Metabolic engineering and protein directed evolution increase the yield of L-phenylalanine synthesized from glucose in Escherichia coli

• Published in: Biotechnology and bioengineering Vol. 87; no. 4; pp. 516 - 524
• Main Authors: Báez-Viveros, José Luis, Osuna, Joel, Hernández-Chávez, Georgina, Soberón, Xavier, Bolívar, Francisco, Gosset, Guillermo
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 20-08-2004; Wiley; Wiley Subscription Services, Inc
• Subjects: Amino acids; Bacteria; Biological and medical sciences; Biotechnology; chorismate mutase-prephenate dehydratase; DAHP synthase; Directed Molecular Evolution - methods; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Evolution; Fundamental and applied biological sciences. Psychology; Glucose; Glucose - metabolism; L-Phenylalanine production; metabolic engineering; Metabolism; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Phenylalanine - biosynthesis; Phenylalanine - genetics; phosphotransferase transport system (PTS); Phosphotransferases - genetics; Phosphotransferases - metabolism; Prephenate Dehydratase - genetics; Prephenate Dehydratase - metabolism; Protein Engineering - methods; Proteins; Recombinant Proteins - metabolism; Signal Transduction - physiology; transketolase; Directed molecular evolution; Escherichia coli; Lyases; Glucose; Synthase; phosphotransferase transport system (PTS); DAHP synthase; Isomerases; Prephenate dehydratase; Production; Bacteria; Phosphotransferases; Enterobacteriaceae; Protein engineering; Phenylalanine; Enzyme; Transferases; Transketolase; L-Phenylalanine production; chorismate mutase-prephenate dehydratase; Chorismate mutase; Intramolecular transferases; Metabolic engineering; Yield; Carbon-oxygen lyases; Hydro-lyases
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.20159

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 (YPhe/Glc). The purpose of this work was to study the effect of PTS (phosphotransferase transport system) inactivation and overexpression of different versions of feedback inhibition resistant chorismate mutase‐prephenate dehydratase (CM‐PDT) on the yield (YPhe/Glc) and productivity of L‐Phe synthesized from glucose. The E. coli JM101 strain and its mutant derivative PB12 (PTS−Glc+ phenotype) were used as hosts. PB12 has an inactive PTS, but is capable of transporting and phosphorylating glucose by using an alternative system constituted by galactose permease (GalP) and glucokinase activities (Glk). JM101 and PB12 were transformed with three plasmids, harboring genes that encode for a feedback inhibition resistant DAHP synthase (aroGfbr), transketolase (tktA) and either a truncated CM‐PDT (pheAfbr) or its derived evolved genes (pheAev1 or pheAev2). Resting‐cells experiments with these engineered strains showed that JM101 and PB12 strains expressing either pheAev1 or pheAev2 genes produced l‐Phe from glucose with YPhe/Glc of 0.21 and 0.33 g/g, corresponding to 38 and 60% of the maximum theoretical yield (0.55 g/g), respectively. In addition, in both engineered strains the reached qPhe high levels of 40 mg/g‐dcw*h. The metabolic engineering strategy followed in this work, including a strain with an inactive PTS, resulted in a positive impact over the YPhe/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 YPhe/Glc. © 2004 Wiley Periodicals, Inc.