A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum

Abstract

BackgroundArginine biosynthesis in Corynebacterium glutamicum consists of eight enzymatic steps, starting with acetylation of glutamate, catalysed by N-acetylglutamate synthase (NAGS). There are different kinds of known NAGSs, for example, “classical” ArgA, bifunctional ArgJ, ArgO, and S-NAGS. However, since C. glutamicum possesses a monofunctional ArgJ, which catalyses only the fifth step of the arginine biosynthesis pathway, glutamate must be acetylated by an as of yet unknown NAGS gene.ResultsArginine biosynthesis was investigated by metabolome profiling using defined gene deletion mutants that were expected to accumulate corresponding intracellular metabolites. HPLC-ESI-qTOF analyses gave detailed insights into arginine metabolism by detecting six out of seven intermediates of arginine biosynthesis. Accumulation of N-acetylglutamate in all mutants was a further confirmation of the unknown NAGS activity. To elucidate the identity of this gene, a genomic library of C. glutamicum was created and used to complement an Escherichia coli ΔargA mutant. The plasmid identified, which allowed functional complementation, contained part of gene cg3035, which contains an acetyltransferase domain in its amino acid sequence. Deletion of cg3035 in the C. glutamicum genome led to a partial auxotrophy for arginine. Heterologous overexpression of the entire cg3035 gene verified its ability to complement the E. coli ΔargA mutant in vivo and homologous overexpression led to a significantly higher intracellular N-acetylglutamate pool. Enzyme assays confirmed the N-acetylglutamate synthase activity of Cg3035 in vitro. However, the amino acid sequence of Cg3035 revealed no similarities to members of known NAGS gene families.ConclusionsThe N-acetylglutamate synthase Cg3035 is able to catalyse the first step of arginine biosynthesis in C. glutamicum. It represents a novel class of NAGS genes apparently present only in bacteria of the suborder Corynebacterineae, comprising amongst others the genera Corynebacterium, Mycobacterium, and Nocardia. Therefore, the name C-NAGS (Corynebacterineae-type NAGS) is proposed for this new family.

Metabolic engineering of microorganisms for the production of L-arginine and its derivatives

“…For species such as S. coelicolor , the OATase is characterized. However, NAGS has not been identified in this bacterium, while new classes of NAGS are continuously being discovered for other species [ 53 ]. For example, the novel type of NAGS (C-NAGS) [ 53 ] encoded by cg3035 from C. glutamicum adds to the diversity of NAGS including (1) the classical NAGS (as in the linear pathway), (2) the bifunctional OATase (as in the recycling pathway), (3) ArgH(A) fusion types ( argH - argA fusion) [ 55 ], and (4) the short versions of NAGS (S-NAGS) [ 56 ].…”

“…However, NAGS has not been identified in this bacterium, while new classes of NAGS are continuously being discovered for other species [ 53 ]. For example, the novel type of NAGS (C-NAGS) [ 53 ] encoded by cg3035 from C. glutamicum adds to the diversity of NAGS including (1) the classical NAGS (as in the linear pathway), (2) the bifunctional OATase (as in the recycling pathway), (3) ArgH(A) fusion types ( argH - argA fusion) [ 55 ], and (4) the short versions of NAGS (S-NAGS) [ 56 ]. Additionally, for species that have both NAGS and OATase such as G. stearothermophilus [ 43 ] and N. gonorrhoeae [ 57 ], there is a functional redundancy and the NAGS function is regarded as anaplerotic to replenish Ac-GLU [ 57 ],[ 58 ].…”

“…In terms of the chromosomal genetic organization, ARG biosynthetic genes are diversely organized in different species, and that from C. glutamicum has been studied the most. In C. glutamicum , the argCJBDFRGH cluster is organized into two operons ( argCJBDFR and argGH ) [ 52 ] and transcription of these operons are regulated by ARG [ 61 ], ArgR [ 62 ] and FarR [ 63 ], while the putative argA ( cg3035 , encoding C-NAGS) is separated from this cluster [ 32 ],[ 52 ],[ 53 ]. FarR regulates transcription of the arg operon by binding to the upstream of argC , argB , argF and argG genes [ 63 ],[ 64 ].…”

Engineering microbial cell factories: Metabolic engineering of Corynebacterium glutamicum with a focus on non‐natural products

“…Ikeda and colleagues have pioneered production of the L‐glutamate family amino acid L‐arginine and introduced key mutations into the wild‐type genome [83]. This strategy has been followed widely [84–89] and classical mutagenesis paired with metabolic engineering led to high‐producing strains [10]. L‐ornithine can be considered a key intermediate of L‐arginine biosynthesis and a number of L‐ornithine producing strains have been obtained [85, 90–92].…”

Improvement of l-citrulline production in Corynebacterium glutamicum by ornithine acetyltransferase

“…Recently, overexpression of the argJ gene showed increased NAGS activity by complementing the C. glutamicum arginine auxotrophic argJ strain [7]. Petri et al [17] discovered that the monofunctional ArgJ only catalyses the fifth step of the citrulline biosynthesis pathway in C. glutamicum, and glutamate was acetylated by N-acetylglutamate synthase Cg3035. Enzyme inhibition tests showed that l-arginine had no influence on OAT activity; however, the ArgJ enzyme was inhibited when 5 mM l-ornithine was added [19].…”