NADPH supply for poly(3-hydroxybutyrate) synthesis concomitant with enzymatic oxidation of phosphite
NADPH supply for poly(3-hydroxybutyrate) synthesis concomitant with enzymatic oxidation of phosphite
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DOI：
10.1016/j.jbiosc.2018.05.026
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发表时间：
2018-12-01
影响因子：
2.8
通讯作者：
Tsuge, Takeharu
中科院分区：
文献类型：
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作者：
Miyahara, Yuki;Oota, Mino;Tsuge, Takeharu
乙酰乙酰辅酶A还原酶（PhaB）参与聚（3-羟基丁酸酯）[P（3 HB）]的生物合成，需要辅酶NADPH作为还原剂。在这项研究中，NADPH供应对P（3 HB）生产的影响进行了研究，在体外和体内使用亚磷酸盐脱氢酶双突变体（PtxD（EAAR）），催化氧化亚磷酸盐与生成的NADH和NADPH。在使用纯化酶的体外试验中，仅当存在亚磷酸盐和PbcD（EAAR）时观察到P（3 HB）聚合，证实NADPH被供应给PhaB。在使用大肠杆菌作为P（3 HB）生产宿主的体内测定中，亚磷酸盐和PtxD（EAAR）的存在不影响正常生长条件下P（3 HB）的产率。而在不生长的E.大肠杆菌细胞与对照相比，表明PtxD（EAAR）介导的NADPH生成与P（3 HB）生物合成偶联。本研究证实了PtxD（EAAR）在体外和体内P（3 HB）合成过程中提供NADPH的用途。(C)2018年，日本生物技术学会。All rights reserved.
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(EAAR)), 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 PbcD(EAAR) 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(EAAR) 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(EAAR)-mediated NADPH generation is coupled with P(3HB) biosynthesis. This study confirmed the use of PtxD(EAAR) for supplying NADPH during P(3HB) synthesis in vitro and in vivo. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.