Modular engineering of L-tyrosine production in Escherichia coli
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Modular engineering of L-tyrosine production in Escherichia coli. / Juminaga, Darmawi; Baidoo, Edward E K; Redding-Johanson, Alyssa M; Batth, Tanveer S; Burd, Helcio; Mukhopadhyay, Aindrila; Petzold, Christopher J; Keasling, Jay D.
In: Applied and Environmental Microbiology, Vol. 78, No. 1, 01.2012, p. 89-98.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Modular engineering of L-tyrosine production in Escherichia coli
AU - Juminaga, Darmawi
AU - Baidoo, Edward E K
AU - Redding-Johanson, Alyssa M
AU - Batth, Tanveer S
AU - Burd, Helcio
AU - Mukhopadhyay, Aindrila
AU - Petzold, Christopher J
AU - Keasling, Jay D
PY - 2012/1
Y1 - 2012/1
N2 - Efficient biosynthesis of L-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for L-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to L-tyrosine on two plasmids. Rational engineering to improve L-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to L-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter L-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways.
AB - Efficient biosynthesis of L-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for L-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to L-tyrosine on two plasmids. Rational engineering to improve L-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to L-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter L-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways.
KW - Alcohol Oxidoreductases/metabolism
KW - Chromatography, High Pressure Liquid
KW - Escherichia coli/metabolism
KW - Escherichia coli Proteins/metabolism
KW - Glucose/metabolism
KW - Metabolic Engineering/methods
KW - Phosphoenolpyruvate/metabolism
KW - Phosphotransferases (Alcohol Group Acceptor)/metabolism
KW - Polymerase Chain Reaction
KW - Proteomics
KW - Sugar Phosphates/metabolism
KW - Tyrosine/biosynthesis
U2 - 10.1128/AEM.06017-11
DO - 10.1128/AEM.06017-11
M3 - Journal article
C2 - 22020510
VL - 78
SP - 89
EP - 98
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
SN - 0099-2240
IS - 1
ER -
ID: 204047139