Effect of pps and aroGfbr overexpression on L-tryptophan production in Corynebacterium pekinense

Abstract:

Abstract:［Objective］In order to redirect carbon flows into aromatic amino acids biosynthesis pathway and further improve the production of L-tryptophan in Corynebacterium pekinense PD-67，two schemes were implemented.First，the supply of phosphoenolpyruvate (PEP)，one of precursors of L-tryptophan biosynthesis，was increased. Second，the feedback inhibition of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase(DS)，a key enzyme in the aromatic amino acids biosynthesis，was relieved and the activity of DS was increased.［Methods］The phosphoenolpyruvate synthase gene (pps) was cloned from C． pekinense PD-67 chromosome by PCR and inserted into expression vector to construct a recombinant plasmid pXPPS; the aroG gene encoding DS isozymes was cloned from Escherichia coli chromosome by PCR and the mutation of Leu175Asp was introduced by site-directed mutagenesis using sequence-overlap extension PCR．The mutated gene named as aroGfbr was cloned to expression vector to construct a recombinant plasmid pXA; and the recombinant plasmid pXAPS co-expressing pps and aroGfbr was constructed．The three recombinant plasmids were transformed into PD-67 to generate the engineering strains PD-67 /pXPS，PD-67/pXA and PD-67/pXAPS，respectively．The fermentation characteristics of the three engineering strains were investigated.［Results］The expression of pps and aroGfbr was confirmed by enzyme activity assays．The deregulation of feedback inhibition of AroGfbr was confirmed by determining DS activity in the presence of three aromatic amino acids．The overexpression of pps and aroGfbr resulted in an increase of L-tryptophan biosynthesis by12. 1% and 26. 8%，respectively，while the co-expression of two genes increased the production of L-tryptophan by 35.9% in the engineering strain PD-67/pXAPS． ［Conclusion］ Both of the overexpressions of the pps gene and aroGfbr gene can increase L-tryptophan biosynthesis，while the production was further improved by the co-expression of the two genes．

Key words:Keywords: Corynebacterium pekinense， phosphoenolpyruvate synthase，3-deoxy-D-arabino-heptulosonate-7-phosphate synthase，feedback inhibition，overexpression，L-tryptophan