Biomedical subjects

M Esperanza Cerdán

Publications and source records attributed to M Esperanza Cerdán.

10 recordsLinked to original sources

PubMed2006

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

BACKGROUND: The beta-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 beta-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. RESULTS: The highest levels of intracellular and extracellular beta-galactosidase were obtained when the segment corresponding to the five domain of K. lactis beta-galactosidase was replaced by the corresponding five domain of the A. niger beta-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 degrees C) for optimum activity differ from values obtained with the native proteins. The stability was higher than the corresponding to the beta-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 beta-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. CONCLUSION: A hybrid protein between K. lactis and A. niger beta-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.

Journal Article↗

PubMed2006

Characterization of the second external alternative dehydrogenase from mitochondria of the respiratory yeast Kluyveromyces lactis.

The mitochondria of the respiratory yeast Kluyveromyces lactis are able to reoxidize cytosolic NADPH. Previously, we characterized an external alternative dehydrogenase, KlNde1p, having this activity. We now characterize the second external alternative dehydrogenase of K. lactis mitochondria, KlNde2p. We examined its role in cytosolic NADPH reoxidation by studying heterologous expression of KlNDE2 in Saccharomyces cerevisiae mutants and by constructing Deltaklnde1 and Deltaklnde2 mutants. KlNde2p uses NADH or NADPH as substrates, its activity in isolated mitochondria is not regulated by exogenously added calcium and it is not down-regulated when the cells grow in glucose versus lactate. KlNde2p shows lower affinity for NADPH than KlNde1p. Both enzymes show similar pH optimum.

Amino Acid Sequence↗

PubMed2005

Functional characterization of KlHEM13, a hypoxic gene of Kluyveromyces lactis.

The KlHEM13 gene of Kluyveromyces lactis encoding the coproporphyrinogen oxidase (EC 1.3.3.3), an oxygen-requiring enzyme that catalyzes the sixth step of heme biosynthesis, was cloned and functionally characterized. The coding and upstream regions of KlHEM13 were analyzed and the putative cis regulatory elements were discussed in relation to the mechanisms of regulation of this hypoxic gene in K. lactis.

Amino Acid Sequence↗

PubMed2004

Functional characterisation and transcriptional regulation of the KlHEM12 gene from Kluyveromyces lactis.

Cloning, sequencing and functional analysis of the Kluyveromyces lactis KlHEM12 gene and its upstream region are reported. The gene encodes for a protein that is highly homologous to uroporphyrinogen decarboxylases from different organisms and complements its mutation in Saccharomyces cerevisiae. Secondary structure prediction allows outlining a topology diagram which is compatible with a (beta/alpha)8-barrel structure. A K. lactis haploid strain carrying a null allele of KlHEM12 showed decreased growth in media not supplemented with hemin (ferriprotoporphyrin IX) and red-fluorescent colonies due to the accumulation of porphyrins. KlHEM12 expression was analysed by Northern blot and promoter fusion to the reporter lacZ gene. Transcription of this gene is not under heme or glucose repression and it is slightly induced by non-fermentable carbon sources through the Hap2/3/4/5 complex.

Amino Acid Sequence↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2004

Engineered autolytic yeast strains secreting Kluyveromyces lactis beta-galactosidase for production of heterologous proteins in lactose media.

Secretion of the heterologous Kluyveromyces lactis beta-galactosidase into culture medium by several Saccharomyces cerevisiae osmotic-remedial thermosensitive-autolytic mutants was assayed and proved that new metabolic abilities were conferred since the constructed strains were able to grow in lactose-containing media. Cell growth became independent of a lactose-uptake mechanism. Higher levels of extra-cellular and intra-cellular beta-galactosidase production, lactose consumption and growth were obtained with the LHDP1 strain, showing a thermosensitive-autolytic phenotype as well as being peptidase-defective. The recombinant strain LHDP1 presented the highest beta-galactosidase yields from biomass and the lowest ethanol levels from lactose. This strain is effective for the heterologous production and release of K. lactis beta-galactosidase into the extra-cellular medium after osmotic shock.

Cheese↗

PubMed2004

Genome-wide analysis of Kluyveromyces lactis in wild-type and rag2 mutant strains.

The use of heterologous DNA arrays from Saccharomyces cerevisiae has been tested and revealed as a suitable tool to compare the transcriptomes of S. cerevisiae and Kluyveromyces lactis, two yeasts with notable differences in their respirofermentative metabolism. The arrays have also been applied to study the changes in the K. lactis transcriptome owing to mutation in the RAG2 gene coding for the glycolytic enzyme phosphoglucose isomerase. Comparison of the rag2 mutant growing in 2% glucose versus 2% fructose has been used as a model to elucidate the importance of transcriptional regulation of metabolic routes, which may be used to reoxidize the NADPH produced in the pentose phosphate pathway. At this transcriptional level, routes related to the oxidative stress response become an interesting alternative for NADPH use.

Fructose↗

PubMed2004

Isolation and transcriptional regulation of the Kluyveromyces lactis FBA1 (fructose-1,6-bisphosphate aldolase) gene.

Cloning and transcriptional regulation of the KlFBA1 gene that codes for the class II fructose-1,6-bisphosphate aldolase of the yeast Kluyveromyces lactis are described. KlFBA1 mRNA diminishes transiently during the shift from hypoxic to fully aerobic conditions and increases in the reversal shift. This regulation is mediated by heme since expression was higher in a mutant defective in heme biosynthesis. KlFBA1 transcription is not induced by calcium-shortage, low temperature, or at stationary phase. These data suggest that KlFBA1 plays a role in the balance between oxidative and fermentative metabolism and that this gene is differentially regulated in K. lactis and Saccharomyces cerevisiae, i.e., a respiratory vs. fermentative yeast.

Cloning, Molecular↗

PubMed2002

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 Deltarox1 deletant compared with the Deltasrb10 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.

Aerobiosis↗

PubMed1995

Yeast phylogenetic relationships based on cytochrome c sequences.

The availability of the KICYC1 sequence was used to establish homologies with other cytochrome c genes from yeasts and the fungus Neurospora crassa. In terms of nucleotide composition, the cytochrome c gene from Kluyveromyces lactis showed a higher homology with Schwanniomyces occidentalis than with Saccharomyces cerevisiae, and this point is discussed in regard to the differences found in the codon usage of these yeasts. The deduced amino acidic composition of the protein facilitated comparison of its sequence with other cytochrome c protein sequences and new assignments of phylogenetic relationships. In this context Kluyveromyces lactis was most closely related to Candida krusei.

Cytochrome c Group↗