INTRODUCTION

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Yeast cells need any one of five unlinked maltose (MAL) loci (MAL1 through MAL4 and MAL6) (8, 27) in order to utilize maltose. Each locus consists of a MALx1 (MALxT) (where x is the locus) gene, encoding maltose permease (9), a MALx2 (MALxS) gene, coding for -glucosidase (maltase) (10), and a MALx3 (MALxR) gene, encoding a positive regulatory protein (28). The MALx1 and MALx2 genes are divergently transcribed from a bidirectional promoter (MAL intergenic region), and the MALx3 regulatory protein interacts with upstream activating sequences in the MAL intergenic region, inducing transcription in the presence of maltose (21). Expression from native MAL loci is maltose induced (inducing conditions), is glucose repressed (repressing conditions), and has a low basal level of expression in the presence of galactose or ethanol (noninducing conditions) (28).

Industrial yeasts are usually polyploid strains of Saccharomyces cerevisiae or closely related species (13). The ability to utilize maltose, and therefore the regulation of the MAL system, is a key factor in many commercial applications, such as baking, brewing, and distilling (4, 6, 31). In a dough consisting of flour, water, yeast, and salt (unsugared or plain dough), the most abundant available sugar is maltose, produced by the action of amylases on damaged starch (4, 33). Some industrial strains are inoculated into unsugared (plain) dough with low MAL activity (maltase and maltose permease enzymes) and have an undesirable decrease in the 2nd-h gassing rate (30). These strains are termed lagging strains. Others, known as nonlagging strains, maintain a high gassing rate in the 2nd h of leavening. Often, lagging strains have other phenotypes that are desirable, such as good gassing ability in high-sugar dough or stability upon storage. Sexual crosses of nonlagging and lagging strains may not yield progeny with all of the desirable traits.

Before recombinant DNA techniques can be used to alter the lagging phenotype of industrial yeast, detailed genetic and biochemical analyses of industrial strains are needed. The MAL loci, maltose utilization phenotypes, and expression of maltase and maltose permease proteins in laboratory strains of yeast (7, 11, 26, 41) have been characterized extensively; however, little is known about the genetic basis of the nonlagging phenotype in industrial strains. Much remains to be learned about the genetic makeup of industrial strains in order to take full advantage of recombinant DNA techniques for strain improvement (1, 36).

Our working hypothesis is that CO₂ production in unsugared dough correlates with MAL activity in industrial strains used for commercial applications. Our objectives were (i) to determine if there are any major differences in regulation of the MAL system between lagging and nonlagging strains and (ii) to identify differences in genetic background that are involved in the nonlagging phenotype of industrial strains.

	MATERIALS AND METHODS

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Strains and plasmids. Industrial baker's strains NL67, NL25, NL89, L38, L83, and L05 were obtained from Burns Philp & Co. Ltd., North Ryde, Sydney, Australia. Strain RMS-14A (MATa trp1 his4 mal⁰ suc⁰) (37), which lacks a functional MALx3 gene, was transformed with a construct that has MALx2 and MALx1 structural genes replaced by the marker genes MEL1 and lacZ, respectively, producing strain PB1 (2). Escherichia coli XL1 Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F' proAB lacI^q ZM15 Tn10 (Tet^r)] from Stratagene (La Jolla, Calif.) was used for cloning and plasmid propagation. The shuttle vector pBEJ17 (16), with G418 resistance as a dominant marker, was used to introduce MALx3 genes into the baker's yeast strains. YRp7 (39) was used for the tryptophan auxotrophic strain PB1.

Media and culture conditions. Bacteria were grown on Luria-Bertani medium (1% peptone, 0.5% yeast extract, 1% NaCl) in the presence of ampicillin (50 µg ml¹) for selection. PB1 Trp⁺ transformants were grown on minimal medium (0.67% yeast nitrogen base without amino acids, supplemented with all auxotrophic requirements except tryptophan) to maintain YRp7 plasmids. For Northern analysis and enzyme assays, industrial baker's yeast strains were grown on YP-based medium (0.5% yeast extract, 1% peptone, 0.3% KH₂PO₄) with or without 220 µg of Geneticin (G418 sulfate; Gibco BRL) ml¹. Fermentable sugars and ethanol were added to a concentration of 2% (wt/vol). Cultures were incubated at 30°C on a platform shaker (200 rpm) and harvested at an optical density at 640 nm of 0.3 to 0.35. Yeast cells tested for gas production were grown in a 100-ml seed culture of YP-based medium which contained 1% sucrose with or without 220 µg of G418 sulfate ml¹. This seed culture was used to inoculate 1 liter of the same media in baffled 2-liter Erlenmeyer flasks. Both cultures were incubated at 30°C on a platform shaker (200 rpm) until late-respiratory phase, when the ethanol level in the medium was between 0.10 and 0.02% (wt/vol). Cultured cells were harvested, washed twice with 0.35 M NaCl, and collected on Whatman paper (no. 3 chromatography) for 30 min. Previously described methods were used to measure gas production in bread dough (25) or alcohol production in synthetic liquid dough (24). Bread dough activities given in the tables are averages of three cultures, each tested in duplicate. Standard errors were less than 10%.

Preparation of MALx3 genes for transformation. We isolated MALx3 genes by colony hybridization from a YRp7-based minilibrary created from BglII-SalI-digested NL67 chromosomal DNA (2). We subcloned MALx3 genes into other vectors after changing the unique PmlI site of YRp7 to a BamHI site. The BamHI-SalI fragments encoding the positive activator genes were inserted into the BamHI-SalI sites of pBEJ17. Yeast transformants were performed by using a Bio 101 (Vista, Calif.) kit according to the manufacturer's instructions.

Northern hybridization. RNA for Northern hybridization was isolated from yeast cells by using TRIZOL reagent (Life Technologies, Inc., Gaithersburg, Md.) according to the manufacturer's instructions. Thirty micrograms of total RNA was resolved on a 1% agarose gel in 1× morpholinepropanesulfonic acid (MOPS) buffer (20 mM MOPS [sodium salt], 5 mM sodium acetate, 1 mM disodium EDTA [pH 7]) containing 6% (wt/vol) formaldehyde. Nonradioactive digoxigenin hybridization was performed according to the manufacturer's instructions (Boehringer Mannheim Australia, Sydney, New South Wales, Australia). The 1-kb HindII fragment containing part of the maltose permease gene (MAL6-1), the 1.5-kb BglII fragment containing part of the maltase gene (MAL6-2), the 0.9-kb EcoRI fragment of the transcriptional-activator gene (MAL6-3), and the 2-kb EcoRI-HindIII fragment containing the yeast actin gene (ACT1) were used as hybridization probes.

Production of cell extracts and determination of protein concentration. Harvested cells were resuspended in breakage buffer (0.1 M citrate buffer [pH 6.5], 0.1 M EDTA, 1 mM dithiothreitol, 0.17 mg of phenylmethylsulfonyl fluoride/ml, 0.7 µM pepstatin) and homogenized for 5 min in the presence of glass beads. The extracts were centrifuged at 4°C for 10 min at 11,000 × g, and the supernatant was used as a cell extract. The protein concentration was determined by the method of Bradford (5).

Enzyme assays. For the determination of maltase activity, cell extract and 50 mM potassium phosphate buffer (pH 6.8) were added to a total volume of 200 µl, followed by the addition of 1 ml of p-nitrophenyl-glucopyranoside (1 mg ml¹). The specific activity of maltase was defined as nanomoles of p-nitrophenol released per minute per milligram of protein at 28°C and pH 6.8. The -galactosidase activity of cell extracts was assayed as described by Miller (23). Specific activity was determined as nanomoles of o-nitrophenol released per minute per milligram of protein at 28°C and pH 7. For -galactosidase activity determination, cell extracts were added to a total volume of 400 µl with assay buffer (39 mM potassium phosphate, 31 mM citric acid [pH 4]), followed by the addition of 100 µl of p-nitrophenol-galactopyranoside (15 mg ml¹). Specific activity was defined as nanomoles of p-nitrophenol released per minute per milligram of protein at 28°C and pH 4. Enzyme activities given are averages of three cultures tested in triplicate. Standard errors for maltase, -galactosidase, and -galactosidase activities were less than 10%.

Transport assays. Maltose transport activity was assayed according to the method of Serrano (38). Yeast cells were suspended in 0.2 M potassium phosphate buffer (pH 6) at a concentration of 90 mg ml¹. The reaction was carried out at 30°C and started by the addition of 30 µl of 10 µCi of -D-[U-¹⁴C]maltose (ICN Biochemicals Australasia Pty. Ltd., Sydney, New South Wales, Australia). Samples were taken at 30-s intervals and stopped by dilution in 4 ml of ice-cold water plus 3.7 mg of iodoacetamide ml¹. The cells were filtered onto Whatman GF/C glass microfiber filters and washed twice with 4 ml of ice-cold water plus 3.7 mg of iodoacetamide ml¹. The radioactivity of filters was determined by liquid scintillation counting using aqueous scintillant; boiled cells were used as a control. Transport activities were defined as picomoles of maltose transported per minute per milligram (dry weight) of yeast cells. Activities given are averages of three cultures tested in triplicate. Standard errors for transport activities were less than 15%.

	RESULTS

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Positive correlation between yeast maltase and maltose permease activities and 2nd-h CO₂ production in unsugared dough. The abilities of six industrial strains of S. cerevisiae to produce CO₂ gas in the 2nd h of a rapid unsugared dough fermentation were strongly correlated to maltase (r = 0.995) and maltose permease (r = 0.963) activities at the time of inoculation in the dough (Table 1). Strains NL67, NL25, and NL89 displayed the gassing characteristics of nonlagging strains (an equal or greater volume of gas was produced in the 2nd h than in the 1st h). By contrast, the volumes of gas produced by L38, L83, and L05 decreased by 60% in the 2nd h, indicating that these are lagging strains. The maltase and maltose permease activities of the three nonlagging strains were at least 7- and 120-fold higher, respectively, than those of the lagging strains (Table 1). These high MAL activities correlated with the 2nd-h gas volumes of the nonlagging strains, which were at least three times higher than those of the lagging strains (Table 1). When inoculated into synthetic dough medium consisting of glucose as the sole carbon source, all strains showed similar levels of gas production over 2 h (data not shown). These findings suggest that differences in maltose utilization affect 2nd-h gassing of lagging and nonlagging baker's yeast.

Nonlagging strains have higher MAL activity under noninduced and induced conditions, but this is highly repressed by glucose. We tested two strains, NL67 (nonlagging) and L38 (lagging), for their maltase and maltose permease activities in mid-log phase in the presence of maltose (inducing), galactose (noninducing), ethanol (noninducing), and glucose (repressing). The major difference between the two strains was seen under noninducing conditions, in which the nonlagging strain produced much higher levels of both activities than the lagging strain (Table 2). Under inducing conditions (maltose), the difference was less marked. Northern analyses of MAL mRNA species indicated that these differences were due to increased transcriptional activity of the MALx2 and MALx1 genes (Fig. 1).

View larger version (125K): [in this window] [in a new window]   FIG. 1.   Northern (RNA) analysis of MALx2, MALx1, and MALx3 mRNA levels in baker's yeast strains L38 and NL67 and their transformants. Cells were growing exponentially under inducing (maltose), noninducing (galactose or ethanol), and repressing (glucose) conditions. Total RNA was loaded in each lane, and the filters were hybridized with labelled probes (MAL6-2, MAL6-1, MAL6-3, and ACT1). All carbon sources were used at a concentration of 2% (wt/wt). Lanes: 1, L38; 2, L38 + BEJ17; 3, L38 + MALx3-VH1; 4, L38 + MALx3-VH7; 5, NL67; 6, NL67 + BEJ17; 7, NL67 + MALx3-VH1; 8, NL67 + MALx3-VH7.

Cloned MALx3 gene from a nonlagging strain results in high noninduced expression of MALx1 and MALx2 genes in a MALx3-negative background. MALx2 and MALx1 gene expression is regulated at transcription by the MALx3 protein (12, 28). We cloned a series of MALx3 genes from the nonlagging NL67 strain. Three polymorphic MALx3 genes, two with novel restriction maps (MALx3-VH7 and MALx3-VH9) and one (MALx3-VH1) corresponding to the published MAL6-3 (MAL6R) gene (18) were isolated. These genes could complement the malx3-negative phenotype of laboratory strain PB1. The MALx3-VH1 and MALx3-VH9 genes were subject to strong maltose induction, with MEL1 expression (MALx2) increased between 90- and 300-fold and lacZ expression (MALx1) increased as much as 440-fold under induced conditions compared with noninduced conditions (Table 3). The PB1 + MALx3-VH7 strain, however, had much higher levels of MALx2 and MALx1 expression under noninduced conditions but could be induced by maltose to the same final levels as the other transformants (Table 3). Induction levels from the MALx3-VH7 gene were only 5- to 13-fold. The MALx3-VH7 gene, therefore, conferred on a malx3 strain a regulation of MALx1 and MALx2 that was qualitatively similar to that seen in nonlagging strains.

View this table: [in this window] [in a new window]   TABLE 3.   Melibiase (MEL1) and -galactosidase (LacZ) activities of PB1 transformants grown in maltose, galactose, or glucosea

The MALx3-VH7 gene product significantly increases the noninduced MAL activity of a lagging strain. We subcloned MALx3-VH7, MALx3-VH50, MALx3-VH23, and MALx3-VH1 into pBEJ17, a 2 µm DNA-based high-copy-number plasmid, to provide enough copies of cloned MALx3 genes to override interference that might arise from the original MALx3 genes in strain L38. The genes that previously gave high noninduced levels of expression (MALx3-VH7 and MALx3-VH50) in PB1 also led to very high noninduced levels in the lagging strain (L38). This was not the case for MALx3-VH1-, MALx3-VH23-, and vector only-transformed L38 (Table 2). These results (for MALx3-VH7) could be attributed to increased transcription of MALx1 and MALx2, (Fig. 1). It is unlikely that this increased transcription is due to the presence of multiple copies of the MALx3 genes, since both MALx3-VH7 and MALx3-VH1 constructs are present at similar copy numbers (see, e.g., the MALx3 transcript levels in Fig. 1), and in the presence of multiple copies of MALx3-VH1, the MALx1 and MALx2 genes retained strong inducibility. The differences observed may be due to differences in the structure or regulation of the transcription factors they encode. This is consistent with the effect of mutations in the MALx3-VH50 gene, which increase noninduced maltase and maltose permease levels beyond those seen in strains with the MALx3-VH7 gene.

Higher noninduced levels of maltase and maltose permease increase the ability of a yeast strain to produce CO₂ in unsugared dough. We tested the effect that cloned MALx3 genes have on fermentation ability in unsugared dough by growing transformed strains in YP with 1% sucrose plus 220 µg of Geneticin/ml. Strains were harvested in late-respiratory phase, maltase and maltose permease activities were assayed, and amounts of gas produced in unsugared dough were measured. The maltase and maltose permease activities of L38 + MALx3-VH7 were approximately 9- and 23-fold higher than those of the control strain, L38 + BEJ17, and resulted in a 2.4-fold increase in 2nd-h gassing (Table 4). Similar effects, but with higher enzyme activities and higher levels of gassing, were seen with L38 + MALx3-VH50.

1

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Fermentation by nonlagging and lagging strains of S. cerevisiae in unsugared synthetic dough medium. Yeast cells were inoculated into unsugared synthetic dough containing 1% sucrose and 5% maltose. Samples were withdrawn at intervals and centrifuged, and supernatants were assayed for ethanol by gas chromatography. Values shown are means of data derived from two experiments. Assays were carried out in triplicate with standard errors of less than 10%. (A) Cycloheximide was added at 0 min; (B) no cycloheximide was added. , L38; , L67.

	DISCUSSION

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We found a strong correlation between a yeast strain's maltase and maltose permease activities and its ability to leaven unsugared dough. These results support the conclusions of Oda and Ouchi (30) that constitutive expression of maltose-utilization genes is crucial to prolonged leavening of unsugared dough. Here we have shown that the nonlagging phenotype is due to the presence in strains of a MALx3 gene activator that allows constitutive expression of the other MAL genes.

There have been several earlier reports that the nonlagging phenotype is dependent on a high level of maltase expression in the presence of glucose (32, 35), which is a feature of the constitutive expression in the system used by Oda and Ouchi (3, 17, 29, 30). We showed that glucose repression characteristics are not important in the nonlagging industrial strain but that a MALx3 gene capable of conferring high constitutive basal levels of expression of the MAL system under noninduced conditions (i.e., on substrates containing neither glucose nor maltose) is the most relevant feature. The MALx3 regulators we identified could still respond to maltose and lead to further increases in MAL gene expression. This ability to undergo maltose induction above the high basal level may also be required in a good baking strain but is not important to the nonlagging phenotype.

These characteristics are consistent with what is required of a strain used commercially in terms of yield and activity. In industrial practice, cane or beet molasses is used for fed-batch growth of yeast. To obtain a high yeast biomass yield, molasses is added incrementally, and biomass increases under conditions supporting respiration. During growth on sucrose or glucose as substrates in high levels of expression of the MAL genes are undesirable because they lead to a selective disadvantage (20). While glucose repression may be partially relieved due to the batch-fed mode of growth, there may be sufficient repression to prevent high levels of expression of the MAL genes. To finish the fermentation, the molasses feed is cut and the yeast is aerated in order to respire the remaining ethanol before the yeast is harvested and packaged (36). These conditions, with neither maltose nor glucose present, are very similar to the noninduced conditions used in our experiments. We suggest that, like the lagging strain, the nonlagging strain, when placed into unsugared dough, can readily ferment the available sugars (glucose, fructose, and sucrose) and produce CO₂ (34). However, due to the MALx3 background of the nonlagging strain, it enters the unsugared dough with significant activities of maltase and maltose permease, thus allowing it to utilize maltose simultaneously. At this stage the concentrations of glucose or fructose present in unsugared dough are not high enough to completely repress expression of the maltase and maltose permease genes or to catabolite inactivate the maltose permease of the nonlagging strain. In support of this, inhibition of protein synthesis at the start of fermentation in unsugared synthetic dough did not prevent a nonlagging strain from fermenting maltose, but it did inhibit the lagging strain.

For a yeast to be useful in leavening unsugared dough, it must contain a MALx3 genetic background that provides for high levels of maltase and maltose permease activities when the yeast enters the dough. These levels enable the strain to continue to produce CO₂ at the same rate even after all the easily assimilated sugars are depleted. Thus, CO₂ is produced at a constant rate typical of the nonlagging phenotype.