Team:TU Darmstadt/Project/Metabolism

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Metabolism

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

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

The first reaction of the pathway is catalyzed by a terephthalic acid 1,2-dioxigenase 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-hydroxisemialdehyd – 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.

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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 ''E. coli'' to convert terephthalic acid in high grade fine and bulk chemicals respectively.
-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 (PET) by enzymes presented on the surface a tripartite tricarboxylate transport system transports the monomers into the cytoplasm. The new metabolic pathway consists of five cytoplasm enzymes and converts terephthalic acid to 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-dioxigenase 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.
-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. XylE catalyzes the reaction from catechol to 2-hydroxisemialdehyd – 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.
-Zusätzlich wird ein E.coli Stamm (MG1655) als Chassis validiert, der Glykol als einige C-Quelle nutzt kann.
 continue to [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science 4. Material Science]