Summary

Un juego de herramientas para la conversión de hidrocarburos en ambientes acuosos

Published: October 02, 2012
doi:

Summary

Un auto sostenible sistema de regulación de las bacterias para la remediación de contaminaciones de aceite ha sido diseñado utilizando piezas estándar de ADN intercambiables (BioBricks). Una ingeniería<em> E. coli</emCepa> se utiliza para degradar alcanos a través de β-oxidación en ambientes acuosos tóxicos. Las respectivas enzimas de diferentes especies mostraron actividad de degradación de alcano. Además, una mayor tolerancia a<em> N</em>-Hexano se obtuvo mediante la introducción de genes de alcano bacterias tolerantes.

Abstract

Este trabajo expone un conjunto de herramientas que permite la conversión de alcanos por Escherichia coli y presenta una prueba de principio de su aplicabilidad. El juego de herramientas se compone de varias piezas estándar intercambiables (BioBricks) 9 abordan la conversión de alcanos, la regulación de la expresión génica y la supervivencia en tóxicos ambientes ricos en hidrocarburos.

Una vía de tres pasos para la degradación de alcano se llevó a cabo en E. coli para permitir la conversión de alcanos a medio y largo de cadena a sus respectivos alcanoles, alcanales y, finalmente, ácidos alcanoicos. Este último se metaboliza a través de la nativo β-oxidación vía. Para facilitar la oxidación de alcanos de cadena media-(C5-C13) y cicloalcanos (C5-C8), cuatro genes (alkB2, rubA3, rubA4 y Rubb) del sistema hidroxilasa alcano de Gordonia sp. TF6 8,21 se transformaron en E. coli. Para la conversión de losalcanos de cadena larga (C15-C36), el gen Lada de Geobacillus thermodenitrificans fue implementado. Para conocer los pasos necesarios adicionales del proceso de degradación, ADH y ALDH (procedente de thermodenitrificans G.) se introdujeron 10,11. La actividad se midió mediante ensayos de células en reposo. Para cada etapa oxidativa, la actividad enzimática se observó.

Para optimizar la eficacia del proceso, la expresión fue inducida sólo bajo condiciones bajas de glucosa: un promotor regulado sustrato, pCaiF, se utilizó. pCaiF está presente en E. coli K12 y regula la expresión de los genes implicados en la degradación de fuentes de carbono distintas a la glucosa.

La última parte del conjunto de instrumentos – focalización supervivencia – se implementó el uso de genes de tolerancia, solventes PhPFDα y β, ambos de Pyrococcus horikoshii OT3. Disolventes orgánicos pueden inducir estrés celular y la disminución de la supervivencia de forma negativa affecting plegamiento de proteínas. Como chaperones, PhPFDα y β mejorar el proceso de plegamiento de proteínas por ejemplo en virtud de la presencia de alcanos. La expresión de estos genes condujeron a una tolerancia mejorada de hidrocarburos se muestra por una tasa de crecimiento mayor (hasta 50%) en la presencia de 10% de n-hexano en el medio de cultivo se observaron.

En resumen, los resultados indican que el kit de herramientas permite E. coli para convertir y tolerar hidrocarburos en ambientes acuosos. Como tal, representa un primer paso hacia una solución sostenible para la recuperación de petróleo mediante un enfoque de la biología 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

1. BioBrick Asamblea BioBricks del Registro de partes biológicas estándar son proporcionados por la sede iGEM. Para construir un nuevo BioBrick de BioBricks existentes, digerir el donante BioBrick (hasta 1,0 g) con las enzimas EcoRI y SpeI para el posicionamiento de las partes aguas abajo de los donantes de la parte aceptor. Se digiere con XbaI y PstI para posicionar la parte donante aguas arriba de la parte aceptor. Añadir una tercera enzima de restricción apropiada que corta en la columna vertebral de l…

Representative Results

Alkane conversion The activity of the three oxidation steps from the alkane to the respective fatty acid was evaluated using resting cell assays and enzyme activity measurements. The results are presented following the pathway reactions (1) alkane hydroxylase, (2) alcohol dehydrogenase and (3) aldehyde dehydrogenase. For the first step, different plasmids were constructed for medium and long-chain alkanes. The plasmid BBa_…

Discussion

El principio BioBrick se utiliza para construir un chasis para la degradación de alcanos y una prueba de principio para los componentes individuales de la caja de herramientas se obtuvo. Se proponen varios ensayos para medir la in vivo e in vitro de la actividad de enzimas de la ruta de degradación de alcanos. El trabajo presentado con éxito demuestra un número de métodos que pueden utilizarse para determinar la actividad enzimática y la expresión en el organismo huésped E. coli despu?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Los experimentos realizados en este video-artículo fueron desarrollados para la competición internacional Genetically Engineered Machine 9. Los autores desean agradecer a los miembros del equipo iGEM Lucas Bergwerff, Pieter van TM Boheemen, Cnossen Jelmer, Hugo F. Cueto Rojas y Ramón van der Valk para la asistencia en la investigación. Agradecemos Han de Winde, Stefan de Kok y Yıldırım Esengul útil para los debates y hosting esta investigación. Este trabajo fue apoyado por la TU Delft University Departamento de Biotecnología, el Laboratorio de Bioinformática Delft, TU Delft Departamento de Bionanoscience, Oil Sands Leadership Initiative (OSLI), Stud studentenuitzendbureau, la Iniciativa de Países Bajos Genómica, Centro Kluyver, Nederlandse Vereniging Biotechnologische (Stichting Biotecnología Nederland) , DSM, Geneart, Greiner Bio-One y 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

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Cite This Article
Brinkman, E. K., Schipper, K., Bongaerts, N., Voges, M. J., Abate, A., Wahl, S. A. A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments. J. Vis. Exp. (68), e4182, doi:10.3791/4182 (2012).

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