Anaerobic growth and potential for amino acid production by nitrate respiration in Corynebacterium glutamicum

Abstract

Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regared as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O2), while no visible colonies were formed in the absence of O2. However, in the presence of nitrate (NO3¯), the organism exhibited limited growth anaerobically with production of nitrite (NO2¯), indicating that C. glutamicum can use nitrate as a final electron acceptor.Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJIcluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate:nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using L-lysine-and L-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.

Characterization of mutations induced by N-methyl-N′-nitro-N-nitrosoguanidine in an industrial Corynebacterium glutamicum strain

“…Complete medium BY [26] was used for cultivation of C. glutamicum. Solid plates were made by the addition of Bacto-Agar (Difco) to 1.6%.…”

Regulation of the nitrate reductase operon narKGHJI by the cAMP-dependent regulator GlxR in Corynebacterium glutamicum

“…It has been previously reported that nitrate reductase activity was not influenced by the oxygen and nitrate levels, although aerobically growing C. glutamicum cells exhibited no significant nitrite production in the presence of oxygen (Takeno et al, 2007). This may be explained by preferential electron flow to oxygen due to the higher electron-accepting affinity of cytochrome oxidase than nitrate reductase enzymes.…”

“…C. glutamicum has long been regarded as an aerobic bacterium because there have been few reports of its growth by anaerobic respiration or fermentation, despite the fact that most other coryneform bacteria are facultative anaerobes (Liebl, 2001). Recently, anaerobic growth of C. glutamicum by nitrate respiration was reported (Nishimura et al, 2007;Takeno et al, 2007). This property is attributed to the presence of a narKGHJI operon.…”

“…These components of nitrate respiration are conserved in a broad range of bacteria (Moreno-Vivián et al, 1999;Zumft, 1997). Nitrite is excreted from the cell as an end product of anaerobic nitrate reduction in C. glutamicum, and neither nitrate nor nitrite is utilized as the sole nitrogen source for its anaerobic or aerobic growth, suggesting that this microorganism lacks an efficient enzymic apparatus to reduce nitrite further (Nishimura et al, 2007;Takeno et al, 2007). This property is distinct from that of well-studied nitratereducing bacteria such as Escherichia coli and Bacillus subtilis, which are able to metabolize nitrate as a nitrogen source to nitrite and further to ammonia (Cole, 1996;Nakano & Zuber, 1998), and of denitrifiers such as Pseudomonas aeruginosa and Paracoccus denitrificans (Zumft, 1997).…”

Combination Strategy to Increase Cyclosporin A Productivity by Tolypocladium niveum Using Random Mutagenesis and Protoplast Transformation

“…It is taken as a given that the amount of antifungal compound in an agar-plug colony assay (APCA) is proportional to the size of the inhibition zone on the plate [21]. To test the feasibility of the APCA method for CyA-producing T. niveum, several paper discs containing various amounts of CyA were first applied to a lawn of Aspergillus niger cultured on a ME medium (malt extract 20 g, glucose 20 g, peptone 1 g, agar 20 g, in 1 l of distilled water) for 2 days.…”