Esther Rodríguez-Belmonte

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.

Metagenomics of Thermophiles with a Focus on Discovery of Novel Thermozymes

Microbial populations living in environments with temperatures above 50°C (thermophiles) have been widely studied, increasing our knowledge in the composition and function of these ecological communities. Since these populations express a broad number of heat-resistant enzymes (thermozymes), they also represent an important source for novel biocatalysts that can be potentially used in industrial processes. The integrated study of the whole-community DNA from an environment, known as metagenomics, coupled with the development of next generation sequencing (NGS) technologies, has allowed the generation of large amounts of data from thermophiles. In this review, we summarize the main approaches commonly utilized for assessing the taxonomic and functional diversity of thermophiles through metagenomics, including several bioinformatics tools and some metagenome-derived methods to isolate their thermozymes.

Metabolic engineering for direct lactose utilization by Saccharomyces cerevisiae

A recombinant strain of Saccharomyces cerevisiae, secreting β-galactosidase from Kluyveromyces lactis, grew efficiently with more than 60 g lactose l−1. The growth rate (0.23 h−1) in a cheese-whey medium was close to the highest reported hitherto for other recombinant S. cerevisiae strains that express intracellular β-galactosidase and lactose-permease genes. The conditions for growth and β-galactosidase secretion in this medium were optimized in a series of factorial experiments. Best results were obtained at 23 °C for 72 h. Since the recombinant strain produced less than 3% ethanol from the lactose, it was also assayed for the production of fructose 1,6-bisphosphate from cheese whey, and 0.06 g l−1 h−1 were obtained.

IDENTIFICATION OF A PUTATIVE METHYLENETETRAHYDROFOLATE REDUCTASE BY SEQUENCE ANALYSIS OF A 6.8 KB DNA FRAGMENT OF YEAST CHROMOSOME VII

We report the sequence analysis of a 6·8 kb DNA fragment from Saccharomyces cerevisiae chromosome VII. This sequence contains five open reading frames (ORFs) greater than 100 amino acids. There is also an incomplete ORF flanking one of the extremes, G2868, which is the 3′ end of the SCS3 gene (Hosaka et al., 1994). The translated sequence of ORF G2882 shows similarity to the human methylenetetrahydrofolate reductase (Goyette et al., 1994). ORF G2889 shows no significant homologies with the sequences compiled in databases. ORF G2893 corresponds to the gene SUP44, coding for the yeast ribosomal protein S4 (All‐Robin et al., 1990). G2873 and G2896 are internal ORFs. The whole sequence of the fragment is available at the EMBL nucleotide sequence database, GenBank and Data Bank of Japan under the Accession Number X94106.

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.

High Mobility Group B Proteins, Their Partners, and Other Redox Sensors in Ovarian and Prostate Cancer

Cancer cells try to avoid the overproduction of reactive oxygen species by metabolic rearrangements. These cells also develop specific strategies to increase ROS resistance and to express the enzymatic activities necessary for ROS detoxification. Oxidative stress produces DNA damage and also induces responses, which could help the cell to restore the initial equilibrium. But if this is not possible, oxidative stress finally activates signals that will lead to cell death. High mobility group B (HMGB) proteins have been previously related to the onset and progressions of cancers of different origins. The protein HMGB1 behaves as a redox sensor and its structural changes, which are conditioned by the oxidative environment, are associated with different functions of the protein. This review describes recent advances in the role of human HMGB proteins and other proteins interacting with them, in cancerous processes related to oxidative stress, with special reference to ovarian and prostate cancer. Their participation in the molecular mechanisms of resistance to cisplatin, a drug commonly used in chemotherapy, is also revised.

Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respiratory to fermentative metabolism by deletion of NDI1.

In this paper, we report the metabolic engineering of the respiratory yeast Kluyveromyces lactis by construction and characterization of a null mutant (Δklndi1) in the single gene encoding a mitochondrial alternative internal dehydrogenase. Isolated mitochondria of the Δklndi1 mutant show unaffected rate of oxidation of exogenous NADH, but no oxidation of matrix NADH; this confirms that KlNdi1p is the only internal NADH dehydrogenase in K. lactis mitochondria. Permeabilized cells of the Δklndi1 mutant do not show oxidation of matrix NADH, which suggests that shuttle systems to transfer the NADH from mitochondrial matrix to cytosol, for being oxidized by external dehydrogenases, are not functional. The Δklndi1 mutation decreases the chronological life span in absence of nutrients. The expression of KlNDI1 is increased by glutathione reductase depletion. The Δklndi1 mutation shifts the K. lactis metabolism from respiratory to fermentative: the Δklndi1 strain shows reduced respiration rate and increased ethanol production from glucose, while it does not grow in non‐fermentable carbon sources such as lactate. The biotechnological benefit of the Δklndi1 mutant for bioethanol production from waste cheese whey lactose was proved.

PICDI, a simple program for codon bias calculation

PICDI is a very simple program designed to calculate the Intrinsic Codon Deviation Index (ICDI). The program is available in Macintosh as well a PC format. Requirements for correct input of the sequences have been kept to a minimum and the analysis of sequences up to 2000 codons is very quick. The ICDI is very useful for estimation of codon bias of genes from species in which optimal codons are not known. The availability of a computer program for its calculation will increase its usefulness in the fields of Molecular Biology and Biotechnology.

The KlSRB10 gene from Kluyveromyces lactis

We report the cloning and sequencing of a gene from Kluyveromyces lactis with high homology to the SRB10 gene (alias UME5, SSN3, GIG2, NUT7, RYE5) from Saccharomyces cerevisiae and other organisms. The KlSRB10 gene is located in a similar configuration to that found in S. cerevisiae, flanked by NOT4 and a gene with high similarity to YPL041c. The translated protein contains 593 amino acids and the characteristic domains of kinases from the CMGC subgroup. The functional relationship to yeast SRB10 is demonstrated by complementation of mutant phenotypes in a haploid S. cerevisiae strain containing a null allele. Submitted to EMBL data library under Accession No. AJ532841. Copyright

Sequence Analysis of a 10 kb DNA Fragment from Yeast Chromosome VII Reveals a Novel Member of the DnaJ Family

We report the sequence analysis of a 10 kb DNA fragment of Saccharomyces cerevisiae chromosome VII. This sequence contains four complete open reading frames (ORFs) of greater than 100 amino acids. There are also two incomplete ORFs flanking the extremes: one of these, G2868, is the 5′ part of the SCS3 gene (Hosaka et al., 1994). ORFs G2853 and G2856 correspond to the genes CEG1, coding for the alfa subunit of the mRNA guanylyl transferase and a 3′ gene of unknown function previously sequenced (Shibagaki et al., 1992). G2864 is identical to SOH1 also reported (Fan and Klein, 1994). The translated sequence of G2861 is similar to the human dnaJ homolog. The nucleotide sequence reported here has been entered in the EMBL Data Library under the Accession Number X87252.