Disruption of Yarrowia lipolytica TPS1 gene encoding trehalose-6-P synthase does not affect growth in glucose but impairs growth at high temperature

Abstract

We have cloned the Yarrowia lipolytica TPS1 gene encoding trehalose-6-P synthase by complementation of the lack of growth in glucose of a Saccharomyces cerevisiae tps1 mutant. Disruption of YlTPS1 could only be achieved with a cassette placed in the 3' half of its coding region due to the overlap of its sequence with the promoter of the essential gene YlTFC1. The Yltps1 mutant grew in glucose although the Y. lipolytica hexokinase is extremely sensitive to inhibition by trehalose-6-P. The presence of a glucokinase, insensitive to trehalose-6-P, that constitutes about 80% of the glucose phosphorylating capacity during growth in glucose may account for the growth phenotype. Trehalose content was below 1 nmol/mg dry weight in Y. lipolytica, but it increased in strains expressing YlTPS1 under the control of the YlTEF1 promoter or with a disruption of YALI0D15598 encoding a putative trehalase. mRNA levels of YlTPS1 were low and did not respond to thermal stresses, but that of YlTPS2 (YALI0D14476) and YlTPS3 (YALI0E31086) increased 4 and 6 times, repectively, by heat treatment. Disruption of YlTPS1 drastically slowed growth at 35°C. Homozygous Yltps1 diploids showed a decreased sporulation frequency that was ascribed to the low level of YALI0D20966 mRNA an homolog of the S. cerevisiae MCK1 which encodes a protein kinase that activates early meiotic gene expression.

Figure 1. Phenotypic complementation of a S.cerevisiae tps1 mutant by expression of the YlTPS1 gene.

The strains indicated were streaked on minimal medium with glucose or galactose as carbon sources and incubated at 30°C for 4 days. Trehalose content of the strains cultured with glucose, except the mutant tps1 cultured in galactose, is given in the table (results of two independent cultures). The multicopy plasmid was pCLF1 and the centromeric one pCLF2.

Figure 2. Relative positions of the TPS1 and TFC1 genes in several yeast species and disruption of the chromosomal copy of the YlTPS1 gene.

a) Diagram of the chromosomal neighborhood of TPS1 in different yeasts species. Notice the unusual relative position of TPS1 and TFC1 in Y. lipolytica. The order and transcriptional direction of the genes correspond to the annotation in Génolevures except for S. cerevisiae for which the annotation of SGD was used . Names of genes appear below each arrow, arrows without name indicate genes of unknown function. Chromosome designation is also indicated. WGD, whole genome duplication. b) Scheme of the chromosomal region around YlTPS1 and its disruption. The location of the coding region of YlTFC1 is also shown. The I-SceI sites flanking the URA3 gene were introduced by PCR as described in Materials and Methods. Short vertical bars indicate the piece of DNA used for the chromosomal disruption. c, d) Southern analysis of two different disruptants: genomic DNA was digested with HindIII (c) or StuI (d) and hybridized with the indicated probe. The sizes of the DNA bands in bp are shown. Attempts to disrupt YlTPS1 placing YlURA3 between the SalI sites or between HindIII and EcoRI were unsuccessful.

Figure 3. mRNA levels corresponding to genes encoding glucose phosphorylating enzymes and proteins related to trehalose metabolism in Y. lipolytica.

Yeasts strains CJM 645, wild type, and CJM 651, Yltps1::URA3, were grown in minimal medium- glucose as indicated in Materials and Methods. mRNA levels were quantified by RT-qPCR as described in Materials and Methods. Three independent cultures were analyzed and three technical replicas were done for each run. Expression of each gene was normalized to that of the YlARP4. The columns represent the mean values of the three biological experiments with bars indicating the standard deviation. The genes studied encode the following proteins: GLK1, glucokinase; HXK1, hexokinase; TPS1, trehalose-6-P synthase; TPS2, trehalose-6-P phosphatase; TPS3, subunit of the trehalose synthase complex; NTH1, neutral trehalase.

Figure 4. Levels of mRNA corresponding to genes YlTPS1, YlTPS2, YlTPS3, MHY1 (YALI0B21582) and HSF1 (YALI0E13948) during thermal stresses.

A sample of an exponentially growing culture of strain CJM 645 in minimal medium glucose (time zero) was taken before its transfer to 40°C or 4°C for 2 or 20 hours. mRNA levels were quantified by RT-qPCR as described in Materials and Methods. Two independent cultures were analyzed and three technical replicas were done for each run. Expression of each gene was normalized to that of YlARP4. Values are shown relative to that of time zero. The columns represent the mean values of the experiments with bars indicating the standard deviation.

Figure 5. Lack of complementation of Sctps1 by YlTPS3.

The S. cerevisiae tps1 mutant (CJM 486) transformed with plasmids pCLF1 (YlTPS1), pCLF7 (YlTPS3) and pDB20 (void) were streaked on minimal medium with glucose or galactose and incubated at 30°C for 4 days.

Figure 6. Effect of YlTPS1 disruption on growth at 35°C.

The strains were streaked on minimal medium glucose plates, incubated at the indicated temperatures and pictures taken 3 and 7 days after inoculation.

Figure 7. Levels of mRNA corresponding to several genes during sporulation.

A wild type diploid and an homozygous tps1 diploid were transferred from minimal medium glucose to V8 sporulation medium (see Materials and Methods). Samples were taken from the initial culture on glucose (time zero) and after 8 days in V8. mRNA levels were quantified by RT-qPCR as described in Materials and Methods. Three independent sporulation experiments were analyzed and three technical replicas were done for each run. Expression of each gene was normalized to that of YlARP4. Levels of mRNA are shown relative to that of time zero. The columns represent the mean values of the experiments with bars indicating the standard deviation.