Poly-(3-hydroxybutyrate) (P3HB) is a promising biodegradable plastic synthesized from acetyl-CoA. One important factor affecting the P3HB production cost is the P3HB yield. Through flux balance analysis of an extended genome-scale metabolic network of E. coli, we found that the introduction of non-oxidative glycolysis pathway (NOG), a previously reported pathway enabling complete carbon conservation, can increase the theoretical carbon yield from 67% to 89%, equivalent to the theoretical mass yield from 0.48 g P3HB/g glucose to 0.64 g P3HB/g glucose. Based on this analysis result, we introduced phosphoketolase and enhanced the NOG pathway in E. coli. The mass yield in the engineered strain was increased from 0.16 g P3HB/g glucose to 0.24 g P3HB/g glucose. We further overexpressed pntAB to enhance the NADPH availability and down-regulated TCA cycle to divert more acetyl-CoA toward P3HB. The final construct accumulated 5.7 g/L P3HB and reached a carbon yield of 0.43 (a mass yield of 0.31 g P3HB/g glucose) in shake flask cultures in shake flask cultures. The introduction of NOG pathway could also be useful for improving yields of many other biochemicals derived from acetyl-coA.