The CORENZ tandem project addressed the challenge of integrating cofactor regeneration into biocatalytic systems for sustainable industrial processes, with a particular focus on CO₂ fixation. Building on the predecessor CASCOO, which demonstrated that naturally occurring decarboxylases can operate in reverse to fix CO₂, CORENZ sought to develop closed‑loop processes that regenerate the consumed cofactors NAD(P)H and ATP. The experimental system centered on the pyruvate:ferredoxin oxidoreductase (PFOR) from Desulfocurvibacter africanus, an enzyme that catalyses the reversible reaction CO₂ + 2 H⁺ + 2 ferredoxin_red + acetyl‑CoA ↔ pyruvate + 2 ferredoxin_ox + CoA. In CASCOO the equilibrium constant (K′ = 2.2 × 10⁻⁴) and ΔG = 20.9 ± 13.1 kJ mol⁻¹ placed the reaction far to the left, favouring decarboxylation. CORENZ introduced two key modifications to shift the balance toward fixation. First, a photochemical upstream module maintained ferredoxin in a reduced state by illuminating a deazaflavin/EDTA system, while a downstream module continuously removed pyruvate by derivatisation with semicarbazide, forming pyruvate‑semicarbazone. Second, the native ferredoxin was replaced by a low‑potential variant (Fdx_ctep) from Chlorobaculum tepidum, whose standard redox potential (E₀′ = –584 mV) is substantially more negative than the native –385 mV, thereby providing a stronger driving force for reduction. These interventions enabled measurable CO₂ fixation in vitro, although the assays were limited to a maximum duration of two hours due to enzyme half‑life constraints. Systematic studies of enzyme stability revealed that activity decays over time, necessitating a continuous production pipeline for fresh enzyme, ferredoxin, and chloroplast preparations. Reproducibility was also challenged by variations in purity and activity of the starting materials, underscoring the need for tighter process control.

The project’s scientific contribution extends beyond the immediate biochemical findings. By integrating kinetic modelling performed at the Max‑Planck Institute for Dynamics of Complex Technical Systems with experimental data from the Institute for Applied Biotechnology at Hochschule Biberach, the team gained insights into the dynamic behaviour of the enzyme system and identified optimisation targets. The modelling effort highlighted the importance of cofactor turnover rates and suggested that immobilisation of enzymes in large‑volume reactors could enhance stability, drawing parallels to industrial processes such as glucose‑isomerase immobilised in packed‑bed reactors operating at 1800 kg reactor⁻¹ and residence times of up to 687 days. Extrapolating the CORENZ results to such scales suggests potential CO₂ conversion rates of 302.4 kg CO₂ per residence time (from one data set) or 95.5 kg CO₂ per residence time (from another), indicating that further scale‑up could render the approach industrially viable.

Collaboration-wise, CORENZ was a two‑partner tandem project between the Max‑Planck Institute for Dynamics of Complex Technical Systems, Magdeburg (project leader Dr. Steffen Klamt) and the Institute for Applied Biotechnology, Hochschule Biberach (project leader and coordinator Dr. Hartmut Grammel). The partnership combined expertise in mathematical modelling and applied microbiology, respectively. The project ran from April 2018 to January 2023, with regular online meetings via MS Teams, group seminars at Biberach, and data exchange between the two sites. The Institute for Applied Biotechnology provided the necessary infrastructure, including bioreactor cultivation, protein purification by IMAC, and quantitative metabolite analysis using an LTQ XL linear ion trap mass spectrometer. Funding was provided through publicly funded research programmes, enabling the project to contribute to the broader goal of establishing bio‑inspired CO₂ recycling pathways and laying the groundwork for future pilot‑scale demonstrations and metabolic engineering of CO₂‑utilising bacteria.