Escherichia coli에서 Ramazzottius varieornatus 물곰 유래 α-carbonic anhydrase의 재조합 발현 및 정제

최예진,조병훈 한국미생물학회 2023 미생물학회지 Vol.59 No.3

Carbonic anhydrase (CA) is an enzyme that catalyzes the reversible hydration of CO2, effectively accelerating carbon capture, utilization, and storage (CCUS) processes to achieve carbon neutrality. The discovery and utilization of stable CA is essential for efficient and cost-effective CCUS. Ramazzottius varieornatus, a species of tardigrade, is resistant to various physical and chemical stresses, and CAs from this species are expected to be stable under these stressful conditions. In this study, for the first time, we attempted the recombinant expression and purification of six α-CAs (RvCAs) from R. varieornatus in Escherichia coli. Additionally, we tested the expression of RvCAs with a fusion of the NEXT tag to enhance the soluble expression. With the exception of RvCA5, all of the recombinant RvCAs showed mostly insoluble expression. Subsequently, RvCA5 and NEXT-RvCA5, which were expressed mostly in soluble forms, were successfully purified. The catalytic activity of RvCA5 was found to be extremely low for both ester hydrolysis and CO2 hydration. Subsequent studies on RvCA5 will be interesting in terms of enzyme activity and stability, and further research on the remaining RvCA enzymes is anticipated to uncover novel stable CAs.

Metabolic Flux Distribution and Thermodynamic Analysis of Green Fluorescent Protein Production in Recombinant Escherichia coli: The Effect of Carbon Source and CO2 Partial Pressure

R. Axayácatl González-García,E. Ines Garcia-Peña,Edgar Salgado-Manjarrez,Juan S. Aranda-Barradas 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.6

Increasing recombinant protein production yieldsfrom bacterial cultures remains an important challenge inbiotechnology. Acetate accumulation due to high dissolvedcarbon dioxide (pCO2) concentrations in the medium hasbeen identified as a factor that negatively affects suchyields. Under appropriate culture conditions, acetate couldbe re-assimilated by bacterial cells to maintain heterologousproteins production. In this work, we developed a simplifiedmetabolic network aiming to establish a reaction rate analysisfor a recombinant Escherichia coli when producing greenfluorescent protein (GFP) under controlled pCO2 concentrations. Because E. coli is able to consume both glucoseand acetate, the analysis was performed in two stages. Ourresults indicated that GFP synthesis is an independentprocess of cellular growth in some culture phases. Additionally,recombinant protein production is influenced bythe available carbon source and the amount of pCO2 in theculture medium. When growing on glucose, the increase inthe pCO2 concentration produced a down-regulation ofcentral carbon metabolism by directing the carbon fluxtoward acetate accumulation; as a result, cellular growthand the overall GFP yield decreased. However, the maximumspecific rate of GFP synthesis occurred with acetate as themain available carbon source, despite the low activity inthe other metabolic pathways. To maintain cellular functions,including GFP synthesis, carbon flux was re-distributedtoward the tricarboxylic acid cycle and the pentose phosphatepathway to produce ATP and NADH. The thermodynamicanalysis allowed demonstrating the feasibility of the simplifiednetwork for describing the metabolic state of a recombinantsystem.

Improving the Productivity of Recombinant Protein in Escherichia coli Under Thermal Stress by Coexpressing GroELS Chaperone System

( So Yeon Kim ),( Niraikulam Ayyadurai ),( Mi Ae Heo ),( Sung Hoon Park ),( Yong Joo Jeong ),( Sun Gu Lee ) 한국미생물 · 생명공학회 2009 Journal of microbiology and biotechnology Vol.19 No.1

Here, we demonstrate that the overexpression of the GroELS chaperone system, which assists the folding of intracellular proteins and prevents aggregation of its biological targets, can enhance the thermotolerance of Escherichia coli strains and facilitate the production of recombinant protein under thermal stress. The overexpression of GroELS led to an about 2-fold higher growth rate of E. coli XL-1 blue than control at 45℃ and induced the growth of the strain even at 50℃, although the growth was not sustained in the second-round culture. The effect of GroELS overexpression was also effective on other E. coli strains such as JM109, DH5α, and BL21. Finally, we have shown that coexpression of GroELS allows us to produce recombinant protein even at 50℃, a temperature at which the protein production based on E. coli is not efficient. This study indicates that the employment of the GroELS overexpression system can expand the range of environmental conditions for E. coli.

폐글리세롤을 탄소원으로 hemA와 otsBA를 공동 발현하는 재조합 대장균 배양을 통한 아미노레블린산 생산

염정미 ( Jingmei Yan ),디엡느구팜 ( Diep Ngoc Pham ),강대경 ( Dae Kyung Kang ),김성배 ( Sung Bae Kim ),김창준 ( Chang Joon Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2016 한국미생물·생명공학회지 Vol.44 No.3

본 연구는 폐글리세롤을 탄소원으로 사용하여 생장저해 없이 아미노레블린산(ALA)을 생산하는 미생물 공정을 개발하는데 그 목적이 있다. 폐글리세롤이 첨가된 배지에서 ALA 생산균주(E. coli/pH-hemA)를 배양하여 얻은 세포밀도와 ALA 생산량은 순수 글리세롤이 첨가된 배지에서 얻은 값보다 각각 1.8배와 1.2배 낮았다. 트레할로스를 첨가(30 g/l 또는 100 g/l) 함에 따라 세포생장과 ALA 생산성이 향상되었다. otsBA를 공동 발현하는 엔지니어링 균주(E. coli/pHhemA/ pS-otsBA)를 제작하여 순수 또는 폐글리세롤이 첨가된 배지에서 그 성능을 평가하였다. 폐글리세롤이 함유된 배지에서 엔지니어링 균주의 세포생장 및 생산성을 향상시키기 위하여 IPTG의 최적 첨가농도 및 시기를 결정하였다. 지수생장 초기에 0.6 mM의 IPTG를 첨가한 배양에서 세포 생장과 ALA 생산량이 최대치를 나타내었다. 이때 얻은 OD600(세포 밀도)와 ALA 농도는 각각 16과 2,121 mg/l였는데, 이는 순수 글리세롤이 첨가된 배지에서 얻은 생산량과 비슷한 값이다. 본 결과는 ALA 생산균주에서 트레할로스 생합성 오페론의 최적 공동 발현을 통해 폐글리세롤에 대한 저항성을 높임으로써 추가적인 전처리 없이 폐글리세롤을 탄소원으로 직접 사용할 수 있음을 보여준다. This study aimed to develop a microbial process for producing aminolevulinic acid (ALA) using crude glycerol. In the culture of ALA-producing cells (Escherichia coli/pH-hemA) in a medium containing crude glycerol, the cell density and production were 1.8-fold and 1.2-fold lower than those obtained from pure glycerol, respectively. However, the cell growth and production were improved by supplementing the medium with trehalose (30 or 100 g/l). Engineered cells (E. coli/pH-hemA/pS-otsBA) were constructed to express otsBA and their culture performance was compared with that of control cells (E. coli/pH hemA/ pSTV28). The effects of isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration and the time of induction were examined to improve the cell growth and ALA production in engineered cells cultured using crude glycerol. When 0.6 mM of IPTG was added at the beginning of the exponential growth phase, the ALA produced by cells was 2,121 mg/l, which was comparable to that from pure glycerol. The results demonstrate that otsBA expression endowed cells with the capacity to tolerate the toxicity of crude glycerol for direct use.

Characterization of a Paenibacillus woosongensis β-Xylosidase/α-Arabinofuranosidase Produced by Recombinant Escherichia coli

( Yeon A. Kim ),( Ki Hong Yoon ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.12

A gene encoding the β-xylosidase/α-arabinofuranosidase (XylC) of Paenibacillus woosangensis was cloned into Escherichia coli. This xylC gene consisted of 1,425 nucleotides, encoding a polypeptide of 474 amino acid residues. The deduced amino acid sequence exhibited an 80% similarity with those of both Clostridium stercorarium β-xy1osidase/α-N-arabinosidase and Bacillus cellulosilyticus α-arabinofuranosidase, belonging to the glycosyl hydrolase family 43. The structural gene was subcloned with a C-terminal His-tag into a pET23a(+) expression vector. The His-tagged XylC, purified from a cell-free extract of a recombinant E. coil BL21(DE3) Codon Plus carrying a xylC gene by affinity chromatography, was active on paranitrophenyl-α-arabinofuranoside (pNPA) as well as paranitrophenyl-β-xylopyranoside (pNPX). However, the enzymatic activities for the substrates were somewhat incongruously influenced by reaction pHs and temperatures. The enzyme was also affected by various chemicals at different levels. SDS (5 mM) inhibited the enzymatic activity for pNPX, while enhancing the enzymatic activity for pNPA. Enzyme activity was also found to be inhibited by addition of pentose or hexose. The Michaelis constant and maximum velocity of the purified enzyme were determined for hydrolysis of pNPX and pNPA, respectively.

Preparation and Antioxidant Activities In Vitro of a Designed Antioxidant Peptide from Pinctada fucata by Recombinant Escherichia coli

( Yanyan Wu ),( Yongkai Ma ),( Laihao Li ),( Xianqing Yang ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.1

An antioxidant peptide derived from Pinctada fucata meat using an Alcalase2.4L enzymatic hydrolysis method (named AOP) and identified by LC-TOF-MS has promising clinical potential for generating cosmetic products that protect skin from sunshine. To date, there have been few published studies investigating the structure-activity relationship in these peptides. To prepare antioxidant peptides better and improve their stability, the design and expression of an antioxidant peptide from Pinctada fucata (named DSAOP) was studied. The peptide contains a common precursor of an expression vector containing an α-helix tandemly linked according to the BamHI restriction sites. The DNA fragments encoding DSAOP were synthesized and subcloned into the expression vector pET-30a (+), and the peptide was expressed mostly as soluble protein in recombinant Escherichia coli. Meanwhile, the DPPH radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging activity of DSAOP IC50 values were 0.136 ± 0.006, 0.625 ± 0.025, and 0.306 ± 0.015 mg/ml, respectively, with 2-fold higher DPPH radical scavenging activity compared with chemosynthesized AOP (p < 0.05), as well as higher superoxide radical scavenging activity compared with natural AOP (p < 0.05). This preparation method was at the international advanced level. Furthermore, pilot-scale production results showed that DSAOP was expressed successfully in fermenter cultures, which indicated that the design strategy and expression methods would be useful for obtaining substantial amounts of stable peptides at low costs. These results showed that DSAOP produced with recombinant Escherichia coli could be useful in cosmetic skin care products, health foods, and pharmaceuticals.

Production of 10-hydroxystearic acid from oleic acid by whole cells of recombinant Escherichia coli containing oleate hydratase from Stenotrophomonas maltophilia

Joo, Y.C.,Seo, E.S.,Kim, Y.S.,Kim, K.R.,Park, J.B.,Oh, D.K. Elsevier Science Publishers 2012 Journal of biotechnology Vol.158 No.1

A putative fatty acid hydratase from Stenotrophomonas maltophilia was cloned and expressed in Escherichia coli. The recombinant enzyme showed the highest hydration activity for oleic acid among the fatty acids tested, indicating that the enzyme is an oleate hydratase. The optimal conditions for the production of 10-hydroxystearic acid from oleic acid using whole cells of recombinant E. coli containing the oleate hydratase were pH 6.5, 35<SUP>o</SUP>C, 0.05% (w/v) Tween 40, 10gl<SUP>-1</SUP> cells, and 50gl<SUP>-1</SUP> oleic acid. Under these conditions, whole recombinant cells produced 49gl<SUP>-1</SUP> 10-hydroxystearic acid for 4h, with a conversion yield of 98% (w/w), a volumetric productivity of 12.3gl<SUP>-1</SUP>h<SUP>-1</SUP>, and a specific productivity of 1.23gg-cells<SUP>-1</SUP>h<SUP>-1</SUP>, which were 18%, 2.5-, and 2.5-fold higher than those of whole wild-type S. maltophilia cells, respectively. This is the first report of 10-hydroxystearic acid production using recombinant cells and the concentration and productivity are the highest reported thus far among cells.

Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli

Sundara Sekar, B.,Mohan Raj, S.,Seol, E.,Ainala, S.K.,Lee, J.,Park, S. Pergamon Press ; Elsevier Science Ltd 2014 INTERNATIONAL JOURNAL OF HYDROGEN ENERGY - Vol.39 No.28

Carbon monoxide (CO) is highly toxic but is an abundant carbon source that can be utilized for the production of hydrogen (H<SUB>2</SUB>). CO-dependent H<SUB>2</SUB> production is catalyzed by a unique enzyme complex composed of carbon monoxide dehydrogenase (CODH) and CO-dependent hydrogenase (CO-H<SUB>2</SUB>ase), both of which contain metal cluster(s). In this study, CODH and the required maturation proteins from the novel facultative anaerobic bacterium Citrobacter amalonaticus Y19 were cloned and heterologously expressed in Escherichia coli. For functional expression of CODH in E. coli, only CooF (ferredoxin-like protein) and CooS (CODH), not the maturation proteins, were needed. The recombinant E. coli BL21(DE3)-cooFS showed a 3.5-fold higher specific CODH activity (4.9 U mg protein<SUP>-1</SUP>) compared to C. amalonaticus Y19 (Y19) (1.4 U mg protein<SUP>-1</SUP>). Purified heterologous CODH from the soluble cell-free extract of the recombinant E. coli showed a specific activity of 170.6 U mg protein<SUP>-1</SUP>. Recombinant E. coli harboring Y19 CODH and maturation proteins did not produce H<SUB>2</SUB> from CO, suggesting that the native hydrogenases present in E. coli could not substitute the Y19 CO-H<SUB>2</SUB>ase for CO-dependent H<SUB>2</SUB> production.

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression

Chung, Jeong Min,Lee, Sangmin,Jung, Hyun Suk Elsevier 2017 Protein expression and purification Vol.133 No.-

<P><B>Abstract</B></P> <P>Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The expression and purification condition for truncated actin binding proteins was optimized from <I>Escherichia coli</I> strain. </LI> <LI> The treatment of sarkosyl-detergent increases the solubility of bacterial recombinant proteins. </LI> <LI> Minimizing concentration of sarkosyl was suggested for enhancing the production of soluble proteins. </LI> </UL> </P>

Temperature Management Strategy for Efficient Gene Expression in a Thermally Inducible Escherichia coli/Bacteriophage System

오정석,박희호,박태현 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.4

In a two-phase operation, E. coli containing λSNU1 (Q-S-) in the chromosome is typically cultured at 33℃ and cloned gene expression is induced by elevating the temperature. At least 40℃ is necessary for complete induction of cloned gene expression; however, temperatures above 40℃ have been shown to inhibit cloned gene expression. This suggests that a three-phase operation, which has an induction phase between the growth and production phases, may result in higher gene expression. In this study, optimal temperature management strategies were investigated for the three-phase operation of cloned gene expression in thermally inducible E. coli/bacteriophage systems. The optimal temperature for the induction phase was determined to be 40℃. When the temperature of the production stage was 33℃, the optimal time period for the induction phase at 40℃ was determined to be 60 min. In contrast, when the temperature of the production phase was 37℃, the optimal period for the induction phase at 40℃ was 20~30 min. When the three-phase temperature and temporal profile were set at a growth phase of 33℃, an induction phase at 40℃ for 30 min, and a production phase at 37℃, the highest level of cloned gene expression was achieved.