Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum

摘要

AIM: To study the effect of co-expression of ppsA, pckA,aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strainfor L-phenylalanineoMETHODS: ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coil and Brevibactenurn flavum to generate constructs pJN2 and pJNS.pJN2 was generated by inserting ppsA and pCKA genes into vector pCZ; whereas pJN5 was obtained by introducing ppSA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes.The recombinant plasmids were then introduced into B.flavum by electroporation and the transformants were used for L-phenylalanine fermentation.RESULTS: Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably, pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly.CONCLUSION: Co-expression of ppsA and pckA. genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pcsA, aroG, pheA and tyrB of E. coil in B. flavum was a feasible approach to construct a strain for phenylalanine production.