Thiamine secretion in yeast YOUSEFHAJ-AHMAD Biology Department, Brock University, St. Catharines, Ont., Canada L2S 3AI AND
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CARLA. BILINSKI, INGERUSSELL,AND GRAHAM G. STEWART Research Department, Labatt Brewing Company Ltd., 150 Simcoe Street, London, Ont., Canada N6A 4M3 Received March 27, 1992 Accepted June 9, 1992 HAJ-AHMAD,Y., BILINSKI, C. A., RUSSELL,I., and STEWART,G. G. 1992. Thiamine secretion in yeast. Can. J . Microbiol. 38: 1156-1 161. To isolate thiamine excretors and (or) overproducers, 188 cultures belonging to nine yeast and three fungal genera were screened. Nine excreted thiamine as determined by both the presence of cross-feeding zones on a thiamine-free agar medium seeded with a thiamine-requiring yeast strain and by the direct detection of excreted thiamine on agar plates. Several of these cultures produced several-fold more intracellular thiamine than the general culture population. Thiamine-requiring strains of Saccharomyces cerevisiae and S. uvarurn (carlsbergensis)were identified and were tentatively assigned to 10 complementation groups. Key words: thiamine, yeast, thiamine secretion, thiamine requiring. HAJ-AHMAD,Y., BILINSKI,C. A., RUSSELL,I., et STEWART,G. G. 1992. Thiamine secretion in yeast. Can. J. Microbiol. 38 : 1156-1 161. Un total de 188 cultures regroupant neuf genres de levures et trois de champignons ont ete verifiees dans le but d'isoler les souches secretrices et (ou) surproductrices de thiamine. Neuf souches secretaient de la thiamine tel qu'observe par la presence de zones d'echanges nutritifs croises sur une gelose sans thiamine ensemencee avec une souche de levure thiamine-dependante ou encore par la detection directe de thiamine secretee dans la gelose. Certaines de ces cultures produisaient plus de thiamine intracellulaire que la population generale cultivee. Des souches de Saccharomyces cerevisiae et de S. uvarurn (carlsbergensis)thiamine-dependantes ont ete identifiees et assignees tentativement a 10 groupes de complementation. Mots cles : thiamine, levures, secretion de thiamine, besoin en thiamine. [Traduit par la redaction]
Introduction Thiamine (vitamin B1) is an essential factor in animal nutrition and in many microorganisms; it is found naturally in a variety of sources such as rice hulls, cereal grains, yeast, liver, eggs, milk, and green leaves (Association of Vitamin Chemists 1951). Although it was originally extracted from rice bran, thiamine currently used for dietary supplements is almost entirely derived from chemical synthesis, mainly because there is no rich source of natural thiamine. Yeast, which is the primary source of vitamin B2 (riboflavin) and B12 (cobalamin), does not produce large amounts of thiamine (Lewis et al. 1944; Lundin 1950; Bunker 1963; White and Spenser 1979). The availability of thiamineexcreting and (or) hyperproducing mutants would make it economically feasible to produce the vitamin via fermentation and would be attractive for the production of thiamineenriched beverages. Although the initial concentration of thiamine in brewer's wort can vary from 150 to 750 j~g/L (Brenner et al. 1973; Graham et al. 1970), active uptake of vitamin B1 by cells reduces this concentration to almost 0 after 48 h fermentation (Brenner et al. 1973). Final beers contain very low amounts of thiamine, ranging from 2 to 100 j~g/L(Brenner et al. 1973); the recommended dietary uptake of vitamin B1 for adults is 5 jLg/kcal. Most beers contain 400-450 kcal/L; therefore, they should be fortified by about 200 j~g/L.Unfortunately, much higher fortification would be necessary to prevent thiamine-related brain damage in alcoholics (Centerwall and Criqui 1978). The availability of thiamine hyperproducers and excretors would ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / lmprime au Canada
also be useful in the baking industry, since dough is poor in vitamin B1 as a consequence of baker's yeast propagation under aerated conditions (Fink and Just 1941a, 1941b). The present study was undertaken to evaluate various yeast genera for their ability to naturally overproduce or excrete thiamine into culture media. The approach taken has led to the identification of thiamine-excreting and thiaminerequiring yeast strains. Thiamine-requiring strains of Saccharornyces cerevisiae and of S. uvarurn (carlsbergensis) were assigned to 10 complementation groups.
Materials and methods Yeast strains and maintenance Yeast strains employed in this study were obtained from the Labatt Culture Collection and were designated according to Lodder (Lodder 1970). Cultures were maintained at 4°C on Wickerham's (Wickerham 1951) malt extract - yeast extract - peptone - glucose (MYPG) medium, which contained the following ingredients per litre of distilled water: malt extract, 3 g; yeast extract, 3 g; peptone, 5 g; glucose, 20 g; and Bacto-Agar (Difco Laboratories), 20 g. Brewer's wort (12" Plato; " Plato = grams of sucrose per 100 mL, equivalent to carbohydrate content at 20°C) sterilized by steam streaming three times for 20-min periods at 100°C was also used as a culture medium. Cross-feeding test The thiamine-requiring haploid yeast Saccharomyces strain 1588 (MATa, thi I , ura I , trp I ; obtained from YGSC strain Y02587) was cultivated in MYPG medium for 48 h at 30°C, harvested by centrifugation, and resuspended in 50% of the original volume of a thiamine-free medium prepared as described earlier (Haj-Ahmad et al. 1989). A 0.25-mL portion of the cell suspension was spread
HAJ-AHMAD ET AL.
TABLE1. Cultures tested and their thiamine content Thiamine content Medium ( m g / ~ ) b
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Species " Candida fennica (1) Candida ishiwadae (1) Candida insectorurn (1) Candida shehatae (1) Candida silvanorurn (1) Candida steatolytica (1) Candida utilis (1) Debaryornyces polyrnorphus (1) Nadsonia fulvescens (1) Endornycopsis sp. (2) Endoyrncopsis fibuligera (2) Hansenula anornala (1) Hypornyces ipornoeae (1) Kluy verornyces rnarxianus (2) Leucosporidiurn capsuligenurn (1) Paecilornyces rnarquandii (1) Paecilornyces varioti (3) Paecilornyces fulvus (1) Pichia tannophilus (1) Pichia stipitis (4) Saccharornyces bayanus (14) Saccharornyces uvarurn (carlsbergensis) (35) Saccharornyces cerevisiae ( 102) Saccharornyces diastaticus (4) Saccharornyces rouxii (1) Saccharornyces dairensis ( 1) Saccharornyces fibuligera (2) Schwanniornyces occidentalis (1)
Synthetic
Brewer's wort
15 15 14 23 18 17 22 26 35 17 15 18 11 15 17 12 15 9 17 26 17
150 134 153 125 131 138 120 159 131 128 153 122 125 125 207 150 156 134 131 125 138
14-lgd 14-201 17 17 15 15 18
125-144 119-256 125 131 128 153 125
Intracellular (mg/g)' Synthetic
Brewer's wort
3 3 3 6 5 3 5 9 3 5 3 6 2 8 5 3 3 3 3 3 5 11-24 5-27 5 12 5 3 8
aValues in parentheses are the number of strains tested using the cross-feeding test (see Materials and methods). b ~ h i a m i n econcentration in cell-free media after 72 h incubation in thiamine-free synthetic medium or in a 12" Plato brewer's wort. T h i a m i n e content in cells grown for 72 h in either a thiamine-free synthetic medium o r in 12" degree Plato brewer's wort. d ~ a l u e represent s the range of thiamine concentration in various strains tested, whereas other single values are based o n measurements carried out on a single strain.
evenly on a dried thiamine-free agar medium that consisted of the following ingredients per litre of deionized, distilled water: vitaminfree Yeast Base (Difco Laboratories), 16.7 g; myoinositol, 2 mg; calcium DL-pantothenate, 0.4 mg; D-biotin, 2 mg; and BactoAgar, 20 g. Individual colonies to be screened were transferred by applicator sticks to thiamine-free agar medium seeded with thiamine-requiring yeast strain 1588. After 3-5 days incubation at 30°C, plates were examined for the presence or absence of crossfeeding zones. Direct measurement of excreted thiamine In addition to the above assay, thiamine excreter yeast colonies were identified using a direct method (Haj-Ahmad et al. 1989). Briefly, yeast colonies to be screened were either grown on (or replica plated on) thiamine-free agar plates. After a day or two of incubation at 30°C, the colonies were removed and excreted thiamine was measured directly on these plates as described by Haj-Ahmad et al. (1989). Colony growth complementation tests Strains found to be thiamine requiring were grown on complete medium and spread individually on thiamine-free agar plates as described above. Subsequently, an inoculum of each thiamine-
requiring yeast strain was transferred to each of the seeded test plates. After 2-3 days incubation at 30°C, the growth of each colony was evaluated by visual inspection and recorded as + (growth) or - (nongrowth). Extraction of thiamine Thiamine was extracted as described by White and Spenser (1979). Cells grown 72 h in 50 mL of culture medium (thiaminefree MYPG and brewer's wort) were harvested by centrifugation at 2000 rpm for 20 min at 4"C, washed in distilled water, and resuspended in 1 mL of 0.1 M HCl. After 30 min acid hydrolysis in a steaming water bath, the samples were centrifuged at 3000 rpm for 10 min. Pellets were given a further acid hydrolysis treatment as above, except with 2 mL of 1.1 M HCl. Supernatants from each round of hydrolysis were pooled and combined with 1.5 mL of 1 M sodium acetate (pH 4.7). Following adjustment to pH 4.7 with 50% (w/v) NaOH, thiamine pyrophosphate was hydrolyzed for 12 h at 30°C in the presence of 5 mg of Clarase concentrate (Miles Laboratories, Inc.). Thiochrorne assay Thiamine was measured quantitatively using a thiochrome assay (Fujiwara and Matsui 1953; Linnett and Walker 1968) as described
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CAN. J. MICROBIOL. VOL. 38, 1992
FIG. 1. A cross-feeding test for thiamine excretion. (A) Excretor colony surrounded by a zone of microcolonies (crossfeeding zone). (B) Nonexcretor colony. by Haj-Ahmad et al. (1989). Briefly, 2 mL of cyanogen bromide (prepared by titrating saturated bromine solution on ice with 20% KCN until colorless), followed by 1 mL of 50% w/v NaOH, was added to 1 mL of treated cell extract. Fluorescent product was extracted by addition of 5 mL of 2-methyl-propan-1-01 (Fisher Scientific). After 2 min of vigorous shaking and centrifugation for 30 s at low speed, fluorescence in the organic phase was measured at 420 nm using a spectrophotofluorometer (G.K. Turner, Model 430) with an excitation wavelength of 370 nm. Thiamine concentrations were determined from a standard curve prepared for each trial with thiamine solutions ranging from 1.2 to 5 mg/mL. The same procedure was employed to quantitate thiamine concentrations in cell-free supernatants.
Results Isolating thiamine excretors Of 188 cultures (Table 1) screened by the cross-feeding and direct assays three gave cross-feeding zones (see Fig. 1) after 5 days incubation; after an additional 1 week at room temperature, cross-feeding zones were evident around an additional six colonies. Fluorescent zones were also visible when the direct test was employed. All nine cultures were designated putative "thiamine excretors" (Table 2). The size of a zone is directly proportional to the amount of thiamine excreted (Haj-Ahmad et al. 1989). In addition, a second test
duced n o zone of microcolonies nor any apparent fluorescence 034).
(Haj-Ahmad et al. 1989) directly detected excreted thiamine on the agar plates (Fig. 2). Zntra- and extra-cellular thiamine Since the intracellular thiamine level in yeast is growthstage dependent, thiamine measurements were always made in cells under comparable physiological conditions. To quantitatively determine thiamine biosynthesis, cells were initially grown overnight in 2 mL of thiamine-free synthetic medium; 1 mL of this culture was used to inoculate 100 mL of thiamine-free medium. After 72 h incubation, 10 mL of the cultures were used to determine the biomass; the remaining 90 mL was used to determine the intracellular thiamine concentration. The extracellular thiamine concentration was measured in triplicate directly in a cell-free medium. Parallel experiments in brewer's wort were carried out to evaluate the yeast cells' behavior vis-a-vis the thiamine level under natural brewing conditions. Data in Tables 1, 2, and 3 indicate that, on average, cells grown in brewer's wort possess approximately 10-fold more intra- and extra-cellular thiamine than cells grown in a thiamine-free synthetic medium. This is expected because brewer's wort is naturally rich in thiamine. In a synthetic medium there was on average sixfold (range from 1 to 16 times) more extracellular (average of 69.2 mg/L) than intracellular (average of 13.0 mg/g) thiamine in thiamine
HAJ-AHMAD ET AL.
TABLE2. Thiamine excretor yeastsa
Thiamine content Medium (mg/L)
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Species
Culture collection No.
Synthetic
Brewer's wort
Intracellular (mg/g) Synthetic
Brewer's wort
Candida shehatae Debaryomyces polymorphus Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Sch wanniomyces occidentalis "Synthetic medium was thiamine-free medium supplemented with 4% glucose. Values given refer to thiamine concentration present after 72 h incubation at 30°C,in media (mg/L) and within cells (mg/g) dry weight.
TABLE3. High thiamine producing strainsa
Thiamine content Medium (mg/L) Species Saccharomyces uvarum (carlsbergensis) Saccharomyces uvarum (carlsbergensis) Saccharomyces uvarum (carlsbergensis) Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae
Intracellular (mg/g)
Culture collection No.
Synthetic
Brewer's wort
Synthetic
Brewer's wort
1234
14
134
24
62
1238
17
125
14
48
1240
14
144
14
47
1161
20
178
12
56
1221
14
119
27
66
1244
15
138
20
42
1248
23
128
23
47
"Synthetic medium was thiamine-free medium supplemented with 4% glucose. Values given refer to thiamine concentration present after 72 h incubation at 30°C in media (mg/L) and within cells (mg/g) dry weight.
excretor yeast (Table 2), whereas in the high thiamine producing yeast (Table 3) there was slightly more intra(19.1 mg/g) than extra-cellular (16.7 mg/L) thiamine (mean of 1 and a range of 0.6-2 times). In brewer's wort, there was on average threefold (range of 2-7 times) more extrathan intra-cellular thiamine, irrespective of the thiamine excretion phenotype. Isolating thiamine hyperproducers To identify thiamine overproducers, individual colonies were transferred to three different agar media: (i) complete;
(ii) thiamine-free; and (iii) thiamine-free seeded with a lawn of thiamine-requiring yeast (strain 1588). After 3-5 days incubation at 30°C, the size of each individual colony was compared within and among the three media. On complete agar plates, all colonies grew to approximately the same size within 3 days (data not shown), whereas colony sizes varied on the thiamine-free agar plates. Colonies that grew at a faster rate on thiamine-free agar plates were predicted to overproduce thiamine and were picked for quantitative measurement of their thiamine content (Table 3). The prediction was correct but only for S. cerevisiae and S. uvarum
CAN. J. MICROBIOL. VOL. 38, 1992
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TABLE 4. Thiamine complementation analysis M
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type
a
t i n g A B B B B C C D E F F F G G G G H I J J 1191 1207 1233 1251 1283 1215 1267 1227 1228 1254 1280 1282 1263 1287 1295 1588 1297 1322 1269 1272
NOTE: Each letter represents a complementation group and strains are denoted by numbers. A plus sign indicates complementation.
(carlsbergensis) (Table 3); Candida utilis (Table 1) grew well but produced little thiamine. A large number (20%) of the cultures tested did not grow on thiamine-free agar plates and were classed as putative thiamine-requiring mutants. Most of these were MATa haploid strains of S. cerevisiae and S. uvarium (carlsbergensis). However, a large number of these thiaminerequiring strains grew on thiamine-free agar plates seeded with strain 1588 (MATa, thi I), suggesting that genetic complementation had occurred. Thiamine complementation groups To place the newly isolated thiamine-requiring strains into complementation groups, and thus determine the number of genes involved in thiamine biosynthesis, a complementation test was devised. Initially, each of the 16 MATa thiamine-requiring yeast strains (Table 4) was grown on complete medium; they were then spread evenly on dry thiamine-free agar plates (plastic square Petri dishes, 6 x 6 cm), as described earlier for the cross-feeding test. Subsequently, each of the 21 MATa strains to be tested was spotted on each of the 16 lawns and plates and was incubated for 2-3 days at 30°C. Colonies could be clearly seen and the results were recorded as " + " or " - ." It is interesting to note that some strains, such as 1289 and 1214, did not complement any of the 21 tested strains, whereas strain 1294 virtually complemented all strains tested (20/21). The data (Table 4) suggest that there are at least 10 different complementation groups, implying the existence of at least 10 different genes in the thiamine biosynthetic pathway. This result is not unexpected given the complexity of thiamine biosynthesis (Brown 1972; Leder 1975; White and Spenser 1979, 1982; Grue-Sorensen et al. 1986).
Discussion Earlier reports (Iwashima and Nose 1976; Iwashima et al. 1973; Iwashima et al. 1977; Nishimura et al. 1982) indicated that yeast actively accumulate thiamine by a specific transport system that is pH, temperature, and energy dependent (Grue-Sorensen et al. 1986; Iwashima et al. 1973; Iwashima et al. 1977; Nishimura et al. 1982). Cernohorsky
et al. (1986) reported on the use of a special strain of S. uvarum (E2917) able to produce nonalcoholic beer with 180 mg thiamine/L. Of various yeast and other fungi examined in the present study for overproduction and excretion of thiamine, strains belonging to S. cerevisiae and S. uvarum (carlsbergensis) were the highest thiamine producers. These findings are in agreement with previously reported data (Bunker 1963). Moreover, the intracellular thiamine content varied among strains within a single species. For example, in S. cerevisiae .the level of intracellular thiamine varied from 5 to 27 pg/g of cells grown in a thiamine-free medium; even in thiamine-rich brewer's wort there was a difference but only twofold (from 38 to 72 pg/g; Table 1). This difference might be due not only to the level of thiamine biosynthesis but also to the level of thiaminases and to thiamine transport efficiency. Of the 188 strains examined, 9 were thiamine excretors as determined by both cross-feeding tests and quantitative measurement. Fatty acids such as potassium caproate, which inhibit thiamine uptake, can also cause intracellular thiamine to exit into the external milieu (Iwashima et al. 1973). Although the precise mechanism by which caproate causes thiamine efflux is unclear, it is likely that passive diffusion dictated by a thiamine concentration gradient is involved. Thus, thiamine excretors may have defects in thiamine uptake and are not necessarily thiamine overproducers. However, the fact that strains isolated in the present study excreted varying levels of thiamine could be attributed to their ability to synthesize varying amounts of thiamine and not only to varying excretion potential; this is supported by the high intracellular thiamine in yeast strains found to excrete a high level of thiamine. Given the efficient yeast transport system for accumulation of thiamine from-culture media b bas hi ma et al. 1973; Grue-Sorensen et al. 1986), the intracellular thiamine concentration is 10 000 times greater than the extracellular concentration (Grue-Sorensen et al. 1986). Consistent with this, the intracellular thiamine level was 2- to 25-fold greater when the cells were grown in 12" Plato brewer's wort than in thiamine-free media. Most of this intracellular thiamine was presumably accumulated from the culture medium. It is not
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HAJ-AHMAD ET AL.
an "evolutionarily" stable strategy to evolve a countertransport system that would excrete thiamine when cells normally expend energy to accumulate it. It is very likely that in the absence of the thiamine carrier, thiamine leaks into the medium via a passive mechanism driven by a thiamine concentration gradient. Based on the size of colonies cultured on thiamine-free agar media, several such hyperproducers were isolated (Table 3). Many (40%) of the S. cerevisiae strains examined were thiamine requiring (Table 4). This might have been due in part to the haploid condition of most of the strains isolated after uv mutagenesis. These strains (Table 4) failed to grow singly on a thiamine-free medium regardless of the incubation period used. The defect rendering them thiaminerequiring is presently unknown. The ability of some of these strains to grow when mixed and plated on thiamine-free medium was most likely due to genetic complementation. Although the data from such a complementation test indicated at least 10 different complementation groups in the thiamine biosynthetic pathway, for a precise determination it would be necessary to mutagenize two isogenic yeast strains of opposite mating type before isolating thiaminerequiring derivatives and testing them for complementation. Recently, 11 genes coding for enzymes in vitamin B12 (cobalamin) biosynthesis have been cloned from Bacillus megaterium (Brey et al. 1986). These genes account for only a fraction of the 20-30 genes thought to be involved in the vitamin's biosynthetic pathway; the thiamine biosynthetic pathway in yeast may be of similar complexity. Use of complementation to evaluate the complexity of thiamine biosynthesis in yeast is critical in assessing the feasibility of applying recombinant DNA technology as a means of constructing thiamine hyperproducing strains.
Acknowledgement The authors thank their colleagues in the Labatt's Research Department for help and advice during the course of this study. Association of Vitamin Chemists, Inc. 1951. Methods of vitamin assay. Interscience Publishers, Inc., New York. Brenner, M.W., Layer, L., and Hsu, W.P. 1973. Tracking yeast autolysis. Am. Soc. Brew. Chem. Proc. 57-62. Brey, R.N., Banner, C.D.B., and Wolf, J.B. 1986. Cloning of multiple genes involved with vitamin B12 biosynthesis in Bacillus megaterium. J . Bacteriol. 167: 623-630. Brown, G.M. 1972. In The vitamins, chemistry, physiology, pathology, methods. Vol. 5. 2nd ed. Edited by W.H. Sebrell and R.S. Harris. Academic Press, New York. pp. 122-129.
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Bunker, H.J. 1963. In Biochemistry of industrial microorganisms. Edited by C. Rainbow and A.H. Rose. Academic Press, New York. pp. 54-58. Centerwall, B.S., and Criqui, M.H. 1978. Prevention of WernickeKorsakoff syndrome: a cost-benefit analysis. N. Engl. J. Med. 299: 285-289. Cernohorsky, V., Curin, J., and Vernerova, J. 1986. Production of non-alcoholic beer with increased level of Vitamin B1. Kvasny Prum. 32: 145-147. Fink, H., and Just, F. 1941a. ~ b e rden Vitamin B1-Gehalt verschiedener Hefen und seine Beeinflussing. Biochem. Z. 308: 15-28. Fink, H., and Just, F. 1941b. ~ b e den r Vitamin B1-Gehalt verschiedener Hefen und seine Beeinflussing. Biochem. Z. 311: 287-306. Fujiwara, M., and Matsui, K. 1953. Determination of thiamine by the thiochrome reaction. Anal. Chem. 25: 810-812. Graham, R.K., Skurray, G.R., and Caiger, P . 1970. Nutritional studies on yeast, during batch and continuous fermentation. I. Changes in vitamin concentration. J. Inst. Brew. 76: 366-371. Grue-Sorensen, G., White, R. L., and Spenser, I .D. 1986. Thiamine biosynthesis in Saccharomyces: origin of the pyrimidine unit. J . Am. Chem. Soc. 108: 146-158. Haj-Ahmad, Y., Russell, I., and Stewart, G.G. 1989. A rapid simple procedure for direct measurement of excreted thiamine from yeast. J. Microbiol. Methods, 10: 297-302. Iwashima, A., and Nose, Y. 1976. Regulation of thiamine transport in Saccharomyces cerevisiae. J . Bacteriol. 128: 855-857. Iwashima, A., Nishino, H., and Nose, Y. 1973. Carrier-mediated transport of thiamine in baker's yeast. Biochim. Biophys. Acta, 330: 222-234. Iwashima, A., Wakabayashi, Y., and Nose, Y. 1977. Inhibition of thiamine transport in Saccharomyces cerevisiae by thiamine disulfides. J. Bacteriol. 131: 1013-1015. Leder, I.G. 1975. Metabolic pathways. Vol. 7. 3rd ed. Edited by D.M. Greenberg. Academic Press, New York. Lewis, J.C., Stubbs, J.J., and Noble, W.M. 1944. Vitamin synthesis by Torula yeast. Arch. Biochem. 389: 380-401. Linnett, P.E., and Walker, J. 1968. Biosynthesis of thiamine. Biochem. J. 109: 161-168. Lodder, J . (Editor). 1970. The yeast. 2nd ed. North-Holland Publishing Co., Amsterdam and London. Lundin, H. 1950. Fat synthesis by micro-organisms and its possible applications in industry. J . Inst. Brew. 56: 17-28. Nishimura, H., Sempuku, K., and Iwashima, A. 1982. Thiamine transport in Saccharomyces cerevisiae protoplasts. J. Bacteriol. 150: 960-962. White, R.L., and Spenser, I.D. 1979. Thiamine biosynthesis in Saccharomyces cerevisiae. Biochem. J . 179: 3 15-325. White, R.L., and Spenser, I.D. 1982. Thiamine biosynthesis in yeast. Origin of the five-carbon unit of the thiazole moiety. J . Am. Chem. Soc. 104: 4934-4943. Wickerham, L. J. 1951. Taxonomy of yeasts. U.S. Department of Agriculture, Washington, D.C. Tech. Bull. No. 1029. pp. 1-56.
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CARLA. BILINSKI, INGERUSSELL,AND GRAHAM G. STEWART Research Department, Labatt Brewing Company Ltd., 150 Simcoe Street, London, Ont., Canada N6A 4M3 Received March 27, 1992 Accepted June 9, 1992 HAJ-AHMAD,Y., BILINSKI, C. A., RUSSELL,I., and STEWART,G. G. 1992. Thiamine secretion in yeast. Can. J . Microbiol. 38: 1156-1 161. To isolate thiamine excretors and (or) overproducers, 188 cultures belonging to nine yeast and three fungal genera were screened. Nine excreted thiamine as determined by both the presence of cross-feeding zones on a thiamine-free agar medium seeded with a thiamine-requiring yeast strain and by the direct detection of excreted thiamine on agar plates. Several of these cultures produced several-fold more intracellular thiamine than the general culture population. Thiamine-requiring strains of Saccharomyces cerevisiae and S. uvarurn (carlsbergensis)were identified and were tentatively assigned to 10 complementation groups. Key words: thiamine, yeast, thiamine secretion, thiamine requiring. HAJ-AHMAD,Y., BILINSKI,C. A., RUSSELL,I., et STEWART,G. G. 1992. Thiamine secretion in yeast. Can. J. Microbiol. 38 : 1156-1 161. Un total de 188 cultures regroupant neuf genres de levures et trois de champignons ont ete verifiees dans le but d'isoler les souches secretrices et (ou) surproductrices de thiamine. Neuf souches secretaient de la thiamine tel qu'observe par la presence de zones d'echanges nutritifs croises sur une gelose sans thiamine ensemencee avec une souche de levure thiamine-dependante ou encore par la detection directe de thiamine secretee dans la gelose. Certaines de ces cultures produisaient plus de thiamine intracellulaire que la population generale cultivee. Des souches de Saccharomyces cerevisiae et de S. uvarurn (carlsbergensis)thiamine-dependantes ont ete identifiees et assignees tentativement a 10 groupes de complementation. Mots cles : thiamine, levures, secretion de thiamine, besoin en thiamine. [Traduit par la redaction]
Introduction Thiamine (vitamin B1) is an essential factor in animal nutrition and in many microorganisms; it is found naturally in a variety of sources such as rice hulls, cereal grains, yeast, liver, eggs, milk, and green leaves (Association of Vitamin Chemists 1951). Although it was originally extracted from rice bran, thiamine currently used for dietary supplements is almost entirely derived from chemical synthesis, mainly because there is no rich source of natural thiamine. Yeast, which is the primary source of vitamin B2 (riboflavin) and B12 (cobalamin), does not produce large amounts of thiamine (Lewis et al. 1944; Lundin 1950; Bunker 1963; White and Spenser 1979). The availability of thiamineexcreting and (or) hyperproducing mutants would make it economically feasible to produce the vitamin via fermentation and would be attractive for the production of thiamineenriched beverages. Although the initial concentration of thiamine in brewer's wort can vary from 150 to 750 j~g/L (Brenner et al. 1973; Graham et al. 1970), active uptake of vitamin B1 by cells reduces this concentration to almost 0 after 48 h fermentation (Brenner et al. 1973). Final beers contain very low amounts of thiamine, ranging from 2 to 100 j~g/L(Brenner et al. 1973); the recommended dietary uptake of vitamin B1 for adults is 5 jLg/kcal. Most beers contain 400-450 kcal/L; therefore, they should be fortified by about 200 j~g/L.Unfortunately, much higher fortification would be necessary to prevent thiamine-related brain damage in alcoholics (Centerwall and Criqui 1978). The availability of thiamine hyperproducers and excretors would ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / lmprime au Canada
also be useful in the baking industry, since dough is poor in vitamin B1 as a consequence of baker's yeast propagation under aerated conditions (Fink and Just 1941a, 1941b). The present study was undertaken to evaluate various yeast genera for their ability to naturally overproduce or excrete thiamine into culture media. The approach taken has led to the identification of thiamine-excreting and thiaminerequiring yeast strains. Thiamine-requiring strains of Saccharornyces cerevisiae and of S. uvarurn (carlsbergensis) were assigned to 10 complementation groups.
Materials and methods Yeast strains and maintenance Yeast strains employed in this study were obtained from the Labatt Culture Collection and were designated according to Lodder (Lodder 1970). Cultures were maintained at 4°C on Wickerham's (Wickerham 1951) malt extract - yeast extract - peptone - glucose (MYPG) medium, which contained the following ingredients per litre of distilled water: malt extract, 3 g; yeast extract, 3 g; peptone, 5 g; glucose, 20 g; and Bacto-Agar (Difco Laboratories), 20 g. Brewer's wort (12" Plato; " Plato = grams of sucrose per 100 mL, equivalent to carbohydrate content at 20°C) sterilized by steam streaming three times for 20-min periods at 100°C was also used as a culture medium. Cross-feeding test The thiamine-requiring haploid yeast Saccharomyces strain 1588 (MATa, thi I , ura I , trp I ; obtained from YGSC strain Y02587) was cultivated in MYPG medium for 48 h at 30°C, harvested by centrifugation, and resuspended in 50% of the original volume of a thiamine-free medium prepared as described earlier (Haj-Ahmad et al. 1989). A 0.25-mL portion of the cell suspension was spread
HAJ-AHMAD ET AL.
TABLE1. Cultures tested and their thiamine content Thiamine content Medium ( m g / ~ ) b
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Species " Candida fennica (1) Candida ishiwadae (1) Candida insectorurn (1) Candida shehatae (1) Candida silvanorurn (1) Candida steatolytica (1) Candida utilis (1) Debaryornyces polyrnorphus (1) Nadsonia fulvescens (1) Endornycopsis sp. (2) Endoyrncopsis fibuligera (2) Hansenula anornala (1) Hypornyces ipornoeae (1) Kluy verornyces rnarxianus (2) Leucosporidiurn capsuligenurn (1) Paecilornyces rnarquandii (1) Paecilornyces varioti (3) Paecilornyces fulvus (1) Pichia tannophilus (1) Pichia stipitis (4) Saccharornyces bayanus (14) Saccharornyces uvarurn (carlsbergensis) (35) Saccharornyces cerevisiae ( 102) Saccharornyces diastaticus (4) Saccharornyces rouxii (1) Saccharornyces dairensis ( 1) Saccharornyces fibuligera (2) Schwanniornyces occidentalis (1)
Synthetic
Brewer's wort
15 15 14 23 18 17 22 26 35 17 15 18 11 15 17 12 15 9 17 26 17
150 134 153 125 131 138 120 159 131 128 153 122 125 125 207 150 156 134 131 125 138
14-lgd 14-201 17 17 15 15 18
125-144 119-256 125 131 128 153 125
Intracellular (mg/g)' Synthetic
Brewer's wort
3 3 3 6 5 3 5 9 3 5 3 6 2 8 5 3 3 3 3 3 5 11-24 5-27 5 12 5 3 8
aValues in parentheses are the number of strains tested using the cross-feeding test (see Materials and methods). b ~ h i a m i n econcentration in cell-free media after 72 h incubation in thiamine-free synthetic medium or in a 12" Plato brewer's wort. T h i a m i n e content in cells grown for 72 h in either a thiamine-free synthetic medium o r in 12" degree Plato brewer's wort. d ~ a l u e represent s the range of thiamine concentration in various strains tested, whereas other single values are based o n measurements carried out on a single strain.
evenly on a dried thiamine-free agar medium that consisted of the following ingredients per litre of deionized, distilled water: vitaminfree Yeast Base (Difco Laboratories), 16.7 g; myoinositol, 2 mg; calcium DL-pantothenate, 0.4 mg; D-biotin, 2 mg; and BactoAgar, 20 g. Individual colonies to be screened were transferred by applicator sticks to thiamine-free agar medium seeded with thiamine-requiring yeast strain 1588. After 3-5 days incubation at 30°C, plates were examined for the presence or absence of crossfeeding zones. Direct measurement of excreted thiamine In addition to the above assay, thiamine excreter yeast colonies were identified using a direct method (Haj-Ahmad et al. 1989). Briefly, yeast colonies to be screened were either grown on (or replica plated on) thiamine-free agar plates. After a day or two of incubation at 30°C, the colonies were removed and excreted thiamine was measured directly on these plates as described by Haj-Ahmad et al. (1989). Colony growth complementation tests Strains found to be thiamine requiring were grown on complete medium and spread individually on thiamine-free agar plates as described above. Subsequently, an inoculum of each thiamine-
requiring yeast strain was transferred to each of the seeded test plates. After 2-3 days incubation at 30°C, the growth of each colony was evaluated by visual inspection and recorded as + (growth) or - (nongrowth). Extraction of thiamine Thiamine was extracted as described by White and Spenser (1979). Cells grown 72 h in 50 mL of culture medium (thiaminefree MYPG and brewer's wort) were harvested by centrifugation at 2000 rpm for 20 min at 4"C, washed in distilled water, and resuspended in 1 mL of 0.1 M HCl. After 30 min acid hydrolysis in a steaming water bath, the samples were centrifuged at 3000 rpm for 10 min. Pellets were given a further acid hydrolysis treatment as above, except with 2 mL of 1.1 M HCl. Supernatants from each round of hydrolysis were pooled and combined with 1.5 mL of 1 M sodium acetate (pH 4.7). Following adjustment to pH 4.7 with 50% (w/v) NaOH, thiamine pyrophosphate was hydrolyzed for 12 h at 30°C in the presence of 5 mg of Clarase concentrate (Miles Laboratories, Inc.). Thiochrorne assay Thiamine was measured quantitatively using a thiochrome assay (Fujiwara and Matsui 1953; Linnett and Walker 1968) as described
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CAN. J. MICROBIOL. VOL. 38, 1992
FIG. 1. A cross-feeding test for thiamine excretion. (A) Excretor colony surrounded by a zone of microcolonies (crossfeeding zone). (B) Nonexcretor colony. by Haj-Ahmad et al. (1989). Briefly, 2 mL of cyanogen bromide (prepared by titrating saturated bromine solution on ice with 20% KCN until colorless), followed by 1 mL of 50% w/v NaOH, was added to 1 mL of treated cell extract. Fluorescent product was extracted by addition of 5 mL of 2-methyl-propan-1-01 (Fisher Scientific). After 2 min of vigorous shaking and centrifugation for 30 s at low speed, fluorescence in the organic phase was measured at 420 nm using a spectrophotofluorometer (G.K. Turner, Model 430) with an excitation wavelength of 370 nm. Thiamine concentrations were determined from a standard curve prepared for each trial with thiamine solutions ranging from 1.2 to 5 mg/mL. The same procedure was employed to quantitate thiamine concentrations in cell-free supernatants.
Results Isolating thiamine excretors Of 188 cultures (Table 1) screened by the cross-feeding and direct assays three gave cross-feeding zones (see Fig. 1) after 5 days incubation; after an additional 1 week at room temperature, cross-feeding zones were evident around an additional six colonies. Fluorescent zones were also visible when the direct test was employed. All nine cultures were designated putative "thiamine excretors" (Table 2). The size of a zone is directly proportional to the amount of thiamine excreted (Haj-Ahmad et al. 1989). In addition, a second test
duced n o zone of microcolonies nor any apparent fluorescence 034).
(Haj-Ahmad et al. 1989) directly detected excreted thiamine on the agar plates (Fig. 2). Zntra- and extra-cellular thiamine Since the intracellular thiamine level in yeast is growthstage dependent, thiamine measurements were always made in cells under comparable physiological conditions. To quantitatively determine thiamine biosynthesis, cells were initially grown overnight in 2 mL of thiamine-free synthetic medium; 1 mL of this culture was used to inoculate 100 mL of thiamine-free medium. After 72 h incubation, 10 mL of the cultures were used to determine the biomass; the remaining 90 mL was used to determine the intracellular thiamine concentration. The extracellular thiamine concentration was measured in triplicate directly in a cell-free medium. Parallel experiments in brewer's wort were carried out to evaluate the yeast cells' behavior vis-a-vis the thiamine level under natural brewing conditions. Data in Tables 1, 2, and 3 indicate that, on average, cells grown in brewer's wort possess approximately 10-fold more intra- and extra-cellular thiamine than cells grown in a thiamine-free synthetic medium. This is expected because brewer's wort is naturally rich in thiamine. In a synthetic medium there was on average sixfold (range from 1 to 16 times) more extracellular (average of 69.2 mg/L) than intracellular (average of 13.0 mg/g) thiamine in thiamine
HAJ-AHMAD ET AL.
TABLE2. Thiamine excretor yeastsa
Thiamine content Medium (mg/L)
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Species
Culture collection No.
Synthetic
Brewer's wort
Intracellular (mg/g) Synthetic
Brewer's wort
Candida shehatae Debaryomyces polymorphus Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Sch wanniomyces occidentalis "Synthetic medium was thiamine-free medium supplemented with 4% glucose. Values given refer to thiamine concentration present after 72 h incubation at 30°C,in media (mg/L) and within cells (mg/g) dry weight.
TABLE3. High thiamine producing strainsa
Thiamine content Medium (mg/L) Species Saccharomyces uvarum (carlsbergensis) Saccharomyces uvarum (carlsbergensis) Saccharomyces uvarum (carlsbergensis) Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae
Intracellular (mg/g)
Culture collection No.
Synthetic
Brewer's wort
Synthetic
Brewer's wort
1234
14
134
24
62
1238
17
125
14
48
1240
14
144
14
47
1161
20
178
12
56
1221
14
119
27
66
1244
15
138
20
42
1248
23
128
23
47
"Synthetic medium was thiamine-free medium supplemented with 4% glucose. Values given refer to thiamine concentration present after 72 h incubation at 30°C in media (mg/L) and within cells (mg/g) dry weight.
excretor yeast (Table 2), whereas in the high thiamine producing yeast (Table 3) there was slightly more intra(19.1 mg/g) than extra-cellular (16.7 mg/L) thiamine (mean of 1 and a range of 0.6-2 times). In brewer's wort, there was on average threefold (range of 2-7 times) more extrathan intra-cellular thiamine, irrespective of the thiamine excretion phenotype. Isolating thiamine hyperproducers To identify thiamine overproducers, individual colonies were transferred to three different agar media: (i) complete;
(ii) thiamine-free; and (iii) thiamine-free seeded with a lawn of thiamine-requiring yeast (strain 1588). After 3-5 days incubation at 30°C, the size of each individual colony was compared within and among the three media. On complete agar plates, all colonies grew to approximately the same size within 3 days (data not shown), whereas colony sizes varied on the thiamine-free agar plates. Colonies that grew at a faster rate on thiamine-free agar plates were predicted to overproduce thiamine and were picked for quantitative measurement of their thiamine content (Table 3). The prediction was correct but only for S. cerevisiae and S. uvarum
CAN. J. MICROBIOL. VOL. 38, 1992
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TABLE 4. Thiamine complementation analysis M
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type
a
t i n g A B B B B C C D E F F F G G G G H I J J 1191 1207 1233 1251 1283 1215 1267 1227 1228 1254 1280 1282 1263 1287 1295 1588 1297 1322 1269 1272
NOTE: Each letter represents a complementation group and strains are denoted by numbers. A plus sign indicates complementation.
(carlsbergensis) (Table 3); Candida utilis (Table 1) grew well but produced little thiamine. A large number (20%) of the cultures tested did not grow on thiamine-free agar plates and were classed as putative thiamine-requiring mutants. Most of these were MATa haploid strains of S. cerevisiae and S. uvarium (carlsbergensis). However, a large number of these thiaminerequiring strains grew on thiamine-free agar plates seeded with strain 1588 (MATa, thi I), suggesting that genetic complementation had occurred. Thiamine complementation groups To place the newly isolated thiamine-requiring strains into complementation groups, and thus determine the number of genes involved in thiamine biosynthesis, a complementation test was devised. Initially, each of the 16 MATa thiamine-requiring yeast strains (Table 4) was grown on complete medium; they were then spread evenly on dry thiamine-free agar plates (plastic square Petri dishes, 6 x 6 cm), as described earlier for the cross-feeding test. Subsequently, each of the 21 MATa strains to be tested was spotted on each of the 16 lawns and plates and was incubated for 2-3 days at 30°C. Colonies could be clearly seen and the results were recorded as " + " or " - ." It is interesting to note that some strains, such as 1289 and 1214, did not complement any of the 21 tested strains, whereas strain 1294 virtually complemented all strains tested (20/21). The data (Table 4) suggest that there are at least 10 different complementation groups, implying the existence of at least 10 different genes in the thiamine biosynthetic pathway. This result is not unexpected given the complexity of thiamine biosynthesis (Brown 1972; Leder 1975; White and Spenser 1979, 1982; Grue-Sorensen et al. 1986).
Discussion Earlier reports (Iwashima and Nose 1976; Iwashima et al. 1973; Iwashima et al. 1977; Nishimura et al. 1982) indicated that yeast actively accumulate thiamine by a specific transport system that is pH, temperature, and energy dependent (Grue-Sorensen et al. 1986; Iwashima et al. 1973; Iwashima et al. 1977; Nishimura et al. 1982). Cernohorsky
et al. (1986) reported on the use of a special strain of S. uvarum (E2917) able to produce nonalcoholic beer with 180 mg thiamine/L. Of various yeast and other fungi examined in the present study for overproduction and excretion of thiamine, strains belonging to S. cerevisiae and S. uvarum (carlsbergensis) were the highest thiamine producers. These findings are in agreement with previously reported data (Bunker 1963). Moreover, the intracellular thiamine content varied among strains within a single species. For example, in S. cerevisiae .the level of intracellular thiamine varied from 5 to 27 pg/g of cells grown in a thiamine-free medium; even in thiamine-rich brewer's wort there was a difference but only twofold (from 38 to 72 pg/g; Table 1). This difference might be due not only to the level of thiamine biosynthesis but also to the level of thiaminases and to thiamine transport efficiency. Of the 188 strains examined, 9 were thiamine excretors as determined by both cross-feeding tests and quantitative measurement. Fatty acids such as potassium caproate, which inhibit thiamine uptake, can also cause intracellular thiamine to exit into the external milieu (Iwashima et al. 1973). Although the precise mechanism by which caproate causes thiamine efflux is unclear, it is likely that passive diffusion dictated by a thiamine concentration gradient is involved. Thus, thiamine excretors may have defects in thiamine uptake and are not necessarily thiamine overproducers. However, the fact that strains isolated in the present study excreted varying levels of thiamine could be attributed to their ability to synthesize varying amounts of thiamine and not only to varying excretion potential; this is supported by the high intracellular thiamine in yeast strains found to excrete a high level of thiamine. Given the efficient yeast transport system for accumulation of thiamine from-culture media b bas hi ma et al. 1973; Grue-Sorensen et al. 1986), the intracellular thiamine concentration is 10 000 times greater than the extracellular concentration (Grue-Sorensen et al. 1986). Consistent with this, the intracellular thiamine level was 2- to 25-fold greater when the cells were grown in 12" Plato brewer's wort than in thiamine-free media. Most of this intracellular thiamine was presumably accumulated from the culture medium. It is not
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HAJ-AHMAD ET AL.
an "evolutionarily" stable strategy to evolve a countertransport system that would excrete thiamine when cells normally expend energy to accumulate it. It is very likely that in the absence of the thiamine carrier, thiamine leaks into the medium via a passive mechanism driven by a thiamine concentration gradient. Based on the size of colonies cultured on thiamine-free agar media, several such hyperproducers were isolated (Table 3). Many (40%) of the S. cerevisiae strains examined were thiamine requiring (Table 4). This might have been due in part to the haploid condition of most of the strains isolated after uv mutagenesis. These strains (Table 4) failed to grow singly on a thiamine-free medium regardless of the incubation period used. The defect rendering them thiaminerequiring is presently unknown. The ability of some of these strains to grow when mixed and plated on thiamine-free medium was most likely due to genetic complementation. Although the data from such a complementation test indicated at least 10 different complementation groups in the thiamine biosynthetic pathway, for a precise determination it would be necessary to mutagenize two isogenic yeast strains of opposite mating type before isolating thiaminerequiring derivatives and testing them for complementation. Recently, 11 genes coding for enzymes in vitamin B12 (cobalamin) biosynthesis have been cloned from Bacillus megaterium (Brey et al. 1986). These genes account for only a fraction of the 20-30 genes thought to be involved in the vitamin's biosynthetic pathway; the thiamine biosynthetic pathway in yeast may be of similar complexity. Use of complementation to evaluate the complexity of thiamine biosynthesis in yeast is critical in assessing the feasibility of applying recombinant DNA technology as a means of constructing thiamine hyperproducing strains.
Acknowledgement The authors thank their colleagues in the Labatt's Research Department for help and advice during the course of this study. Association of Vitamin Chemists, Inc. 1951. Methods of vitamin assay. Interscience Publishers, Inc., New York. Brenner, M.W., Layer, L., and Hsu, W.P. 1973. Tracking yeast autolysis. Am. Soc. Brew. Chem. Proc. 57-62. Brey, R.N., Banner, C.D.B., and Wolf, J.B. 1986. Cloning of multiple genes involved with vitamin B12 biosynthesis in Bacillus megaterium. J . Bacteriol. 167: 623-630. Brown, G.M. 1972. In The vitamins, chemistry, physiology, pathology, methods. Vol. 5. 2nd ed. Edited by W.H. Sebrell and R.S. Harris. Academic Press, New York. pp. 122-129.
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Bunker, H.J. 1963. In Biochemistry of industrial microorganisms. Edited by C. Rainbow and A.H. Rose. Academic Press, New York. pp. 54-58. Centerwall, B.S., and Criqui, M.H. 1978. Prevention of WernickeKorsakoff syndrome: a cost-benefit analysis. N. Engl. J. Med. 299: 285-289. Cernohorsky, V., Curin, J., and Vernerova, J. 1986. Production of non-alcoholic beer with increased level of Vitamin B1. Kvasny Prum. 32: 145-147. Fink, H., and Just, F. 1941a. ~ b e rden Vitamin B1-Gehalt verschiedener Hefen und seine Beeinflussing. Biochem. Z. 308: 15-28. Fink, H., and Just, F. 1941b. ~ b e den r Vitamin B1-Gehalt verschiedener Hefen und seine Beeinflussing. Biochem. Z. 311: 287-306. Fujiwara, M., and Matsui, K. 1953. Determination of thiamine by the thiochrome reaction. Anal. Chem. 25: 810-812. Graham, R.K., Skurray, G.R., and Caiger, P . 1970. Nutritional studies on yeast, during batch and continuous fermentation. I. Changes in vitamin concentration. J. Inst. Brew. 76: 366-371. Grue-Sorensen, G., White, R. L., and Spenser, I .D. 1986. Thiamine biosynthesis in Saccharomyces: origin of the pyrimidine unit. J . Am. Chem. Soc. 108: 146-158. Haj-Ahmad, Y., Russell, I., and Stewart, G.G. 1989. A rapid simple procedure for direct measurement of excreted thiamine from yeast. J. Microbiol. Methods, 10: 297-302. Iwashima, A., and Nose, Y. 1976. Regulation of thiamine transport in Saccharomyces cerevisiae. J . Bacteriol. 128: 855-857. Iwashima, A., Nishino, H., and Nose, Y. 1973. Carrier-mediated transport of thiamine in baker's yeast. Biochim. Biophys. Acta, 330: 222-234. Iwashima, A., Wakabayashi, Y., and Nose, Y. 1977. Inhibition of thiamine transport in Saccharomyces cerevisiae by thiamine disulfides. J. Bacteriol. 131: 1013-1015. Leder, I.G. 1975. Metabolic pathways. Vol. 7. 3rd ed. Edited by D.M. Greenberg. Academic Press, New York. Lewis, J.C., Stubbs, J.J., and Noble, W.M. 1944. Vitamin synthesis by Torula yeast. Arch. Biochem. 389: 380-401. Linnett, P.E., and Walker, J. 1968. Biosynthesis of thiamine. Biochem. J. 109: 161-168. Lodder, J . (Editor). 1970. The yeast. 2nd ed. North-Holland Publishing Co., Amsterdam and London. Lundin, H. 1950. Fat synthesis by micro-organisms and its possible applications in industry. J . Inst. Brew. 56: 17-28. Nishimura, H., Sempuku, K., and Iwashima, A. 1982. Thiamine transport in Saccharomyces cerevisiae protoplasts. J. Bacteriol. 150: 960-962. White, R.L., and Spenser, I.D. 1979. Thiamine biosynthesis in Saccharomyces cerevisiae. Biochem. J . 179: 3 15-325. White, R.L., and Spenser, I.D. 1982. Thiamine biosynthesis in yeast. Origin of the five-carbon unit of the thiazole moiety. J . Am. Chem. Soc. 104: 4934-4943. Wickerham, L. J. 1951. Taxonomy of yeasts. U.S. Department of Agriculture, Washington, D.C. Tech. Bull. No. 1029. pp. 1-56.
