Occurrence of FFZ genes in yeasts and correlation with fructophilic behaviour.

Fructophily has been described in yeasts as the ability to utilize fructose preferentially when fructose and glucose are available in the environment. In Zygosaccharomyces bailii and Zygosaccharomyces rouxii, fructophilic behaviour has been associated with the presence of a particular type of high-capacity and low-affinity fructose transporters designated Ffz. In this study, a PCR screening was performed in several yeasts using degenerate primers suitable to detect FFZ-like genes. In parallel, fructophilic character was evaluated in the same strains by comparing the relative consumption rate of fructose and glucose. For all the strains in which FFZ-like genes were detected, fructophilic behaviour was observed (25 strains). Results show that FFZ genes are ubiquitous in the Zygosaccharomyces and Starmerella clades. Strains of Lachancea fermentati, Torulaspora microellipsoides and Zygotorulaspora florentina were not fructophilic and did not harbour FFZ genes. It is of note that these new species were recently removed by taxonomists from the Zygosaccharomyces clade, supporting the view that the presence of FFZ-like genes is a main characteristic of Zygosaccharomyces. Among the strains tested, only Hanseniaspora guilliermondii NCYC2380 was an exception, having a preference for fructose in medium with high sugar concentrations, despite no FFZ-like genes being detected in the screening. Furthermore, this study supports the previous idea of the emergence of a new family of hexose transporters (Ffz facilitators) distinct from the Sugar Porter family.

The high-capacity specific fructose facilitator ZrFfz1 is essential for the fructophilic behavior of Zygosaccharomyces rouxii CBS 732T.

Zygosaccharomyces rouxii is a fructophilic yeast that consumes fructose preferably to glucose. This behavior seems to be related to sugar uptake. In this study, we constructed Z. rouxii single-, double-, and triple-deletion mutants in the UL4 strain background (a ura3 strain derived from CBS 732(T)) by deleting the genes encoding the specific fructose facilitator Z. rouxii Ffz1 (ZrFfz1), the fructose/glucose facilitator ZrFfz2, and/or the fructose symporter ZrFsy1. We analyzed the effects on the growth phenotype, on kinetic parameters of fructose and glucose uptake, and on sugar consumption profiles. No growth phenotype was observed on fructose or glucose upon deletion of FFZ genes. Deletion of ZrFFZ1 drastically reduced fructose transport capacity, increased glucose transport capacity, and eliminated the fructophilic character, while deletion of ZrFFZ2 had almost no effect. The strain in which both FFZ genes were deleted presented even higher consumption of glucose than strain Zrffz1Δ, probably due to a reduced repressing effect of fructose. This study confirms the molecular basis of the Z. rouxii fructophilic character, demonstrating that ZrFfz1 is essential for Z. rouxii fructophilic behavior. The gene is a good candidate to improve the fructose fermentation performance of industrial Saccharomyces cerevisiae strains.

Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae.

In previous work, we developed a Saccharomyces cerevisiae strain (DLG-K1) lacking the main monosaccharide transporters (hxt-null) and displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities. This strain proved to be a useful chassis strain to study new glucose/xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Proteins with high amino acid sequence similarity (78-80%) to SsXUT1 were identified from Spathaspora passalidarum and Spathaspora arborariae genomes. The characterization of these putative transporter genes (SpXUT1 and SaXUT1, respectively) was performed in the same chassis strain. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested (including glucose and xylose), but after being grown in maltose, the uptake of 14C-glucose and 14C-xylose was detected. While SsXUT1 lacks lysine residues with high ubiquitinylation potential in its N-terminal domain and displays only one in its C-terminal domain, both SpXUT1 and SaXUT1 transporters have several such residues in their C-terminal domains. A truncated version of SpXUT1 gene, deprived of the respective 3'-end, was cloned in DLG-K1 and allowed growth and fermentation in glucose or xylose. In another approach, two arrestins known to be involved in the ubiquitinylation and endocytosis of sugar transporters (ROD1 and ROG3) were knocked out, but only the rog3 mutant allowed a significant improvement of growth and fermentation in glucose when either of the XUT permeases were expressed. Therefore, for the efficient heterologous expression of monosaccharide (e.g., glucose/xylose) transporters in S. cerevisiae, we propose either the removal of lysines involved in ubiquitinylation and endocytosis or the use of chassis strains hampered in the specific mechanism of membrane protein turnover.

The osmotolerant fructophilic yeast Zygosaccharomyces rouxii employs two plasma-membrane fructose uptake systems belonging to a new family of yeast sugar transporters.

Owing to its high resistance to weak-acid preservatives and extreme osmotolerance, Zygosaccharomyces rouxii is one of the main spoilage yeasts of sweet foods and beverages. In contrast with Saccharomyces cerevisiae, Z. rouxii is a fructophilic yeast; it consumes fructose faster than glucose. So far, to our knowledge, no specific Z. rouxii proteins responsible for this fructophilic behaviour have been characterized. We have identified two genes encoding putative fructose transporters in the Z. rouxii CBS 732 genome. Heterologous expression of these two Z. rouxii ORFs in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic analysis of sugar transport showed that both proteins are functionally expressed at the plasma membrane: ZrFfz1 is a high-capacity fructose-specific facilitator (K(m)∼400 mM and V(max)∼13 mmol h(-1) g(-1)) and ZrFfz2 is a facilitator transporting glucose and fructose with similar capacity and affinity (K(m)∼200 mM and V(max)∼4 mmol h(-1) g(-1)). These two proteins together with the Zygosaccharomyces bailii Ffz1 fructose-specific transporter belong to a new family of sugar transport systems mediating the uptake of hexoses via the facilitated diffusion mechanism, and are more homologous to drug/H(+) antiporters (regarding their primary protein structure) than to other yeast sugar transporters of the Sugar Porter family.

Bronchiectasis in rheumatoid arthritis. A clinical appraisial.

Bronchiectasis is defined as irreversibly damaged and dilated bronchi and is one of the most common pulmonary manifestations in patients with rheumatoid arthritis (RA). The model of RA-associated autoimmunity induced in some individuals by chronic bacterial infection in bronchiectasis is becoming increasingly acceptable, although a genetic predisposition to RA-associated bronchiectasis has also been demonstrated. Bronchiectasis should be suspected in RA patients with chronic cough and sputum production or frequent respiratory infections and the diagnosis must be confirmed by thoracic high-resolution computed tomography. Management of patients with RA-associated bronchiectasis includes a multimodal treatment approach. Similar to all patients with non-cystic fibrosis bronchiectasis, patients with RA-associated bronchiectasis benefit from a pulmonary rehabilitation program, including an exercise/muscle strengthening program and an education program with a specific session on airway clearance techniques. Prophylactic antibiotics are recommended for patients with frequent (3 or more infective exacerbations per year) or severe infections requiring hospitalization/intravenous antibiotics and inhaled corticosteroids and long-acting ß2-agonists should be used in patients with non-cystic fibrosis bronchiectasis and associated airway hyper-responsiveness. In patients with RA-associated bronchiectasis the use of immunomodulatory drugs has to be carefully considered, as they are essential to control disease activity, despite being associated with an increased infectious risk. Pneumococcal and influenza vaccines are advised to all patients with RA-associated bronchiectasis in order to reduce the risk of infection. Patients with RA-associated bronchiectasis have a poorer prognosis than those with either RA or bronchiectasis alone and require regular follow-up, under the joint care of a rheumatologist and a pulmonologist.

Hexose and pentose transport in ascomycetous yeasts: an overview.

The biochemical characterization of sugar uptake in yeasts started five decades ago and led to the early production of abundant kinetic and mechanistic data. However, the first accurate overview of the underlying sugar transporter genes was obtained relatively late, due mainly to the genetic complexity of hexose uptake in the model yeast Saccharomyces cerevisiae. The genomic era generated in turn a massive amount of information, allowing the identification of a multitude of putative sugar transporter and sensor-encoding genes in yeast genomes, many of which are phylogenetically related. This review aims to briefly summarize our current knowledge on the biochemical and molecular features of the transporters of hexoses and pentoses in yeasts, when possible establishing links between previous kinetic studies and genomic data currently available. Emphasis is given to recent developments concerning the identification of d-xylose and l-arabinose transporter genes, which are thought to be key players in the optimization of S. cerevisiae strains for bioethanol production from lignocellulose hydrolysates.

Integrated Process for Bioenergy Production and Water Recycling in the Dairy Industry: Selection of Kluyveromyces Strains for Direct Conversion of Concentrated Lactose-Rich Streams into Bioethanol.

Dairy industries have a high environmental impact, with very high energy and water consumption and polluting effluents. To increase the sustainability of these industries it is urgent to implement technologies for wastewater treatment allowing water recycling and energy savings. In this study, dairy wastewater was processed by ultrafiltration and nanofiltration or ultrafiltration and reverse osmosis (UF/RO) and retentates from the second membrane separation processes were assessed for bioenergy production. Lactose-fermenting yeasts were tested in direct conversion of the retentates (lactose-rich streams) into bioethanol. Two Kluyveromyces strains efficiently fermented all the lactose, with ethanol yields higher than 90% (>0.47 g/g yield). Under severe oxygen-limiting conditions, the K. marxianus PYCC 3286 strain reached 70 g/L of ethanol, which is compatible with energy-efficient distillation processes. In turn, the RO permeate is suitable for recycling into the cleaning process. The proposed integrated process, using UF/RO membrane technology, could allow water recycling (RO permeate) and bioenergy production (from RO retentate) for a more sustainable dairy industry.

A New Pathway for Mannitol Metabolism in Yeasts Suggests a Link to the Evolution of Alcoholic Fermentation.

The yeasts belonging to the Wickerhamiella and Starmerella genera (W/S clade) share a distinctive evolutionary history marked by loss and subsequent reinstatement of alcoholic fermentation mediated by horizontal gene transfer events. Species in this clade also share unusual features of metabolism, namely the preference for fructose over glucose as carbon source, a rare trait known as fructophily. Here we show that fructose may be the preferred sugar in W/S-clade species because, unlike glucose, it can be converted directly to mannitol in a reaction with impact on redox balance. According to our results, mannitol is excreted to the growth medium in appreciable amounts along with other fermentation products such as glycerol and ethanol but unlike the latter metabolites mannitol production increases with temperature. We used comparative genomics to find genes involved in mannitol metabolism and established the mannitol biosynthesis pathway in W/S-clade species Starmerella bombicola using molecular genetics tools. Surprisingly, mannitol production seems to be so important that St. bombicola (and other W/S-clade species) deploys a novel pathway to mediate the conversion of glucose to fructose, thereby allowing cells to produce mannitol even when glucose is the sole carbon source. Using targeted mutations and 13C-labeled glucose followed by NMR analysis of end-products, we showed that the novel mannitol biosynthesis pathway involves fructose-6-phosphate as an intermediate, implying a key role for a yet unknown fructose-6-P phosphatase. We hypothesize that mannitol production contributed to mitigate the negative effects on redox balance of the ancient loss of alcoholic fermentation in the W/S clade. Presently, mannitol also seems to play a role in stress protection.

Evidence for loss and reacquisition of alcoholic fermentation in a fructophilic yeast lineage.

Fructophily is a rare trait that consists of the preference for fructose over other carbon sources. Here, we show that in a yeast lineage (the Wickerhamiella/Starmerella, W/S clade) comprised of fructophilic species thriving in the high-sugar floral niche, the acquisition of fructophily is concurrent with a wider remodeling of central carbon metabolism. Coupling comparative genomics with biochemical and genetic approaches, we gathered ample evidence for the loss of alcoholic fermentation in an ancestor of the W/S clade and subsequent reinstatement through either horizontal acquisition of homologous bacterial genes or modification of a pre-existing yeast gene. An enzyme required for sucrose assimilation was also acquired from bacteria, suggesting that the genetic novelties identified in the W/S clade may be related to adaptation to the high-sugar environment. This work shows how even central carbon metabolism can be remodeled by a surge of HGT events.

Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose-H+ symporter.

Candida intermedia PYCC 4715 was previously shown to grow well on xylose and to transport this sugar by two different transport systems: high-capacity and low-affinity facilitated diffusion and a high-affinity xylose-proton symporter, both of which accept glucose as a substrate. Here we report the isolation of genes encoding both transporters, designated GXF1 (glucose/xylose facilitator 1) and GXS1 (glucose/xylose symporter 1) respectively. Although GXF1 was isolated by functional complementation of an HXT-null (where Hxt refers to hexose transporters) Saccharomyces cerevisiae strain, isolation of the GXS1 cDNA required partial purification and micro-sequencing of the transporter, identified by its relative abundance in cells grown on low xylose concentrations. Both genes were expressed in S. cerevisiae and the kinetic parameters of glucose and xylose transport were determined. Gxs1 is the first yeast xylose/glucose-H+ symporter to be characterized at the molecular level. Comparison of its amino acid sequence with available sequence data revealed the existence of a family of putative monosaccharide-H+ symporters encompassing proteins from several yeasts and filamentous fungi.

Characterization of new polyol/H+ symporters in Debaryomyces hansenii.

Debaryomyces hansenii is a halotolerant yeast that produces and assimilates a wide variety of polyols. In this work we evaluate polyol transport in D. hansenii CBS 767, detecting the occurrence of polyol/H(+) (and sugar/H(+)) symporter activity, through the transient extracellular alkalinization of unbuffered starved cell suspensions. From the D. hansenii genome database, we selected nine ORFs encoding putative transporter proteins to clone in a centromeric plasmid with C-terminal GFP tagging and screened for polyol/H(+) symporters by heterologous expression in Saccharomyces cerevisiae. Five distinct D. hansenii polyol/H(+) symporters were identified and characterized, with different specificities and affinities for polyols, namely one glycerol-specific (DhStl1), one D-galactitol-specific (DhSgl1, Symporter galactitol/H(+) 1), one D-(+)-chiro-inositol-specific (DhSyi1, Symporter D-(+)-chiro-inositol/H(+) 1), one for D-sorbitol/D-mannitol/ribitol/D-arabitol/D-galactitol (DhSyl1, Symporter Polyols 1) and another for D-sorbitol/D-mannitol/ribitol/D-arabitol (DhSyl2, Symporter Polyols 2). This work contributed to the annotation of new yeast polyol transporters, including two specific for uncommon substrates as galactitol and D-(+)-chiro-inositol.

Exploring three PIPs and three TIPs of grapevine for transport of water and atypical substrates through heterologous expression in aqy-null yeast.

Aquaporins are membrane channels that facilitate the transport of water and other small molecules across the cellular membranes. We examined the role of six aquaporins of Vitis vinifera (cv. Touriga nacional) in the transport of water and atypical substrates (other than water) in an aqy-null strain of Saccharomyces cerevisiae. Their functional characterization for water transport was performed by stopped-flow fluorescence spectroscopy. The evaluation of permeability coefficients (Pf) and activation energies (Ea) revealed that three aquaporins (VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2) are functional for water transport, while the other three (VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1) are non-functional. TIPs (VvTnTIP1;1 and VvTnTIP2;2) exhibited higher water permeability than VvTnPIP2;1. All functional aquaporins were found to be sensitive to HgCl2, since their water conductivity was reduced (24-38%) by the addition of 0.5 mM HgCl2. Expression of Vitis aquaporins caused different sensitive phenotypes to yeast strains when grown under hyperosmotic stress generated by KCl or sorbitol. Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance. Their sequence analyses revealed the presence of signature sequences for transport of ammonia, boron, CO2, H2O2 and urea. The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H2O2, suggesting the contribution of Vitis aquaporins in the transport of these species.

ZrFsy1, a high-affinity fructose/H+ symporter from fructophilic yeast Zygosaccharomyces rouxii.

Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H(+) symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H(+) symporter, with Km 0.45 ± 0.07 mM and Vmax 0.57 ± 0.02 mmol h(-1) (gdw)(-1). We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2%) and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H(+) symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.

The expression in Saccharomyces cerevisiae of a glucose/xylose symporter from Candida intermedia is affected by the presence of a glucose/xylose facilitator.

Two glucose/xylose transporter genes from Candida intermedia were recently cloned and characterized: GXF1, which encodes a glucose/xylose facilitator; and GXS1, which encodes a glucose/xylose proton symporter. Here we report the functional expression of these transporters in Saccharomyces cerevisiae. While Gxf1p seems to be fully functional in S. cerevisiae, the symporter Gxs1p exhibits very low glucose/xylose transport activity, which could not be ascribed to insufficient production of the protein or incorrect subcellular localization. In addition, co-expression of glucose/xylose facilitators with Gxs1p strongly reduced GXS1 mRNA levels, and consequently symport activity, in glucose-grown, but not in ethanol-grown, cells. The observed decrease in GXS1 transcript levels seems to be related to an enhanced glucose influx mediated by glucose facilitator protein(s), and not to a specific interaction between Gxs1p and other transporters. We found GXS1 mRNA levels to be severely reduced as a result of glucose addition, and we show that this effect takes place at the level of GXS1 mRNA stability. Our results suggest that a decrease in mRNAs encoding high-affinity/active sugar transport systems may be a widespread and conserved mechanism in yeasts, limiting expression of these proteins whenever their activity is dispensable.

Diabetes fosforada idiopática do adulto: a propósito de um caso clínico / Acquired hypophosphatemic osteomalacia: case report

O fósforo é elemento essencial e muitas vezes pouco valo rizado na prática clínica. A diabetes fosforada idiopática do adulto (DFIA) é causa de alteraçao da homeostase do fósforo, resultante de defeito da reabsorçao deste elemento no túbulo proximal, de causa nao conhecida. Caracteriza-se clinicamente por raquialgias, radiculalgias, fadiga, dores ósseas e articulares e sintomas neuropsiquiátricos. As características laboratoriais típicas sao: hipofos fatemia, hiperfosfatúria e índices de reabsorçao tubular do fósforo que demons tram diminuiçao da reabsorçao renal desse elemento. A hipofosfatemia moderada que se verifica na DFIA provoca alteraçao do metabolismo ósseo, que resulta em osteopenia. A terapeutica com fósforo e vitamina D leva a normalizaçao da fosfa temia e dos índices de reabsorçao renal de fósforo, a melhoria clínica e o aumento da densidade mineral óssea. Os autores escrevem o caso de paciente com DFIA e discutem alguns pontos de etiopatogenia, clínica e terapeutica dessa doença