María Esperanza Cerdán

Respirofermentative metabolism in Kluyveromyces lactis:: Insights and perspectives☆

Yeasts do not form a homogeneous group as far as energy-yielding metabolism is concerned and the fate of pyruvate, a glycolytic intermediate, determines the type of energy metabolism. Kluyveromyces lactis has become an alternative to the traditional yeast Saccharomyces cerevisiae owing to its industrial applications as well as to studies on mitochondrial respiration. In this review we summarize the current knowdeledge about the K. lactis respirofermentative metabolism, taking into account the respiratory capacity of this yeast and the molecular mechanisms controlling its regulation, giving an up-to-date picture.

New Extremophilic Lipases and Esterases from Metagenomics

Lipolytic enzymes catalyze the hydrolysis of ester bonds in the presence of water. In media with low water content or in organic solvents, they can catalyze synthetic reactions such as esterification and transesterification. Lipases and esterases, in particular those from extremophilic origin, are robust enzymes, functional under the harsh conditions of industrial processes owing to their inherent thermostability and resistance towards organic solvents, which combined with their high chemo-, regio- and enantioselectivity make them very attractive biocatalysts for a variety of industrial applications. Likewise, enzymes from extremophile sources can provide additional features such as activity at extreme temperatures, extreme pH values or high salinity levels, which could be interesting for certain purposes. New lipases and esterases have traditionally been discovered by the isolation of microbial strains producing lipolytic activity. The Genome Projects Era allowed genome mining, exploiting homology with known lipases and esterases, to be used in the search for new enzymes. The Metagenomic Era meant a step forward in this field with the study of the metagenome, the pool of genomes in an environmental microbial community. Current molecular biology techniques make it possible to construct total environmental DNA libraries, including the genomes of unculturable organisms, opening a new window to a vast field of unknown enzymes with new and unique properties. Here, we review the latest advances and findings from research into new extremophilic lipases and esterases, using metagenomic approaches, and their potential industrial and biotechnological applications.

Respirofermentative metabolism in Kluyveromyces lactis: Ethanol production and the Crabtree effect

Abstract Kluyveromyces lactis is a yeast widely used in processes related to milk whey use and lactose fermentation. However, contradictory information about some aspects related to the respirofermentative metabolism of this yeast is found in the literature. We have studied ethanol production and oxygen use in discontinuous and continuous cultures of K. lactis under hypoxic and aerobic conditions. Growth in nonfermentable carbon sources reflects a more efficient respiratory capacity of K. lactis in relation to Saccharomyces cerevisiae ; however, in both species, similar glucose fermentation levels under aerobic oxygen-limited conditions are found. Continuous K. lactis cultures in fully oxidative conditions show the oxygen and substrate uptake rates typical of a respiration-unlimited Crabtree-negative yeast; however, a small residual fermentation is present even when respiration is not limited. Some aspects of the Crabtree effect in K. lactis are discussed. The impossibility of including K. lactis in any group of the metabolism-based classification from Alexander and Jeffries (1990) has led us to the formulation of a new group which incorporates the peculiarities of this and other related yeasts.

Hot Spring Metagenomics

Hot springs have been investigated since the XIX century, but isolation and examination of their thermophilic microbial inhabitants did not start until the 1950s. Many thermophilic microorganisms and their viruses have since been discovered, although the real complexity of thermal communities was envisaged when research based on PCR amplification of the 16S rRNA genes arose. Thereafter, the possibility of cloning and sequencing the total environmental DNA, defined as metagenome, and the study of the genes rescued in the metagenomic libraries and assemblies made it possible to gain a more comprehensive understanding of microbial communities—their diversity, structure, the interactions existing between their components, and the factors shaping the nature of these communities. In the last decade, hot springs have been a source of thermophilic enzymes of industrial interest, encouraging further study of the poorly understood diversity of microbial life in these habitats.

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus.

BackgroundIn spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyceslactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties.ResultsThe highest GOX expression levels (1552 units of secreted protein per gram dry cell weight) were achieved using an episomal system, in which the INU1 promoter and terminator were used to drive heterologous gene expression, together with the INU1 prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from Saccharomyces cerevisiae to drive heterologous protein expression led to higher expression levels in K. lactis than in K. marxianus, the use of INU1 genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that K. marxianus has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e.g. K. lactis and S. cerevisiae. Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity.ConclusionsTaken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins.

Disruption of six novel Saccharomyces cerevisiae genes reveals that YGL129c is necessary for growth in non-fermentable carbon sources, YGL128c for growth at low or high temperatures and YGL125w is implicated in the biosynthesis of methionine.

Six open reading frames (ORFs) from chromosome VII, YGL131c, YGL129c, YGL128c, YGL125w, YGL124c and YGL121c, were disrupted by deletion cassettes with short flanking regions homologous to the target locus (SFH). YGL129c is necessary for growth in non‐fermentable carbon sources, YGL128c for growth at low or high temperatures and YGL125w is implicated in the biosynthesis of methionine. With regard to the other ORFs, basic phenotypic analyses did not reveal any significant clues about their function. Copyright

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.

Genome-wide analysis of the yeast transcriptome upon heat and cold shock

DNA arrays were used to measure changes in transcript levels as yeast cells responded to temperature shocks. The number of genes upregulated by temperature shifts from 30 ℃ to 37℃ or 45℃ was correlated with the severity of the stress. Pre-adaptation of cells, by growth at 37 ℃ previous to the 45℃ shift, caused a decrease in the number of genes related to this response. Heat shock also caused downregulation of a set of genes related to metabolism, cell growth and division, transcription, ribosomal proteins, protein synthesis and destination. Probably all of these responses combine to slow down cell growth and division during heat shock, thus saving energy for cell rescue. The presence of putative binding sites for Xbp1p in the promoters of these genes suggests a hypothetical role for this transcriptional repressor, although other mechanisms may be considered. The response to cold shock (4℃) affected a small number of genes, but the vast majority of those genes induced by exposure to 4 ℃ were also induced during heat shock; these genes share in their promoters cis-regulatory elements previously related to other stress responses.

Haem regulation of the mitochondrial import of the Kluyveromyces lactis 5-aminolaevulinate synthase: an organelle approach

The enzyme 5‐aminolaevulinate acid synthase (ALAS) catalyses the first reaction in the haem biosynthetic pathway. In eukaryotes this protein is translated by cytosolic ribosomes and then targeted to the mitochondria. We present evidence that in the yeast Kluyveromyces lactis haem exerts a feedback control upon the import of the ALAS into mitochondria. The ALAS from K. lactis (KlALAS) contains two haem regulatory motifs (HRM) in the mitochondrial targeting signal. Mutagenesis experiments reveal the involvement of these HRM in the response of the KlALAS to haem. Copyright

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.