The Establishment of Plasmid Vectors for Producing Rhamnolipids in Escherichia coli

Young-Joon KO,Jeong-Seon KIM,Da-Hye KIM,Soo-Jin KIM 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Rhamnolipids are a class of biosurfactants which are composed of rhamnose and B-hydroxylated fatty acid chain. Rhamnolipids can be widely used in various fields such as food, biomedical engineering, and agriculture, etc. Pseudomonas aeruginosa is still well known as a producer of rhamnolipids, but this microorganism causes health concerns due to its pathogenicity. Therefore, Heterologous production of rhamnolipids using non-pathogenic strains has been studied to improve the production of rhamnolipids. RhlA, RhlB, and RhlC are key enzymes for biosynthesis of rhamnolipids in Pseudomonas aeruginosa. Here, we constructed plasmid vectors for producing rhamnolipids in E. coli by introducing codon optimized gene of rhamnolipids biosynthesis. These plasmid vectors can be applied to produce rhamnolipids in non-pathogenic E. coli. Furthermore, the heterologous expression of rhl genes will be a background system to increase the yield of rhamnolipids in E. coli by carrying out genetic, metabolic engineering tools.

Rhamnolipids 생성 미생물 고정 담체를 이용한 분리막 오염 제어에 관한 연구

박유찬 ( Yu-chan Park ),김민경 ( Min-kyung Kim ),조진우 ( Jin-woo Cho ) 한국폐기물자원순환학회(구 한국폐기물학회) 2021 한국폐기물자원순환학회 춘계학술발표논문집 Vol.2021 No.-

활성슬러지를 활용한 하수처리 방법은 용존 오염물질 처리에 있어 높은 효율을 보여 전 세계적으로 사용되어지고 있다. 그 중에서도 분리막과 활성슬러지 반응조를 결합한 MBR(분리막 생물반응조, Membrane bioreactor)은 고농도의 활성슬러지를 생물반응조 내부에 유지시킬 수 있을 뿐만 아니라 기존의 활성슬러지 공정과 다르게 중력식 침전지를 필요로 하지 않아 요구하는 시설부지가 적다는 장점이 있다. 이러한 장점에 힘입어 MBR은 우리나라뿐만 아니라 전 세계적인 하수처리 시장에서 그 존재감을 꾸준히 늘려 가고 있는 추세이다. 하지만 미생물이 분리막 표면에 생물막을 형성하여 발생하는 막 오염 현상이 불가피하여 성공적인 MBR 가동에 있어서 이러한 막 오염 현상에 대한 적절한 대처는 반드시 필요하다. 이러한 대처로서 생물학적 계면활성제의 일종인 Rhamnolipids를 MBR에 투입하였을 때 분리막 오염을 효과적으로 저감시킨 것을 확인한 연구가 보고되었다. Pseudomonas sp.에 의해 생성되는 Rhamnolipids는 Glycolipid의 한 종류로서 주로 Rhamnose moiety와 lipid moiety로 구성되어있다. Rhamnolipids는 생태독성이 적고 생분해가 되는 환경 친화적인 특징 때문에 요식업, 농업, 화장품업 등 여러 분야에서 사용되고 있다. 이러한 특징과 세정력 때문에 생물학적 반응조를 운용하는 MBR의 막오염 제어에 있어 Rhamnolipids의 투입은 적절한 효과를 보여주었다. 그러나 Rhamnolipids의 지속적인 사용은 낮은 경제성으로 인해 상업적으로 적용시키는 것이 제한적이다. 이에 따라 본 연구에서는 Rhamnolipids를 생성하는 특정 미생물을 담체에 고정시켜 MBR에 투입하는 방식을 제안하였다. 미생물을 담체에 고정시켜줌으로서 반응조 내부에서 지속적으로 Rhamnolipids를 생성시켜 주어서 Rhamnolipids를 계속해서 투입해 줄 필요성을 제거하였고, 담체를 통한 물리적인 막오염 제어 효과도 기대하였다. 본 연구에서는 랩규모 장치의 MBR을 설계하여 람노리피드를 생성하는 미생물을 고정시킨 담체의 주입 유무를 다르게 하여 비교함으로서 그에 따른 막오염 제어 가능성을 알아보았다.

생물계면활성제 람노리피드를 이용한 심해저 광업 잔재물 내 중금속 정화

김효진,김경련 대한환경공학회 2019 대한환경공학회지 Vol.41 No.10

Objectives : Rhamnolipid for the remediation of heavy metals in deep-sea mining tailings originated from manganese nodules was mainly focused in this research. As continued development depletes land-based metal resources, deep-sea natural resources such as manganese nodules collected from the Clarion-Clipperton Fracture Zone in the Pacific Ocean are inevitably drawing attention as useful alternative resources. The London protocol 1996 (LP) under International Maritime Organization (IMO) and the International Sea-bed Authority (ISA), however, will come up with the environmental guidelines of deep-sea mining tailings by early 2020s. Accordingly, there are urgently required to develop technologies that remediate heavy metals of deep-sea mining tailings. In this research, rhamnolipids which were known as biosurfactants was used to extracting agent for heavy metals in deep-sea mining tailings. Methods : Sample (20-63 μm, manganese nodules) made of 2% (w/v) liquid using ultrapure water were stirred and mixed with a rhamnolipid solution (0.05%, 0.1%, 0.2%, 0.3%) for various elapsed time, to evaluate the possibility of remediation of heavy metals in deep-sea mining tailings using rhamnolipid. Each mixture was analyzed for heavy metals eluted into the liquid after solid-liquid separation. Results and Discussion : Rhamnolipid was effective in removal of heavy metals and environmentally friendly. The extracted concentration of copper (Cu), zinc (Zn), nickel (Ni), and cadmium (Cd) were correspondingly enhanced with specific elapsed time and rhamnolipid’s concentrations, also. Conclusions : The results of this research should be used as basic data for the actual development of the remediation technology for deep-sea mining tailings using rhamnolipid in the future. 목적 : 본 연구는 생물계면활성제인 람노리피드(rhamnolipid)를 이용한 세척 처리에 의한 심해저 광업 잔재물 내중금속 정화 가능성을 평가하였다. 급격한 개발로 육상의 금속자원이 고갈됨에 따라, 대체자원으로 태평양의 클라리온-클립톤 구역(The Clarion-Clipperton Fracture Zone)의 망간 단괴 등 심해저 광물자원이 주목받고 있다. 하지만 국제해사기구(IMO) 산하 런던의정서(London Protocol 1996; LP)가 국제해저기구(ISA)와 심해저 광물자원 개발을위한 국제환경기준안을 2020년 초까지 수립할 예정이기 때문에, 심해저 광물자원 개발 시 발생하는 잔재물에 대한 정화(Remediation) 기술 또한 개발이 시급하다. Rhamnolipid는 생분해성을 가지는 친환경 생물계면활성제로, 토양 및 광산폐수에서 중금속 제거 효과를 입증한 바 있다. 본 연구에서는 rhamnolipid를 이용하여 심해저 광물자원내 중금속 정화 가능성을 평가하고, 경과시간 및 rhamnolipid 농도에 따른 다양한 조건에서의 중금속 용출 특성을평가하였다. 방법 : Rhamnolipid를 이용한 심해저 광물자원 내 중금속 정화 가능성을 평가하기 위하여, 초순수를 사용하여 2% (w/v)의 액상으로 만든 시료(20-63 μm, 망간 단괴)와 rhamnolipid용액(0.05%, 0.1%, 0.2%, 0.3% (w/v))을 다양한 경과시간 동안 교반・혼합하였다. 각각의 혼합물은 고상과 액상을 분리하여 액상에 용출된 중금속을 분석하였다. 결과 및 토의 : 연구 결과, 우선 처리대상물질로 선정한 구리, 아연, 니켈, 카드뮴에 대하여 용출된 중금속의 농도는경과시간 및 rhamnolipid 농도에 비례하여 증가하는 경향을 보였다. 결론 : 다양한 처리 조건에서 시료의 용출 특성을 기초로, 향후 rhamnolipid를 이용한 실제 심해저 광업 잔재물 정화 기술 개발을 위한 기초 자료로써 활용될 것이다.

Effect of Rhamnolipids on Degradation of Anthracene by Two Newly Isolated Strains, Sphingomonas sp. 12A and Pseudomonas sp. 12B

( Chang Zheng Cui ),( Chi Zeng ),( Xia Wan ),( Dong Chen ),( Jia Yao Zhang ),( Ping Shen ) 한국미생물 · 생명공학회 2008 Journal of microbiology and biotechnology Vol.18 No.1

Anthracene is a PAH that is not readily degraded, plus its degradation mechanism is still not clear. Thus, two strains of anthracene-degrading bacteria were isolated from longterm petroleum-polluted soil and identified as Sphingomonas sp. 12A and Pseudomonas sp. 12B by a 16S rRNA sequence analysis. To further enhance the anthracenedegrading ability of the two strains, the biosurfactants produced by Pseudomonas aeruginosa W₃ were used, which were characterized as rhamnolipids. It was found that these rhamnolipids dramatically increased the solubility of anthracene, and a reverse-phase HPLC assay showed that the anthracene degradation percentage after 18 days with Pseudomonas sp. 12B was significantly enhanced from 34% to 52%. Interestingly, their effect on the degradation by Sphingomonas sp. 12A was much less, from 35% to 39%. Further study revealed that Sphingomonas sp. 12A also degraded the rhamnolipids, which may have hampered the effect of the rhamnolipids on the anthracene degradation.

Effect of Biosurfactant Addition on the Biodegradation of Phenanthrene in Soil-water System

Kyung Hee Shin,Ju Yong Kim,Kyoung Woong Kim 대한환경공학회 2008 Environmental Engineering Research Vol.13 No.1

The extent of solubility enhancement by biosurfactant was examined at various pHs prior to the biodegradation experiments. The molar solubilization ratio (MSR) was calculated from the batch solubilization experiments and the highest MSR was detected at pH 5. The effect of the biosurfactant, rhamnolipids, on the phenanthrene mineralization in soil-water system was investigated. The strain 3Y was selected for the mineralization assay and large amounts of phenanthrene were degraded at neutral pH in soil-water system without the biosurfactant. The addition of 150 mg/L rhamnolipids showed no effect on mineralization of phenanthrene in soil-water system, and total mineralization rates after 6 weeks incubation at each pH showed no differences in presence and absence of rhamnolipids. Our result indicated that the toxic effect of rhamnolipids can disappear when soil particles exist, and also the enhanced solubility of phenanthrene does not work for mineralization enhancement in this soil-water system.

Metabolic Profiling and Biological Activities of Bioactive Compounds Produced by Pseudomonas sp. Strain ICTB-745 Isolated from Ladakh, India

( Kamal ),( Ahmed ),( Anver Basha Shaik ),( C. Ganesh Kumar ),( Poornima Mongolla ),( P. Usha Rani ),( K. V. S ),( Rama Krishna ),( Suman Kumar Mamidyala ),( Joveeta Joseph ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.1

In an ongoing survey of the bioactive potential of microorganisms from Ladakh, India, the culture medium of a bacterial strain of a new Pseudomonas sp., strain ICTB-745, isolated from an alkaline soil sample collected from Leh, Ladakh, India, was found to contain metabolites that exhibited broad-spectrum antimicrobial and biosurfactant activities. Bioactivity-guided purification resulted in the isolation of four bioactive compounds. Their chemical structures were elucidated by 1H and 13C NMR, 2D-NMR (HMBC, HSQC, 1H,1H-COSY, and DEPT- 135), FT-IR, and mass spectroscopic methods, and were identified as 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), rhamnolipid-1 (RL-1), and rhamnolipid-2 (RL-2). These metabolites exhibited various biological activities like antimicrobial and efficient cytotoxic potencies against different human tumor cell lines such as HeLa, HepG2, A549, and MDA MB 231. RL-1 and RL-2 exhibited a dose-dependent antifeedant activity against Spodoptera litura, producing about 82.06% and 73.66% antifeedant activity, whereas PCA showed a moderate antifeedant activity (63.67%) at 60 μg/cm2 area of castor leaf. Furthermore, PCA, RL-1, and RL-2 exhibited about 65%, 52%, and 47% mortality, respectively, against Rhyzopertha dominica at 20 μg/ml. This is the first report of rhamnolipids as antifeedant metabolites against Spodoptera litura and as insecticidal metabolites against Rhyzopertha dominica. The metabolites from Pseudomonas sp. strain ICTB-745 have interesting potential for use as a biopesticide in pest control programs.

Rhamnolipid Production in Batch and Fed-batch Fermentation Using Pseudomonas aeruginosa BYK-2 KCTC 18012P

Lee, Kyung-Mi,Hwang, Sun-Hee,Ha, Soon-Duck,Jang, Jae-Hyuk,Lim, Dong-Jung,Kong, Jai-Yul The Korean Society for Biotechnology and Bioengine 2004 Biotechnology and Bioprocess Engineering Vol.9 No.4

The optimization of culture conditions for the bacterium Pseudomonas aeruginosa BYK-2 KCTC 18012P, was performed to increase its rhamnolipid production. The optimum level for carbon, nitrogen sources, temperature and pH, for rhamnolipid production in a flask, were identified as 25 g/L fish oil, 0.01% (w/v) urea, 25 and pH 7.0, respectively. Optimum conditions for batch culture, using a 7-L jar fermentor, were 200 rpm of agitation speed and a 2.0 L/min aeration rate. Under the optimum conditions, on fish oil for 216 h, the final cell and rhamnolipid concentrations were 5.3 g/L and 17.0 g/L respectively. Fed-batch fermentation, with different feeding conditions, was carried out in order to increase, cell growth and rhamnolipid production by the Pseudomonas aeruginosa, BYK-2 KCTC 18012P. When 2.5 g of fish oil and 100 mL basal salts medium, containing 0.01 % (w/v) urea, were fed intermittently during the fermentation, the final cell and rhamnolipid concentrations at 264 h, were 6.1 and 22.7 g/L respectively. The fed-batch culture resulted in a 1.2-fold increase in the dry cell mass and a 1.3-fold increase in rhamnolipid production, compared to the production of the batch culture. The rhamnolipid production-substrate conversion factor (0.75 g/g) was higher than that of the batch culture (0.68 g/g).

The Establishment of Recombinant Vectors for Producing Fatty-acid Variable Rhamnolipids

Young-Joon KO,Yi-Seul KIM,Da-Hye KIM,Yu-Jin KIM,Soo-Jin KIM 한국생물공학회 2023 한국생물공학회 학술대회 Vol.2023 No.10

The Establishment of Non-pathogenic Rhamnolipids Producing Escherichia coli

Young-Joon KO,Jeong-Seun KIM,Da-Hye KIM,Raman JEGADEESH,Soo-Jin KIM 한국생물공학회 2022 한국생물공학회 학술대회 Vol.2022 No.4

분리막 오염 제어를 위한 Rhamnolipids 생성 미생물이 고정화된 코팅 비드에 관한 연구

박유찬(Yu Chan Park),조진우(Jin Woo Cho),김민경(Min Kyung Kim) 대한환경공학회 2021 대한환경공학회 학술발표논문집 Vol.2021 No.11