In this study, Plackett-Burman and central composite designs were applied to improve polyhydroxybutyrate (PHB) production from alkaline pretreatment liquor (APL) by Cupriavidus necator DSM 545 using a supplement system consisting of oxidative enzymes (laccase, aryl alcohol oxidase (AAO)), mediators (ABTS, HOBT), DMSO, silica nanoparticle Aerosol R816 and surfactant Tween 80. First, screening experiments under Plackett-Burman design showed R816, ABTS and Tween 80 could significantly enhance PHB production. Additional experiments showed that HOBT and DMSO could be removed, and laccase and AAO were needed to remain in the system. Second, a central composite design was applied to obtain the optimum supplemental levels of R816, ABTS and Tween 80. Under optimum conditions, theoretical maximum PHB production (1.9 g/L) was close to experimental PHB production (2.1 g/L). With the supplement system, a 10-fold increase was achieved compared to PHB production (0.2 g/L) without any supplements.