Biomedical subjects

Eiichiro Kimura

Publications and source records attributed to Eiichiro Kimura.

7 recordsLinked to original sources

PubMed2006

Altered metabolic flux due to deletion of odhA causes L-glutamate overproduction in Corynebacterium glutamicum.

L-glutamate overproduction in Corynebacterium glutamicum, a biotin auxotroph, is induced by biotin limitation or by treatment with certain fatty acid ester surfactants or with penicillin. We have analyzed the relationship between the inductions, 2-oxoglutarate dehydrogenase complex (ODHC) activity, and L-glutamate production. Here we show that a strain deleted for odhA and completely lacking ODHC activity produces L-glutamate as efficiently as the induced wild type (27.8 mmol/g [dry weight] of cells for the ohdA deletion strain compared with only 1.0 mmol/g [dry weight] of cells for the uninduced wild type). This level of production is achieved without any induction or alteration in the fatty acid composition of the cells, showing that L-glutamate overproduction can be caused by the change in metabolic flux alone. Interestingly, the L-glutamate productivity of the odhA-deleted strain is increased about 10% by each of the L-glutamate-producing inductions, showing that the change in metabolic flux resulting from the odhA deletion and the inductions have additive effects on L-glutamate overproduction. Tween 40 was indicated to induce drastic metabolic change leading to L-glutamate overproduction in the odhA-deleted strain. Furthermore, optimizing the metabolic flux from 2-oxoglutarate to L-glutamate by tuning glutamate dehydrogenase activity increased the l-glutamate production of the odhA-deleted strain.

Corynebacterium glutamicum↗

PubMed2006

Temperature-sensitive cloning vector for Corynebacterium glutamicum.

We constructed a temperature-sensitive form of the Corynebacterium glutamicum ATCC13869 cryptic plasmid, pBL1. The C. glutamicum/Escherichia coli shuttle vector pSFK6, which is composed of pBL1 and the E. coli cloning vector pK1, was mutagenized in vitro by treatment with hydroxylamine, and introduced into C. glutamicum cells. A mutant plasmid, which was stably maintained at 25 degrees C but not at 34 degrees C, was isolated from the cells. Sequencing the plasmid, which was named p48K, revealed four substitutions in the Rep protein coding region. Moreover, site-directed single-nucleotide substitutions showed that a G to A transition at position 2,920, which resulted in a Pro-47 to Ser substitution in the Rep protein, was responsible for its temperature-sensitive replication. Pro-47 is conserved among the Rep proteins of the pIJ101/pJV1 family of plasmids. This temperature-sensitive cloning vector will be useful for disrupting genes in this industrially important bacterium.

Amino Acid Sequence↗

PubMed2005

Comparative study of flux redistribution of metabolic pathway in glutamate production by two coryneform bacteria.

In amino acid production by coryneform bacteria, study on relationship between change in enzyme activities and production of a target amino acid is important. In glutamate production, Kawahara et al. discovered that the effect of decrease in 2-oxoglutamate dehydrogenase complex (ODHC) on glutamate production is essential (Kawahara et al., Biosci. Biotechnol. Biochem. 61(7) (1997) 1109). Significant reduction of the ODHC activity was observed in the cells under the several glutamate-productive conditions in Corynebacterium glutamicum. Recent progress in metabolic engineering enables us to quantitatively compare the flux redistribution of the different strains after change in enzyme activity precisely. In this paper, relationship between flux redistribution and change in enzyme activities after biotin deletion and addition of detergent (Tween 40) was studied in two coryneform bacteria, C. glutamicum and a newly isolated strain, Corynebacterium efficiens (Fudou et al., Int. J. Syst. Evol. Microbiol. 52(Part 4) 1127), based on metabolic flux analysis (MFA). It was observed that in both species the specific activities of isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) did not significantly change throughout the fermentation, while that of the ODHC significantly decreased after biotin depletion and Tween 40 addition. Flux redistribution clearly occurred after the decrease in ODHC specific activity. The difference in glutamate production between C. glutamicum and C. efficiens was caused by the difference in the degree of decrease in ODHC specific activity. The difference in Michaelis-Menten constants or K(m) value between ICDH, GDH, and ODHC explained the mechanism of flux redistribution at the branch point of 2-oxoglutarate. It was found that the K(m) values of ICDH and ODHC were much lower than that of GDH for both strains. It was quantitatively proved that the ODHC plays the most important role in controlling flux distribution at the key branch point of 2-oxoglutarate in both coryneform bacteria. Flux redistribution mechanism was well simulated by a Michaelis-Menten-based model with kinetic parameters. The knowledge of the mechanism of flux redistribution will contribute to improvement of glutamate production in coryneform bacteria.

Bacterial Proteins↗

PubMed2003

Metabolic engineering of glutamate production.

Since the discovery of Corynebacterium glutamicum as an efficient glutamate-overproducing microorganism in 1957, the production of L-amino acids by the fermentative method has become one of the most important research-target of industrial microbiology. Several research groups have developed metabolic engineering principles for L-amino acid-producing C. glutamicum strains over the last four decades. The mechanism of L-glutamate-overproduction by the microorganism is very unique and interesting. L-Glutamate overproduction by this bacterium, a biotin auxotroph, is induced by a biotin limitation and suppressed by an excess of biotin. Addition of a surfactant or penicillin is known to induce L-glutamate overproduction under excess biotin. After the development of the general molecular biology tools such as cloning vectors and DNA transfer technique, genes encoding biosynthetic enzymes were isolated. With those genes and tools, recombinant DNA technology can be applied in analysis of biosynthetic pathways and strain construction of C. glutamicum. In this review, key points of the L-glutamate biosynthetic pathways are summarized and the recent studies about triggering mechanism of L-glutamate overproduction by C. glutamicum are introduced. Then the metabolic flux analysis of L-glutamate overproduction is explored.

Cloning, Molecular↗

PubMed2003

Comparative complete genome sequence analysis of the amino acid replacements responsible for the thermostability of Corynebacterium efficiens.

Corynebacterium efficiens is the closest relative of Corynebacterium glutamicum, a species widely used for the industrial production of amino acids. C. efficiens but not C. glutamicum can grow above 40 degrees C. We sequenced the complete C. efficiens genome to investigate the basis of its thermostability by comparing its genome with that of C. glutamicum. The difference in GC content between the species was reflected in codon usage and nucleotide substitutions. Our comparative genomic study clearly showed that there was tremendous bias in amino acid substitutions in all orthologous ORFs. Analysis of the direction of the amino acid substitutions suggested that three substitutions are important for the stability of the C. efficiens proteins: from lysine to arginine, serine to alanine, and serine to threonine. Our results strongly suggest that the accumulation of these three types of amino acid substitutions correlates with the acquisition of thermostability and is responsible for the greater GC content of C. efficiens.

Amino Acid Sequence↗

PubMed2002

Triggering mechanism of L-glutamate overproduction by DtsR1 in coryneform bacteria.

The mechanism of L-glutamate-overproduction by Corynebacterium glutamicum, a biotin auxotroph, is very unique and interesting. L-Glutamate overproduction by this bacterium is induced by biotin-limitation and suppressed by an excess of biotin. Addition of a surfactant or penicillin is also induces L-glutamate overproduction even under excess biotin. After the development of general molecular biological tools such as cloning vectors and DNA transfer techniques, genes encoding biosynthetic enzymes were isolated. With those genes and tools, recombinant DNA technology can be applied to the analysis of biosynthetic pathways and the construction of C. glutamicum strains. In this review, recent studies on the triggering mechanism of L-glutamate overproduction by C. glutamicum are discussed. Disruption of the dtsR1 gene, which encodes a putative component of a biotin-containing enzyme complex that is involved in fatty acid synthesis, causes constitutive overproduction of L-glutamate. As in the case of biotin-limitation, i.e., addition of a surfactant or penicillin, dtsR1-disruption also reduces the activity of the 2-oxoglutarate dehydrogense complex (ODHC). These results indicate that the DtsR1 level affects the activity of ODHC. In our recent studies, a novel regulatory factor that suppresses the expression of DtsR1 was determined. Based on these findings, the triggering mechanism of L-glutamate overproduction is expected to be clarified in more detail.

Journal Article↗

PubMed2002

Corynebacterium efficiens sp. nov., a glutamic-acid-producing species from soil and vegetables.

Three glutamic-acid-producing coryneform strains were isolated from soil and vegetable samples. Chemotaxonomic investigations indicated that these strains belonged to the genus Corynebacterium. Phylogenetic studies, based on 16S rDNA analysis, demonstrated that the three strains formed a distinct cluster within the genus Corynebacterium and that their nearest relatives were Corynebacterium glutamicum and Corynebacterium callunae, also known as glutamic-acid-producing species. The data from 16S rDNA sequence and DNA-DNA relatedness studies clearly indicated that the three isolates represented a new species within the genus Corynebacterium. All of the isolates could grow at 45 degrees C and produced acid from dextrin; these were the most significant characteristics differentiating the three isolates from their neighbours. On the basis of the data presented here, it is proposed that the three glutamic-acid-producing isolates together be classified as Corynebacterium efficiens sp. nov., the type strain of which is YS-314T (= AJ 12310T = JCM 11189T = DSM 44549T).

Corynebacterium↗