Factors influencing the utilisation of l -malate by yeasts

FEMS MicrobiologyLeners 72 (1990) 17-22 Published by Elsevier

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FEMSLE04163

Factors influencing the utilisation of L-malate by yeasts Susan B. Rodriguez and Roy J. Thorntc,n

Depart~nt of Microbwlog~and Genetics, Ma~seyUniversity, Palmerston North. New Zealand Received9 May 1990 • Accepted4 June 1990 Key words: Malate utilisation; Yeasts; Catabolite repression

1. SUMMARY The utilisation of L-malate and the effect of glucose concentration on malate utilisation under semi-anaerobic conditions were investigated in three yeasts unable to grow on malate as sole carbon source (Saccharomyt~s cereeisiae, Schizosaccharnmyees malidevorans, Zygosaccharomyces bailii) and two yeasts able to utilise the TCA cycle intermediate as sole carbon source (Pichia stJpitis and Pachysolen lannophilus). Utilisation of malate by both Schiz. malidevorans and Z. bailii was reduced at high and low levels of glucose. In the absence of glucose, P. slipitis and Pa. lannophilus utilised malate rapidly; however, their utilisation was drastically reduced in the presence of glucose, suggesting that malatc utilisation is urtder catabolite repression.

2. INTRODUCTION Yeasts have been divided into two classes based on their ability to utihse exogenously supplied tricarboxylic acid (TCA) cycle intermediates as

CorresFondence to." S.B, Rodrigue~ Present address: Biolechnology Division,Department of Scientificand Industrial Research, PalmerstonNorth, New Zealand.

sole source of carbon and energy [11. Since the TCA cycle is intact in those yeasts unable to grow on these organic acids, Barnett and Kornberg [1] postulated the existence of "permeability barriers' to TCA cycle intermediates in the " K " - yeasts. The K - yeasts, Saccharomyces cereoisiae, Zygosaccharomyces bailii and Schizosaceharomyces pombe, can metabolise the TCA cycle intermediate, malic acid, if glucose is present [2-4]. K + yeasts can utilise one or more of the TCA cycle intermediates as sole carbon and energy source. The utilisation of L-malate by yeasts has been investigated mainly in the context of winemaking, since malate depletion is important for wine deacidification. One study of 40 strains of Saccharomyces reported 3-45% utilisation of malate in grape juice 15]. In a survey of 300 strains of Saccharomyces, utilisalion of malate in a synthetic medium at pH 3,0 ranged from insignificant to 40%, with the majority utilising approximately 20% [2]. Species of Schizosaeeharomyces utifise 25-100% of the malate in grape juice [5,6]. Z. bailii can completely utilise malate [31. Under winemaking or anaerobic conditions, malate is converted to ethanol and CO2 [7], The enzyme in yeasts responsible for the first step of malate degradation, malic enzyme (EC 1.1.1.38 or 40) converts malate to pyruvate and CO2. Enzymes of the alcoholic fermentation pathway,

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pyruvate decarboxylase and alcohol dehydrogenase, convert pyruvate to acetaldehyde and finally to ethanol. Malie enzyme is constitutive in Z. bailii [3] and S. cerevisiae [8], and inducible in Sehiz. pornbe [9]. Transport of malate by K - yeasts has been investigated in Z. bailii [10], S. ~revisiae [I0] and Schiz. pombe [11]. Evidence icom these studies indicates that malate enters S. cerer,isiae by passive diffusion and is transported into Z. bailii and Schiz. pombe by facilitated diffusion and active transport, respectively. Thus. in two K - yeasts, the cell membrane is not a permeability barrier for malate. It is difficult to draw conclusions from previous studies on malate utilisation by K - yeasts since the experiments were carried out under different conditions of cultivation. Also, the physiological basis for the difference between K - and K + yeasts has not been determined. In the present study, we compared the ability of three K - and two K + yeasts to utilise malate under similar conditions of cultivation, and we examine the effect to glucose concentration on malate utilisation in the two types of yeasts.

3. MATERIALS A N D METHODS 3.1. Yeast strains Pichia stipitis N R R L Y-7124 (M. Miranda, Department of Food Science and Technology, University of California, Davis); Schizosaccharomyces malidevorans 442 (B. Rankine, Australian Wine Research Institute, Adelaide); Saccharomyces cerevisiae MD26 (Montana Wines, New Zealand); Zygosaceharomyces bailii RIB0 (R. Eschenbruch, DSIR Research Station, Te Kauwhata, New Zealand). Pachysolen mnnophilus 2530 (A. James, National Research Council, Ottawa, Canada). 3.2. Media MYGP contained 3 g / l malt extract, 3 g / l yeast extract, 10 g/1 glucose and 5 g / I peptone. Minimal malate glucose (MMG) medium contained 100 g / l glucose, 3 g / l L-malic acid and 6.7 g / l Difco yeast nitrogen base without amino acids. The pH was adjusted to 3.0 with KOH, Minimal

malate (MM) medium was M M G medium in which the glucose was omitted. A mixture of amino acids, adenine and uracil was added to M M G medium for Schiz. malidevorans cultures to prevent ]ysis in late exponential phase. 3.3. Culture conditions Inocula were grown in MYGP broth for 24-36 h at 25°C. Viable cell numbers were determined using a hemacytometer with a methylene blue stain [12]. Experimental cultures were inoculated at a viable cell concentration of 105 per ml. Cultures were grown semi-anaerobically, i.e. 100 ml medium in 250 mi flasks with fermentation traps. Flasks were incubated at 2 5 ° C with gentle shaking (100 rpm) to keep cells suspended. Duplicate cultures were used for all experiments. 3. 4. Analyses L-Malate and glucose concentrations of cultures were determined by HPLC analysis [13].

4. RESULTS A N D DISCUSSION In the presence of high concentrations of glucose, Schiz. malidevorans and Z. bailii utifised substantial amounts of malic acid, 2.4 g / l (18 mM) in both cases (Figs. 1, 2). Both Z. bailii and Schiz. malidevorans utilised malate and glucose simultaneously, although the latter did so more rapidly than the former. S. cerevisiae utilised 1.2

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Fig. 4. Glucose and malate utilisation by P. stipiris NRRL Y-7124 in MMG. The values represent averages of duplicate cultures. MMG medium contains glucose and malate; MM medium contains only malate.

g / l (9 mM) of malic acid in 8 days, and only a small amount was utilised after the glucose in the medium was depleted (Fig. 3). The difference in degree of malate utilisation between S. cerevisiae and the other two K - yeasts is compatible with the systems of malate transport in these three yeasts• Malate enters cells of S. cerevisiae by passive diffusion [1O]. In Z. bailii, inducible malate transport occurs by facilitated diffusion [10]. In cultures of Z. ba#ii gzowing on glucose and malate, the intracellular concentration of malate is 10-15 times higher than that of S. ceree/siae [3], Malate enters Sehiz. pombe, and presumably its close relative, Schfz. malidevorans, by active transport [11]. The medium in this study was adjusted to pH 3.0. The maximum rate of transport of malate in Z. ~JliJ occurs at approximately pH 3.0 [10], that

in Schiz. pombe, at approximately 3.5 [11]. Since the pK~t and pK,2 of malate are 3.40 and 5.11, respectively, most of the acid would be in the undissoci,~ted form in M M G and MM medium. The maximum rate of diffusion of malate through the cell membrane would be expected when the acid was in the undissociated form, although undissociated dicarboxylic acids diffuse across the cell membrane very slowly as compared with undissociated monocarboxylic fatty acids [14]. Thus, a relatively high rate of diffusion of malate into ,5'. cerevisiae would he expected at pH 3.0. A mutant of Schiz. malidevorans demonstrates the importance of the relationship between the dissociation constant of an acid and its movement across the cell membrane: the mutant is dependent on malate

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Fig. 6. Effect of glucoseconcentration on L-nlalateutilisation by Schiz. malidevorans 442. The values represent averagesof duplicatecultures. (D. glucose;lit malale).

Fig. 8. Effectof glucoseconcentraliorloil L-malaleutilisation by S. cerevisiae MD26. The values represent averages of duplicatecultures. {~2,glucose;I1, malate).

as well as glucose for growth and cannot grow at a pH value above 5.2 [13]. A decrease in malate utilisation was observed at the lowest glucose concentration (28 raM) in Schiz. malidevorans and Z. bailii when the glucose concentration of the MMG medium was varied (Figs. 6, 7). Varying the glucose concentration did not affect malate utilisation in cultures of S, cerevisiae (Fig. 8). A glucose effect would not be expected in S. cerevisiae since malate enters this yeast by passive diffusion and the malic enzyme is constitutive. Glucose dependence of malate utilisation in Schiz. malidevo-ans may be a requirement for an energy source since transport is probably active and constitutive as it is in Schiz.

pombe. This suggestion was supported by the observation that Schiz. malidevorans utilised a similar amount of malate when grown in M M G in which fructose was substituted for glucose. The glucose dependence of malate utilisation in Z. bailii may be function of the malate transport protein being induced by growth on glucose [10]. in this study, Z. bailii was also grown in MMG medium substituted with five alternative carbon sources: fructose (555 raM), mannose (555 raM), galactose (555 raM), glycerol (1086 mM) and ethanol (434 raM). As has been previously observed [10], malate was poorly ulilised when Z. bailii, a fructophilic yeast, was grown on fructose instead of glucose, 38% vs. 89% utilisation, in MM + mannose, MM + galactose, MM + glycerol, and MM + ethanol, Z. bailii utilised 42, 66, 69 and 83"7o respectively of the malate. Similar amounts of malate are used in fructose and mannose cultures. In S. cerevisiae cultures, 44% of the malic acid (560 mM glucose) was utilised. Since malate is known to diffuse into S. eerevisiae and into fructose-grown Z. bailii cells, it appears that it also does so in mannose cultures of 2"-. bailii. The malate transport protein appears to be induced by growth on galaetose and glycerol as well as glucose. Ethanol may increase the utilisation of malate by changing the permeability of the cell membrane. The K + yeast, P. stipitis, did not significantly utilise malic acid (0.3 g/1, 2.2 mM) in the presence

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of a high concentration of glucose (Fig. 4). In fact. P. stipitis utilised less malate than S. cerevisiae in which malate enters the cell by diffusion, In the presence of glucose, Pa. tannophilus utilised less malie acid than Z. bailii and Schiz. malidevorans, but more than 5. eereoisiae (1.9 g/l, 14 mM) (Fig. 5). A difference in degree of repression of malate utilisation was observed in the two K + yeasts (Fig. 9, 10). In the presence of 448 mM glucose, malate utilisation was 90% repressed in P. stipitis and 55% repressed in Pa. tannophilus. The difference m'~y b,~ due to transport: possibly subject to tatabolite repression in P. stipitis and not in Pa. tannophilus. Thus, when glucose is present, malate cannot enter P. snpitis except by passive

diffusion, but it can be transported into Pa. tannophilus. The complete utilisation of malate in the absence of glucose can be explained by catabolite derepressed transport in P. stipitis and a eatabolite derepressed malie enzyme in Pa. tannophilus. Varying the glucose concentration in the medium revealed a concentration (112 mM) below which malate utilisation, possibly the malic enzyme, in Pa. tannophilus was derepressed (Fig. 10). The critical level of glucose for derepression of malate transport in P. stipitis was not observed at the lowest glucose concentration (28 mM) used in this study (Fig. 9). The utilisation of L-malate by yeasts has been studied due to the importance of malate depletion in winemaking. Since the wine yeasts of most interest are K - yeasts, little is understood about malate atilisation by K + yeasts, Catabolite derepressed mutants of the K + yeasts, P. sttpitis and Pa. tannophilus, have been isolated and are currently under investigation. We suggest that the source of the difference between K - and K + yeasts may be found in parallel evolutionary development. The cell types which were able to selectively utilise only the most favorable carbon and energy source available had a growth advantage over other cell types. Thus, a system of repression of utilisation of relatively unfavorable energy compounds by glucose or one of its metabolites evolved in the K + yeasts. A system of permeability barriers which prevents access to the dicarboxylate metabolizing enzymes evolved in the K - yeasts.

ACKNOWLEDGEMENTS

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S.B.R. ackaowledges the award of a New Zealand University Grants Committee Postdoctoral Fellowship which supported this research. We are grateful to Rosanne Eustace for technical assistance.

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[2] Fuck, E, and Radler, F. (1972) Arch. MikrobioL 87, 149-164. [3] Kuczynski, J.T. and Radler. ~'. (1982) Arch. Microbiol. 131,266-270. {4] Temperli, A., Kunsch, U., Mayer. K.D, and Busch, L (1965) Biocldm. Biophys. Acta 110, 630-632. [5] Rankine. B.C. (1966).I. Sci, Food Agrlc 17. 312-316. [6] Peynaud, E., Domercq. S., Boidron. A.-M.. LafanLafourcade. S. and Guimbcrteau, G. (1964) Arch. Mikrobiol. 48, 150-165. [7] Mayvr, K. and Temperli, A. (1963) Arch. MikrobioL 46, 321-328. [8] Fuck, E.. Stark, G. and Radler, F. (1973) Arch. Mikrobiol. 89. 223-231.

[9] Osothsilp, C. and Subden, R.E. (1986} Can. J. Microbiol. 32, 481-486. [10] BaranowskL K. and Radler. F. (1984} Antoni¢ van Leeuwenhoek 50, 329-340. []1] Osothsilp. C. and Suhden, R.E. (]986) J, Bacteriol. 168, 1439-1443. [12] Anon. (1981) Report of the subcommittee on microbiology. J. Am. Soc. Brew. Chem. 39, 86-89. [13l godfiguez, S.B. and Thornton. RJ. (1989) Arch. Microbiol. 152. 564-566. [14] Suomalainen, H, and Oura. E. {1971) In: The Yeasts, Vol. 2 (Rose. A,H. and Harrison. J.S., eds.), pp. 36-48, Academic press, New York.