Femtosecond laser irradiation was employed to induce mutations in Rhizopus oryzae, leading to increases in fumaric acid production. Compared to the parental strain, mutant strain FM19 exhibited an increase in titer and yield of 56.3% and 36.6%, respectively, corresponding to a titer of 49.4 g/L and a yield of 0.56 g fumaric acid per g glucose. Metabolic profiling by gas chromatography-mass spectrometry revealed that higher levels of carbon (Embden-Meyerhof-Parnas and tricarboxylic acid cycle) and amino acid metabolism were operating in the high-yielding strain: particularly, 4-aminobutyric acid and 5-aminolevulinic acid were increased 10.33- and 7.22-fold, respectively, compared with parental strain during stationary phase. These findings provided new insights into metabolic characterization of high-yielding fumaric acid R. oryzae. (C) 2012 Elsevier Ltd. All rights reserved.