Aug 16, 1990 - Utilization of L-malic acid by yeast strain Hansenula anomala IGC 4380 is subject ... behavior of mutant strains of H. anomala derepressed with.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1990, p. 3402-3404
Vol. 56, No. 11
0099-2240/90/113402-03$02.00/0 Copyright C 1990, American Society for Microbiology
Inverse Diauxy in the Yeast Hansenula anomala: Mutants Derepressed for Malic Acid Utilization in the Presence of Glucose MANUELA CORTE-REAL,' C. LEAO,1* AND N. VAN UDEN2 Laboratory of Biology, University of Minho, 4719 Braga Codex,' and Laboratory of Microbiology, Gulbenkian Institute of Science, 2781 Oeiras Codex,' Portugal Received 3 April 1990/Accepted 16 August 1990
Utilization of L-malic acid by yeast strain Hansenula anomala IGC 4380 is subject to glucose repression. Derepressed mutants were obtained with UV light by use of the nonmetabolizable glucose analog 2-deoxyglucose as a selective agent. Three mutant strains degraded L-malic acid in the presence of up to 30% (wt/vol) glucose and are of potential interest for the biological deacidification of grape must. The mutant strains, as compared with the parent strain, displayed inverse diauxy in glucose-malate medium, glucose being metabolized only after malate consumption had been completed.
lizable glucose analog 2-deoxyglucose at 0.4% (wt/vol) prevented growth of the parent strain on malic acid medium while inhibiting only slightly growth of the strain on glucose medium, indicating that the prevention of growth on malic acid medium was the result of repression rather than inhibition. The pH indicator bromocresol purple was used for the preliminary detection of the yeast colonies on the plates of selective medium that metabolized malic acid. The plates were irradiated with UV light during a period long enough (15 s under our conditions) to reduce the number of viable cells in the inoculum to about 15%. Incubation was at 25°C in the dark. After about 3 weeks, the plates displayed irregular areas of growth above the agar surface, with a change in color of the pH indicator from green to purple resulting from a pH increase as a consequence of malic acid utilization. Secondary plating on the same medium produced isolated colonies which produced a color change from which 30 strains were picked for further study. These strains were maintained on slants of selective medium. Test for derepression. To test for derepression, the strains were first grown on 2% (wt/vol) glucose-1% (wt/vol) peptone-2% (wt/vol) yeast extract-2% (wt/vol) agar. From each culture a small amount was transferred to a 250-ml Erlenmeyer flask containing 100 ml of MV medium with DL-malic acid (0.5%, wt/vol) and glucose at different concentrations (2 to 30%, wt/vol) (pH 5.5). Incubation was at 25°C with
The yeast Hansenula anomala is able to use L-malic acid oxidatively as the sole carbon and energy source. It does not ferment malate (7) and transports malate and other dicarboxylates across its plasma membrane by an active proton symport mechanism (2). H. anomala commonly occurs among the microbial flora of grapes and grape must and frequently participates in the early stage of wine fermentation before ethanol-resistant Saccharomyces strains become dominant (3, 4). It occurred to us that H. anomala might be used to decrease the malic acid concentration of grape must, when desirable, as an alternative to other biological deacidification methods (for a review of such methods, see reference 1). However, malic acid transport and metabolism by this yeast are repressed by glucose (2) and are therefore not operational when the yeast is growing in grape must and during early vinification. In the present study we report on the preparation and behavior of mutant strains of H. anomala derepressed with respect to the use of malic acid in the presence of glucose. MATERIALS AND METHODS Microorganism. Parent strain IGC 4380 (CBS 1982) of the yeast H. anomala, a haploid mating type, was maintained on glucose (2% [wt/vol])-peptone (1% [wt/vol])-yeast extract
(0.5% [wt/vol])-agar (2% [wt/vol]). Production of derepressed mutants. Mutant strains of H. anomala IGC 4380 resistant to glucose repression of malic acid transport and metabolism were produced by adapting methods described earlier (5, 9, 10). The parent strain was grown with shaking at 25°C in a liquid mineral medium with vitamins and 2% (wt/vol) glucose (MV medium) (8). In the mid-exponential phase, the cells were harvested by centrifugation, washed, and suspended in sterile distilled water. Of this suspension, 0.1-ml samples containing approximately 7 x 107 viable cells were spread on the surface of plates with selective medium. The selective medium was MV medium (8) without glucose and with the following additions: 2-deoxyglucose, 0.4% (wt/vol); DL-malic acid, 0.5% (wt/vol); bromocresol purple, 0.24% (wt/vol); agar, 2% (wt/vol); pH adjusted to 5.5. Preliminary experiments had shown that the nonmetabo*
shaking. After about 18 h of growth and while the glucose concentration was still high, the cells were harvested by centrifugation, washed twice with ice-cold water, and suspended in ice-cold water to a final concentration of about 25 mg (dry weight) per ml. The activity of the proton-malate symporter was tested either by measurement of the uptake of labeled L-[U-'4C]malic acid or labeled [2,3-'4C]succinic acid at a saturating concentration or by measuring proton movements associated with malate or succinate uptake. The methods used for testing malate transport were described earlier (2). Estimation of amounts of glucose and L-malic acid. The amount of glucose was estimated by the glucose oxidase method (Boehringer-Mannheim Test-Combination; Boehringer-Mannheim GmbH, Mannheim, Federal Republic of Germany). The amount of acid was estimated by the enzymatic method previously (6) described.
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FIG. 1. Fermentation profiles at pH 5.5 of wild-type strain H. anomala IGC 4380 (a) and mutant strain H. anomala IGC 4380-41 (b) in MV medium, glucose (30% [wt/vol]), and DL-malic acid (0.5% [wt/vol]). Symbols: 0, cell density (optical density at 640 nm); 0, glucose concentration (percent [weight/volume]); *, L-malic acid concentration (percent [weight/volume]); C1, relative activity of the proton malate symport measured at a saturating concentration of succinic acid (0.2 mM).
Reproducibility of the results. All experiments were repeated at least three times, and the data reported here are the average values. RESULTS AND DISCUSSION By using the parent strain as a control, the 30 presumptive mutant strains were tested for derepression with respect to malic acid utilization in the presence of glucose. Seven of the strains behaved as the parent strain did. During growth in the glucose-malic acid medium, malic acid had not been consumed and proton-malate transport activity had not developed in samples taken when the glucose concentration was about 2% (wt/vol). However, in cultures of the 23 other strains, L-malic acid disappeared from the medium and active proton-malate transport developed while glucose was present.
Four of the isolated mutant strains (IGC 4380-30, IGC 4380-39, IGC 4380-40, and IGC 4380-41) were tested for derepression of malate utilization in glucose-malic acid medium at high glucose concentrations ranging from 10 to
30% (wt/vol). In contrast to the wild-type strain and with the exception of strain IGC 4380-39, activity of the proton/ malate symport and utilization of malic acid during the first growth phase were observed in the other three strains while the glucose concentration was about 10, 15, 20, or 30% (wt/vol). Fermentation profiles were obtained for the parent strain and for the mutant strain IGC 4380-41 (Fig. 1). The parent strain displayed repressed behavior, and growth on malic acid and active malate transport were not observed in the presence of glucose (Fig. la). In contrast to the results obtained earlier with low initial glucose concentrations (2), no growth on malate occurred after the glucose had been consumed. This difference was possibly a result of the very high initial glucose concentration (30% [wt/vol]) employed since changes in the medium composition would be expected to have occurred during glucose consumption. Possible changes include production of toxic ethanol concentrations via fermentation and exhaustion of a limiting nutrient. These possibilities were not further investigated.
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The mutant strain transported and consumed malic acid in the presence of glucose (Fig. lb). Unexpectedly, glucose was not consumed simultaneously with malic acid but only after the latter had disappeared from the medium. Thus the mutant strain displayed a growth behavior which was the inverse of the behavior of the parent strain and for which we propose the name inverse diauxy, i.e., the sequential utilization for growth of malic acid and then glucose. The biochemical mechanisms underlying the observed inverse diauxy remain to be identified. From an applied point of view, one expects that strains of H. anomala with the diauxy behavior depicted in Fig. lb might be able to deacidify grape must without fermenting the sugars. We are now exploring this possibility by use of microvinifications. ACKNOWLEDGMENTS This work was supported by a research grant (contract 8719) from the Junta Nacional de Investigacao Cientifica e Tecnol6gica, Lisbon, Portugal, and grant DPE 5542-G-SS-8071-00 from the Agency for International Development, Washington, D.C. LITERATURE CITED 1. Beelman, R. B., and J. F. Gallander. 1979. Wine deacidification. Adv. Food Res. 25:1-23. 2. Corte-Real, M., and C. Leao. 1990. Transport of malic acid and
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other dicarboxylic acids in the yeast Hansenula anomala. Appl. Environ. Microbiol. 56:1109-1113. Kunkee, R. E., and M. A. Amerine. 1970. Yeasts in wine making, p. 5-60. In A. H. Rose and J. S. Harrison (ed.), The yeasts-1970, vol. 3. Academic Press, Inc., New York. Lafon-Lafourcade, S. 1983. Wine and brandy, p. 81-163. In H. J. Rehm and G. Reed (ed.), Biotechnology-1983, vol. 5. Verlag Chemie, Weinheim, Federal Republic of Germany. Laires, S., I. Spencer-Martins, and N. van Uden. 1983. Use of D-glucosamine and 2-deoxyglucose in the selective isolation of mutants of the yeast Lipomyces starkeyi derepressed for the production of extracellular endodextranase. Z. Allg. Mikrobiol. 23:600-603. McCloskey, L. P. 1980. Enzymatic assay for malic and malolactic fermentations. Am. J. Enol. Vitic. 31:212-215. Ruiz-Amil, M., G. Torrontegui, E. Palacian, L. Catalina, and M. Losada. 1965. Properties and function of yeast pyruvate carboxylase. J. Biol. Chem. 240:3485-3492. van Uden, N. 1967. Transport-limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition. Arch. Mikrobiol. 58:155-158. van Uden, N., C. Cabeca-Silva, A. Madeira-Lopes, and I. Spencer-Martins. 1980. Selective isolation of derepressed mutants of an ot-amylase yeast by the use of 2-deoxyglucose. Biotechnol. Bioeng. 22:651-654. Zimmermann, F. K., and I. Scheel. 1977. Mutants of Saccharomyces cerevisiae resistant to carbon catabolite repression. Mol. Gen. Genet. 155:75-82.
