title: Integration of E. coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis.
abstract: OBJECTIVE
  To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine.
  
  
  METHODS
  By nitrosoguanidine mutagenesis, five p-fluorophenylalanine (FP)-resistant mutants of C.glutamicum FP were selected. The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR. Kanamycin resistance gene (Km) and the P(BF) -aroG-pheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pTK and pTGAK, respectively. Then, they were transformed into C.glutamicum FP respectively by electroporation. Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis. The transformed strains were used for L-phenylalanine fermentation and enzyme assays.
  
  
  RESULTS
  Engineering strains of C.glutamicum (Tyr(-)) were obtained. Compared with the original strain, the transformed strain C. glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold, and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, respectively.
  
  
  CONCLUSIONS
  Integration of tandem aroG-pheA genes into tyrA locus of C. glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production.
authors.affiliation: Department of Microbiology, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China.
authors.forename: Dong-Xin
authors.initials: DX
authors.lastname: Liu
authors.forename: Chang-Sheng
authors.initials: CS
authors.lastname: Fan
authors.forename: Ju-Hong
authors.initials: JH
authors.lastname: Tao
authors.forename: Guo-Xin
authors.initials: GX
authors.lastname: Liang
authors.forename: Shan-E
authors.initials: SE
authors.lastname: Gao
authors.forename: Hai-Jiao
authors.initials: HJ
authors.lastname: Wang
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Da-Xin
authors.initials: DX
authors.lastname: Song
mesh_terms: D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D002826:Chorismate Mutase / Q000235:genetics* / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D010649:Phenylalanine / Q000096:biosynthesis*; D010957:Plasmids / Q000235:genetics; D011302:Prephenate Dehydratase / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C071216:P-protein, E coli; D011994:Recombinant Proteins; C076398:TyrA protein, Bacteria; D010649:Phenylalanine; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D011302:Prephenate Dehydratase; D002826:Chorismate Mutase