INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Saccharomyces cerevisiae may utilize a variety of carbon sources, but glucose and fructose are preferred. When one of these sugars is present, carbon catabolite repression occurs and the enzymes required for utilization of the alternative carbon sources are synthesized at low rates or not at all (12, 13, 14). Carbon catabolite repression alters transcription and is regulated mainly by the Mig1p protein (13, 14, 23), a transcriptional repressor of glucose-repressible genes involved in metabolic processes other than glucose fermentation (such as utilization of the alternative carbon sources sucrose, maltose, or galactose; gluconeogenesis; and respiratory metabolism [13, 14]). Transcription of genes required for growth in nonfermentable carbon sources is activated by the Hap complex, which is repressed by Mig1p (5, 14).

S. cerevisiae baker's yeasts commonly are grown in molasses, which contains sucrose as the primary carbon source, and genotypes with the highest growth rate and productivity in molasses are favored (8, 9, 10, 11). Further increases in invertase expression and redirection of the respiro-fermentative flux through the deregulation of Mig1p or Hap complex (5) would improve utilization of molasses and production of sweet doughs by these strains. Expression of the SUC genes, which code for the invertase required for catabolism of sucrose and raffinose, is repressed at high levels of glucose (12, 13, 14). Various regulatory regions have been identified in the SUC2 promoter. Mig1p binds to SUC2A and SUC2B (activation sequences) in the presence of glucose. Repression mediated by the upstream repression sequence for SUC2 (URS_SUC2) occurs in the absence of glucose (14).

In dough without addition of sugar, the principal fermentable sugar for yeast is maltose, liberated from the starch of the flour by amylases. The leavening ability of sponge dough is closely related to maltose fermentability (4, 20). Maltose utilization requires a MAL locus and transcription of the structural genes for permease (MALT) and maltase (MALS), which are induced by maltose and catabolite repressed by glucose (20, 27, 39). Both genes share a common intergenic upstream activation sequence (UAS) region whose activation results in the coordinate expression of MALT and MALS (39). Thus, glucose plays two roles in maltose utilization: it interferes with induction of the MAL transcriptional activator by maltose and it represses the expression of the permease and the maltase (12, 14, 20).

2-Deoxy-D-glucose (DOG), a toxic glucose analog, has frequently been used to isolate glucose-deregulated mutants (19, 21, 22, 38, 40). Mutants isolated in galactose and DOG from industrial S. cerevisiae strains ferment equimolar mixtures of glucose and galactose to ethanol rapidly and completely (2) and have altered sugar transport activity (29, 32).

Bread manufacturers have ongoing interest in new strains of baker's yeasts (S. cerevisiae), especially those that increase dough fermentation rates and yield a high quality final product. Deoxyglucose-resistant (DOG^r) mutants isolated on maltose and DOG, with MAL deregulated phenotypes, have improved leavening ability in lean doughs (30, 31, 33). However, constitutive synthesis of MAL genes in these strains, generally osmosensitive, has very little effect in doughs supplemented with sucrose (34, 35, 36).

Our objective was to produce baker's yeast strains that could more efficiently ferment sweet doughs by isolating DOG^r mutants during growth in a medium with DOG and raffinose rather than maltose (30). These mutants, deregulated for both invertase and maltase, have many of the properties needed for commercial application, including (i) they are spontaneous in origin and are not subject to recombinant DNA technology regulations, (ii) they have been stable during storage and cultivation for 5 years, and (iii) they ferment sweet doughs faster than the wild type.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Strains. We selected baker's yeast strain V1 (8), because of its high fermentative capacity (8) and its high frequency of sporulation, tetrad formation (over 50%), and spore viability (about 60%) (11). We used the laboratory yeast YNN295 obtained from Alko Ltd. (Helsinki, Finland), as the control for karyotype electrophoresis, and strain S288C (American Type Culture Collection, Manassas, Va.; previously at Yeast Genetic Stock Center, Berkeley, Calif.) as the laboratory control for enzymatic activities. A wine strain, IFI256 (Instituto de Fermentaciones Industriales, Madrid, Spain), was used as an industrial yeast control in enzymatic assays, and a commercial baker's yeast (L'Hirondelle, Lesaffre, France) was used as the control for baking abilities.

Media. Yeasts were grown in complete YP medium (1% Difco [Detroit, Mich.] yeast extract, 2% Bacto Peptone) supplemented with 2% glucose (YPD), 10% glucose (YPD10), 2% maltose (YPM), 2% sucrose (YPS), 3% glycerol (YPG), or 1% glucose plus 1% maltose (YPDM) or in minimal medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate) supplemented with either 2% glucose (SD), 2% raffinose (SR), or 2% raffinose and 0.05 to 0.1% 2-deoxy-D-glucose (SRdog) (2). Beet molasses (72% sucrose) obtained from Unión Alcoholera Española, S.A. (Granada, Spain), diluted 20 times (3.6% sucrose) was used too. Media were solidified with 2% agar.

Enzymes and chemicals. Proteinase K and sucrose were obtained from Merck, A.G. (Darmstadt, Germany), and Zymolyase 20000 was obtained from Seikagako kogyo Co. Ltd. (Tokyo, Japan). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.).

Culture conditions. Cells were cultivated as described in reference (9). The growth rate, µ, was determined by measuring the increase in A₆₆₀ for laboratory media and A₆₉₀ for media containing molasses (10).

Total cell number and viability. Cell number was estimated by diluting the samples in water, measuring A₆₆₀, and counting cells under the microscope in a hemacytometer. Viability was determined by spreading samples on YPD and counting colonies after 3 to 4 days of incubation at 30°C.

Mutant isolation and stabilization. Baker's yeast strain V1 was pregrown overnight in YPD10, harvested during the exponential phase of growth (10⁷ cells/ml), washed with sterile distilled water, and plated on SRdog selective medium (0.05% DOG). Raffinose was used instead of sucrose because sucrose is easily hydrolyzed in cultures of strain V1, resulting in glucose, which competes with the toxin. Colonies appearing between 6 and 12 days were subcultured on the same medium, and those still growing vigorously were tested for growth on SRdog with 0.05 or 0.1% DOG. Mutant stability was tested by growth in liquid nonselective complete medium (YPD) for 100 generations. When cultures reached the late exponential phase (5 × 10⁷ cells/ml), 0.5 ml was transferred to fresh YPD. Samples were taken periodically, washed, and plated on SR and on SRdog solid media to determine the percentage of colonies still resistant to DOG.

Assimilation of glucose and maltose. Cells growing at 30°C in YPDM were harvested in mid-log phase (10⁷ cells/ml), resuspended in YPDM, incubated at 30°C for 3 h, and finally resuspended in 3 ml of YPDM and incubated at 30°C with agitation (300 rpm). At specified times, aliquots of 0.5 ml were taken and centrifuged at 5,000 × g for 5 min. The amount of the remaining glucose and maltose in the supernatant was determined by reverse-phase high-performance liquid chromatography (HPLC). The system consisted of a chromatograph (model lambda Max 481; Waters Co., Milford, Mass.) equipped with a column, two pumps (Waters 501 and Waters 510), and a Waters detector (model 481). HPLC analyses were performed with an HPX-42A column (Bio-Rad Laboratories, Richmond, Calif.) maintained at 60°C. Water was used as the eluent at a flow rate of 0.6 ml/min. Maltose and glucose were detected on the basis of their absorbance at 210 nm and identified by comparison with glucose and maltose standards.

Analytical procedures. Cells were permeabilized by adding a toluene-ethanol (1:1) solution, and the mixture was agitated with vigorous stirring for 5 min (8). Alternatively, cells were broken by the addition of 1 g of glass beads (0.5-mm diameter) and shaken in a vortex for five periods of 1 min (crude extracts) (8).

Enzyme assays of invertase, maltase, and 2-deoxy-D-glucose-6P phosphatase. Invertase and maltase were assayed by monitoring glucose production in a YSI27 glucose analyzer (Yellow Spring Instruments, Yellow Spring, Ohio). The standard assay mixture contained 20 µl of permeabilized cells or crude extract (8) obtained from 20 ml of either YPD, YPDM, YPS, or molasses (repressed conditions) or YPM or SR (derepressed conditions) media and 20 µl of a 5% (wt/vol) solution of either sucrose or maltose. Maltase also was assayed with p-nitrophenyl--D-glucopyranoside (pNPG) as previously described (8, 9, 10, 11). One unit of activity was defined as the amount of enzyme required to liberate 1 nmol glucose per µg protein per min at 30°C.

Protein determination. Total protein was determined in permeabilized cells following the procedure of Lowry et al. (24). In other experiments, protein was determined according to the Bradford assay (6) using the Bio-Rad protein assay dye reagent and bovine serum albumin as a protein standard.

Electrophoretic karyotype. The basic procedure for chromosomal DNA preparation was that of Naumov et al. (25). The gel was prepared with 0.5% TBE buffer and 1% agarose. The system used was a CHEF-DRII gel electrophoresis apparatus from Bio-Rad. Electrophoresis was carried out as previously described (8, 9, 10, 11).

Determination of the capacity to leaven dough. To determine the leavening capacity under laboratory conditions, 20-ml tubes containing 7 ml of distilled water plus 4 g of wheat flour, supplemented or not with 5% glucose, were inoculated with 0.1 g (wet weight [1.5 × 10⁷ cells/mg]) of the yeasts previously grown in YPD (9, 10, 11). Tubes were incubated without shaking at 30°C, and the increase in volume was monitored every 10 min for 2 to 3 h (plain doughs) or for 3 to 4 h (sweet doughs) (9, 10, 11).

Baking. Yeasts were grown in molasses until late stationary phase (about 30 g/liter [wet weight]). Elaboration of sweet bakery products was carried out by mixing wheat flour (W, 230 to 240 × 10³erg) with 20% sucrose, 2% salt, 5% lard, 2 eggs, and 4.5% yeast (wet weight) with regard to the flour's weight, of either the parental strain V1; the mutants DOG9, DOG21, or DOG24; or a commercial strain, C (L'Hirondelle). The mixture was incubated at 40°C and 60% humidity for 3 h and then baked at 210°C for 10 min. The products were assessed with regards to their texture and organoleptic properties by 30 to 40 nonexperts following standard procedures for quality evaluation (3).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Selection of DOG^r mutants. Strain V1 was grown in YPD10 (repression conditions) and then spread on SRdog solid medium with 0.05% DOG at a concentration of 10⁸ cells per plate. The spontaneous frequency of DOG^r mutants was approximately 3 × 10⁸. Of 113 mutants, 84 had increased maltase activity under repression conditions. Twenty of these mutants had approximately the same growth rate (µ) as the wild type in SD, and were examined further. Eight of these mutants used maltose preferentially when incubated in YPDM, and the rate of maltose and glucose assimilation was very low. The other 12 mutants could rapidly assimilate both glucose and maltose. These 12 mutants were grown for 100 generations in YPD to assess stability. After 100 generations, three mutants had between 80 and 100% DOG^r cells. These three mutants are available from Colección Española de Cultivos Tipo (Burjassot, Valencia, Spain) as DOG9-CECT 11840, DOG21-CECT 11841, and DOG24-CECT 11842.

Characterization of stable DOG^r mutants. (i) Genetic stability. In all three mutants there were numerous changes in the number and position of the chromosomal bands with respect to the V1 parental strain (Fig. 1). DOG24 had the most rearrangement, followed by DOG9 and DOG21. The mutants were maintained at 80°C in glycerol and at 4°C on solid YPD for 5 years, transferring the YPD cultures every 2 months. During these 5 years, samples were taken periodically and grown on YPD and spread on SR and SRdog solid media, and the electrophoretic karyotype was determined again. No new changes were observed in the mutants, thus confirming their karyotypic and phenotypic stability.

View larger version (96K): [in this window] [in a new window]   FIG. 1.   Electrophoretic karyotype of the baker's yeast V1 and the mutants DOG9, DOG21, and DOG24. Strain YNN295 was included as control. The chromosome numbers corresponding to strain YNN295 are indicated on the left.

(ii) Growth rate in laboratory and molasses media. Both the yield (28 to 30 g of cells/liter [wet weight]) in YPD and molasses and the growth rate, µ, in the laboratory media (SD, YPD, YPM, and YPDM) were similar in the parental V1 strain and DOG21 (Table 1). DOG9 and DOG24 mutants grew slower in molasses and in some of the laboratory media (Table 1) and only produced 23 to 25 g of cells/liter (wet weight) in the media employed.

(iii) Invertase, maltase, and phosphatase activities. Invertase specific activity was higher, compared to strain V1, in DOG21 and DOG24 when glucose or sucrose was present but was lower in YPM medium. In YPD, YPDM, YPS, and molasses, invertase was only partly derepressed in the mutants (Table 2). Under the same conditions (Table 2), maltase was partly derepressed in the three mutants in YPDM; in DOG9 in molasses; and in DOG21, in YPD, YPS, and molasses. Phosphatase activity, resulting in the dephosphorylation of DOG-6P was very high in DOG24, detectable in DOG21, and absent in the remaining strains (Table 2).

(iv) Leavening ability. Under laboratory conditions, both DOG21 and DOG24 produced more CO₂ than V1, with 2.5% final yeast concentration and 5% glucose-supplemented doughs. The V1 strain fermented nonsupplemented doughs faster than any of the mutants (Table 3 and Fig. 2).

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Leavening capacity of the baker's strain V1 () and the DOGr mutants DOG9 (), DOG21 (), and DOG24 () measured as increase in dough volume under laboratory conditions. (A) Fermentation of plain doughs; (B) fermentation of doughs supplemented with 5% of glucose. Data represent mean values (n = 6) and standard error of less than 10%. Mean values of the average rates in either A or B, followed by the same letter, do not differ significantly (P < 0.01) (41).

(v) Baking. The pieces of sweet dough and yeast weighed between 50 and 55 g. Baked pieces were sampled by 30 to 40 nonexpert tasters and evaluated for taste, flavor, and texture (3). Data are mean values (n = 6), with a standard error of less than 10%. The DOG21 mutant was the best strain with regards to each of the three parameters (8.6 ± 0.4, on a scale of 1 to 10) (Fig. 3), followed by the commercial strain (average, 7.6 ± 0.2), DOG9 (average, 7.0 ± 0.1), and V1 (average, 6.2 ± 0.3). DOG24 was the worst (average, 6.0 ± 0.2), although V1 and DOG24 do not differ significantly (3, 41). DOG21 also was the best with respect to external and internal aspects of the pieces: browning, volume and density, elasticity, color, consistency, suitability for slicing, and regularity and size of the alveoli (pore structure) (Fig. 3).

View larger version (78K): [in this window] [in a new window]   FIG. 3.   Cross-sections of pieces fermented with strain V1; mutants DOG9, DOG21, and DOG24; and the commercial C strain. Pieces fermented with mutant DOG21 showed the best volume, elasticity, regularity and size of the alveoli, and suitability for slicing (smooth section shown). These characteristics get increasingly worse in C, V1, and DOG24, so that pore structure of pieces fermented with DOG24 was the most irregular (very rough section shown).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The paramount consideration in baker's yeast production traditionally has been its quality, as expressed by fermentation characteristics in dough substrates (35). For fast dough fermentation, the yeast properties required are invertase activity to hydrolyze the higher glucofructans as rapidly as possible and high potential maltose fermentation rate. There has been significant interest in strains with deregulated maltase and maltase permease activities. There also has been interest in the fermentation of sweet doughs, in which sucrose is added to the mix, and in the construction of novel strains with deregulated, enhanced invertase activity (33, 35).

The most desirable property of these new industrial strains is genetic stability and physiological reproducibility (30, 31). The mutants finally selected in this study were very stable, and their growth rates, µ (Table 1), and fermentative capacities (Tables 2 and 3) were in many cases improved with regards to the wild type. The mutant with the best properties was DOG21, which can be immediately used in industrial processes.

DOG^r mutants fermented sweet doughs faster than the wild type but were slower at fermenting plain doughs (Table 3). This fermentative capacity correlated with maltase and invertase activity in V1 and the mutants (Table 2 and 3): in YPDM, the activity of maltase plus invertase of any of the mutants was higher than that of V1, liberating more glucose to be fermented in sweet doughs, so that more CO₂ is produced.

Changes in the activity of invertase and maltase in the mutants could result from mutations in structural genes such as SUC or MAL. Increasing the copy number of the SUC4 promoter also increases expression of the invertase genes, suggesting that transcriptional regulatory (negative) factors may become limiting (18). This copy number increase could explain the high invertase levels found in baker's yeasts, where multiple SUC genes on different chromosomes are known (10, 26, 28). The mutants are partially derepressed for invertase and maltase in the presence of glucose but have equal or lower activity in its absence (Table 2). These results suggest that, in addition to SUC genes, MAL loci also may be affected and that no further amplification of SUC genes has occurred. DOG^r mutants had only a slight increase in either maltase and/or invertase activity when glucose was present (Table 2). The reason for this might be that the parental V1 strain was polyploid (almost triploid: 2.7n) (8, 9, 10, 11) and that there are multiple copies of SUC and MAL genes (26) on different chromosomes in V1 (10, 16, 17). The mutant phenotype could be masked by the wild-type copies, and complete glucose repression insensitivity would not be expected (Table 2). DOG^r mutants also could have mutations in regulatory genes, such as MIG (12, 13, 14, 37). Mig1p derepresses SUC2 and MAL genes (13, 14), and overexpression of SUC2 in mig mutants does not increase growth rate, µ, or yield in molasses (14), as occurs in DOG^r strains (Table 1). In addition, in S. cerevisiae two genes that confer DOG resistance (4, 19), DOG^R1 and DOG^R2 (33), encode isoenzymes with DOG-6P phosphatase activity (34). DOG21 and DOG24 had significant DOG-6P phosphatase specific activity, but there was no activity by the wild type or the DOG9 mutant (Table 2). The lack of phosphatase activity in DOG9 suggests that there are multiple mechanisms for resistance to DOG. Further genetic analysis is needed to determine the number of mutated loci. However, in the V1 parent chromosomal reorganization (Ty-mediated translocations and Y'-mediated ectopic recombinations) occurs at a very high frequency during meiosis (9, 17); thus, genetic analysis of these phenotypes will be difficult.

The contribution of maltase productivity of yeasts to the leavening ability in flour doughs has long ago been estimated (28, 33, 34); invertase productivity in fructo-oligosaccharide-containing dough also has been reported (33, 34). Recombinant DNA techniques have been used to construct such strains, but these organisms are not acceptable for commercial use due to regulatory restrictions. We describe improvements of sweet baker's products fermented with carbon catabolite-deregulated yeast mutants. By using resistance to DOG in the presence of raffinose, instead of maltose, as a selection tool, we isolated and evaluated naturally occurring mutant strains of baker's yeast V1 that have increased levels of invertase and maltase. These mutants have sufficient potential to be produced commercially, because their cell yields are similar to those of commercial yeasts, they are spontaneous in origin, their fermentation abilities on sweet dough are improved, and in sensory evaluation the quality and flavor of bread fermented by these mutants was excellent.