Dicarboxylic acids are widely used organic acids in food, pharmaceutical and chemical industries. Pyruvate carboxylase plays a pivotal role in production of dicarboxylic acids. In this study, we have constructed a synthetic pathway of fumaric acid by deleting the FUM1 gene in Saccharomyces cerevisiae cells, lacking both PDC1 and ADH1, with reduced carbon flow towards ethanol.Furthermore, overexpression of the heterologous Rhizopus oryzae pyruvate carboxylase (RoPYC) gene in S. cerevisiae cells lacking PDC1, ADH1 and FUM1 rendered a fermentation titer of fumaric acid of 194.0±4.0 mg/L. Based on this model, we have demonstrated that each of three mutations (N315F, R485P and N1078F) or codon optimization of RoPYC significantly increased production of fumaric acid. A maximal titer of fumaric acid of 465.5±6.5 mg/L in a flask was achieved by combining the R485P mutation and the codon optimization of RoPYC in this model. Furthermore, in a 7-L bioreactor, we have observed that supplementation of CO2 has a positive impact on fumaric acid production.