Improved fermentation performance of a lager yeast after repair of its AGT1 maltose and maltotriose transporter genes

Abstract

The use of more concentrated, so-called high-gravity and very-high-gravity (VHG) brewer's worts for the manufacture of beer has economic and environmental advantages. However, many current strains of brewer's yeasts ferment VHG worts slowly and incompletely, leaving undesirably large amounts of maltose and especially maltotriose in the final beers. alpha-Glucosides are transported into Saccharomyces yeasts by several transporters, including Agt1, which is a good carrier of both maltose and maltotriose. The AGT1 genes of brewer's ale yeast strains encode functional transporters, but the AGT1 genes of the lager strains studied contain a premature stop codon and do not encode functional transporters. In the present work, one or more copies of the AGT1 gene of a lager strain were repaired with DNA sequence from an ale strain and put under the control of a constitutive promoter. Compared to the untransformed strain, the transformants with repaired AGT1 had higher maltose transport activity, especially after growth on glucose (which represses endogenous alpha-glucoside transporter genes) and higher ratios of maltotriose transport activity to maltose transport activity. They fermented VHG (24 degrees Plato) wort faster and more completely, producing beers containing more ethanol and less residual maltose and maltotriose. The growth and sedimentation behaviors of the transformants were similar to those of the untransformed strain, as were the profiles of yeast-derived volatile aroma compounds in the beers.

FIG. 1.

Diagram of the integration cassettes used in this study. The short and long cassettes contain, respectively, 387 and 705 nucleotides of the AGT1 promoter from strain A15.

FIG. 2.

Southern analyses of strain A15 and integrants 1, 2, and 14. Lanes 1 and 15, molecular weight marker II (23,130, 9,416, 6,557, 4,361, 2,322, and 2,027 bp); lanes 2 and 16, molecular weight marker III (21,226, 5,148, 4,973, 4,268, 3,530, and 2,027 bp); lanes 3, 7, and 11, strain A15; lanes 4, 8, and 12, integrant 1; lanes 5, 9, and 13, integrant 2; lanes 6, 10, and 14, integrant 14. Chromosomal DNA was restricted with EcoRI (lanes 3 to 6), XbaI (lanes 7 to 10), or XmnI (lanes 11 to 14); separated in 0.8% agarose gel; blotted onto a nylon filter; and probed with a 984-bp AGT1 PCR fragment. The band sizes (in kilobases) predicted from the SGDB sequence of native AGT1 loci are as follows: EcoRI, 6.3; XbaI, 4.8; XmnI, 4.1. For repaired loci, they are as follows: EcoRI, 5.3; XbaI, 6.3; XmnI, 3.1. Tandem integration of cassette DNA would give a 9.6-kb or larger XbaI band(s).

FIG. 3.

Expression of AGT1 and other α-glucoside transporter and maltase genes during batch growth of strain A15 and integrant 1 (Int1), Int2, and Int14 on 20 g · liter⁻¹ glucose, maltose, or maltotriose at 18°C. Yeast samples were collected after 13 h (upper row), while sugars were still present at about 7 g · liter⁻¹, or at 36 h (lower row), when cells were in stationary phase. Samples were lysed and analyzed by TRAC with probes specific for ACT1 (actin control); the transporter genes MTT1, AGT1, and MALx1; and the maltase gene MALx2. Shown are the fluorescence signals from specifically bound probes. The signals from the MALx2 probe were divided by 10 (MALx2/10). Results are averages ± ranges of data from two replicate growths of each yeast strain on each sugar.

FIG. 4.

Attenuation profiles during the fermentation of 15°P (upper panel) and 24°P (lower panel) worts by duplicate growths of strain A15 (A15A, A15B), duplicate growths of integrant 1 (Int 1 A, Int 1 B), and single growths of integrants 2 and 14 (Int 2, Int 14). The 15 and 24°P worts were pitched with, respectively, 5.0 or 8.0 g of fresh yeast mass · liter⁻¹ at 10°C, and fermentations were continued at 14°C. The 24°P fermentations were shifted to 18°C at 20 h. Insets show detail during the last 4 or 5 days.

FIG. 5.

Yeast in suspension during the fermentations shown in Fig. 4 of 15°P (upper panel) and 24°P (lower panel) worts by strain A15 and integrant 1 (Int 1), Int 2, and Int 14. The plots show the increases in yeast mass in suspension caused by growth during the first 2 to 3 days, followed by decreases as the yeast sediments in the static fermentations. Experimental details are the same as in the legend to Fig. 4.

FIG. 6.

Effect of cropping and repitching strain A15 and integrant 1. Duplicate growths of strain A15 (A15A, A15B) and integrant 1 (Int 1 A, Int 1 B) were pitched at 8.0 g of fresh yeast mass · liter⁻¹ into 24°P wort, each fermentation being performed in duplicate. At 89 h, one of each duplicate pair was stopped and the yeast was cropped as described in Materials and Methods. The attenuation profile of the other duplicate is shown in the main part of the figure. The cropped yeasts were again pitched at 8.0 g of fresh yeast mass · liter⁻¹ into 24°P wort, and the attenuation profiles of these fermentations are shown in the inset. App., apparent.

FIG. 7.

Volatile aroma compounds in beers from series A 24°P fermentations. Results are normalized to a standard beer containing 35 g of ethanol · liter⁻¹. Error bars for integrant (Int) 1 and strain A15 show the range between the average and highest values for duplicate fermentations with independently grown yeast lots.