APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY Metabolic engineering of Escherichia coli to enhance phenylalanine production N. Yakandawala & T. Romeo & A. D. Friesen & S. Madhyastha Received: 3 July 2007 / Revised: 15 November 2007 / Accepted: 28 November 2007 / Published online: 15 December 2007 # Springer-Verlag 2007 Abstract 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 muta- tions (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 phenyl- alanine 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] pro- duced 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. Introduction Phenylalanine, together with glutamate, methionine, and lysine is one of the most important commercially produced amino acids. It is used predominantly for the production of the low-calorie sweetener aspartame (L-phenylalanyl-L- aspartyl-methyl ester), which is used in soft drinks and confectionary products (Bongaerts et al. 2001). The biosynthesis of phenylalanine is initiated by the condensa- tion reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form 3-deoxy-D-arabino- heptulosonate-7-phosphate (DAHP). This is the committed step of the shikimate pathway and is catalyzed by three DAHP synthases (Fig. 1). These isoenzymes are encoded by the genes aroF , aroG, and aroH, whose products are feedback inhibited by tyrosine, phenylalanine, and trypto- phan, respectively. A second PEP molecule is incorporated at a later step to form 5-enolpyruvoylshikimate-3-phosphate (EPSP). Furthermore, the central pathway branches to permit the biosynthesis of phenylalanine, tyrosine, and tryptophan when EPSP is converted to chorismate (Fig. 1). In phenylalanine synthesis, prephenate formation from chorismate is catalyzed by the bifunctional enzyme cho- rismate mutase-p-prephenate dehydratase (PheA). PheA is feedback inhibited by phenylalanine and it also catalyzes Appl Microbiol Biotechnol (2008) 78:283–291 DOI 10.1007/s00253-007-1307-z N. Yakandawala : S. Madhyastha (*) Kane Biotech Inc., 5-1250 Waverley Street, Winnipeg, MB, Canada R3T 6C6 e-mail: srim@kanebiotech.com T. Romeo Department of Microbiology and Immunology, Emory University School of Medicine, 3105 Rollins Research Centre, 1510 Clifton Road N.E., Atlanta, GA 30322, USA A. D. Friesen Medicure Inc., 4-1200 Waverley Street, Winnipeg, MB, Canada R3T 0P4