Supplementary Material for: Fermentative Cyclohexane Carboxylate Formation in <b><i>Syntrophus aciditrophicus</i></b>

Short-chain fatty acids such as acetic, propionic, butyric or lactic acids are typical primary fermentation products in the anaerobic feeding chain. Fifteen years ago, a novel fermentation type was discovered in the obligately anaerobic Deltaproteobacterium <i>Syntrophus aciditrophicus</i>. During fermentative growth with crotonate and/or benzoate, acetate is formed in the oxidative branch and cyclohexane carboxylate in the reductive branch. In both cases cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA) is a central intermediate that is either formed by a class II benzoyl-CoA reductase (fermentation of benzoate) or by reverse reactions of the benzoyl-CoA degradation pathway (fermentation of crotonate). Here, we summarize the current knowledge of the enzymology involved in fermentations yielding cyclohexane carboxylate as an excreted product. The characteristic enzymes involved are two acyl-CoA dehydrogenases specifically acting on Ch1,5CoA and cyclohex-1-ene-1-carboxyl-CoA. Both enzymes are also employed during the syntrophic growth of <i>S. aciditrophicus</i> with cyclohexane carboxylate as the carbon source in coculture with a methanogen. An investigation of anabolic pathways in <i>S. aciditrophicus</i> revealed a rather unusual pathway for glutamate synthesis involving a <i>Re</i>-citrate synthase. Future work has to address the unresolved question concerning which components are involved in reoxidation of the NADH formed in the oxidative branch of the unique cyclohexane carboxylate fermentation pathway in <i>S. aciditrophicus</i>.