Manuel Becerra

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.

Enzyme encapsulation on chitosan microbeads

Abstract The influence of two variables (cross-linking and protein concentration) on the activity shown by α-amylase and invertase immobilized on chitosan microbeads was studied by means of full factorial experimental designs. Microencapsulation on chitosan beads has shown to be an effective immobilization method for both enzymes and observed differences in their behaviour are explained mainly by the molecular weight of their substrates.

Lactose bioconversion by calcium-alginate immobilization of Kluyveromyces lactis cells

Abstract A new β-galactosidase based biocatalyst consisting of whole Kluyveromyces lactis cells entrapped in calcium alginate beads has been developed. Formulative parameters and their effects on the enzymatic activity were studied by a 23 full factorial experimental design. Enzymatic activity showed a bimodal trend progressing with time and appeared to be influenced by the structure and dimension of the surrounding gel. Small particles became the choice for scale-up purposes. Remarkably, it was demonstrated that β-galactosidase activity per unit of cell biomass was higher in alginate-immobilized than in free-growing cells in the same medium. Milk whey saccharification by the ethanol-permeabilized cells was studied in packed bed bioreactors. Permeabilization increased the lactose hydrolysis rate and prevented ethanol fermentation allowing 99.5% of milk whey lactose to be hydrolyzed at 30°C for 30 h.

Micro-scale purification of β-galactosidase from Kluyveromyces lactis reveals that dimeric and tetrameric forms are active

β-Galactosidase was purified from Kluyveromyces lactis. Polyclonal antibodies raised against it cross-reacted against the β-galactosidase of Kluyveromyces fragilis but not against β-galactosidases of bacterial, fungal or animal origin. The enzyme is composed of 124 kDa monomers and is active in both dimeric and tetrameric form.

Yeast β-galactosidase in solid-state fermentations

A simple and economical system is proposed for producing β-galactosidase with the yeast Kluyveromyces lactis in solid-state fermentations on corn grits or wheat bran moistened with deproteinized milk whey. By means of a full-factorial design, we studied some conditions that improve this production. Results of liquid- and solid-state cultures were compared with the latter proving to be more effective. We have demonstrated that changing from liquid- to solid-state cultures does not promote β-galactosidase secretion by Kluyveromyces lactis.

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.

Dealing with different methods for Kluyveromyces lactis β-galactosidase purification

Several micro-scale chromatography-based procedures for purification of the β-galactosidase from the yeast Kluyveromyces lactis were assayed. Purified enzyme was suitable to be used as antigen to induce polyclonal antibodies production. Specific staining of non-denaturing PAGE gels with chromogenic substrates allowed the determination of the number of subunits forming the native enzyme.

Biobutanol from cheese whey

At present, due to environmental and economic concerns, it is urgent to evolve efficient, clean and secure systems for the production of advanced biofuels from sustainable cheap sources. Biobutanol has proved better characteristics than the more widely used bioethanol, however the main disadvantage of biobutanol is that it is produced in low yield and titer by ABE (acetone-butanol-ethanol) fermentation, this process being not competitive from the economic point of view. In this review we summarize the natural metabolic pathways for biobutanol production by Clostridia and yeasts, together with the metabolic engineering efforts performed up to date with the aim of either enhancing the yield of the natural producer Clostridia or transferring the butanol production ability to other hosts with better attributes for industrial use and facilities for genetic manipulation. Molasses and starch-based feedstocks are main sources for biobutanol production at industrial scale hitherto. We also review herewith (and for the first time up to our knowledge) the research performed for the use of whey, the subproduct of cheese making, as another sustainable source for biobutanol production. This represents a promising alternative that still needs further research. The use of an abundant waste material like cheese whey, that would otherwise be considered an environmental pollutant, for biobutanol production, makes economy of the process more profitable.

Ixr1p regulates oxygen‐dependent HEM13 transcription

In Saccharomyces cerevisiae, HEM13 encodes the enzyme coproporphyrinogen III oxidase, which catalyzes the rate-limiting step in heme biosynthesis. HEM13 is a regulated hypoxic gene repressed by Rox1p and Mot3p under aerobic conditions. In this study, we further investigate the hypoxic expression of HEM13, focusing on the promoter regions that are functionally important during hypoxia and on the effect of deleting the transcriptional regulators Sut1p, Sut2p, Upc2p, Ecm22p and Ixr1p. Ixr1p is necessary for the high expression of HEM13 under hypoxic conditions and its function is exerted in vivo through the HEM13 promoter region extending from -577 to -419. Ixr1p binds in vivo to the HEM13 promoter both under aerobic and under hypoxic conditions. Purified Ixr1p binds in vitro to two sequences extending from -534 to -509 and from -497 to -450, respectively. These DNA regions compete for Ixr1p binding and the consensus KTTSAAYKGTTYASA is important for the regulatory protein to interact. These results suggest that the regulation of HEM13 expression is dependent on two proteins with high mobility group (HMG) domains: Rox1p and Ixr1p. Their interactions with the HEM13 promoter might change in the transition from aerobiosis to hypoxia.

The yeast hypoxic responses, resources for new biotechnological opportunities

Recent advances in the knowledge of molecular mechanisms that control the adaptation to low oxygen levels in yeast and their biotechnological applications, including bioproduct synthesis, such as ethanol, glutathione or recombinant proteins, as well as pathogenic virulence, are reviewed. Possible pathways and target genes, which might be of particular interest for the improvement of biotechnological applications, are evaluated.