Characterization of synthetic DNA bar codes in Saccharomyces cerevisiae gene-deletion strains.
Ontology highlight
ABSTRACT:
SUBMITTER: Eason RG
PROVIDER: S-EPMC491991 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Central carbon metabolism is controlled by modulating the protein abundance profiles of enzymes that maintain the essential systems in living organisms. In this study, metabolic adaptation mechanisms in the model organism Saccharomyces cerevisiae were investigated by direct determination of enzyme abundance levels in 30 wild type and mutant strains. We performed a targeted proteome analysis using S. cerevisiae strains that lack genes encoding the enzymes responsible for central carbon metabolism. Our analysis revealed that at least 30% of the observed variations in enzyme abundance levels could be explained by global regulatory mechanisms. A enzyme-enzyme co-abundance analysis revealed that the abundances of enzyme proteins involved in the trehalose metabolism and glycolysis changed in a coordinated manner under the control of the transcription factors for global regulation. The remaining variations were derived from local mechanisms such as a mutant-specific increase in the abundances of remote enzymes. The proteome data also suggested that, although the functional compensation of the deficient enzyme was attained by using more resources for protein biosynthesis, available resources for the biosynthesis of the enzymes responsible for central metabolism were not abundant in S. cerevisiae cells. These results showed that global and local regulation of enzyme abundance levels shape central carbon metabolism in S. cerevisiae by using a limited resource for protein biosynthesis.
| S-EPMC5328394 | biostudies-literature
Project description:We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cerevisiae with high efficiency. Preparation of the disruption gene cassette in this approach was simply performed by overlap extension-PCR of an invert repeat of a partial or complete sequence of the targeted gene with URA3. Integration of the prepared disruption gene cassette to the designated position of a target gene leads to the formation of a mutagenesis cassette within the yeast genome, which consists of a URA3 gene flanked by the targeted gene and its inverted repeat between two short identical direct repeats. The inherent instability of the inverted sequences in close proximity facilitates the self-excision of the entire mutagenesis cassette deposited in the genome and promotes homologous recombination resulting in a seamless deletion via a single transformation. This rapid assembly circumvents the difficulty during preparation of disruption gene cassettes composed of two inverted repeats of the URA3, which requires the engineering of unique restriction sites for subsequent digestion and T4 DNA ligation in vitro. We further identified that the excision of the entire mutagenesis cassette flanked by two DRs in the transformed S. cerevisiae is dependent on the length of the inverted repeat of which a minimum of 800 bp is required for effective gene deletion. The deletion efficiency improves with the increase of the inverted repeat till 1.2 kb. Finally, the use of gene-specific inverted repeats of target genes enables simultaneous gene deletions. The procedure has the potential for application on other yeast strains to achieve precise and efficient removal of gene sequences.
| S-EPMC3748122 | biostudies-literature
Project description:BackgroundMobile Genetic Elements (MGEs) are selfish DNA integrated in the genomes. Their detection is mainly based on consensus-like searches by scanning the investigated genome against the sequence of an already identified MGE. Mobilomics aims at discovering all the MGEs in a genome and understanding their dynamic behavior: The data for this kind of investigation can be provided by comparative genomics of closely related organisms. The amount of data thus involved requires a strong computational effort, which should be alleviated.ResultsOur approach proposes to exploit the high similarity among homologous chromosomes of different strains of the same species, following a progressive comparative genomics philosophy. We introduce a software tool based on our new fast algorithm, called regender, which is able to identify the conserved regions between chromosomes. Our case study is represented by a unique recently available dataset of 39 different strains of S.cerevisiae, which regender is able to compare in few minutes. By exploring the non-conserved regions, where MGEs are mainly retrotransposons called Tys, and marking the candidate Tys based on their length, we are able to locate a priori and automatically all the already known Tys and map all the putative Tys in all the strains. The remaining putative mobile elements (PMEs) emerging from this intra-specific comparison are sharp markers of inter-specific evolution: indeed, many events of non-conservation among different yeast strains correspond to PMEs. A clustering based on the presence/absence of the candidate Tys in the strains suggests an evolutionary interconnection that is very similar to classic phylogenetic trees based on SNPs analysis, even though it is computed without using phylogenetic information.ConclusionsThe case study indicates that the proposed methodology brings two major advantages: (a) it does not require any template sequence for the wanted MGEs and (b) it can be applied to infer MGEs also for low coverage genomes with unresolved bases, where traditional approaches are largely ineffective.
| S-EPMC3684551 | biostudies-literature
Project description:Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase-mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimize genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (rapid evolution detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with the haploid progeny of industrial yeast strains to produce stress-tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate the optimal SCRaMbLE induction times of different Cre-recombinase expression systems for the development of industrial strains.
| S-EPMC7761333 | biostudies-literature
Project description:BackgroundDespite the latest advancements in metabolic engineering for genome editing and characterization of host performance, the successful development of robust cell factories used for industrial bioprocesses and accurate prediction of the behavior of microbial systems, especially when shifting from laboratory-scale to industrial conditions, remains challenging. To increase the probability of success of a scale-up process, data obtained from thoroughly performed studies mirroring cellular responses to typical large-scale stimuli may be used to derive crucial information to better understand potential implications of large-scale cultivation on strain performance. This study assesses the feasibility to employ a barcoded yeast deletion library to assess genome-wide strain fitness across a simulated industrial fermentation regime and aims to understand the genetic basis of changes in strain physiology during industrial fermentation, and the corresponding roles these genes play in strain performance.ResultsWe find that mutant population diversity is maintained through multiple seed trains, enabling large scale fermentation selective pressures to act upon the community. We identify specific deletion mutants that were enriched in all processes tested in this study, independent of the cultivation conditions, which include MCK1, RIM11, MRK1, and YGK3 that all encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). Ecological analysis of beta diversity between all samples revealed significant population divergence over time and showed feed specific consequences of population structure. Further, we show that significant changes in the population diversity during fed-batch cultivations reflect the presence of significant stresses. Our observations indicate that, for this yeast deletion collection, the selection of the feeding scheme which affects the accumulation of the fermentative by-product ethanol impacts the diversity of the mutant pool to a higher degree as compared to the pH of the culture broth. The mutants that were lost during the time of most extreme population selection suggest that specific biological processes may be required to cope with these specific stresses.ConclusionsOur results demonstrate the feasibility of Bar-seq to assess fermentation associated stresses in yeast populations under industrial conditions and to understand critical stages of a scale-up process where variability emerges, and selection pressure gets imposed. Overall our work highlights a promising avenue to identify genetic loci and biological stress responses required for fitness under industrial conditions.
| S-EPMC7437010 | biostudies-literature
Project description:The multifunctional Saccharomyces cerevisiae Pif1 DNA helicase affects the maintenance of telomeric, ribosomal, and mitochondrial DNAs, suppresses DNA damage at G-quadruplex motifs, influences the processing of Okazaki fragments, and promotes breakage induced replication. All of these functions require the ATPase/helicase activity of the protein. Owing to Pif1's critical role in the maintenance of mitochondrial DNA, pif1Δ strains quickly generate respiratory deficient cells and hence grow very slowly. This slow growth makes it difficult to carry out genome-wide synthetic genetic analysis in this background. Here, we used a partial loss of function allele of PIF1, pif1-m2, which is mitochondrial proficient but has reduced abundance of nuclear Pif1. Although pif1-m2 is not a null allele, pif1-m2 cells exhibit defects in telomere maintenance, reduced suppression of damage at G-quadruplex motifs and defects in breakage induced replication. We performed a synthetic screen to identify nonessential genes with a synthetic sick or lethal relationship in cells with low abundance of nuclear Pif1. This study identified eleven genes that were synthetic lethal (APM1, ARG80, CDH1, GCR1, GTO3, PRK1, RAD10, SKT5, SOP4, UMP1, and YCK1) and three genes that were synthetic sick (DEF1, YIP4, and HOM3) with pif1-m2.
| S-EPMC4683662 | biostudies-literature
Project description:Protein decay rates are regulated by degradation machinery that clears unnecessary housekeeping proteins and maintains appropriate dynamic resolution for transcriptional regulators. Turnover rates are also crucial for fluorescence reporters that must strike a balance between sufficient fluorescence for signal detection and temporal resolution for tracking dynamic responses. Here, we use components of the Escherichia coli degradation machinery to construct a Saccharomyces cerevisiae strain that allows for tunable degradation of a tagged protein. Using a microfluidic platform tailored for single-cell fluorescence measurements, we monitor protein decay rates after repression using an ssrA-tagged fluorescent reporter. We observe a half-life ranging from 91 to 22 min, depending on the level of activation of the degradation genes. Computational modeling of the underlying set of enzymatic reactions leads to GFP decay curves that are in excellent agreement with the observations, implying that degradation is governed by Michaelis-Menten-type interactions. In addition to providing a reporter with tunable dynamic resolution, our findings set the stage for explorations of the effect of protein degradation on gene regulatory and signalling pathways.
| S-EPMC1943424 | biostudies-literature
Project description:Cellular metabolism is directly or indirectly associated with various cellular processes by producing a variety of metabolites. Metabolic alterations may cause adverse effects on cell viability. However, some alterations potentiate the rescue of the malfunction of the cell system. Here, we found that the alteration of glucose metabolism suppressed genome instability caused by the impairment of chromatin structure. Deletion of the TDH2 gene, which encodes glyceraldehyde 3-phospho dehydrogenase and is essential for glycolysis/gluconeogenesis, partially suppressed DNA damage sensitivity due to chromatin structure, which was persistently acetylated histone H3 on lysine 56 in cells with deletions of both HST3 and HST4, encoding NAD+-dependent deacetylases. tdh2 deletion also restored the short replicative lifespan of cells with deletion of sir2, another NAD+-dependent deacetylase, by suppressing intrachromosomal recombination in rDNA repeats increased by the unacetylated histone H4 on lysine 16. tdh2 deletion also suppressed recombination between direct repeats in hst3∆ hst4∆ cells by suppressing the replication fork instability that leads to both DNA deletions among repeats. We focused on quinolinic acid (QUIN), a metabolic intermediate in the de novo nicotinamide adenine dinucleotide (NAD+) synthesis pathway, which accumulated in the tdh2 deletion cells and was a candidate metabolite to suppress DNA replication fork instability. Deletion of QPT1, quinolinate phosphoribosyl transferase, elevated intracellular QUIN levels and partially suppressed the DNA damage sensitivity of hst3∆ hst4∆ cells as well as tdh2∆ cells. qpt1 deletion restored the short replicative lifespan of sir2∆ cells by suppressing intrachromosomal recombination among rDNA repeats. In addition, qpt1 deletion could suppress replication fork slippage between direct repeats. These findings suggest a connection between glucose metabolism and genomic stability.
| S-EPMC7713361 | biostudies-literature
Project description:Diastatic strains of Saccharomyces cerevisiae are common contaminants in beer fermentations and are capable of producing an extracellular STA1-encoded glucoamylase. Recent studies have revealed variable diastatic ability in strains tested positive for STA1, and here, we elucidate genetic determinants behind this variation. We show that poorly diastatic strains have a 1162-bp deletion in the promoter of STA1. With CRISPR/Cas9-aided reverse engineering, we show that this deletion greatly decreases the ability to grow in beer and consume dextrin, and the expression of STA1. New PCR primers were designed for differentiation of highly and poorly diastatic strains based on the presence of the deletion in the STA1 promoter. In addition, using publically available whole genome sequence data, we show that the STA1 gene is prevalent among the 'Beer 2'/'Mosaic Beer' brewing strains. These strains utilize maltotriose efficiently, but the mechanisms for this have been unknown. By deleting STA1 from a number of highly diastatic strains, we show here that extracellular hydrolysis of maltotriose through STA1 appears to be the dominant mechanism enabling maltotriose use during wort fermentation in STA1+ strains. The formation and retention of STA1 seems to be an alternative evolutionary strategy for efficient utilization of sugars present in brewer's wort. The results of this study allow for the improved reliability of molecular detection methods for diastatic contaminants in beer and can be exploited for strain development where maltotriose use is desired.
| S-EPMC6719335 | biostudies-literature
Project description:Grape must or freshly pressed grape juice is a complex chemical matrix that impacts the efficiency of yeast fermentation. The composition of natural grape must (NGM) can be variable; thus, to ensure reproducibility, a synthetic grape must (SGM) with defined composition is commonly used. The aim of this work was to create conditions to advance the use of Saccharomyces cerevisiae laboratory strains for wine fermentation studies, considering previous results obtained for enological strains fermenting NGM under simulated winery conditions. We designed a new SGM formulation, ISA-SGM, by introducing specific modifications to a commonly used formulation, putting together previous reports. We added glucose and fructose in equal amounts (125 g/l) and 50 parts per million (ppm) sulfur dioxide (SO2, corresponding to standard enological treatment), and we optimized the concentrations of malic acid (3 g/l), citric acid (0.3 g/l), and tartaric acid (3 g/l). Using ISA-SGM, we obtained similar fermentative profiles for the wine strain ISA1000, the prototrophic strain S288C, and its auxotrophic derivative BY4741. In this case, the concentrations of supplements were optimized to 120 mg/l L-uracil, 80 mg/l L-methionine, 400 mg/l L-leucine, and 100 mg/l L-histidine. All these strains tested in ISA-SGM presented a similar fermentative performance as ISA1000 in NGM. ISA-SGM formulation is a promising new tool to allow the use of the auxotrophic BY strains in the detailed assessment of the alcoholic fermentation process under simulated winery conditions, and it provides a foundation to extract relevant physiological conclusions in future research on enological yeast traits.
| S-EPMC4052690 | biostudies-literature