Poly-beta-hydroxybutyrate (PHB) biosynthesis in Ralstonia eutropha from gluconate as a carbon source is carried out through the Entner-Doudoroff (ED) pathway and the pentose-phosphate (PP) pathway generating NADPH and glyceraldehyde-3-phosphate that flows to acetyl-CoA, actively in the unbalanced PHB accumulation phase. The gnd gene encoding 6-phosphogluconate dehydrogenase (6PGDH) and the tktA gene encoding the transketolase (TK) in PP pathway of E. coli were transformed into R. eutropha H16 to modify the metabolic flux of gluconate to the PHB biosynthesis. Over-generated NADPH by the amplified gnd gene tended to depress the cell growth and PHB concentration. Meanwhile, the amplified tktA gene significantly increased both PHB biosynthesis and cell growth as a result of the effective flow of glyceraldehyde-3-phosphate into acetyl-CoA along with the concomitant supplementation of NADPH. The amplified tktA gene also activated the enzyme activities directly associated with PHB biosynthesis. The transformant R. eutropha harboring tktA gene was cultivated using pH-stat-fed-batch to achieve the overproduction of PHB.