Disruption of the ABC transporter genes PDR5, YOR1, and SNQ2, and their participation in improved fermentative activity of a sake yeast mutant showing pleiotropic drug resistance

Clotrimazole-resistant mutants from sake yeasts show improved fermentative activity in sake mash and pleiotropic drug resistance (PDR). The PDR mechanism is interpreted by overexpression of ATP-binding cassette (ABC) transporters, which extrude various kinds of drugs out of a cell. In a clotrimazole-resistant mutant, CTZ21, isolated from the haploid sake yeast HL69, the levels of mRNA for three major ABC transporter genes, PDR5, SNQ2, and YOR1, markedly increased. These three genes of CTZ21 were disrupted to investigate which participated in the improved fermentative activity of CTZ21. The fermentative activities of Δpdr5 and Δsnq2 strains of CTZ21 were reduced to that of HL69 in the initial and middle stages of fermentation. In the last stage, however, the sake meter [(1/gravity-1) × 1443] of the Δpdr5 and Δsnq2 strains rose faster than that of HL69. On the other hand, a Δyor1 strain of CTZ21 fermented sake mash in a manner nearly identical to that of CTZ21 until the last stage of fermentation. But in the last stage, fermentation of the Δyor1 slowed down compared with that of CTZ21. A Δyor1 strain of HL69 also exhibited much reduced fermentative activity in the middle and last fermentation stages. The YOR1 gene seems necessary for sake fermentation to be completed efficiently. The ATP content in sake mash brewed with CTZ21 was drastically decreased throughout the whole fermentation period. This low ATP level was restored to a medium level in the cases of both the Δpdr5 and Δsnq2 strains of CTZ21. In contrast, the Δyor1 of CTZ21 exhibited a low ATP level in sake mash in the same manner as CTZ21. These results suggest that the low ATP level of CTZ21 contributes to a certain extent its improved fermentative activity in the initial and middle stages of sake fermentation.     

Deletion of the N-terminal domain of the yeast vacuolar (Na+,K+)/H+ antiporter Vnx1p improves salt tolerance in yeast and transgenic Arabidopsis

Cation/proton antiporters play a major role in the control of cytosolic ion concentrations in prokaryotes and eukaryotes organisms. In yeast, we previously demonstrated that Vnx1p is a vacuolar monovalent cation/H⁺ exchanger showing Na⁺/H⁺ and K⁺/H⁺ antiporter activity. We have also shown that disruption of VNX1 results in an almost complete abolishment of vacuolar Na⁺/H⁺ exchange, but yeast cells overexpressing the complete protein do not show improved salinity tolerance. In this study, we have identified an autoinhibitory N-terminal domain and have engineered a constitutively activated version of Vnx1p, by removing this domain. Contrary to the wild type protein, the activated protein has a pronounced effect on yeast salt tolerance and vacuolar pH. Expression of this truncated VNX1 gene also improves Arabidopsis salt tolerance and increases Na⁺ and K⁺ accumulation of salt grown plants thus suggesting a biotechnological potential of activated Vnx1p to improve salt tolerance of crop plants.