Junko Ohnishi

A genome-based approach to create a minimally mutated Corynebacterium glutamicum strain for efficient l-lysine production

Based on the progress in genomics, we have developed a novel approach that employs genomic information to generate an efficient amino acid producer. A comparative genomic analysis of an industrial l-lysine producer with its natural ancestor identified a variety of mutations in genes associated with l-lysine biosynthesis. Among these mutations, we identified two mutations in the relevant terminal pathways as key mutations for l-lysine production, and three mutations in central metabolism that resulted in increased titers. These five mutations when assembled in the wild-type genome led to a significant increase in both the rate of production and final l-lysine titer. Further investigations incorporated with transcriptome analysis suggested that other as yet unidentified mutations are necessary to support the l-lysine titers observed by the original production strain. Here we describe the essence of our approach for strain reconstruction, and also discuss mechanisms of l-lysine hyperproduction unraveled by combining genomics with classical strain improvement.

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

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 regarded 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 (

Transcriptome Analysis Reveals Global Expression Changes in an Industrial L-Lysine Producer of Corynebacterium glutamicum

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Disruption of malate:quinone oxidoreductase increases L-lysine production by Corynebacterium glutamicum.

), the organism exhibited limited growth anaerobically with production of nitrite (

The Cgl1281-encoding putative transporter of the cation diffusion facilitator family is responsible for alkali-tolerance in Corynebacterium glutamicum

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A novel gnd mutation leading to increased l-lysine production in Corynebacterium glutamicum

), 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 narK2GHJI cluster 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.