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Kim, Min Woo,Lee, Bo-Rahm,You, SungYong,Kim, Eun-Jung,Kim, Ji-Nu,Song, Eunjung,Yang, Yung-Hun,Hwang, Daehee,Kim, Byung-Gee Springer-Verlag 2018 Applied microbiology and biotechnology Vol.102 No.7
<P>Most secondary metabolism in Actinobacteria is controlled by multi-layered, gene-regulatory networks. These regulatory mechanisms are not easily identified due to their complexity. As a result, when a strong transcriptional regulator (TR) governs activation of biosynthetic pathways of target antibiotics such as actinorhodin (ACT), additional enhancement of the biosynthesis is difficult in combination with other TRs. To find out any 'synergistic transcriptional regulators (sTRs)' that show an additive effect on the major, often strong, transcriptional regulator (mTR), here, we performed a clustering analysis using the transcriptome datasets of an mTR deletion mutant and wild-type strain. In the case of ACT biosynthesis in Streptomyces coelicolor, PhoU (SCO4228) and RsfA (SCO4677) were selected through the clustering analysis, using AfsS (SCO4425) as a model mTR, and experimentally validated their roles as sTRs. Furthermore, through analysis of synergistic effects, we were able to suggest a novel regulation mechanism and formulate a strategy to maximize the synergistic effect. In the case of the double TR mutant strain (Delta rsfA pIBR25::afsS), it was confirmed that the increase of cell mass was the major cause of the synergistic effect. Therefore, the strategy to increase the cell mass of double mutant was further attempted by optimizing the expression of efflux pump, which resulted in 2-fold increase in the cell mass and 24-fold increase in the production of ACT. This result is the highest ACT yield from S. coelicolor ever reported.</P>
Kim Goon-Tae,Devi Shivani,Sharma Amitesh,Cho Kyung-Hee,Kim Su-Jung,Kim Bo-Rahm,Kwon Sang-Ho,Park Tae-Sik 생화학분자생물학회 2022 Experimental and molecular medicine Vol.54 No.-
Endoplasmic reticulum (ER) stress is induced by various conditions, such as inflammation and the presence of excess nutrients. Abnormal accumulation of unfolded proteins leads to the activation of a collective signaling cascade, termed the unfolded protein response (UPR). ER stress is reported to perturb hepatic insulin response metabolism while promoting insulin resistance. Here, we report that ER stress regulates the de novo biosynthesis of sphingolipids via the activation of serine palmitoyltransferase (SPT), a rate-limiting enzyme involved in the de novo biosynthesis of ceramides. We found that the expression levels of Sptlc1 and Sptlc2, the major SPT subunits, were upregulated and that the cellular concentrations of ceramide and dihydroceramide were elevated by acute ER stress inducers in primary hepatocytes and HepG2 cells. Sptlc2 was upregulated and ceramide levels were elevated by tunicamycin in the livers of C57BL/6J wild-type mice. Analysis of the Sptlc2 promoter demonstrated that the transcriptional activation of Sptlc2 was mediated by the spliced form of X-box binding protein 1 (sXBP1). Liver-specific Sptlc2 transgenic mice exhibited increased ceramide levels in the liver and elevated fasting glucose levels. The insulin response was reduced by the inhibition of the phosphorylation of insulin receptor β (IRβ). Collectively, these results demonstrate that ER stress induces activation of the de novo biosynthesis of ceramide and contributes to the progression of hepatic insulin resistance via the reduced phosphorylation of IRβ in hepatocytes.
Increased vulnerability to physical stress by inactivation of NdgR in Streptomyces coelicolor.
Lee, Bo-Rahm,Yi, Da-Hye,Song, Eunjung,Bhatia, Shashi Kant,Lee, Ju Hee,Kim, Yun-Gon,Park, Sung-Hee,Lee, Yoo Kyung,Kim, Byung-Gee,Yang, Yung-Hun Humana Press 2015 Applied biochemistry and biotechnology Vol.175 No.8
<P>The antibiotic production and spore formation process in Streptomyces coelicolor need complex decision making processes by several regulatory units. These regulatory units are involved in both primary and secondary metabolism. As a result, most regulators have several functions, and those are worthwhile themes to study about different functions of a known regulator. In this study, a deletion mutant of ndgR, which encodes the nitrogen-dependent growth regulator, was examined by the cell viability test, TEM, and growth in N-acetylglucosamine/asparagine (GlcNAc/Asn) liquid medium. The results of the study show that NdgR is also involved in the structure of the cell membrane affecting survival under physical shocks. Deletion of ndgR leads to abnormal cell membrane resulting in the vulnerable cells to physical stress caused by shaking with beads in liquid culture condition. This empirical observation is the first meaningful explanation to why ndgR mutant could not grow well in a liquid minimal medium due to the defect of N-acetylglucosamine (GlcNAc) utilization and phospholipid synthesis.</P>
앱타머와 단백질간 가교를 이용한 바이오마커 진단 방법 개발
이보람(Bo-rahm Lee),김진우(Ji-nu Kim),김병기(Byung-Gee Kim) 한국생물공학회 2011 KSBB Journal Vol.26 No.4
The detection of biomarkers is an important issue for disease diagnosis. However, many systems are not suitable to detect the biomarker itself directly. For direct detection of biomarker proteins in human serum, a new affinity-capture method using aptamers combined with the mass spectrometry was suggested. Since signals from protein samples cannot be amplified, modified chromatin immunoprecipitation (ChIP) and subsequent cross-linking with formaldehyde between aptamers and target proteins were used not to lose the captured target proteins, which allowed us to perform a harsh washing step to remove the non-specifically bound proteins. As a model system, a thrombin aptamer was used as a bait and thrombin as a target protein. Using our modified ChIP and affinity-capture method, non-specific binding proteins on the beads decreased significantly, suggesting that our new method is efficient and can be applied to developing diagnosis systems for various biomarkers.
Rajesh, Thangamani,Song, Eunjung,Kim, Ji-Nu,Lee, Bo-Rahm,Kim, Eun-Jung,Park, Sung-Hee,Kim, Yun-Gon,Yoo, Dongwon,Park, Hyung-Yeon,Choi, Yun-Hui,Kim, Byung-Gee,Yang, Yung-Hun Springer International 2012 Applied microbiology and biotechnology Vol.93 No.4
<P>Phosphomannose isomerases (PMIs) in bacteria and fungi catalyze the reversible conversion of d-fructose-6-phosphate to d-mannose-6-phosphate during biosynthesis of GDP-mannose, which is the main intermediate in the mannosylation of important cell wall components, glycoproteins, and certain glycolipids. In the present study, the kinetic parameters of PMI from Streptomyces coelicolor were obtained, and its function on antibiotic production and sporulation was studied. manA (SCO3025) encoding PMI in S. coelicolor was deleted by insertional inactivation. Its mutant (S. coelicolora dagger manA) was found to exhibit a bld-like phenotype. Additionally, S. coelicolora dagger manA failed to produce the antibiotics actinorhodin and red tripyrolle undecylprodigiosin in liquid media. To identify the function of manA, the gene was cloned and expressed in Escherichia coli BL21 (DE3). The purified recombinant ManA exhibited PMI activity (K (cat)/K (m) (mM(-1) s(-1) = 0.41 for d-mannose-6-phosphate), but failed to show GDP-d-mannose pyrophosphorylase [GMP (ManC)] activity. Complementation analysis with manA from S. coelicolor or E. coli resulted in the recovery of bld-like phenotype of S. coelicolora dagger manA. SCO3026, another ORF that encodes a protein with sequence similarity towards bifunctional PMI and GMP, was also tested for its ability to function as an alternate ManA. However, the purified protein of SCO3026 failed to exhibit both PMI and GMP activity. The present study shows that enzymes involved in carbohydrate metabolism could control cellular differentiation as well as the production of secondary metabolites.</P>
Yang, Yung-Hun,Song, Eunjung,Kim, Ji-Nu,Lee, Bo-Rahm,Kim, Eun-Jung,Park, Sung-Hee,Kim, Woo-Seong,Park, Hyung-Yeon,Jeon, Jong-Min,Rajesh, Thangamani,Kim, Yun-Gon,Kim, Byung-Gee Springer-Verlag 2012 Applied microbiology and biotechnology Vol.96 No.1
<P>gamma-Butyrolactones in Streptomyces are well recognized as bacterial hormones, and they affect secondary metabolism of Streptomyces. gamma-Butyrolactone receptors are considered important regulatory proteins, and various gamma-butyrolactone synthases and receptors have been reported in Streptomyces. Here, we characterized a new regulator, SCO0608, that interacted with SCB1 (gamma-butyrolactone of Streptomyces coelicolor) and bound to the scbR/A and adpA promoters. The SCO0608 protein sequences are not similar to those of any known gamma-butyrolactone binding proteins in Streptomyces such as ScbR from S. coelicolor or ArpA from Streptomyces griseus. Interestingly, SCO0608 functions as a repressor of antibiotic biosynthesis and spore formation in R5 complex media. We showed the existence of another type of gamma-butyrolactone receptor in Streptomyces, and this SCO0608 was named ScbR-like gamma-butyrolactone binding regulator (SlbR) in S. coelicolor.</P>