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Proteomic investigation on the pyk-F gene knockout Escherichia coli for aromatic amino acid production

Paper ID Volume ID Publish Year Pages File Format Full-Text
17895 42704 2007 11 PDF Available
Title
Proteomic investigation on the pyk-F gene knockout Escherichia coli for aromatic amino acid production
Abstract

Escherichia coli and many other microorganisms synthesize aromatic amino acids through the condensation reaction between phosphoenol pyruvate (PEP) and erythrose-4-phosphate (E4P) to form 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) and through a series of reactions to form phenylalinine, tyrosine and tryptophan. The proteomic response of a pyruvate kinase (pyk-F) mutant of Escherichia coli was quantitatively analyzed by two-dimensional electrophoresis with matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. Protein expressions of the mutant strain were compared with the wild type E. coli, where 24 protein spots were found to be expressed differentially. Ten protein spots, which remarkably increased in the mutant E. coli, were identified by MALDI-TOF-MS analysis. Evidently, main metabolic enzymes from the aromatic amino acid biosynthetic pathway, such as DAHP synthase (aro F, G, H), shikimate dehydrogenase (aro E), shikimate kinase (aroK, L), chorismate synthase (aro C), prephenate dehydrogenase (pheA), aminotransferase, anthranilate synthetase, tryptophan synthetase showed significant up-regulation in the mutant as compared to the wild type strain. Accumulation of PEP and E4P may direct the metabolic flow towards the biosynthetic route of chorismate and shikimate, key metabolic precursors of aromatic amino acids. The level of chorismate synthase was more than 10-fold higher in the mutant strain. In addition, we measured the enzyme activities of anthranilate synthetase (AS), prephenate dehydrogenase (PRDH), chorismate synthase (CM) and shikimate dehydrogenase (SKDH) to confirm the proteome result. It showed that 10 times overexpression of the PRDH and two to three times increase in the activities of AS, CM and SKDH are consistent with proteome results. Overexpression of these enzymes play the important role in increasing the production of phenylalanine, tyrosine and tryptophan in the E. coli mutant. Comparative proteome analyses of the wild type and the mutant showed that aromatic amino acid overproduction accompanied the significant changes in the synthesis level of key metabolic enzymes involved in aromatic amino acid biosynthesis and degradation pathway.

Keywords
pyk-F gene knockout Escherichia coli; Proteome; Aromatic amino acid biosynthesis; Shikimate pathway; Two-dimensional gel electrophoresis (2DE) and MALDI-TOF-MS
First Page Preview
Proteomic investigation on the pyk-F gene knockout Escherichia coli for aromatic amino acid production
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 41, Issue 4, 3 September 2007, Pages 455–465
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering