title: Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum.
abstract: Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and (13)C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products.
authors: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Hamburg, Germany.
authors: Zhen
authors: Z
authors: Chen
authors: Rajesh Reddy
authors: RR
authors: Bommareddy
authors: Doinita
authors: D
authors: Frank
authors: Sugima
authors: S
authors: Rappert
authors: An-Ping
authors: AP
authors: Zeng
mesh_terms: D001224:Aspartic Acid / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D005246:Feedback; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007553:Isotope Labeling; D008239:Lysine / Q000096:biosynthesis*; D008293:Malates / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D064688:Metabolic Flux Analysis; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation
chemical_list: D002247:Carbon Isotopes; D004791:Enzyme Inhibitors; D008293:Malates; D050505:Mutant Proteins; D001224:Aspartic Acid; D009249:NADP; C030298:malic acid; D010730:Phosphoenolpyruvate Carboxylase; D008239:Lysine