• Microbiology and Biotechnology Letters
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• v.13 no.3
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• pp.279-283
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• 1985

As a method of breeding L-lysine producing strains, the intergeneric protoplast fusion between Brevibacterium flavum and Corynebacterium glutamicum was performed. As a results, Brevibacterium flavum ATCC 21528 R showed 99％ of protoplast formation and 10％ of regeneration frequencies when treated with 400$\mu\textrm{g}$/$m\ell$ of lysozyme for 12hrs. In Corynebacterium glutamicum ATCC 21514 S, 99％ and 12％ were obtained by treatment of 300$\mu\textrm{g}$/$m\ell$ lysozyme for 12 hrs. In intergeneric protoplast fusion between Brevibacterium flavum ATCC 21528 R and Corynebacterium glutamicum ATCC 21831 S, 1.0$\times$10$^{-6}$ of recombinant frequency per regenerable cells was observed by use of PEG 6000, 30％(w/v). Among the strains obtained KR$_{43}$ strain showed 12％ higher productivity of L-lysine than the parental cell. Then, the activity of aspartokinase of KR$_{43}$ was about 13％ higher than the parental cell.

• Microbiology and Biotechnology Letters
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• v.15 no.2
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• pp.104-111
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• 1987

As a trial method of breeding L-lysine producing strains, the intraspecific protoplast fusion bet-ween Brevibacterium flavum ATCC 21528R and Brevibacterium flavum ATCC 21529S and the intergeneric protoplast fusion between Brevibacterium flavum ATCC 21528R and Corynebacterium glutamicum ATCC 13058S were performed. The optimum conditions for protoplast formation of these strains were examined and the effect of plasma expander on regeneration and/or fusion was also observed. Both fusants No. CH23 and No. CH4l showed higher productivity of L-lysine than those of parental cells under the optimum cultural conditions at a rate of 21％ and 8.9％, respectively. And, activity of several enzymes in L-lysine biosynthetic pathway including aspartokinase, a rate-limiting enzyme, was determined. Besides, metabolic control mechanism of L-lysine biosynthesis in fusant No. CH23 and in No. CH41 was investigated to compare with that of parental strains.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.3
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• pp.225-229
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• 2005

To investigate impediments to plasmid transformation in Brevibacterium flavum BF4 and B. lactofermentum BL1, cell surface barriers were determined by measuring growth inhibition whilst enzymatic barriers were determined by comparing DNA methylation properties. B. lactofermentum was more sensitive to growth inhibition by glycine than B. flavum. Release of cellular proteins during sonication was more rapid for B. lactofermentum than for B. flavum. Plasmid DNA (pCSL 17) isolated from B. flavum transformed recipient $McrBC^+$ strains of Escherichia coli with lower efficiency than $McrBC^-$. McrBC digestion of this DNA confirmed that B. flavum contain methylated cytidines in the target sequence of McrBc sequences but B. lactofermentum contained a different methylation pattern. DNA derived from the B. lactofermentum transformed recipient $EcoKR^+$ strains of E. coli with lower efficiency than $EcoKR^-$, indicating the presence of methylated adenosines in the target sequence of EcoK sequences. The present data describe the differences in the physical and enzymatic barriers between two species of corynebacteria and also provide some insight into the successful foreign gene expression in corynebacteria.

• Applied Biological Chemistry
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• v.34 no.2
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• pp.174-179
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• 1991

Protoplast fusion was carried out between Brevibacterium flavum and Corynebacterium glutamicum. For the protoplast fusion, various mutants were isolated from Brevibacterium flavum ATCC 21493 and Corynebacteriurn glutamicum ATCC 21831. The optimum conditions for protoplast fusion of these mutants were examined. In the present work, the authors obtained a fusant, MWF 9031, by the intergeneric protoplast fusion between Brevibacterium flavum 108-125 and Corynebacterium glutamicum 41-214A, which was excellent in L-arginine fermentation. Fusant MWF 9031 was found to accumulate a large amount of L-arginine reached 32.5 mg/ml with a medium containing 10% glucose. The fusant possessed intermediate characteristics between the parental strains and the stability was found to retain for 60 days.

• Journal of Applied Biological Chemistry
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• v.48 no.4
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• pp.218-221
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• 2005

Nucleotide sequence of Brevibacterium flavum ptsG gene capable of complementing Escherichia coli ZSC113 mutations defective to glucose permease activity of phosphotransferase system was completely determined, and the gene product was compared with other glucose-specific enzyme II ($EII^{Glc}$). A ptsG gene of B. flavum consisted of open reading frame of 2,025 nucleotides putatively encoding polypeptide of 675 amino acid residues and TAA stop codon. Deduced amino acid sequence of B. flavum ($EII^{Glc}$) had high homology with ($EIIs^{Glc}$) of Corynebacterium glutamicum, C. efficiens, and B. lactofermentum. Arrangement of structural domains, IIBCA, of B. flanum ($EII^{Glc}$) protein was identical to that of EIIs belonging to glucose-phosphotransferase system.

• Korean Journal of Microbiology
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• v.23 no.3
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• pp.190-196
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• 1985

For frequency improvement of protoplast fusion in Brevibacterium flavum, Brevibacterium lactofermentum lactofermentum and Corynebacterium glutamicum, the effect of plasma expanders on fusion and cell wall regeneration, compatison between direct and two-step selection method, tendency of fusion frequency according to pH of fusion fluid and polyethylene glycol concentration were examined. By addition of 3% polyvinyl pyrrolidone to cell wall regeneration medium, regeneration frequencies were expressed 23 (Brevibacterium lactofermentum), 10.4 (Brevibacterium flavum) and 2.7 (Corynebacterium glutamicum) times higher than those of none polyvinyl pyrrolidone medium respectively.

• Korean Journal of Microbiology
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• v.26 no.2
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• pp.93-100
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• 1988

In order to understand the regulation of glutamate dehydrogenase(GDH) synthesis in Brevibacterium flavum, we have isolated a mutant lacking NADP-linked GDH activity by ethlmethane sulfonate treatment. The $gdh^-$ mutant was grown on the minimal plate with 1mM ammonium chloride and not that with 300mM ammonium chloride. The cell-free extracts from $gdh^-$ mutant and prototroph were also examined with glutamine synthetase(GS) and glutamate synthase (GOGAT) production by niteogen sources. The growth of $gdh^-$ mutant in presence of 20mM ammonium chloride means that GOGAT synthesis is sufficient to allow growth in this condition. GS production of $gdh^-$ mutant as well as parental strain was induced by 1mM urea and ammonium tartrate, but it was repressed by higher concentration of ammonia, and also induced by 20mM to 50mM glutamate as a substrate. It was special attention that GOGAT synthesis from $gdh^-$ strain was more repressed by higher concentration of ammonia than prototroph as described in E. coli system.

• Microbiology and Biotechnology Letters
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• v.2 no.2
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• pp.103-109
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• 1974

In the cource of the studies on L-glutamic acid production from acetic acid, 383 strains capable of assimilating acetate as sole source of carbon were isolated from 279 kinds of soil sample. Out of them, 5 strains which produced relatively larger amount of L-glutamate from acetate were selected and named Brevibacterium flavum nov. sp. D1005B, Corynebacterium glutamicum nov. sp. D1025A, Brevib. flavum nov. sp. D2209B, Coryneb. acetoacidophilum nov. sp. D2212B and Coryneb. acetoacidophilum nov. sp. D2349A respectively.

• Journal of Microbiology and Biotechnology
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• v.5 no.4
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• pp.188-193
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• 1995

A Brevibacterium flavum gene coding for glucose permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned by complementing the Escherichia coli ZSCl13 mutations affecting a ptsG gene with the B. flavum genomic library. From the E. coli clone grown as red colony on a MacConkey plate supplemented with glucose as an additional carbon source, a recombinant plasmid was isolated and named pBFT93. The plasmid pBFT93 was identified as carrying a 3.6-kb fragment of B. flavum chromosomal DNA which enables the E. coli transformant to use glucose or man nose as a sole carbon source in an M9 minimal medium. The non-metabolizable sugar analogues, 2-deoxy-D-glucose (2-DG) and methyl-$\alpha$-D-glucopyranoside (MeGlc) affected the growth of ZSCl13 cells carrying the plasmid pBFT93 on minimal medium supplemented with non-PTS carbohydrate, glycerol, as a sole cabon source, while the analogues did not repress the growth of ZSCl13 cells without pBFT93. It was also found that both $2-deoxy-D-[U-^{14}C]glucose{\;}and{\;}methyl-{\alpha}-D-[U-^{14}C]glucopyranoside$ could be effectively transported into ZSCl13 cells transformed with plasmid pBFT93. Several in vivo complementation studies suggested that the B. flavum DNA in pBFT93 encodes a glucose permease specific for glucose and mannose.

• Korean Journal of Food Science and Technology
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• v.23 no.5
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• pp.561-567
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• 1991

This study was designed to investigate the productivity of L-methionine by the method of protoplast fusion between Brevibacterium flavum ATCC 14067 and Corynebacterium glutamicm ATCC 13032, and then L-methionine production was performed to continuous fermentation using the immobilized fusant cells. Mutants B. flavum K 104($thr\;met\;Km^{r}\;Et^{r}\;Sm^{r}\;Tm^{r}\;as\;genetic\;marker$) and C. glutamicum B 70($thr\;Hos\;Km^{r}\;Et^{r}\;Sm^{r}\;Tm^{r}as\;genetic\;marker$) were isolated by MNNG treatment. On the other hand, protoplast of mutants were formed to treat with lysis solution containing $500{\mu}g/ml$ of lysozyme. The ratios of protoplast formation and regeneration were 99% and $64{\sim}66%$ respectively. Fusion frequency between B. flavum K 104 and C. glutamicum B 70 showed the $3.5{\times}10^{5}$ in the 35% polyethylene glycol(PEG6000) containing 3% PVP solution. The productivity of L-methionine by fusant BFCG 37 immobilized with sodium alginate was 0.89 g/l the batch fermentation and was $18.75mg/^{1}hr\;^{1}$ on the continuous fermentation at $30^{\circ}C$ for 72 hr.