Effect of aromatic amino acid transport gene knock-out on L-tryptophan accumulation in Corynebacterium pekinense PD-67

Abstract:

Abstract:［Objective］A Corynebacterium pekinense PD-67 mutant with aromatic amino acid transport system gene(aroP) in-frame deletion was constructed to decrease the uptake of L-tryptophan and reduce the intracellular pool of L-tryptophan，further to deregulate the feedback regulation of L-tryptophan and increase the extracellular accumulation． The effects of aroP knock-out as well as anthranilate synthetase (EC4. 1. 3. 27; AS) gene overexpression on L-tryptophan accumulation of the mutant were investigated． ［Methods］The aroP gene was cloned from C． pekinense PD-67 chromosome and ligated to integration vector，and then deleted about 600bp fragment by restriction endonuclease digestion． The mutant C．pekinense PD-67-ΔaroP was screened by homologous recombination． The mutant phenotype can be reversed by complementation with aroP gene from the expression vector． AS gene was cloned and ligated to expression vector to construct a recombinant plasmid． The plasmid was transformed into PD-67ΔaroP to generate the engineering strain PD-67ΔaroP/pXAS．The fermentation characteristics of the mutant and the engineering strain were investigated． ［Results］The aroP gene in-frame deletion was screened and confirmed by PCR analysis and the AS gene expression was confirmed by determination of enzyme activity． The aroP knock-out resulted in increase of L-tryptophan accumulation by 65% compared with that of the parent strain，while the expression of AS gene resulted in increase of L-tryptophan yield on cell mass by 25. 6% in engineered strain． ［Conclusion］The aroP gene knock-out of the strain PD-67 improved L-tryptophan accumulation． The expression of AS gene could further improve L-tryptophan yield on cell mass in engineered strain．