An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichia coli

Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, generation fermentation products is more cost-effective. Aiming to find a key piece for the (poly)-3-hydroxybutyrate (PHB) as product, here we characterize an acetoacetyl-CoA reductase, isolated from Candidatus Accumulibacter phosphatis-enriched mixed culture, showing (kcatNADH/KMNADH)/(kcatNADPH/KMNADPH)>500. Further kinetic analyses indicate that, at physiological concentrations, this enzyme clearly prefers NADH, presenting strongest NADH preference so far observed among reductases. Structural that residues between E37 P41 have important role preference. Moreover, operon was assembled combining phaCA genes Cupriavidus necator gene encoding NADH-preferring reductase. Escherichia coli cells expressing showed continuous accumulation PHB under oxygen limiting conditions titer increased when decreasing specific consumption rate. Taken together, these results show it possible generate product in E. coli, opening opportunities further protein/metabolic engineering strategies envisioning efficient anaerobic PHB.