Metabolic Engineering and Protein Directed Evolution Increase the Yield of L-Phenylalanine Synthesized From Glucose in Escherichia coli Jose ´ Luis Ba ´ez-Viveros, Joel Osuna, Georgina Herna ´ndez-Cha ´vez, Xavier Sobero ´n, Francisco Bolı ´var, Guillermo Gosset Departamento de Ingenierı ´a Celular y Biocata ´lisis, Instituto de Biotecnologı ´a, Universidad Nacional Auto ´noma de Me ´xico, Apdo Postal 510-3. Cuernavaca, Morelos 62250, Me ´xico; telephone: 52-777-3291648; fax: 52-777-3172388; e-mail: gosset @ibt.unam.mx Received 13 November 2003; accepted 23 March 2004 Published online 26 July 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20159 Abstract: L-phenylalanine (L-Phe) is an aromatic amino acid with diverse commercial applications. Technologies for industrial microbial synthesis of L-Phe using glucose as a starting raw material currently achieve a relatively low conversion yield (Y Phe/Glc ). The purpose of this work was to study the effect of PTS (phosphotransferase trans- port system) inactivation and overexpression of differ- ent versions of feedback inhibition resistant chorismate mutase-prephenate dehydratase (CM-PDT) on the Y Phe/Glc and productivity of L-Phe synthesized from glucose. The E. coli JM101 strain and its mutant derivative PB12 (PTS À Glc + phenotype) were used as hosts. PB12 has an inactive PTS, but is capable of transporting and phospho- rylating glucose by using an alternative system constituted by galactose permease (GalP) and glucokinase activities (Glk). JM101 and PB12 were transformed with three plas- mids, harboring genes that encode for a feedback inhibi- tion resistant DAHP synthase (aroG fbr ), transketolase (tktA) and either a truncated CM-PDT (pheA fbr ) or its derived evolved genes (pheA ev1 or pheA ev2 ). Resting-cells experi- ments with these engineered strains showed that JM101 and PB12 strains expressing either pheA ev1 or pheA ev2 genes produced L-Phe from glucose with Y Phe/Glc of 0.21 and 0.33 g/g, corresponding to 38 and 60% of the max- imum theoretical yield (0.55 g/g), respectively. In addition, in both engineered strains the q Phe reached high levels of 40 mg/g-dcw * h. The metabolic engineering strategy fol- lowed in this work, including a strain with an inactive PTS, resulted in a positive impact over the Y Phe/Glc , enhancing it nearly 57% compared with its PTS + counterpart. This is the first report wherein PTS inactivation was a successful strategy to improve the Y Phe/Glc . B 2004 Wiley Periodicals, Inc. Keywords: Escherichia coli; phosphotransferase transport system (PTS); L-Phenylalanine production; metabolic en- gineering; DAHP synthase; transketolase; chorismate mutase-prephenate dehydratase INTRODUCTION Aromatic amino acids are important examples of chemical products that can be produced by microorganisms using renewable raw materials such as D-glucose. Of particular interest is L-phenylalanine production (L-Phe), since this compound has many applications in the food and pharma- ceutical industries. The market volume of L-Phe ascends to f11,000 metric tons per annum. This amino acid is mainly used as a nutritional supplement and as a precursor for the synthesis of food additives and pharmaceuticals (Bongaerts et al., 2001). Considering the size of its market, aspartame (L-aspartyl-L-phenylalanine methyl ester) is the most impor- tant compound having L-Phe as a precursor. Nowadays, aspartame is the most widely used low-calorie sweetener, with an estimated world market of USD $1.5 billion. The chemical synthesis of L-Phe has clear disadvantages when compared with the biosynthesis using recombinant microorganisms; for instance, chemical synthesis employs nonrenewable toxic raw materials and generates racemic mixtures of D and L Phe isomers, which complicate the puri- fication processes (Frost and Lievense, 1994). In contrast, the biocatalytic synthesis of L-Phe constitutes an attractive alternative since it is a clean technology that uses renew- able resources such as simple carbohydrates (D-glucose), generating less environmental pollution. Nevertheless, the challenge to overcome in the biochemical processes is to improve the modest yield coefficient (conversion efficiency of product/substrate) and productivities of current technol- ogies (Patnaik et al., 1995; Berry, 1996). Figure 1 shows a simplified metabolic map of the Esch- erichia coli central metabolism including the main in- termediates and direct precursors involved in aromatic amino acid biosynthesis: phosphoenolpyruvate (PEP) and erythrose 4-phosphate (EP4). Among the constraints limit- ing the capacity of E. coli for producing high levels of L-Phe are the highly controlled regulation on genes and B 2004 Wiley Periodicals, Inc. Correspondence to: Guillermo Gosset Contract grant sponsor: Consejo Nacional de Ciencia y Tecnologı ´a, Me ´xico (CONACyT); CONACyT and UNAM (fellowships to J.L.B.V) Contract grant number: NC-230