title: C4-dicarboxylic acid production by overexpressing the reductive TCA pathway.
abstract: As C4-dicarboxylic acids could replace C4-petrochemicals, the reductive tricarboxylic acid (TCA) pathway was overexpressed in Pichia pastoris for production of the C4-dicarboxylic acids. Three expression cassettes which carried the pyruvate carboxylase gene (pc), the cytoplasmic malate dehydrogenase gene (mdh1) and the retargeted fumarase gene (Tfum) were integrated into the chromosomal DNA of P. pastoris GS115 alone or jointly. Multicopy integrations were screened using quantitative PCR for C4-dicarboxylic acid overaccumulation. The results showed that the highest titer in 96 h of fumaric, malic and succinic acid (0.76, 42.28 and 9.42 g l(-1)) was obtained by co-expression of pc and mdh1 in P. pastoris. This is the first report about multiple genes engineered in P. pastoris for C4-dicarboxylic acid production. The strain Pp-PC-MDH1, moreover, has a significant potential to produce malic acid in aerobic conditions.
authors: Beijing Bioprocess Key Laboratory, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, PR China.
authors: Ting
authors: T
authors: Zhang
authors: Beijing Bioprocess Key Laboratory, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, PR China.
authors: Chengyu
authors: C
authors: Ge
authors: Beijing Bioprocess Key Laboratory, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, PR China Amoy - BUCT Industrial of Bio-technovation Institute, Amoy, 361022, PR China dengli@mail.buct.edu.cn.
authors: Li
authors: L
authors: Deng
authors: Beijing Bioprocess Key Laboratory, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, PR China.
authors: Tianwei
authors: T
authors: Tan
authors: Beijing Bioprocess Key Laboratory, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, PR China.
authors: Fang
authors: F
authors: Wang
mesh_terms: D002952:Citric Acid Cycle / Q000235:genetics; D003998:Dicarboxylic Acids / Q000378:metabolism*; D005649:Fumarate Hydratase / Q000096:biosynthesis* / Q000235:genetics; D005650:Fumarates / Q000378:metabolism; D016680:Genome, Bacterial; D059765:Homologous Recombination; D008291:Malate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D008293:Malates / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D000432:Methanol / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000502:physiology; D011766:Pyruvate Carboxylase / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003998:Dicarboxylic Acids; D005650:Fumarates; D008293:Malates; C030298:malic acid; D008291:Malate Dehydrogenase; D005649:Fumarate Hydratase; D011766:Pyruvate Carboxylase; D000432:Methanol
keywords: Pichia pastoris; homologous recombination; methanol induction; real-time PCR