Two Fission Yeast Rab7 Homologs, Ypt7 and Ypt71, Play Antagonistic Roles in the Regulation of Vacuolar Morphology

Small guanine triphosphatases (GTPases) of the Rab family are key regulators of membrane trafficking events between the various subcellular compartments in eukaryotic cells. Rab7 is a conserved protein required in the late endocytic pathway and in lysosome biogenesis. A Schizosaccharomyces pombe ( S. pombe ) homolog of Rab7, Ypt7, is necessary for trafficking from the endosome to the vacuole and for homotypic vacuole fusion. Here, we identified and characterized a second fission yeast Rab7 homolog, Ypt71. Ypt71 is localized to the vacuolar membrane. Cells deleted for ypt71 ⁺ exhibit normal growth rates and morphology. Interestingly, a ypt71 null mutant contains large vacuoles in contrast with the small fragmented vacuoles found in the ypt7 null mutant. Furthermore, the ypt71 mutation does not enhance or alleviate the temperature sensitivity or vacuole fusion defect of ypt7 Δ cells. Like ypt7 Δ cells, overexpression of ypt71 ⁺ caused fragmentation of vacuoles and inhibits vacuole fusion under hypotonic conditions. Thus, the two S. pombe Rab7 homologs act antagonistically in regulating vacuolar morphology. Analysis of a chimeric Ypt7/Ypt71 protein showed that Rab7-directed vacuole dynamics, fusion versus fission, largely depends on the medial region of the protein, including a part of RabSF3/α3-L7.     

Three new species of bipolar budding yeasts of the genus Hanseniaspora and its anamorph Kloeckera isolated in Thailand

In the course of a survey of yeast biodiversity in the natural substrates in Thailand, eight strains were found to represent three hitherto undescribed species of Hanseniaspora/Kloeckera . They were isolated from insect frass, flower, lichen, rotted fruit and rotted wood. Based on the morphological and physiological characteristics, and sequences of D1/D2 domain, six strains represent a single species of the genus Hanseniaspora , described as Hanseniaspora thailandica sp. nov. (type BCC 14938^T=NBRC 104216^T=CBS 10841^T), and another strain as Hanseniaspora singularis sp. nov. (type BCC 15001^T=NBRC 104214^T=CBS 10840^T). A further strain, which belongs to Kloeckera and does not produce ascospores, is described as Kloeckera hatyaiensis sp. nov. (type BCC 14939^T=NBRC 104215^T=CBS 10842^T). Strains belonging to H. thailandica sp. nov. differed by 17–19 nucleotide substitutions from Hanseniaspora meyeri , the closest species. DNA reassociation between the two taxa showed 30–48% relatedness. Kloeckera hatyaiensis sp. nov. and H. singularis sp. nov. differed by eight and 16 nucleotide substitutions with one gap from the nearest species, Hanseniaspora clermontiae and Hanseniaspora valbyensis , respectively.     

The role of acetaldehyde and glycerol in the adaptation to ethanol stress of Saccharomyces cerevisiae and other yeasts

Ethanol inhibition is a commonly encountered stress condition during typical yeast fermentations and often results in reduced fermentation rates and production yields. While past studies have shown that acetaldehyde addition has a significant ameliorating effect on the growth of ethanol-stressed Saccharomyces cerevisiae , this study investigated the potential ameliorating effect of acetaldehyde on a wide range of ethanol-stressed yeasts. Acetaldehyde does not appear to be a universal ameliorating agent for yeasts exposed to ethanol stress. It is also shown that as a result of an ethanol stress, most yeasts rapidly produce glycerol as an alternative means of NAD⁺ regeneration rather than having a specific requirement for glycerol. The results strongly suggest that both ethanol and acetaldehyde exposure have a direct effect on the cellular NAD⁺/NADH ratio, which can manifest itself as modulations in glycerol production.     

Development of host and vector for high-efficiency transformation and gene disruption in Debaryomyces hansenii

Debaryomyces hansenii is one of the most osmotolerant and halotolerant yeasts. The molecular mechanisms underlying its extreme osmotolerance and halotolerance have drawn considerable attention in the recent past. However, progress in this regard has been limited due to lack of availability of a transformation system and molecular tools to study the functions of the genes in D. hansenii . Here, we have described the development of an efficient transformation system for D. hansenii that is based on a histidine auxotrophic recipient strain and the DhHIS4 gene as the selectable marker. By screening the D. hansenii genomic library, we have isolated several autonomous replication sequences that can be used for constructing a replicating vector. Moreover, our study is the first to demonstrate gene disruption in D. hansenii by homologous recombination.     

Knocking out the MFE-2 gene of Candida bombicola leads to improved medium-chain sophorolipid production

The nonpathogenic yeast Candida bombicola synthesizes sophorolipids. These biosurfactants are composed of the disaccharide sophorose linked to a long-chain hydroxy fatty acid and have potential applications in the food, pharmaceutical, cosmetic and cleaning industries. In order to expand the range of application, a shift of the fatty acid moiety towards medium-chain lengths would be recommendable. However, the synthesis of medium-chain sophorolipids by C. bombicola is a challenging objective. First of all, these sophorolipids can only be obtained by fermentations on unconventional carbon sources, which often have a toxic effect on the cells. Furthermore, medium-chain substrates are partially metabolized in the β-oxidation pathway. In order to redirect unconventional substrates towards sophorolipid synthesis, the β-oxidation pathway was blocked on the genome level by knocking out the multifunctional enzyme type 2 (MFE-2) gene. The total gene sequence of the C. bombicola MFE-2 (6033 bp) was cloned (GenBank accession number EU371724 ), and the obtained nucleotide sequence was used to construct a knock-out cassette. Several knock-out mutants with the correct geno- and phenotype were evaluated in a fermentation on 1-dodecanol. All mutants showed a 1.7–2.9 times higher production of sophorolipids, indicating that in those strains the substrate is redirected towards the sophorolipid synthesis.     

Functional interaction between the ubiquitin-specific protease 25 and the SYK tyrosine kinase

The SYK non-receptor tyrosine kinase is a key effector of immune receptors signaling in hematopoietic cells. Here, we identified and characterized a novel interaction between SYK and the ubiquitin-specific protease 25 (USP25). We report that the second SH2 domain of SYK physically interacts with a tyrosine-rich, C-terminal region of USP25 independently of tyrosine phosphorylation. Moreover, we showed that SYK specifically phosphorylates USP25 and alters its cellular levels. This study thus uncovers a new SYK substrate and reveals a novel SYK function, namely the regulation of USP25 cellular levels.     

Integrated single‐cell analysis shows Pichia pastoris secretes protein stochastically

The production of heterologous proteins by secretion from cellular hosts is an important determinant for the cost of biotherapeutics. A single‐cell analytical method called microengraving was used to examine the heterogeneity in secretion by the methylotrophic yeast Pichia pastoris. We show that constitutive secretion of a human Fc fragment by P. pastoris is not cell‐cycle dependent, but rather fluctuates between states of high and low productivity in a stochastic manner. Biotechnol. Bioeng. 2010;106: 319–325. © 2010 Wiley Periodicals, Inc.     

Flow cytometry‐based methods for assessing soluble scFv activities and detecting antigens in solution

Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast‐display libraries. Yeast‐display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from non‐immune libraries is the conversion of highly active yeast‐displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast‐display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeast‐displayed and ‐secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of their ability to competitively inhibit the binding of biotinylated antigen to yeast‐displayed scFv. The second is an epitope binning assay that uses secreted scFv to identify additional yeast‐displayed scFv that bind non‐overlapping or non‐competing epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast‐displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast‐displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast‐display libraries. Biotechnol. Bioeng. 2010;105: 973–981. © 2009 Wiley Periodicals, Inc.