Effect of phosphoenolpyruvate carboxylase gene knock-out on metabolism in Corynebacterium pekinense PD-67

Abstract:

Abstract:[Objective] In order to optimize precursor supply for L-tryptophan biosynthesis, a Corynebacterium pekinense PD-67 mutant with phosphoenolpyruvate carboxylase gene(ppc) in-frame deletion was constructed. The effect of ppc knock-out on physiological characteristics of the mutant was investigated. [Methods] The upstream and downstream fragments of ppc were cloned from C. pekinense PD-67 chromosome and ligated to integration vector. The mutant C. pekinense PD-67-Δppc was screened by homologous recombination. The physiological characteristics of the mutant were investigated by fermentation experiments and measurement of pyruvate carboxylase (PCx) and pyruvate kinase (PK). [Results] The mutant with ppc gene in-frame deletion was screened and confirmed by PCR check and phosphoenolpyruvate carboxylase determination. The mutant exhibited slow growth and less cell mass, 80% as much as the parent strain. The ppc knock-out resulted in decrease of L-tryptophan accumulation and overproduction of pyruvate-related amino acids, which accompanied by increase of PK activity and the decrease of PCx activity, in C. pekinense PD-67. [Conclusion] The knock-out of ppc gene affected the metabolism of the strain to some extent. Only by blocking the anaplerotic pathway PEPCx participated was insufficient to increase the accumulation of L-tryptophan in C. pekinense PD-67.