FIG. 5.

Heterologous overexpression of the human aquaporin gene hAQP1 improves freeze tolerance. (A) Overexpression of the wild-type gene, but not of the mutant allele hAQP1-A73M, improves freeze tolerance in a laboratory strain. IGC, FGC, and RGC were determined for strain BY4743 overexpressing either wild-type hAQP1 or the poorly active mutant hAQP1-A73M versus strain BY4743 transformed with an empty plasmid. Cells were either frozen (for 1 day at −30°C) (FGC) or not frozen (i.e., cooled on ice) (IGC) 40 min after the onset of fermentation by addition of 100 mM glucose. After thawing, glucose consumption was measured for 4 h to assess residual yeast activity. RGC is calculated as (FGC/IGC) × 100. Representative results are shown. Compared to that of BY4743 containing an empty plasmid, hAQP1 and hAQP1-A73M expression strains showed RGCs that were 2.3 (±0.2) and 1.5 (±0.1) times higher, respectively. (B) Overexpression of the human aquaporin gene hAQP1 improves freeze tolerance in industrial strains. IGC, FGC, and RGC were determined for strains AT25 and S47 overexpressing wild-type hAQP1 versus the respective strains transformed with an empty plasmid. The procedure described for panel A was used, except that cells were frozen or cooled on ice 30 min after the onset of fermentation. Representative results are shown. Compared to those of AT25 and S47 containing empty plasmids, hAQP1 expression strains showed RGCs that were 1.5 (±0.1) and 2.0 (±0.0) times higher, respectively.