Codon optimization of the synthetic 3-ketosphinganine reductase (3KSR) protein for enhancing sphingolipid biosynthetic enzyme expression

Um Hyun-Ju,Shin Woo-ri,Sekhon Simranjeet Singh,Woo Sung Min,김영창,안지영,김양훈 대한독성 유전단백체 학회 2021 Molecular & cellular toxicology Vol.17 No.4

Background Sphinganine is a key precursor for the synthesis of ceramide during sphingolipid biosynthesis in eukaryotes. In the sphinganine biosynthetic pathway, serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase (3KSR) catalyze the condensation of serine and palmitoyl-CoA to form 3-ketosphinganine, and the conversation of 3-ketosphinganine to dehydrosphingosine (DHS), respectively. Objective Phytosphingosine among cosmeceutical ingredients exists primarily on the surface of the skin and serves as a potent anti-microbial and anti-inflammatory agent. To overexpressing produce phytosphingosine in bacteria, we designed and synthesized the oligonucleotide of two major genes (3KSR and SPT) involved in the biosynthesis of sphingolipid for industrial applications. Results The genes encoding SPT from the Gram-negative bacterium Sphingobium chungbukense DJ77 and 3KSR from the eukaryotic Saccharomyces cerevisiae were expressed in Escherichia coli BL21 (DE3) to produce DHS efficiently. Codon optimization was utilized for 3KSR expression in E. coli to facilitate the expression of eukaryotic proteins in the bacterial host. The synthetic 3KSR (3sKSR) protein activity was improved approximately 1.8 times compared to the wild-type 3KSR. Additionally, we successfully overexpressed the spt gene obtained from S. chungbukense DJ77, which can be used in efficient production of the sphenoid bases, with codon-optimized 3sKSR. The enhanced accumulation of DHS by SPT-3sKSR suggests that the codon-optimized enzyme (3sKSR) is more efficient for heterogeneous expression in E. coli. Conclusion Our results demonstrate that sphingolipid metabolism can be configured in engineered E. coli by the simultaneous overexpression of two key enzymes, SPT and 3sKSR, thereby producing sphingosine. Background Sphinganine is a key precursor for the synthesis of ceramide during sphingolipid biosynthesis in eukaryotes. In the sphinganine biosynthetic pathway, serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase (3KSR) catalyze the condensation of serine and palmitoyl-CoA to form 3-ketosphinganine, and the conversation of 3-ketosphinganine to dehydrosphingosine (DHS), respectively. Objective Phytosphingosine among cosmeceutical ingredients exists primarily on the surface of the skin and serves as a potent anti-microbial and anti-inflammatory agent. To overexpressing produce phytosphingosine in bacteria, we designed and synthesized the oligonucleotide of two major genes (3KSR and SPT) involved in the biosynthesis of sphingolipid for industrial applications. Results The genes encoding SPT from the Gram-negative bacterium Sphingobium chungbukense DJ77 and 3KSR from the eukaryotic Saccharomyces cerevisiae were expressed in Escherichia coli BL21 (DE3) to produce DHS efficiently. Codon optimization was utilized for 3KSR expression in E. coli to facilitate the expression of eukaryotic proteins in the bacterial host. The synthetic 3KSR (3sKSR) protein activity was improved approximately 1.8 times compared to the wild-type 3KSR. Additionally, we successfully overexpressed the spt gene obtained from S. chungbukense DJ77, which can be used in efficient production of the sphenoid bases, with codon-optimized 3sKSR. The enhanced accumulation of DHS by SPT-3sKSR suggests that the codon-optimized enzyme (3sKSR) is more efficient for heterogeneous expression in E. coli. Conclusion Our results demonstrate that sphingolipid metabolism can be configured in engineered E. coli by the simultaneous overexpression of two key enzymes, SPT and 3sKSR, thereby producing sphingosine.

Molecular identification and characterization of an intracellular poly[D(-)-3-Hydroxybutyrate] depolymerase gene from Sphingobium chungbukense DJ77

Hyun-Ju Um,Young-Chang Kim,Yang-Hoon Kim 한국생물공학회 2008 한국생물공학회 학술대회 Vol.2008 No.4

Cloning, Heterologous Expression of Interleukin 6 (IL-6) for Development of Rheumatoid Arthritis Early Diagnosis Aptasensor

Hyun-Ju UM,Sang-Hee LEE,Jihee YOON,Jiho MIN,Yang-Hoon KIM 한국생물공학회 2011 한국생물공학회 학술대회 Vol.2011 No.10

Cloning, Heterologous Expression of Tumour Necrosis Factor (TNF) Biomarker for Development of Rheumatoid Arthritis Early Diagnosis Aptasensor

Hyun-Ju UM,Jihee YOON,Jiho MIN,Yang-Hoon KIM 한국생물공학회 2011 한국생물공학회 학술대회 Vol.2011 No.4

Effect of degumming method on the structure and properties of solution, film, and electrospun fiber of regenerated silk

Hyun Ju Kim,In Chul Um 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

Silk fibroin can be damaged or degraded during degumming process. Therefore, it is expected that different structure and properties of silk fibroin can be obtained by different degumming method. However, effect of degumming method on the structure and properties of regenerated silk has not been considered, yet. In this study, Effect of degumming method on the structure and properties of solution, film, and electrospun fiber of regenerated silk was examined. Order of viscosity of regenerated silk solution as follows : Urea method > Acid method, HTHP method > Soap/soda method, Soda method. This viscosity difference among the degumming method strongly influenced the electro-spinning performance of regenerated silk perpared from different degumming method. Also, solution turbidity, crystallinity index (from FTIR), mechanical properties of silk were remarkably affected by degumming method.

201210

Um, Hyun Ju 한국콘텐츠학회 2012 ICCC International Digital Design Invitation Exhib Vol.8 No.2

Expression patterns of gangliosides in lipopolysaccharid-stimulated normal PK-15 cells and gangliosides knock-down PK15 cells

Ju-Taek Lee,Jae-Sung Ryu,Ghislain Moussavou,Malg-Um Lim,Hyun-Ki Min,Yoon-Ju Na,Su-Bin Lee,In-Sun Kim,Ju-Hyoung Lee,Ji-Su Kim,Sun-Uk Kim,Kyu-Tae Chang,Young-Kug Choo 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

Glycosphingolipids including gangliosides play important regulatory roles in cell prolifera-tion and differentiation. UDP-glucose:ceramide glucosyltransferase(Ugcg) catalyze the initial step in glycosphingolipids biosynthesis pathway. In this study, Ugcg expression was reduced to approximately 80% by short hairpin RNAs(shRNAs) to evaluate the roles of glycosphingolipids in proliferation and gangliosdies differentiation of PK15. HPTLC/immunofluorescence analyses of shRNA transfected PK15 revealed that treatment with Ugcg-shRNA decreased expression of major gangliosides, GM1 and GD1b. Furthermore, MTT and western blot/immunofluorescence analyses demonstreated that inhibition of the Ugcg expression in PK15 resulted in decrease of cell proliferation. In addition to we showed that inflammatory respon-ses in PK15 and gangliosides knock-down PK15 cells stimulated with Escherichia coli lipopolysaccharide(LPS). The gangliosides GM3, GM2, GD3, GD1a and GD1b were detected in normal PK15 cell. However almost gangliosides undetected in gangliosides knock-down PK15 cell. In addition we found that the expression levels of TNF-α, indu-cible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and we measured the production of NO. The expression of inflammatory proteins, TNF- α, iNOS and COX-2 the elevated concentration in gangliosides knock-down PK15 cell. These results suggest that gangliosides interact with components of the proinflammatory response pathway and might, therefore, be relevant for designing future therapeutic strategies intended to prolong xenograft survival.

The Selection of Human CD1d Molecule Binding Aptamer for Activating the NKT Cell

Hyun-Ju UM,Sang-Hee LEE,Jiho MIN,Yang-Hoon KIM 한국생물공학회 2013 한국생물공학회 학술대회 Vol.2013 No.4

시각화 유형에 따른 에너지 피드백의 효과

엄주현 ( Um Ju Hyun ),윤재영 ( Yun Ray Jaeyoung ) 디자인융복합학회 2017 디자인융복합연구 Vol.16 No.2

본 연구는 사용자의 에너지 절감 행동 유도를 위하여 가정의 전기 에너지 사용량을 두 가지 시각 유형의 에너지 피드백 디자인으로 제작하고, 이에 따라 인구통계학 요인별 피드백 전략 도출을 목표로 한다. 선행 연구 고찰을 통해 유형화한 이성 소구 피드백과 감성 소구 피드백은 각각 바(bar) 그래프와 북극곰 위젯(widget) 디스플레이로 디자인하였다. 피드백 유형의 시각적인 차이에 근거한 사용자의 이해도, 선호도, 절전의도를 알아보기 위하여 설문지 실험 후 결과를 도출하였다. 실험 결과, 이성 소구 피드백은 낮은 연령과 공학/사회과학 분야의 사람에게 높은 이해도를 보였다. 감성 소구 피드백은 낮은 연령일수록 높은 이해도를 보였으며 여성이 남성보다, 인문/예술 분야가 다른 분야보다 선호도가 높게 나타났다. 최종적으로 연구 결과를 바탕으로 네 가지 에너지 피드백 전략을 도출하였다. This study aims to draw up energy feedback strategies on demographic factors through two visualization types of energy feedback for inducing energy saving behaviors. Through the background study, we analyzed visual types for energy feedback design and these types are classified into Rational Appeal Feedback and Emotional Appeal Feedback. Each of feedback types is designed as Bar Graph and Polar Bear Widget Display. This study draws conclusions through online survey method for evaluating user`s understanding, preference and behavior intention based on visual distinction. Firstly, the understanding of rational appeal feedback among under age 20 is better than 30 age or older. People in engineering or social sciences major/occupation have a high degree of understanding of rational appeal feedback. Secondly, emotional appeal feedback draw up high understanding with low age. In the case of preference, women were higher than men, and the major/occupation of humanities and arts had a relatively higher average value than other fields. Thirdly, as a result of analyzing the correlation between variables of each feedback type, there were high correlation between understanding, preference, and behavior intention of each feedback type. Finally, feedback strategy for each type of users is derived according to the experimental results.

북한 의약품 생산체계의 형성과정에 대한 고찰

엄주현(Um Ju Hyun),박혜경(Hyekyung Park) 대한약학회 2018 약학회지 Vol.62 No.4

This study looked at the formation process of North Korea s pharmaceutical production system based on data disclosed by the North between 1945 and 2017 in order to find ways to cooperate in the inter-Korean cooperation era. Since 1945, North Korea has established the principle of free treatment and free supply of medicines, and has organized state – owned pharmaceutical factories and drug dispensaries to supply pharmaceuticals to national health care institutions first. As large-scale pharmaceutical plants and small and medium-sized pharmaceutical factories were established by region, and drug distribution centers were expanded, the basic demand for pharmaceuticals was finally met in 1980. However, as the inter-national environment deteriorated since 1990, the nation has been unable to produce and supply medicines, and patients have been procuring their own medicines through market systems. After Kim Jong Eun s inauguration in 2012, North Korea is trying to recover to the mid-1980s through reconstruction of the self-sustaining structure and acquisition of foreign cur-rency through development and production of Koryo Pharmaceutical. It is necessary to prepare for the mutual benefit of the North and South in the field of medicine supply through inter-Korean exchange cooperation based on the understanding of reality of North Korea.