Miyahara Yuki, Oota Mino, Tsuge Takeharu
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan; Department of Materials Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
J Biosci Bioeng. 2018 Dec;126(6):764-768. doi: 10.1016/j.jbiosc.2018.05.026. Epub 2018 Jun 15.
Acetoacetyl-CoA reductase (PhaB), involved in poly(3-hydroxybutyrate) [P(3HB)] biosynthesis, requires the coenzyme NADPH as a reducing agent. In this study, the effect of NADPH supply on P(3HB) production was investigated in vitro and in vivo using a phosphite dehydrogenase double mutant (PtxD), which catalyzes oxidation of phosphite to phosphate with the generation of NADH and NADPH. In an in vitro assay using purified enzymes, P(3HB) polymerization was observed only when phosphite and PtxD were present, confirming that NADPH was supplied to PhaB. In an in vivo assay using Escherichia coli as a production host for P(3HB), the presence of phosphite and PtxD did not influence the yield of P(3HB) under normal growth conditions. However, P(3HB) yield increased 3.2-fold in non-growing E. coli cells compared to the control, suggesting that PtxD-mediated NADPH generation is coupled with P(3HB) biosynthesis. This study confirmed the use of PtxD for supplying NADPH during P(3HB) synthesis in vitro and in vivo.
参与聚（3-羟基丁酸酯）[P(3HB)]生物合成的乙酰乙酰辅酶A还原酶（PhaB）需要辅酶NADPH作为还原剂。在本研究中，使用亚磷酸脱氢酶双突变体（PtxD）在体外和体内研究了NADPH供应对P(3HB)产量的影响，该突变体催化亚磷酸氧化为磷酸并生成NADH和NADPH。在使用纯化酶的体外测定中，仅当存在亚磷酸和PtxD时才观察到P(3HB)聚合，证实了NADPH被供应给PhaB。在使用大肠杆菌作为P(3HB)生产宿主的体内测定中，在正常生长条件下，亚磷酸和PtxD的存在不影响P(3HB)的产量。然而，与对照相比，在不生长的大肠杆菌细胞中P(3HB)产量增加了3.2倍，这表明PtxD介导的NADPH生成与P(3HB)生物合成相关。本研究证实了在体外和体内P(3HB)合成过程中使用PtxD供应NADPH。
