Dokument

Summary:
Glutaric and glutaconic acids serve as monomers for the production of polymers. Glutaric acid (pentanedioic acid) might be used for polyester synthesis, related to the biodegradable Ecoflex available from BASF. Glutaconic acid (pentenedioic acid) could be applied for the formation of polyamides by polymerization with diamines. Furthermore this α,β-unsaturated dicarboxylic acid is suitable for radical polymerization. Therefore we became interested in the biological production of these dicarboxylic acids. The ideal material for biotechnological production of glutaconic acid would be glutamic acid, obtained by sugar fermentation. The chemical deamination of this α-amino acid to glutaconate is not executable. In contrary to this, strictly anaerobic bacteria, as are Acidaminococcus fermentans and Clostridium symbiosum can easily ferment glutamate to ammonia, acetate, butyrate, CO2 and H2 via 2-oxoglutarate, (R)-2-hydroxyglutarate, (R)-2-hydroxyglutaryl-CoA, and glutaconyl-CoA. Inhibition of the subsequent decarboxylation to crotonyl-CoA would lead to glutaconate. We achieved this aim on another route, the conversion of Escherichia coli into a glutaconate producer by introducing six genes encoding (R)-2-hydroxyglutarate dehydrogenase (HgdH), glutaconate CoA-transferase (GctAB), and the extremely oxygen sensitive activator of the dehydratase (HgdC) from A. fermentans as well as the also oxygen sensitive (R)-2-hydroxyglutaryl-CoA dehydratase (HgdAB) from C. symbiosum. Hence, within 5 h after induction of gene expression the recombinant E. coli produced 2.7 ± 0.2 mM glutaconate on a medium containing 1.5% peptone, 0.3 % yeast extract, 100 mM NaCl, 5 mM glucose, 3 mM cysteine, 10 mM glutamate, 2 mM ferric citrate, 0.2 mM riboflavin, and antibiotics. Interestingly, initially the concentration of glutamate decreased by 30% but later regained its original level, whereas glucose was almost quantitatively converted to two ethanol. The reduction of glutaconyl-CoA to glutaryl-CoA is catalyzed by an enzyme involved in the synthesis of cyclohexanecarboxylate and benzoate in Syntrophus aciditrophicus. Preliminary experiments indicate that coexpression of the genes encoding glutaryl-CoA dehydrogenase and electron-transferring flavoprotein (EtfAB) from S. aciditrophicus in E. coli yield an enzyme system that together with hydrogenase catalyzes the bifurcation of 2 NAD(P)H to glutaconyl-CoA and ferredoxin. Thus glutaryl-CoA and H2 were formed though at a very low rate.

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