Un Toolkit per permettere la conversione di idrocarburi in ambienti acquosi
Summary
Un sistema di regolazione automatica sostenibile batterica per la bonifica di inquinamenti del petrolio è stato progettato utilizzando le normali parti di DNA intercambiabili (BioBricks). L'ingegneria<em> E. coli</emCeppo> è stato usato per degradare alcani tramite β-ossidazione tossici in ambienti acquosi. Gli enzimi rispettivi di specie diverse hanno mostrato un'attività alcano degrado. Inoltre, una maggiore tolleranza<em> N</em>-Esano è stato ottenuto con l'introduzione di geni di alcano-tolleranti batteri.
Abstract
Questo lavoro propone una serie di strumenti che consente la conversione di alcani da Escherichia coli e presenta una prova di principio della sua applicabilità. Il toolkit è costituito da più parti standard intercambiabili (BioBricks) 9 riguardanti la conversione di alcani, regolazione dell'espressione genica e della sopravvivenza in idrocarburi tossici ambienti ricchi.
A tre fasi pathway per la degradazione alcano è stato attuato in E. coli per consentire la conversione di alcani media e lunga catena ai loro rispettivi alcanoli, alkanals e infine alcanoico-acidi. Questi ultimi sono stati metabolizzati tramite il nativo di β-ossidazione percorso. Per facilitare l'ossidazione di alcani a catena media (C5-C13) e cicloalcani (C5-C8), quattro geni (alkB2, rubA3, rubA4 e Rubb) del sistema idrossilasi alcano da Gordonia sp. TF6 8,21 sono stati trasformati in E. coli. Per la conversione dialcani a catena lunga (C15-C36), il gene Lada da thermodenitrificans Geobacillus è stato attuato. Per i passaggi necessari ulteriori del processo di degradazione, ADH e ALDH (provenienti da thermodenitrificans G.) sono stati introdotti 10,11. L'attività è stata misurata mediante saggi su cellule quiescenti. Per ogni passaggio ossidativo, l'attività enzimatica è stata osservata.
Per ottimizzare l'efficienza del processo, l'espressione è indotta soltanto in condizioni di glucosio basso: un substrato regolato promotore, pCaiF, è stato utilizzato. pCaiF è presente in E. coli K12 e regola l'espressione dei geni coinvolti nella degradazione del glucosio non fonti di carbonio.
L'ultima parte del toolkit – mira sopravvivenza – è stato implementato utilizzando geni di tolleranza solventi, PhPFDα e β, sia dal Pyrococcus horikoshii OT3. Solventi organici possono indurre stress cellulare e diminuiscono la capacità di sopravvivenza di negativo affecting ripiegamento delle proteine. Come accompagnatori, PhPFDα e β migliorare il ripiegamento ad esempio processi di proteina in presenza di alcani. L'espressione di questi geni ha portato ad un miglioramento della tolleranza idrocarburo mostrato da una crescita maggiore (fino al 50%) in presenze del 10% di n-esano in mezzo di coltura sono stati osservati.
Riassumendo, i risultati indicano che il toolkit consente di E. coli da convertire e tollerare idrocarburi in ambienti acquosi. In quanto tale, rappresenta un primo passo verso una soluzione sostenibile per l'olio-bonifica utilizzando un approccio biologia sintetica.
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
Il principio BioBrick viene utilizzato per costruire un telaio per la degradazione di alcani e una prova di principio per i singoli componenti del toolkit è stato ottenuto. Parecchi saggi sono proposto di misurare in vivo e in vitro di alcano pathway enzimi degradativi. Il lavoro presentato dimostra con successo un certo numero di metodi che possono essere utilizzati per determinare le attività enzimatiche e espressione nell'ospite microrganismo E. coli dopo l'implementazione di BioB…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Gli esperimenti condotti in questo video-articolo sono stati sviluppati per il concorso internazionale macchina Genetically Engineered 9. Gli autori desiderano ringraziare i membri del team iGEM Luca Bergwerff, Pieter van TM Boheemen, Jelmer Cnossen, Hugo F. Cueto Rojas e Ramon van der Valk per l'assistenza nella ricerca. Ringraziamo Han de Winde, Stefan de Kok e Esengül Yıldırım per le discussioni utili e che ospitano questa ricerca. Questo lavoro è stato sostenuto dal TU Delft University Dipartimento di Biotecnologie, Il laboratorio di Bioinformatica Delft, TU Delft Dipartimento di Bionanoscience, Oil Sands Leadership Initiative (OSLI), Stud studentenuitzendbureau, Paesi Bassi Genomics Initiative, Kluyver Centro, Nederlandse Vereniging Biotechnologische (Stichting Biotecnologie 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 |
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