Variations in the energy metabolism of biotechnologically relevant heterofermentative lactic acid bacteria during growth on sugars and organic acids
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- Zaunmüller, T., Eichert, M., Richter, H. et al. Appl Microbiol Biotechnol (2006) 72: 421. doi:10.1007/s00253-006-0514-3
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Heterofermentative lactic acid bacteria (LAB) such as Leuconostoc, Oenococcus, and Lactobacillus strains ferment pentoses by the phosphoketolase pathway. The extra NAD(P)H, which is produced during growth on hexoses, is transferred to acetyl-CoA, yielding ethanol. Ethanol fermentation represents the limiting step in hexose fermentation, therefore, part of the extra NAD(P)H is used to produce erythritol and glycerol. Fructose, pyruvate, citrate, and O2 can be used in addition as external electron acceptors for NAD(P)H reoxidation. Use of the external acceptors increases the growth rate of the bacteria. The bacteria are also able to ferment organic acids like malate, pyruvate, and citrate. Malolactic fermentation generates a proton potential by substrate transport. Pyruvate fermentation sustains growth by pyruvate disproportionation involving pyruvate dehydrogenase. Citrate is fermented in the presence of an additional electron donor to acetate and lactate. Thus, heterofermentative LAB are able to use a variety of unusual fermentation reactions in addition to classical heterofermentation. Most of the reactions are significant for food biotechnology/microbiology.
KeywordsSugar fermentationLactic acid bacteriaHeterofermentativeOrganic acids
Sugars are the characteristic substrates for lactic acid bacteria (LAB). Homofermentative LAB ferment hexoses by the reactions of glycolysis and lactate dehydrogenase to lactic acid. At slow growth and low glycolytic flux rates the homofermentative bacteria shift to mixed acid fermentation with formate, acetate, ethanol, and lactate as the products. The change is caused by regulation of the lactate dehydrogenase and pyruvate formate lyase activities, which are subject to control by the catabolic and anabolic flux rates and changes in the NADH/NAD ratios (Melchiorsen et al. 2002; Garrigues et al. 1997, 2001). Overall, limiting catabolism leads to mixed acid fermentation and nonlimiting catabolism with high glycolytic flux to homofermentative metabolism.
Heterofermentative LAB play an important role in production, preservation, and improvement of food, mainly of plant origin (for reviews, see Steinkraus 1993; Ross et al. 2002; Caplice and Fitzgerald 1999; Mills et al. 2005; Moreno-Arribas and Polo 2005). The products of the fermentation of sugars and organic acids are essential in this process. Sugars are the preferred substrates of the heterofermentative LAB. Heterofermentative LAB grow in plant-associated environments like degrading plant material (Leuconostoc strains), grape must and wine (Oenococcus oeni, Lactobacillus hilgardii, Lactobacillus brevis) or sourdough (Lactobacillus sanfranciscensis and Lactobacillus fermentum) (Rodas et al. 2005; Randazzo et al. 2005). The same biotopes also house facultatively heterofermentative LAB like Lactobacillus plantarum and Lactobacillus pentosus. The plant material contains, in addition to hexoses (glucose, fructose, mannose, and lactose), considerable amounts of pentoses (xylose and ribose). Hexoses and pentoses are fermented by the heterofermentative LAB by the phosphoketolase (or oxidative pentose–phosphate) pathway. Excess hexoses are also used for the production of exopolysaccharides like dextran, levan, and fructan by various groups of LAB (Cerning 1990). A second class of substrates fermented by many heterofermentative LAB is organic acids, which is common in plant material, and arginine. Thus, malate, citrate, or pyruvate are fermented either on their own or as a cosubstrate with hexoses (for reviews, see Mills et al. 2005; Salou et al. 1994; Lolkema et al. 1995; Wagner et al. 2005).
The review deals with peculiarities of the carbon metabolism of obligately heterofermentative LAB. Heterofermentative fermentation of hexose includes limiting steps, which are overcome by modifications of the fermentation reaction or by the use of electron acceptors like fructose, O2, pyruvate, or citrate. In the same way, fermentation reactions of organic acids and of arginine will be discussed. The role of the fermentation reactions for biotechnology will be addressed. Only obligately heterofermentative bacteria will be considered with emphasis on Leuconostoc mesenteroides and O. oeni.
Special features of hexose fermentation by O. oeni and L. mesenteroides
Fermentation and phosphoketolase pathway
Fermentation balances and growth parameters of O. oeni on sugars (with and without electron acceptors) under various conditions
ATP/gluc or ribosea (calculated)
→1 Lac+1 ace
→1 Lac+1 EtOH+1 CO2
→1 Lac+1 ace+2 Mtl+1 CO2
→3 Lac+1 ace+1 CO2
→3 Lac+3 ace+1 3CO2
→ 1 Lac+1 ace+2 H2O2+1 CO2
Studies with O. oeni, L. mesenteroides, and other heterofermentative LAB showed that the slow growth on glucose is caused by the low activity of the ethanol pathway in the reoxidation of the extra two NAD(P)H (Maicas et al. 2002; Richter et al. 2001, 2003a). The key enzymes of the phosphoketolase pathway including glucose-6P dehydrogenase and 6P-gluconate dehydrogenase have activities, which are distinctly above the activities required for maintaining glucose fermentation (Richter et al. 2001). The acetaldehyde dehydrogenase activity of the bifunctional acetaldehyde/ethanol dehydrogenase of the ethanol pathway, on the other hand, has activities, which are only at the same level, or lower, as the rate of glucose fermentation. When the cellular contents of HSCoA, and thus of acetyl-CoA, were limiting, the activity of acetaldehyde dehydrogenase dropped further by a factor of approx. 2 and limited catabolism and growth. Limitation of HSCoA (and consequently acetyl-CoA) can be produced by shortage of d-pantothenate (Richter et al. 2001), an essential precursor of HSCoA in O. oeni and other LAB (Garvie 1967; http://www.jgi.doe.gov). The result is a decrease in production of ethanol and a partial shift to alternative, HSCoA-independent production of erythritol (Veigha-Da-Cunha et al. 1992, 1993; Richter et al. 2001) (Fig. 1). The ethanol/erythritol shift is reversed by the supply of sufficient d-pantothenate (Richter et al. 2001). During growth on C5-sugars like ribose, when the ethanol pathway is not required, d-pantothenate depletion has no significant effect on the fermentation pattern. Overall, the results show that the reoxidation of the extra two NAD(P)H from glucose oxidation in the ethanol pathway limits heterofermentative growth on glucose due to low acetaldehyde dehydrogenase activity.
d-Pantothenate as a specific growth factor for hexose fermentation
d-Pantothenate is required as a growth factor and precursor for HSCoA biosynthesis in many LAB (Garvie 1967). HSCoA is needed for the activation of fatty acids in biosynthetic and catabolic pathways. Ethanol formation from acetyl-CoA in the heterofermentative pathway is inhibited during d-pantothenate (and HSCoA) limitation, before general growth and cell synthesis is affected (Richter et al. 2001). It was suggested that 4′-O-(β-d-glucopyranosyl)-d-pantothenic acid, which is found in tomato and fruit juice (“tomato juice growth factor”), is the actual growth factor (Amachi et al. 1970). A minimal concentration of 50 μg/l was required for efficient growth on glucose. The glycosylated d-pantothenate could be produced from uridine 5′-diphosphate-glucose by plant glycosyltransferases. d-Pantothenate, however, has the same biological activity (50 to 80 μg/l) (Richter and Unden, unpublished report). O. oeni and L. mesenteroides lack the gene products for the synthesis of d-pantothenate from the precursors 2-acetolactate, l-valine, and β-alanine, which explains the need for d-pantothenate (http://www.jgi.doe.gov). The genes encoding the enzymes for the subsequent conversion of d-pantothenate to HSCoA are present with the exception of a structural gene for a PanF type pantothenate permease. d-Pantothenate therefore plays a specific role in the heterofermentative metabolism of O. oeni and other LAB (Richter et al. 2001).
Endogenous alternative pathways for [H] reoxidation
Heterofermentative LAB use alternative pathways for reoxidizing [H] to bypass the limiting ethanol pathway. Part of the extra NAD(P)H can be consumed by reduction of erythrose-4P to erythritol-4P and formation of erythritol as an end product (Fig. 1). Erythritol fermentation was shown in O. oeni, L. mesenteroides, and L. sanfranciscensis (Veigha-Da-Cunha et al. 1993; Stolz et al. 1995; Richter et al. 2001). Erythrose-4P is obtained (together with acetyl-P) from fructose-6P by d-xylulose 5-phosphate/d-fructose 6-phosphoketolase (Xfp). Various heterofermentative LAB and Bifidobacterium are able to use fructose-6P as a minor substrate for phosphoketolase, in addition to the principle substrate xylulose-5P (Veigha-Da-Cunha et al. 1993; Meile et al. 2001; Yin et al. 2005; Mills et al. 2005). The activity of the erythritol pathway is low and does not significantly increase the rate of glucose fermentation (Richter et al. 2001). Under pantothenate limitation or in resting cells up to 0.2 mol erythritol are formed per mole glucose. The residual enzymes of the erythritol pathway (erythritol-4P dehydrogenase) were not identified, and in the genomes of O. oeni and L. mesenteroides there are no genes with similarity to the eryB and eryA genes of Brucella abortus encoding erythritol-1P dehydrogenase and erythritol kinase (Sangari et al. 2000; Sperry and Robertson 1975). Glycerol which is also a minor alternative product of NAD(P)H reoxidation, is obtained by reduction of glyceraldehyde-3P to glycerol-1P followed by dephosphorylation (Fig. 1) (Veigha-Da-Cunha et al. 1993). Candidate genes encoding glycerol-1P dehydrogenase and phosphatase are found in the genome of O. oeni and L. mesenteroides (http://www.jgi.doe.gov) (Fig. 1). Biochemically, the pathway resembles that of erythritol formation, and erythritol and glycerol could be formed by the same or similar enzymes.
Cofermentation of hexoses with electron acceptors
When fructose is present as the only hexose, it serves both as a substrate for the phosphoketolase pathway and as an electron acceptor (Richter et al. 2003a,b). Under fructose limitation most of the fructose is fermented by the heterofermentative pathway similar to glucose (Table 1). However, under fructose excess conditions and in resting cells, up to two to three of the fructose are used as electron acceptor and large amounts of mannitol are produced. Therefore, fructose is fermented either by the phosphoketolase or by a combined phosphoketolase/mannitol pathway. The heterofermentative pathway yields one ATP per hexose, whereas in the phosphoketolase/mannitol pathway the ATP yield decreases to two to three ATP per hexose. Channeling of fructose in either pathway is regulated at the level of phosphoglucose isomerase by 6P-gluconate and erythrose-4P (Richter et al. 2003b). Mannitol dehydrogenase from Leuconostoc pseudomesenteroides and other bacteria can be used, together with an appropriate electron donor, to produce d-mannitol from fructose in metabolically engineered bacteria (Gaspar et al. 2004; Kaup et al. 2003; Hahn et al. 2003).
Overall, growth of O. oeni and other heterofermentative LAB on hexoses is stimulated by the presence of electron acceptors, which provide a by-pass for NAD(P)H reoxidation in the ethanol pathway. The erythritol and glycerol pathways are only of limited capacity, whereas the pathways using the external acceptors O2, pyruvate, citrate, and fructose have a much higher capacity and significantly increase the growth rate of the bacteria. The redox reactions are cytoplasmic without involvement of electron transport or generation of a proton potential.
Growth by fermentation of organic acids and arginine
Fermentation of organic acids plays an important role in the energy metabolism of heterofermentative LAB (Radler 1958, 1966; Radler and Brohl 1984; Stolz et al. 1995). Fermentation of citrate and malate is widespread under LAB, whereas fermentation of fumarate and pyruvate is found only in some bacteria.
Malate (“malolactic”) fermentation
Many LAB, including O. oeni and L. mesenteroides, use citrate as substrate (electron acceptor) for cometabolism with sugars like glucose, fructose, lactose, or xylose providing NADH (citrate+2 [H]→lactate+acetate+CO2) (Salou et al. 1994; Schmitt et al. 1997; Hache et al. 1999; Starrenburg and Hugenholtz 1991; Drinan et al. 1976) (see Fig. 2). Only few LAB are able to grow on citrate as the sole substrate (Medina de Figueroa et al. 2000). Transport of citrate and of the products lactate and acetate is central to the function of citrate fermentation (Fig. 3). Citrate uptake is catalyzed by the CitP or MaeP carrier in an electrogenic precursor/product (Hcitrate2−/lactate−) antiport, which generates an electrochemical gradient over the membrane (Ramos et al. 1994; Marty-Teysset et al. 1995, 1996; Konings 2002). The electrochemical gradient is (similar to that from malolactic fermentation) not sufficient for growth of the bacteria.
The product of citrate cleavage, oxaloacetate, is decarboxylated by soluble oxaloacetate decarboxylase, which is related to malate decarboxylase, yielding pyruvate (Marty-Teysset et al. 1996; Mills et al. 2005; Sender et al. 2004). Pyruvate is used as electron acceptor for NADH from hexose oxidation. Thus, citrate fermentation depends on the presence of an electron donor, i.e., hexose, which supplies NADH for pyruvate reduction. The lactate produced in this way is important for efficient driving of the citrate/lactate antiport (Salou et al. 1994; Konings 2002).
Part of the pyruvate derived from citrate is converted to acetoin and 2,3-butanediol by condensation of two molecules of pyruvate to 2-acetolactate (Fig. 4), followed by decarboxylation to acetoin and reduction to 2,3-butanediol (Ramos et al. 1994; Nielsen and Richelieu 1999). 2-Acetolactate can be converted to diacetyl by decarboxylation and chemical (nonenzymatic) oxidation with O2. Channeling of pyruvate into the acetoin pathway is significant in LAB when the bacteria are incubated with citrate in the absence of other carbon sources. In the presence of excess NADH (e.g., from hexoses) the pyruvate is reduced essentially to lactate (Ramos et al. 1994). 2-Acetolactate is the source of diacetyl, a flavor compound in wine and other products treated by LAB (Mills et al. 2005; Nielsen and Richelieu 1999; Schmitt et al. 1997; Bartowsky and Henschke 2004).
The cit gene clusters of L. mesenteroides and O. oeni comprise genes for citrate lyase (citDEF), citrate lyase ligase (citC), oxaloacetate decarboxylase (mae) and the citrate carrier (maeP or citM) (Mills et al. 2005; Martin et al. 2000, and references therein). The clusters also contain the citX and citG genes, which are homologous to the corresponding genes of Klebsiella for the synthesis of the phosphoribosyl-dephospho-SCoA prosthetic group of citrate lyase (Schneider et al. 2002). Putative genes for acetolactate synthase and acetolactate decarboxylase are found in O. oeni and L. mesenteroides (Fig. 4).
Arginine fermentation by the arginine deiminase pathway (1 arginine→1 ornithine+1 CO2+1 NH3) yields one ATP per arginine from carbamoyl-P by substrate level phosphorylation. The pathway includes an arginine/ornithine antiporter (ArcD), arginine deiminase (ArcA), ornithine carbamoyl transferase (ArcB), and carbamate kinase (ArcC). The fermentation is found in obligately heterofermentative LAB as L. sanfranciscensis, some strains of O. oeni, L. buchneri, L. hilgardii, but also in the facultatively heterofermentative L. plantarum (Arena et al. 2002; De Angelis et al. 2002; Spano et al. 2004; Mira De Orduna et al. 2001; Tonon and Lonvaud-Funel 2000; Mills et al. 2005). For some of the bacteria the respective genes, enzymes, and fermentation products were demonstrated. Grape must and wine contain significant amounts of arginine, and arginine fermentation supports viability and growth of the bacteria (Mira De Orduna et al. 2001; Tonon and Lonvaud-Funel 2000), but growth rate and yield is low. Formation of NH3 and of citrulline is undesirable in wine due to the increase in pH and the formation of potentially toxic derivatives.
Overall, the heterofermentative LAB show during growth on hexoses a considerable number of variations in the classical phosphoketolase pathway, which serve to by-pass the rate limiting ethanol pathway for NAD(P)H reoxidation. The alternatives include the formation of small amounts of acetate plus erythritol or glycerol. In the presence of external electron acceptors like fructose, O2, pyruvate, or citrate, the extra NAD(P)H is transferred to the acceptors, resulting in the excretion of large amounts of acetate, which is no longer reduced to ethanol. At the same time the growth rates of the bacteria increase due to omission of the limiting pathway. In addition, the bacteria are able to ferment various organic acids (citrate, malate, and pyruvate) and arginine, but only part of the organic acids support growth when present as the sole substrate.
The work in the authors’ laboratory was supported by Innovationsstiftung Rheinland-Pfalz (Grant no. 15202-38 62 61/675).