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  • 1.

    【2hr】Engineering the expression system for Komagataella phaffii (Pichia pastoris): an attempt to develop a methanol-free expression system

    包量

    机译 设计Komagataella phaffii（Pichia pastoris）的表达系统：尝试开发不含甲醇的表达系统
    摘要：
  • 2.

    【2hr】Corrigendum to: Piotr P. Slonimski – The Warrior Pope: The discovery of mitochondrial (petite) mutants and split genes

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    机译 更正于：皮奥·P·斯隆尼姆斯基（Piotr P. Slonimski）–战士教皇：线粒体（小）突变体和分裂基因的发现
    • 作者：
    • 刊名：FEMS Yeast Research
    • 2019年第4期
    摘要：
  • 3.

    【2hr】Effects of overexpression of STB5 in Saccharomyces cerevisiae on fatty acid biosynthesis, physiology and transcriptome

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    机译 酿酒酵母中STB5过表达对脂肪酸生物合成，生理和转录组的影响
    摘要：
  • 4.

    【2hr】Genetics is the logic of life (at least of mine)

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    机译 遗传学是生命的逻辑（至少是我的生命）
    摘要：I retrace my path from math to medicine to biochemistry to yeast genetics, my focus on infectious diseases of yeast and finally prions. My discovery of yeast prions relied on my particular focus on the logical relations of non-chromosomal genetic elements and the chromosomal genes involved in their propagation and expression. Pursuing an understanding of yeast prions involved structural biology based on genetics, solid-state NMR, population genetics and more genetics.
  • 5.

    【2hr】A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains

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    机译 用于快速CRISPR-SpCas9辅助构建己糖运输缺陷型酿酒酵母菌株的工具包
    摘要：Hexose transporter-deficient yeast strains are valuable testbeds for the study of sugar transport by native and heterologous transporters. In the popular Saccharomyces cerevisiae strain EBY.VW4000, deletion of 21 transporters completely abolished hexose transport. However, repeated use of the LoxP/Cre system in successive deletion rounds also resulted in major chromosomal rearrangements, gene loss and phenotypic changes. In the present study, CRISPR/SpCas9 was used to delete the 21 hexose transporters in an S. cerevisiae strain from the CEN.PK family in only three deletion rounds, using 11 unique guide RNAs. Even upon prolonged cultivation, the resulting strain IMX1812 (CRISPR-Hxt0) was unable to consume glucose, while its growth rate on maltose was the same as that of a strain equipped with a full set of hexose transporters. Karyotyping and whole-genome sequencing of the CRISPR-Hxt0 strain with Illumina and Oxford Nanopore technologies did not reveal chromosomal rearrangements or other unintended mutations besides a few SNPs. This study provides a new, ‘genetically unaltered’ hexose transporter-deficient strain and supplies a CRISPR toolkit for removing all hexose transporter genes from most S. cerevisiae laboratory strains in only three transformation rounds.
  • 6.

    【2hr】Reassessment of requirements for anaerobic xylose fermentation by engineered, non-evolved Saccharomyces cerevisiae strains

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    机译 工程化，非进化酿酒酵母菌株对厌氧木糖发酵要求的重新评估
    摘要：Expression of a heterologous xylose isomerase, deletion of the GRE3 aldose-reductase gene and overexpression of genes encoding xylulokinase (XKS1) and non-oxidative pentose-phosphate-pathway enzymes (RKI1, RPE1, TAL1, TKL1) enables aerobic growth of Saccharomyces cerevisiae on d-xylose. However, literature reports differ on whether anaerobic growth on d-xylose requires additional mutations. Here, CRISPR-Cas9-assisted reconstruction and physiological analysis confirmed an early report that this basic set of genetic modifications suffices to enable anaerobic growth on d-xylose in the CEN.PK genetic background. Strains that additionally carried overexpression cassettes for the transaldolase and transketolase paralogs NQM1 and TKL2 only exhibited anaerobic growth on d-xylose after a 7–10 day lag phase. This extended lag phase was eliminated by increasing inoculum concentrations from 0.02 to 0.2 g biomass L−1. Alternatively, a long lag phase could be prevented by sparging low-inoculum-density bioreactor cultures with a CO2/N2-mixture, thus mimicking initial CO2 concentrations in high-inoculum-density, nitrogen-sparged cultures, or by using l-aspartate instead of ammonium as nitrogen source. This study resolves apparent contradictions in the literature on the genetic interventions required for anaerobic growth of CEN.PK-derived strains on d-xylose. Additionally, it indicates the potential relevance of CO2 availability and anaplerotic carboxylation reactions for anaerobic growth of engineered S. cerevisiae strains on d-xylose.
  • 7.

    【2hr】Lager-brewing yeasts in the era of modern genetics

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    机译 现代遗传学时代的啤酒发酵酵母
    摘要：
  • 8.

    【2hr】Anaerobic growth of Saccharomyces cerevisiae CEN.PK113-7D does not depend on synthesis or supplementation of unsaturated fatty acids

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    机译 酿酒酵母CEN.PK113-7D的厌氧生长不依赖于不饱和脂肪酸的合成或补充
    摘要：
  • 9.

    【2hr】Elucidation of secondary alcohol metabolism in Starmerella bombicola and contribution of primary alcohol oxidase FAO1

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    机译 阐明星状夜蛾中的仲醇代谢和伯醇氧化酶的作用FAO1
    摘要：
  • 10.

    【2hr】PHO15 genes of Candida albicans and Candida parapsilosis encode HAD-type phosphatases dephosphorylating 2-phosphoglycolate

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    机译 白色念珠菌和副念珠菌的PHO15基因编码脱磷酸化2-磷酸乙醇酸的HAD型磷酸酶
    摘要：Most of the phosphatases of human fungal pathogens Candida albicans and C. parapsilosis have never been experimentally characterised, although dephosphorylation reactions are central to many biological processes. PHO15 genes of these yeasts have been annotated as the sequences encoding 4-nitrophenyl phosphatase, on the basis of homology to PHO13 gene from the bakers’ yeast Saccharomyces cerevisiae. To examine the real function of these potential phosphatases from Candida spp., CaPho15p and CpPho15p were prepared using expression in Escherichia coli and characterised. They share the hallmark motifs of the haloacid dehalogenase superfamily, readily hydrolyse 4-nitrophenyl phosphate at pH 8–8.3 and require divalent cations (Mg2+, Mn2+ or Co2+) as cofactors. CaPho15p and CpPho15p did not dephosphorylate phosphopeptides, but rather hydrolysed molecules related to carbohydrate metabolism. The preferred substrate for the both phosphatases was 2-phosphoglycolate. Among the other molecules tested, CaPho15 showed preference for glyceraldehyde phosphate and ß-glycerol phosphate, while CpPho15 dephosphorylated mainly 1,3-dihydroxyacetone phosphate. This type of substrate specificity indicates that CaPho15 and CpPho15 may be a part of metabolic repair system of C. albicans and C. parapsilosis.
  • 11.

    【2hr】Geotrichum candidum gene expression and metabolite accumulation inside the cells reflect the strain oxidative stress sensitivity and ability to produce flavour compounds

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    机译 念珠菌基因表达和代谢产物在细胞内的积累反映了菌株的氧化应激敏感性和产生风味化合物的能力
    摘要：Geotrichum candidum is a fungus-like yeast widely used as a starter culture for cheese ripening for its proteolytic and lipolytic activities and its contribution to the cheese flavours. The sequenced strain G. candidum CLIB 918 was isolated from cheese Pont-L’Evêque. This strain's ability to produce volatile compounds was compared to the ability of a known strong sulphur compound producer G. candidum strain (Gc203). The aminotransferase-coding genes BAT2 and ARO8 were identified to be involved in methionine catabolism. The production of volatile compounds indicated that the sequenced strain was a moderate producer compared to the strong producer strain. The major volatile compounds were produced from sulphur amino acid, branched-chain amino acid and fatty acid metabolisms. Metabolite content of the cells showed that the ability of the strain to produce volatile compounds was inversely proportional to its ability to store amino acids inside the cells. Reduced glutathione, hypotaurine and taurine intracellular concentrations and volatile fatty aldehyde production indicated the role of oxidative stress sensitivity in flavour production. The increase in expression of several genes in a Reblochon-type cheese at the end of ripening confirmed that oxygen and iron were key factors regulating cheese flavour production.
  • 12.

    【2hr】Reconstitution of human peroxisomal β-oxidation in yeast

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    机译 酵母中人过氧化物酶体β-氧化的重建
    摘要：We report the permanent introduction of the human peroxisomal β-oxidation enzymatic machinery required for straight chain degradation of fatty acids into the yeast, Saccharomyces cerevisiae. Peroxisomal β-oxidation encompasses four sequential reactions that are confined to three enzymes. The genes encoding human acyl-CoA oxidase 1, peroxisomal multifunctional enzyme type 2 and 3-ketoacyl-CoA thiolase were introduced into the genomic loci of their yeast gene equivalents. The human β-oxidation genes were individually tagged with sequence coding for GFP and expression of the protein chimeras as well as their targeting to peroxisomes was confirmed. Functional complementation of the β-oxidation pathway was assessed by growth on media containing fatty acids of different chain lengths. Yeast cells exhibited distinctive substrate specificities depending on whether they expressed the human or their endogenous β-oxidation machinery. The genetic engineering of yeast to contain a ‘humanized’ organelle is a first step for the in vivo study of human peroxisome disorders in a model organism.
  • 13.

    【2hr】A protocol for introduction of multiple genetic modifications in Saccharomyces cerevisiae using CRISPR/Cas9

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    机译 使用CRISPR / Cas9在酿酒酵母中引入多种遗传修饰的协议
    摘要：Here, two methods are described for efficient genetic modification of Saccharomyces cerevisiae using CRISPR/Cas9. The first method enables the modification of a single genetic locus using in vivo assembly of a guide RNA (gRNA) expression plasmid without the need for prior cloning. A second method using in vitro assembled plasmids that could contain up to two gRNAs was used to simultaneously introduce up to six genetic modifications (e.g. six gene deletions) in a single transformation step by transforming up to three gRNA expression plasmids simultaneously. The method is not only suitable for gene deletion but is also applicable for in vivo site-directed mutagenesis and integration of multiple DNA fragments in a single locus. In all cases, the strain transformed with the gRNA expression plasmids was equipped with a genomic integration of Spcas9, leading to strong and constitutive expression of SpCas9. The protocols detailed here have been streamlined to be executed by virtually any yeast molecular geneticist.
  • 14.

    【2hr】A brief overview of the Swi1 prion—[SWI+]

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    机译 Swi1 pr病毒的简要概述-[SWI +]
    摘要：Prion and prion-like phenomena are involved in the pathology of numerous human neurodegenerative diseases. The budding yeast, Saccharomyces cerevisiae, has a number of endogenous yeast prions—epigenetic elements that are transmitted as altered protein conformations and often manifested as heritable phenotypic traits. One such yeast prion, [SWI+], was discovered and characterized by our laboratory. The protein determinant of [SWI+], Swi1 was found to contain an amino-terminal, asparagine-rich prion domain. Normally, Swi1 functions as part of the SWI/SNF chromatin remodeling complex, thus, acting as a global transcriptional regulator. Consequently, prionization of Swi1 leads to a variety of phenotypes including poor growth on non-glucose carbon sources and abolishment of multicellular features—with implications on characterization of [SWI+] as being detrimental or beneficial to yeast. The study of [SWI+] has revealed important knowledge regarding the chaperone systems supporting prion propagation as well as prion–prion interactions with [PSI+] and [RNQ+]. Additionally, an intricate regulatory network involving [SWI+] and other prion elements governing multicellular features in yeast has begun to be revealed. In this review, we discuss the current understanding of [SWI+] in addition to some possibilities for future study.
  • 15.

    【2hr】Laboratory evolution for forced glucose-xylose co-consumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae

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    机译 强制性葡萄糖-木糖共消耗的实验室演变能够鉴定突变，这些突变可通过木糖发酵啤酒酵母来改善混合糖发酵。
    摘要：Simultaneous fermentation of glucose and xylose can contribute to improved productivity and robustness of yeast-based processes for bioethanol production from lignocellulosic hydrolysates. This study explores a novel laboratory evolution strategy for identifying mutations that contribute to simultaneous utilisation of these sugars in batch cultures of Saccharomyces cerevisiae. To force simultaneous utilisation of xylose and glucose, the genes encoding glucose-6-phosphate isomerase (PGI1) and ribulose-5-phosphate epimerase (RPE1) were deleted in a xylose-isomerase-based xylose-fermenting strain with a modified oxidative pentose-phosphate pathway. Laboratory evolution of this strain in serial batch cultures on glucose–xylose mixtures yielded mutants that rapidly co-consumed the two sugars. Whole-genome sequencing of evolved strains identified mutations in HXK2, RSP5 and GAL83, whose introduction into a non-evolved xylose-fermenting S. cerevisiae strain improved co-consumption of xylose and glucose under aerobic and anaerobic conditions. Combined deletion of HXK2 and introduction of a GAL83G673T allele yielded a strain with a 2.5-fold higher xylose and glucose co-consumption ratio than its xylose-fermenting parental strain. These two modifications decreased the time required for full sugar conversion in anaerobic bioreactor batch cultures, grown on 20 g L−1 glucose and 10 g L−1 xylose, by over 24 h. This study demonstrates that laboratory evolution and genome resequencing of microbial strains engineered for forced co-consumption is a powerful approach for studying and improving simultaneous conversion of mixed substrates.
  • 16.

    【2hr】Potentiating Hsp104 activity via phosphomimetic mutations in the middle domain

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    机译 通过中间域中的磷酸化突变增强Hsp104的活性
    摘要：Hsp104 is a hexameric AAA + ATPase and protein disaggregase found in yeast, which can be potentiated via mutations in its middle domain (MD) to counter toxic phase separation by TDP-43, FUS and α-synuclein connected to devastating neurodegenerative disorders. Subtle missense mutations in the Hsp104 MD can enhance activity, indicating that post-translational modification of specific MD residues might also potentiate Hsp104. Indeed, several serine and threonine residues throughout Hsp104 can be phosphorylated in vivo. Here, we introduce phosphomimetic aspartate or glutamate residues at these positions and assess Hsp104 activity. Remarkably, phosphomimetic T499D/E and S535D/E mutations in the MD enable Hsp104 to counter TDP-43, FUS and α-synuclein aggregation and toxicity in yeast, whereas T499A/V/I and S535A do not. Moreover, Hsp104T499E and Hsp104S535E exhibit enhanced ATPase activity and Hsp70-independent disaggregase activity in vitro. We suggest that phosphorylation of T499 or S535 may elicit enhanced Hsp104 disaggregase activity in a reversible and regulated manner.
  • 17.

    【2hr】Th17 cells differentiated with mycelial membranes of Candida albicans prevent oral candidiasis

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    机译 白色念珠菌菌丝体膜分化的Th17细胞可预防口腔念珠菌病
    摘要：Candida albicans is a human commensal that causes opportunistic infections. Th17 cells provide resistance against mucosal infection with C. albicans; however, the T cell antigens remain little known. Our final goal is to find effective T cell antigens of C. albicans that are responsible for immunotherapy against candidiasis. Here, we prepared fractions including cytosol, membrane and cell wall from yeast and mycelial cells. Proteins derived from a membrane fraction of mycelial cells effectively induced differentiation of CD4+ T cells into IL-17A-producing Th17 cells. To confirm the immunological response in vivo of proteins from mycelial membrane, we performed adoptive transfer experiments using ex vivo stimulated CD4+ T cells from IL-17A-GFP reporter mice. Mycelial membrane-differentiated CD4+ Th17 cells adoptively transferred intravenously prevented oral candidiasis by oral infection of C. albicans, compared with control anti-CD3-stimulated CD4+ T cells. This was confirmed by the clinical score and the number of neutrophils on the infected tissues. These data suggest that effective T cell antigens against candidiasis could be present in the membrane protein fraction of mycelial cells. The design of novel vaccination strategies against candidiasis will be our next step.
  • 18.

    【2hr】Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid

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    机译 使用宽宿主范围Cas9 / gRNA共表达质粒编辑克鲁维酵母和绪形酵母中的基因组
    摘要：While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade− mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.
  • 19.

    【2hr】Whi2 signals low leucine availability to halt yeast growth and cell death

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    机译 Whi2信号表明亮氨酸利用率低，以阻止酵母生长和细胞死亡
    摘要：Cells are exquisitely tuned to environmental ques. Amino acid availability is rapidly sensed, allowing cells to adjust molecular processes and implement short or long-term metabolic shifts accordingly. How levels of most individual amino acids may be sensed and subsequently signaled to inform cells of their nutrient status is largely unknown. We made the unexpected observation that small changes in the levels of specific amino acids can have a profound effect on yeast cell growth, leading to the identification of yeast Whi2 as a negative regulator of cell growth in low amino acids. Although Whi2 was originally thought to be fungi-specific, Whi2 appears to share a conserved structural domain found in a family of 25 largely uncharacterized human genes encoding the KCTD (potassium channel tetramerization domain) protein family. Insights gained from yeast Whi2 are likely to be revealing about human KCTDs, many of which have been implicated or demonstrated to cause disease when mutated. Here we report new evidence that Whi2 responds to specific amino acids in the medium, particularly low leucine levels. We also discuss the known pathways of amino acid signaling and potential points of regulation by Whi2 in nutrient signaling in yeast and mammals.
  • 20.

    【2hr】Characterization of the APSES-family transcriptional regulators of Histoplasma capsulatum

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    机译 荚膜组织胞浆菌APSES家族转录调节子的表征
    摘要：The fungal APSES protein family of transcription factors is characterized by a conserved DNA-binding motif facilitating regulation of gene expression in fungal development and other biological processes. However, their functions in the thermally dimorphic fungal pathogen Histoplasma capsulatum are unexplored. Histoplasma capsulatum switches between avirulent hyphae in the environment and virulent yeasts in mammalian hosts. We identified five APSES domain-containing proteins in H. capsulatum homologous to Swi6, Mbp1, Stu1 and Xbp1 proteins and one protein found in related Ascomycetes (APSES-family protein 1; Afp1). Through transcriptional analyses and RNA interference-based functional tests we explored their roles in fungal biology and virulence. Mbp1 serves an essential role and Swi6 contributes to full yeast cell growth. Stu1 is primarily expressed in mycelia and is necessary for aerial hyphae development and conidiation. Xbp1 is the only factor enriched specifically in yeast cells. The APSES proteins do not regulate conversion of conidia into yeast and hyphal morphologies. The APSES-family transcription factors are not individually required for H. capsulatum infection of cultured macrophages or murine infection, nor do any contribute significantly to resistance to cellular stresses including cell wall perturbation, osmotic stress, oxidative stress or antifungal treatment. Further studies of the downstream genes regulated by the individual APSES factors will be helpful in revealing their functional roles in H. capsulatum biology.
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