Molecular analysis of maltotriose transport and utilization by Saccharomyces cerevisiae

Abstract

Efficient fermentation of maltotriose is a desired property of Saccharomyces cerevisiae for brewing. In a standard wort, maltotriose is the second most abundant sugar, and slower uptake leads to residual maltotriose in the finished product. The limiting factor of sugar metabolism is its transport, and there are conflicting reports on whether a specific maltotriose permease exists or whether the mechanisms responsible for maltose uptake also carry out maltotriose transport. In this study, radiolabeled maltotriose was used to show that overexpression of the maltose permease gene, MAL61, in an industrial yeast strain resulted in an increase in the rate of transport of maltotriose as well as maltose. A strain derived from W303-1A and lacking any maltose or maltotriose transporter but carrying a functional maltose transport activator (MAL63) was developed. By complementing this strain with permeases encoded by MAL31, MAL61, and AGT1, it was possible to measure their specific transport kinetics by using maltotriose and maltose. All three permeases were capable of high-affinity transport of maltotriose and of allowing growth of the strain on the sugar. Maltotriose utilization from the permease encoded by AGT1 was regulated by the same genetic mechanisms as those involving the maltose transcriptional activator. Competition studies carried out with two industrial strains, one not containing any homologue of AGT1, showed that maltose uptake and maltotriose uptake were competitive and that maltose was the preferred substrate. These results indicate that the presence of residual maltotriose in beer is not due to a genetic or physiological inability of yeast cells to utilize the sugar but rather to the lower affinity for maltotriose uptake in conjunction with deteriorating conditions present at the later stages of fermentation. Here we identify molecular mechanisms regulating the uptake of maltotriose and determine the role of each of the transporter genes in the cells.

FIG. 1.

Regulation of permeases. (a) Cells were grown under noninducing conditions, and maltose (white) and maltotriose (black) permease activities were assayed. Activity is represented as the fold increase, with the basal level as the reference point. (b) Northern hybridization analysis of strains PB1+MAL63 and PB1+VH50 with probes to MALx1, AGT1, and ACT1 (RNA control).

FIG. 2.

Enhanced expression of Mal61p. (a and b) Strains L38 and L38+PDC1 were grown on YEP-2% galactose medium, i.e., under noninducing conditions. The uptake of maltose (a) and maltotriose (b) (concentration, 0.5 mM) was measured. The rates of uptake of maltose and maltotriose in L38+PDC1 were 51 and 32 nmol min⁻¹ mg of dry weight⁻¹, respectively. (c) Northern hybridization analysis with probes to MALx1, AGT1, and ACT1 (RNA control).

FIG.3.

Growth of W303R/MAL31, W303R/MAL61, W303R/AGT1, and W303RKO on different sugars. The cells were pregrown on YEP-2% maltose medium and streaked on solid minimal media supplemented with the appropriate auxotrophic requirements and containing the following sugar: glucose (A), maltose (B), maltotriose (C), α-methylglucoside (D), melezitose (E), trehalose (F), and turanose (G). The plates were incubated at 30°C for up to 3 days. The key to strain locations is given in the lower left-hand corner of the figure.

FIG. 4.

Growth kinetics of W303R/MAL31, W303R/MAL61, W303R/AGT1, and W303RKO. Symbols: ♦, W303R/MAL31; ▴, W303R/MAL61; ○, W303R/AGT1; •, W303RKO; □, W303R/MAL61 with a permease but no integrated regulator; ▪, W303R/MAL61 grown with glucose. Results for W303RKO in panel a are the same as those for W303R/MAL61 with a permease but no integrated regulator. Strains were inoculated on minimal medium with auxotrophic requirements and maltose (a) or maltotriose (b). Cell density measurements (OD₆₀₀) were taken at regular intervals.

FIG. 5.

Transport of maltose and maltotriose in W303R/MAL31, W303R/MAL61, W303R/AGT1, and W303RKO. Strains were grown in YEP-2% glucose medium; cells were harvested at an OD₆₀₀ of 0.4, washed twice, resuspended in an equal volume of YEP- 2% maltose medium, and harvested after 4 h. Maltose (a) and maltotriose (b) (0.5 mM) permease activities of strains W303R/MAL31 (♦), W303R/MAL61 (▴), and W303R/AGT1 (○) were assayed over a period of 150 s. W303RKO (a) and W303R/MAL61 (b) cells were grown in YEP- 2% glucose medium, and permease activity was assayed over a period of 150 s (□).

FIG. 6.

Analysis of intracellular material of W303R/MAL61. Strains were grown in YEP-2% glucose medium; cells were harvested at an OD₆₀₀ of 0.4, washed twice, resuspended in an equal volume of YEP-2% maltose or YEP-2% glucose medium, and harvested after 4 h. Maltose and maltotriose permease activity assays were performed. The intracellular material was extracted and run on a silica gel 60 TLC plate; the solvent used was n-butanol-acetic acid-water (6:3:1). Control samples were run by using solutions of radiolabeled sugars. The chromatogram was visualized by using a PhosphorImager. A repeat of labeled glucose is contained in lane 12.

FIG. 7.

Effect of competing sugars on maltose and maltotriose permease activities in an industrial strain. A lager strain (black) and an ale strain (white) were grown on YEP-2% maltose medium and used to measure the uptake of 0.5 mM radiolabeled maltose (a) and maltotriose (b) in the presence of 5 mM maltotriose and maltose, respectively. The rate of uptake at 100% corresponds to activity in the presence of no competitior (only reaction buffer). (a) Maltose permease activities in the presence of no competitor in the lager and ale strains, 10 and 7.2 nmol min⁻¹ mg of dry weight⁻¹, respectively. (b) Maltotriose permease activities in the presence of no competitor in the lager and ale strains, 6 and 3.5 nmol min⁻¹ mg of dry weight⁻¹, respectively. Results are the mean of triplicate determinations.