Dicarboxylic acids (DCAs) can be obtained by oxidizing alkanes by Candida tropicalis. Through alpha-monocarboxylic acids (MCAs), alpha- and omega-oxidation yield alpha- or omega-DCAs, respectively. However, both MCAs and DCAs may be degraded to acetyl-CoA by beta-oxidation, resulting in a limited DCA yield. Acetyl-CoA can be transported into the mitochondrion for the TCA cycle by carnitine acetyltransferase (CAT), by which the energy generation and beta-oxidation are connected. In this paper, we present a method to reconstruct the metabolic pathway by inhibiting the acetyl-CoA transportation system. Metabolic engineering is applied on the acetyl-CoA transportation system, but not the key enzymes in beta-oxidation. Starting with the original strain W10-1, cat heterozygote CZ-15 and cat homozygote CKC-11 were obtained by gene knockout. The CAT specific activity in CZ-15 was about 50% lower than that in W10-1, resulting in a 21.0% increase of the DCA concentration, and a 12% increase of the molar conversion of alkane, reaching 61.6%. However, no CAT activity was detected in CKC-11, and CKC-11 could not grow on alkane. These results indicate that inhibition of beta-oxidation via reconstruction of the transportation process between organelles can facilitate DCA production, but that totally blocking the & betagr;-oxidation would be harmful for energy supply. We thus provide a novel insight into regulation of the beta-oxidation system and metabolic flux. Further understanding of beta-oxidation and the acetyl-CoA transportation system in Candida tropicalis is reached through examination of fermentation data by metabolic flux analysis.