• 한국미생물·생명공학회지
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• 제47권1호
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• pp.78-86
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• 2019

대사공학을 이용하여 N-아세틸글루코사민(GlcNAc)을 생산하는 재조합 Corynebacterium glutamicum을 개발하였다. 먼저 GlcNAc를 생산하는 기반균주를 제작하기 위하여, N-acetylglucosamine-6-phosphate deacetylase와 glucosamine-6-phosphate deaminase를 암호화하는 nagAB와 N-acetylmannosamine-6-phosphate epimerase를 암호화하는 nanE를 C. glutamicum ATCC 13032에서 순차적으로 결손하여, 최종적으로 KG208 균주를 제작하였다. 또한, glucosamine-6-phosphate synthase를 암호화하는 C. glutamicum 유래의 glmS와 glucosamine-6-phosphate N-acetyltransferase를 암호화하는 Saccharomyces cerevisiae 유래의 gna1을 각각 여러 발현벡터에 클로닝하였다. 여러 발현 조합의 플라스미드들 중에서 pCXI40-glmS와 pCEI40-gna1을 함유한 제조합균주 KG440은 삼각플라스크 발효에서 1.77 g/l의 GlcNAc와 0.63 g/l의 글루코사민을 생산하였다.

• Journal of Applied Biological Chemistry
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• 제58권3호
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• pp.227-232
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• 2015

A microarray study has been employed to understand changes of gene expression in E. coli KD43162 resistant to ampicillin, ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, cefazolin, cefepime, aztreonam, imipenem, meropenem, gentamicin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, fosfomycin, and trimethoprim-sulfamethoxazole except for amikacin using disk diffusion assay. Using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and MALDI-TOF MS analyses, 36 kDa of outer membrane proteins (OMPs) was found to be deleted in the multidrug resistant E. coli KD 43162. Microarray analysis was used to determine up- and down-regulated genes in relation to multidrug resistant E. coli KD43162. Among the up-regulated genes, these genes were corresponded to express the proteins as penicillin-binding proteins (PBPs), tartronate semialdehyde reductase, ethanolamine utilization protein, shikimate kinase I, allantoinase, predicted SAM-dependent methyltransferase, L-glutamine: D-fructose-6-phosphate aminotransferase (GFAT), phospho-glucosamine mutase, predicted N-acetylmannosamine kinase, and predicted N-acetylmannosamine-6-P epimerase. Up-regulation of PBPs, one of primary target sites of antibiotics, might be responsible for the multidrug resistance in E. coli with increasing amount of target sites. Up-regulation of GFAT enzyme may be related to the up-regulation of PBPs because GFAT produces N-acetylglucosamine, a precursor of peptidoglycans. One of GFAT inhibitors, azaserine, showed a potent inhibition on the growth of E. coli KD43162. In conclusion, up-regulation of PBPs and GFATs with the loss of 36 kDa OMP refers the multidrug resistance in E. coli KD 43162.

• 한국미생물·생명공학회지
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• 제32권2호
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• pp.142-148
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• 2004

에리스로포이에틴은 인간 적혈구분화의 조절인자로 작용한다. 유전자 재조합 인간에리스로포이에틴(rhEPO)은 동물세포에서 생산되고 있는 재조합 당단백질의 하나이며 당쇄부분이 전체 분자량의 40％를 차지한다. 시알산 함량은 체내약물 투여 지속기간과 직접적인 연관이 있어 시알산 함량은 의약용 당단백질의 중요한 성질로 여겨진다. 본 연구에서는 CHO세포 배양액에 시알산 생합성 전구물질인 N-acetylmannosamine(ManNAc)과 sialidase 저해제인 2-deoxy-2,3-hyo-N-acetylneuraminic acid(NeuAc2en)를 첨가하여 rhEPO의 sialic acid함량을 증가시킬 수 있었다. 특히, 배양액에 20 mM ManNAc/0.5 mM NeuAc2en를 첨가할 때 대조구에 비하여 약 10배의 시알산 함량이 증가하였으며 세포성장이나 배양액의 rhEPO생산량에는 영향이 없었다. rhEPO의 정제시 시알산 함량이 11∼15％인 다당쇄 rhEPO분획을 얻었으며, 배양액 내에 20 mM ManNAc와 0.5 mM NeuAc2en를 동시에 첨가함으로 대조구에 비하여 시말산함량이 높은다당쇄 rhEPO의 생산성이 50％ 증가하였다.

• 생명과학회지
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• 제24권3호
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• pp.311-317
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• 2014

Rimed vacuole을 가진 원위 근육병(distal myopathy with rimmed vacuoles, DMRV)은 제2형 유전성 봉입체 근육병으로도 불리며 초기 성인기에 발병하여 원위부의 근력약화를 보이는 임상양상과 rimmed vacuole의 근육병리소견을 특징으로 하는 상염색체 열성의 근육병이다. 이러한 DMRV의 원인은 UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) 유전자의 돌연변이임이 밝혀져 있다. 저자들은 원위부 근력약화를 호소하는 환자에서 전체 엑솜 염기서열분석을 이용하여 GNE 유전자의 복합 이형접합성 돌연변이(p.Asp176Val 및 p.Val572Leu)를 확인하여 DMRV를 진단할 수 있었다. 본 연구는 근육병의 정확한 분자진단에 있어서 전체 엑솜 염기서열분석의 유용성을 보여주었기에 이를 보고하는 바이다.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1024-1032
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• 2008

Efficient expression of the Salmonella Typhimurium tdc ABCDEG operon involved in the degradation of L-serine and L-threonine requires TdcA, the transcriptional activator of the tdc operon. We found that the tdcA gene was transiently activated when the bacterial growth condition was changed from aerobic to anaerobic, but this was not observed if Salmonella was grown anaerobically from the beginning of the culture. Expression kinetics of six tdc genes after anaerobic shock demonstrated by a real-time PCR assay showed that the tdc CDEG genes were not induced in the tdcA mutant but tdcB maintained its inducibility by anaerobic shock even in the absence of tdcA, suggesting that an additional unknown transcriptional regulation may be working for the tdcB expression. We also investigated the effects of nucleoid-associated proteins by primer extension analysis and found that H-NS repressed tdcA under anaerobic shock conditions, and fis mutation delayed the peak expression time of the tdc operon. DNA microarray analysis of genes regulated by TdcA revealed that the genes involved in N-acetylmannosamine, maltose, and propanediol utilization were significantly induced in a tdcA mutant. These findings suggest that Tdc enzymes may playa pivotal role in energy metabolism under a sudden change of oxygen tension.

• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.115-122
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• 2021

Phenol-soluble modulins (PSMs) are responsible for regulating biofilm formation, persister cell formation, pmtR expression, host cell lysis, and anti-bacterial effects. To determine the effect of psm deletion on methicillin-resistant Staphylococcus aureus, we investigated psm deletion mutants including Δpsmα, Δpsmβ, and Δpsmαβ. These mutants exhibited increased β-lactam antibiotic resistance to ampicillin and oxacillin that was shown to be caused by increased N-acetylmannosamine kinase (nanK) mRNA expression, which regulates persister cell formation, leading to changes in the pattern of phospholipid fatty acids resulting in increased anteiso-C15:0, and increased membrane hydrophobicity with the deletion of PSMs. When synthetic PSMs were applied to Δpsmα and Δpsmβ mutants, treatment of Δpsmα with PSMα1-4 and Δpsmβ with PSMβ1-2 restored the sensitivity to oxacillin and slightly reduced the biofilm formation. Addition of a single fragment showed that α1, α2, α3, and β2 had an inhibiting effect on biofilms in Δpsmα; however, β1 showed an enhancing effect on biofilms in Δpsmβ. This study demonstrates a possible reason for the increased antibiotic resistance in psm mutants and the effect of PSMs on biofilm formation.