Mutations of the Corynebacterium glutamicum NCgl1221 gene, encoding a mechanosensitive channel homolog, induce L-glutamic acid production

Abstract

Corynebacterium glutamicum is a biotin auxotroph that secretes L-glutamic acid in response to biotin limitation; this process is employed in industrial L-glutamic acid production. Fatty acid ester surfactants and penicillin also induce L-glutamic acid secretion, even in the presence of biotin. However, the mechanism of L-glutamic acid secretion remains unclear. It was recently reported that disruption of odhA, encoding a subunit of the 2-oxoglutarate dehydrogenase complex, resulted in L-glutamic acid secretion without induction. In this study, we analyzed odhA disruptants and found that those which exhibited constitutive L-glutamic acid secretion carried additional mutations in the NCgl1221 gene, which encodes a mechanosensitive channel homolog. These NCgl1221 gene mutations lead to constitutive L-glutamic acid secretion even in the absence of odhA disruption and also render cells resistant to an L-glutamic acid analog, 4-fluoroglutamic acid. Disruption of the NCgl1221 gene essentially abolishes L-glutamic acid secretion, causing an increase in the intracellular L-glutamic acid pool under biotin-limiting conditions, while amplification of the wild-type NCgl1221 gene increased L-glutamate secretion, although only in response to induction. These results suggest that the NCgl1221 gene encodes an L-glutamic acid exporter. We propose that treatments that induce L-glutamic acid secretion alter membrane tension and trigger a structural transformation of the NCgl1221 protein, enabling it to export L-glutamic acid.

FIG. 1.

Fermentation kinetics of the odhA mutant and revertant in the presence of excess biotin. The wild-type, C. glutamicum ATCC 13869, and the l-glutamic acid-producing odhA mutant 2A-1 and its odhA⁺ derivative 2A-1R were grown with excess biotin at 31.5°C. (a) Growth; (b) residual glucose; (c) l-glutamate accumulation. Symbols: filled circles, wild-type; filled triangles, odhA mutant 2A-1; open squares, 2A-1R.

FIG. 2.

Secondary structure of C. glutamicum NCgl1221 and E. coli YggB and their mutation sites. (a) Membrane topology of C. glutamicum NCgl1221 as predicted by the PHDhtm program. Arrows indicate positions of NCgl1221 gene mutations. Circled numbers indicate residues located on the borders of transmembrane segments. (b) Proposed membrane topology of E. coli YggB based on the PhoA fusion assay (24). Arrows indicate positions of GOF mutations. Circled numbers indicate residues used for the PhoA fusion assay.

FIG. 3.

Fluoroglutamic acid resistance of NCgl1221 gene mutants. (a) Serial dilutions (1/10 each) of cultures of wild-type C. glutamicum ATCC 13869 or NCgl1221 gene mutants were spotted onto minimal medium with or without 7.5 mM 4-fluoroglutamic acid, and the plates were incubated at 31.5°C. The upper numbers give cell titers estimated from the OD₆₂₀ of cultures. (b) Cultures of wild-type C. glutamicum ATCC 13869 and its NCgl1221 gene mutants were inoculated into fresh minimal medium with or without 4-fluoroglutamic acid, and the cultures were shaken at 31.5°C. When the OD₆₂₀ of each strain without 4-fluoroglutamic acid reached 1.0, cell numbers were examined by plating appropriate dilutions on CM2B plates. Colonies were counted after 2 days of incubation at 31.5°C. Symbols: filled circles, wild-type; open triangles, NCgl1221 gene deletion mutant; open squares, NCgl1221(A111V); X cross, NCgl1221(W15CSLW). Error bars indicate standard deviations (n = 3).

FIG. 4.

Effect of NCgl1221 gene deletion on l-glutamate production and the intracellular l-glutamate pool. Wild-type C. glutamicum ATCC 13869 and its NCgl1221 gene deletion derivative were grown on biotin-depleted medium. (a) Growth; (b) residual glucose (symbols: filled circles, wild type; open circles, NCgl1221 gene deletion mutant); (c) extracellular and intracellular l-glutamate; (d) extracellular and intracellular l-aspartate (symbols: filled circles, extracellular pool of the wild type; open circles, extracellular pool of the NCgl1221 gene deletion mutant; filled triangle, intracellular pool of the wild type; open triangle, intracellular pool of NCgl1221 gene deletion mutant).

FIG. 5.

Model of induction of l-glutamate production in C. glutamicum. Treatments inducing l-glutamate production, such as biotin limitation and detergent, alter membrane tension by inhibiting fatty acid biosynthesis. Addition of penicillin also leads to a change of membrane tension by inhibiting cell wall biosynthesis. Alteration of membrane tension triggers a structural transformation of NCgl1221 that enables it to catalyze l-glutamic acid excretion.