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.