Proteomic Characterization of the Outer Membrane Vesicle of <i>Pseudomonas putida</i> KT2440

Choi, Chi-Won,Park, Edmond Changkyun,Yun, Sung Ho,Lee, Sang-Yeop,Lee, Yeol Gyun,Hong, Yeonhee,Park, Kyeong Ryang,Kim, Sang-Hyun,Kim, Gun-Hwa,Kim, Seung Il American Chemical Society 2014 JOURNAL OF PROTEOME RESEARCH Vol.13 No.10

<P>Outer membrane vesicles (OMVs) are produced by various pathogenic Gram-negative bacteria such as <I>Escherichia coli</I>, <I>Pseudomonas aeruginosa</I>, and <I>Acinetobacter baumannii</I>. In this study, we isolated OMVs from a representative soil bacterium, <I>Pseudomonas putida</I> KT2440, which has a biodegradative activity toward various aromatic compounds. Proteomic analysis identified the outer membrane proteins (OMPs) OprC, OprD, OprE, OprF, OprH, OprG, and OprW as major components of the OMV of <I>P. putida</I> KT2440. The production of OMVs was dependent on the nutrient availability in the culture media, and the up- or down-regulation of specific OMPs was observed according to the culture conditions. In particular, porins (e.g., benzoate-specific porin, BenF-like porin) and enzymes (e.g., catechol 1,2-dioxygenase, benzoate dioxygenase) for benzoate degradation were uniquely found in OMVs prepared from <I>P. putida</I> KT2440 that were cultured in media containing benzoate as the energy source. OMVs of <I>P. putida</I> KT2440 showed low pathological activity toward cultured cells that originated from human lung cells, which suggests their potential as adjuvants or OMV vaccine carriers. Our results suggest that the protein composition of the OMVs of <I>P. putida</I> KT2440 reflects the characteristics of the total proteome of <I>P. putida</I> KT2440.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2014/jprobs.2014.13.issue-10/pr500411d/production/images/medium/pr-2014-00411d_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr500411d'>ACS Electronic Supporting Info</A></P>

Markerless gene knockout and integration to express heterologous biosynthetic gene clusters in <i>Pseudomonas putida</i>

Choi, Kyeong Rok,Cho, Jae Sung,Cho, In Jin,Park, Dahyeon,Lee, Sang Yup Academic Press 2018 Metabolic engineering Vol.47 No.-

<P><B>Abstract</B></P> <P> <I>Pseudomonas putida</I> has gained much interest among metabolic engineers as a workhorse for producing valuable natural products. While a few gene knockout tools for <I>P. putida</I> have been reported, integration of heterologous genes into the chromosome of <I>P. putida</I>, an essential strategy to develop stable industrial strains producing heterologous bioproducts, requires development of a more efficient method. Current methods rely on time-consuming homologous recombination techniques and transposon-mediated random insertions. Here we report a RecET recombineering system for markerless integration of heterologous genes into the <I>P. putida</I> chromosome. The efficiency and capacity of the recombineering system were first demonstrated by knocking out various genetic loci on the <I>P. putida</I> chromosome with knockout lengths widely spanning 0.6–101.7 kb. The RecET recombineering system developed here allowed successful integration of biosynthetic gene clusters for four proof-of-concept bioproducts, including protein, polyketide, isoprenoid, and amino acid derivative, into the target genetic locus of <I>P. putida</I> chromosome. The markerless recombineering system was completed by combining Cre/<I>lox</I> system and developing efficient plasmid curing systems, generating final strains free of antibiotic markers and plasmids. This markerless recombineering system for efficient gene knockout and integration will expedite metabolic engineering of <I>P. putida</I>, a bacterial host strain of increasing academic and industrial interest.</P> <P><B>Highlights</B></P> <P> <UL> <LI> RecET system for markerless recombineering of <I>P. putida</I> was constructed. </LI> <LI> Curing systems for RecET and Cre vectors were developed. </LI> <LI> Large region of chromosome up to 101.7 kb could be deleted. </LI> <LI> A single-step recombineering system was developed using a donor plasmid. </LI> <LI> Violacein biosynthetic gene cluster (7.4 kb) could be markerlessly integrated. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Pseudomonas putida BJ10의 Tetrachloroethylene (PCE) 분해 특성

최명훈,김재수,이상섭,Choi, Myung-Hoon,Kim, Jai-Soo,Lee, Sang-Seob 한국미생물학회 2008 미생물학회지 Vol.44 No.4

BTEX 분해능을 가진 BJ10세균을 이용하여 호기조건에서 toluene 첨가 시 tetrachloroethylene (PCE) 분해에 관한 연구를 수행하였다. BJ10은 형태학적 특징, 생리 생화학적 특징, 16S rRNA 염기서열 분석 및 지방산 분석 결과에 따라 Pseudomonas putida로 동정되었다. BJ10의 PCE 저농도 5 mg/L에서 PCE 분해 실험 결과(toluene 첨가 기질 농도 50mg/L, 균초기 접종농도 1.0g/L, 온도 $30^{\circ}C$, pH7 그리고 DO $3.0{\sim}4.2\;mg/L$), 10일간 52.8%의 분해 효율을 보였으며, PCE 분해 속도는 5.9 nmol/hr로 나타났다. 또한 BJ10의 PCE 고농도 100 mg/L에서 PCE 분해 실험 결과 (toluene 첨가 기질 농도 50 mg/L, 균 초기 접종 농도 1.0 g/L, 온도 $30^{\circ}C$, pH 7 그리고 DO $3.0{\sim}4.2\;mg/L$), 10일간 20.3%의 분해 효율을 보였으며, PCE 분해 속도는 46.0 nmol/hr로 나타났다. Toluene 첨가 농도에 따른 PCE 분해 효율 증감 효과를 알아보기 위하여, 동일한 배양 조건하에 10 mg/L의 PCE에 toluene ($5{\sim}200\;mg/L$)을 첨가하여 분해 실험을 실시한 결과, toluene 200 mg/L 첨가시 10일간 57.0%의 PCE가 분해되어 가장 높은 제거 효율을 보였다. 또한 PCE 5.5 mg/L(총 7.6 mg/L)를 추가적으로 주입하여 동일조건하에서 PCE 분해를 확인하였으며 결과적으로 8일 동안 63.0%의 PCE가 분해되었다. 이 때의 PCE 분해 속도는 13.5 nmol/hr로 초기의 분해속도(8.1 nmol/hr)보다 증가되었다. In this study, biological PCE degradation by using a BTEX degrading bacterium, named BJ10, under aerobic conditions in the presence of toluene was examined. According to morphological, physiological characteristics, 16S rDNA sequencing and fatty acid analysis, BJ10 was classified as Pseudomonas putida. As a result of biological PCE degradation at low PCE concentrations (5 mg/L), PCE removal efficiency by P. putida BJ10 was 52.8% for 10 days, and PCE removal rate was 5.9 nmol/hr (toluene concentration 50 mg/L, initial cell density 1.0 g (wet weight)/L, temperature 30, pH 7 and DO $3.0{\sim}4.2\;mg/L$. At high PCE concentration (100 mg/L), PCE removal efficiency by P. putida BJ10 was 20.3% for 10 days, and PCE removal rate was 46.0 nmol/hr under the same conditions. The effects of various toluene concentration (5, 25, 50, 100, 200 mg/L) on PCE degradation were examined under the same incubation conditions. The highest PCE removal efficiency of PCE was 57.0% in the initial PCE concentration of 10 mg/L in the presence of 200 mg/L toluene for 10 days. Furthermore, the additional injection of 5.5 mg/L PCE (total 7.6 mg/L) made 63.0% degradation for 8 days in the presence of 50 mg/L toluene under the same conditions. Its removal rate was 13.5 nmol/hr, which was better than the initial removal rate (8.1 nmol/hr).

Synergistic effects of biogenic manganese oxide and Mn(II)-oxidizing bacterium <i>Pseudomonas putida</i> strain MnB1 on the degradation of 17 α-ethinylestradiol

Tran, Thi Nhung,Kim, Do-Gun,Ko, Seok-Oh Elsevier 2018 Journal of hazardous materials Vol.344 No.-

<P><B>Abstract</B></P> <P>While biogenic manganese oxide (BMO) generated via the oxidation of Mn(II) by the Mn-oxidizing bacteria (MOB) have received attention, the relative roles of biological activity by MOB themselves were not clearly investigated. In this study, the synergistic effects of BMO and MOB <I>Pseudomonas putida</I> strain MnB1 on the degradation of 17α-ethinylestradiol (EE2) was investigated. Experiments with BMO in the presence of <I>P. putida</I> MnB1 showed 15-fold higher removal than that with BMO alone, suggesting that EE2 degradation was mediated by the biological activity of MOB as well as abiotic reaction by BMO. Trapping experiments with pyrophosphate (PP) proved that Mn(III) intermediate formed during the biological process from Mn (II) to Mn (IV) contribute much to the EE2 removal. Also, sharp decreases in EE2 removal were observed when microbial activity was inactivated by heat treatment or sodium azide. From this study, the EE2 removal mechanisms by BMO in the presence <I>P. putida</I> MnB1 are described as follows: (1) abiotic oxidation of EE2 by BMO occurs. (2) <I>P. putida</I> MnB1 indirectly oxidizes EE2 by transferring electrons from the Mn (III) intermediate. (3) <I>P. putida</I> MnB1 continuously re-oxidizes the Mn(II) released from the oxidative degradation of EE2 by BMO, generating new Mn(III)-intermediates or BMO.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The presence of <I>P. putida</I> MnB1 significantly enhanced the removal of EE2 by BMO. </LI> <LI> EE2 degradation was mediated by the biological activity of MOB as well as abiotic reaction by BMO. </LI> <LI> A linear correlation was established between biological Mn(III)-intermediates and EE2 removal rate. </LI> <LI> EE2 oxidation by BMO and <I>P. putida</I> MnB1 involves the reduction of Mn(III)-enzyme complexes and BMO to Mn(II). </LI> <LI> The Mn(II) from Mn(III)-enzyme complexes and BMO is re-oxidized by <I>P. putida</I> MnB1, providing additional EE2 oxidation. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Engineering <i>Pseudomonas putida</i> KT2440 to convert 2,3-butanediol to mevalonate

Yang, Jeongmo,Im, Yeongeun,Kim, Tae Hwan,Lee, Myeong Jun,Cho, Sukhyeong,Na, Jeong-geol,Lee, Jinwon,Oh, Byung-keun Elsevier 2020 Enzyme and microbial technology Vol.132 No.-

<P><B>Abstract</B></P> <P>Biological production of 2,3-butanediol (2,3-BDO), a C4 platform chemical, has been studied recently, but the high cost of separation and purification before chemical conversion is substantial. To overcome this obstacle, we have conducted a study to convert 2,3-BDO to mevalonate, a terpenoid intermediate, using recombinant <I>Pseudomonas putida</I> and this biological process won’t need the separation and purification process of 2,3-BDO. The production of mevalonate when 2,3-BDO was used as a substrate was 6.61 and 8.44 times higher than when glucose and glycerol were used as substrates under the same conditions, respectively. Lower aeration contributed to higher yields of mevalonate in otherwise identical conditions. The maximum mevalonate production on the shaking flask scale was about 2.21 g/L, in this study (product yield was 0.295, 27% of theoretical yield (1.10)). This study was the first successful attempt for mevalonate production by <I>P. putida</I> using 2,3-BDO as the sole carbon source and presented a new metabolic engineering tool and biological process for mevalonate synthesis.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Pseudomonas putida</I> KT2440 can metabolize 2,3-butanediol as a sole carbon source. </LI> <LI> 2,3-butandiol was converted to mevalonate by engineered <I>P. putida</I> KT2440 successfully. </LI> <LI> <I>atoB</I> gene expression and aeration optimization enhanced the mevalonate production. </LI> </UL> </P>

Effect of alkalinity on the removal of nutrients in the co-culture system of Chlorella vulgaris and Pseudomonas putida under light/dark condition

( Ghulam Mujtaba ),이기세 한국공업화학회 2014 한국공업화학회 연구논문 초록집 Vol.2014 No.1

The co-culture system of microalgae and bacteria is an alternative biosystem for the wastewater treatment. Microorganisms used were the freshwater green microalga Chlorella vulgaris and a bacterium Pseudomonas putida. Experiments were performed in 250ml-Erlenmeyer flasks with 200ml culture volume using synthetic wastewater. Removal of nutrients was checked in the absence or presence of alkalinity (NaHCO₃) under light and dark conditions. Light is important for the removal of nitrogen and phosphorous in the presence of NaHCO₃. While, dark condition is more useful for the removal of COD. Co-culture system performed well in order to remove nutrients. Growth of C. vulgaris was better in light with NaHCO₃.

Removal of nutrients and COD from wastewater using symbiotic co-culture of bacterium Pseudomonas putida and immobilized microalga Chlorella vulgaris

Mujtaba, G.,Rizwan, M.,Lee, K. Korean Society of Industrial and Engineering Chemi 2017 Journal of industrial and engineering chemistry Vol.49 No.-

<P>Simultaneous removal of nutrients (ammonium and phosphate) and COD was investigated by the co-culture consortium of microalga Chlorella vulgaris and bacterium Pseudomonas putida. The co-culture system showed higher removal of both nutrients and COD than the each axenic culture, indicating that nutrients uptake capability of C vulgaris was enhanced in the presence of P. putida. The best performance in the removal of nitrogen, phosphorus, and COD was obtained through the co-culture with suspended P. putida and immobilized C vulgaris, demonstrating that the employment of immobilization of one species is more synergistic than suspended co-culture system in nutrients removal from wastewater. (C) 2017 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.</P>

Gene Expression Systems in Pseudomonas putida: an Alternative Microbial Cell Factory for Production of Platform Chemicals

Sung Kuk LEE 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Recent concerns over sustainability of our existing petroleum-based economy have raised interest in harnessing microbial organisms to produce chemicals from renewable biomasses. This microbial conversion is hampered by the difficulty in designing and constructing effective synthetic metabolic pathways and inhibitory effect of substrates and products. The Pseudomonas putida KT2440 strain recognized as safe microbial host for recombinant DNA constructs, has become a next-generation-synthetic-biology chassis or industrial workhorse with its metabolic versatility and applicability. The strain exhibits excellent properties, including a remarkable tolerance to oxidative stress, organic solvents, and aromatic compounds; no or reduced byproduct formation; and a high yield of NADPH through the Entner-Doudoroff (ED) pathway. Our Lab has been working to develop elemental technologies for a new synthetic platform Pseudomonas strain for biorefinery applications like 1) coconsumption of biomass derived substrates like glucose, xylose, cellobiose or levulinic acid 2) metabolic engineering of central metabolic pathways, 3) metabolic engineering of target biosynthesis pathways and 4) development of substrate or product based gene expression tools. Inducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter systems were developed and further evaluated in Pseudomonas putida KT2440 by analyzing the expression of green fluorescent protein (GFP) as a reporter protein. For application, metabolic engineered P. putida strain was developed to produce 4-hydroxyvalerate (4HV), a versatile chemical used for the synthesis of various commodities and fine chemicals. The final engineered strain produced a maximum of 50 g/L 4HV with 97% conversion from levulinic acid. For the future studies, these technologies will be merged into biorefineries that produce valuable compounds [3-hyroxypropionate, dicarboxylic acids, 2,3-butanediol, fatty acids].

Biochemical characterization of ferredoxin-NADP+ reductase interaction with flavodoxin in Pseudomonas putida

( Jin Ki Yeom ),( Woo Jun Park ) 생화학분자생물학회 2012 BMB Reports Vol.45 No.8

Flavodoxin (Fld) has been demonstrated to bind to ferredoxin- NADP+ reductase A (FprA) in Pseudomonas putida. Two residues (Phe256, Lys259) of FprA are likely to be important for interacting with Fld based on homology modeling. Sitedirected mutagenesis and pH-dependent enzyme kinetics were performed to further examine the role of these residues. The catalytic efficiencies of FprA-Ala259 and FprA-Asp259 proteins were two-fold lower than those of the wild-type FprA. Homology modeling also strongly suggested that these two residues are important for electron transfer. Thermodynamic properties such as entropy, enthalpy, and heat capacity changes of FprA-Ala259 and FprA-Asp259 were examined by isothermal titration calorimetry. We demonstrated, for the first time, that Phe256 and Lys259 are critical residues for the interaction between FprA and Fld. Van der Waals interactions and hydrogen bonding were also more important than ionic interactions for forming the FprA-Fld complex. [BMB Reports 2012; 45(8): 476-481]

Pseudomonas putida를 고정화시킨 생물활성탄의 유기물 및 영양염류 분해에 대한 온도의 영향

박형진(Hyungjin Park),김영기(Young-Kee Kim) 한국생물공학회 2020 KSBB Journal Vol.35 No.1

In this study, we aim to evaluate the degrading performance of organics and nutrients by a biological activated carbon (BAC), which was manufactured by immobilizing Pseudomonas putida having ability to degrade aromatic carbon compound on activated carbon, a material to use remediation of organic pollutants contaminated lake sediment. A culture with BAC showed higher degrading performance (93.1%, 54.4%, and 95.5% for removal of organics, total nitrogen, and total phosphorus, respectively) compared to those of suspended culture with only P. putida (68.0%, 33.9%, and 24.1% for removal of organics, total nitrogen, and total phosphorus, respectively). In order to show application ability for real domestic lake environment, culture experiments to remove organic compounds and nutrients were performed at temperature of 10, 18, and 26oC, respectively. Effective removal performance of organics and nutrients was confirmed at a culture temperature of 18oC and over. Also, feasibility of BAC to remediate persistent organics contaminated sediment was confirmed by the result of phenol degrading culture experiment (94.3% phenol removal after 12 h).