Summary

वातावरण में जलीय हाइड्रोकार्बन रूपांतरण सक्षम टूलकिट

Published: October 02, 2012
doi:

Summary

एक स्थायी तेल प्रदूषण के remediation के लिए जीवाणु प्रणाली को विनियमित ऑटो मानक विनिमेय डीएनए भागों (BioBricks) का उपयोग कर बनाया गया था. एक इंजीनियर<em> ई. कोलाई</em> तनाव विषाक्त वातावरण में जलीय β ऑक्सीकरण के माध्यम से alkanes नीचा करने के लिए इस्तेमाल किया गया था. विभिन्न प्रजातियों में से संबंधित एंजाइमों alkane गिरावट गतिविधि दिखाया. इसके अतिरिक्त, एक वृद्धि की सहिष्णुता<emN></em> Hexane alkane सहिष्णु बैक्टीरिया के जीन से शुरू करने से हासिल की थी.

Abstract

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.

A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.

To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.

The last part of the toolkit – targeting survival – was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.

Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

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 विधानसभा मानक जैविक भागों की रजिस्ट्री से BioBricks iGEM मुख्यालय द्वारा प्रदान की जाती हैं. मौजूदा BioBricks से एक नया BioBrick का निर्माण, EcoRI और SpeI एंजाइमों के साथ दाता स्वीकर्ता भाग का हिस्सा नीचे की ओर की स्थिति…

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

BioBrick सिद्धांत alkanes की गिरावट के लिए एक हवाई जहाज़ के पहिये का निर्माण करने के लिए प्रयोग किया जाता है और टूलकिट के एक घटक के लिए सिद्धांत का एक सबूत प्राप्त किया गया था. कई assays के लिए vivo में और alkane अपमानजनक म?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

इस वीडियो लेख में प्रदर्शन प्रयोगों अंतरराष्ट्रीय आनुवंशिक रूप से इंजीनियर मशीन 9 प्रतियोगिता के लिए विकसित किए गए लेखकों iGEM टीम के सदस्यों के लिए ल्यूक Bergwerff, पीटर टीएम वैन Boheemen, Jelmer Cnossen, ह्यूगो एफ Cueto रोजास और Ramon वैन der Valk का शुक्रिया अदा करना चाहते हैं अनुसंधान में सहायता. हम उपयोगी विचार – विमर्श और इस शोध की मेजबानी के लिए हान डी winde, Stefan De Kok और Esengül Yildirim धन्यवाद. यह काम टीयू डेल्फ़्ट विश्वविद्यालय जैव प्रौद्योगिकी विभाग, डेल्फ़्ट बायोइनफॉरमैटिक्स प्रयोगशाला, टीयू डेल्फ़्ट Bionanoscience के विभाग, तेल रेत नेतृत्व पहल (OSLI), स्टड studentenuitzendbureau, नीदरलैंड जीनोमिक्स पहल, Kluyver केंद्र, Nederlandse Biotechnologische Vereniging (Stichting बायोटैक्नोलॉजी Nederland) द्वारा समर्थित किया गया , DSM, Geneart, Greiner जैव एक और 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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