Activating Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Combination for Improvement of Succinate Production

Abstract

d Phosphoenolpyruvate (PEP) carboxylation is an important step in the production of succinate by Escherichia coli. Two enzymes, PEP carboxylase (PPC) and PEP carboxykinase (PCK), are responsible for PEP carboxylation. PPC has high substrate affinity and catalytic velocity but wastes the high energy of PEP. PCK has low substrate affinity and catalytic velocity but can conserve the high energy of PEP for ATP formation. In this work, the expression of both the ppc and pck genes was modulated, with multiple regulatory parts of different strengths, in order to investigate the relationship between PPC or PCK activity and succinate production. There was a positive correlation between PCK activity and succinate production. In contrast, there was a positive correlation between PPC activity and succinate production only when PPC activity was within a certain range; excessive PPC activity decreased the rates of both cell growth and succinate formation. These two enzymes were also activated in combination in order to recruit the advantages of each for the improvement of succinate production. It was demonstrated that PPC and PCK had a synergistic effect in improving succinate production.

Combinatorial optimization of CO2 transport and fixation to improve succinate production by promoter engineering

“…To address this issue, promoter engineering including promoter library construction and characterization is essential for generating the dynamic range necessary to enable fine-tuned gene expression (Keasling, 2012;Young and Alper, 2010), and it has become an important solution to optimal expression of genes and combinatorial optimization of metabolic pathways (Dahl et al, 2013;Du et al, 2012;Liu et al, 2014). In E. coli, promoter engineering has been successfully applied to optimize single gene expression to improve the synthesis of succinic acid (Tan et al, 2013;Thakker et al, 2011).…”

“…In E. coli, the CO 2 fixation represents the committed step in succinate synthesis (Kim et al, 2004;Tan et al, 2013). CO 2 was used by cell involves a two-step process-uptake process from extracellular to intracellular and fixation process of intracellular metabolism.…”

Enhancing succinic acid biosynthesis in Escherichia coli by engineering its global transcription factor, catabolite repressor/activator (Cra)

“…3c, PCK activity was higher in Tang1541 and Tang1544 than in Tang1505 (pTrc99A) or Tang1534 (Cra). This higher PCK and PPC activity enhanced CO 2 fixation (Table 3), which is considered the key step in succinic acid synthesis32. The mutation of Cra did not significantly alter MDH activity (Fig.…”

Efficient production of succinic acid from macroalgae hydrolysate by metabolically engineered Escherichia coli

“…Byproduct pathways were knocked out (i.e., lactate dehydrogenase and pyruvate-formate lyase) (Bunch et al, 1997). Key limited-reactions were enhanced (i.e., phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase) (Tan et al, 2013;Wang et al, 2011). Exogenous synthetic routes were introduced (pyruvate carboxylase) (Sanchez et al, 2005).…”