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The goal of this work was to constructEscherichia coli strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineeredE. coli strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33–38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine ofN -acetylglutamate synthetase (NAGS), the product of theargA gene and the first enzyme in the pathway of arginine biosynthesis inE. coli . To circumvent this inhibition, theargA genes fromE. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain “arg boxes,” which titrate the ArgR repressor protein, with or without theE. coli carAB genes encoding carbamyl phosphate synthetase and theargI gene for ornithine transcarbamylase. The free arginine production rates of “arg -derepressed”E. coli cells overexpressing plasmid-encodedcarAB ,argI , and fbrargA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt)argA . The expression system with fbrargA produced 7- to 35-fold more arginine than a system overexpressingcarAB andargI genes on a plasmid in a strain with a wtargA gene on the chromosome. The arginine biosynthetic capacity ofarg -derepressed DH5α strains with plasmids containing only the fbrargA gene was similar to that of cells with plasmids also containing thecarAB andargI genes. Plasmids containing wt or fbrargA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbrargA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.

applied and environmental microbiology

Use of Inducible Feedback-Resistant N -Acetylglutamate Synthetase ( argA ) Genes for Enhanced Arginine Biosynthesis by Genetically Engineered Escherichia coli K-12 Strains