Cloning of Catechol 2,3-Dioxygenase Gene and Construction of a Stable Genetically Engineered Strain for Degrading Crude Oil

Xie, Yun; Yu, Feng; Wang, Qi; Gu, Xin; Wu-ling, Chen

doi:10.1007/s12088-013-0411-2

Cited by 26 publications

(10 citation statements)

References 13 publications

(8 reference statements)

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“…3). The enzyme C23O is a key enzyme in the degradation of aromatic rings intradiol and extradiol dioxygenase [7] and in the neutralization of petroleum-based compounds [4], thereby, breaking down the toxic compounds in the crude oil into harmless chemicals. The screening for the C23O gene in bacteria further authenticates their involvement in the degradation of crude oil.…”

Section: Discussionmentioning

confidence: 99%

“…In order to identify the transformants bearing the correct inserts from the selected clones, colony PCR was performed in 96 wells PCR plates using M13 primers [4]. Each 25 μl reaction mixture contained 0.5U Taq polymerase enzyme, 1X PCR buffer, 0.4 pmol primers and 0.2 mM of each dNTP.…”

Section: Identification Of True Transformantsmentioning

confidence: 99%

“…When crude oil typically gets into the soil they are absorbed by the microorganisms and broken down by enzymes into less toxic compounds, such as methyl catechol, which are further degraded into harmless chemicals by catechol 2, 3 dioxygenase. While catechol 2, 3 dioxygenase is not the only enzyme used by microorganisms to neutralize petroleum-based compounds, it is a key enzyme responsible for the biodegradation of aromatic hydrocarbons [4]. The different types of dioxygenases which catalyze the scission of aromatic hydrocarbons include catechol 1, 2dioxygenase (C12O), catechol 2, 3dioxygenase (C23O), protocatechuate 3, 4dioxygenase and protocatechuate 4, 5dioxygenase.…”

Section: Introductionmentioning

confidence: 99%

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Molecular Identification of Crude Oil-Degrading Bacteria and Screening for Catechol 2, 3 Dioxygenase (C23O) Gene

Olukunle

2020

BJI

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Aims: To identify crude oil-degrading bacteria isolated from polluted soils and waters and screen the presence of catechol 2, 3 dioxygenase (C23O) gene encoding oil-degradation in the strains with the highest degradative activity. Study Design: Laboratory-experimental design was used in this study. Place and Duration of Study: Crude oil polluted soils and waters were collected from Awoye, Mese and Oluwa villages in Ondo State, Nigeria and three different flow stations (Agbada-Aluu shell, Obite, and Bonny) in Rivers State, Nigeria. Methodology: The identities of the isolates were confirmed by extracting their total genomic DNA using standard DNA protocols while a portion of 16S bacterial gene of their DNA was amplified by polymerase chain reaction (PCR) using the primers E9F and U1510R and sequenced using Sanger method. Degenerate primers were used to isolate and identify the gene encoding C23O, responsible for the degradation of oil. Molecular cloning of the gene was done by transforming into Escherichia coli DHα. The correct inserts from the selected clones were performed by colony PCR. The isolated gene was sequenced with a Dye terminator sequencing kit and the product was analyzed with Prism DNA sequencer. Results: The results obtained from the conserved sequence of the 16S rRNA coupled with the nucleotide sequence revealed ten (10) crude oil-degrading bacteria, with CFfab 14 and CFfab 12 having the highest and lowest degrading activity of 78.92 ± 0.9 Unit/mL/h and 43.89 ± 1.3 Unit/mL/h on day 3 respectively. Conclusion: The gene C23O responsible for the production of catechol 2, 3 dioxygenase was isolated from strains CFfab 5, CFfab 14 and CFfab 15. The nucleotide base sequence of the gene was determined to be 238 bp. It is expected that in bioremediation, indigenous microorganisms from polluted environments should be screened for the possible existence of this unique gene sequence for effectiveness. Further studies could be conducted on the possibility of cloning this C23O gene into other bacteria for more efficiency and effectiveness in the bioremediation process.

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Section: Discussionmentioning

confidence: 99%

Section: Identification Of True Transformantsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular Identification of Crude Oil-Degrading Bacteria and Screening for Catechol 2, 3 Dioxygenase (C23O) Gene

Olukunle

2020

BJI

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“…The type of intermediates detected during fluoranthene degradation suggests that both ortho-and meta-cleavage pathways were active in the consortium (and postulated in Figure S2), which might be due to the presence of catechol dioxygenases genes in all the three strains. The previous studies have reported the presence of catechol dioxygenases genes in Mycobacterium, Pseudomonas and Burkholderia (Kang et al 2016;Xie et al 2014;Suzuki et al 2002;Lopez et al 2005;Cenci et al 1999;Juhasz et al 1997).…”

Section: Stoichiometry Profile Of Fluoranthene Degradationmentioning

confidence: 95%

Characterizing the bacterial consortium ASDF capable of catabolic degradation of fluoranthene and other mono- and poly-aromatic hydrocarbons

Vaidya¹,

Patel²,

Jain³

et al. 2020

3 Biotech

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In this study, a bacterial consortium ASDF was developed, capable of degrading fluoranthene (a non-alternant poly-aromatic hydrocarbon). It comprised of three bacterial strains: Pseudomonas sp. ASDF1, Burkholderia sp. ASDF2 and Mycobacterium sp. ASDF3 capable of degrading 100 mg/L of fluoranthene under experimentally defined and optimum conditions (37 °C, pH 7.0, 150 rpm) within 7 days. Consortium had metabolized fluoranthene as sole source of carbon and energy with maximum degradation rate of 0.52 mg/L/h and growth rate of 0.054/h. Fluoranthene degradation is an aerobic process, therefore with increasing the gyratory shaking from 50 to 150 rpm, degradation was concurrently enhanced by 7.1-fold. The synthetic surfactants SDS and CTAB had antagonistic effect on fluoranthene degradation (decreased up to 2.8-fold). The proficiency of consortium was assessed for its inherent ability to degrade seven other hydrocarbons both individually as well as in mixture. The degradation profile was studied using HPLC and the detection of two degraded intermediates (salicylic acid and derivatives of phthalic acid) suggested that fluoranthene degradation might have occurred via ortho-and meta-cleavage pathways. The competency of consortium was further validated through simulated microcosm studies, which showed 96% degradation of fluoranthene in soil ecosystem under the ambient conditions. Hence, the study suggested that the consortium ASDF has an inherent potential for its wide applicability in bioremediation of hydrocarbon-contaminated sites.

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“…BS3 was developed with insertion of xylE gene encoding for catechol 2,3-dioxygenase enzyme from Pseudomonas putida strain BNF1 responsible for biodegradation of hydrocarbons, which are aromatic in nature. This engineered strain expressed enzyme with broad substrate specificity, hence exhibiting a superior efficacy to degrade a variety of n-alkanes and other aromatic hydrocarbons when compared to its wild strain [ 94 ]. Engineered psychrophilic recombinant Antarctic Pseudoalteromonas haloplanktis TAC125 successfully expressed toluene-o-xylene monooxygenase (capable of degrading a wide range of aromatics) along with its inherent laccase-like protein to address the remediation of cold and marine xenobiotic loaded effluents [ 95 ].…”

Section: Bioengineering Of Microorganisms For Soil Health Restoration By Remediationmentioning

confidence: 99%

Bioengineered microbes for soil health restoration: present status and future

et al. 2021

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According to the United Nations Environment Programme (UNEP), soil health is declining over the decades and it has an adverse impact on human health and food security. Hence, soil health restoration is a need of the hour. It is known that microorganisms play a vital role in remediation of soil pollutants like heavy metals, pesticides, hydrocarbons, etc. However, the indigenous microbes have a limited capacity to degrade these pollutants and it will be a slow process. Genetically modified organisms (GMOs) can catalyze the degradation process as their altered metabolic pathways lead to hypersecretions of various biomolecules that favor the bioremediation process. This review provides an overview on the application of bioengineered microorganisms for the restoration of soil health by degradation of various pollutants. It also sheds light on the challenges of using GMOs in environmental application as their introduction may affect the normal microbial community in soil. Since soil health also refers to the potential of native organisms to survive, the possible changes in the native microbial community with the introduction of GMOs are also discussed. Finally, the future prospects of using bioengineered microorganisms in environmental engineering applications to make the soil fertile and healthy have been deciphered. With the alarming rates of soil health loss, the treatment of soil and soil health restoration need to be fastened to a greater pace and the combinatorial efforts unifying GMOs, plant growth-promoting rhizobacteria, and other soil amendments will provide an effective solution to soil heath restoration ten years ahead.

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Cloning of Catechol 2,3-Dioxygenase Gene and Construction of a Stable Genetically Engineered Strain for Degrading Crude Oil

Cited by 26 publications

References 13 publications

Molecular Identification of Crude Oil-Degrading Bacteria and Screening for Catechol 2, 3 Dioxygenase (C23O) Gene

Molecular Identification of Crude Oil-Degrading Bacteria and Screening for Catechol 2, 3 Dioxygenase (C23O) Gene

Characterizing the bacterial consortium ASDF capable of catabolic degradation of fluoranthene and other mono- and poly-aromatic hydrocarbons

Bioengineered microbes for soil health restoration: present status and future

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