M. Esperanza Cerdán

The yeast transcriptome in aerobic and hypoxic conditions: effects of hap1, rox1, rox3 and srb10 deletions

The transcriptome of Saccharomyces cerevisiae was screened using the high‐density membrane hybridization method, under aerobic and hypoxic conditions, in wild‐type and mutant backgrounds obtained by the disruption of the genes encoding the regulatory proteins Hap1, Rox1 and the Srb10 and Rox3 subunits of RNA polymerase II holoenzyme. None of the mutations studied was able to fully overcome the wild‐type hypoxic response. Deletion of the hap1 gene changed the expression profiles of individual open reading frames (ORFs) under both aerobic and hypoxic conditions. Major changes associated with rox3 deletion were related to the hypoxic activation. Rox3 also caused a repressor effect (oxygen‐independent) on a subset of genes related to subtelomeric proteins. With regard to the effect brought about by the deletion of rox1 and srb10, correspondence cluster analysis revealed that the transcriptome profile in aerobic conditions is very similar in the wild‐type and both deletion strains. In contrast, however, differences were found during hypoxia between the subgroup formed by wild‐type and the Δrox1 deletant compared with the Δsrb10 deletant. An analysis of selected ORFs responding to hypoxia, in association with a dependence on the regulatory factors studied, made it possible to identify the clusters that are related to different regulatory circuits.

Codon usage in Kluyveromyces lactis and in yeast cytochrome c-encoding genes

Codon usage (CU) in Kluyveromyces lactis has been studied. Comparison of CU in highly and lowly expressed genes reveals the existence of 21 optimal codons; 18 of them are also optimal in other yeasts like Saccharomyces cerevisiae or Candida albicans. Codon bias index (CBI) values have been recalculated with reference to the assignment of optimal codons in K. lactis and compared to those previously reported in the literature taking as reference the optimal codons from S. cerevisiae. A new index, the intrinsic codon deviation index (ICDI), is proposed to estimate codon bias of genes from species in which optimal codons are not known; its correlation with other index values, like CBI or effective number of codons (Nc), is high. A comparative analysis of CU in six cytochrome-c-encoding genes (CYC) from five yeasts is also presented and the differences found in the codon bias of these genes are discussed in relation to the metabolic type to which the corresponding yeasts belong. Codon bias in the CYC from K. lactis and S. cerevisiae is correlated to mRNA levels.

Sugar metabolism, redox balance and oxidative stress response in the respiratory yeast Kluyveromyces lactis

A lot of studies have been carried out on Saccharomyces cerevisiae, an yeast with a predominant fermentative metabolism under aerobic conditions, which allows exploring the complex response induced by oxidative stress. S. cerevisiae is considered a eukaryote model for these studies. We propose Kluyveromyces lactis as a good alternative model to analyse variants in the oxidative stress response, since the respiratory metabolism in this yeast is predominant under aerobic conditions and it shows other important differences with S. cerevisiae in catabolic repression and carbohydrate utilization. The knowledge of oxidative stress response in K. lactis is still a developing field. In this article, we summarize the state of the art derived from experimental approaches and we provide a global vision on the characteristics of the putative K. lactis components of the oxidative stress response pathway, inferred from their sequence homology with the S. cerevisiae counterparts. Since K. lactis is also a well-established alternative host for industrial production of native enzymes and heterologous proteins, relevant differences in the oxidative stress response pathway and their potential in biotechnological uses of this yeast are also reviewed.

Transcript analysis of 1003 novel yeast genes using high-throughput northern hybridizations

The expression of 1008 open reading frames (ORFs) from the yeast Saccharomyces cerevisiae has been examined under eight different physiological conditions, using classical northern analysis. These northern data have been compared with publicly available data from a microarray analysis of the diauxic transition in S.cerevisiae. The results demonstrate the importance of comparing biologically equivalent situations and of the standardization of data normalization procedures. We have also used our northern data to identify co‐regulated gene clusters and define the putative target sites of transcriptional activators responsible for their control. Clusters containing genes of known function identify target sites of known activators. In contrast, clusters comprised solely of genes of unknown function usually define novel putative target sites. Finally, we have examined possible global controls on gene expression. It was discovered that ORFs that are highly expressed following a nutritional upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not. These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs.

Transcript analysis of 250 novel yeast genes from chromosome XIV

The European Functional Analysis Network (EUROFAN) is systematically analysing the function of novel Saccharomyces cerevisiae genes revealed by genome sequencing. As part of this effort our consortium has performed a detailed transcript analysis for 250 novel ORFs on chromosome XIV. All transcripts were quantified by Northern analysis under three quasi‐steady‐state conditions (exponential growth on rich fermentative, rich non‐fermentative, and minimal fermentative media) and eight transient conditions (glucose derepression, glucose upshift, stationary phase, nitrogen starvation, osmo‐stress, heat‐shock, and two control conditions). Transcripts were detected for 82% of the 250 ORFs, and only one ORF did not yield a transcript of the expected length (YNL285w). Transcripts ranged from low (62%), moderate (16%) to high abundance (2%) relative to the ACT1 mRNA. The levels of 73% of the 206 chromosome XIV transcripts detected fluctuated in response to the transient states tested. However, only a small number responded strongly to the transients: eight ORFs were induced upon glucose upshift; five were repressed by glucose; six were induced in response to nitrogen starvation; three were induced in stationary phase; five were induced by osmo‐stress; four were induced by heat‐shock. These data provide useful clues about the general function of these ORFs and add to our understanding of gene regulation on a genome‐wide basis. Copyright

Secretion and properties of a hybrid Kluyveromyces lactis-Aspergillus niger β-galactosidase

BackgroundThe β-galactosidase from Kluyveromyces lactis is a protein of outstanding biotechnological interest in the food industry and milk whey reutilization. However, due to its intracellular nature, its industrial production is limited by the high cost associated to extraction and downstream processing.The yeast-system is an attractive method for producing many heterologous proteins. The addition of a secretory signal in the recombinant protein is the method of choice to sort it out of the cell, although biotechnological success is not guaranteed. The cell wall acting as a molecular sieve to large molecules, culture conditions and structural determinants present in the protein, all have a decisive role in the overall process.Protein engineering, combining domains of related proteins, is an alternative to take into account when the task is difficult. In this work, we have constructed and analyzed two hybrid proteins from the β-galactosidase of K. lactis, intracellular, and its Aspergillus niger homologue that is extracellular. In both, a heterologous signal peptide for secretion was also included at the N-terminus of the recombinant proteins. One of the hybrid proteins obtained has interesting properties for its biotechnological utilization.ResultsThe highest levels of intracellular and extracellular β-galactosidase were obtained when the segment corresponding to the five domain of K. lactis β-galactosidase was replaced by the corresponding five domain of the A. niger β-galactosidase. Taking into account that this replacement may affect other parameters related to the activity or the stability of the hybrid protein, a thoroughly study was performed. Both pH (6.5) and temperature (40°C) for optimum activity differ from values obtained with the native proteins. The stability was higher than the corresponding to the β-galactosidase of K. lactis and, unlike this, the activity of the hybrid protein was increased by the presence of Ni2+. The affinity for synthetic (ONPG) or natural (lactose) substrates was higher in the hybrid than in the native K. lactis β-galactosidase. Finally, a structural-model of the hybrid protein was obtained by homology modelling and the experimentally determined properties of the protein were discussed in relation to it.ConclusionA hybrid protein between K. lactis and A. niger β-galactosidases was constructed that increases the yield of the protein released to the growth medium. Modifications introduced in the construction, besides to improve secretion, conferred to the protein biochemical characteristics of biotechnological interest.

Regulation of cytochrome c expression in the aerobic respiratory yeast Kluyveromyces lactis

Transcriptional regulation of the KlCYC1 gene from the aerobic respiratory yeast Kluyveromyces lactis has been studied. The KlCYC1 gene produces two transcripts of different sizes, in contrast with the single transcripts found for CYC1 and CYC7 from Saccharomyces cerevisiae, and for the CYC gene from Schwanniomyces occidentali. Both KlCYC1 transcripts respond in the same way to the regulatory signals studied here. The transcription of KlCYC1 is regulated by oxygen and this control is mediated by heme. The KlCYC1 gene is also subject to catabolite repression. Heterologous expression in S. cerevisiae mutants reveals that the factors HAP1 and HAP2 take part in the regulatory mechanism.

Heterologous expression of an esterase from Thermus thermophilus HB27 in Saccharomyces cerevisiae.

In this work, a system for high-level secretion by Saccharomyces cerevisiae of the Thermus thermophilus HB27 putative esterase YP_004875.1 was constructed. The recombinant protein was purified and partially characterised. Its lipolytic activity dropped abruptly when the acyl chain length of the substrate increased from 12 to 18 carbon atoms, and variation of the reaction rate as function of substrate concentration followed Michaelis-Menten kinetics. These results suggested that the enzyme was an esterase. The recombinant enzyme was N-glycosylated and both the glycosylated and non-glycosylated forms showed activity. Compared to the native enzyme, thermal stability (half-life of 4.3h at 85 degrees C) was higher, optimum temperature (40 degrees C) was lower and optimum pH (7.5-8.5) was similar. These characteristics support potential biotechnological applications of the recombinant esterase.

Production and characterization of two N-terminal truncated esterases from Thermus thermophilus HB27 in a mesophilic yeast: effect of N-terminus in thermal activity and stability.

Two N-terminally truncated variants of the esterase E34Tt from Thermus thermophilus HB27 (YP_004875.1) were expressed in Kluyveromyces lactis. Production and biochemical properties of both recombinant proteins were investigated. The esterase activity was greatly increased compared to the wild-type strain. In particular, the extracellular production of the ΔN16 variant (KLEST-3S) was 50-fold higher than that obtained with T. thermophilus HB27. Response surface methodology was applied to describe the pH and temperature dependence of both activity and stability. When compared with the wild type esterase, the optimal temperature of reaction decreased 35 and 15 °C for ΔN16 and ΔN26, respectively. KLEST-3S showed a maximum of activity at pH 7.5 and 47.5 °C, and maximal stability at pH 8.1 and 65 °C. KLEST-5A (ΔN26) did not show an absolute maximum of activity. However, best results were obtained at 40 °C and pH 8.5. KLEST-5A showed also a lower stability. In the presence of a surfactant, both proteins showed lower stability at 85 °C (t(½)< 5 min) than the wild-type enzyme (t(½)=135 min). However, in the absence of detergent, the stability of KLEST-3S was higher (t(½)=230 min, at 85 °C) than that of the mutant KLEST-5A (12 min) or the wild type enzyme (19 min). Minor differences were observed in the substrate specificity. Our results suggest that the N-terminal segment is critical for maintaining the hyperthermophilic function and stability.

Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 by oxygen levels: a model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation.

Ixr1p from Saccharomyces cerevisiae has been previously studied because it binds to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. Ixr1p is also a transcriptional regulator of anaerobic/hypoxic genes, such as SRP1/TIR1, which encodes a stress-response cell wall manoprotein, and COX5B, which encodes the Vb subunit of the mitochondrial complex cytochrome c oxidase. However, factors controlling IXR1 expression remained unexplored. In the present study we show that IXR1 mRNA levels are controlled by oxygen availability and increase during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1-Ssn6. Ixr1p itself is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (nucleotides -557 to -376). EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is also required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H2O2. IXR1 expression is moderately activated by H2O2 and this induction is Yap1p-dependent. A model of IXR1 regulation as a relay for sensing different signals related to change in oxygen availability is proposed. In this model, transcriptional adaptation from aerobiosis to hypoxia depends on ROX1 and IXR1 cross-regulation.