M. Isabel González-Siso

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.

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.

Ixr1p and the control of the Saccharomyces cerevisiae hypoxic response

In Saccharomyces cerevisiae, adaptation to hypoxia/anaerobiosis requires the transcriptional induction or derepression of multiple genes organized in regulons controlled by specific transcriptional regulators. Ixr1p is a transcriptional regulatory factor that causes aerobic repression of several hypoxic genes (COX5B, TIR1, and HEM13) and also the activation of HEM13 during hypoxic growth. Analysis of the transcriptome of the wild-type strain BY4741 and its isogenic derivative Δixr1, grown in aerobic and hypoxic conditions, reveals differential regulation of genes related not only to the hypoxic and oxidative stress responses but also to the re-adaptation of catabolic and anabolic fluxes in response to oxygen limitation. The function of Ixr1p in the transcriptional regulation of genes from the sulfate assimilation pathway and other pathways producing α-keto acids is of biotechnological importance for industries based on yeast-derived fermentation products.

Proteomic Analyses Reveal that Sky1 Modulates Apoptosis and Mitophagy in Saccharomyces cerevisiae Cells Exposed to Cisplatin

Sky1 is the only member of the SR (Serine–Arginine) protein kinase family in Saccharomyces cerevisiae. When yeast cells are treated with the anti-cancer drug cisplatin, Sky1 kinase activity is necessary to produce the cytotoxic effect. In this study, proteome changes in response to this drug and/or SKY1 deletion have been evaluated in order to understand the role of Sky1 in the response of yeast cells to cisplatin. Results reveal differential expression of proteins previously related to the oxidative stress response, DNA damage, apoptosis and mitophagy. With these precedents, the role of Sky1 in apoptosis, necrosis and mitophagy has been evaluated by flow-cytometry, fluorescence microscopy, biosensors and fluorescence techniques. After cisplatin treatment, an apoptotic-like process diminishes in the ∆sky1 strain in comparison to the wild-type. The treatment does not affect mitophagy in the wild-type strain, while an increase is observed in the ∆sky1 strain. The increased resistance to cisplatin observed in the ∆sky1 strain may be attributable to a decrease of apoptosis and an increase of mitophagy.

Two Proteins with Different Functions Are Derived from the KlHEM13 Gene

ABSTRACT Two proteins that differ at the N terminus (l-KlCpo and s-KlCpo) are derived from KlHEM13, a single-copy-number gene in the haploid genome of Kluyveromyces lactis. Two transcriptional start site (tss) pools are detectable using primer extension, and their selection is heme dependent. One of these tss pools is located 5′ of the first translation initiation codon (TIC) in the open reading frame of KlHEM13, while the other is located between the first and second TICs. In terms of functional significance, only s-KlCpo complements the heme deficiency caused by the Δhem13 deletion in K. lactis. Data obtained from immune detection in subcellular fractions, directed mutagenesis, chromatin immunoprecipitation (ChIP) assays, and the functional relevance of ΔKlhem13 deletion for KlHEM13 promoter activity suggest that l-KlCpo regulates KlHEM13 transcription. A hypothetical model of the evolutionary origins and coexistence of these two proteins in K. lactis is discussed.