Um kit de ferramentas para permitir a conversão de hidrocarbonetos em ambientes aquosos
Summary
A auto sustentável regulação sistema bacteriano para a remediação de contaminações de óleo foi desenhado usando o padrão partes do DNA intercambiáveis (BioBricks). Uma engenharia<em> E. coli</emEstirpe> foi usada para degradar a alcanos via β-oxidação em ambientes aquosos tóxicos. As enzimas respectivos de diferentes espécies mostraram atividade de degradação alcano. Além disso, um aumento da tolerância ao<em> N</em>-Hexano foi conseguida através da introdução de genes a partir de alcano-tolerantes bactérias.
Abstract
Este trabalho apresenta um conjunto de ferramentas que permite a conversão de alcanos por Escherichia coli e apresenta uma prova de princípio de sua aplicabilidade. O kit de ferramentas padrão consiste em várias peças intercambiáveis (BioBricks) 9 abordando a conversão de alcanos, a regulação da expressão gênica e de sobrevivência em tóxicos hidrocarbonetos ambientes ricos.
Um percurso de três passos para a degradação alcano foi implementada em E. coli para permitir a conversão de alcanos de média e de cadeia longa para as suas respectivas alcanois, e, finalmente, alkanals alcanóico-ácidos. Este último foi metabolizada pela via β-oxidação nativa. Para facilitar a oxidação de alcanos de cadeia média (C5-C13) e os cicloalcanos (C5-C8), quatro genes (alkB2, rubA3, rubA4 e Rubb) do sistema de alcano-hidroxilase a partir de Gordonia sp. TF6 8,21 foram transformados em E. coli. Para a conversão dealcanos de cadeia longa (C15-C36), o gene a partir de Lada Geobacillus thermodenitrificans foi implementada. Para os passos necessários adicionais do processo de degradação, ADH e ALDH (proveniente G. thermodenitrificans) foram introduzidos 10,11. A actividade foi medida por ensaios de células em repouso. Para cada passo de oxidação, a actividade da enzima foi observada.
Para optimizar a eficiência do processo, a expressão foi induzida somente em condições de glucose baixa: um promotor de substrato-regulado, pCaiF, foi usado. pCaiF está presente em E. coli K12 e regula a expressão dos genes envolvidos na degradação de fontes não-glicose de carbono.
A última parte do kit de ferramentas – visando a sobrevivência – foi implementado usando genes de tolerância de solventes, PhPFDα e β, ambos da Pyrococcus horikoshii OT3. Os solventes orgânicos podem induzir stress celular e diminuição da capacidade de sobrevivência por negativamente affecting enovelamento de proteínas. Como chaperones, PhPFDα e β melhorar o processo de dobragem de proteínas, por exemplo sob a presença de alcanos. A expressão destes genes conduziu a uma tolerância de hidrocarboneto melhorado mostrado por um aumento da taxa de crescimento (até 50%) na presença de 10% de n-hexano no meio de cultura foram observados.
Resumindo, os resultados indicam que o kit de ferramentas permite E. coli para converter e tolerar hidrocarbonetos em ambientes aquosos. Como tal, representa um primeiro passo para uma solução sustentável para o óleo-remediação utilizando uma abordagem de biologia sintética.
Introduction
Oil pollution is among the most serious causes of environmental contamination, and greatly affects ecosystems, businesses and communities 3. Solutions are for example required to battle the continuous oil pollution originating from the oil sands tailing waters in Alberta, Canada. During the process of oil extraction from oil sands, bitumen, a semi-solid oxidized form of oil, is removed using thermal recovery techniques that consume about 3.1 barrels of water per single barrel of oil 1. Oil contaminated process water, mainly originating from a local river, is stored in tailing ponds after bitumen extraction. A more effective recycling of process water in order to reduce the need for freshwater uptake is needed. To facilitate the bitumen extraction and to ensure that downstream sites meet water quality guidelines for the protection of aquatic ecosystems, process water treatments are rapidly evolving 3.
To treat pollution of organic compounds, bioremediation technologies employing microorganisms are presently encouraged 1. Alkanes are the most abundant family of hydrocarbons in crude oil, containing 5 to 40 carbon atoms per molecule 7, 21. Many bacteria are known to degrade alkanes of various lengths via sequential oxidation of the terminal methyl group forming first alcohols, then aldehydes and finally fatty acids 8. Within this iGEM project several enzymes from different organisms were expressed and characterized, and made available via the BioBrick standard and Registry of Standard Biological Parts.
The well-studied alkane hydroxylase system of Gordonia sp. TF6 facilitates the initial oxidation step of C5-C13 alkanes along with that of C5-C8 cycloalkanes using a minimum of four components: alkB2 (alkane 1-monooxygenase), rubA3, rubA4 (two rubredoxins) and RubB (rubredoxin reductase) 8, 21. Oxidation of long-chain alkanes (ranging from C15 up to C36) is reported to be performed by ladA, a flavoprotein alkane monooxygenase from Geobacillus thermodinitrificans NG-80-2 7, 15, 18, 22. LadA forms a catalytic complex with flavin mononucleotide (FMN) that utilizes atomic oxygen for oxidation. This results in the conversion of alkanes into the corresponding primary alkanol. The alcohols are further oxidized by alcohol and aldehyde dehydrogenases to fatty acids, which readily enter the β-oxidation pathway 7, 21. A zinc-independent alcohol dehydrogenase from the thermophillic bacterium Geobacillus thermoleovorans B23 oxidizes medium-chain alkanols into their respective alkanals, using NAD+ as a cofactor 10. Aldehyde dehydrogenase from the same bacterium is able to catalyze the NAD+-dependent final step in the medium-chain oxidation 11.
In order to reduce induction costs and to maintain optimal proliferation of the bacterial system, the promoter pCaiF from E.coli was characterized. This promoter can regulate expression of the hydrocarbon degradation pathway components, and is regulated by cAMP-Crp levels, which in turn depend on glucose levels 6. At high extracellular glucose concentrations in the environment the cellular cAMP (cyclic Adenosine Mononucleotide Phosphate) level was low through the inhibition of adenylyl cyclase as a side effect of PTS mediated glucose transport. Conversely, during limitation (low glucose concentrations) the cAMP level increased and Crp bound to cAMP forming the complex, cAMP-Crp, which bound pCaiF and activated transcription of the downstream components 6, 14.
Wildtype E. coli can only tolerate moderate concentrations of hydrocarbons. To complete the toolkit, tolerance to hydrocarbons had to be addressed. Several organic solvent-tolerant bacteria are known to survive in water-solvent two-phase systems 12. Molecular components known to increase tolerance are chaperones that facilitate the correct folding of proteins. The prefoldin system from Pyrococcus horikoshii OT3, consisting of the proteins phPFDα and phPFDβ, was shown to increase hydrocarbon-tolerance 17.
The alkane conversion toolkit was constructed following the BioBrick principle, which is documented at the Registry of Standard Biological Parts 9. BioBricks are plasmids containing a specific functional insert that is flanked by 4 predefined restriction sites. The BioBrick inserts can be extended flexibly, allowing the construction of biological systems with new functions.
Protocol
Representative Results
Discussion
O princípio biotijolo é utilizado para construir um chassis para a degradação dos alcanos e uma prova de princípio para os componentes individuais da caixa de ferramentas foi obtido. Os ensaios são propostas diversas para medir a in vivo e in vitro a actividade de enzimas da via de alcano degradantes. O trabalho apresentado demonstra com sucesso um número de métodos que podem ser usados para determinar as actividades enzimáticas e de expressão do organismo hospedeiro E. coli ap…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Os experimentos realizados neste vídeo-artigo foram desenvolvidos para a competição internacional Máquina Genetically Engineered 9. Os autores gostariam de agradecer aos membros da equipe iGEM Lucas Bergwerff, Pieter van TM Boheemen, Jelmer Cnossen, Hugo F. Cueto Rojas e Ramon van der Valk para a assistência na pesquisa. Agradecemos Han de Winde, Stefan de Kok e Esengül Yıldırım para discussões úteis e hospedagem desta pesquisa. Este trabalho foi financiado pela TU Delft University Departamento de Biotecnologia, a Delft Bioinformática laboratório, TU Delft Departamento de Bionanoscience, areias petrolíferas Leadership Initiative (OSLI), Stud studentenuitzendbureau, Iniciativa Holanda Genomics, Centro Kluyver, Biotechnologische Nederlandse Vereniging (Stichting Biotecnologia Nederland) , DSM, Geneart, Greiner Bio-One e Genencor.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| E. coli K12 | New England Biolabs | C2523H | |
| Octane | Fluka | 74822 | |
| Hexadecane | Fluka | 52209 | |
| octanol-1 | Fluka | 95446 | |
| dodecanol-1 | Sigma-Aldrich | 126799 | |
| Hexane | Sigma-Aldrich | 296090 | |
| NADH | Sigma | N4505 | |
| FMN | Sigma | F2253 | |
| MgSO4 | J.T. Baker Casno | 7487 889 | |
| Triton X-100 | Sigma-Aldrich | T8787 | |
| T4 ligase | New England Biolabs | M0202L | |
| Gas chromatograph | |||
| Cell disrupter | LA Biosystems | CD-019 | |
| Spectrophotometer | Amersham pharmacia | spec 2000 | |
| Plate reader | Tecan | Magellan v7.0 | |
| Incubator | Innova, 44 | ||
| BioBrick K398014: BBa_J23100-BBa_J61100-alkB2-BBa_J61100-rubA3-BBa_J61100-rubA4– BBa_J61100-rubB |
Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts | BBa_K398014 | Alkane Hydroxylase System Resistance: Chloramphenicol |
| BioBrick K398027: BBa_R0040-BBa_B0034-ladA | Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts | BBa_K398027 | ladA Protein Generator Resistance: Chloramphenicol |
| BioBrick K398018: BBa_J23100-BBa_J61101-ADH | Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts | BBa_K398018 | ADH generator Resistance: Chloramphenicol |
| BioBrick K398030: BBa_R0040-BBa_B0034-ALDH | Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts | BBa_K398030 | ALDH generator Resistance: Chloramphenicol |
| BioBrick K398326: pCaiF | Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts | BBa_K398326 | pCaiF promoter Resistance: Chloramphenicol |
| BioBrick K398331: pCaiF-BBa_B0032-BBa_I13401 | Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts | BBa_K398331 | pCaiF measurement device Resistance: Chloramphenicol |
| BioBrick K398406: BBa_J23002-BBa_J61107-phPFDα-BBa_J61107- | Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts | BBa_K398406 | Solvent tolerance cluster Resistance: Chloramphenicol |
References
- Allen, E. W. Process water treatment in Canada’s oil sands industry: I: Target pollutants and treatment objectives. J. Environ. Eng. Sci. 7, 123-138 (2008).
- Alon, U. . An Introduction to Systems Biology: Design Principles of Biological Circuits. , (2007).
- Center, O. P. . Understanding Oil Spills and Oil Spill Response. , (1999).
- Eichler, K., Buchet, A., Lemke, R., Kleber, H. P., Mandrand-Berthelot, M. A. Identification and characterization of the caiF gene encoding a potential transcriptional activator of carnitine metabolism in Escherichia coli. J. Bacteriol. 178, 1248-1257 (1995).
- Feng, L., Wang, W., Cheng, J., Ren, Y., Zhao, G., Gao, C., Tang, Y., Liu, X., Han, W., Peng, X., et al. Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir. Proc. Natl. Acad. Sci. U.S.A. 104 (13), 5602-5607 (2007).
- Fujii, T., Narikawa, T., Takeda, K., Kato, J. Biotransformation of various alkanes using the Escherichia coli expressing an alkane hydroxylase system from Gordonia sp. TF6. Biosci. Biotechnol. Biochem. 68 (10), 2171-2177 (2004).
- Kato, T., Miyanaga, A., Haruki, M., Imanaka, T., Morikawa, M., Gene Kanaya, S. cloning of an alcohol dehydrogenase from thermophilic alkane-degrading Bacillus thermoleovorans B23. J. Biosci. Bioeng. 91 (1), 100-102 (2001).
- Kato, T., Miyanaga, A., Kanaya, S., Morikawa, M. Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading Geobacillus thermoleovorans B23. Extremophiles. 14, 33-39 (2010).
- Kieboom, J., De Bont, J. a. M. . Bacterial Stress Responses. , (2000).
- Kotte, O., Zaugg, J. B., Heinemann, M. Bacterial adaptation through distributed sensing of metabolic fluxes. Mol Syst Biol. 6, 355 (2010).
- Kremling, A., Bettenbrock, K., Gilles, E. D. Analysis of global control of Escherichia coli carbohydrate uptake. BMC Syst. Biol. 1, (2007).
- Li, L., Liu, X., Yang, W., Xu, F., Wang, W., Feng, L., Bartlam, M., Wang, L., Rao, Z. Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase. J. Mol. Biol. 376 (2), 453-465 (2008).
- Lin, H. Y., Mathiszik, B., Xu, B., Enfors, S. O., Neubauer, P. Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli. Biotechnol. Bioeng. 73 (5), 347-357 (2001).
- Okochi, M., Kanie, K., Kurimoto, M., Yohda, M., Honda, H. Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance. Appl. Microbiol. Biotechnol. 79 (3), 443-449 (2008).
- Rehm, H. J., Reiff, I. Mechanisms and occurrence of microbial oxidation of long-chain alkanes. Adv. Biochem. Eng. / Biotechnol. 19, 175-215 (1981).
- Rojo, F. Degradation of alkanes by bacteria. Environ. Microbiol. 11 (10), 2477-2490 (2009).
- Van Beilen, J. B., Panke, S., Lucchini, S., Franchini, A. G., Rothlisberger, M., Witholt, B. Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes. Microbiology. 147, 1621-1630 (2001).
- Wang, L., Tang, Y., Wang, S., Liu, R. L., Liu, M. Z., Zhang, Y., Liang, F. L., Feng, L. Isolation and characterization of a novel thermophilic Bacillus strain degrading long-chain n-alkanes. Extremophiles. 10 (4), 347-356 (2006).
