Single cell-strain부터 유래된 무세포 효소 시스템을 이용한 톨루엔 및 아세트산 분해

장재현 ( Jae Hyun Jang ),김예지 ( Yeji Kim ),노태용 ( Tae Yong Roh ),박중곤 ( Joong Kon Park ) 한국화학공학회 2016 Korean Chemical Engineering Research(HWAHAK KONGHA Vol.54 No.5

본 연구에서는 톨루엔 분해 균주인 Pseudomonas putida와 아세트산 분해 균주인 Cupriavidus necator에 무세포 효소 시스템(cell-free enzyme system)을 적용하여 톨루엔과 아세트산에 대한 분해 가능성을 확인하는 실험을 수행하였다. P. putida는 톨루엔 존재 하에서만 toluene dioxygenase를 생성하여 톨루엔을 cis-toluene dihydrodiol로 분해하며, C. necator는 acetyl coenzyme A synthetase-1을 생성하여 아세트산을 acetyl CoA로 전환시켜 생존에 필요한 ATP나 생분해성(biodegradable) 고분자인 Polyhydroxyalkanoate (PHA)를 합성한다. P. putida의 톨루엔 분해 효소인 toluene dioxygenase는 유도효소이기 때문에 toluene dioxygenase 생성 전과 후로 나누어 실험을 진행하였다. P. putida의 톨루엔 분해능력 확인을 위한 gas chromatography (GC) 분석 결과, 대조군과 toluene dioxygenase 생성 전인 실험군 1에서는 검출된 톨루엔의 양이 거의 유사하였으나, toluene dioxygenase 생성 후인 실험군 2에서는 검출된 톨루엔의 양이 대조군 및 실험군 1에 비해 감소하였다. 또한 C. necator의 아세트산 분해능력 확인을 위한 gas chromatography-mass spectrometer (GC-MS) 분석 결과, 무세포 효소 시스템을 적용한 실험군에서는 아세트산에 대한 피크가 검출되지 않았다. 따라서 P. putida와 C. necator는 무세포 효소 시스템 적용 후에도 톨루엔 및 아세트산 분해 능력이 유지되었으나, P. putida는 무세포 효소 시스템을 적용하기 전에 유도 효소를 생성하는 과정이 필요하다. This study deals with the possible degradation of toluene and acetic acid when subjected to cell free enzyme system from the toluene degrading bacteria Pseudomonas putida and acetic acid degrading bacteria Cupriavidus necator. P. putida produces toluene dioxygenase only under the existence of toluene in culture medium and toluene is degraded to cis-toluene dihydrodiol by this enzyme. C. necator produces acetyl coenzyme A synthetase-1 and converts acetic acid to acetyl CoA in order to synthesize ATP to need for growth or PHA which is biodegradable polymer. In case of toluene degradation, the experiment was conducted before and after production of toluene dioxygenase as this enzyme, produced by P. putida, is an inducible enzyme. Toluene was detected using gas chromatography (GC). Similar amount of toluene was found in control group and before production of toluene dioxygenase (experimental group 1). However, reduction in toluene was detected after the production of toluene dioxygenase (experimental group 2). Acetic acid was detected through application of gas chromatography-mass spectrometer (GC-MS). The results showed the acetic acid peak was not detected in the experimental group to apply cell-free enzyme system. These results show that the cell-free enzyme system obtained from P. putida and C. necator retained the ability to degrade toluene and acetic acid. However, P. putida needs to produce the inducible enzyme before preparation of the cell free enzyme system.

Cell-free synthesis of functional cell-penetrating scFv in a cell-free protein synthesis system derived from Escherichia coli

박유진,( Christy Catherine ),( Devi Kasi ),이경호,신승민,김용성,김동명 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.0

In this study we attempted to express the scFv antibodies that had been engineered to penetrate into the cells and give signal through split GFP complementation. Cell-free protein synthesis system provides a high accessibility to the translational mechanism, thereby providing optimal environments to increase the productivity and solubility of scFv. To assist the formation of correct disulfide bonds, we have changed redox conditions of the cell extract and reaction mixture using GSSG/GSH redox buffer. To increase the solubility, we used a cell extract enriched with DsbA, DsbC, FkpA and SurA. We also used the leader sequence ubiquitin,in front of the target scFv and achieved a high level expression of functional scFv antibodies. The functionality has confirmed by reading the fluorescence signal of transfected cells in a black plate assay.

Simplified and cost-effective cell-free protein synthesis system using continuous-exchange bioreactor

이경호,권진호,김동명 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.0

Cell-free protein synthesis (CFPS) is a powerful tool for rapid production of recombinant proteins as well as for functional analysis of genes.The unique flexibility and throughput of cell-free protein synthesis have made this technology a promising alternative to conventional cell-based protein production methods. Despite the versatility of CFPS,however, it requires further improvements in protein productivity. One of the measures for improving yield is a continuous-exchange cell-free (CECF) system. The CECF system supports extended syntheses of target proteins through continuous supply and removal of substrate materials and by-products, respectively. However, previous CECF platforms have suffered from the complexity of reaction devices and unacceptably high reagent costs. In this study, we tried to simplify CECF system with cost-effective substrates. First, we designed a simplified CECF device using common lab materials. In addition, we prepared a concentrated master mix solution to simplify the reaction schemes. Finally, nucleoside triphosphates (NTP) were replaced with nucleoside monophosphates(NMP) that are far more cost-effective. These simplified CECF platform with cost-effective substrates led to a significant increase yield of protein synthesis with a fraction of the cost required for conventional CECF reactions.

Encapsulated Yeast Cell-free System: A Strategy for Cost-effective and Sustainable Production of Bio-ethanol in Consecutive Batches

Muhammad Wajid Ullah,Waleed Ahmad Khattak,Mazhar Ul-Islam,Shaukat Khan,박중곤 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.3

This study was intended to develop an encapsulated yeast cell-free system (EyCFS) by confining yeast cell-free lysate within a calcium alginate capsule. The system was evaluated for bio-ethanol production at elevated temperatures and was compared to a bare yeast cell-free system (ByCFS). Fermentation of 10 g/L glucose with shaking (150 rpm), using 2 mg/mL cell-free proteins in the ByCFS produced 3.31 g/L bio-ethanol, corresponding to 65% of the maximal theoretical yield, at 45°C and pH 7.0. On the contrary, the EyCFS produced 4.12 g/L bioethanol, corresponding to 81% of the maximal theoretical yield, under the same experimental conditions. The EyCFS also retained 32% of its original activity after 15 consecutive batches. We observed an 11% increase in bio-ethanol production after replenishment of cofactors (ATP and NADH) and ATPase. The weight-based total turnover number (TTNw; 0.82 × 103), cost ratio (R value; 1.22), and yield (80.4%) indicated the economic suitability of the EyCFS for large-scale production. Connecting the EyCFS with an encapsulated saccharification system through separate hydrolysis and fermentation (SHF) resulted in production of 4.87 g/L bio-ethanol, corresponding to 87.6% of the maximal theoretical yield. This system resolved serious limitations of conventional simultaneous saccharification and fermentation in bare cell-free systems. These data demonstrates the superiority of the proposed system in terms of thermal stability, yield, efficacy, and cost-effectiveness.

배아줄기세표의 인슐린 분비세포로의 유도 분화에 대한 연구

성지혜,임천규,최혜원,이형송,신현상,전진현,윤현수,궁미경,Sung, Ji-Hye,Lim, Chun-Kyu,Choi, Hye-Won,Lee, Hyoung-Song,Shin, Hyeon-Sang,Jun, Jin-Hyun,Yoon, Hyun-Soo,Koong, Mi-Kyoung 대한생식의학회 2004 Clinical and Experimental Reproductive Medicine Vol.31 No.4

Objective: Embryonic stem (ES) cells could be differentiated into the specific cell types by alternation of culture condition and modification of gene expression. This study was performed to evaluate the differentiation protocol for mouse and human ES cells to insulin secreting cells. Methods: Undifferentiated mouse (JH-I) and human (Miz-hESI) ES cells were cultured on STO feeder layer, and embryoid bodies (EBs) were formed by suspension culture. For the differentiation, EBs were cultured by sequential system with three stage protocol. The differentiating ES cells were collected and marker gene expressions were analyzed by seIni-quantitative RT-PCR in each stage. Amount of secreted insulin levels in culture media of human ES cells were measured by human insulin specific RIA kit. Results: During the differentiation process of human ES cells, GATA-4, a-fetoprotein, glucose transporter-2 and Ngn-3 expression were increased whereas OctA was decreased progressively. Insulin and albuInin mRNAs were expressed from stage IT in mouse ES cells and from stage III in human ES cells. We detected 3.0~7.9 IlU/rnl secretion of insulin from differentiated human ES cells by in vitro culture for 36 days. Conclusion: The sequential culture system could induce the differentiation of mouse and human ES cells into insulin secreting cells. This is the fIrst report of differentiation of human ES cells into insulin secreting cells by in vitro culture with serum and insulin free medium.

Synthesis of (R,R)-2,3-butanediol from starch in a hybrid cell-free reaction system

Tong Yi,임혜진,이소정,이경호,김동명 한국공업화학회 2018 Journal of Industrial and Engineering Chemistry Vol.67 No.-

In this study, we demonstrate the conversion of starch to (R,R)-2,3-butanediol (2,3-BD) in a hybrid cell-free synthesis system containing a mixture of lysates derived from Escherichia coli (E. coli) and cyanobacteria. A sufficient pool of pyruvate required for the synthesis of 2,3-BD was generated by combining metabolic pathways of cyanobacteria and E. coli. Successful synthesis of 2,3-BD was achieved by additional modifications of the hybrid cell-free system with the enzymes required to convert pyruvate to 2,3-BD. The results demonstrate a new approach to harness biological pathways to expand the scope of cell-free metabolic engineering by cross-species combinations of cell lysates.

Designing Cell-free Biology for Analysis and Diagnosis

Dong-Myung KIM 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Cell-free synthesis harnesses synthetic apparatus of cells in open environment. In principle, the constituents of cell-free synthesis can be individually manipulated without being limited by the requirement for maintaining cell viability and integrity. For example, in analogy to reconnecting a short circuit, an incomplete reaction mixture can be activated to produce protein outputs upon complementation with the missing components. We probed the possibility of extending this concept for the analysis of various chemical targets as well as important biomarkers. This presentation will also include a discussion how the concept of cell-free biology can be further developed into a universal platform for on-site analysis and point-of-care diagnostics.

무세포 단백질합성 시스템 기반의 epoxide hydrolase 발현 및 활성 분석

이옥경,김희숙,이은열,Lee, Ok-Kyung,Kim, Hee-Sook,Lee, Eun-Yeol 한국생명과학회 2005 생명과학회지 Vol.15 No.5

Coupled transcription/translation cocktail을 이용하여 R. glutinis EH 유전자를 in vitro에서 합성하고 활성을 평가하였다. SDS-PAGE 및 immunoblotting을 통하여 45 kDa 크기의 EH 단백질이 발현되었음을 확인하였고, NBP assay 및 chiral GC 분석을 통해 발현된 단백질이 (R)-styrene oxide에 대한 입체선택성이 있음을 확인하였다. 따라서 무세포 단백질 합성 시스템을 이용하여 입체선택성을 유지시킨 EH 유전자 발현이 가능하며, 이러한 방법은 putative EH 유전자 탐색 등에 효율적으로 응용될 것이다. Cell-free expression is a powerful tool for rapid protein analysis, enabling an efficient identification of gene without cumbersome procedure of transformation and cell culture. Epoxide hydrolase (EH) gene of Rhodotorula glutinis was simply amplified by PCR, and the resultant gene was expressed in vitro using a coupled Transcription/translation system. The cell-free expressed EH protein mixture exhibited the enantioselective hydrolysis activity toward (R)-styrene oxide, representing that cell-free protein synthesis system can be used for the rapid expression of an enantioselective enzyme for an efficient identification of the chiral activity.

Detergent-assisted Enhancement of the Translation Rate during Cell-free Synthesis of Peptides in an Escherichia coli Extract

고서영,이경호,김동명 한국생물공학회 2018 Biotechnology and Bioprocess Engineering Vol.23 No.6

The open nature of cell-free synthesis allows customization of the reaction conditions for given target molecules using diverse biological and non-biological substances. This study demonstrates that non-ionic detergents can be used to enhance translation during the synthesis of peptides in a cell-free system derived from an Escherichia coli extract. The yield of the antimicrobial peptide Cecropin P1 was markedly increased in the presence of detergents. The stimulatory effect of detergents was not limited to the Cecropin P1 peptide, but the detergent also enhanced the translation of other antimicrobial peptides. Furthermore, the enhanced translation rate by detergents was maintained for extended periods by a continuous exchange cell-free synthesis reaction, leading to production of antimicrobial peptides with markedly improved yields.

Synthesis of (R,R)-2,3-butanediol from starch in a hybrid cell-free reaction system

Yi, Tong,Lim, Hye Jin,Lee, So Jeong,Lee, Kyung-Ho,Kim, Dong-Myung THE KOREAN SOCIETY OF INDUSTRIAL AND ENGINEERING 2018 JOURNAL OF INDUSTRIAL AND ENGINEERING CHEMISTRY -S Vol.67 No.-

<P><B>Abstract</B></P> <P>In this study, we demonstrate the conversion of starch to (R,R)-2,3-butanediol (2,3-BD) in a hybrid cell-free synthesis system containing a mixture of lysates derived from <I>Escherichia coli</I> (<I>E. coli</I>) and cyanobacteria. A sufficient pool of pyruvate required for the synthesis of 2,3-BD was generated by combining metabolic pathways of cyanobacteria and <I>E. coli.</I> Successful synthesis of 2,3-BD was achieved by additional modifications of the hybrid cell-free system with the enzymes required to convert pyruvate to 2,3-BD. The results demonstrate a new approach to harness biological pathways to expand the scope of cell-free metabolic engineering by cross-species combinations of cell lysates.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>