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Metabolic engineering and protein directed evolution increase the yield of L ‐phenylalanine synthesized from glucose in Escherichia coli
Author(s) -
BáezViveros José Luis,
Osuna Joel,
HernándezChávez Georgina,
Soberón Xavier,
Bolívar Francisco,
Gosset Guillermo
Publication year - 2004
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20159
Subject(s) - metabolic engineering , biochemistry , phenylalanine , glucokinase , escherichia coli , chorismate mutase , yield (engineering) , chemistry , pep group translocation , plasmid , mutant , aromatic amino acids , amino acid , gene , materials science , metallurgy
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 transport system) inactivation and overexpression of different versions of feedback inhibition resistant chorismate mutase‐prephenate dehydratase (CM‐PDT) on the yield ( Y Phe/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 ( aroG fbr ), transketolase ( tktA ) and either a truncated CM‐PDT ( pheA fbr ) or its derived evolved genes ( pheA ev1 or pheA ev2 ). Resting‐cells experiments 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 maximum theoretical yield (0.55 g/g), respectively. In addition, in both engineered strains the reached q Phe 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 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 . © 2004 Wiley Periodicals, Inc.