The overexpression transformant of D. hansenii had much higher AHP expression levels than its wild type counterpart when grown under 3.5 M NaCl and in the presence of the inducer methanol (Fig. 7A). Without any salt the overexpression trasnsformant showed a comparable growth to that of the wild type strain with or without the presence of methanol in the culture media (Fig. 8). Growth of both the wild type strain and the overexpression transformant was inhibited by 3.5 M NaCl (Fig. 8B). However, only the overexpression transformant

showed enhanced growth in the presence of the inducer methanol. Thus, overexpression and suppression of DhAHP reduce the salt tolerance of D. hansenii, respectively. The small enhancements in growth in the overexpression transformant under high salt, as compared to the wild type RAD001 in vitro strain, is expected as expression of endogenous NADPH-oxidase inhibitor DhAHP can be largely induced by salt in this halophilic organism (Fig. 5). Figure 7 Relative levels of DhAHP transcript of three yeasts and their DhAHP overexpression transformants. Cells of D. hansenii

(A), S. cerevisiae (B) and P. methanolica (C) were grown in media containing 3.5, 2.0 and 2.5 M NaCl, respectively, in the presence or absence of methanol for 72 min, and their DhAHP transcripts determined by real-time RT-PCR. For each species, the level for the wild type strain grown in media without methanol was taken as 1. Since the wild type strains of S.c. and P.m do not contain DhAHP their DhAHP transcript Unoprostone levels were low while their overexpression SGC-CBP30 purchase transformants showed high levels of expression relatively. Data presented were means +/- S.D. from 3–4 replicates of measurement. Figure 8 Growth of D. hansenii and its DhAHP overexpression transformant as affected by salt. Cells were cultured in YM11 media with or without

3.5 M NaCl and in the presence or absence of methanol for 5 days. W-M: wild type strain, without methanol, W+M: wild type strain, with 0.5% methanol, T-M: transformant, without methanol, T+M: transformant with 0.5% methanol. Data presented were means +/- S.D. from 3–4 replicates of measurement. Overexpression of DhAHP in S. cerevisiae and P. methanolica The function of DhAHP was further tested by overexpression of the gene in the two salt-sensitive yeasts S. cerevisiae and P. methanolica. As expected, the levels of DhAHP transcript in the wild type strains of the two species were very low even under high salt conditions, but its expression levels in the overexpression transformants increased drastically, especially in the presence of the inducer methanol (Figs. 7B, 7C). The salt tolerance of the overexpression transformants of the two yeasts was evaluated by culture in YPD medium containing 2.0 M NaCl for S. cerevisiae (Fig. 9b) and in YPAD medium containing 2.5 M NaCl for P. methanolica, relative to those of their wild type counterparts (Fig. 10b).