A structural perspective of human RNA polymerase III.

RNA polymerase III (Pol III) is a large multisubunit complex conserved in all eukaryotes that plays an essential role in producing a variety of short non-coding RNAs, such as tRNA, 5S rRNA and U6 snRNA transcripts. Pol III comprises of 17 subunits in both yeast and human with a 10-subunit core and seven peripheral subunits. Because of its size and complexity, Pol III has posed a formidable challenge to structural biologists. The first atomic cryogenic electron microscopy structure of yeast Pol III leading to the canonical view was reported in 2015. Within the last few years, the optimization of endogenous extract and purification procedure and the technical and methodological advances in cryogenic electron microscopy, together allow us to have a first look at the unprecedented details of human Pol III organization. Here, we look back on the structural studies of human Pol III and discuss them in the light of our current understanding of its role in eukaryotic transcription.

Yeast GH30 Xylanase from Sugiyamaella lignohabitans Is a Glucuronoxylanase with Auxiliary Xylobiohydrolase Activity.

Xylanases are the enzymes that catalyze the breakdown of the main hemicellulose present in plant cell walls. They have attracted attention due to their biotechnological potential for the preparation of industrially interesting products from lignocellulose. While many xylanases have been characterized from bacteria and filamentous fungi, information on yeast xylanases is scarce and no yeast xylanase belonging to glycoside hydrolase (GH) family 30 has been described so far. Here, we cloned, expressed and characterized GH30 xylanase SlXyn30A from the yeast Sugiyamaella lignohabitans. The enzyme is active on glucuronoxylan (8.4 U/mg) and rhodymenan (linear ß-1,4-1,3-xylan) (3.1 U/mg) while its activity on arabinoxylan is very low (0.03 U/mg). From glucuronoxylan SlXyn30A releases a series of acidic xylooligosaccharides of general formula MeGlcA2Xyln. These products, which are typical for GH30-specific glucuronoxylanases, are subsequently shortened at the non-reducing end, from which xylobiose moieties are liberated. Xylobiohydrolase activity was also observed during the hydrolysis of various xylooligosaccharides. SlXyn30A thus expands the group of glucuronoxylanases/xylobiohydrolases which has been hitherto represented only by several fungal GH30-7 members.

Heterologous expression of 4α-glucanotransferase: overproduction and properties for industrial applications.

4α-Glucanotransferase (4α-GTase) is unique in its ability to form cyclic oligosaccharides, some of which are of industrial importance. Generally, low amount of enzymes is produced by or isolated from their natural sources: animals, plants, and microorganisms. Heterologous expressions of these enzymes, in an attempt to increase their production for applicable uses, have been widely studied since 1980s; however, the expressions are mostly performed in the prokaryotic bacteria, mostly Escherichia coli. Site-directed mutagenesis has added more value to these expressed enzymes to display the desired properties beneficial for their applications. The search for further suitable properties for food application leads to an extended research in expression by another group of host organism, the generally-recognized as safe host including the Bacillus and the eukaryotic yeast systems. Herein, our review focuses on two types of 4α-GTase: the cyclodextrin glycosyltransferase and amylomaltase. The updated studies on the general structure and properties of the two enzymes with emphasis on heterologous expression, mutagenesis for property improvement, and their industrial applications are provided.

The safety of a Kluyveromyces lactis strain lineage for enzyme production.

Kluyveromyces lactis is broadly considered as a safe yeast in food and a suitable organism for the production of food enzymes. The K. lactis enzyme production strains of DSM are used to produce a variety of enzymes, for example beta-galactosidase (lactase), chymosin and esterase. All of these production strains are derived from the same lineage, meaning they all originate from the same ancestor strain after classical mutagenesis and/or genetic engineering. Four different enzyme preparations produced with strains within this lineage were toxicologically tested. These enzyme preparations were nontoxic in repeated-dose oral toxicity studies performed in rats and were non-genotoxic in vitro. These studies confirm the safety of the DSM K. lactis strains as a production platform for food enzymes, as well as the safety of the genetic modifications made to these strains through genetic engineering or classical mutagenesis. The outcome of the toxicity studies can be extended to other enzyme preparations produced by any strain from this lineage through read across. Therefore, no new toxicity studies are required for the safety evaluation, as long as the modifications made do not raise safety concerns. Consequently, this approach is in line with the public ambition to reduce animal toxicity studies.

Beta-glucosidase activity of wine yeasts and its impacts on wine volatiles and phenolics: A mini-review.

Beta-glucosidase is an important enzyme for the hydrolysis of grape glycosides in the course of winemaking. Yeasts are the main producers of ß-glucosidase in winemaking, therefore play an important role in determining wine aroma and flavour. This article discusses common methods for ß-glucosidase evaluation, the ß-glucosidase activity of different Saccharomyces and non- Saccharomyces yeasts and the influences of winemaking conditions, such as glucose and ethanol concentration, low pH environment, fermentation temperature and SO2 level, on their activity. This review further highlights the roles of ß-glucosidase in promoting the release of free volatile compounds especially terpenes and the modification of wine phenolic composition during the winemaking process. Furthermore, this review proposes future research direction in this area and guides wine professionals in yeast selection to improve wine quality.

Unchecked nick ligation can promote localized genome re-replication.

Single-stranded DNA breaks, or nicks, are amongst the most common forms of DNA damage in cells. They can be repaired by ligation; however, if a nick occurs just ahead of an approaching replisome, the outcome is a collapsed replication fork comprising a single-ended double-strand break and a 'hybrid nick' with parental DNA on one side and nascent DNA on the other (Figure 1A). We realized that in eukaryotic cells, where replication initiates from multiple replication origins, a fork from an adjacent origin can promote localized re-replication if the hybrid nick is ligated. We have modelled this situation with purified proteins in vitro and have found that there is, indeed, an additional hazard that eukaryotic replisomes face. We discuss how this problem might be mitigated.

Lipid metabolism has been good to me.

My career in research has flourished through hard work, supportive mentors, and outstanding mentees and collaborators. The Carman laboratory has contributed to the understanding of lipid metabolism through the isolation and characterization of key lipid biosynthetic enzymes as well as through the identification of the enzyme-encoding genes. Our findings from yeast have proven to be invaluable to understand regulatory mechanisms of human lipid metabolism. Several rewarding aspects of my career have been my service to the Journal of Biological Chemistry as an editorial board member and Associate Editor, the National Institutes of Health as a member of study sections, and national and international scientific meetings as an organizer. I advise early career scientists to not assume anything, acknowledge others' accomplishments, and pay it forward.

RNA helicase-mediated regulation of snoRNP dynamics on pre-ribosomes and rRNA 2'-O-methylation.

RNA helicases play important roles in diverse aspects of RNA metabolism through their functions in remodelling ribonucleoprotein complexes (RNPs), such as pre-ribosomes. Here, we show that the DEAD box helicase Dbp3 is required for efficient processing of the U18 and U24 intron-encoded snoRNAs and 2'-O-methylation of various sites within the 25S ribosomal RNA (rRNA) sequence. Furthermore, numerous box C/D snoRNPs accumulate on pre-ribosomes in the absence of Dbp3. Many snoRNAs guiding Dbp3-dependent rRNA modifications have overlapping pre-rRNA basepairing sites and therefore form mutually exclusive interactions with pre-ribosomes. Analysis of the distribution of these snoRNAs between pre-ribosome-associated and 'free' pools demonstrated that many are almost exclusively associated with pre-ribosomal complexes. Our data suggest that retention of such snoRNPs on pre-ribosomes when Dbp3 is lacking may impede rRNA 2'-O-methylation by reducing the recycling efficiency of snoRNPs and by inhibiting snoRNP access to proximal target sites. The observation of substoichiometric rRNA modification at adjacent sites suggests that the snoRNPs guiding such modifications likely interact stochastically rather than hierarchically with their pre-rRNA target sites. Together, our data provide new insights into the dynamics of snoRNPs on pre-ribosomal complexes and the remodelling events occurring during the early stages of ribosome assembly.

Bayesian genome scale modelling identifies thermal determinants of yeast metabolism.

The molecular basis of how temperature affects cell metabolism has been a long-standing question in biology, where the main obstacles are the lack of high-quality data and methods to associate temperature effects on the function of individual proteins as well as to combine them at a systems level. Here we develop and apply a Bayesian modeling approach to resolve the temperature effects in genome scale metabolic models (GEM). The approach minimizes uncertainties in enzymatic thermal parameters and greatly improves the predictive strength of the GEMs. The resulting temperature constrained yeast GEM uncovers enzymes that limit growth at superoptimal temperatures, and squalene epoxidase (ERG1) is predicted to be the most rate limiting. By replacing this single key enzyme with an ortholog from a thermotolerant yeast strain, we obtain a thermotolerant strain that outgrows the wild type, demonstrating the critical role of sterol metabolism in yeast thermosensitivity. Therefore, apart from identifying thermal determinants of cell metabolism and enabling the design of thermotolerant strains, our Bayesian GEM approach facilitates modelling of complex biological systems in the absence of high-quality data and therefore shows promise for becoming a standard tool for genome scale modeling.

Happy Birthday: 30 Years of RNA Helicases.

RNA helicases are ubiquitous, highly conserved RNA-binding enzymes that use the energy derived from the hydrolysis of nucleoside triphosphate to modify the structure of RNA molecules and/or the functionality of ribonucleoprotein complexes. Ultimately, the action of RNA helicases results in changes in gene expression that allow the cell to perform crucial functions. In this chapter, we review established and emerging concepts for DEAD-box and DExH-box RNA helicases. We mention examples from both eukaryotic and prokaryotic systems, in order to highlight common themes and specific actions.

Synthesis, α-Glucosidase inhibition and molecular docking studies of tyrosol derivatives.

To find a potent α-glucosidase inhibitor, 24 tyrosol derivatives with different substituents located at the meta, ortho, or para position of the phenyl group have been synthesised via the Mitsunobu reaction, characterised by 1H NMR, 13C NMR, ESI-MS and IR and evaluated for inhibition. The derivatives possessed varying degrees of in vitro inhibitory activity against α-glucosidase and a relationship between the structure and activity was subsequently established for all compounds. Two of these compounds with substituents at the para position showed significant inhibitory effects surpassing that of the control standard acarbose. Molecular docking studies performed to better understand the binding interactions between the enzyme and the two most active compounds showed substantial binding within the active site of α-glucosidase. Taken together, these results indicate that the position of the substituent plays a crucial role in this inhibition and may facilitate the development of new α-glucosidase inhibitors.

Hands on Native Mass Spectrometry Analysis of Multi-protein Complexes.

By maintaining intact multi-protein complexes in the gas-phase, native mass spectrometry provides their molecular weight with very good accuracy compared to other methods (typically native PAGE or SEC-MALS) (Marcoux and Robinson, Structure 21:1541-1550, 2013). Besides, heterogeneous samples, in terms of both oligomeric states and ligand-bound species can be fully characterized. Here we thoroughly describe the analysis of several oligomeric protein complexes ranging from a 16 = kDa dimer to a 801-kDa tetradecameric complex on different instrumental setups.

Isolation and molecular identification of industrially important enzyme producer yeasts from tree barks and fruits.

This study aimed to identify the yeast strains associated with the tree bark samples collected from the Aegean and Marmara regions and from rotten fruit samples. Fifty-one yeast strains were successfully isolated and screened for their abilities to produce industrially important extracellular enzymes. Thirty isolates demonstrated ability to produce at least two different enzymes and were selected for subsequent molecular identification using sequence analysis of ITS region and D1/D2 domain of the 26S rDNA. The most prevalent strains belonged to Papiliotrema laurentii (%23), Papiliotrema terrestris (%13) and Candida membranifaciens (%10). Papiliotrema laurentii and Papiliotrema terrestris recorded the highest enzymatic activities for all the screened enzymes. To the best of our knowledge, this is the first report that identifies the yeast strains associated with the tree barks of Turkey and among the limited comprehensive studies that screened considerable number of isolates for their ability to produce several industrially important enzymes.

Evolutionary Overview of Molecular Interactions and Enzymatic Activities in the Yeast Cell Walls.

Fungal cell walls are composed of a polysaccharide network that serves as a scaffold in which different glycoproteins are embedded. Investigation of fungal cell walls, besides simple identification and characterization of the main cell wall building blocks, covers the pathways and regulations of synthesis of each individual component of the wall and biochemical reactions by which they are cross-linked and remodeled in response to different growth phase and environmental signals. In this review, a survey of composition and organization of so far identified and characterized cell wall components of different yeast genera including Saccharomyces, Candida, Kluyveromyces, Yarrowia, and Schizosaccharomyces are presented with the focus on their cell wall proteomes.

Conditional Protein Rescue by Binding-Induced Protective Shielding.

Synthetic protein-level circuits offer an extra layer of cellular control on top of conventional gene-level circuits. Here, we describe a technology that allows conditional protein rescue (CPR) from proteasomal degradation using different protein inputs as masking agents. A target protein is fused to a degron tag and an affinity sensor domain. The use of nanobodies as the sensor domain offers a generalizable strategy to execute a wide range of protein-level circuits with ease. The utility of this new strategy was successfully demonstrated to distinguish cancer cells out of a healthy population using the HPV-specific E7 protein as a cellular marker. Because CPR can be programmed to execute more complex Boolean logic designs using cell-specific proteomes, this platform offers a highly modular and scalable framework for a wide range of applications based on synthetic protein circuits.

Yeasts Associated with Various Amazonian Native Fruits.

Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.

Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells.

The dynamic cytoskeletal network of microtubules and actin filaments can be disassembled by drugs. Cytoskeletal drugs work by perturbing the monomer-polymer equilibrium, thus changing the size and number of macromolecular crowders inside cells. Changes in both crowding and nonspecific surface interactions ("sticking") following cytoskeleton disassembly can affect the protein stability, structure, and function directly or indirectly by changing the fluidity of the cytoplasm and altering the crowding and sticking of other macromolecules in the cytoplasm. The effect of cytoskeleton disassembly on protein energy landscapes inside cells has yet to be observed. Here we have measured the effect of several cytoskeletal drugs on the folding energy landscape of two FRET-labeled proteins with different in vitro sensitivities to macromolecular crowding. Phosphoglycerate kinase (PGK) was previously shown to be more sensitive to crowding, whereas variable major protein-like sequence expressed (VlsE) was previously shown to be more sensitive to sticking. The in-cell effects of drugs that depolymerize either actin filaments (cytochalasin D and latrunculin B) or microtubules (nocodazole and vinblastine) were compared. The crowding sensor protein CrH2-FRET verified that cytoskeletal drugs decrease the extent of crowding inside cells despite also reducing the overall cell volume. The decreased compactness and folding stability of PGK could be explained by the decreased extent of crowding induced by these drugs. VlsE's opposite response to the drugs shows that depolymerization of the cytoskeleton also changes sticking in the cellular milieu. Our results demonstrate that perturbation of the monomer-polymer cytoskeletal equilibrium, for example, during natural cell migration or stresses from drug treatment, has off-target effects on the energy landscapes of proteins in the cell.

Production of ethanol and xylanolytic enzymes by yeasts inhabiting rotting wood isolated in sugarcane bagasse hydrolysate.

Yeasts associated with rotting wood from four Atlantic Rain forest sites in Brazil were investigated using a culture medium based on sugarcane bagasse hydrolysate. A total of 330 yeast strains were isolated. Pichia manshurica, Candida pseudolambica, and Wickerhamomyces sp. 3 were the most frequently isolated species. Fourteen novel species were obtained in this study. All isolates were tested for their ability to ferment d-xylose and to produce xylanases. In the fermentation assays using d-xylose (30 g L-1), the main ethanol producers were Scheffersomyces stipitis (14.08 g L-1), Scheffersomyces sp. (7.94 g L-1) and Spathaspora boniae (7.16 g L-1). Sc. stipitis showed the highest ethanol yield (0.42 g g-1) and the highest productivity (0.39 g L-1h-1). The fermentation results using hemicellulosic hydrolysate showed that Sc. stipitis was the best ethanol producer, achieving a yield of 0.32 g g-1, while Sp. boniae and Scheffersomyces sp. were excellent xylitol producers. The best xylanase-producing yeasts at 50 °C belonged to the species Su. xylanicola (0.487 U mg-1) and Saitozyma podzolica (0.384 U mg-1). The results showed that rotting wood collected from the Atlantic Rainforest is a valuable source of yeasts able to grow in sugarcane bagasse hydrolysate, including species with promising biotechnological properties.

Hopane-type triterpenes from Cnidoscolus spinosus and their bioactivities.

Chemical investigation of the aerial parts of Cnidoscolus spinosus resulted in the isolation of relatively infrequent hopane-type triterpenes, 3ß-acetoxy-hop-22(29)-ene (1), first reported here as natural product, together with 3-oxo-hop-22(29)-ene (2), and 3ß-hydroxy-hop-22(29)-ene (3). ß-Amyrin palmitate and three phytosterols were also characterized. The structures of the compounds were established using spectroscopic methods, and those of 1 and 2 were confirmed by crystallographic analysis. Selected biological activities for the isolated hopane-type triterpenes were tested through a series of assays for determining the cytotoxic, anti-inflammatory, α-glucosidase inhibition and antiparasitic activities. Compounds 1-3 did not show cytotoxic activity, compound 1 displayed an important inhibitory effect in the mouse ear induced inflammation assay, and significantly inhibited the yeast α-glucosidase activity in vitro and in silico. Additionally, compounds 2 and 3 showed marginal activities against Trypanosoma cruzi and Leishmania mexicana. Therefore, the bioactivities of hopane-type triterpenes deserve further investigation, particularly their anti-inflammatory properties.

Exploiting the Diversity of Saccharomycotina Yeasts To Engineer Biotin-Independent Growth of Saccharomyces cerevisiae.

Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiaeBIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiaeIMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.