Citrate metabolism in lactic acid bacteria: is there a beneficial effect for Oenococcus oeni in wine?

Abstract

Lactic acid bacteria (LAB) are Gram positive bacteria frequently used in the food industry for fermentation, mainly transformation of carbohydrates into lactic acid. In addition, these bacteria also have the capacity to metabolize citrate, an organic acid commonly found in food products. Its fermentation leads to the production of 4-carbon compounds such as diacetyl, resulting in a buttery flavor desired in dairy products. Citrate metabolism is known to have several beneficial effects on LAB physiology. Nevertheless, a controversial effect of citrate has been described on the acid tolerance of the wine bacterium Oenococcus oeni. This observation raises questions about the effect of citrate on the capacity of O. oeni to conduct malolactic fermentation in highly acidic wines. This review aims to summarize the current understanding of citrate metabolism in LAB, with a focus on the wine bacterium O. oeni. Metabolism with the related enzymes is detailed, as are the involved genes organized in cit loci. The known systems of cit locus expression regulation are also described. Finally, the beneficial effects of citrate catabolism on LAB physiology are reported and the negative impact observed in O. oeni is discussed.

Keywords: Oenococcus oeni; citrate; citrate locus; lactic acid bacteria; metabolic engineering; proton motive force.

Figure 1

Organization of the citrate cluster from different LAB species. The citrate locus of Lactococcus lactis bv. diacetylactis, Weissella paramesenteroides, Enterococcus faecalis and Oenococcus oeni are represented and the size of each one is indicated. Thin arrows localize identified promotors (except for O. oeni in which putative promotors are represented). Air-pin structures represent identified rho-independent terminators. Dashed arrow represents a pseudogene founded on the citrate locus of L lactis bv. diacetylactis.

Figure 2

Citrate Lyase synthesis and catabolism of citrate mediated by this enzyme. Genes encoding the different citrate lyase (CL) subunits and the three additional proteins involved in this metabolism are showed. Arrows with solid lines represent enzymatic reactions, arrows with dashed lines represent the provenance or redirection of metabolites to other pathways. (1) CitX (apo-citrate lyase phosphoribosyl-dephospho-coenzyme A) and CitG (triphosphoribosyl-dephospho-coenzyme A synthase) synthetize the prosthetic group from ATP and dephospho-coenzyme A. (2) Activation by acetylation of the prosthetic group by CitC (acetate:SH-citrate lyase ligase). (3) Exchange of the acetyl group with the citryl group of citrate catalyzed by the α-subunit of the CL (acetyl-ACP:citrate ACP-transferase) and formation of acetate. (4) Clivage of the citryl group by the β-subunit of the CL (citryl-S-ACP oxaloacetate-lyase) releasing oxaloacetate and enable the acetyl group regeneration.

Figure 3

Citrate metabolism and becoming of pyruvate in Oenococcus oeni. Pyruvate produced by the citrate catabolism (green part) can be metabolized into D-lactate (blue pathway) for the reoxidation of co-factors generated by sugar metabolism (Pentose-P pathway, represented in gray). It can also be used for the production of acetate through the pyruvate dehydrogenase pathway (represented in pink) or through the pyruvate oxidase pathway (represented in orange). Finally, two molecules of pyruvate (symbolized by the number 2 on the figure) can be metabolized into α-acetolactate under the action of the acetolactate synthase (ALS) (dark blue pathway) which leads to the production of diacetyl, acetoin and 2,3-butanediol. Genes encoding the different enzymes are indicated in colored boxes. When no name was attributed to a gene, its locus tag from the PSU-1 strain is indicated. As the transport mechanism of acetate has not been characterized in O. oeni, question marks are represented.

Figure 4

Generation of PMF in Lactococcus lactis bv. diacetylactis and in Oenococcus oeni by citrate metabolism. 1/ Internalization of citrate by either the permeases CitP or MaeP leads to the creation of an electrical gradient, inside negative (ΔΨ). Citrate forms are represented in gray, the majority form founded in each environment of the two LAB according to the extracellular pH is in bold 2/ Citrate metabolism drives the consumption of scalar protons leading to the creation of a ΔpH gradient, inside alkaline.