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

氧气供应意味着更高的生产成本和最大理论产量的降低。因此，发酵产物的产生更具成本效益。旨在找到用于生产（聚）-3-羟基丁酸酯（PHB），为发酵产物的关键部分，在这里我们表征乙酰乙酰基-CoA还原酶，从分离的暂定Accumulibacter phosphatis富集混合培养，示出了（ķ_猫^NADH /K _M ^NADH )/( k_猫^NADPH /K _M ^NADPH)>500。进一步的动力学分析表明，在生理浓度下，该酶明显偏爱 NADH，表现出迄今为止在乙酰乙酰辅酶 A 还原酶中观察到的最强的 NADH 偏爱。结构和动力学分析表明，E37 和 P41 之间的残基对观察到的 NADH 偏好具有重要作用。此外，还组装了一个操纵子，结合了来自Cupriavidus necator的phaCA基因和编码这种 NADH 偏好的乙酰乙酰辅酶 A 还原酶的基因。大肠杆菌表达该组装操纵子的细胞在氧限制条件下显示出 PHB 的持续积累，并且当降低比耗氧率时，PHB 滴度增加。综上所述，这些结果表明可以在大肠杆菌中产生 PHB 作为发酵产物，这为设想更有效的 PHB 厌氧生产的进一步蛋白质/代谢工程策略开辟了机会。