Characterization of hexose transporters in Yarrowia lipolytica reveals new groups of Sugar Porters involved in yeast growth.

Sugar assimilation has been intensively studied in the model yeast S. cerevisiae, and for two decades, it has been clear that the homologous HXT genes, which encode a set of hexose transporters, play a central role in this process. However, in the yeast Yarrowia lipolytica, which is well-known for its biotechnological applications, sugar assimilation is only poorly understood, even though this yeast exhibits peculiar intra-strain differences in fructose uptake: some strains (e.g., W29) are known to be slow-growing in fructose while others (e.g., H222) grow rapidly under the same conditions. Here, we retrieved 24 proteins of the Sugar Porter family from these two strains, and determined that at least six of these proteins can function as hexose transporters in the heterologous host Saccharomyces cerevisiae EBY.VW4000. Transcriptional studies and deletion analysis in Y. lipolytica indicated that two genes, YHT1 and YHT4, are probably the main players in both strains, with a similar role in the uptake of glucose, fructose, and mannose at various concentrations. The other four genes appear to constitute a set of 'reservoir' hexose transporters with an as-yet unclear physiological role. Furthermore, through examining Sugar Porters of the entire Yarrowia clade, we show that they constitute a dynamic family, within which hexose transport genes have been duplicated and lost several times. Our phylogenetic analyses support the existence of at least three distinct evolutionary groups of transporters which allow yeasts to grow on hexoses. In addition to the well-known and widespread Hxt-type transporters (which are not essential in Y. lipolytica), we highlight a second group of transporters, represented by Yht1, which are phylogenetically related to sensors that play a regulatory role in S. cerevisiae, and a third group, represented by Yht4, previously thought to contain only high-affinity glucose transporters related to Hgt1of Kluyveromyces lactis.

Biotransformation of acetophenone and its halogen derivatives by Yarrowia lipolytica strains.

The ability of 16 strains of Yarrowia lipolytica to biotransform acetophenone and its derivatives has been studied. Thirteen of these strains were derived from a wild-type strain Y. lipolytica A-101; six had the invertase gene (SUC2) from Saccharomyces cerevisiae integrated into their genome, as well as the damaged or undamaged gene encoding orotidine-5'-phosphate decarboxylase (URA3), three had integrated the damaged URA3 gene into their genome and three were UV acetate-negative mutants, not able to growth on acetate as the sole carbon source. The other tested strains included two wild strains, A-101 and PMR-1, and an adenine auxotroph ATCC 32-338A. All strains were capable of reducing acetophenone to the R-alcohol in high enantiomeric excess (80-89 %). In all of the cultures tested, reversibility of the reduction was observed, which led to an increase in the enantiomeric excess. nantioselective reduction of the acetophenone halogen derivatives revealed that the nature and location of the halogen atom had a significant influence on the enantioselectivity of the reduction. In the culture of ATCC 32-338A, after a 3-day biotransformation of 2,4'-dibromoacetophenone the enantiopure R-alcohol was obtained at a rate of 100 % of substrate conversion. In conclusion, using these invertase-containing strains or uracyl auxotrophs provided no additional benefit in terms of biotransformation capacity over the parental strain.

[Fructose transporter in yeasts]. / Transportery fruktozy w komórkach drozdzy.

Study of hexoses transporter started with discovery of galactose permease in Saccharomyces cerevisiae. Glucose, fructose and mannose assimilation is assumed by numerous proteins encoded by different genes. To date over 20 hexoses transporters, belonging to Sugar Porter family and to Major Facilitator Superfamily, were known. Genome sequence analysis of Candida glabrata, Kluyveromyces lactis, Yarrowia lipolytica, S. cerevisaie and Debaryomyces hansenii reveled potential presence of 17-48 sugar porter proteins. Glucose transporters in S. cerevisiae have been already characterized. In this paper, hexoses transporters, responsible for assimilation of fructose by cells, are presented and compared. Fructose specific transporter are described for yeasts: Zygosaccharomyces rouxii, Zygosaccharomyces bailli, K. lactis, Saccharomyces pastorianus, S. cerevisiae winemaking strain and for fungus Botritys cinerea and human (Glut5p). Among six yeasts transporters, five are fructose specific, acting by facilitated diffusion or proton symport. Yeasts monosaccharides transporter studies allow understanding of sugars uptake and metabolism important aspects, even in higher eukaryotes cells.

Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications.

Yarrowia lipolytica requires the expression of a heterologous invertase to grow on a sucrose-based substrate. This work reports the construction of an optimized invertase expression cassette composed of Saccharomyces cerevisiae Suc2p secretion signal sequence followed by the SUC2 sequence and under the control of the strong Y. lipolytica pTEF promoter. This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate. Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context. The strain JMY2593, expressing this new cassette with an optimized secretion signal sequence and a strong promoter, produces 4,519 U/l of extracellular invertase in bioreactor experiments compared to 597 U/l in a strain expressing the former invertase construction. The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.

New Look on Antifungal Activity of Silver Nanoparticles (AgNPs).

The progress of research on silver nanoparticles (AgNPs) has led to their inclusion in many consumer products (chemicals, cosmetics, clothing, water filters, and medical devices) as a biocide. Despite the widespread use of AgNPs, their biocidal activity is not yet fully understood and is usually associated with various factors (size, composition, surface, red-ox potential, and concentration) and, obviously, specific features of microorganisms. There are merely a few studies concerning the interaction of molds with AgNPs. Therefore, the determination of the minimal AgNPs concentration required for effective growth suppression of five fungal species (Paecilomyces variotii, Penicillium pinophilum, Chaetomium globosum, Trichoderma virens, and Aspergillus brasiliensis), involved in the deterioration of construction materials, was particularly important. Inhibition of bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli) and yeasts (Candida albicans and Yarrowia lipolytica) was also assessed as the control of AgNPs effectiveness. AgNPs at the concentrations of 9-10.7 ppm displayed high inhibitory activity against moulds, yeast, and bacteria. The TEM images revealed that 20 nm AgNPs migrated into bacterial, yeast, and fungal cells but aggregated in larger particles (50-100 nm) exclusively inside eukaryotic cells. The aggregation of 20 nm AgNPs and particularly their accumulation in the cell wall, observed for A. brasiliensis cells, are described here for the first time.The progress of research on silver nanoparticles (AgNPs) has led to their inclusion in many consumer products (chemicals, cosmetics, clothing, water filters, and medical devices) as a biocide. Despite the widespread use of AgNPs, their biocidal activity is not yet fully understood and is usually associated with various factors (size, composition, surface, red-ox potential, and concentration) and, obviously, specific features of microorganisms. There are merely a few studies concerning the interaction of molds with AgNPs. Therefore, the determination of the minimal AgNPs concentration required for effective growth suppression of five fungal species (Paecilomyces variotii, Penicillium pinophilum, Chaetomium globosum, Trichoderma virens, and Aspergillus brasiliensis), involved in the deterioration of construction materials, was particularly important. Inhibition of bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli) and yeasts (Candida albicans and Yarrowia lipolytica) was also assessed as the control of AgNPs effectiveness. AgNPs at the concentrations of 9­10.7 ppm displayed high inhibitory activity against moulds, yeast, and bacteria. The TEM images revealed that 20 nm AgNPs migrated into bacterial, yeast, and fungal cells but aggregated in larger particles (50­100 nm) exclusively inside eukaryotic cells. The aggregation of 20 nm AgNPs and particularly their accumulation in the cell wall, observed for A. brasiliensis cells, are described here for the first time.

Draft Genome Sequence of Yarrowia lipolytica Strain A-101 Isolated from Polluted Soil in Poland.

Yarrowia lipolytica is an early diverging species of the Saccharomycotina subphylum, which is recognized as a valuable host for many biotechnological applications exploiting its oleaginous capacities. The 20.5-Mb genome of the Polish Y. lipolytica strain A-101 will greatly help decipher the genetic basis of the regulation of its lipid metabolism.

Simultaneous production of citric acid and invertase by Yarrowia lipolytica SUC+ transformants.

Simultaneous production of citric acid (CA) and invertase by Yarrowia lipolytica A-101-B56-5 (SUC(+) clone) growing from sucrose, mixture of glucose and fructose, glucose or glycerol was investigated. Among the tested substrates the highest concentration of CA was reached from glycerol (57.15 g/L) with high yield (Y(CA/S)=0.6g/g). When sucrose was used, comparable amount of CA was secreted (45 g/L) with slightly higher yield (Y(CA/S)=0.643 g/g). In all cultures amount of isocitrate (ICA) was below 2% of total citrates. Considering invertase production, the best carbon source appeared to be sucrose (72380 U/L). The highest yield of CA and invertase biosynthesis calculated for 1g of biomass was obtained for cells growing from glycerol (9.9 g/g and 4325 U/g, respectively). Concentrates of extra- and intracellular invertase of the highest activity were obtained from sucrose as substrate (0.5 and 1.8 × 10(6)U/L, respectively).

RAPD with microsatellite as a tool for differentiation of Candida genus yeasts isolated in brewing.

Fifteen wild yeast strains were isolated in two factories of a lager brewing company in Poland. Their identification with API 32C system showed mainly the presence of Candida sake species (7/15). To differentiate the isolates, randomly amplified polymorphic DNA (RAPD) with (GTG)(5), (GAC)(5), (GACA)(4) microsatellite primers and M13 core sequence (5'-GAG GGT GGC GGT TCT-3') were chosen. The results of patterns similarity are presented as dendrograms for each RAPD analysis and for overall patterns. On the overall patterns, all isolates identified as C. sake, except Strain No. 1, were regrouped in one cluster. Collection strain C. sake CBS 617 was similar in 46% to the cluster with six isolates (Strain Nos. 3, 6, 8, 11, 13, 14). The second reference strain C. sake CBS 159 and the Strain No. 1 were regrouped with other Candida species (collection strains) showing, respectively, only 20% and 42% of similarity to other C. sake strains. The similarity based on the overall dendrogram between isolate Nos. 3, 6, 8, 11, 13, 14 and C. sake CBS 617 was 49%. Between those strains and other Candida, the similarity was only 37%.