The YlTPS1 disruptants grew in glucose in contrast with the behavior of the tps1 mutants of S. cerevisiae. The distinct phenotype could be caused by a difference in the glucose SB431542 phosphorylating equipment between the two yeasts, by a lack of significant activity of Tps1 or both. We found that in glucose grown cultures of Y. Masitinib cost lipolytica expression of the gene YALI0E15488 encoding a glucokinase (Flores and Gancedo, unpublished results) exceeded that of the gene encoding hexokinase (Figure 3). Also enzyme measurements showed that glucokinase constitutes the main phosphorylating activity in Y. lipolytica (Table 2). Since glucokinase is insensitive to inhibition by T6P, the growth in glucose of the Yltps1 mutant may be explained by the scarce contribution of hexokinase to the glucose phosphorylating activity. Moreover, disruption of YlTPS1 slightly decreased the proportion of hexokinase activity (Table 2). Concentration of hexose mono or bis- phosphates and ATP were not affected with respect to that of a wild type during growth in glucose (Table 3) in contrast to what happens in a S. cerevisiae tps1 mutant which accumulates those compounds and loses ATP upon glucose addition. This result is consistent with a lack of control of the glycolytic flux by T6P in Y. lipolytica. The YlTPS1 disruptants showed a slightly shorter duplication time than the wild type (Wt 14964 min, tps1 13964 min, tps1/pCLF5 15168 min, means of four experiments) (Figure S1). No immediate explanation can be provided for this difference. Levels of trehalose in Y. lipolytica grown in glucose up to stationary phase or in glycerol were below 1 nmol/mg dry weight. A possible explanation for this result could be that the genes encoding the trehalose biosynthetic pathway enzymes were not expressed during growth in glucose, therefore we measured the expression of those genes in Y.lipolytica. In the Ge´nolevuress database, YALI0D14476 is annotated as similar to S. cerevisiae TPS2 and YALI0E31086 shows the highest homology with S. cerevisiae TPS3/TSL1. All these genes were expressed during growth in glucose although the levels of YlTPS2 and YlTPS3 were low when compared to that of YlTPS1 (Figure 3). Although a western blot analysis of a fusion of Tps1 to the HA epitope indicated that the protein was expressed during growth in glucose (results not shown) its activity was very low (Table 4).