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Papers by Michele Bianchi

Microorganisms, Apr 21, 2023

Properties and In Vivo Anti-Ageing Effects of Lactoplantibacillus plantarum PFA2018AU Strain Isol... more Properties and In Vivo Anti-Ageing Effects of Lactoplantibacillus plantarum PFA2018AU Strain Isolated from Carrots on Caenorhabditis elegans.

Molecular Biology and its Application to Medical Mycology, 1993

The yeast Saccharomyces cerevisiae is probably the most thoroughly understood amongst eukaryotic ... more The yeast Saccharomyces cerevisiae is probably the most thoroughly understood amongst eukaryotic organisms and an excellent model for the study of eukaryotic cells in general; indeed, the term “yeast” is often used as a synonim for this species. Recently, however, other yeasts have attracted the attention of researchers as a result of their distinct biological and metabolic properties which open up new possibilities of biological utilization. Among these so called “non-conventional” yeasts, Kluyveromyces lactis has attracted a special interest because of several peculiar characteristics, including the easiness of mass cultivation, its status of safe organism and its very good secretion properties. K.lactis is a budding yeast and, like S.cerevisiae, lends itself easily to genetic analysis. It differs, however, from S.cerevisiae for several important metabolic properties and is a “petite negative” yeast, i.e. a species in which no mitochondrial respiratory deficient mutants have been found. It has a considerably smaller number of chromosomes than S.cerevisiae, which are however larger in size. Pulsed field gel electrophoresis reveals six DNA bands ranging in size roughly between 1.2 and 2.8 megabasepairs (Sor and Fukuhara, 1989). Several genes are being mapped by hybridization on the chromosomes and results are being compared with linkage maps of known mutations obtained by tetrad analysis.

Frontiers in Microbiology

Aguirre-López et al. K. lactis Paralogous Diversification Pathway phenotype is only fully complem... more Aguirre-López et al. K. lactis Paralogous Diversification Pathway phenotype is only fully complemented when transformed with the syntenic KlLEU4 mitochondrial isoform. KlLEU4 and KlLEU4BIS underwent a different diversification pathways than that leading to ScLEU4/ScLEU9. KlLEU4 could be considered as the functional ortholog of ScLEU4, since its encoded isozyme can complement both the Scleu4 Scleu9 leucine auxotrophy and the Scleu4 ScLEU9 complex phenotype.

Frontiers in Microbiology, 2021

In unicellular organisms like yeasts, which do not have specialized tissues for protection agains... more In unicellular organisms like yeasts, which do not have specialized tissues for protection against environmental challenges, the presence of cellular mechanisms to respond and adapt to stress conditions is fundamental. In this work, we aimed to investigate the response to environmental light in Kluyveromyces lactis. Yeast lacks specialized light-sensing proteins; however, Saccharomyces cerevisiae has been reported to respond to light by increasing hydrogen peroxide level and triggering nuclear translocation of Msn2. This is a stress-sensitive transcription factor also present in K. lactis. To investigate light response in this yeast, we analyzed the different phenotypes generated by the deletion of the hypoxia responsive and lipid biosynthesis transcription factor KlMga2. Alterations in growth rate, mitochondrial functioning, ROS metabolism, and fatty acid biosynthesis provide evidence that light was a source of stress in K. lactis and that KlMga2 had a role in the light-stress resp...

Fems Yeast Research, Feb 27, 2019

A network of connections among hypoxia, respiration, growth and ageing, mediated by a single prot... more A network of connections among hypoxia, respiration, growth and ageing, mediated by a single protein regulating lipid homeostasis, has been established in yeast.

Journal of Basic Microbiology, 1988

A transformation system derived from the circular plasmid pKD1 has been developed for Kluyveromyc... more A transformation system derived from the circular plasmid pKD1 has been developed for Kluyveromyces lactis. The principle is essentially equivalent to that of the 2 μ/Saccharomyces cerevisiae transformation system. The main features of the system are presented. Using a pKD1‐based DNA bank of K. lactis, the KEX1 gene involved in the killer system was isolated by complementation.

Springer eBooks, 2003

This experiment is intended to establish a correlation between elevated dosage of heterologous ge... more This experiment is intended to establish a correlation between elevated dosage of heterologous genes and high-level production of the corresponding proteins in an inducible system for vector copy number amplification in Kluyveromyces lactis.

Yeast, Mar 30, 1999

The yeast Kluyveromyces lactis has a single structural gene coding for pyruvate decarboxylase (Kl... more The yeast Kluyveromyces lactis has a single structural gene coding for pyruvate decarboxylase (KlPDC1). In order to study the regulation of the expression of KlPDC1, we have sequenced (EMBL Accession No. Y15435) its promoter and have fused the promoter to the reporter gene lacZ from E. coli. Transcription analysis in a Klpdc1 strain showed that KlPDC1 expression is subject to autoregulation. The PDC1 gene from Saccharomyces cerevisiae was able to complement the Rag phenotype of the Klpdc1 mutant strain and it could also repress transcription of the KlPDC1-lacZ fusion on glucose. A deletion analysis of the promoter region was performed to study carbon source-dependent regulation and revealed that at least two cis-acting regions are necessary for full induction of gene expression on glucose. Other cis-elements mediate repression on ethanol.

Fems Yeast Research, May 1, 2009

In yeast, many environmental stimuli are sensed and signaled by the MAP kinases pathways. In a pr... more In yeast, many environmental stimuli are sensed and signaled by the MAP kinases pathways. In a previous work, we showed that cesium chloride activates the HOG pathway and modulates the transcription of several genes, especially those involved in cell wall biosynthesis and organization. The response to cesium was largely overlapping with the response to salt and osmotic stress. However, when low cesium chloride concentrations were used, a specific response was eventually elicited. The cesium-specific response involved the Yaf9 protein and its activity of chromatin remodeling and transcription regulation. In this paper we show that the osmotic activity of cesium salt is detected and signaled by the two branches downstream of the Sln1 and Sho1 sensors of the HOG pathway, that seem to possess different but exchangeables functions in cesium signaling. However, the cesium-specific response mediated by Yaf9, that counteracts the efficiency of the HOG pathway, is not routed by these sensors. In addition, the cesium response also involves the cell wall integrity (CWI) pathway, which is activated by low concentration of cesium chloride. Mutations blocking the CWI pathway show sensitivity to this salt.

Current Genetics, Mar 1, 1991

Plasmid factors involved in the stable propagation of pKDl-derived vectors in Kluyveromyces lacti... more Plasmid factors involved in the stable propagation of pKDl-derived vectors in Kluyveromyces lactis transformants have been identified. Three genes (A, B and C) have been found to be present in pKDI: the interruption of the B and C genes led to high plasmid instability. Stability could be restored in trans when host cells contained pKD1 as the resident plasmid (pKDI + strains). The A gene, which codes for a site-specific recombinase, did not affect plasmid partitioning. Vectors bearing only the pKD1 replication origin (or a chromosomal ARS), and no other pKD1 sequence, were very unstable both in the presence and absence of the resident plasmid in host cells. These vectors could be stabilized in pKD1 + strains, but not in pKDI ~ strains, by the insertion of a 200 bp-long pKDI sequence. This sequence, called the cis-acting stability locus (CSL), together with the products of the B and C genes, ensured plasmid partitioning at cell divison. Possible hairpin structures and direct repeats were regularly spaced within the CSL. This region, and the corresponding cis-acting stabilizing elements of other yeast plasmids, did not have sequence homology but shared some structural regularities.

Current Genetics, Jul 1, 1987

The circular plasmid pKD1 (or 1.6 gm DNA) has recently been isolated from Kluyveromyces drosophil... more The circular plasmid pKD1 (or 1.6 gm DNA) has recently been isolated from Kluyveromyces drosophilarum. This plasmid appears to have a functional organization analogous to that of the 2 g DNA of Saccharomyces cerevisiae, although the respective nucleotide sequences show little homology, pKD1 can be transferred toKluyverornyces lactis where it is replicated stably. Using recombinant molecules derived from pKD1, a practical transformation system has been developed for Kluyveromyces lactis, with an efficiency and stability comparable to the 2#-based Saccharomyces cerevisiae transformation system.

Journal of Enzyme Inhibition and Medicinal Chemistry, Nov 7, 2019

The 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme of the mevalonate pathway for the synt... more The 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme of the mevalonate pathway for the synthesis of cholesterol in mammals (ergosterol in fungi), is inhibited by statins, a class of cholesterol lowering drugs. Indeed, statins are in a wide medical use, yet statins treatment could induce side effects as hepatotoxicity and myopathy in patients. We used Saccharomyces cerevisiae as a model to investigate the effects of statins on mitochondria. We demonstrate that statins are active in S.cerevisiae by lowering the ergosterol content in cells and interfering with the attachment of mitochondrial DNA to the inner mitochondrial membrane. Experiments on murine myoblasts confirmed these results in mammals. We propose that the instability of mitochondrial DNA is an early indirect target of statins.

Nucleic Acids Research, 1984

ITn Saccharomyces cerevisiae most mitochondrial tRNA genes are cluste red in a 9 kbp region betwe... more ITn Saccharomyces cerevisiae most mitochondrial tRNA genes are cluste red in a 9 kbp region between the. cap and oxil genes. Polygenic transcripts of this region have been previously identified. A transcriptional initiation site at a TTATAAGTA box, located upstream from the tRNAcYS gene, has now been detected by SI mapping experiments and by the capping of primary tran scripts. Results are consistent with the hypothesis that this box represents the initiation site for transcription of a cluster of tRNA genes, while the adjacent tRNAthr is cotranscribed with the 21S rRNA. Results obtained with various strains are compared, and the efficiency of this sequence as a tran scriptional initiation site in different mitochondrial contexts is discussed.

Biochimica Et Biophysica Acta - Molecular And Cell Biology Of Lipids, Mar 1, 2019

Sterols are essential lipids, involved in many biological processes. In Saccharomyces cerevisiae,... more Sterols are essential lipids, involved in many biological processes. In Saccharomyces cerevisiae, the enzymes of the ergosterol biosynthetic pathway (Erg proteins) are localised in different cellular compartments. With the aim of studying organelle interactions, we discovered that Erg27p resides mainly in Lipid Droplets (LDs) in respiratory competent cells, while in absence of respiration, is found mostly in the ER. The results presented in this paper demonstrate an interplay between the mitochondrial respiration and ergosterol production: on the one hand, rho° cells show lower ergosterol content when compared with wild type respiratory competent cells, on the other hand, the ergosterol biosynthetic pathway influences the mitochondrial status, since treatment with ketoconazole, which blocks the ergosterol pathway, or the absence of the ERG27 gene, induced rho° production in S. cerevisiae. The loss of mitochondrial DNA in the ∆erg27 strain is fully suppressed by exogenous addition of ergosterol. These data suggest the notion that ergosterol is essential for maintaining the mitochondrial DNA attached to the inner mitochondrial membrane.

Microbial Cell Factories, Jun 7, 2014

Background: Microbial lipids represent a valuable alternative feedstock for biodiesel production ... more Background: Microbial lipids represent a valuable alternative feedstock for biodiesel production when oleaginous microbes are cultured with inexpensive substrates in processes exhibiting high yield and productivity. In this perspective, crude glycerol is among the most promising raw materials for lipid production, because it is the costless residual of biodiesel production. Thus, cultivation of oleaginous yeasts in glycerol-based media is attracting great interest and natural biodiversity is increasingly explored to identify novel oleaginous species recycling this carbon source for growth and lipid production. Results: Thirty-three yeasts strains belonging to 19 species were screened for the ability to grow and produce intracellular lipids in a pure glycerol-based medium with high C/N ratio. A minority of them consumed most of the glycerol and generated visible lipid bodies. Among them, Candida freyschussii ATCC 18737 was selected, because it exhibited the highest lipid production and glycerol conversion yield. Lipid production in this strain was positively affected by the increase of C/N ratio, but growth was inhibited by glycerol concentration higher than 40 g/L. In batch cultures, the highest lipid production (4.6 g/L), lipid content of biomass (33% w/w), and lipid volumetric productivity (0.15 g/L/h) were obtained with 40 g/L glycerol, during the course of a 30-h process. Fed-batch cultivation succeeded in preventing substrate inhibition and in achieving a high cell-density culture. The improved lipid production and volumetric productivity reached the remarkable high level of 28 g/L and 0.28 g/L/h, respectively. The lipids accumulated by C. freyschussii ATCC 18737 have similar fatty acid composition of plant oil indicating their potential use as biodiesel feedstock. Calculated physicochemical properties of a biodiesel produced with the lipids from C. freyschussii ATCC 18737 are expected to meet the European and American standards, being equal to those of rapeseed and palm biodiesel.

FEMS Microbiology Letters, 2021

Transient modification of the environment involves the expression of specific genes and degradati... more Transient modification of the environment involves the expression of specific genes and degradation of mRNAs and proteins. How these events are linked is poorly understood. CCR4-NOT is an evolutionary conserved complex involved in transcription initiation and mRNA degradation. In this paper, we report that the yeast Not4 localizes in cytoplasmic foci after cellular stress. We focused our attention on the functional characterization of the C-terminus of the Not4 protein. Molecular dissection of this region indicates that the removal of the last 120 amino acids, does not affect protein localization and function, in that the protein is still able to suppress the thermosensitivity observed in the not4Δ mutant. In addition, such shortened form of Not4, as well its absence, increases the transcription of stress-responsive genes conferring to the cell high resistance to the oxidative stress. On the contrary, the last C-terminal 211 amino acids are required for proper Not4 localization at c...

FEMS Yeast Research, 2019

Glucose is the preferred nutrient for most living cells and is also a signaling molecule that mod... more Glucose is the preferred nutrient for most living cells and is also a signaling molecule that modulates several cellular processes. Glucose regulates the expression of glucose permease genes in yeasts through signaling pathways dependent on plasma membrane glucose sensors. In the yeast Kluyveromyces lactis, sufficient levels of glucose induction of the low-affinity glucose transporter RAG1 gene also depends on a functional glycolysis, suggesting additional intracellular signaling. We have found that the expression of RAG1 gene is also induced by hypoxia in the presence of glucose, indicating that glucose and oxygen signaling pathways are interconnected. In this study we investigated the molecular mechanisms underlying this crosstalk. By analyzing RAG1 expression in various K. lactis mutants, we found that the bHLH transcriptional activator Sck1 is required for the hypoxic induction of RAG1 gene. The RAG1 promoter region essential for its hypoxic induction was identified by promoter ...

Yeast, Mar 15, 2000

Six ORFs of unknown function located on chromosome VII of Saccharomyces cerevisiae were disrupted... more Six ORFs of unknown function located on chromosome VII of Saccharomyces cerevisiae were disrupted in two different genetic backgrounds, and the phenotype of the generated mutants was analysed. Disruptions of ORFs YGR256w, YGR272c, YGR273c, YGR275w and YGR276c were carried out using the disruption marker kanMX4 anked by short homology regions, whereas ORF YGR255c was inactivated with a long¯anking homology (LFH) disruption cassette (Wach et al., 1994). Tetrad analysis of the heterozygous disruptants revealed that ORF YGR255c, previously identi®ed as COQ6 and encoding a protein involved in the biosynthesis of coenzime Q (Tzagoloff and Dieckmann, 1990), is an essential gene. The same analysis also revealed that sporulation of the ygr272cD heterozygous diploid produced two small colonies per ascus that were also G418-resistant, indicating that the inactivation of ORF YGR272c could result in a slower growth rate. This result was con®rmed by growth tests of the haploid disruptants and by complementation of the phenotype after transformation with a plasmid carrying the cognate gene. No phenotypes could be associated to the inactivation of ORFs YGR256w, YGR273c, YGR275w and YGR276c. Two of these genes have recently been further characterized: ORF YGR255w, renamed RTT102, encodes a regulator of the Ty1-element transposition, whereas ORF YGR276c was found to encode the 70 kDa RNase H activity and was renamed RNH70 (Frank et al.

International Journal of Molecular Sciences

Living organisms on the surface biosphere are periodically yet consistently exposed to light. The... more Living organisms on the surface biosphere are periodically yet consistently exposed to light. The adaptive or protective evolution caused by this source of energy has led to the biological systems present in a large variety of organisms, including fungi. Among fungi, yeasts have developed essential protective responses against the deleterious effects of light. Stress generated by light exposure is propagated through the synthesis of hydrogen peroxide and mediated by regulatory factors that are also involved in the response to other stressors. These have included Msn2/4, Crz1, Yap1, and Mga2, thus suggesting that light stress is a common factor in the yeast environmental response.

Microorganisms, Apr 21, 2023

Properties and In Vivo Anti-Ageing Effects of Lactoplantibacillus plantarum PFA2018AU Strain Isol... more Properties and In Vivo Anti-Ageing Effects of Lactoplantibacillus plantarum PFA2018AU Strain Isolated from Carrots on Caenorhabditis elegans.

Molecular Biology and its Application to Medical Mycology, 1993

The yeast Saccharomyces cerevisiae is probably the most thoroughly understood amongst eukaryotic ... more The yeast Saccharomyces cerevisiae is probably the most thoroughly understood amongst eukaryotic organisms and an excellent model for the study of eukaryotic cells in general; indeed, the term “yeast” is often used as a synonim for this species. Recently, however, other yeasts have attracted the attention of researchers as a result of their distinct biological and metabolic properties which open up new possibilities of biological utilization. Among these so called “non-conventional” yeasts, Kluyveromyces lactis has attracted a special interest because of several peculiar characteristics, including the easiness of mass cultivation, its status of safe organism and its very good secretion properties. K.lactis is a budding yeast and, like S.cerevisiae, lends itself easily to genetic analysis. It differs, however, from S.cerevisiae for several important metabolic properties and is a “petite negative” yeast, i.e. a species in which no mitochondrial respiratory deficient mutants have been found. It has a considerably smaller number of chromosomes than S.cerevisiae, which are however larger in size. Pulsed field gel electrophoresis reveals six DNA bands ranging in size roughly between 1.2 and 2.8 megabasepairs (Sor and Fukuhara, 1989). Several genes are being mapped by hybridization on the chromosomes and results are being compared with linkage maps of known mutations obtained by tetrad analysis.

Frontiers in Microbiology

Aguirre-López et al. K. lactis Paralogous Diversification Pathway phenotype is only fully complem... more Aguirre-López et al. K. lactis Paralogous Diversification Pathway phenotype is only fully complemented when transformed with the syntenic KlLEU4 mitochondrial isoform. KlLEU4 and KlLEU4BIS underwent a different diversification pathways than that leading to ScLEU4/ScLEU9. KlLEU4 could be considered as the functional ortholog of ScLEU4, since its encoded isozyme can complement both the Scleu4 Scleu9 leucine auxotrophy and the Scleu4 ScLEU9 complex phenotype.

Frontiers in Microbiology, 2021

In unicellular organisms like yeasts, which do not have specialized tissues for protection agains... more In unicellular organisms like yeasts, which do not have specialized tissues for protection against environmental challenges, the presence of cellular mechanisms to respond and adapt to stress conditions is fundamental. In this work, we aimed to investigate the response to environmental light in Kluyveromyces lactis. Yeast lacks specialized light-sensing proteins; however, Saccharomyces cerevisiae has been reported to respond to light by increasing hydrogen peroxide level and triggering nuclear translocation of Msn2. This is a stress-sensitive transcription factor also present in K. lactis. To investigate light response in this yeast, we analyzed the different phenotypes generated by the deletion of the hypoxia responsive and lipid biosynthesis transcription factor KlMga2. Alterations in growth rate, mitochondrial functioning, ROS metabolism, and fatty acid biosynthesis provide evidence that light was a source of stress in K. lactis and that KlMga2 had a role in the light-stress resp...

Fems Yeast Research, Feb 27, 2019

A network of connections among hypoxia, respiration, growth and ageing, mediated by a single prot... more A network of connections among hypoxia, respiration, growth and ageing, mediated by a single protein regulating lipid homeostasis, has been established in yeast.

Journal of Basic Microbiology, 1988

A transformation system derived from the circular plasmid pKD1 has been developed for Kluyveromyc... more A transformation system derived from the circular plasmid pKD1 has been developed for Kluyveromyces lactis. The principle is essentially equivalent to that of the 2 μ/Saccharomyces cerevisiae transformation system. The main features of the system are presented. Using a pKD1‐based DNA bank of K. lactis, the KEX1 gene involved in the killer system was isolated by complementation.

Springer eBooks, 2003

This experiment is intended to establish a correlation between elevated dosage of heterologous ge... more This experiment is intended to establish a correlation between elevated dosage of heterologous genes and high-level production of the corresponding proteins in an inducible system for vector copy number amplification in Kluyveromyces lactis.

Yeast, Mar 30, 1999

The yeast Kluyveromyces lactis has a single structural gene coding for pyruvate decarboxylase (Kl... more The yeast Kluyveromyces lactis has a single structural gene coding for pyruvate decarboxylase (KlPDC1). In order to study the regulation of the expression of KlPDC1, we have sequenced (EMBL Accession No. Y15435) its promoter and have fused the promoter to the reporter gene lacZ from E. coli. Transcription analysis in a Klpdc1 strain showed that KlPDC1 expression is subject to autoregulation. The PDC1 gene from Saccharomyces cerevisiae was able to complement the Rag phenotype of the Klpdc1 mutant strain and it could also repress transcription of the KlPDC1-lacZ fusion on glucose. A deletion analysis of the promoter region was performed to study carbon source-dependent regulation and revealed that at least two cis-acting regions are necessary for full induction of gene expression on glucose. Other cis-elements mediate repression on ethanol.

Fems Yeast Research, May 1, 2009

In yeast, many environmental stimuli are sensed and signaled by the MAP kinases pathways. In a pr... more In yeast, many environmental stimuli are sensed and signaled by the MAP kinases pathways. In a previous work, we showed that cesium chloride activates the HOG pathway and modulates the transcription of several genes, especially those involved in cell wall biosynthesis and organization. The response to cesium was largely overlapping with the response to salt and osmotic stress. However, when low cesium chloride concentrations were used, a specific response was eventually elicited. The cesium-specific response involved the Yaf9 protein and its activity of chromatin remodeling and transcription regulation. In this paper we show that the osmotic activity of cesium salt is detected and signaled by the two branches downstream of the Sln1 and Sho1 sensors of the HOG pathway, that seem to possess different but exchangeables functions in cesium signaling. However, the cesium-specific response mediated by Yaf9, that counteracts the efficiency of the HOG pathway, is not routed by these sensors. In addition, the cesium response also involves the cell wall integrity (CWI) pathway, which is activated by low concentration of cesium chloride. Mutations blocking the CWI pathway show sensitivity to this salt.

Current Genetics, Mar 1, 1991

Plasmid factors involved in the stable propagation of pKDl-derived vectors in Kluyveromyces lacti... more Plasmid factors involved in the stable propagation of pKDl-derived vectors in Kluyveromyces lactis transformants have been identified. Three genes (A, B and C) have been found to be present in pKDI: the interruption of the B and C genes led to high plasmid instability. Stability could be restored in trans when host cells contained pKD1 as the resident plasmid (pKDI + strains). The A gene, which codes for a site-specific recombinase, did not affect plasmid partitioning. Vectors bearing only the pKD1 replication origin (or a chromosomal ARS), and no other pKD1 sequence, were very unstable both in the presence and absence of the resident plasmid in host cells. These vectors could be stabilized in pKD1 + strains, but not in pKDI ~ strains, by the insertion of a 200 bp-long pKDI sequence. This sequence, called the cis-acting stability locus (CSL), together with the products of the B and C genes, ensured plasmid partitioning at cell divison. Possible hairpin structures and direct repeats were regularly spaced within the CSL. This region, and the corresponding cis-acting stabilizing elements of other yeast plasmids, did not have sequence homology but shared some structural regularities.

Current Genetics, Jul 1, 1987

The circular plasmid pKD1 (or 1.6 gm DNA) has recently been isolated from Kluyveromyces drosophil... more The circular plasmid pKD1 (or 1.6 gm DNA) has recently been isolated from Kluyveromyces drosophilarum. This plasmid appears to have a functional organization analogous to that of the 2 g DNA of Saccharomyces cerevisiae, although the respective nucleotide sequences show little homology, pKD1 can be transferred toKluyverornyces lactis where it is replicated stably. Using recombinant molecules derived from pKD1, a practical transformation system has been developed for Kluyveromyces lactis, with an efficiency and stability comparable to the 2#-based Saccharomyces cerevisiae transformation system.

Journal of Enzyme Inhibition and Medicinal Chemistry, Nov 7, 2019

The 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme of the mevalonate pathway for the synt... more The 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme of the mevalonate pathway for the synthesis of cholesterol in mammals (ergosterol in fungi), is inhibited by statins, a class of cholesterol lowering drugs. Indeed, statins are in a wide medical use, yet statins treatment could induce side effects as hepatotoxicity and myopathy in patients. We used Saccharomyces cerevisiae as a model to investigate the effects of statins on mitochondria. We demonstrate that statins are active in S.cerevisiae by lowering the ergosterol content in cells and interfering with the attachment of mitochondrial DNA to the inner mitochondrial membrane. Experiments on murine myoblasts confirmed these results in mammals. We propose that the instability of mitochondrial DNA is an early indirect target of statins.

Nucleic Acids Research, 1984

ITn Saccharomyces cerevisiae most mitochondrial tRNA genes are cluste red in a 9 kbp region betwe... more ITn Saccharomyces cerevisiae most mitochondrial tRNA genes are cluste red in a 9 kbp region between the. cap and oxil genes. Polygenic transcripts of this region have been previously identified. A transcriptional initiation site at a TTATAAGTA box, located upstream from the tRNAcYS gene, has now been detected by SI mapping experiments and by the capping of primary tran scripts. Results are consistent with the hypothesis that this box represents the initiation site for transcription of a cluster of tRNA genes, while the adjacent tRNAthr is cotranscribed with the 21S rRNA. Results obtained with various strains are compared, and the efficiency of this sequence as a tran scriptional initiation site in different mitochondrial contexts is discussed.

Biochimica Et Biophysica Acta - Molecular And Cell Biology Of Lipids, Mar 1, 2019

Sterols are essential lipids, involved in many biological processes. In Saccharomyces cerevisiae,... more Sterols are essential lipids, involved in many biological processes. In Saccharomyces cerevisiae, the enzymes of the ergosterol biosynthetic pathway (Erg proteins) are localised in different cellular compartments. With the aim of studying organelle interactions, we discovered that Erg27p resides mainly in Lipid Droplets (LDs) in respiratory competent cells, while in absence of respiration, is found mostly in the ER. The results presented in this paper demonstrate an interplay between the mitochondrial respiration and ergosterol production: on the one hand, rho° cells show lower ergosterol content when compared with wild type respiratory competent cells, on the other hand, the ergosterol biosynthetic pathway influences the mitochondrial status, since treatment with ketoconazole, which blocks the ergosterol pathway, or the absence of the ERG27 gene, induced rho° production in S. cerevisiae. The loss of mitochondrial DNA in the ∆erg27 strain is fully suppressed by exogenous addition of ergosterol. These data suggest the notion that ergosterol is essential for maintaining the mitochondrial DNA attached to the inner mitochondrial membrane.

Microbial Cell Factories, Jun 7, 2014

Background: Microbial lipids represent a valuable alternative feedstock for biodiesel production ... more Background: Microbial lipids represent a valuable alternative feedstock for biodiesel production when oleaginous microbes are cultured with inexpensive substrates in processes exhibiting high yield and productivity. In this perspective, crude glycerol is among the most promising raw materials for lipid production, because it is the costless residual of biodiesel production. Thus, cultivation of oleaginous yeasts in glycerol-based media is attracting great interest and natural biodiversity is increasingly explored to identify novel oleaginous species recycling this carbon source for growth and lipid production. Results: Thirty-three yeasts strains belonging to 19 species were screened for the ability to grow and produce intracellular lipids in a pure glycerol-based medium with high C/N ratio. A minority of them consumed most of the glycerol and generated visible lipid bodies. Among them, Candida freyschussii ATCC 18737 was selected, because it exhibited the highest lipid production and glycerol conversion yield. Lipid production in this strain was positively affected by the increase of C/N ratio, but growth was inhibited by glycerol concentration higher than 40 g/L. In batch cultures, the highest lipid production (4.6 g/L), lipid content of biomass (33% w/w), and lipid volumetric productivity (0.15 g/L/h) were obtained with 40 g/L glycerol, during the course of a 30-h process. Fed-batch cultivation succeeded in preventing substrate inhibition and in achieving a high cell-density culture. The improved lipid production and volumetric productivity reached the remarkable high level of 28 g/L and 0.28 g/L/h, respectively. The lipids accumulated by C. freyschussii ATCC 18737 have similar fatty acid composition of plant oil indicating their potential use as biodiesel feedstock. Calculated physicochemical properties of a biodiesel produced with the lipids from C. freyschussii ATCC 18737 are expected to meet the European and American standards, being equal to those of rapeseed and palm biodiesel.

FEMS Microbiology Letters, 2021

Transient modification of the environment involves the expression of specific genes and degradati... more Transient modification of the environment involves the expression of specific genes and degradation of mRNAs and proteins. How these events are linked is poorly understood. CCR4-NOT is an evolutionary conserved complex involved in transcription initiation and mRNA degradation. In this paper, we report that the yeast Not4 localizes in cytoplasmic foci after cellular stress. We focused our attention on the functional characterization of the C-terminus of the Not4 protein. Molecular dissection of this region indicates that the removal of the last 120 amino acids, does not affect protein localization and function, in that the protein is still able to suppress the thermosensitivity observed in the not4Δ mutant. In addition, such shortened form of Not4, as well its absence, increases the transcription of stress-responsive genes conferring to the cell high resistance to the oxidative stress. On the contrary, the last C-terminal 211 amino acids are required for proper Not4 localization at c...

FEMS Yeast Research, 2019

Glucose is the preferred nutrient for most living cells and is also a signaling molecule that mod... more Glucose is the preferred nutrient for most living cells and is also a signaling molecule that modulates several cellular processes. Glucose regulates the expression of glucose permease genes in yeasts through signaling pathways dependent on plasma membrane glucose sensors. In the yeast Kluyveromyces lactis, sufficient levels of glucose induction of the low-affinity glucose transporter RAG1 gene also depends on a functional glycolysis, suggesting additional intracellular signaling. We have found that the expression of RAG1 gene is also induced by hypoxia in the presence of glucose, indicating that glucose and oxygen signaling pathways are interconnected. In this study we investigated the molecular mechanisms underlying this crosstalk. By analyzing RAG1 expression in various K. lactis mutants, we found that the bHLH transcriptional activator Sck1 is required for the hypoxic induction of RAG1 gene. The RAG1 promoter region essential for its hypoxic induction was identified by promoter ...

Yeast, Mar 15, 2000

Six ORFs of unknown function located on chromosome VII of Saccharomyces cerevisiae were disrupted... more Six ORFs of unknown function located on chromosome VII of Saccharomyces cerevisiae were disrupted in two different genetic backgrounds, and the phenotype of the generated mutants was analysed. Disruptions of ORFs YGR256w, YGR272c, YGR273c, YGR275w and YGR276c were carried out using the disruption marker kanMX4 anked by short homology regions, whereas ORF YGR255c was inactivated with a long¯anking homology (LFH) disruption cassette (Wach et al., 1994). Tetrad analysis of the heterozygous disruptants revealed that ORF YGR255c, previously identi®ed as COQ6 and encoding a protein involved in the biosynthesis of coenzime Q (Tzagoloff and Dieckmann, 1990), is an essential gene. The same analysis also revealed that sporulation of the ygr272cD heterozygous diploid produced two small colonies per ascus that were also G418-resistant, indicating that the inactivation of ORF YGR272c could result in a slower growth rate. This result was con®rmed by growth tests of the haploid disruptants and by complementation of the phenotype after transformation with a plasmid carrying the cognate gene. No phenotypes could be associated to the inactivation of ORFs YGR256w, YGR273c, YGR275w and YGR276c. Two of these genes have recently been further characterized: ORF YGR255w, renamed RTT102, encodes a regulator of the Ty1-element transposition, whereas ORF YGR276c was found to encode the 70 kDa RNase H activity and was renamed RNH70 (Frank et al.

International Journal of Molecular Sciences

Living organisms on the surface biosphere are periodically yet consistently exposed to light. The... more Living organisms on the surface biosphere are periodically yet consistently exposed to light. The adaptive or protective evolution caused by this source of energy has led to the biological systems present in a large variety of organisms, including fungi. Among fungi, yeasts have developed essential protective responses against the deleterious effects of light. Stress generated by light exposure is propagated through the synthesis of hydrogen peroxide and mediated by regulatory factors that are also involved in the response to other stressors. These have included Msn2/4, Crz1, Yap1, and Mga2, thus suggesting that light stress is a common factor in the yeast environmental response.

Sterols are essential lipids, involved in many biological processes. In Saccharomyces cerevisiae,... more Sterols are essential lipids, involved in many biological processes. In Saccharomyces cerevisiae, the enzymes of the ergosterol biosynthetic pathway (Erg proteins) are localized in different cellular compartments. With the aim of studying organelle interactions, we discovered that Erg27p resides mainly in Lipid Droplets (LDs) in respiratory competent cells, while in absence of respiration, is found mostly in the ER. The results presented in this paper demonstrate an interplay between the mitochondrial respiration and ergosterol production: on the one hand, rho°cells show lower ergosterol content when compared with wild type respiratory competent cells, on the other hand, the ergosterol biosynthetic pathway influences the mitochondrial status, since treatment with ketocona-zole, which blocks the ergosterol pathway, or the absence of the ERG27 gene, induced rho°production in S. cerevisiae. The loss of mitochondrial DNA in the Δerg27 strain is fully suppressed by exogenous addition of ergosterol. These data suggest the notion that ergosterol is essential for maintaining the mitochondrial DNA attached to the inner mitochondrial membrane.