A disruption of YlTPS3 abolished trehalose accumulation upon heat treatment raising the question that Tps3 could be another T6P synthase an hypothesis that cannot be ruled out due to the similarity between the YlTPS1 and YlTPS3 sequences. This possibility was made unlikely by the absence of trehalose in a Yltps1 mutant after heat shock and by the lack of complementation of the glucose negative phenotype of a S. cerevisiae tps1 mutant by the YlTPS3 cDNA (Figure 5). We suggest that YlTps3 is necessary to maintain the stability of the trehalose synthase complex during heat shock in Y. lipolytica. This reveals a difference with S. cerevisiae where the absence of Tps3 does not affect trehalose content during heat shock. Disruption of YlTPS1 severely decreased growth at 35uC, only small colonies were visible after 7 days at this temperature (Figure 6). A plasmid carrying YlTPS1 restored a wild type phenotype. The S.cerevisiae TPS1 gene slightly improved growth of the Yltps1 mutant at 35uC. Treatment of Y.lipolytica at 4uC during 2 or 20 hours did not modify significantly the mRNA levels corresponding to the genes of the trehalose biosynthetic pathway (Figure 4), in contrast with the behavior of TPS1 and TPS2 in S. cerevisiae whose expression increase upon a treatment below 10uC. In S. cerevisiae transcription factors Hsf1 and Msn2/4 are implicated in the response to heat shock and other stresses. In Y. lipolytica the protein Mhy1 (YALI0B21582p) shows high similarity in its zinc finger domain to that of Msn2/4 and also binds STRE sequences. A BLAST search of the Y. lipolytica database for genes encoding homologues of ScHSF1 yielded gene YALI0E13948. Levels of mRNA corresponding to those genes increased about 3 times after heat shock (Figure 4) consistent with their possible implication in heat shock regulated 537049-40-4 processes. When Y. lipolytica diploids homozygous for the tps1 mutation (CJM 724) were placed in SB431542 sporulation conditions the sporulation frequency was reduced with respect to that of wild type (CJM 722) or heterozygous TPS1/tps1 (CJM 723) diploids. A similar behaviour in tps1/tps1 diploids in S. cerevisiae was ascribed to a decreased expression of MCK1, a gene that stimulates expression of IME1 which encodes a transcriptional activator of sporulation.