Enzymatic reduction of carbon dioxide (CO2) to methanol (CH3OH) can be accomplished using a designed set-up of three oxidoreductases utilizing reduced pyridine nucleotide (NADH) as cofactor for the reducing equivalents electron supply. For this enzyme system to function efficiently a balanced regeneration of the reducing equivalents during reaction is required. Herein, we report the optimization of the enzymatic conversion of formaldehyde (CHOH) to CH3OH by alcohol dehydrogenase, the final step of the enzymatic redox reaction of CO2 to CH3OH, with kinetically synchronous enzymatic cofactor regeneration using either glucose dehydrogenase (System I) or xylose dehydrogenase (System II). A mathematical model of the enzyme kinetics was employed to identify the best reaction set-up for attaining optimal cofactor recycling rate and enzyme utilization efficiency. Targeted process optimization experiments were conducted to verify the kinetically modeled results. Repetitive reaction cycles were shown to enhance the yield of CH3OH, increase the total turnover number (TTN) and the biocatalytic productivity rate (BPR) value for both system I and II whilst minimizing the exposure of the enzymes to high concentrations of CHOH. System II was found to be superior to System I with a yield of 8 mM CH3OH, a TTN of 160 and BPR of 24 mu mol CH3OH/U center dot h during 6 hr of reaction. The study demonstrates that an optimal reaction set-up could be designed from rational kinetics modeling to maximize the yield of CH3OH, whilst simultaneously optimizing cofactor recycling and enzyme utilization efficiency.