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Xindi Wang, Ruoming Xu, Yichen Wang, Ziyue Liu, Ronghui Lou,
Published: 12 July 2021
Yeast; doi:10.1002/yea.3660

Abstract:
The PCR-based gene targeting method, which can delete a specific gene or introduce tags, has been widely utilized to study gene function in fission yeast. One of the critical steps in this method is to design primers for amplifying DNA fragments of deletion or tagging modules and for checking the integration of those DNA fragments at designated loci. Although the primer design tool PPPP is available for Schizosaccharomyces pombe, there is no such publicly available application for the other three fission yeast species, S. cryophilus, S. japonicus, and S. octosporus. Likewise, no application enabling DNA/protein sequence retrieval for these three fission yeast species is available either. Therefore, access to such functionality would substantially assist in retrieval of gene sequences of interest and primer design in these fission yeast species. In this report, we describe two applications for fission yeast study: Yesprit and Yeaseq. Yesprit is a primer design tool for strain construction using the PCR-based method, and Yeaseq is a sequence viewer that can acquire the DNA/protein sequences of specific genes. Both tools can be run on the Windows, macOS, and Linux platforms. We believe that the Yesprit and Yeaseq will facilitate research using the four fission yeast species.
Qingguo Guo, Na Meng, Guanzhi Fan, Dong Sun, Yuan Meng, ,
Published: 11 July 2021
Yeast; doi:10.1002/yea.3659

Abstract:
The cell wall is a dynamic organelle which is tightly controlled for cell morphology, viability, and pathogenesis. It was previously shown that exocytosis is involved in the secretion of some components and enzymes of the cell wall. However, how the secretory pathway affects the cell wall integrity and assembly remains unclear. Here we show that the secretory pathway mutant (sec) cells were sensitive to cell wall antagonists in Saccharomyces cerevisiae, and they were lysed at restrictive conditions but can be rescued by osmotic stabilizers, indicating their cell walls were disrupted. Although glucans were reduced at the cell surface in sec mutants as speculated, the other two main cell wall components, chitins and mannoproteins, were accumulated at the cell surface. We also found that both the protein level and the phosphorylation level of Slt2 increased in sec mutants. These results suggest that the exocytic pathway has a critical role in cell wall assembly. Our study will help to understand the mechanism of cell wall formation.
Published: 1 July 2021
Yeast, Volume 38, pp 438-438; doi:10.1002/yea.3487

Published: 1 July 2021
Yeast, Volume 38, pp 389-390; doi:10.1002/yea.3488

Published: 1 July 2021
Yeast, Volume 38; doi:10.1002/yea.3656

Abstract:
Andy Warhol-like portrait of budding yeasts by Jacob L. Steenwyk (https://jlsteenwyk.com/arts.html). For further details, readers are referred to the article by Steenwyk on page 55 of issue 1.
Rashmi Karki, Swechha Rimal,
Published: 28 June 2021
Yeast; doi:10.1002/yea.3657

Abstract:
N-linked glycosylation is one type of posttranslational modification that proteins undergo during expression. The following describes the effects of N-linked glycosylation on high-level membrane protein expression in yeast with an emphasis on Saccharomyces cerevisiae. N-linked glycosylation is highlighted here as an important consideration when preparing membrane protein gene constructs for expression in S. cerevisiae, which continues to be used as a workhorse in both research and industrial applications. Non-native N-linked glycosylation commonly occurs during the heterologous expression of mammalian proteins in many yeast species which can have important immunological consequences when used in the production of biotherapeutic proteins or peptides. Further, non-native N-linked glycosylation can lead to improper protein folding and premature degradation, which can impede high-level expression yields and hinder downstream analysis. Multiple strategies are presented in this article, which suggest different methods that can be implemented to circumvent the unwanted consequences of N-linked glycosylation during the expression process. These considerations may have long-term benefits for high-level protein production in S. cerevisiae across a broad spectrum of expression targets with special emphasis placed on G-protein coupled receptors, one of the largest families of membrane proteins.
Eugenio Meza, Ana Joyce Muñoz‐Arellano, Magnus Johansson, , Dina Petranovic
Published: 28 June 2021
Yeast; doi:10.1002/yea.3658

Abstract:
All living cells, including yeast cells, are challenged by different types of stresses in their environments and must cope with challenges such as heat, chemical stress, or oxidative damage. By reversibly adjusting the physiology while maintaining structural and genetic integrity, cells can achieve a competitive advantage and adapt environmental fluctuations. The yeast Saccharomyces cerevisiae has been extensively used as a model for study of stress responses due to the strong conservation of many essential cellular processes between yeast and human cells. We focused here on developing a tool to detect and quantify early responses using specific transcriptional responses. We analyzed the published transcriptional data on S. cerevisiae DBY strain responses to 10 different stresses in different time points. The principal component analysis (PCA) and the Pearson analysis were used to assess the stress response genes that are highly expressed in each individual stress condition. Except for these stress response genes, we also identified the reference genes in each stress condition, which would not be induced under stress condition and show stable transcriptional expression over time. We then tested our candidates experimentally in the CEN.PK strain. After data analysis, we identified two stress response genes (UBI4 and RRP) and two reference genes (MEX67 and SSY1) under heat shock (HS) condition. These genes were further verified by real-time PCR at mild (42°C), severe (46°C), to lethal temperature (50°C), respectively.
Pengli Ma, Shigeo Takashima, Chikako Fujita, Saya Yamada, Yusuke Oshima, Hao‐Liang Cai, , Yasuyoshi Sakai, Takashi Hayakawa, Masaya Shimada, et al.
Published: 5 June 2021
Yeast; doi:10.1002/yea.3655

Abstract:
In this study, we analyzed the intracellular fatty acid profiles of Komagataella phaffii during methylotrophic growth. K. phaffii grown on methanol had significantly lower total fatty acid contents in the cells compared with glucose-grown cells. C18 and C16 fatty acids were the predominant fatty acids in K. phaffii, although the contents of odd-chain fatty acids such as C17 fatty acids were also relatively high. Moreover, the intracellular fatty acid composition of K. phaffii changed in response to not only carbon sources but also methanol concentrations: C17 fatty acids and C18:2 content increased significantly as methanol concentration increased, while C18:1 and C18:3 contents were significantly lower in methanol-grown cells. The intracellular content of unidentified compounds (CnH2nO4), on the other hand, was significantly greater in cells grown on methanol. As the intracellular contents of these CnH2nO4 compounds were significantly higher in a gene-disrupted strain for glutathione peroxidase (gpx1Δ) than in the wild-type strain, we presume that the CnH2nO4 compounds are fatty acid peroxides. These results indicate that K. phaffii can coordinate intracellular fatty acid composition during methylotrophic growth in order to adapt to high-methanol conditions, and that certain fatty acid species such as C17:0, C17:1, C17:2 and C18:2 may be related to the physiological functions by which K. phaffii adapts to high-methanol conditions.
Published: 1 June 2021
Yeast, Volume 38, pp 388-388; doi:10.1002/yea.3485

Published: 1 June 2021
Yeast, Volume 38, pp 337-338; doi:10.1002/yea.3486

Published: 1 June 2021
Yeast, Volume 38; doi:10.1002/yea.3654

Abstract:
Andy Warhol-like portrait of budding yeasts by Jacob L. Steenwyk (https://jlsteenwyk.com/arts.html). For further details, readers are referred to the article by Steenwyk on page 55 of issue 1.
Sabnam Parbin, Subha Damodharan,
Published: 28 May 2021
Yeast; doi:10.1002/yea.3653

Abstract:
Posttranslational modifications play a crucial role in regulating gene expression. Among these modifications, arginine methylation has recently attracted tremendous attention due to its role in multiple cellular functions. This review discusses the recent advances that have established arginine methylation as a major player in determining cytoplasmic messenger RNA (mRNA) fate. We specifically focus on research that implicates arginine methylation in regulating mRNA translation, decay, and RNA granule dynamics. Based on this research, we highlight a few emerging future avenues that will lead to exciting discoveries in this field.
Xiaobing Li, Emmanuelle Cordat, Manfred J. Schmitt,
Published: 25 May 2021
Yeast; doi:10.1002/yea.3652

Abstract:
Human kidney anion exchanger 1 (kAE1) facilitates simultaneous efflux of bicarbonate and absorption of chloride at the basolateral membrane of α-intercalated cells. In these cells, kAE1 contributes to systemic acid–base balance along with the proton pump v-H+-ATPase and the cytosolic carbonic anhydrase II. Recent electron microscopy analyses in yeast demonstrate that heterologous expression of several kAE1 variants causes a massive accumulation of the anion transporter in intracellular membrane structures. Here, we examined the origin of these kAE1 aggregations in more detail. Using various biochemical techniques and advanced light and electron microscopy, we showed that accumulation of kAE1 mainly occurs in endoplasmic reticulum (ER) membranes which eventually leads to strong unfolded protein response (UPR) activation and severe growth defect in kAE1 expressing yeast cells. Furthermore, our data indicate that UPR activation is dose dependent and uncoupled from the bicarbonate transport activity. By using truncated kAE1 variants, we identified the C-terminal region of kAE1 as crucial factor for the increased ER stress level. Finally, a redistribution of ER-localized kAE1 to the cell periphery was achieved by boosting the ER folding capacity. Our findings not only demonstrate a promising strategy for preventing intracellular kAE1 accumulation and improving kAE1 plasma membrane targeting but also highlight the versatility of yeast as model to investigate kAE1-related research questions including the analysis of structural features, protein degradation and trafficking. Furthermore, our approach might be a promising strategy for future analyses to further optimize the cell surface targeting of other disease-related PM proteins, not only in yeast but also in mammalian cells.
, Maurizio Bettiga
Published: 17 May 2021
Yeast, Volume 38, pp 391-400; doi:10.1002/yea.3651

Abstract:
Acetic acid stress represents a frequent challenge to counteract for yeast cells under several environmental conditions and industrial bioprocesses. The molecular mechanisms underlying its response have been mostly elucidated in the budding yeast Saccharomyces cerevisiae, where acetic acid can be either a physiological substrate or a stressor. This review will focus on acetic acid stress and its response in the context of cellular transport, pH homeostasis, metabolism and stress-signalling pathways. This information has been integrated with the results obtained by multi-omics, synthetic biology and metabolic engineering approaches aimed to identify major cellular players involved in acetic acid tolerance. In the production of biofuels and renewable chemicals from lignocellulosic biomass, the improvement of acetic acid tolerance is a key factor. In this view, how this knowledge could be used to contribute to the development and competitiveness of yeast cell factories for sustainable applications will be also discussed.
Published: 12 May 2021
Yeast, Volume 38, pp 339-351; doi:10.1002/yea.3650

Abstract:
Much like other living organisms, yeast cells have a limited lifespan, both in terms of the maximal length of time a cell can stay alive (chronological lifespan) as well as the maximal number of cell divisions it can undergo (replicative lifespan). Over the past years, intensive research revealed that the lifespan of yeast depends both on the genetic background of the cells as well as on environmental factors. Specifically, the presence of stress factors, reactive oxygen species and the availability of nutrients profoundly impact lifespan, and signaling cascades involved in the response to these factors, including the TOR and cAMP/PKA pathways, play a central role. Interestingly, yeast lifespan also has direct implications for its use in industrial processes. In beer brewing, for example, the inoculation of finished beer with live yeast cells, a process called “bottle conditioning” helps improve the product’s shelf life by clearing undesirable carbonyl compounds such as furfural and 2‐methylpropanal that cause staling. However, this effect depends on the reductive metabolism of living cells and is thus inherently limited by the cells’ chronological lifespan. Here, we review the mechanisms underlying chronological lifespan in yeast. We also discuss how this insight connects to industrial observations and ultimately opens new routes towards superior industrial yeasts that can help improve a product’s shelf life, and thus contribute to a more sustainable industry.
Angelo Wong, Ernest Moses Lam, Cheryl Pai, Annika Gunderson, Tamar E. Carter,
Published: 6 May 2021
Yeast; doi:10.1002/yea.3565

Abstract:
Regulation of mRNA steady state levels is important in controlling gene expression particularly in response to environmental stimuli. This allows cells to rapidly respond to environment changes. The highly conserved nonsense‐mediated mRNA decay (NMD) pathway was initially identified as a pathway that degrades aberrant mRNAs. NMD is now recognized as a pathway with additional functions including precisely regulating the expression of select natural mRNAs. Majority of these natural mRNAs encode fully functional proteins. Regulation of natural mRNAs by NMD is activated by NMD targeting features and environmental cues. Here, we show that Saccharomyces cerevisiae strains from three genetic backgrounds respond differentially to NMD depending on the environmental stimuli. We found that wild type and NMD mutant W303a, BY4741 and RM11‐1a yeast strains respond similarly to copper in the environment but respond differentially to toxic cadmium. Furthermore, the PCA1 alleles encoding different mRNAs from W303a and RM11‐1a strains are regulated similarly by NMD in response to the bio‐metal copper but differentially in response to toxic cadmium.
Published: 2 May 2021
Yeast, Volume 38; doi:10.1002/yea.3563

Abstract:
Andy Warhol‐like portrait of budding yeasts by Jacob L. Steenwyk (https://jlsteenwyk.com/arts.html). For further details, readers are referred to the article by Steenwyk on page 55 of issue 1.
Published: 2 May 2021
Yeast, Volume 38, pp 293-294; doi:10.1002/yea.3484

Published: 2 May 2021
Yeast, Volume 38, pp 336-336; doi:10.1002/yea.3483

Ellie Gibbs, Justine Hsu, Kathryn Barth,
Published: 28 April 2021
Yeast; doi:10.1002/yea.3564

Abstract:
Variations in cell wall composition and biomechanical properties can contribute to the cellular plasticity required during complex processes such as polarized growth and elongation in microbial cells. This study utilizes atomic force microscopy (AFM) to map the cell surface topography of fission yeast, Schizosaccharomyces pombe, at the pole regions and to characterize the biophysical properties within these regions under physiological, hydrated conditions. High‐resolution images acquired from AFM topographic scanning reveal decreased surface roughness at the cell poles. Force extension curves acquired by nanoindentation probing with AFM cantilever tips under low applied force revealed increased cell wall deformation and decreased cellular stiffness (cellular spring constant) at cell poles (17 ± 4 mN/m) relative to the main body of the cell that is not undergoing growth and expansion (44 ± 10 mN/m). These findings suggest that the increased deformation and decreased stiffness at regions of polarized growth at fission yeast cell poles provide the plasticity necessary for cellular extension. This study provides a direct biophysical characterization of the S. pombe cell surface by AFM, and it provides a foundation for future investigation of how the surface topography and local nanomechanical properties vary during different cellular processes.
Hanna Viktória Rácz, Fezan Mukhtar, Alexandra Imre, , , Tamás Rátonyi, János Nagy, ,
Published: 12 April 2021
Yeast; doi:10.1002/yea.3562

Abstract:
Populations of microbes are constantly evolving heterogeneity that selection acts upon, yet heterogeneity is non‐trivial to assess methodologically. The necessary practice of isolating single cell colonies and thus, subclone lineages for establishing, transferring, and using a strain results in single‐cell bottlenecks with a generally neglected effect on the characteristics of the strain itself. Here, we present evidence that various subclone lineages for industrial yeasts sequenced for recent genomic studies show considerable differences, ranging from loss‐of‐heterozygosity to aneuploidies. Subsequently, we assessed whether phenotypic heterogeneity is also observable in industrial yeast, by individually testing subclone lineages obtained from products. Phenotyping of industrial yeast samples and their newly isolated subclones showed that single‐cell bottlenecks during isolation can indeed considerably influence the observable phenotype. Next, we decoupled fitness distributions on the level of individual cells from clonal interference by plating single cell colonies and quantifying colony area distributions. We describe and apply an approach using statistical modeling to compare the heterogeneity in phenotypes accross samples and subclone lineages. One strain was further used to show how individual subclonal lineages are remarkably different not just in phenotype, but also in the level of heterogeneity in phenotype. With these observations, we call attention to the fact that choosing an initial clonal lineage from an industrial yeast strain may vastly influence downstream performances and observations on karyotype, phenotype, and also on heterogeneity.
Published: 4 April 2021
Yeast, Volume 38, pp 290-290; doi:10.1002/yea.3481

Published: 4 April 2021
Yeast, Volume 38; doi:10.1002/yea.3560

Abstract:
Andy Warhol‐like portrait of budding yeasts by Jacob L. Steenwyk (https://jlsteenwyk.com/arts.html). For further details, readers are referred to the article by Steenwyk on page 55 of issue 1.
Published: 4 April 2021
Yeast, Volume 38, pp 241-242; doi:10.1002/yea.3482

, Katsuaki Kuroki
Published: 2 April 2021
Yeast; doi:10.1002/yea.3561

Abstract:
Zygosaccharomyces sp. is an industrially important yeast for the production traditional fermented foods in Japan. At present, however, there is no easy method for mating Zygosaccharomyces sp. strains in the laboratory; furthermore, little is known about the expression of mating‐type‐specific genes in this yeast. Here, mating was observed when Zygosaccharomyces sp. was subjected to nitrogen‐starvation conditions. The expression of mating‐type‐specific genes, Zygo STE6 and Zygo MFα1, was induced under nitrogen‐starvation conditions, as confirmed by lacZ reporter assay. This expression was mating‐type‐specific: Zygo STE6 was expressed specifically for mating‐type a, whereas and Zygo MFα1 was expressed specifically for mating‐type α. Yeast strains Z. rouxii DL2 and DA2, derived from type strain Z. rouxii CBS732, did not show mating even under nitrogen‐starvation conditions. Gene sequencing revealed that the Zygo STE12 in Z. rouxii CBS732 has a frameshift mutation. Under nitrogen starvation, mating was observed in both DL2 and DA2 transformed with the wild‐type Zygo STE12. The expression of Zygo STE6 in Z. rouxii DL2 transformed with wild‐type Zygo STE12 under nitrogen‐starvation conditions was confirmed by lacZ reporter assay. Collectively, these results revealed that, under nitrogen‐starvation conditions, Zygosaccharomyces sp. can mate and mating‐type‐specific genes are expressed. Furthermore, Zygo Ste12 is essential for both mating and the expression of mating‐type‐specific genes in Zygosaccharomyces sp.
Published: 9 March 2021
Yeast, Volume 38, pp 239-239; doi:10.1002/yea.3479

Published: 9 March 2021
Yeast, Volume 38, pp 185-186; doi:10.1002/yea.3480

Published: 9 March 2021
Yeast, Volume 38; doi:10.1002/yea.3557

Abstract:
Andy Warhol‐like portrait of budding yeasts by Jacob L. Steenwyk (https://jlsteenwyk.com/arts.html). For further details, readers are referred to the article by Steenwyk on page 55 of issue 1.
Yuki Yoshikawa, Ryo Nasuno,
Published: 1 March 2021
Yeast, Volume 38, pp 414-423; doi:10.1002/yea.3558

Abstract:
The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), which is required for various redox systems involving antioxidative stress enzymes, is thus important for stress tolerance mechanisms. Here, we analyzed the stress response of the NADPH‐depleted cells of Saccharomyces cerevisiae. A cell viability assay showed that the NADPH depletion induced by disruption of the ZWF1 gene encoding glucose‐6‐phosphate dehydrogenase, which is the major determinant of the intracellular NADPH/NADP+ ratio, enhanced the tolerance of S. cerevisiae to both oxidative and nitrosative stresses. The subsequent analyses demonstrated that the antioxidative transcriptional factor Yap1 was activated and the cytosolic catalase Ctt1, whose expression is regulated by Yap1, was upregulated in zwf1Δ cells irrespective of the presence or absence of stress stimuli. Moreover, deletion of the YAP1 or CTT1 gene inhibited the increased stress tolerance of zwf1Δ cells, indicating that Ctt1 dominantly contributed to the higher stress tolerance of zwf1Δ cells. Our findings suggest that an NADPH‐independent mechanism enhances oxidative and nitrosative stress tolerance in ZWF1‐lacking yeast cells.
Kalaivani Paramasivan, Aneesha A, Nabarupa Gupta,
Published: 1 March 2021
Yeast, Volume 38, pp 424-437; doi:10.1002/yea.3559

Abstract:
In the present study, the adaptive evolution of a metabolically engineered Saccharomyces cerevisiae strain in the presence of an enzyme inhibitor terbinafine for enhanced squalene accumulation via serial transfer leads to the development of robust strains. After adaptation for nearly 1500 h, a strain with higher squalene production efficiency was identified at a specific growth rate of 0.28 h−1 with a final squalene titer of 193 mg/L, which is 16.5‐fold higher than the BY4741 and 3‐fold higher over the metabolically engineered SK22 strain. Whole‐genome sequencing comparison between the reference strain and the evolved variant SK23 has led to the identification of 462 single‐nucleotide variants (SNVs) between both strains, with 102 SNVs affecting metabolism‐related genes. It was also established that F420I mutation of ERG1 in S. cerevisiae improves squalene synthesis. Further, the effect of increased squalene on lipid droplet and neutral lipid pattern in the evolved mutant strains was investigated by fluorescent techniques proving that the neutral lipid content and clustering of lipid droplets increase with an increase in squalene.
Published: 23 February 2021
Yeast, Volume 38, pp 183-183; doi:10.1002/yea.3477

Published: 23 February 2021
Yeast, Volume 38, pp 129-130; doi:10.1002/yea.3478

Published: 23 February 2021
Yeast, Volume 38; doi:10.1002/yea.3555

Published: 19 February 2021
Yeast, Volume 38, pp 401-413; doi:10.1002/yea.3556

Abstract:
Unicellular organisms, like yeast, have developed mechanisms to overcome environmental stress conditions like nutrient starvation. Autophagy and sporulation are two such mechanisms employed by yeast cells. Autophagy is a well‐conserved, catabolic process that degrades excess and unwanted cytoplasmic materials and provides building blocks during starvation conditions. Thus, autophagy maintains cellular homeostasis at basal conditions and acts as a survival mechanism during stress conditions. Sporulation is an essential process that, like autophagy, is triggered due to stress conditions in yeast. It involves the formation of ascospores that protect the yeast cells during extreme conditions and germinate when the conditions are favorable. Studies show that autophagy is required for the sporulation process in yeast. However, the exact mechanism of action is not clear. Furthermore, several of the core autophagy gene knockouts do not sporulate and at what stage of sporulation they are involved is not clear. Besides, many overlapping proteins function in both sporulation and autophagy and it is unclear how the pathway‐specific roles of these proteins are determined. All these observations suggest that the two processes cross‐talk. Individually, some key features from both the processes remain to be studied with respect to the source of membrane for autophagosomes, prospore membrane (PSM) formation, and closure of the membranes. Therefore, it becomes crucial to study the cross‐talk between autophagy and sporulation. In this review, the cross‐talk between the two pathways, the common protein machineries have been discussed.
, David R. Olsen
Published: 12 February 2021
Yeast, Volume 38, pp 382-387; doi:10.1002/yea.3554

Abstract:
The methylotrophic yeast Pichia pastoris (reclassified as Komagataella phaffii) is a versatile protein expression system, yet many commonly used promoters have attributes undesirable for fermentation or its optimization. Hence, the copper‐inducible CUP1 gene promoter from the related yeast Saccharomyces cerevisiae was used to express human gelatin. Multimerization of a potential copper response element in the CUP1 promoter, a S. cerevisiae Ace1p binding site, significantly increased gelatin expression. Expression was induced by copper in a dose‐dependent fashion and was not dependent on cell density. Gelatin was additionally induced in standard copper‐containing fermentation basal salts media. Removal of a S. cerevisiae heat shock factor (Hsf1p) binding site reduced copper‐dependent gelatin induction suggesting that a similar protein may regulate this promoter in P. pastoris. This engineered copper inducible promoter expands the yeast recombinant protein production tool kit.
, Jin Zhang, , Andrea Lee, Anne Houlès, ,
Published: 9 February 2021
Yeast, Volume 38, pp 367-381; doi:10.1002/yea.3553

Abstract:
Hydrogen sulfide is a common wine fault, with a rotten‐egg odour, which is directly related to yeast metabolism in response to nitrogen and sulfur availability. In grape juice, sulfate is the most abundant inorganic sulfur compound, which is taken up by yeast through two high‐affinity sulfate transporters, Sul1p and Sul2p and a low affinity transporter, Soa1p. Sulfate contributes to H2S production under nitrogen limitation, by being reduced via the Sulfur Assimilation Pathway (SAP). Therefore, yeast strains with limited H2S are highly desirable. We report on the use of toxic analogs of sulfate following ethyl methane sulfate treatment, to isolate six wine yeast mutants that produce no or reduced H2S and SO2 during fermentation in synthetic and natural juice. Four amino acid substitutions (A99V, G380R, N588K, E856K) in Sul1p were found in all strains except D25‐1 which had heterozygous alleles. Two changes were also identified in Sul2p (L268S and A470T). The Sul1p (G380R) and Sul2p (A470T) mutations were chosen for further investigation as these residues are conserved amongst SLC26 membrane proteins (including sulfate permeases). The mutations were introduced into EC1118 using Crispr cas9 technology, and shown to reduce accumulation of H2S and not result in increased SO2 production during fermentation of model medium (chemically defined grape juice) or Riesling juice. The Sul1p (G380R) and Sul2p (A470T) mutations are newly reported as causal mutations. Our findings contribute to knowledge of the genetic basis of H2S production as well as the potential use of these strains for winemaking and in yeast breeding programs.
Kunalika Jain, Neha Khetan, Shivani A. Yadav, Saravanan Palani,
Published: 6 February 2021
Yeast, Volume 38, pp 352-366; doi:10.1002/yea.3552

Abstract:
Positioning the nucleus at the bud‐neck during Saccharomyces cerevisiae mitosis involves pulling forces of cytoplasmic dynein localized in the daughter cell. While genetic analysis has revealed a complex network positioning the nucleus, quantification of the forces acting on the nucleus and the number of dyneins driving the process has remained difficult. To better understand the collective forces involved in nuclear positioning, we compare a model of dyneins driven microtubule (MT) pulling, MT pushing and cytoplasmic drag to experiments. During S. cerevisiae mitosis, MTs interacting with the cortex nucleated by the daughter SPB (SPB‐D) are longer than the mother SPB (SPB‐M), increasing further during spindle elongation in anaphase. Interphasic SPB mobility is effectively diffusive, while the mitotic mobility is directed. By optimizing a computational model of the mobility of the nucleus due to diffusion and MTs pushing at the cell membrane to experiment, we estimate the viscosity governing the drag force on nuclei during positioning. A force‐balance model of mitotic SPB mobility compared to experimental mobility, suggests even one or two dynein dimers are sufficient to move the nucleus in the bud‐neck. Using stochastic computer simulations of a budding cell, we find punctate dynein localization can generate sufficient force to reel in the nucleus to the bud‐neck. Compared to uniform motor localization, puncta involve fewer motors suggesting a functional role for motor cluster‐ ing. Stochastic simulations also suggest a higher number of force generators than predicted by force‐balance may be required to ensure the robustness of spindle positioning.
Laxmi Shanker Rai, Lasse Van Wijlick, Marie‐Elisabeth Bougnoux, ,
Published: 3 February 2021
Yeast, Volume 38, pp 243-250; doi:10.1002/yea.3550

Abstract:
The yeast Candida albicans is primarily a commensal of humans that colonizes the mucosal surfaces of the gastrointestinal and genital tracts. Yet, C. albicans can under certain circumstances undergo a shift from commensalism to pathogenicity. This transition is governed by fungal factors such as morphological transitions, environmental cues for instance relationships with gut microbiota and the host immune system. C. albicans utilizes distinct sets of regulatory programs to colonize or infect its host and to evade the host defense systems. Moreover, an orchestrated iron acquisition mechanism operates to adapt to specific niches with variable iron availability. Studies on regulatory networks and morphogenesis of these two distinct modes of C. albicans growth, suggest that both yeast and hyphal forms exist in both growth patterns and the regulatory circuits are inter‐connected. Here, we summarize current knowledge about C. albicans commensal‐to‐pathogen shift, its regulatory elements and their contribution to human disease.
Kousuke Toyomura, Taisuke Hisatomi
Published: 14 January 2021
Yeast, Volume 38, pp 326-335; doi:10.1002/yea.3549

Abstract:
We have previously isolated heterothallic haploid strains from original homothallic diploids of two yeast species in the family Saccharomycetaceae. In this study, heterothallic haploid strains were isolated from an original homothallic diploid of Saccharomyces kudriavzevii type strain, followed by investigation of sexual interactions among these yeast strains, in addition to S. cerevisiae laboratory strains. It has been shown that prezygotic reproductive isolation was observed between Kazachstania naganishii and S. cerevisiae with α‐factor mating pheromones representing crossaction with each other beyond the genus boundary. Using heterothallic strains, postzygotic reproductive isolation system was shown to reside in the genus Saccharomyces by mass mating and cell‐cell contact experiments. In mass mating experiments, crossaction of α‐factor and a‐factor mating pheromones and sexual agglutination effectively occurred beyond species boundaries among S. kudriavzevii, S. paradoxus, and S. cerevisiae. When the fates of cell‐cell pairs from these Saccharomyces yeast species were systematically chased one by one, interspecific F1 hybrids were effectively produced, while sporulations were partially prohibited, with spore germination perfectly blocked in the hybrids. These results indicated that postzygotic reproductive isolation definitively resides among these Saccharomyces yeast species and that disorder of chromosome organization had to some extent occurred in interspecific F1 hybrids.
Chitwadee Phithakrotchanakoon, Aekkachai Puseenam, Worarat Kruasuwan, Somsak Likhitrattanapisal, Narumon Phaonakrop, Sittiruk Roytrakul, Supawadee Ingsriswang, Sutipa Tanapongpipat,
Published: 14 January 2021
Yeast, Volume 38, pp 316-325; doi:10.1002/yea.3548

Abstract:
The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein production. However, overproduction of heterologous protein can induce cellular stress and limit the level of its secretion. To improve the secretion of heterologous protein, we identified the candidate proteins with altered production during production of heterologous protein in O. thermomethanolica by using a label‐free comparative proteomic approach. Four hundred sixty‐four proteins with various biological functions showed differential abundance between O. thermomethanolica expressing fungal xylanase (OT + Xyl) and a control strain. The induction of proteins in transport and proteasomal proteolysis was prominently observed. Eight candidate proteins involved in cell wall biosynthesis (Chs3, Gas4), chaperone (Sgt2, Pex19), glycan metabolism (Csf1), protein transport (Ypt35), and vacuole and protein sorting (Cof1, Npr2) were mutated by a CRISPR/Cas9 approach. An Sgt2 mutant showed higher phytase and xylanase activity compared with the control strain (13%–20%), whereas mutants of other genes including Cof1, Pex19, Gas4, and Ypt35 showed lower xylanase activity compared with the control strain (15%–25%). In addition, an Npr2 mutant showed defective growth, while overproduction of Npr2 enhanced xylanase activity. These results reveal genes that can be mutated to modulate heterologous protein production and growth of O. thermomethanolica TBRC656.
Published: 1 January 2021
Yeast, Volume 38, pp 1-2; doi:10.1002/yea.3476

Jacob L. Steenwyk
Published: 1 January 2021
Yeast, Volume 38; doi:10.1002/yea.3551

Abstract:
Andy Warhol‐like portrait of budding yeasts by Jacob L. Steenwyk (https://jlsteenwyk.com/arts.html). For further details, readers are referred to the article by Steenwyk on page 55 of this issue.
K.T. Nishant, Dominika Wloch‐Salamon, Kenneth H. Wolfe,
Published: 1 January 2021
Yeast, Volume 38, pp 3-4; doi:10.1002/yea.3547

Abstract:
Click on the article title to read more.
Published: 1 January 2021
Yeast, Volume 38, pp 127-127; doi:10.1002/yea.3475

Siyu Sun,
Published: 22 December 2020
Yeast, Volume 38, pp 12-29; doi:10.1002/yea.3545

Abstract:
Cellular quiescence, the temporary and reversible exit from proliferative growth, is the predominant state of all cells. However, our understanding of the biological processes and molecular mechanisms that underlie cell quiescence remains incomplete. As with the mitotic cell cycle, budding and fission yeast are preeminent model systems for studying cellular quiescence owing to their rich experimental toolboxes and the evolutionary conservation across eukaryotes of pathways and processes that control quiescence. Here, we review current knowledge of cell quiescence in budding yeast and how it pertains to cellular quiescence in other organisms, including multicellular animals. Quiescence entails large-scale remodeling of virtually every cellular process, organelle, gene expression, and metabolic state that is executed dynamically as cells undergo the initiation, maintenance, and exit from quiescence. We review these major transitions, our current understanding of their molecular bases and highlight unresolved questions. We summarize the primary methods employed for quiescence studies in yeast and discuss their relative merits. Understanding cell quiescence has important consequences for human disease as quiescent single-celled microbes are notoriously difficult to kill and quiescent human cells play important roles in diseases such as cancer. We argue that research on cellular quiescence will be accelerated through the adoption of common criteria, and methods, for defining cell quiescence. An integrated approach to studying cell quiescence, and a focus on the behavior of individual cells, will yield new insights into the pathways and processes that underlie cell quiescence leading to a more complete understanding of the life cycle of cells.
Shawna Miles, Graham T. Bradley,
Published: 22 December 2020
Yeast, Volume 38, pp 30-38; doi:10.1002/yea.3546

Abstract:
A subset of Saccharomyces cerevisiae cells in a stationary phase culture achieve a unique quiescent state characterized by increased cell density, stress tolerance, and longevity. Trehalose accumulation is necessary but not sufficient for conferring this state, and it is not recapitulated by abrupt starvation. The fraction of cells that achieve this state varies widely in haploids and diploids and can approach 100%, indicating that both mother and daughter cells can enter quiescence. The transition begins when about half the glucose has been taken up from the medium. The high affinity glucose transporters are turned on, glycogen storage begins, the Rim15 kinase enters the nucleus and the accumulation of cells in G1 is initiated. After the diauxic shift (DS), when glucose is exhausted from the medium, growth promoting genes are repressed by the recruitment of the histone deacetylase Rpd3 by quiescence‐specific repressors. The final division that takes place post‐DS is highly asymmetrical and G1 arrest is complete after 48 h. The timing of these events can vary considerably, but they are tightly correlated with total biomass of the culture, suggesting that the transition to quiescence is tightly linked to changes in external glucose levels. After 7 days in culture, there are massive morphological changes at the protein and organelle level. There are global changes in histone modification. An extensive array of condensin‐dependent, long‐range chromatin interactions lead to genome‐wide chromatin compaction that is conserved in yeast and human cells. These interactions are required for the global transcriptional repression that occurs in quiescent yeast.
Published: 11 December 2020
Yeast, Volume 37, pp 658-658; doi:10.1002/yea.3423

Published: 11 December 2020
Yeast, Volume 37, pp 623-624; doi:10.1002/yea.3424

Clara Navarrete, August T. Frost, Laura Ramos‐Moreno, Mette R. Krum,
Published: 10 December 2020
Yeast, Volume 38, pp 302-315; doi:10.1002/yea.3544

Abstract:
Debaryomyces hansenii is traditionally described as a halotolerant non‐conventional yeast, and has served as a model organism for the study of osmo‐ and salt tolerance mechanisms in eukaryotic systems for the past 30 years. However, unravelling of D. hansenii´s biotechnological potential has always been difficult due to the persistent limitations in the availability of efficient molecular tools described for this yeast. Additionally, there is a lack of consensus and contradictory information along the recent years that limits a comprehensive understanding of its central carbon metabolism, mainly due to a lack of physiological studies in controlled and monitored environments. Moreover, there is little consistency in the culture conditions (media composition, temperature and pH among others) used by different groups, which makes it complicated when trying to get prevalent conclusions on behavioural patterns. In this work, we present for the first time a characterization of D. hansenii in batch cultivations using highly controlled lab‐scale bioreactors. Our findings contribute to a more complete picture of the central carbon metabolism and the external pH influence on the yeast’s ability to tolerate high Na+ and K+ concentrations, pointing to a differential effect of both salts, as well as a positive effect in cell performance when low environmental pH values are combined with a high sodium concentration in the media. Finally, a novel survival strategy at very high salinity (2 M) is proposed for this yeast, as well as potential outcomes for its use in industrial biotechnology applications.
Published: 8 December 2020
Yeast, Volume 38, pp 295-301; doi:10.1002/yea.3543

Abstract:
Studies have reported on the ability of green fluorescent proteins to photoconvert into a red fluorescent form under various conditions, such as the presence of oxidants, hypoxia, as well as under benign conditions using irradiation with a 405 nm laser. Here we show that in Saccharomyces cerevisiae yeast green fluorescent protein (S65T) fused to different cellular proteins can easily photoconvert into a red form when cells are grown in media with non‐fermentable carbon sources. This photoconversion occurs during standard microscopy between glass slide and coverslip, but is completely prevented by imaging on pads of solid medium or in a large volume of medium on an inverted microscope. The observed effect was due to rapid hypoxia of cells with respiratory metabolism in standard conditions for upright microscopy. Photoconversion could be prevented by antioxidant treatment, suggesting that it proceeds via the effects of reactive oxidative species emerging in response to oxygen deficiency. Our results show the need for caution during upright microscopy imaging in conditions where there is active respiration, and demonstrate simple approaches to prevent unwanted GFP photoconversion. They also provide easy means of performing photoconversion experiments on existing GFP‐bearing cell lines, at least in the case of yeast.
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