• Journal of Microbiology and Biotechnology
• /
• 제19권5호
• /
• pp.439-446
• /
• 2009

The biosynthesis study of antibiotics saframycin (SFM) in Streptomyces lavendulae and safracin (SAC) in Pseudomonas fluorescens demonstrated that 3-hydroxy-S-methyl-O-methyltyrosine (3hSmOmTyr), a nonproteinogenic amino acid, is the precursor of the tetrahydroisoquinoline molecular core. In the biosynthetic gene cluster of SAC/SFM, sacD/sfmD encodes a protein with high homology to each other but no sequence similarity to other known enzymes; sacF/sfmM2 and sacG/sfmM3 encode methyltransferases for C-methylation and O-methylation; and sacE/sfinF encodes a small protein with significant sequence similarity to the MbtH-like proteins, which are frequently found in the biosynthetic pathways of non ribosomal peptide antibiotics and siderophores. To address their function, the biosynthetic cassette of 3h5mOmTyr was heterologously expressed in S. coelicolor and P. putida, and an in-frame deletion and complementation in trans were carried out. The results revealed that (i) SfmD catalyzes the hydroxylation of aromatic rings; (ii) sacD/sacF/sacG in the SAC gene cluster and sfmD/sfmM2/sfmM3 in the SFM cluster are sufficient for the biosynthesis of 3h5mOmTyr; and (iii) the mbtH-like gene is not required for the biosynthesis of the 3h5mOmTyr precursor.

• Journal of Microbiology and Biotechnology
• /
• 제30권2호
• /
• pp.187-195
• /
• 2020

To understand the molecular mechanism involved in the survivability of cold-tolerant lactic acid bacteria was of great significance in food processing, since these bacteria play a key role in a variety of low-temperature fermented foods. In this study, the cold-stress response of probiotic Lactobacillus plantarum K25 isolated from Tibetan kefir grains was analyzed by iTRAQ proteomic method. By comparing differentially expressed (DE) protein profiles of the strain incubated at 10℃ and 37℃, 506 DE proteins were identified. The DE proteins involved in carbohydrate, amino acid and fatty acid biosynthesis and metabolism were significantly down-regulated, leading to a specific energy conservation survival mode. The DE proteins related to DNA repair, transcription and translation were up-regulated, implicating change of gene expression and more protein biosynthesis needed in response to cold stress. In addition, two-component system, quorum sensing and ABC (ATP-binding cassette) transporters also participated in cell cold-adaptation process. These findings provide novel insight into the cold-resistance mechanism in L. plantarum with potential application in low temperature fermented or preserved foods.

• Mycobiology
• /
• 제43권1호
• /
• pp.31-36
• /
• 2015

We have previously isolated ${\varepsilon}$-COP, the ${\alpha}$-COP interactor in COPI of Aspergillus nidulans, by yeast two-hybrid screening. To understand the function of ${\varepsilon}$-COP, the $aneA^+$ gene for ${\varepsilon}$-COP/AneA was deleted by homologous recombination using a gene-specific disruption cassette. Deletion of the ${\varepsilon}$-COP gene showed no detectable changes in vegetative growth or asexual development, but resulted in decrease in the production of the fruiting body, cleistothecium, under conditions favorable for sexual development. Unlike in the budding yeast Saccharomyces cerevisiae, in A. nidulans, over-expression of ${\varepsilon}$-COP did not rescue the thermo-sensitive growth defect of the ${\alpha}$-COP mutant at $42^{\circ}C$. Together, these data show that ${\varepsilon}$-COP is not essential for viability, but it plays a role in fruiting body formation in A. nidulans.

• 생명과학회지
• /
• 제20권2호
• /
• pp.176-182
• /
• 2010

벼의 생산에 있어 가장 큰 문제 요인 중 하나인 벼도열병의 발생 원인균인 벼도열병균은 다양한 기작에 의해 방제 약제에 대한 내성을 가지는 것으로 알려져 있다. 막 운반단백질인 ABC transporter의 경우 환경으로부터의 다양한 독성 물질들을 배출하는 것으로 알려져 있다. 이미 알려진 벼도열병균의 게놈 서열로부터 생물정보학적 분석을 통하여 ABC transporter 단백질의 도메인 특성을 보이는 33개의 유전자군 서열을 예측하였다. 이중 3개의 경우는 이미 알려진 유전자로 판명되었다. Southern Hybridization 분석에 적용한 20개의 유전자들이 모두 게놈상에 단일 copy로 존재함을 확인하였다. 새로 예측된 30개의 유전자중 11개는 RT-PCR을 통하여 전사단계에서의 유전자 발현이 확인되었다.

• 생명과학회지
• /
• 제20권10호
• /
• pp.1563-1568
• /
• 2010

암(dark) 상태에서 재배한 대두의 하배축 길이 생장의 분자 기작을 연구하기 위한 일환으로 암 상태에서 재배한 대두 하배축으로부터 cDNA library를 제작한 후 ESTs를 구축하였다. 이들 ESTs 중 색소체 ABC 단백질과 아미노산 서열이 매우 유사한 clone을 선발한 후 이 유전자의 전체염기서열을 결정하였다. GmNAP1 단백질은 엽록체로 향하는 transit peptide 서열이 존재한다. 빛에 의해 GmNAP1 유전자 전사가 어떻게 변화되는지 알아보기 위해 지속적인 적색광, 근적색광 그리고 암 상태에서 성장시키면서 유전자의 전사량을 확인하였다. 이 색소체 NAP1는 엽록소의 전구 물질인 protoporphytin IX를 세포질에서 엽록체로 이동시키는 기능을 한다. 대두에서 분리된 GmNAP1 유전자의 기능을 확인하기 위하여 35S 프로모터 뒤에 GmNAP1 유전자를 접합한 후 애기장대에 형질전환하였다. 형질전환 된 애기장대의 엽록소 함량은 야생형의 엽록소 함량보다 훨씬 높게 측정되었다.

• 한국미생물·생명공학회지
• /
• 제48권2호
• /
• pp.185-192
• /
• 2020

The global carbon storage regulator (Csr) system is conserved in bacteria and functions as a regulator in the exponential and stationary phases of growth in batch culture. The Csr system plays a role in the central carbon metabolism, virulence, motility, resistance to oxidative stress, and biofilm formation. Although the Csr was extensively studied in Gram negative bacteria, it has been reported only in the control of motility in Bacillus subtilis among Gram positive bacteria. The goal of this study was to explore the role of the csrA gene of Bacillus licheniformis M2-7 on motility and the bacterial ability to use hydrocarbons as carbon source. We deleted the csrA gene of B. licheniformis M2-7 using the plasmid pCsr-L, harboring the spectinomycin cassette obtained from the plasmid pHP45-omega2. Mutants were grown on culture medium supplemented with 2% glucose or 0.1% gasoline and motility was assessed by electron microscopy. We observed that CsrA negatively regulates motility by controlling the expression of the hag gene and the synthesis of flagellin. Notably, we showed the ability of B. licheniformis to use gasoline as a unique carbon source. Our results demonstrated that CsrA is an indispensable regulator for the growth of B. licheniformis M2-7 on gasoline.

• Molecules and Cells
• /
• 제43권1호
• /
• pp.48-57
• /
• 2020

The microalga Chlamydomonas reinhardtii accumulates triacylglycerols (TAGs) in lipid droplets under stress conditions, such as nitrogen starvation. TAG biosynthesis occurs mainly at the endoplasmic reticulum (ER) and requires fatty acid (FA) substrates supplied from chloroplasts. How FAs are transferred from chloroplast to ER in microalgae was unknown. We previously reported that an Arabidopsis thaliana ATP-binding cassette (ABC) transporter, AtABCA9, facilitates FA transport at the ER during seed development. Here we identified a gene homologous to AtABCA9 in the C. reinhardtii genome, which we named CrABCA2. Under nitrogen deprivation conditions, CrABCA2 expression was upregulated, and the CrABCA2 protein level also increased. CrABCA2 knockdown lines accumulated less TAGs and CrABCA2 overexpression lines accumulated more TAGs than their untransformed parental lines. Transmission electron microscopy showed that CrABCA2 was localized in swollen ER. These results suggest that CrABCA2 transports substrates for TAG biosynthesis to the ER during nitrogen starvation. Our study provides a potential tool for increasing lipid production in microalgae.

• Journal of Microbiology and Biotechnology
• /
• 제31권8호
• /
• pp.1154-1162
• /
• 2021

The transcriptional capacities of target genes are strongly influenced by promoters, whereas few studies have focused on the development of robust, high-performance and cross-species promoters for wide application in different bacteria. In this work, four novel promoters (P_k.rtufB, P_k.r1, P_k.r2, and P_k.r3) were predicted from Ketogulonicigenium robustum and their inconsistency in the -10 and -35 region nucleotide sequences indicated they were different promoters. Their activities were evaluated by using green fluorescent protein (gfp) as a reporter in different species of bacteria, including K. vulgare SPU B805, Pseudomonas putida KT2440, Paracoccus denitrificans PD1222, Bacillus licheniformis and Raoultella ornithinolytica, due to their importance in metabolic engineering. Our results showed that the four promoters had different activities, with P_k.r1 showing the strongest activity in almost all of the experimental bacteria. By comparison with the commonly used promoters of E. coli (tufB, lac, lacUV5), K. vulgare (Psdh, Psndh) and P. putida KT2440 (JE111411), the four promoters showed significant differences due to only 12.62% nucleotide similarities, and relatively higher ability in regulating target gene expression. Further validation experiments confirmed their ability in initiating the target minCD cassette because of the shape changes under the promoter regulation. The overexpression of sorbose dehydrogenase and cytochrome c551 by P_k.r1 and P_k.r2 resulted in a 22.75% enhancement of 2-KGA yield, indicating their potential for practical application in metabolic engineering. This study demonstrates an example of applying bioinformatics to find new biological components for gene operation and provides four novel promoters with broad suitability, which enriches the usable range of promoters to realize accurate regulation in different genetic backgrounds.

• Nutrition Research and Practice
• /
• 제18권2호
• /
• pp.194-209
• /
• 2024

BACKGROUND/OBJECTIVES: High levels of plasma low-density lipoprotein (LDL) cholesterol are an important determinant of atherosclerotic lesion formation. The disruption of cholesterol efflux or reverse cholesterol transport (RCT) in peripheral tissues and macrophages may promote atherogenesis. The aim of the current study was to examine whether bioactive ellagic acid, a functional food component, improved RCT functionality and high-density lipoprotein (HDL) function in diet-induced atherogenesis of apolipoproteins E (apoE) knockout (KO) mice. MATERIALS/METHODS: Wild type mice and apoE KO mice were fed a high-cholesterol Paigen diet for 10 weeks to induce hypercholesterolemia and atherosclerosis, and concomitantly received 10 mg/kg ellagic acid via gavage. RESULTS: Supplying ellagic acid enhanced induction of apoE and ATP-binding cassette (ABC) transporter G1 in oxidized LDL-exposed macrophages, facilitating cholesterol efflux associated with RCT. Oral administration of ellagic acid to apoE KO mice fed on Paigen diet improved hypercholesterolemia with reduced atherogenic index. This compound enhanced the expression of ABC transporters in peritoneal macrophages isolated from apoE KO mice fed on Paigen diet, indicating increased cholesterol efflux. Plasma levels of cholesterol ester transport protein and phospholipid transport protein involved in RCT were elevated in mice lack of apoE gene, which was substantially reduced by supplementing ellagic acid to Paigen diet-fed mice. In addition, ellagic acid attenuated hepatic lipid accumulation in apoE KO mice, evidenced by staining of hematoxylin and eosin and oil red O. Furthermore, the supplementation of 10 mg/kg ellagic acid favorably influenced the transcriptional levels of hepatic LDL receptor and scavenger receptor-B1 in Paigen diet-fed apoE KO mice. CONCLUSION: Ellagic acid may be an athero-protective dietary compound encumbering diet-induced atherogenesis though improving the RCT functionality.

• 대한소아신경학회지
• /
• 제23권2호
• /
• pp.45-50
• /
• 2015

목적: X-ALD는 Xq28에 위치한 ABCD1 유전자의 돌연변이로 긴사슬지방산이 신경 조직과 부신에 축적되어 일어나는 퇴행 뇌질환이며, 소아기 대뇌형의 경우 빠르고 심한 임상 경과를 보인다. 이 과정에서 산화스트레스도 조직 손상에 영향을 주는 것으로 알려져 있다. 골수이식이나 로렌조 기름 등이 치료 방법으로 이용되나 치료의 위험성과 효과에서 한계를 보이는 실정이다. 이에 저자들은 X-ALD 환자에게 채취한 섬유모세포를 이용하여, X-ALD의 치료 가능성을 알아보기 위하여 VPA의 효과를 연구해보고자 하였다. 방법: X-ALD 환자의 피부에서 채취한 섬유모세포와 정상인의 피부에서 채취한 섬유모세포를 배양하였다. 배양된 섬유모세포에 VPA를 처리한 후 RNA발현 정도를 통해 ABCD2 발현을 확인하고 유동세포계측법으로 활성산소종을 측정하였다. 결과: VPA을 처리한 후 정상과 X-ALD 섬유모세포 모두에서 ABCD2의 mRNA 발현이 증가하였다. 특히 X-ALD 섬유모세포에서 ABCD2 유전자 mRNA 발현이 2.22배로 정상의 1.76배보다 더 증가하였다. 유동세포계측법으로 활성산소종을 확인한 결과 대조군에서 13.7, VPA를 처리한 군에서는 각각 0.25 mM에서 8.67, 0.5 mM에서 9.37, 1 mM에서 5.83을 나타내었다. 결론: X-ALD 환자에서 VPA의 항산화능을 이용하여 신경손상을 막을 수 있는 가능성이 있을 것으로 보이며, 이를 실제 환자에 적용하는 연구가 필요할 것이다.