Cyclohexane Carboxylate and Benzoate Formation from Crotonate in Syntrophus aciditrophicus

Abstract

ABSTRACT The anaerobic, syntrophic bacterium Syntrophus aciditrophicus grown in pure culture produced 1.4 ± 0.24 mol of acetate and 0.16 ± 0.02 mol of cyclohexane carboxylate per mole of crotonate metabolized. [U- 13 C]crotonate was metabolized to [1,2- 13 C]acetate and [1,2,3,4,5,7- 13 C]cyclohexane carboxylate. Cultures grown with unlabeled crotonate and [ 13 C]sodium bicarbonate formed [6- 13 C]cyclohexane carboxylate. Trimethylsilyl (TMS) derivatives of cyclohexane carboxylate, cyclohex-1-ene carboxylate, benzoate, pimelate, glutarate, 3-hydroxybutyrate, and acetoacetate were detected as intermediates by comparison of retention times and mass spectral profiles to authentic standards. With [U- 13 C]crotonate, the m/z -15 ion of TMS-derivatized glutarate, 3-hydroxybutyrate, and acetoacetate each increased by +4 mass units, and the m/z -15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +6 mass units. With [ 13 C]sodium bicarbonate and unlabeled crotonate, the m/z -15 ion of TMS derivatives of glutarate, pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +1 mass unit, suggesting that carboxylation occurred after the synthesis of a four-carbon intermediate. With [1,2- 13 C]acetate and unlabeled crotonate, the m/z -15 ion of TMS-derivatized 3-hydroxybutyrate, acetoacetate, and glutarate each increased by +0, +2, and +4 mass units, respectively, and the m/z -15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, cyclohexane carboxylate, and 2-hydroxycyclohexane carboxylate each increased by +0, +2, +4, and +6 mass units. The data are consistent with a pathway for cyclohexane carboxylate formation involving the condensation of two-carbon units derived from crotonate degradation with CO 2 addition, rather than the use of the intact four-carbon skeleton of crotonate.