The global regulatory system of Escherichia coli, carbon storage regulator (Csr), was engineered to increase the intracellular concentration of phosphoenolpyruvate. We examined the effects of csrA and csrD mutations and csrB overexpression on phenylalanine production in E. coli NST37 (NST). Overexpression of csrB led to significantly greater phenylalanine production than csrA and csrD mutations (2.33 vs 1.67 and 1.61 g l(-1), respectively; P<0.01). Furthermore, the overexpression of csrB was confirmed by the observed increase in csrB transcription level. We also determined the effect of overexpressing transketolase A (TktA) or glucose-6-phosphate dehydrogenase (Zwf) in NST and the csrA mutant of NST (NSTCSRA) on phenylalanine production. The NSTCSRA strain overexpressing TktA (NSTCSRA [pTktA]) produced significantly more phenylalanine than that of Zwf (2.39 vs 1.61 g l(-1); P>0.01). Furthermore, we examined the effect of overexpressing TktA, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (AroF(FR)), and chorismate mutase/prephenate dehydratase (PheA(FR)) together in NSTCSRA (NSTCSRA [pTkaFpA]). It is interesting to note that NSTCSRA [pTkaFpA] produced significantly less phenylalanine than both NSTCSRA [pTktA] and NST overexpressing csrB (NST [pCsrB]) (1.84 vs 2.39 and 2.33 g l(-1), respectively; P<0.01). Thus, csrB overexpression or csrA mutation in combination with tktA overexpression was more effective than previous approaches that targeted the glycolytic or aromatic pathway enzymes for enhancing phenylalanine production.