The coupling of PHB generation with NADH reoxidation is required to generate PHB as a fermentation product. A fundamental trait to accomplish this feature is to express a functional NADH-preferring acetoacetyl-CoA reductase, engaged in PHB accumulation. One way to obtain such a reductase is by engineering the cofactor preference of the acetoacetyl-CoA reductase encoded by the phaB1 gene from Cupriavidus necator (AARCn1). Aiming to have a deeper understanding of the structural determinants of the cofactor preference in AARCn1, and to obtain an NADH-preferring acetoacetyl-CoA reductase derived from this protein, some engineered enzymes were expressed, purified and kinetically characterized, together with the parental AARCn1. One of these engineered enzymes, Chimera 5, experimentally showed a selectivity ratio ((kcat/KM)NADH/(kcat/KM)NADPH) ≈ 18, which is 160 times higher than the selectivity ratio experimentally observed in the parental AARCn1. A thermodynamic-kinetic approach was employed to estimate the cofactor preference and flux capacity of Chimera 5 under physiological conditions. According to this approach, Chimera 5 could prefer NADH over NADPH between 25 and 150 times. Being a derivative of AARCn1, Chimera 5 should be readily functional in Escherichia coli and C. necator. Moreover, with the expected expression level, its activity should be enough to sustain PHB accumulation fluxes similar to the fluxes previously observed in these biotechnologically relevant cell factories.@en