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

The mammalian urea cycle is the main chemical pathway for the “detoxification” of ammonia by conversion to urea which is efficiently eliminated in the urine. Hyperammonemia, a clinical problem with severe consequences for the central nervous system, is usually caused by liver disease or inherited metabolic disorders. This work was undertaken to engineer Escherichia coli strains for enhanced incorporation of ammonia into arginine. The availability of such a biological system could then be used for development of therapy for the removal of ammonia in hyperammonemic patients. For example, these bacteria can be used to colonize the intestine for the purpose of incorporating free intestinal ammonia into arginine. As arginine contains three nitrogen atoms compared to the one atom in glutamate, the “flux” through this biosynthetic pathway, if enhanced, would result in incorporation of a large number of nitrogen atoms into organic compounds. In E. coli, biosynthesis of arginine from glutamate is carried out by a series of reactions initiated by the acetylation of glutamate by N-acetylglutamate synthetase (NAGS) encoded by argA (2) (Fig. ​(Fig.1).1). Arginine biosynthesis is regulated via transcriptional repression of the arg regulon and by feedback inhibition of NAGS by arginine (2, 6, 10, 19). l-Arginine represses argA expression with a ratio greater than 250 and inhibits NAGS activity (Ki = 0.02 mM) (9). In order to develop a system for enhanced biosynthesis of arginine by E. coli, effective transcriptional derepression of arg biosynthetic genes and feedback-resistant (fbr) NAGS enzymes are required. Previous attempts to overproduce arginine in Serratia marcescens by using this strategy were unsuccessful, since the bacteria carrying the chromosomal fbr argA mutations were unstable, giving rise to argA mutants with reduced activity or with altered affinity for glutamate (8, 15, 16). Recently, we have reported the use of a plasmid system in E. coli for enhanced biosynthesis of arginine by means of derepression of the arginine regulon and simultaneous overexpression of the E. coli carAB genes encoding carbamyl phosphate synthetase and the argI gene for ornithine transcarbamylase on a plasmid (18). Arginine production in these bacteria was 6- to 16-fold higher than controls, but only if exogenous ornithine was added to the incubation medium, since ornithine production was limited due to feedback inhibition of NAGS by arginine. In order to circumvent the requirement of exogenous ornithine, fbr NAGS activity was needed. The argA genes from the three fbr NAGS E. coli strains of Eckhardt and Leisinger (5), as well as from two newly isolated fbr argA strains, were incorporated into the previously engineered arginine-producing systems, and the modified expression systems were investigated with respect to arginine production and strain stability. Different single-base substitutions in argA genes were found in each of the fbr NAGS strains. FIG. 1 Pathway of arginine biosynthesis in E. coli. (P), overexpression of the genes on engineered plasmids; wt, feedback sensitive; fbr, feedback resistant; AcSCoA, acetyl coenzyme A; HSCoA, coenzyme A.