P

glyceraldehyde-3-phosphate lactic acid acetyl phosphate phospho- L-— CoA transacetylase pi acetyl CoA

acetaldehyde U- NADH + H dehydrogenase S-^. nad acetaldehyde alcohol dehydrogenase ^ nad

ethanol

Figure 2-11. The phosphoketolase pathway used by heterofermentative lactic acid bacteria.

48 Microbiology and Technology of Fermented Foods Box 2—3. The Heterolactic Fermentation: Dealing with Pyruvate

Lactic acid bacteria, as previously noted, are either homofermentative, heterofermentative, or facultative heterofermentative (where both pathways are present). However, even obligate homofermentative strains have the potential to produce acetic acid, ethanol, acetoin, CO2 and end-products other than lactic acid.These alternative fermentation end-products are formed, however, only under conditions in which pyruvate concentrations are elevated.

Such a scenario occurs when the rate of intracellular pyruvate formation exceeds the rate at which pyruvate is reduced to lactate (via lactate dehydrogenase). The pyruvate can arise from sugars (see below), but also from amino acids. In either case, the excess pyruvate must be removed because it could otherwise become toxic.Moreover,when excess pyruvate is generated as a result of sugar metabolism, the cells must also have a means for re-oxidizing the NADH formed from glycolysis (upstream in the pathway). Several different pathways in lactic acid bacteria appear to serve this function (Figure 1; Cocaign-Bousquet et al., 1996; Garrigues et al., 1997). In addition, at least one of these alternative pathways include a substrate level phosphorylation reaction and, therefore, provides the cells with additional ATP.

2,3-butanediol

acetoin

a-acetolactate t-

a-acetolactate carbon dioxide ^^jjayrth^ formate acetate ^ pyruvate dehydrogenase ^ pyruvate-formate lyase ^ acetate acetaldehyde ^ _____' ^ acetaldehyde ethanol ethanol lactate dehydrogenase

lactate

Figure 1. Heterolactic end products from pyruvate metabolism.

Sugar Transport by Lactic Acid Bacteria

Although the catabolic pathways used by lactic acid bacteria to ferment sugars constitute a major part of the overall metabolic process, the first step (and perhaps an even more important one) involves the transport of the substrate across the cytoplasmic membrane.Transport is important for several reasons, not the least of which is that the cell membrane is impermeable to polar solutes (e.g., sugars, amino acids, peptides). In the absence of transport systems, these solutes would be unable to transverse the membrane and gain entry into the cell. Second, the cell devotes a considerable amount of its total energy resources to support active transport. Third, some sugars are phosphorylated during the transport event, which then dictates the catabolic pathway used by that organism. Finally, transport systems may serve a regulatory role, influencing expression and activity of other transport systems.

Box 2—3. The Heterolactic Fermentation: Dealing with Pyruvate (Continued)

What are the conditions or environments that result in pyruvate accumulation and induction of alternate pathways? There are several possible situations where these reactions occur. First, glycolysis is subject to several levels of regulation, such that when fermentation substrates are limiting, the glycolytic flux tends to be diminished (Axelsson, 2004). Specifically, when the concentration of fructose-1,6-diphosphate (a glycolytic regulator that forms early during glycolysis), is low, the activity of lactate dehydrogenase (an allosteric enzyme that occurs at the end of glycolysis) is reduced.Thus, pyruvate accumulates.The glycolytic flux also may be decreased during growth on less preferred carbon sources, such as galactose, again resulting in excess pyruvate. At the same time that the lactate dehydrogenase activity is decreased, the enzyme, pyruvate-formate lyase, is activated. This enzyme splits pyruvate to form formate and acetyl CoA. The latter is subsequently reduced to ethanol or phosphorylated to acetyl phosphate (both reactions releasing CoA). Importantly, the acetyl phosphate can be used as part of a substrate level phosphorylation reaction (via acetate kinase), resulting in the formation of an ATP.

Under aerobic environments, however, pyruvate-formate lyase is inactive, and other pathways become active. In the pyruvate dehydrogenase pathway, for example, pyruvate is decarboxy-lated by pyruvate dehydrogenase, and acetate and CO2 are formed. NADH that would normally reduce pyruvate also is oxidized directly by molecular oxygen when the environment is aerobic, rendering it unavailable for the lactate dehydrogenase reaction. Finally, excess pyruvate can be diverted to end-products that may have a more functional role in certain fermented dairy products. Specifically, pyruvate can serve as a substrate for a-acetolactate synthase to form a-acetolactate.The a-acetolactate may then be further oxidized to form diacetyl, which has desirable aroma properties.

References

Axelsson,L. 2004.Lactic acid bacteria:classification and physiology,pp. 1—66.In Salminen, S.,A.von Write, and A. Ouwehand. Lactic Acid Bacteria Microbiological and Functional Aspects Third Edition. Marcel Dekker, Inc. New York, New York. Cocaign-Bousquet, M., C. Garrigues, P. Loubiere, and N. Lindley. 1996. Physiology of pyruvate metabolism in

Lactococcus lactis.Antonie Van Leeuwenhoek 70:253-267. Garrigues, C., P. Loubiere, N. Lindley, and M. Cocaign-Bousquet. 1997. Control of shift from homolactic acid to mixed-acid fermentation in Lactooccus lactis: predominant role of the NADH/NAD+ ratio. J. Bacte-riol. 179:5282-5287.

The phosphoenolpyruvate-dependent phosphotransferase system

In general, several different systems are used by lactic acid bacteria to transport carbohydrates, depending on the species and the specific sugar. The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) is used by most mesophilic, homofermentative lactic acid bacteria, including lactococci and pediococci for transport of lactose and glucose. In contrast, other lactic acid bacteria transport sugars via symport-type or ATP-dependent systems (described below). Another group of transporters are the precursor-

product exchange systems that provide an altogether different (and more efficient) means for transporting sugars (Box 2-4).

The PTS consists of both cytoplasmic and membrane-associated proteins that sequentially transfer a high-energy phosphate group from PEP, the initial donor,to the incoming sugar (Figure 2-12).The phosphorylation step coincides with the vectorial movement of the sugar substrate across the membrane, resulting in the in-tracellular accumulation of a sugar-phosphate. Two of the cytoplasmic proteins of the PTS, Enzyme I and HPr (for histidine-containing protein), are non-specific and serve all of the sugar

Box 2—4. Something For Nothing, or How Lactic Acid Bacteria Conserve Energy Via "Precursor-product" Exchange Systems

Transport of nutrients from the environmental medium across the cytoplasmic membrane and into the cytoplasm is one of the most important of all microbial processes. For many microorganisms, including lactic acid bacteria, nearly 20% of the genome is devoted to transport functions, and cell membranes are literally crammed with a hundred or more transport permeases, translocators, and accessory proteins (Lorca et al., 2005). The function of transporters, of course, is to provide a means for the cell to selectively permit solutes to move, back and forth as the case may be, across a generally impermeable membrane.

For most nutrients (and other transport substrates), however, transport is not cheap, in that energy (e.g.,ATP) is usually required to perform "vectorial" work (i.e., to move molecules from one side of a membrane to another).This is because nutrient transport generally occurs against a concentration gradient, meaning that the concentration of the substrate is usually much lower in the outside medium than it is on the inside (i.e., within the cytoplasm).Although passive or facilitated diffusion, where the concentration gradient (higher outside than inside) drives transport occurs occasionally, this is not the normal situation, and instead some sort of active, energy-requiring process is required.

The metabolic cost of transport is especially high for lactic acid bacteria, given the limited and generally inefficient means by which these bacteria make energy.After all, glycolysis generates only two molecules of ATP per molecule of glucose or hexose fermented. If ATP or its equivalent (e.g.,phosphoenolpyruvate or the proton motive force) is required to "move" mono-and disaccharides across the cytoplasmic membrane, the net energy gain by the cell may be reduced by as much as 50%. On the other hand, if the driving force for solute transport does not depend on an energy source, then the cell can conserve that un-spent energy or use it for other reactions.

By what means can cells transport solutes without having to spend energy? In many lactic acid bacteria, metabolism of substrate (or precursor) molecules results in a large amount of metabolic products that must be excreted from the cell. In some cases, these products are not further metabolized; in other cases, they may even be toxic if allowed to accumulate intracellu-larly. In any event, a rather large concentration gradient is formed, where the inside concentration of "product" is much higher than the outside concentration. Efflux of "product" molecules, therefore, is driven by the concentration gradient in a downhill manner.The carrier for efflux in these situations, however, actually has affinity not just for the product, but also for the substrate. Moreover, it operates in opposing directions, such that product excretion (via the downhill concentration gradient) can drive uphill uptake of the precursor substrate (against the concentration gradient).These so-called precursor-product exchange systems, therefore, represent a novel means by which cells can accumulate nutrients and metabolic substrates without having to consume much-needed sources of energy.

Examples of Precursor-product Exchange Systems in Lactic Acid Bacteria

Precursor-product exchange systems are widely distributed in lactic acid bacteria, and are used to transport fermentation substrates, amino acids, and organic acids (Konings, 2002).These systems can be electroneutral, without a net change in the electric charge across the cell membrane, or electrogenic, where a charge is generated across the membrane.For example, a neutral sugar precursor (lactose) exchanged for a neutral sugar product (galactose) is electroneutral, as is the exchange of the amino acid arginine for ornithine (Figure 1, panels A and C). However, a di-anionic precursor (citrate) exchanged for a mono-anionic product (lactate) is electrogenic, since it results in an increase of the transmembrane electric charge (Figure 1, panel B).

Box 2—4. Something For Nothing, or How Lactic Acid Bacteria Conserve Energy Via "Precursor-product" Exchange Systems (Continued)

lactose galactose out H+^

vtS^

LacS

H+ * lactose b-galactosidase glucose galactose

^EM pathway lactic acid

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