Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution

Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. NADPH in the mutant stress 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. INTRODUCTION The phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate (OAA) node is one of the most important links between glycolysis/gluconeogenesis and the tricarboxylic acid (TCA) cycle (1). A set of reactions at this node direct the carbon flux into appropriate directions, making it a highly relevant switch point for carbon flux distribution within the central metabolism (Fig. 1). In most bacterias, the cells regenerate OAA through anaplerotic pathways either from PEP by phosphoenolpyruvate carboxylase 334951-92-7 IC50 (PEPC) or from pyruvate by pyruvate carboxylase (Personal computer). 334951-92-7 IC50 possesses both PEPC and Personal computer for replenishing OAA consumed for biosynthesis during mobile development or OAA-derived amino acidity creation in commercial fermentations (2). The OAA source has been regarded as a bottleneck for lysine creation and different strategies have been applied to increase OAA availability such as overexpression of PEPC (3, 4) or PC (5, 6), deletion of pyruvate kinase (7), or phosphoenolpyruvate carboxykinase (PEPCK) (8). FIG 1 Simplified scheme of allosteric regulation of the central metabolism and lysine synthesis pathways in activity than PC, it has been reported that it contributed only ca. 10% of the total oxaloacetate synthesis in glucose-growing cells of (9). Overexpression of PC in increased lysine accumulation by 50% (6), while the overexpression of PEPC showed only marginal effect on lysine production (3, 4). The contradictory phenomenon of the PEPC for its high activity with its low effect on lysine production may be due to the fact that PEPC is subjected to the rigid feedback inhibitions of aspartate and malate (4) (Fig. 1), whereas PC is only slightly inhibited by aspartate (10). Thus, deregulation of the feedback inhibition of PEPC by aspartate and malate may enhance the anaplerotic function of PEPC and improve lysine production. This conjecture, however, has not been verified thus far. In the present work, we report for the first time a rational deregulation 334951-92-7 IC50 of the feedback inhibition of PEPC and demonstrate that it can significantly improve lysine production in has not been crystallized until now, a homology modeling of its three-dimensional structure was generated by using the software Modeller ( based on the structure of PEPC (PDB code 1FIY). PEPC shared 33.5% identity with the amino acid sequence of PEPC, and the residues involved in aspartate binding are conserved. Bacterial strains and plasmids. The strains and plasmids used in the present study are listed in Table 1. JM109 was used for the construction of plasmids, and BL21(DE3)-RIL cells (Stratagene) was used as a host for enzyme overexpression and purification. LC298, a lysine-producing strain that has a deregulated aspartokinase (11), was used as the host to construct LP917. TABLE 1 Strains and plasmids used in the present study Molecular cloning and enzyme overexpression. The wild-type gene encoding PEPC was amplified by PCR from the genomic DNA of ATCC 13032 using the primers 5-GCGCGCCCATATGACTGATTTTTTACGCGATGACAT-3 and 5-TATAGTCGACCTAGCCGGAGTTGCGCAGCGCAGTGG-3 and inserted into NdeI-XhoI sites of pET-28a(+) (Novagen). The expression vector was designated pEppc. Six PEPC mutants with selected point mutations were constructed by using a QuikChange site-directed mutagenesis kit (Stratagene) according to the standard protocol. Wild-type PEPC and its mutants were overexpressed in BL21(DE3) RIPL (Stratagene). The cells were cultured in Luria-Bertani (LB) medium at 37 C until the optical density at 600 nm (OD600) reached 0.6, and 0.1 mmol of IPTG (isopropyl–d-thiogalactopyranoside) was added to induce enzyme overexpression for Rabbit polyclonal to ABCG5 another 12 h at 20C. Purified enzymes were obtained by using a Ni2+-nitrilotriacetic acid (NTA) column (GE Healthcare Bio-Sciences, Piscataway, NJ). Protein content was quantified by the method of Bradford (14). Determination of the enzyme activities of PEPC and its mutants. The enzyme activity of the purified PEPC was assayed by a coupling reaction catalyzed by malate dehydrogenase at 25C as previously described (15,C17). The standard reaction mixture contained 100 mM Tris-HCl (pH 7.5), 10 mM MnSO4, 10 mM NaHCO3, 2 mM PEP, 0.1 mM NADH, and 1.5 IU of malate dehydrogenase. The decrease of NADH.