Abstract:
: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.
journal_name
Appl Environ Microbioljournal_title
Applied and environmental microbiologyauthors
Juminaga D,Baidoo EE,Redding-Johanson AM,Batth TS,Burd H,Mukhopadhyay A,Petzold CJ,Keasling JDdoi
10.1128/AEM.06017-11subject
Has Abstractpub_date
2012-01-01 00:00:00pages
89-98issue
1eissn
0099-2240issn
1098-5336pii
AEM.06017-11journal_volume
78pub_type
杂志文章abstract::We previously reported the isolation of a 21.5-kb genomic DNA fragment from Pseudomonas sp. strain ADP, which contains the atzA gene, encoding the first metabolic step for the degradation of the herbicide atrazine (M. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Mic...
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doi:10.1128/AEM.63.3.916-923.1997
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doi:10.1128/aem.66.8.3481-3486.2000
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pub_type: 杂志文章
doi:10.1128/AEM.50.2.529-531.1985
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