Integration of E. coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis.
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.
Liu
Dong-Xin
DX
Department of Microbiology, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China.
Fan
Chang-Sheng
CS
Tao
Ju-Hong
JH
Liang
Guo-Xin
GX
Gao
Shan-E
SE
Wang
Hai-Jiao
HJ
Li
Xin
X
Song
Da-Xin
DX
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
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