[Objective] To obtain the proteins of acetyl-CoA synthase (ACS_MU) and PHA synthase (PhaC_MU) fromMassilia sp. UMI-21 by structuring anin vitro recombinant expression system, and to elucidate their roles in the biosynthesis of polyhydroxybutyrate (PHB) using the one-phase reaction system (OPRS). [Methods] Seamless cloning was employed to ligate the acetyl-CoA synthase geneacs_MU and the PHA synthase genephaC_MU amplified fromMassilia sp. UMI-21 to the pQE-80L plasmid to construct the recombinant plasmids. The recombinant plasmids were transformed intoEscherichia coli BL21(DE3), and the recombinant strains were obtained. ACS_MU and PhaC_MU were purified using a 6×His tag, and their activities were determined by the 5, 5′-dithiobis-(2-nitrobenzoic acid) (DTNB) method. With 3HB as a substrate, the one-phase reaction system (OPRS) was employed to validate the functions of ACS_MU and PhaC_MU in the biosynthesis of PHB. [Results] The recombinant strains BL21-pQE-80L-acs_MU and BL21-pQE-80L-phaC_MU were successfully engineered, with the ACS_MU and PhaC_MU yields of 24.8 mg/L and 25.6 mg/L, respectively. The specific activity of ACS_MU was (0.148±0.011) U/mg, and that of PhaC_MU for (R)-3HBCoA was (0.102±0.011) U/mg. Nuclear magnetic resonance hydrogen spectroscopy (¹H-NMR) results showed that products from the all three PHB synthesis pathways, ACS_Pt-PCT_CP-PhaC_Re, ACS_MU-PCT_CP-PhaC_Re, and ACS_MU-PCT_CP-PhaC_MU, in OPRS were PHB. The yields of PHBvia the three pathways were 0.62, 0.76, and 0.64 g/L, respectively. [Conclusion] The genesacs_MU andphaC_MU can be overexpressed in theE.coli expression system to yield active soluble proteins. Compared with the ACS_Pt-PCT_CP-PhaC_Re pathway, substitution of ACS_Pt with ACS_MU increased the PHB yield by 22.58%. The yield of PHB was contingent upon the stability of acetyl-CoA synthase (ACS), which provided acetyl-CoA for reaction under identical PhaC. Replacing PhaC_Re with PhaC_MU decreased the PHB yield by 15.79% compared with ACS_MU-PCT_CP-PhaC_Re. The polymerase PhaC plays a crucial role in PHB synthesis under identical precursor concentrations.