Table 1.
Primers used in this study.
Fig 1.
K1801M showed improved growth on maltose medium.
Growth of K1801 and K1801M on various maltose-containing media. Each strain was streaked on SD, SD+EtOH (vehicle control), SD+Antimycin A, and MA (SMal+antimycin A) plates. Each plate was incubated at 30°C for 2–3 days.
Fig 2.
K1801M showed enhanced expression of MAL11p-lacZ reporter gene.
Cells carrying MAL11p-lacZ reporter plasmid were harvested after overnight cultivation in YPD medium, then transferred to YPD or YPM medium, and further cultured at 30°C for 2 h. β-galactosidase activity was measured using yeast β-galactosidase assay kit. X2180-1A, which does not have a functional MALR [5], was used as a negative control.
Table 2.
Chromosomally encoded MALR genes in each yeast.
Fig 3.
Phylogenetic and sequence analysis of YRP196W-2 in K1801 and K1801M.
a An unrooted phylogenetic tree of selected MALR genes was constructed using the Neighbor-Joining Method (1000 replicates) in the CLUSTALW multiple alignment program [21, 22]. Bar = 0.01 nucleotide substitutions per site. b Sequence comparison of MAL63 [3] and YRP196W-2. c DNA (upper line) and protein (bottom line) sequences of YRP196W-2 and YRP196W-2L. The asterisk indicates the nucleotide insertion detected in K1801M.
Fig 4.
Expression of YRP196W-2 /-2L and MAL11 target gene in K1801M.
a Gene expression in K1801 and K1801M. Cells were harvested after overnight cultivation in YPD medium, then transferred to YPD (indicated as Glc) or YPM (indicated as Mal) and cultured at 30°C. After 6 h, total RNA was extracted and RT-PCR was performed using the indicated primers. The mRNA transcript level of each target gene was standardized by normalizing the value to that obtained for the control gene TDH3, and then presented as the value relative to that observed for K1801 grown on YPD. Data are presented as mean and standard deviation from three independent experiments. b Cells harboring a YPR196W-2p-lacZ reporter in a K1801 background were harvested after overnight cultivation in YPD medium, then transferred to YPD or YPM and cultured at 30°C for 6 h. β-galactosidase activity was measured using yeast β-galactosidase assay kit. c Restoration of maltose fermentation ability by YPR196W-2L. Cells carrying the indicated plasmid were streaked on SD, SD+EtOH (vehicle control), SD+Antimycin A, and MA (SMal+antimycin A) plates. Each plate was incubated at 30°C for 2–3 days.
Fig 5.
The maltose fermentation ability of ypr196W-2Δ sake yeast.
a Growth of K1801 and its derivatives on maltose-containing media. Each strain was streaked on SD, SD+EtOH (vehicle control), SD+Antimycin A, and MA (SMal+antimycin A) plates. Each plate was incubated at 30°C for 2–3 days. b Maltose fermentation test of ypr196W-2Δ strain. Cells were harvested after overnight cultivation in SD medium, then resuspended in SMal medium at 1 × 107 cells/mL and cultured at 20°C. Sugar content in the medium was measured at the indicated times.
Fig 6.
Maltose fermentation ability of cells carrying TDH3p-driven YRP196W-2.
Maltose fermentation test of cells carrying TDH3p-YPR196W-2. Cells carrying each plasmid were harvested after overnight cultivation in SD medium, then transferred into SMal medium at 1 × 107 cells/mL and cultured at 20°C. Sugar content in the medium was measured at the indicated times.
Fig 7.
Growth in the presence of maltose of K7 family strains carrying YRP196W-2L.
Each strain was streaked on SD, SD+EtOH (vehicle control), SD+Antimycin A, and MA (SMal+antimycin A) plates, and plates were incubated at 30°C for 2–3 days.
Fig 8.
Cells expressing YPR196W-2L exhibit rapid maltose incorporation compared to control in a small-scale sake test fermentation.
a Sake mash that contained yeast cells with control vector or YPR196W-2L were prepared triplicate as described in the Materials and Methods section. Glucose and maltose content in the test fermentation. Mash was sampled at the indicated time point, and the supernatant was collected by centrifugation and assessed for sugar content. b At Day 3, total RNA was extracted and RT-PCR was performed using the indicated primers. The mRNA transcript level of each target gene was standardized by normalizing the value to that obtained for the control gene TDH3, and then presented as the value relative to that observed for control (empty vector) strain. Data are presented as mean and standard deviation from three independent experiments.
Fig 9.
An ancestral brewing yeast is presumed to have harbored a functional Mal63p ortholog. During the evolution or human selection of this ancestral strain, MAL63 experienced the deletion of a residue at nucleotide position 917 of the ORF. The resulting gene, MAL73, encodes a truncated MALR protein with attenuated trans-activation activity. As shown in this study, K1801M, isolated from K1801 on the basis of enhanced maltose fermentation ability, harbors a MAL73 gene in which a single nucleotide was inserted at ORF residue 914, resulting in a frameshifted extension of the ORF. The resulting MAL73L gene encodes a functionally restored MALR with improved trans-activation activity compared to the parental Mal73p.