Carbohydrate/citrate cometabolism in Lactococcus lactis results in the formation of the flavor compound acetoin. Resting cells of strain IL1403(pFL3) rapidly consumed citrate while producing acetoin when substoichiometric concentrations of glucose or L-lactate were present. A proton motive force was generated by electrogenic exchange of citrate and lactate catalyzed by the citrate transporter CitP and proton consumption in decarboxylation reactions in the pathway. In the absence of glucose or L-lactate, citrate consumption was biphasic. During the first phase, hardly any citrate was consumed. In the second phase, citrate was converted rapidly, but without the formation of acetoin. Instead, significant amounts of the intermediates pyruvate and ␣-acetolactate, and the end product acetate, were excreted from the cells. It is shown that the intermediates and acetate are excreted in exchange with the uptake of citrate catalyzed by CitP. The availability of exchangeable substrates in the cytoplasm determines both the rate of citrate consumption and the end product profile. It follows that citrate metabolism in L. lactis IL1403(pFL3) splits up in two routes after the formation of pyruvate, one the well-characterized route yielding acetoin and the other a new route yielding acetate. The flux distribution between the two branches changes from 85:15 in the presence of L-lactate to 30:70 in the presence of pyruvate. The proton motive force generated was greatest in the presence of L-lactate and zero in the presence of pyruvate, suggesting that the pathway to acetate does not generate proton motive force.

Citrate metabolism plays an important role in many food fermentation processes involving lactic acid bacteria (LAB) (11). Citrate is the precursor of carbon dioxide and the flavor compounds diacetyl and acetoin that contribute to the organoleptic properties of fermented foods. The flavor compounds are formed from the central metabolite pyruvate in the cytoplasm. Citrate metabolism feeds directly into the pyruvate pool (Fig. 1). Following uptake by the secondary citrate transporter CitP, citrate lyase (CL) converts citrate to acetate and oxaloacetate. Acetate is not further metabolized and leaves the cells, while oxaloacetate is decarboxylated to pyruvate by a soluble oxaloacetate decarboxylase encoded by the mae gene and termed CitM (25). In a recent study it was demonstrated that in the lactic acid bacterium Lactococcus lactis IL1403(pFL3), part of the pyruvate formed from citrate was converted into acetoin and part was converted into acetate in parallel pathways (23). In resting cells, the distribution over the two end products strongly depended on the conditions. A high turnover rate through the pathway favored the formation of acetoin over acetate.The citrate transporter CitP plays a pivotal role in the kinetics of the pathway. Under physiological conditions, when citrate is cometabolized with a carbohydrate, CitP operates in the fast citrate/L-lactate exchange mode; citrate is taken up and at the same time that L-lactate, the product of glycolysis, is excreted (precursor/product exchange) (2,17,19,23). In the absence of L-lactate, CitP operates in the much slower unidirectional H ϩ -symport mode (19), which makes uptake the rate-determining step for the flux through the pathway (23). It was shown that, in addition to L-lactate, CitP has an affinity for intermediates/products of the pathway, which results in the typical biphasic consumption of citrate in the absence of L-lactate. During the first phase, citrate is taken up slowly in symport with an H ϩ , but as intermediates/products accumulate in the cytoplasm, the transporter switches to the fast exchange mode, in which citrate is taken up in exchange with pyruvate, ␣-acetolactate, and/or acetate, resulting in much higher consumption rates in the second phase. Therefore, the kinetics and product profile of citrate metabolism are determined largely by the availability of exchangeable substrates for the transporter CitP.The physiological function of citrate metabolism in LAB is in metabolic energy generation and acid stress resistance (8,18,20,28). The citrate metabolic pathway generates proton motive force (PMF) (3,19,20) by an indirect proton pumping mechanism, by which the two components of the PMF, membrane potential (⌬) and pH gradient (⌬pH), are generated in separate steps (13,14). The exchange of divalent citrate and monovalent lactate catalyzed by CitP generates membrane po-