Team:TU Darmstadt/Project/Metabolism

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Latest revision as of 03:35, 27 September 2012

Metabolism

The group metabolism is employed to create a new metabolic pathway in E. coli. This pathway enables it to convert terephthalic acid (TPA) in high grade fine and bulk chemicals respectively.

Sasoh M, Masai E, Ishibashi S, Hara H, Kamimura N, Miyauchi K, Fukuda M.; Characterization of the terephthalate degradation genes of Comamonas sp. strain E6.; Appl Environ Microbiol. 2006 Mar;72(3):1825-32.

After degradation of polyethylene therephalate (PET) by enzymes presented on the surface, a tripartite tricarboxylate transport system transfers the monomers into the cytoplasm. The new metabolic pathway consists of five cytoplasmatic enzymes and converts terephthalic acid into catechol.

The first reaction of the pathway is catalyzed by a terephthalic acid 1,2-dioxygenase system (TERDOS). This system is a natural enzyme complex from C. testosteroni – KF1 and consists of three enzymes (TphA1, TphA2 and TphA3). TERDOS catalyzes the NAD+ depended reaction from terephthalic acid to (1R,2S)-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD). The second reaction converts the DCD to protocatechuic acid. The enzyme involved in the reaction is called TphB, a dihydrodiol decarboxylase. Protocatechuic acid is a central microbiological metabolite. The final metabolic step is the decarboxylation of protocatechuic acid by AroY.

A sixth enzyme was introduced to cope with the need of verification. XylE catalyzes the reaction from catechol to 2-hydroxysemialdehyd – cis - muconic acid (2-HSM). 2-HSM has a yellowish colour in solution and can be detected by a simple photometric analysis.

We constructed a gene cassette coding for TphA1, TphA2, TphA3, TphB, AroY and XylE. The expression of XylE only is regulated by an L(+)-arabinose inducible promoter while the remaining genes are regulated by a promoter for a constitutive expression. Thus catechol production can easily be verified by simply inducing a culture sample with L(+)-arabinose and incubating it for 2 hours afterwards. This could proof as an excellent system for the production of catechol from terephthalate acid. Detailed information is available in our labjournal.

During our experiments we successfully overexpressed all proteins of our metabolic pathway and confirmed the suggested oligomerizations of the homodimer of TphB, the homotrimer of TphA3 and the homopentamer of AroY. Furthermore we characterised the BioBrick XylE from the parts registry and discovered protocatechuic acid to be an alternative substrate for XylE - including a striking difference in its kinetics. As our results show, anaerobic conditions are crucial for this final step because AroY proved to be sensitive to oxygen exposure. Finally we used XylE to prove AroY's activity in catalyzing our final step from protocatechuate to catechol.

For further reading continue to 4. Material Science.

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 == Metabolism ==
-Die Gruppe Metabolismus beschäftigt sich mit der Metabolisierung von Ethylenglykol und der Umsetzung von Terephthalsäure (TPA) in hochwertige Fein- und Bulkchemikalien.
+The group metabolism is employed to create a new metabolic pathway in ''E. coli''. This pathway enables it to convert terephthalic acid ([http://en.wikipedia.org/wiki/Terephthalic_acid TPA]) in high grade fine and bulk chemicals respectively.
+[[File:TPA_Degradation.png|350px|thumb|right|Sasoh M, Masai E, Ishibashi S, Hara H, Kamimura N, Miyauchi K, Fukuda M.; Characterization of the terephthalate degradation genes of Comamonas sp. strain E6.; Appl Environ Microbiol. 2006 Mar;72(3):1825-32.]]
-Das bei der Spaltung von Polyethylenterephthalat (PET) durch zelloberflächenpräsentierten Fusionsproteine entstandene TPA wird mit spezifischen Transporter in das Zellinnere gebracht. Im Zytosol befinden sich Enzyme aus dem Bakterium Comamonas testosteroni KF-1 die giftige TPA zu weniger toxischen Metaboliten umsetzen. Ein TPA Oxidase setzt TPA zu Dihydroxy Cyclohexadiene Dicarboxylat (PCA) um. Durch anschließende Decarboxylierung entsteht Protocatechoat (PCA). Dies lässt sich in einem weiteren Schritt zu Catechol einer wichtigen Bulk-Chemikalie umsetzen. Zum Nachweis einer erfolgreichen Umsetzung wird das Catechol mithilfe einer Arabinose induzierbaren spezifischen Dihydroxygenase zu cis,cis-Muconsäure (2-HMS) gespalten, diese lässt sich quantitativ photometrisch nachweisen.
+After degradation of polyethylene therephalate ([http://en.wikipedia.org/wiki/Polyethylene_terephthalate PET]) by enzymes presented on the surface, a tripartite tricarboxylate transport system transfers the monomers into the cytoplasm. The new metabolic pathway consists of five cytoplasmatic enzymes and converts terephthalic acid into catechol.
--HMS ist ein leuchtend gelber Farbstoff und wird in E.coli nicht verstoffwechselt, somit reichert sich 2-HMS sich in den Zellen an.
+The first reaction of the pathway is catalyzed by a terephthalic acid 1,2-dioxygenase system (TERDOS). This system is a natural enzyme complex from ''C. testosteroni – KF1'' and consists of three enzymes ([http://partsregistry.org/Part:BBa_K808011 TphA1], [http://partsregistry.org/Part:BBa_K808012 TphA2] and [http://partsregistry.org/Part:BBa_K808013 TphA3]). TERDOS catalyzes the NAD+ depended reaction from terephthalic acid to (1R,2S)-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD).
+The second reaction converts the DCD to protocatechuic acid. The enzyme involved in the reaction is called [http://partsregistry.org/Part:BBa_K808010 TphB], a dihydrodiol decarboxylase. Protocatechuic acid is a central microbiological metabolite. The final metabolic step is the decarboxylation of protocatechuic acid by [http://partsregistry.org/Part:BBa_K808014 AroY].
-Die für die oben beschreibenden Schritte benötigten Gene Stammen aus Comamonas testosteroni KF-1 und Klebsiella pneumonie subsp. Pneumonie, sie werden in E.Coli MG1655 eingebracht (Chemisch).
+A sixth enzyme was introduced to cope with the need of verification. [http://partsregistry.org/Part:BBa_K316003 XylE] catalyzes the reaction from catechol to 2-hydroxysemialdehyd – cis - muconic acid (2-HSM). 2-HSM has a yellowish colour in solution and can be detected by a simple photometric analysis.
-Zusätzlich wird ein E.coli Stamm (MG1655) als Chassis validiert, der Glykol als einige C-Quelle nutzt kann.
+We constructed a gene cassette coding for [http://partsregistry.org/Part:BBa_K808011 TphA1], [http://partsregistry.org/Part:BBa_K808012 TphA2], [http://partsregistry.org/Part:BBa_K808013 TphA3], [http://partsregistry.org/Part:BBa_K808010 TphB], [http://partsregistry.org/Part:BBa_K808014 AroY] and [http://partsregistry.org/Part:BBa_K316003 XylE]. The expression of XylE only is regulated by an L(+)-arabinose inducible promoter while the remaining genes are regulated by a promoter for a constitutive expression. Thus catechol production can easily be verified by simply inducing a culture sample with L(+)-arabinose and incubating it for 2 hours afterwards. This could proof as an excellent system for the production of catechol from terephthalate acid. Detailed information is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism labjournal].
-continue to [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science 4. Material Science]
+During our experiments we successfully overexpressed all proteins of our metabolic pathway and confirmed the suggested oligomerizations of the homodimer of [http://partsregistry.org/Part:BBa_K808010 TphB], the homotrimer of [http://partsregistry.org/Part:BBa_K808013 TphA3] and the homopentamer of [http://partsregistry.org/Part:BBa_K808014 AroY]. Furthermore we characterised the BioBrick [http://partsregistry.org/Part:BBa_K316003 XylE] from the parts registry and discovered protocatechuic acid to be an alternative substrate for [http://partsregistry.org/Part:BBa_K316003 XylE] - including a striking difference in its kinetics. As our results show, anaerobic conditions are crucial for this final step because [http://partsregistry.org/Part:BBa_K808014 AroY] proved to be sensitive to oxygen exposure. Finally we used [http://partsregistry.org/Part:BBa_K316003 XylE] to prove [http://partsregistry.org/Part:BBa_K808014 AroY]'s activity in catalyzing our final step from protocatechuate to catechol.
+For further reading continue to [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science 4. Material Science].