Dataset Information

Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1.

ABSTRACT: We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we transferred this respiratory phenotype in the presence of up to 50 g/L glucose to a yeast strain, V5 hxt1-7Delta, in which only HXT1-7 had been deleted. In this study, we compared the transcriptome of the resultant strain, V5.TM6*P, with that of its wild-type parent, V5, at different glucose concentrations.cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4 (in complex with Hap2, 3, 5), Cat8 and Mig1, and used this in combination with verified binding consensus sequences to identify genes likely to be regulated by one or more of these. Of the induced genes in our dataset, 77% had binding sites for the Hap complex, with 72% having at least two. In addition, 13% were found to have a binding site for Cat8 and 21% had a binding site for Mig1. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell.Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88% of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three proteins: Hap4, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.

SUBMITTER: Bonander N

PROVIDER: S-EPMC2536679 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1.

Bonander Nicklas N Ferndahl Cecilia C Mostad Petter P Wilks Martin D B MD Chang Celia C Showe Louise L Gustafsson Lena L Larsson Christer C Bill Roslyn M RM

BMC genomics 20080731

<h4>Background</h4>We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we transferred this respiratory phenotype in the presence of up to 50 g/L glucose to a yeast strain, V5 hxt1-7Delta, in which only HXT1-7 had been deleted. In this study, we compared the transcriptome of the resultant strain, V5.TM6*P, with that of its wild-type parent, ...[more]

PMID: 18671860

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Project description:Background We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we demonstrated the transferability of this respiratory phenotype in the presence of up to 100 g/L glucose to a yeast strain in which only HXT1-7 had been deleted. In this study, we wanted to examine the basis of the respiratory phenotype of the resultant strain, V5.TM6*P, by comparing its transcriptome with that of its parent, V5, at different glucose concentrations. Results cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. Highly complete collections of known genes of the TCA cycle, glyoxylate cycle and respiratory chain were identified, consistent with a respiratory metabolism. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4, Cat8 and Mig1, and using this in combination with verified binding consensus sequences, to identify genes likely to be regulated by one or more of these transcription factors. Of the induced genes of our dataset, 77 % had binding sites for Hap2/3/5 (Hap4 is an activator of this complex), with 72 % having at least two (the latter set being more induced than the former). This is relevant since Hap4 is known to be involved in the transcriptional activation of respiratory genes and other mitochondrial functions. In addition, 13 % of genes were found to have a binding site for Cat8, which together with its complexes with Sip4 have previously been identified as mediating de-repression of a number of genes during the diauxic shift. Finally, 21 % of genes had a binding site for Mig1 which is a transcriptional repressor involved in glucose repression. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This important result indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell. Conclusions Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88 % of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three transcription factors; Hap2/3/5, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.

Project description:Backgroundl-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains.ResultsChemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (Km = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10-3 and 1.8 g L-1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L-1 l-arabinose and 20 g L-1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2.ConclusionIts high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

Dataset Information

Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1.

Publications

Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1.

Similar Datasets

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