RESUMEN
This chapter describes the computational pipeline for the processing and visualization of Protec-Seq data, a method for purification and genome-wide mapping of double-stranded DNA protected by a specific protein at both ends. In the published case, the protein of choice was Saccharomyces cerevisiae Spo11, a conserved topoisomerase-like enzyme that makes meiotic double-strand breaks (DSBs) to initiate homologous recombination, ensuring proper segregation of homologous chromosomes and fertility. The isolated DNA molecules were thus termed double DSB (dDSB) fragments and were found to represent 34 to several hundred base-pair long segments that are generated by Spo11 and are enriched at DSB hotspots, which are sites of topological stress. In order to allow quantitative comparisons between dDSB profiles across experiments, we implemented calibrated chromatin immunoprecipitation sequencing (ChIP-Seq) using the meiosis-competent yeast species Saccharomyces kudriavzevii as calibration strain. Here, we provide a detailed description of the computational methods for processing, analyzing, and visualizing Protec-Seq data, comprising the download of the raw data, the calibrated genome-wide alignments, and the scripted creation of either arc plots or Hi-C-style heatmaps for the illustration of chromosomal regions of interest. The workflow is based on Linux shell scripts (including wrappers for publicly available, open-source software) as well as R scripts and is highly customizable through its modular structure.
Asunto(s)
Roturas del ADN de Doble Cadena , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Programas Informáticos , Meiosis/genética , Genoma Fúngico , Mapeo Cromosómico/métodos , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Biología Computacional/métodos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismoRESUMEN
This study examined the impact of live bread yeast (Saccharomyces cerevisiae) on the nutritional characteristics of Asian dried noodles. Micronutrient analysis of fermented noodles revealed a 6.9% increase in the overall amino acid content, a 37.1% increase in the vitamin B content and a 63.0% decrease in the phytic acid level. Molecular weight analysis of starch and protein contents revealed moderate decrease in the fermented noodles. The in vitro digestion of fermented noodles showed a slightly faster initial acidification, four-fold decrease in the initial shear viscosity (from 8.85 to 1.94 Pa·s). The initial large food particle count (>2 mm diameter) was 19.5% lower in the fermented noodles. The fermented noodles contained slightly higher free sugar content (73.5 mg g-1 noodle) during the gastric digestion phase. The overall nutrition and digestion results indicate nutritional improvement and digestion-easing attributes in the fermented noodles.
Asunto(s)
Digestión , Fermentación , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Nutrientes/metabolismo , Nutrientes/análisis , Humanos , Aminoácidos/metabolismo , Aminoácidos/análisis , Pan/análisis , Pan/microbiología , Modelos Biológicos , China , Pueblos del Este de AsiaRESUMEN
Probiotic viability, metabolite concentrations, physicochemical parameters, and volatile compounds were characterized in Gueuze beers formulated with probiotic lactic acid bacteria (LAB) and yeast. Additionally, the sensory profile of the beers and the resistance of the probiotics to digestion were determined. The use of 2 International Bitterness Units resulted in high concentrations of probiotic LAB but a decline in probiotic yeast as pH decreased. Secondary fermentation led to the consumption of maltose, citric acid, and malic acid, and the production of lactic and propionic acids. Carbonation and storage at 4 °C had minimal impact on probiotic viability. The addition of probiotic LAB resulted in a distinct aroma profile with improved sensory characteristics. Our results demonstrate that sour beers produced with probiotic LAB and a probiotic yeast, and fermented using a two-step fermentation process, exhibited optimal physicochemical parameters, discriminant volatile compound profiles, promising sensory characteristics, and high probiotic concentrations after digestion.
Asunto(s)
Cerveza , Fermentación , Probióticos , Gusto , Compuestos Orgánicos Volátiles , Cerveza/análisis , Cerveza/microbiología , Probióticos/metabolismo , Probióticos/análisis , Compuestos Orgánicos Volátiles/metabolismo , Compuestos Orgánicos Volátiles/química , Humanos , Digestión , Lactobacillales/metabolismo , Lactobacillales/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Viabilidad MicrobianaRESUMEN
Epigenetic inheritance of heterochromatin requires transfer of parental H3-H4 tetramers to both daughter duplexes during replication. Three recent papers exploit yeast genetics coupled to inheritance assays and AlphaFold2-multimer predictions coupled to biochemistry to reveal that a replisome component (Mrc1/CLASPIN) is an H3-H4 tetramer chaperone important for parental histone transfer to daughters.
Asunto(s)
Replicación del ADN , Aprendizaje Profundo , Histonas , Saccharomyces cerevisiae , Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Heterocromatina/metabolismo , Epigénesis GenéticaRESUMEN
Fumonisin B1 (FB1) targets sphingolipid biosynthesis, inhibiting ceramide synthases. In this issue of Structure, Zhang et al.1 determined the cryoelectron microscopic structures of yeast ceramide synthase in complex with FB1 and its acylated derivative, acyl-FB1, revealing a two-step "ping-pong" mechanism for the N-acylation of FB1 and how it inhibits ceramide synthase.
Asunto(s)
Microscopía por Crioelectrón , Fumonisinas , Oxidorreductasas , Fumonisinas/química , Fumonisinas/metabolismo , Oxidorreductasas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/antagonistas & inhibidores , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Acilación , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Esfingolípidos/metabolismo , Esfingolípidos/químicaRESUMEN
This study aimed to assess the impact of Saccharomyces cerevisiae and different non-Saccharomyces cerevisiae (Zygosaccharomyces bailii, Hanseniaspora opuntiae and Zygosaccharomyces rouxii) on the volatile compounds and sensory properties of low-alcohol pear beverages fermented from three varieties of pear juices (Korla, Laiyang and Binzhou). Results showed that all three pear juices were favorable matrices for yeasts growth. Non-Saccharomyces cerevisiae exhibited a higher capacity for acetate ester production compared to Saccharomyces cerevisiae, resulting in a significant enhancement in sensory complexity of the beverages. PCA and sensory analysis demonstrated that pear varieties exerted a stronger influence on the crucial volatile components and aroma characteristics of the fermented beverages compared to the yeast species. CA results showed different yeast strains exhibited suitability for the fermentation of specific pear juice varieties.
Asunto(s)
Fermentación , Odorantes , Pyrus , Saccharomyces cerevisiae , Compuestos Orgánicos Volátiles , Compuestos Orgánicos Volátiles/metabolismo , Compuestos Orgánicos Volátiles/análisis , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Pyrus/microbiología , Pyrus/química , Odorantes/análisis , Jugos de Frutas y Vegetales/análisis , Jugos de Frutas y Vegetales/microbiología , Gusto , Humanos , Zygosaccharomyces/metabolismo , Zygosaccharomyces/crecimiento & desarrollo , Hanseniaspora/metabolismo , Hanseniaspora/crecimiento & desarrollo , Frutas/microbiología , Frutas/química , SaccharomycetalesRESUMEN
Yeast assimilable nitrogen (YAN) is one of the important factors affecting yeast growth and metabolism. However, the nitrogen requirement of indigenous commercial S. cerevisiae NX11424 is unclear. In this study, metabolomics was used to analyze the metabolite profiles of the yeast strain NX11424 under high (433 mg/L) and low (55 mg/L) YAN concentrations. It was found that yeast biomass exhibited different trends under different YAN conditions and was generally positively correlated with the initial YAN concentration, while changes of key biomarkers of yeast strain NX11424 at different stages of fermentation showed a similar trend under high and low YAN concentrations. The YAN concentration affected the metabolite levels of the yeast strain NX11424, which resulted in the significant difference in the levels of pyruvic acid, α-oxoglutarate, palmitoleic acid, proline, butane-2,3-diol, citrulline, ornithine, galactinol, citramalic acid, tryptophan, alanine, phosphate and phenylethanol, mainly involving pathways such as central carbon metabolism, amino acid metabolism, fatty acid metabolism, purine metabolism, and energy metabolism. Yeast strain NX11424 could utilize proline to produce protein under a low YAN level. The intracellular level of citrulline and ornithine under high YAN concentration was higher than that under low YAN level. Yeast strain NX11424 is more suitable for fermentation at lower YAN level. The results obtained here will help to rational utilize of YAN by S. cerevisiae NX11424, and is conducive to precise control of the alcohol fermentation and improve wine quality.
Asunto(s)
Fermentación , Metabolómica , Nitrógeno , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Nitrógeno/metabolismo , Vino/análisis , Vino/microbiología , Biomasa , Aminoácidos/metabolismoRESUMEN
Fino Sherry wine undergoes biological aging carried out by a velum of flor yeast within a traditional dynamic system known as "criaderas and solera". The complex microbiota of biofilm-forming Saccharomyces cerevisiae strains play a crucial role in shaping the distinctive organoleptic profile of these types of wines. For this reason, the aim of this study is to analyze the changes produced by different flor yeast strains in the volatilome and the aminogram of different wines from the criaderas and solera system during biological aging in the laboratory, simulating a flor yeast velum condition at different stages of the system. Results suggest that each strain metabolizes wine differently, finding that depending on the wine, some strains are better suited for the process than others. In addition, it is found that the content of biogenic amines in Fino Sherry wines, previously attributed to malolactic bacteria, varies according to the yeast strain metabolizing the wine, suggesting that flor yeast could be used to modify biogenic amines content during biological aging. Results indicate that the use of selected flor yeast starters in biological aging may be of interest to modulate some parameters during Fino Sherry wine aging.
Asunto(s)
Fermentación , Saccharomyces cerevisiae , Compuestos Orgánicos Volátiles , Vino , Vino/análisis , Vino/microbiología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Compuestos Orgánicos Volátiles/metabolismo , Compuestos Orgánicos Volátiles/análisis , Compuestos de Nitrógeno/metabolismo , Aminas Biogénicas/metabolismo , Aminas Biogénicas/análisisRESUMEN
Environmental conditions significantly impact the metabolism of Saccharomyces cerevisiae, a Crabtree-positive yeast that maintains a fermentative metabolism in high-sugar environments even in the presence of oxygen. Although the introduction of oxygen has been reported to induce alterations in yeast metabolism, knowledge of the mechanisms behind these metabolic adaptations in relation to redox cofactor metabolism and their implications in the context of wine fermentation remains limited. This study aimed to compare the intracellular redox cofactor levels, the cofactor ratios, and primary metabolite production in S. cerevisiae under aerobic and anaerobic conditions in synthetic grape juice. The molecular mechanisms underlying these metabolic differences were explored using a transcriptomic approach. Aerobic conditions resulted in an enhanced fermentation rate and biomass yield. Total NADP(H) levels were threefold higher during aerobiosis, while a decline in the total levels of NAD(H) was observed. However, there were stark differences in the ratio of NAD+/NADH between the treatments. Despite few changes in the differential expression of genes involved in redox cofactor metabolism, anaerobiosis resulted in an increased expression of genes involved in lipid biosynthesis pathways, while the presence of oxygen increased the expression of genes associated with thiamine, methionine, and sulfur metabolism. The production of fermentation by-products was linked with differences in the redox metabolism in each treatment. This study provides valuable insights that may help steer the production of metabolites of industrial interest during alcoholic fermentation (including winemaking) by using oxygen as a lever of redox metabolism.
Asunto(s)
Fermentación , Oxidación-Reducción , Oxígeno , Saccharomyces cerevisiae , Vino , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Oxígeno/metabolismo , Vino/microbiología , Vino/análisis , Anaerobiosis , Vitis/microbiología , Vitis/metabolismo , NAD/metabolismo , Etanol/metabolismo , NADP/metabolismo , Aerobiosis , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Coenzimas/metabolismoRESUMEN
The traditional lignocellulose pretreatment by deep eutectic solvent (DES) was usually conducted under higher acidic, alkaline and high temperature conditions, which leads to the severe degradation of xylan, decreasing the subsequent reducing sugar concentration by enzymatic hydrolysis and further ethanol fermentation. It is essential to develop an effective DES that selectively removes lignin while preventing excessive xylan degradation during lignocellulose pretreatment. An effective ethylene glycol-assisted ternary DES was designed to treat corn straw (CS) at 100 °C for 6 h. 65.51 % lignin removal was achieved, over 93.46 % cellulose and 50.22 % xylan were retained in pretreated CS with excellent enzymatic digestibility (glucan conversion of 77.05 % and xylan conversion of 71.72 %), total sugar conversion could reach 75.93 %, implying the unique capacity to selectively remove lignin while preserving carbohydrate components. Furthermore, the universality of the selective removal of lignin and effective retention of xylan by ternary DES has been successfully proven by other polyols. The enzymatic hydrolysate of ternary DES-pretreated CS fermented over our genetically engineered yeast strain SFA1OE gave a high ethanol yield of 0.488 g/g total reducing sugar, demonstrating the effectiveness of the polyol-assisted ternary DES pretreatment in achieving high-efficiency cellulosic ethanol production.
Asunto(s)
Disolventes Eutécticos Profundos , Etanol , Fermentación , Lignina , Xilanos , Zea mays , Lignina/química , Etanol/química , Etanol/metabolismo , Xilanos/química , Hidrólisis , Zea mays/química , Disolventes Eutécticos Profundos/química , Polímeros/química , Saccharomyces cerevisiae/metabolismo , Celulosa/química , Solventes/químicaRESUMEN
Alcoholic fermentation is one of man's most efficient food preservation processes, and innovations in this area are a trend in food science and nutrition. In addition to the classic Saccharomyces yeasts, various other species may have desirable characteristics for obtaining fruit wines. This study investigated the profile of non-Saccharomyces commercial yeasts compared with S. cerevisiae regarding pineapple wine's chemical composition and bioaccessibility. The fermentation profile of the yeasts Lachancea thermotolerans, Brettanomyces bruxellensis, Brettanomyces lambicus, and S. cerevisiae was evaluated for sugar and alcohol content, and the pineapple wines obtained were analyzed for amino acids, phenolics, and organic acids by HPLC and volatile profile by GC/MS. All yeast strains were able to produce ethanol and glycerol at acceptable levels. L. thermotolerans produced higher levels of lactic acid (0.95 g/L) and higher consumption of free amino acids. B. bruxellensis produced higher levels of individual phenolics and ethanol 109 g/L. The alcoholic fermentation process improved the bioaccessibility of phenolics such as catechin (237 %), epigallocatechin gallate (81 %), procyanidin B1 (61 %) and procyanidin B2 (61 %). The yeasts differed in their volatile profiles, with Brettanomyces and Lachancea producing higher levels of compounds associated with pineapple aroma, such as ester ethyl butyrate (260-270 µg/L). These results demonstrate the importance of choosing the yeast strain for the conduction of alcoholic fermentation and that the yeasts Brettanomyces and Lachancea showed technological potential in obtaining pineapple wines. This study contributes to developing processes for obtaining fruit wines by highlighting two non-Saccharomyces yeast species with technological potential for alcoholic fermentations.
Asunto(s)
Ananas , Etanol , Fermentación , Saccharomyces cerevisiae , Vino , Vino/análisis , Ananas/química , Saccharomyces cerevisiae/metabolismo , Etanol/metabolismo , Etanol/análisis , Fenoles/análisis , Fenoles/metabolismo , Aminoácidos/análisis , Aminoácidos/metabolismo , Brettanomyces/metabolismo , Saccharomycetales/metabolismo , Compuestos Orgánicos Volátiles/análisis , Cromatografía de Gases y Espectrometría de Masas , Frutas/químicaRESUMEN
Considerable effort is required to build mathematical models of large protein regulatory networks. Utilizing computational algorithms that guide model development can significantly streamline the process and enhance the reliability of the resulting models. In this article, we present a perturbation approach for developing data-centric Boolean models of cell cycle regulation. To evaluate networks, we assign a score based on their steady states and the dynamical trajectories corresponding to the initial conditions. Then, perturbation analysis is used to find new networks with lower scores, in which dynamical trajectories traverse through the correct cell cycle path with high frequency. We apply this method to refine Boolean models of cell cycle regulation in budding yeast and mammalian cells.
Asunto(s)
Algoritmos , Ciclo Celular , Modelos Biológicos , Humanos , Animales , Redes Reguladoras de Genes , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomycetales/metabolismoRESUMEN
BACKGROUND: Metabolic engineering enables the sustainable and cost-efficient production of complex chemicals. Efficient production of terpenes in Saccharomyces cerevisiae can be achieved by recruiting an intermediate of the mevalonate pathway. The present study aimed to evaluate the engineering strategies of S. cerevisiae for the production of taxadiene, a precursor of taxol, an antineoplastic drug. RESULT: SCIGS22a, a previously engineered strain with modifications in the mevalonate pathway (MVA), was used as a background strain. This strain was engineered to enable a high flux towards farnesyl diphosphate (FPP) and the availability of NADPH. The strain MVA was generated from SCIGS22a by overexpressing all mevalonate pathway genes. Combining the background strains with 16 different episomal plasmids, which included the combination of 4 genes: tHMGR (3-hydroxy-3-methylglutaryl-CoA reductase), ERG20 (farnesyl pyrophosphate synthase), GGPPS (geranyl diphosphate synthase) and TS (taxadiene synthase) resulted in the highest taxadiene production in S. cerevisiae of 528 mg/L. CONCLUSION: Our study highlights the critical role of pathway balance in metabolic engineering, mainly when dealing with toxic molecules like taxadiene. We achieved significant improvements in taxadiene production by employing a combinatorial approach and focusing on balancing the downstream and upstream pathways. These findings emphasize the importance of minor gene expression modification levels to achieve a well-balanced pathway, ultimately leading to enhanced taxadiene accumulation.
Asunto(s)
Ingeniería Metabólica , Ácido Mevalónico , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ingeniería Metabólica/métodos , Ácido Mevalónico/metabolismo , Alquenos/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Diterpenos/metabolismo , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , NADP/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SesquiterpenosRESUMEN
Microbial secretions, such as metabolic enzymes, are often considered to be cooperative public goods as they are costly to produce but can be exploited by others. They create incentives for the evolution of non-producers, which can drive producer and population productivity declines. In response, producers can adjust production levels. Past studies suggest that while producers lower production to reduce costs and exploitation opportunities when under strong selection pressure from non-producers, they overproduce secretions when these pressures are weak. We challenge the universality of this trend with the production of a metabolic enzyme, invertase, by Saccharomyces cerevisiae, which catalyses sucrose hydrolysis into two hexose molecules. Contrary to past studies, overproducers evolve during evolutionary experiments even when under strong selection pressure from non-producers. Phenotypic and competition assays with a collection of synthetic strains - engineered to have modified metabolic attributes - identify two mechanisms for suppressing the benefits of invertase to those who exploit it. Invertase overproduction increases extracellular hexose concentrations that suppresses the metabolic efficiency of competitors, due to the rate-efficiency trade-off, and also enhances overproducers' hexose capture rate by inducing transporter expression. Thus, overproducers are maintained in the environment originally thought to not support public goods production.
Asunto(s)
Saccharomyces cerevisiae , beta-Fructofuranosidasa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , beta-Fructofuranosidasa/metabolismo , beta-Fructofuranosidasa/genética , Sacarosa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Evolución Biológica , Hexosas/metabolismo , Regulación hacia Arriba , Regulación Fúngica de la Expresión GénicaRESUMEN
In metazoans mitochondrial DNA (mtDNA) or retrotransposon cDNA released to cytoplasm are degraded by nucleases to prevent sterile inflammation. It remains unknown whether degradation of these DNA also prevents nuclear genome instability. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. In non-dividing stationary phase cells, Pol4-mediated non-homologous end-joining increases, resulting in frequent insertions of 1-3 nucleotides, and insertions of mtDNA (NUMTs) or retrotransposon cDNA. Yeast EndoG (Nuc1) nuclease limits insertion of cDNA and transfer of very long mtDNA ( >10 kb) to the nucleus, where it forms unstable circles, while promoting the formation of short NUMTs (~45-200 bp). Nuc1 also regulates transfer of extranuclear DNA to nucleus in aging or meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating extranuclear DNA preserve genome stability.
Asunto(s)
ADN Mitocondrial , Inestabilidad Genómica , Retroelementos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Retroelementos/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , Reparación del ADN por Unión de Extremidades , Roturas del ADN de Doble Cadena , Meiosis/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genéticaRESUMEN
Chromatin remodelling complexes (CRC) are ATP-dependent molecular machines important for the dynamic organization of nucleosomes along eukaryotic DNA. CRCs SWI/SNF, RSC and INO80 can move positioned nucleosomes in promoter DNA, leading to nucleosome-depleted regions which facilitate access of general transcription factors. This function is strongly supported by transcriptional activators being able to interact with subunits of various CRCs. In this work we show that SWI/SNF subunits Swi1, Swi2, Snf5 and Snf6 can bind to activation domains of Ino2 required for expression of phospholipid biosynthetic genes in yeast. We identify an activator binding domain (ABD) of ATPase Swi2 and show that this ABD is functionally dispensable, presumably because ABDs of other SWI/SNF subunits can compensate for the loss. In contrast, mutational characterization of the ABD of the Swi2-related ATPase Sth1 revealed that some conserved basic and hydrophobic amino acids within this domain are essential for the function of Sth1. While ABDs of Swi2 and Sth1 define separate functional protein domains, mapping of an ABD within ATPase Ino80 showed co-localization with its HSA domain also required for binding actin-related proteins. Comparative interaction studies finally demonstrated that several unrelated activators each exhibit a specific binding pattern with ABDs of Swi2, Sth1 and Ino80.
Asunto(s)
Adenosina Trifosfatasas , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN , Unión Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Factores de Transcripción , Activación Transcripcional , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Regulación Fúngica de la Expresión Génica , Dominios Proteicos , Proteínas Nucleares , Proteínas de Ciclo Celular , Factores de Transcripción con Motivo Hélice-Asa-Hélice BásicoRESUMEN
Expressing plant metabolic pathways in microbial platforms is an efficient, cost-effective solution for producing many desired plant compounds. As eukaryotic organisms, yeasts are often the preferred platform. However, expression of plant enzymes in a yeast frequently leads to failure because the enzymes are poorly adapted to the foreign yeast cellular environment. Here, we first summarize the current engineering approaches for optimizing performance of plant enzymes in yeast. A critical limitation of these approaches is that they are labour-intensive and must be customized for each individual enzyme, which significantly hinders the establishment of plant pathways in cellular factories. In response to this challenge, we propose the development of a cost-effective computational pipeline to redesign plant enzymes for better adaptation to the yeast cellular milieu. This proposition is underpinned by compelling evidence that plant and yeast enzymes exhibit distinct sequence features that are generalizable across enzyme families. Consequently, we introduce a data-driven machine learning framework designed to extract 'yeastizing' rules from natural protein sequence variations, which can be broadly applied to all enzymes. Additionally, we discuss the potential to integrate the machine learning model into a full design-build-test cycle.
Asunto(s)
Ingeniería Metabólica , Ingeniería Metabólica/métodos , Plantas , Enzimas/genética , Enzimas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Aprendizaje Automático , Redes y Vías Metabólicas/genéticaRESUMEN
The fermentative model yeast Saccharomyces cerevisiae has been extensively used to study the genetic basis of stress response and homeostasis. In this study, we performed quantitative trait loci (QTL) analysis of the high-temperature fermentation trait of the progeny from the mating of the S. cerevisiae natural isolate BCC39850 (haploid#17) and the laboratory strain CEN.PK2-1C. A single QTL on chromosome X was identified, encompassing six candidate genes (GEA1, PTK2, NTA1, NPA3, IRT1, and IML1). The functions of these candidates were tested by reverse genetic experiments. Deletion mutants of PTK2, NTA1, and IML1 showed growth defects at 42 °C. The PTK2 knock-out mutant also showed significantly reduced ethanol production and plasma membrane H+ ATPase activity and increased sensitivity to acetic acid, ethanol, amphotericin B (AMB), and ß-1,3-glucanase treatment. The CRISPR-Cas9 system was used to construct knock-in mutants by replacement of PTK2, NTA1, IML1, and NPA3 genes with BCC39850 alleles. The PTK2 and NTA1 knock-in mutants showed increased growth and ethanol production titers at 42 °C. These findings suggest an important role for the PTK2 serine/threonine protein kinase in regulating plasma membrane H+ ATPase activity and the NTA1 N-terminal amidase in protein degradation via the ubiquitin-proteasome system machinery, which affects tolerance to heat stress in S. cerevisiae.
Asunto(s)
Etanol , Fermentación , Calor , Sitios de Carácter Cuantitativo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Etanol/metabolismoRESUMEN
The yeast pre1-1(ß4-S142F) mutant accumulates late 20S proteasome core particle precursor complexes (late-PCs). We report a 2.1 Å cryo-EM structure of this intermediate with full-length Ump1 trapped inside, and Pba1-Pba2 attached to the α-ring surfaces. The structure discloses intimate interactions of Ump1 with ß2- and ß5-propeptides, which together fill most of the antechambers between the α- and ß-rings. The ß5-propeptide is unprocessed and separates Ump1 from ß6 and ß7. The ß2-propeptide is disconnected from the subunit by autocatalytic processing and localizes between Ump1 and ß3. A comparison of different proteasome maturation states reveals that maturation goes along with global conformational changes in the rings, initiated by structuring of the proteolytic sites and their autocatalytic activation. In the pre1-1 strain, ß2 is activated first enabling processing of ß1-, ß6-, and ß7-propeptides. Subsequent maturation of ß5 and ß1 precedes degradation of Ump1, tightening of the complex, and finally release of Pba1-Pba2.
Asunto(s)
Microscopía por Crioelectrón , Complejo de la Endopetidasa Proteasomal , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Subunidades de Proteína/metabolismo , Subunidades de Proteína/química , Modelos Moleculares , Conformación Proteica , Péptidos/metabolismo , Péptidos/química , Unión Proteica , Chaperonas MolecularesRESUMEN
Double-strand breaks (DSBs) in DNA are challenging to repair. Cells employ at least three DSB-repair mechanisms, with a preference for non-homologous end joining (NHEJ) over homologous recombination (HR) and microhomology-mediated end joining (MMEJ). While most eukaryotic DNA is transcribed into RNA, providing complementary genetic information, much remains unknown about the direct impact of RNA on DSB-repair outcomes and its role in DSB-repair via end joining. Here, we show that both sense and antisense-transcript RNAs impact DSB repair in a sequence-specific manner in wild-type human and yeast cells. Depending on its sequence complementarity with the broken DNA ends, a transcript RNA can promote repair of a DSB or a double-strand gap in its DNA gene via NHEJ or MMEJ, independently from DNA synthesis. The results demonstrate a role of transcript RNA in directing the way DSBs are repaired in DNA, suggesting that RNA may directly modulate genome stability and evolution.