Gene_locus Report for: yeast-ym60

Ymr210w was identified as a MAG (Monoacylglycerol) lipase. The accumulation of the phospholipids in the ymr210wDelta was not clearly understood. It was expressed in S. cerevisiae using pYES2/CT vector and His-tag purified recombinant protein confirmed TAG lipase activity. To further evaluate the role of YMR210w, ester hydrolase activity was also confirmed with pNP-acetate, pNP-butyrate and pNP-palmitate. GC-MS lipid profiling of ymr210wDelta showed an increase in the 15:0 Pentadecanoic acid by 76% among the total lipids. Phospholipid, Erucic acid 22:1 (delta13) showed 43% increase while steryl esters showed significant changes with 16:0 hexadecanoic acid augmentations by 80% and 18:0 Octadecanoic acid by 165% when compared to wild type (WT). Increase in the steryl ester and TAG content supports the accumulation of lipid bodies in ymr210wDelta strain when compared with WT cells.

In silico analysis of the uncharacterized open reading frame YMR210w in Saccharomyces cerevisiae revealed that it possesses both an alpha/beta hydrolase domain (ABHD) and a typical lipase (GXSXG) motif. The purified protein displayed monoacylglycerol (MAG) lipase activity and preferred palmitoyl-MAG. Overexpression of YMR210w in the known MAG lipase mutant yju3Delta clearly revealed that the protein had MAG lipase activity, hence we named the ORF MGL2. Overexpression of YMR210w decreased the cellular triacylglycerol levels. Analysis of the overexpressed strains showed reduction in the lipid droplets number and size. Phenotype studies revealed that the double deletion yju3Deltamgl2Delta displayed a growth defect that was partially restored by MGL2 overexpression.

Fatty acid ethyl esters are secondary metabolites produced by Saccharomyces cerevisiae and many other fungi. Their natural physiological role is not known but in fermentations of alcoholic beverages and other food products they play a key role as flavor compounds. Information about the metabolic pathways and enzymology of fatty acid ethyl ester biosynthesis, however, is very limited. In this work, we have investigated the role of a three-member S. cerevisiae gene family with moderately divergent sequences (YBR177c/EHT1, YPL095c/EEB1, and YMR210w). We demonstrate that two family members encode an acyl-coenzymeA:ethanol O-acyltransferase, an enzyme required for the synthesis of medium-chain fatty acid ethyl esters. Deletion of either one or both of these genes resulted in severely reduced medium-chain fatty acid ethyl ester production. Purified glutathione S-transferase-tagged Eht1 and Eeb1 proteins both exhibited acyl-coenzymeA:ethanol O-acyltransferase activity in vitro, as well as esterase activity. Overexpression of Eht1 and Eeb1 did not enhance medium-chain fatty acid ethyl ester content, which is probably due to the bifunctional synthesis and hydrolysis activity. Molecular modeling of Eht1 and Eeb1 revealed the presence of a alpha/beta-hydrolase fold, which is generally present in the substrate-binding site of esterase enzymes. Hence, our results identify Eht1 and Eeb1 as novel acyl-coenzymeA:ethanol O-acyltransferases/esterases, whereas the third family member, Ymr210w, does not seem to play an important role in medium-chain fatty acid ethyl ester formation.

Ymr210w was identified as a MAG (Monoacylglycerol) lipase. The accumulation of the phospholipids in the ymr210wDelta was not clearly understood. It was expressed in S. cerevisiae using pYES2/CT vector and His-tag purified recombinant protein confirmed TAG lipase activity. To further evaluate the role of YMR210w, ester hydrolase activity was also confirmed with pNP-acetate, pNP-butyrate and pNP-palmitate. GC-MS lipid profiling of ymr210wDelta showed an increase in the 15:0 Pentadecanoic acid by 76% among the total lipids. Phospholipid, Erucic acid 22:1 (delta13) showed 43% increase while steryl esters showed significant changes with 16:0 hexadecanoic acid augmentations by 80% and 18:0 Octadecanoic acid by 165% when compared to wild type (WT). Increase in the steryl ester and TAG content supports the accumulation of lipid bodies in ymr210wDelta strain when compared with WT cells.

In silico analysis of the uncharacterized open reading frame YMR210w in Saccharomyces cerevisiae revealed that it possesses both an alpha/beta hydrolase domain (ABHD) and a typical lipase (GXSXG) motif. The purified protein displayed monoacylglycerol (MAG) lipase activity and preferred palmitoyl-MAG. Overexpression of YMR210w in the known MAG lipase mutant yju3Delta clearly revealed that the protein had MAG lipase activity, hence we named the ORF MGL2. Overexpression of YMR210w decreased the cellular triacylglycerol levels. Analysis of the overexpressed strains showed reduction in the lipid droplets number and size. Phenotype studies revealed that the double deletion yju3Deltamgl2Delta displayed a growth defect that was partially restored by MGL2 overexpression.

Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (approximately 2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1118 chromosome. These regions encompass 34 genes involved in key wine fermentation functions. Phylogeny and synteny analyses showed that 1 of these regions originated from a species closely related to the Saccharomyces genus, whereas the 2 other regions were of non-Saccharomyces origin. We identified Zygosaccharomyces bailii, a major contaminant of wine fermentations, as the donor species for 1 of these 2 regions. Although natural hybridization between Saccharomyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and non-Saccharomyces species. We show that the regions identified are frequent and differentially distributed among S. cerevisiae clades, being found almost exclusively in wine strains, suggesting acquisition through recent transfer events. Overall, these data show that the wine yeast genome is subject to constant remodeling through the contribution of exogenous genes. Our results suggest that these processes are favored by ecologic proximity and are involved in the molecular adaptation of wine yeasts to conditions of high sugar, low nitrogen, and high ethanol concentrations.

Many industrial strains of Saccharomyces cerevisiae have been selected primarily for their ability to convert sugars into ethanol efficiently despite exposure to a variety of stresses. To begin investigation of the genetic basis of phenotypic variation in industrial strains of S. cerevisiae, we have sequenced the genome of a wine yeast, AWRI1631, and have compared this sequence with both the laboratory strain S288c and the human pathogenic isolate YJM789. AWRI1631 was found to be substantially different from S288c and YJM789, especially at the level of single-nucleotide polymorphisms, which were present, on average, every 150 bp between all three strains. In addition, there were major differences in the arrangement and number of Ty elements between the strains, as well as several regions of DNA that were specific to AWRI1631 and that were predicted to encode proteins that are unique to this industrial strain.

We sequenced the genome of Saccharomyces cerevisiae strain YJM789, which was derived from a yeast isolated from the lung of an AIDS patient with pneumonia. The strain is used for studies of fungal infections and quantitative genetics because of its extensive phenotypic differences to the laboratory reference strain, including growth at high temperature and deadly virulence in mouse models. Here we show that the approximately 12-Mb genome of YJM789 contains approximately 60,000 SNPs and approximately 6,000 indels with respect to the reference S288c genome, leading to protein polymorphisms with a few known cases of phenotypic changes. Several ORFs are found to be unique to YJM789, some of which might have been acquired through horizontal transfer. Localized regions of high polymorphism density are scattered over the genome, in some cases spanning multiple ORFs and in others concentrated within single genes. The sequence of YJM789 contains clues to pathogenicity and spurs the development of more powerful approaches to dissecting the genetic basis of complex hereditary traits.

Fatty acid ethyl esters are secondary metabolites produced by Saccharomyces cerevisiae and many other fungi. Their natural physiological role is not known but in fermentations of alcoholic beverages and other food products they play a key role as flavor compounds. Information about the metabolic pathways and enzymology of fatty acid ethyl ester biosynthesis, however, is very limited. In this work, we have investigated the role of a three-member S. cerevisiae gene family with moderately divergent sequences (YBR177c/EHT1, YPL095c/EEB1, and YMR210w). We demonstrate that two family members encode an acyl-coenzymeA:ethanol O-acyltransferase, an enzyme required for the synthesis of medium-chain fatty acid ethyl esters. Deletion of either one or both of these genes resulted in severely reduced medium-chain fatty acid ethyl ester production. Purified glutathione S-transferase-tagged Eht1 and Eeb1 proteins both exhibited acyl-coenzymeA:ethanol O-acyltransferase activity in vitro, as well as esterase activity. Overexpression of Eht1 and Eeb1 did not enhance medium-chain fatty acid ethyl ester content, which is probably due to the bifunctional synthesis and hydrolysis activity. Molecular modeling of Eht1 and Eeb1 revealed the presence of a alpha/beta-hydrolase fold, which is generally present in the substrate-binding site of esterase enzymes. Hence, our results identify Eht1 and Eeb1 as novel acyl-coenzymeA:ethanol O-acyltransferases/esterases, whereas the third family member, Ymr210w, does not seem to play an important role in medium-chain fatty acid ethyl ester formation.

Systematic sequencing of the genome of Saccharomyces cerevisiae has revealed thousands of new predicted genes and allowed analysis of long-range features of chromosomal organization. Generally, genes and predicted genes seem to be distributed evenly throughout the genome, having no overall preference for DNA strand. Apart from the smaller chromosomes, which can have substantially lower gene density in their telomeric regions, there is a consistent average of one open reading frame (ORF) approximately every two kilobases. However, one of the most surprising findings for a eukaryote with approximately 6,000 genes was the amount of apparent redundancy in its genome. This redundancy occurs both between individual ORFs and over more extensive chromosome regions, which have been duplicated preserving gene order and orientation. Here we report the entire nucleotide sequence of chromosome XIII, the sixth-largest S. cerevisiae chromosome, and demonstrate that its features and organization are consistent with those observed for other S. cerevisiae chromosomes. Analysis revealed 459 ORFs, 284 have not been identified previously. Both intra- and interchromosomal duplications of regions of this chromosome have occurred.