Use of inducible feedback-resistant N-acetylglutamate synthetase (argA) genes for enhanced arginine biosynthesis by genetically engineered Escherichia coli K-12 strains - PubMed (original) (raw)

Use of inducible feedback-resistant N-acetylglutamate synthetase (argA) genes for enhanced arginine biosynthesis by genetically engineered Escherichia coli K-12 strains

B S Rajagopal et al. Appl Environ Microbiol. 1998 May.

Abstract

The goal of this work was to construct Escherichia coli strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. 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 of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain "arg boxes," which titrate the ArgR repressor protein, with or without the E. coli carAB genes encoding carbamyl phosphate synthetase and the argI gene for ornithine transcarbamylase. The free arginine production rates of "arg-derepressed" E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt) argA. The expression system with fbr argA produced 7- to 35-fold more arginine than a system overexpressing carAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5 alpha strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carAB and argI genes. Plasmids containing wt or fbr argA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.

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Figures

FIG. 1

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.

FIG. 2

FIG. 2

pABIA (linked as an operon) (a) and pA vectors (b) engineered for this study. The vectors contained the gene(s) downstream from a control region which includes the trc promoter and a lac operator. The constructs also contained an arg box cloned from the argI gene for binding and titration of the arginine repressor. Ori, ColE1 replication origin.

FIG. 3

FIG. 3

Arginine biosynthesis in E. coli DH5α containing the engineered plasmids or the parent vectors. The relevant expressed genes of vectors are shown in Table 2. The induced cultures were incubated with glutamine (20 mM) or with glutamine (20 mM) plus ornithine (5 mM) for 3 h, and total arginine production was determined and reported as nanomoles of arginine per milligram (dry weight) per hour.

FIG. 4

FIG. 4

Multiple-sequence alignment of argA homologs. The sequences used were argA homologs from E. coli (1) (arga_ecoli), Pseudomonas aeruginosa (4) (arga_psea), and Pseudomonas putida (4) (arga_psepu). The argA homologs were aligned by using the PILEUP and PRETTY programs of the Genetics Computer Group. The three prokaryotic NAGS sequences showed 45% identity and an additional 12% similarity. Identical residues are shaded, and homologous residues detected by the PAM250 matrix of amino acid similarity (3) (determined by using the SEQVU program and manual editing) are boxed (functionally similar amino acids follow: D and E; F and Y; G and W; N and D; K and R; Q and E; L and M; I and V; and A, S, and T). The mutations H15Y (argA214), Y19C (argA215), S54N (argA218), R58H (argA213), G287S (argA216), and Q432R (argA219) are indicated above the sequence. Gaps introduced to optimize alignment are indicated by hyphens.

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