple assumptions; standardized benchmarks and long-duration tests are lacking.
• Mechanistic details of inner-membrane electron carriers (e.g., role of CymA analogs, NapC, hydrogenases) and the relative contributions of nuo versus ndh in reverse electron transport need further genetic and biochemical dissection.