• 한국미생물·생명공학회지
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• 제37권4호
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• pp.405-407
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• 2009

A Bacillus subtilis mannanase gene was subcloned into an Escherichia coli- Corynebacterium lactofermentum shuttle vector pHE83, and the resultant plasmid pHE83M was transferred into an endogenous plasmid-free strain of C. lactofermentum. Mannanase produced by the recombinant C. lactofermentum (pHE83M) was secreted extracellulary at the level of 86%, and the remaining activity of mannanase was detected in the cell-free extract. The maximum mannanase productivity of the recombinant strain reached 8.1 unit/mL in the culture filtrate of LB medium. According to the zymogram of mannanase on SDS-PAGE, it was found that the mannanase produced by the recombinant C. lactofermentum could be maintained stably with a migration identical to the mannanase produced by the parental strain, B. subtilis WL-3.

• Journal of Microbiology and Biotechnology
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• 제7권5호
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• pp.287-292
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• 1997

The role of glyoxylate bypass in lysine production by Corynebacterium glutamicum ssp. lactofermentum ATCC21799 was analyzed by using cloned aceA and aceB genes which encode enzymes catalyzing the bypass. Introduction of a plasmid carrying aceA and aceB to the strain increased enzyme activities of the bypass to approximately 5 fold on acetate minimal medium. The strain with amplified glyoxylate bypass excreted 25% more lysine to the growth medium than the parental strain, apparently due to the increased availability of intracellular oxaloacetate. The final cell yield was lower in the strain with amplified glyoxylate bypass. These changes were specific to the lysine-producing C. glutamicum ssp. lactofermentum ATCC21799, since the lysine-nonproducing wild type Corynebacterium glutamicum strain grew faster and achieved higher cell yield when the glyoxylate bypass was amplified. These findings suggest that the lysine producing C. glutamicum ssp. lactofermentum ATCC21799 has the ability to efficiently channel oxaloacetate, the TCA cycle intermediate, to the lysine biosynthesis pathway whereas lysine-nonproducing strains do not. Our results show that amplification of the glyoxylate bypass efficiently increases the intracellular oxaloacetate in lysine producing Corynebacterium species and thus results in increased lysine production.

• 미생물학회지
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• 제23권3호
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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.

• Journal of Microbiology and Biotechnology
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• 제6권5호
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• pp.315-320
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• 1996

The role of glyoxylate bypass in a lysine-producing Corynebacterium lactofermentum strain was analyzed. Unlike the wild type, the strain expressed enzymes of glyoxylate bypass during growth in the fermentation broth containing glucose as the carbon source. To evaluate the importance of glyoxylate bypass in the strain, we disrupted chromosomal aceA by using a cloned fragment of the gene. Site-specific disruption of aceA which codes for the isocitrate lyase, the first enzyme of the bypass, was confirmed by Southern blot analysis. The aceA mutant strain completely lost isocitrate lyase activity and ability to grow in a minimal medium containing acetate as the sole carbon source. The mutant strain was similar to its parental strain in growth characteristics and produced comparable amounts of lysine in shake flasks containing glucose as the carbon source. The amount of oxaloacetate accumulated in the fermentation medium was similar for both strains, suggesting that expression of glyoxylate bypass does not necessarily lead to the increase in intracellular oxaloacetate. These data clearly demonstrate that glyoxylate bypass does not function as one of the routes of carbon supply for lysine production in the strain. It appears that the leakiness of the glyoxylate bypass in the strain might be the result of a secondary mutation which arose during previous strain development by random mutagenesis.

• Journal of Microbiology and Biotechnology
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• 제5권5호
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• pp.250-256
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• 1995

The ddh gene encoding meso-diaminopimelate (meso-DAP)-dehydrogenase (DDH) in Brevibacterium lactofermentum was isolated by complementation of the Escherichia coli dapD mutation. It was supposed from subcloning experiments and complementation tests that the evidence for DDH activity appeared in about 2.5 kb Xhol fragmented genome. The 2.5 kb Xhol fragment containing the ddh gene was sequenced, and an open reading frame of 960 bp encoding a polypeptide comprising 320 amino acids was found. Computer analysis indicated that the deduced amino acid of the B. lactofermentum ddh gene showed a high homology with that of the Corynebacterium glutamicum ddh gene.

• Journal of Microbiology and Biotechnology
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• 제9권5호
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• pp.582-588
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• 1999

A Brevibacterium lactofermentum gene coding for a glucose-specific permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned, by complementing an Escherichia coli mutation affecting a ptsG gene with the B. lactofermentum genomic library, and completely sequenced. The gene was identified as a ptsG, which enables an E. coli transformant to transport non-metabolizable glucose analogue 2-deoxyglucose (2DG). The ptsG gene of B. lactofermentum consists of an open reading frame of 2,025 nucleotides encoding a polypeptide of 674 amino acid residues and a TAA stop codon. The 3' flanking region contains two stem-loop structures which may be involved in transcriptional termination. The deduced amino acid sequence of the B. lactofermentum enzyme $II^{GIe}$ specific to glucose ($EII^{GIe}$) has a high homology with the Corynebacterium glutamicum enzyme $II^{Man}$ specific to glucose and mannose ($EII^{Man}$), and the Brevibacterium ammoniagenes enzyme $II^{GIc}$ specific to glucose ($EII^{GIc}$). The 171-amino-acid C-terminal sequence of the $EII^{Glc}$ is also similar to the Escherichia coli enzyme $IIA^{GIc}$ specific to glucose ($IIA^{GIc}$). It is interesting that the arrangement of the structural domains, IIBCA, of the B. lactofermentum $EII^{GIc}$ protein is identical to that of EIIs specific to sucrose or $\beta$-glucoside. Several in vivo complementation studies indicated that the B. lactofermentum $EII^{Glc}$ protein could replace both $EII^{ Glc}$ and $EIIA^{Glc}$ in an E. coli ptsG mutant or crr mutant, respectively.

• Journal of Microbiology and Biotechnology
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• 제11권5호
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• pp.819-824
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• 2001

The principal DNA modification systems of the amino-acid-producing bacteria Corynebacterium glutamicum AS019, Brevibacterium flavum BF4, and B. lactofermentum BL1 was investigated using two approaches; digestion of plasmid DNA isolated from these species TseI and Fnu4HI, and sequence analysis of the putative methyltransferase target sites following the derivatization of DNA using metabisulfite treatment. The C. glutamicum and B. flavum strains showed similar digestion patterns to the two enzymes, indicating that the target for cytidine methyltransferase recognizes 5'-GCSGC-3'(where S is either G or C). Mapping the methylated cytidine sites by bisulfite derivatization, followed by PCR amplification and sequencing, was only possible when the protocol included an additional step eliminating any underivatized DNA after PCR amplification, thereby indicating that the derivatization was not $100\%$ efficient. This may have been due to the high G0C content of this genus. It was confirmed that C. glutamicum AS019 and B. flavum BF4 methylated the cytidine in the $Gm^5CCGC$ sequences, yet there were no similar patterns of methylation in B. lactofermentum, which was consistent with the distinctive degradation pattern seen for the above enzymes. These findings demonstrate the successful application of a modified bisulfite derivatization method with the Corynebacterium species for determining methylation patterns, and showed that different species in the geneus contain distinctive restriction and modification systems.

• Journal of Applied Biological Chemistry
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• 제48권4호
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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.

• 한국식품과학회지
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• 제21권1호
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• pp.154-163
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• 1989

L-Lysine 생산 균주인 B. flavum ATCC 21528, B. lactofermentum ATCC 21086 및 C. glutamicum 820을 이용하여 L-lysine 생산성이 우수한 균주를 분리할 목적으로 N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) $250{\mu}g/ml$ 농도로 처리한 다음 $300{\mu}g/ml$의 penicillin-G로 변이주를 농축하여 B. flavum $37-2(Hos^-,\;Kan^r,\;AEC^r)$, B. lactofermentum $6-2(Ile^-,\;Val^-,\;Str^r,\;ACE^r)$ 및 C. glutamicum $57-5(Met^-,\;Thr^-,\;Rif^r,\;AEC^r)$ 등의 변이주를 분리하였다. 분리된 변이주의 원형질체 형성은 lysozyme $500{\mu}g/ml$를 함유한 LS 용액으로 6시간 처리시 원형질체의 형성율은 97-99%였으며, 세포벽 재생율은 0.5M sodium succinate를 함유한 RCM에 0.7% osft agar를 중층하였을 때 33-37%를 나타내었다. 또 각각의 원형질체를 동량 혼합후 30% PEG 6,000에서 융합을 시킨 다음 분리된 융합주 BBFL 21, BCFG 37 및 BCLG 59는 $1.25{\times}10^{-6}{\sim}5.83{\times}10^{-7}$의 융합 빈도를 나타내었다. 분리된 융합주 BBFL 21은 LPB에서 $30^{\circ}C$, 72hr 배양하였을때 $411.1ng/ml{\cdot}hr$로 높은 L-lysine 생산성을 나타내었다. 발효 방법을 개선할 목적으로 융합주 BBFL 21를 sodium alginate, polyacrylamide, agar, x-carrageenan등으로 고정화 하여 회분식 발효를 행한 바 $413ng/ml{\cdot}hr$로 sodium alginate로 처리했을 때 가장 좋았다. 고정화 균체를 이용하여 관형 발효기를 제작하여 연속 발효를 행한 바 $416.7ng/ml{\cdot}hr$의 가장 높은 L-lysine 생산성을 나타내어 회분식 보다 높은 수율을 얻었다.