Isolation and characterization of a gene specific to lager brewing yeast that encodes a branched-chain amino acid permease

Abstract

We found two types of branched-chain amino acid permease gene (BAP2) in the lager brewing yeast Saccharomyces pastorianus BH-225 and cloned one type of BAP2 gene (Lg-BAP2), which is identical to that of Saccharomyces bayanus (by-BAP2-1). The other BAP2 gene of the lager brewing yeast (cer-BAP2) is very similar to the Saccharomyces cerevisiae BAP2 gene. This result substantiates the notion that lager brewing yeast is a hybrid of S. cerevisiae and S. bayanus. The amino acid sequence homology between S. cerevisiae Bap2p and Lg-Bap2p was 88%. The transcription of Lg-BAP2 was not induced by the addition of leucine to the growth medium, while that of cer-BAP2 was induced. The transcription of Lg-BAP2 was repressed by the presence of ethanol and weak organic acid, while that of cer-BAP2 was not affected by these compounds. Furthermore, Northern analysis during beer fermentation revealed that the transcription of Lg-BAP2 was repressed at the beginning of the fermentation, while cer-BAP2 was highly expressed throughout the fermentation. These results suggest that the transcription of Lg-BAP2 is regulated differently from that of cer-BAP2 in lager brewing yeasts.

FIG. 1

The structures of the plasmids. (A) PYCGPY is a centromeric vector containing yeast centromere sequence (CEN4), yeast autonomously replicating sequence (ARS1), yeast glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p), the G418 resistance gene (G418r), the yeast pyruvate kinase promoter (PYK1p), the yeast glyceraldehyde-3-phosphate dehydrogenase terminator (TDH3t), and the ampicillin resistance gene (Amp^r). (B and C) BAP2 and Lg-BAP2 were inserted in the SacI-BamHI site of PYCGPY and named PYCGPYBP2 and PYCGPYLgBP, respectively.

FIG. 2

Genomic Southern hybridization with S. cerevisiae BAP2 probe (nucleotides 180 to 891). Genomic DNA was digested with XbaI. Lane 1, S. cerevisiae X2180-1A; lane 2, lager brewing yeast BH-225; lane 3, S. bayanus IFO1127.

FIG. 3

Genomic Southern hybridization with the Lg-BAP2 probe. (A) Genomic DNA was digested with EcoRV (lanes 1 to 3) and with XbaI (lanes 4 to 6). Lane 1 and lane 4, S. cerevisiae X2180-1A; lane 2 and lane 5, lager brewing yeast BH-225; lane 3 and lane 6, S. bayanus IFO1127. (B) Genomic DNA was digested with SpeI. Lane 1, lager brewing yeast BH-225; lane 2, S. bayanus IFO1127.

FIG. 4

Amino acid sequence homology between the Lg-Bap2 protein of the lager brewing yeast BH-225 and the Bap2 protein of S. cerevisiae. The numbers indicate the amino acid positions. Amino acid sequence identities between two proteins are shaded.

FIG. 5

Growth phenotypes of strains X2180-1A (wild type), YK006 (Δgap1), YK007 (Δgap1 Δssy1), YK008 (Δgap1 Δssy1, pYCGPY [vector]), YK009 (Δgap1 Δssy1, pYCGPYBP2 [PYK1p-BAP2]), and YK010 (Δgap1 Δssy1, pYCGPYLgBP [BPPYK1p-Lg-BAP2]) after 3 days of growth on an SLD agar plate which contained leucine as the sole nitrogen source.

FIG. 6

The transcription of BAP2 homologues in the lager brewing yeast BH-225 after addition of leucine was analyzed by Northern blotting. Cells were pregrown overnight on SD medium at 30°C. From these precultures, main cultures were inoculated at an optical density at 600 nm of 0.5 in fresh SD medium and grown subsequently to an optical density at 600 nm of 0.65 (for 4 h) at 30°C. Then, leucine was added to a final concentration of 2 mM. Total RNA was isolated at different time points after leucine addition and hybridized with BAP2, Lg-BAP2, and ACT1 as probes. ACT1 was used as a loading control.

FIG. 7

(A) The transcription of BAP2 homologues in the lager brewing yeast BH-225 in response to nitrogen starvation was analyzed by Northern blotting. Total RNA was isolated after cultivation for 4 h in YPM medium (lane 1) and after transfer to SPM medium and was cultivated for 2 h (lane 2) and hybridized with BAP2, Lg-BAP2, DUR1, and ACT1 as probes. The blot that was hybridized with Lg-BAP2 probe was overexposed for comparison of the transcriptional level in these conditions. ACT1 was used as a loading control, and DUR1 was used as a control nitrogen starvation-induced gene. (B) The transcription of BAP2 homologues in the lager brewing yeast BH-225 in response to various stresses was analyzed by Northern blotting. Total RNA was isolated after incubation in YPM medium at 30°C (lane 1); in YPM medium containing 8% ethanol at 30°C (lane 2); in YPM medium containing 1 mM sorbate (pH 4.5) at 30°C (lane 3); in YPM medium containing 27% maltose at 30°C (lane 4); and in YPM medium at 37°C (lane 5) and hybridized with BAP2, Lg-BAP2, HSP30, and ACT1 as probes. ACT1 was used as a loading control, and HSP30 was used as a control stress-induced gene.

FIG. 8

The transcription of BAP2 homologues in the lager brewing yeast BH-225 during wort fermentation was analyzed by Northern blotting. Total RNA was isolated after 1, 2, 3, 4, 5, 6, and 7 days of the fermentation period and hybridized with BAP2, Lg-BAP2, and ACT1 as probes. ACT1 was used as a loading control.