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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-deltappc 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.