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 ). 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 ). 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 ). 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.
TABLE 1 Fermentation activity in relation to maltase and maltose permease activities in industrial strains of baker’syeasta 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 ). 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. ).
TABLE 2 Maltase and maltose permease activities of baker’s yeast strains L38 and NL67 and theirtransformantsa
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) (more ...)
In the presence of glucose, the maltase and maltose permease activities of both strains were reduced to very low levels (Table ). It appears, therefore, that neither the glucose repression characteristics of maltase and maltose permease activities nor the level of glucose present in unsugared dough prior to fermentation is important in determining the nonlagging phenotype.
Cloned MALx3 gene from a nonlagging strain results in high noninduced expression of MALx1 and MALx2 genes in a MALx3-negative background. MALx2
gene expression is regulated at transcription by the MALx3 protein (12
). We cloned a series of MALx3
genes from the nonlagging NL67 strain. Three polymorphic MALx3
genes, two with novel restriction maps (MALx3–VH7
) and one (MALx3–VH1
) corresponding to the published MAL6-3
) gene (18
) were isolated. These genes could complement the malx3
-negative phenotype of laboratory strain PB1. The MALx3–VH1
genes were subject to strong maltose induction, with MEL1
) increased between 90- and 300-fold and lacZ
) increased as much as 440-fold under induced conditions compared with noninduced conditions (Table ). The PB1 + MALx3–VH7 strain, however, had much higher levels of MALx2
expression under noninduced conditions but could be induced by maltose to the same final levels as the other transformants (Table ). 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
that was qualitatively similar to that seen in nonlagging strains.
Melibiase (MEL1) and β-galactosidase (LacZ) activities of PB1 transformants grown in maltose, galactose, or glucosea
The MALx3–VH7 gene was mutated to produce MALx3–VH50 (Lys364Glu, Lys371Gly, Phe375Leu), which showed higher noninduced levels in PB1 (Table ). We also fused the promoter and the 1st 954 bp of the MALx3–VH7 structural gene with the carboxyl terminus of the MALx3–VH1 gene. This construct (MALx3–VH23) regulated the marker genes in strain PB1 with strong maltose induction, but the fully induced activities of melibiase and β-galactosidase were approximately 45 and 60% higher than those seen with all other MALx3 genes (Table ). These results suggest that NL67 contains a novel MALx3 gene that leads to significantly higher MAL activity under noninduced conditions and that this gene may be responsible for the nonlagging phenotype.
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 ). These results (for MALx3–VH7) could be attributed to increased transcription of MALx1 and MALx2, (Fig. ). 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. ), 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.
In the presence of glucose, the activities of maltase and maltose permease in all strains were very low. However, in strains carrying the MALx3–VH7 and MALx3–VH50 genes, there was at least a 10-fold increase in the expression of the MALx2 gene (Table ).
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 ). Similar effects, but with higher enzyme activities and higher levels of gassing, were seen with L38 + MALx3–VH50.
TABLE 4 Fermentation activity in relation to maltase and maltose permease activities in recombinant strains of baker’syeasta
Strain L38 + PDC1 was developed by integrating a MAL6-1 structural gene fused to the PDC1 promoter at the TRP1 locus of strain L38. This strain showed a maltose permease activity 150-fold higher than that of the control strain but showed no increase in maltase activity. The higher maltose permease activity resulted in a 2.4-fold increase in 2nd-h gas production in unsugared dough (Table ).
Even though both L38 + MALx3–VH7 and L38 + PDC1 had higher 2nd-h gas production, these levels were still lower than those of the nonlagging strain NL67 (Table ). Transforming L38 + PDC1 with the BEJ17 + MALx3–VH7 plasmid (L38 + PDC1 + VH7) increased the maltase activity ninefold (Table ). This combination of maltase and maltose permease increases led to a 3.5-fold increase in 2nd-h gas production, which approaches the activity of the transformed nonlagging control (Table ). Thus, all strains with significant increases in noninduced maltase and maltose permease activities had corresponding increases in 2nd-h gas production. L38 + MALx3–VH23 had higher maltase and maltose permease activities in the presence of maltose (Table ), but no significant increase in 2nd-h gas production was evident in unsugared dough (Table ).
We added 150 μg of cycloheximide ml−1 to unsugared synthetic dough before the addition of yeast. When cycloheximide was added at the start of the fermentation, the nonlagging strain, NL67, was still producing gas 250 min into the fermentation, whereas the lagging strain (L38) was unable to produce gas beyond 110 min (Fig. ). This result suggests that the nonlagging strain entered the synthetic dough with sufficient levels of maltase and maltose permease proteins to utilize maltose without the need for further protein synthesis.
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 (more ...)