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

被引:107
作者
Báez-Viveros, JL [1 ]
Osuna, J [1 ]
Hernández-Chávez, G [1 ]
Soberón, X [1 ]
Bolívar, F [1 ]
Gosset, G [1 ]
机构
[1] Univ Nacl Autonoma Mexico, Inst Biotechnol, Dept Ingn Celular & Biocatalisis, Cuernavaca 62250, Morelos, Mexico
关键词
Escherichia coli; phosphotransferase transport system (PTS); L-Phenylalanine production; metabolic engineering; DAHP synthase; transketolase; chorismate mutase-prephenate dehydratase;
D O I
10.1002/bit.20159
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 clehydratase (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(-)Gic(+) 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 (GaIP) and glucokinase activities (Glk). JM101 and PB12 were transformed with three plasmids, harboring genes that encode for a feedback inhibition resistant DAHIP 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 q(Phe) reached 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. (C) 2004 Wiley Periodicals, Inc.
引用
收藏
页码:516 / 524
页数:9
相关论文
共 34 条
[1]   TIGHTLY REGULATED TAC PROMOTER VECTORS USEFUL FOR THE EXPRESSION OF UNFUSED AND FUSED PROTEINS IN ESCHERICHIA-COLI [J].
AMANN, E ;
OCHS, B ;
ABEL, KJ .
GENE, 1988, 69 (02) :301-315
[2]   Determination of 3-deoxy-D-arabino-heptulosonate 7-phosphate productivity and yield from glucose in Escherichia coli devoid of the glucose phosphotransferase transport system [J].
Báez, JL ;
Bolívar, F ;
Gosset, G .
BIOTECHNOLOGY AND BIOENGINEERING, 2001, 73 (06) :530-535
[3]   Improving production of aromatic compounds in Escherichia coli by metabolic engineering [J].
Berry, A .
TRENDS IN BIOTECHNOLOGY, 1996, 14 (07) :250-256
[4]   CONSTRUCTION AND CHARACTERIZATION OF NEW CLONING VEHICLES .2. MULTIPURPOSE CLONING SYSTEM [J].
BOLIVAR, F ;
RODRIGUEZ, RL ;
GREENE, PJ ;
BETLACH, MC ;
HEYNEKER, HL ;
BOYER, HW ;
CROSA, JH ;
FALKOW, S .
GENE, 1977, 2 (02) :95-113
[5]   Metabolic engineering for microbial production of aromatic amino acids and derived compounds [J].
Bongaerts, J ;
Krämer, M ;
Müller, U ;
Raeven, L ;
Wubbolts, M .
METABOLIC ENGINEERING, 2001, 3 (04) :289-300
[6]   Phosphoenolpyruvate availability and the biosynthesis of shikimic acid [J].
Chandran, SS ;
Yi, J ;
Draths, KM ;
von Daeniken, R ;
Weber, W ;
Frost, JW .
BIOTECHNOLOGY PROGRESS, 2003, 19 (03) :808-814
[7]   Metabolic consequences of phosphotransferase (PTS) mutation in a phenylalanine-producing recombinant Escherichia coli [J].
Chen, RZ ;
Hatzimanikatis, V ;
Yap, WMGJ ;
Postma, PW ;
Bailey, JE .
BIOTECHNOLOGY PROGRESS, 1997, 13 (06) :768-775
[8]   IDENTIFICATION AND REMOVAL OF IMPEDIMENTS TO BIOCATALYTIC SYNTHESIS OF AROMATICS FROM D-GLUCOSE - RATE-LIMITING ENZYMES IN THE COMMON PATHWAY OF AROMATIC AMINO-ACID BIOSYNTHESIS [J].
DELL, KA ;
FROST, JW .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1993, 115 (24) :11581-11589
[9]   BIOCATALYTIC SYNTHESIS OF AROMATICS FROM D-GLUCOSE - THE ROLE OF TRANSKETOLASE [J].
DRATHS, KM ;
POMPLIANO, DL ;
CONLEY, DL ;
FROST, JW ;
BERRY, A ;
DISBROW, GL ;
STAVERSKY, RJ ;
LIEVENSE, JC .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1992, 114 (10) :3956-3962
[10]   Pathway engineering for the production of aromatic compounds in Escherichia coli [J].
Flores, N ;
Xiao, J ;
Berry, A ;
Bolivar, F ;
Valle, F .
NATURE BIOTECHNOLOGY, 1996, 14 (05) :620-623