Biodecomposition of Phenanthrene and Pyrene by a Genetically Engineered Escherichia coli

Background: Genetically engineered microorganisms (GEMs) can be used for bioremediation of the biological pollutants into nonhazardous or less-hazardous substances, at lower cost. Polycyclic aromatic hydrocarbons (PAHs) are one of these contaminants that associated with a risk of human cancer development. Genetically engineered E. coli that encoded catechol 2,3- dioxygenase (C230) was created and investigated its ability to biodecomposition of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) measurement. We revised patents documents relating to the use of GEMs for bioremediation. This approach have already been done in others studies although using other genes codifying for same catechol degradation approach. Objective: In this study, we investigated biodecomposition of phenanthrene and pyrene by a genetically engineered Escherichia coli. Methods: Briefly, following the cloning of C230 gene (nahH) into pUC18 vector and transformation into E. coli Top10F, the complementary tests, including catalase, oxidase and PCR were used as on isolated bacteria from spiked soil. Results: The results of HPLC measurement showed that in spiked soil containing engineered E. coli, biodegradation of phenanthrene and pyrene comparing to autoclaved soil that inoculated by wild type of E. coli and normal soil group with natural microbial flora, were statistically significant (p<0.05). Moreover, catalase test was positive while the oxidase tests were negative. Conclusion: These findings indicated that genetically manipulated E. coli can provide an effective clean-up process on PAH compounds and it is useful for bioremediation of environmental pollution with petrochemical products.

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Degradation of phenanthrene and pyrene using genetically engineered dioxygenase producing Pseudomonas putida in soil

Genetika ◽

10.2298/gensr1603837m ◽

2016 ◽

Vol 48 (3) ◽

pp. 837-858 ◽

Cited By ~ 2

Author(s):

Gashtasb Mardani ◽

Amir Mahvi ◽

Morteza Hashemzadeh-Chaleshtori ◽

Simin Nasseri ◽

Mohammad Dehghani ◽

...

Keyword(s):

High Performance ◽

Human Cancer ◽

Hplc Method ◽

Genetically Engineered ◽

Hazardous Substances ◽

Natural Soil ◽

Spiked Soil ◽

Polycyclic Aromatic ◽

Hplc Measurement ◽

Normal Microbial Flora

Bioremediation use to promote degradation and/or removal of contaminants into nonhazardous or less-hazardous substances from the environment using microbial metabolic ability. Pseudomonas spp. is one of saprotrophic soil bacterium and can be used for biodegradation of polycyclic aromatic hydrocarbons (PAHs) but this activity in most species is weak. Phenanthrene and pyrene could associate with a risk of human cancer development in exposed individuals. The aim of the present study was application of genetically engineered P. putida that produce dioxygenase for degradation of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) method. The nahH gene that encoded catechol 2,3-dioxygenase (C23O) was cloned into pUC18 and pUC18-nahH recombinant vector was generated and transformed into wild P. putida, successfully. The genetically modified and wild types of P. putida were inoculated in soil and pilot plan was prepared. Finally, degradation of phenanthrene and pyrene by this bacterium in spiked soil were evaluated using HPLC measurement technique. The results were showed elimination of these PAH compounds in spiked soil by engineered P. putida comparing to dishes containing natural soil with normal microbial flora and inoculated autoclaved soil by wild type of P. putida were statistically significant (p<0.05). Although adding N and P chemical nutrients on degradation ability of phenanthrene and pyrene by engineered P. putida in soil were not statistically significant (p>0.05) but it was few impact on this process (more than 2%). Additional and verification tests including catalase, oxidase and PCR on isolated bacteria from spiked soil were indicated that engineered P. putida was alive and functional as well as it can affect on phenanthrene and pyrene degradation via nahH gene producing. These findings indicated that genetically engineered P. putida generated in this work via producing C23O enzyme can useful and practical for biodegradation of phenanthrene and pyrene as well as petroleum compounds in polluted environments.

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Optimizing a production strategy for a nonspecific nuclease from Yersinia enterocolitica subsp. palearctica in genetically engineered Escherichia coli

FEMS Microbiology Letters ◽

10.1093/femsle/fnz208 ◽

2019 ◽

Vol 366 (24) ◽

Author(s):

Yan Ge ◽

Senlin Guo ◽

Tao Liu ◽

Chen Zhao ◽

Duanhua Li ◽

...

Keyword(s):

Escherichia Coli ◽

Yersinia Enterocolitica ◽

Inclusion Bodies ◽

Genetically Engineered ◽

Biological Research ◽

Dilution Method ◽

E Coli ◽

Protein Renaturation ◽

Engineered Escherichia Coli ◽

Pharmaceutical Production

ABSTRACT A nuclease from Yersinia enterocolitica subsp. palearctica (Nucyep) is a newly found thermostable nonspecific nuclease. The heat-resisting ability of this nuclease would be extremely useful in biological research or pharmaceutical production. However, the application of this nuclease is limited because of its poor yield. This research aimed to improve Nucyep productivity by producing a novel genetically engineered Escherichia coli and optimizing the production procedures. After 4 h of induction by lactose, the new genetically engineered E. coli can express a substantial amount of Nucyep in the form of inclusion bodies. The yield was approximately 0.3 g of inclusion bodies in 1 g of bacterial pellets. The inclusion bodies were extracted by sonication and solubilized in an 8 M urea buffer. Protein renaturation was successfully achieved by dilution method. Pure enzyme was obtained after subjecting the protein solution to anion exchange. The Nucyep showed its nonspecific and heat resistant properties as previously reported (Boissinot et al. 2016). Through a quantification method, its activity was determined to be 1.3 × 10 6 Kunitz units (K.U.)/mg. These results can serve as a reference for increasing Nucyep production.

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Fed-Batch Cultivation and Adding Supplements to Increase Yield of β-1,3-1,4-Glucanase by Genetically Engineered Escherichia coli

Catalysts ◽

10.3390/catal11020269 ◽

2021 ◽

Vol 11 (2) ◽

pp. 269

Author(s):

Lijuan Zhong ◽

Zheng Liu ◽

Yinghua Lu

Keyword(s):

Escherichia Coli ◽

Feeding Strategy ◽

Batch Cultivation ◽

Genetically Engineered ◽

Fed Batch ◽

Glucanase Activity ◽

E Coli ◽

Constant Rate ◽

Engineered Escherichia Coli ◽

Fed Batch Cultivation

The aim of this study was to analyze the major influence factors of culture medium on the expression level of β-1,3-1,4-glucanase, and to further develop an optimized process for the extracellular production of β-glucanase at a bioreactor scale (7 L) with a genetically engineered Escherichia coli (E. coli) JM109-pLF3. In this study, batch cultivation and fed-batch cultivation including the constant rate feeding strategy and the DO-stat (DO: Dissolved Oxygen) feeding strategy were conducted. At a 7 L bioreactor scale for batch cultivation, biomass reached 3.14 g/L and the maximum β-glucanase activity was 506.94 U/mL. Compared with batch cultivation, the addition of glycerol, complex nitrogen and complete medium during fed-batch cultivation increased the production of biomass and β-1,3-1,4-glucanase. The maximum biomass and β-glucanase activity, which were 7.67 g/L and 1680 U/mL, respectively, that is, 2.45 and 3.31 times higher than those obtained with batch cultivation, were obtained by feeding a complex nitrogen source at a constant rate of 1.11 mL/min. Therefore, these nutritional supplements and strategies can be used as a reference to enhance the production of other bioproducts from E. coli.

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Biosynthesis of morin-3-O-rhamnopyranoside in a genetically engineered Escherichia coli

Vietnam Journal of Biotechnology ◽

10.15625/1811-4989/15/1/12331 ◽

2018 ◽

Vol 15 (1) ◽

pp. 161-168

Author(s):

Nguyễn Huy Thuần ◽

Nguyễn Thành Trung ◽

Đồng Văn Quyền ◽

Vũ Thị Thu Hằng ◽

Jae Kyung Sohng

Keyword(s):

Escherichia Coli ◽

Liquid Chromatography ◽

High Performance ◽

Extraction Methods ◽

Biosynthetic Gene Cluster ◽

Culture Broth ◽

Genetically Engineered ◽

E Coli ◽

Biotransformation Process ◽

Tandem Mass Spectrometric

Flavonoids are significant secondary metabolites of vascular plants. They have been proven to possess numerous types of anti-bacterial, anti-tumor, anti-oxidant and anti-inflammatory bioactivities etc. Thereby, there are many flavonoids extracted and purified from plants and have been used for biological tests. Nevertheless, the traditional extraction methods require a large amount of initial sample, tedious and professional techniques and low yield of target compound. Until present, the advanced development of genetic engineerings and recombinant protein as platform allow synthesis of high amount of different types of flavoid in short time. Escherichia coli is one of the most popular host for whole-cell biotransformation of flavonoids and their derivatives due to its simple genetical, physiological system and fast growth. In this paper, we described the biosynthetic method of morin-3-O-rhamnopyranoside from a genetically engineered E. coli using morin as substrate. In particular, the E. coli harboring biosynthetic gene cluster of activated TDP-L-rhamnose and gene encoding for glycosyltransferase was used as host for biotransformation. Morin was fed into the culture broth of recombinant E. coli for rhamnosylation. The formation of morin-3-O-rhamnopyranoside was then quantitied and qualified using Thin Layer Chromatography (TLC), Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC-ESI-MS/MS) assays. The highest yield of product (56.5 µM) was achieved after 48h, 32 oC and pH = 7.2-8.0. This result showed that morin-3-O-rhamnopyranoside was succesfully synthesized in the genetically engineered E. coli. Furthermore, metabolic engineering of intra-cellular system for improving absorption of substrate as well as excretion of product or enhancement of glycosyltransferase activity may increase the final yield of biotransformation process.

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Escherichia coli as a genetic tool

Journal of Hygiene ◽

10.1017/s0022172400060708 ◽

1985 ◽

Vol 95 (3) ◽

pp. 611-618

Author(s):

Naomi Datta

Keyword(s):

Escherichia Coli ◽

Academic Research ◽

Genetically Engineered ◽

Cloning And Expression ◽

Biological Research ◽

New Techniques ◽

E Coli ◽

Genetic Tool ◽

The Past ◽

Made In

SUMMARYThe study of Escherichia coli and its plasmids and bacteriophages has provided a vast body of genetical information, much of it relevant to the whole of biology. This was true even before the development of the new techniques, for cloning and analysing DNA, that have revolutionized biological research during the past decade. Thousands of millions of dollars are now invested in industrial uses of these techniques, which all depend on discoveries made in the course of academic research on E. coli. Much of the background of knowledge necessary for the cloning and expression of genetically engineered information, as well as the techniques themselves, came from work with this organism.

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