Redox-reactions play a key role in organic chemistry, and the shift towards a sustainable production need biological solutions. Enzyme-catalyzed redox reactions rely on natural cofactos such as NAD(P)H/+ which are used in catalytic amounts and continuously recycled. However, the large-scale, cell-free application of enzymatic redox chemistry remains mostly confined to high-value products due to the low atom economy of the conventional cofactor recycling systems and the need of excess sacrificial reagents. This also complicates the downstream process and drives up production costs. Using H2/H+ as a terminal co-substrate offers a promising alternative as it significantly simplifies these reactions and enables their use in medium and low-cost compounds for the first time. During the first RP of CirculH2, we have made notable progress that contributes to achieving this impact.
Development of an Efficient, Scalable, and Robust Catalyst & the Upscaling of the Hydrogenase Production. Scientific advancements have been made in discovering and engineering novel hydrogenases. Ancestral sequence reconstruction has identified new hydrogenases, and enzyme engineering efforts are enhancing properties such as O2 resistance, thermostability and functional expression. This directly addresses the need for a catalyst that can sustain harsh conditions in industrial setting. Additionally, steps are underway to establish a microfluidic high-throughput screening platform for hydrogenases, aimed at improving their stability for practical application.
For the upscaling of the hydrogenases, a conceptual design study for the FeFe hydrogenase apoenzyme production has been completed identifying the cost drivers and changes have been proposed to reduce the costs and for scalability. With the proposed design, up to a 500-fold cost reduction opportunity from lab-scale production is detected. Furthermore, the production of NiFe hydrogenase has been successfully scaled up to a 300 l pilot scale. This progress directly supports the industrial relevance and scalability of hydrogenase production.
Implementation of the CirculH2 Technology for Specialty and Commodity Chemicals Production. We are working intensively to implement the hydrogenases into biotransformation and to use the H2/H+ as a terminal co-substrate. A major progress that contributes to this, is our H2-driven nicotinamide adenine dinucleotide phosphate (NADPH, reduced form) regeneration system, in which a FeFe hydrogenase and FNR are co-immobilized in a system that has shown to be O2-tolerant and scalable, while achieving efficient NADPH recycling in 1 l reactor volume. This is a critical step toward industrial applications. We also managed to implement a H2-driven H2O2 generation to drive oxidative processes relevant to industry.
Moreover, the industrial potential of the CirculH2 technology is evident in our model reactions for the specialty chemical production. The H2-driven NAD(P)H regeneration drives CAR-mediated reductions, which has been successfully demonstrated on an analytical scale. Using the hydrogenase-driven NADPH regeneration system we susccessfully implemented the CAR-mediated reduction of Piperonylic acid. In another application, we were able to demonstrate the (S)-citronellol synthesis, a valuable fragrance compound, using H2-based cofactor recycling and reaching full conversion.
That the H2-driven technology can advance the European competitiveness in sustainable and circular chemical production, is evident in our environmental assessment. The results support this impact and demonstrate that using H2 as a sacrificial substrate significantly reduces CO2 generation compared to conventional alternatives such as glucose or H2O2. This is a critical quantitative indicator of CirculH2’s contribution to decarbonisation and resource efficiency.
