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C1 Compound Biosensors: Design, Functional Study, and Applications
Lee, Jin-Young,Sung, Bong Hyun,Oh, So-Hyung,Kwon, Kil Koang,Lee, Hyewon,Kim, Haseong,Lee, Dae-Hee,Yeom, Soo-Jin,Lee, Seung-Goo MDPI AG 2019 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.20 No.9
<P>The microbial assimilation of one-carbon (C1) gases is a topic of interest, given that products developed using this pathway have the potential to act as promising substrates for the synthesis of valuable chemicals via enzymatic oxidation or C–C bonding. Despite extensive studies on C1 gas assimilation pathways, their key enzymes have yet to be subjected to high-throughput evolution studies on account of the lack of an efficient analytical tool for C1 metabolites. To address this challenging issue, we attempted to establish a fine-tuned single-cell–level biosensor system constituting a combination of transcription factors (TFs) and several C1-converting enzymes that convert target compounds to the ligand of a TF. This enzymatic conversion broadens the detection range of ligands by the genetic biosensor systems. In this study, we presented new genetic enzyme screening systems (GESSs) to detect formate, formaldehyde, and methanol from specific enzyme activities and pathways, named FA-GESS, Frm-GESS, and MeOH-GESS, respectively. All the biosensors displayed linear responses to their respective C1 molecules, namely, formate (1.0–250 mM), formaldehyde (1.0–50 μM), and methanol (5–400 mM), and they did so with high specificity. Consequently, the helper enzymes, including formaldehyde dehydrogenase and methanol dehydrogenase, were successfully combined to constitute new versatile combinations of the C1-biosensors.</P>
Kwon, Kil Koang,Yeom, Soo-Jin,Lee, Dae-Hee,Jeong, Ki Jun,Lee, Seung-Goo Elsevier 2018 Biochemical and biophysical research communication Vol.495 No.1
<P><B>Abstract</B></P> <P>Successful utilization of cellulose as renewable biomass depends on the development of economically feasible technologies, which can aid in enzymatic hydrolysis. In this study, we developed a whole-cell biosensor for detecting cellulolytic activity that relies on the recognition of cellobiose using the transcriptional factor CelR from <I>Thermobifida fusca</I> and transcriptional activation of its downstream <I>gfp</I> reporter gene. The fluorescence intensity of whole-cell biosensor, which was named as cellobiose-detectible genetic enzyme screening system (CBGESS), was directly proportional to the concentration of cellobiose. The strong fluorescence intensity of CBGESS demonstrated the ability to detect cellulolytic activity with two cellulosic substrates, carboxymethyl cellulose and <I>p</I>-nitrophenyl β-D-cellobioside in cellulase-expressing <I>Escherichia coli</I>. In addition, CBGESS easily sensed crystalline cellulolytic activity when commercial Celluclast 1.5L was dropped on an Avicel plate. Therefore, CBGESS is a powerful tool for detecting cellulolytic activity with high sensitivity in the presence of soluble or insoluble cellulosic substrates. CBGESS may be further applied to excavate novel cellulases or microbes from both genetic libraries and various environments.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A novel biosensor for detecting cellulolytic activity is developed using cellobiose-dependent transcriptional regulator. </LI> <LI> CBGESS shows quantitative response to cellobiose with hyper sensitivity and specificity. </LI> <LI> Cellulase activity including hydrolysis of crystalline cellulose is measured by CBGESS <I>via</I> intracellular fluorescence. </LI> </UL> </P>
한기원(Ki Won Han),이수원(Soo Won Lee),한광수(Koang Soo Han),이대진(Dae Jin Lee),이병의(Byung Eui Lee),장원철(Won Cheoul Jang) 한국독성학회 2003 Toxicological Research Vol.19 No.2
We investigated antitumor activities of the ethanol extract from mushroom Phellinus linteus and Phellinus baumii on mulberry, oak and elm. In vitro test, the ethanol extract of mushroom cultivated on oak of Phellinus linteus showed highest activities about SK-OV-3, HCT15, XF498, SKMEL-2 and A549. SK-OV-3 cell line showed 100% cytotoxicity in 100 mg/ml and HCT15 (98.39%), XF498 (89.62%), SK-MEL-2 (84.07%) and A549 (79.92%) cytotoxicity respectively. Also IC50 showed 3.99 ㎍/ml to SK-OV-3 cell line and HCT15 (4.37 ㎍/ml), A549 (5.48 ㎍/ml), SK-MEL-2 (6.72 ㎍/ml), XF 498 (6.88 ㎍/ml). As those results, cultivated oak of Phellinus linteus showed a very low IC50 value against SK-OV-3, HCT15, XF498, SK-MEL-2 and A549 cancer cell lines.
만성 췌장염이 의심되는 환자에서 대변 및 혈청 Elastase 1 측정의 임상적 의의
김정권,안득수,김대곤,이수택,최광호 대한소화기학회 1999 대한소화기학회지 Vol.34 No.4
Background/Aims: Indirect pancreatic function tests available currently are unreliable in clinical practice for early chronic pancreatitis because of their low sensitivity in mild and moderate exocrine pancreatic insufficiency. In addition, the measurement of serum amylase and lipase serum levels of levels is not useful in the diagnosis of chronic pancreatitis. Methods: We evaluated the sensitivity and specificity of fecal elastase 1 determination in diagnosing pancreatic insufficiency. Concentrations of fecal and serum elastase 1 were measured with enzyme linked immunosorbent assay (ELISA) in 25 patients with chronic pancreatitis, 29 patients with acute pancreatitis and 29 normal controls. Results: The mean concentration of fecal elastase 1 was 136.8±24.7 μg/g stool in chronic pancreatitis patients, 644.2±52.1 μg/g stool in acute pancreatitis and 649.8±56.7 μg/g stool in control group. When the cut-off value was set at 300 μg/g stool, the sensitivity and specificity of stool elastase 1 determination w and 94.8%, respectively, in patients with chronic pancreatitis. Significant correlations were found between serum amylase level and serum lipase level (r=0.923, p$lt;0.01). Fecal elastase 1 level was not correlated with serum level of amylase or lipase. Conclusions: Fecal elastase 1 determination may be a highly sensitive and specific exocrine pancreatic function test in diagnosing chronic pancreatitis.
A Genetically Encoded Biosensor for the Detection of Levulinic Acid
Kim Tae Hyun,Woo Seung-Gyun,Kim Seong Keun,Yoo Byeong Hyeon,Shin Jonghyeok,Rha Eugene,Kim Soo Jung,Kwon Kil Koang,Lee Hyewon,Kim Haseong,Kim Hee-Taek,Sung Bong-Hyun,Lee Seung-Goo,Lee Dae-Hee 한국미생물·생명공학회 2023 Journal of microbiology and biotechnology Vol.33 No.4
Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production from lignin and poly(ethylene terephthalate). We also created a genetically encoded biosensor responsive to LA, which can be used for screening and evolving the LA biosynthesis pathway genes, by employing an LvaR transcriptional regulator of Pseudomonas putida KT2440 to express a fluorescent reporter gene. The LvaR regulator senses LA as a cognate ligand. The LA biosensor was first examined in an Escherichia coli strain and was found to be non-functional. When the host of the LA biosensor was switched from E. coli to P. putida KT2440, the LA biosensor showed a linear correlation between fluorescence intensity and LA concentration in the range of 0.156–10 mM LA. In addition, we determined that 0.156 mM LA was the limit of LA detection in P. putida KT2440 harboring an LA-responsive biosensor. The maximal fluorescence increase was 12.3-fold in the presence of 10 mM LA compared to that in the absence of LA. The individual cell responses to LA concentrations reflected the population-averaged responses, which enabled high-throughput screening of enzymes and metabolic pathways involved in LA biosynthesis and sustainable production of LA in engineered microbes.