Quantifying absolute gene expression profiles reveals distinct regulation of central carbon metabolism genes in yeast

In addition to controlled expression of genes by specific regulatory circuits, the abundance of proteins and transcripts can also be influenced by physiological states of the cell such as growth rate and metabolism. Here we examine the control of gene expression by growth rate and metabolism, by analyzing a multi-omics dataset consisting of absolute-quantitative abundances of the transcriptome, proteome, and amino acids in 22 steady-state yeast cultures. We find that transcription and translation are coordinately controlled by the cell growth rate via RNA polymerase II and ribosome abundance, but they are independently controlled by nitrogen metabolism via amino acid and nucleotide availabilities. Genes in central carbon metabolism, however, are distinctly regulated and do not respond to the cell growth rate or nitrogen metabolism as all other genes. Understanding these effects allows the confounding factors of growth rate and metabolism to be accounted for in gene expression profiling studies

Nitrogen limitation reveals large reserves in metabolic and translational capacities of yeast

Cells maintain reserves in their metabolic and translational capacities as a strategy to quickly respond to changing environments. Here we quantify these reserves by stepwise\ua0reducing nitrogen availability in yeast steady-state chemostat cultures, imposing severe restrictions on total cellular protein and transcript content. Combining multi-omics analysis with metabolic modeling, we find that seven metabolic superpathways maintain >50% metabolic capacity in reserve, with glucose metabolism maintaining >80% reserve capacity. Cells maintain >50% reserve in translational capacity for 2490 out of 3361 expressed genes (74%), with a disproportionately large reserve dedicated to translating metabolic proteins. Finally, ribosome reserves contain up to 30% sub-stoichiometric ribosomal proteins, with activation of reserve translational capacity associated with selective upregulation of 17 ribosomal proteins. Together, our dataset provides a quantitative link between yeast physiology and cellular economics, which could be leveraged in future cell engineering through targeted proteome streamlining

Suppressors of amyloid-β toxicity improve recombinant protein production in yeast by reducing oxidative stress and tuning cellular metabolism

High-level production of recombinant proteins in industrial microorganisms is often limited by the formation of misfolded proteins or protein aggregates, which consequently induce cellular stress responses. We hypothesized that in a yeast Alzheimer\u27s disease (AD) model overexpression of amyloid-β peptides (Aβ42), one of the main peptides relevant for AD pathologies, induces similar phenotypes of cellular stress. Using this humanized AD model, we previously identified suppressors of Aβ42 cytotoxicity. Here we hypothesize that these suppressors could be used as metabolic engineering targets to alleviate cellular stress and improve recombinant protein production in the yeast Saccharomyces cerevisiae. Forty-six candidate genes were individually deleted and twenty were individually overexpressed. The positive targets that increased recombinant α-amylase production were further combined leading to an 18.7-fold increased recombinant protein production. These target genes are involved in multiple cellular networks including RNA processing, transcription, ER-mitochondrial complex, and protein unfolding. By using transcriptomics and proteomics analyses, combined with reverse metabolic engineering, we showed that reduced oxidative stress, increased membrane lipid biosynthesis and repressed arginine and sulfur amino acid biosynthesis are significant pathways for increased recombinant protein production. Our findings provide new insights towards developing synthetic yeast cell factories for biosynthesis of valuable proteins

Dataset for suppressors of amyloid-beta toxicity and their functions in recombinant protein production in yeast

The production of recombinant proteins at high levels often induces stress-related phenotypes by protein misfolding or aggregation. These are similar to those of the yeast Alzheimer\u27s disease (AD) model in which amyloid-beta peptides (A beta 42) were accumulated [1,2] . We have previously identified suppressors of A beta 42 cytotoxicity via the genome-wide synthetic genetic array (SGA) [3] and here we use them as metabolic engineering targets to evaluate their potentiality on recombinant protein production in yeast Saccharomyces cerevisiae. In order to investigate the mechanisms linking the genetic modifications to the improved recombinant protein production, we perform systems biology approaches (transcriptomics and proteomics) on the resulting strain and intermediate strains. The RNAseq data are preprocessed by the nf-core/RNAseq pipeline and analyzed using the Platform for Integrative Analysis of Omics (PIANO) package [4] . The quantitative proteome is analyzed on an Orbitrap Fusion Lumos mass spectrometer interfaced with an Easy-nLC1200 liquid chromatography (LC) system. LC-MS data files are processed by Proteome Discoverer version 2.4 with Mascot 2.5.1 as a database search engine. The original data presented in this work can be found in the research paper titled "Suppressors of Amyloid-beta Toxicity Improve Recombinant Protein Produc-tion in yeast by Reducing Oxidative Stress and Tuning Cellu-lar Metabolism", by Chen et al. [5]

Strain improvement of Pichia kudriavzevii TY13 for raised phytase production and reduced phosphate repression

In this work, we present the development and characterization of a strain of Pichia kudriavzevii (TY1322), with highly improved phytate-degrading capacity. The mutant strain TY1322 shows a biomass-specific phytate degradation of 1.26 mmol g−1 h−1 after 8 h of cultivation in a high-phosphate medium, which is about 8 times higher compared with the wild-type strain. Strain TY1322 was able to grow at low pH (pH 2), at high temperature (46\ub0C) and in the presence of ox bile (2% w/v), indicating this strain\u27s ability to survive passage through the gastrointestinal tract. The purified phytase showed two pH optima, at pH 3.5 and 5.5, and one temperature optimum at 55\ub0C. The lower pH optimum of 3.5 matches the reported pH of the pig stomach, meaning that TY1322 and/or its phytase is highly suitable for use in feed production. Furthermore, P. kudriavzevii TY1322 tolerates ethanol up to 6% (v/v) and shows high osmotic stress tolerance. Owing to the phenotypic characteristics and non-genetically modified organisms nature of TY1322, this strain show great potential for future uses in (i) cereal fermentations for increased mineral bioavailability, and (ii) feed production to increase the phosphate bioavailability for monogastric animals to reduce the need for artificial phosphate fortification

Abundant Lysine Methylation and N-Terminal Acetylation in Sulfolobus islandicus Revealed by Bottom-Up and Top-Down Proteomics

International audienceProtein posttranslational methylation has been reported to occur in archaea, including members of the genus Sulfolobus, but has never been characterized on a proteome-wide scale. Among important Sulfolobus proteins carrying such modification are the chromatin proteins that have been described to be methylated on lysine side chains, resembling eukaryotic histones in that aspect. To get more insight into the extent of this modification and its dynamics during the different growth steps of the thermoacidophylic archaeon S. islandicus LAL14/1, we performed a global and deep proteomic analysis using a combination of high-throughput bottom-up and proteomics approaches on a single high-resolution mass spectrometer. 1,931 methylation sites on 751 proteins were found by the bottom-up analysis, with methylation sites on 526 proteins monitored throughout three cell culture growth stages: early-exponential, mid-exponential and stationary. The top-down analysis revealed 3,978 proteoforms arising from 681 proteins, including 292 methylated proteoforms, 85 of which were comprehensively characterized. Methylated proteoforms of the five chromatin proteins (Alba1, Alba2, Cren7, Sul7d1, Sul7d2) were fully characterized by a combination of bottom-up and top-down data. The top-down analysis also revealed an increase of methylation during cell growth for two chromatin proteins, which had not been evidenced by bottom-up. These results shed new light on the ubiquitous lysine methylation throughout the S. islandicus proteome. Furthermore, we found that S. islandicus proteins are frequently acetylated at the N-terminus, following the removal of the N-terminal methionine. This study highlights the great value of combining bottom-up and top-down proteomics for obtaining an unprecedented level of accuracy in detecting differentially-modified intact proteoforms. The data have been deposited to the ProteomeXchange with identifiers PXD003074 and PXD004179

OPTIMIZATION OF LEACHING MODE IN HYDROGENIC URANIUM DEPOSITS WITH HIGH PERMEABILITY SAND BENCHMARK

The relevance of the study is caused by the increase in the volume of uranium production at the deposits of the hydrogen type, the most common type on the territory of both the Republic of Kazakhstan. Аt the present stage there is a need to optimize the in-situ leaching to increase uranium ore extraction. The goal of the research is to substantiate the possibility of optimizing the regulation of the in-situ leaching mode at uranium deposits with a mature sandy benchmark and high permeability by reducing the rate of diffuse mass transfer to increase recoverability and prevent secondary ore genesis. Objects: boreholes logs and laboratory data of boreholes of block X of the Moinkum deposit of the Chu-Sarysu uranium ore province. Methods: physical and mathematical modeling, induction logging, gamma ray logging, caliper logging, temperature logging, correlation and analysis of log data using Curve Editor, GIK. The hardware includes PIK-50, KSP-43 instruments, NS caliper, TEG electro thermometer, logging station, BSK interface and switching unit. Results. Data of electric logging, gamma ray logging, and thermometry and caliper log data of the boreholes of block X of the Moinkum deposit, obtained by resistivity logging, intrinsic polarization potential logging and induction logging methods were correlated with subsequent analysis. The geological and technological conditions of ore extraction are revealed on the basis of the analysis. The features of mineralization and patterns of technogenic impact on the ore-bearing rock are analyzed. Optimization solutions were developed for intensification of ore extraction and control of secondary technogenic ore genesis based on the permeability factors of the rocks of the ore-bearing strata. The feasibility of controlling the diffusion rate in leaching by changing the thermo baric regime and the viscosity of the solution at the experimental boreholes of the Moinkum deposit of the Chu-Sarysu uranium ore province was proved. The paper introduces the method for calculating the optimal parameters of the leaching mode based on the characteristics of diffusion in rocks with different permeability

Cytokine responses to LPS in reprogrammed monocytes are associated with the transcription factor PU.1

Myeloid-derived suppressor cells (MDSCs) are functionally immunosuppressive cellsthat arise and expand during extensive inflammatory conditions by increasedhematopoietic output or reprogramming of immune cells. In sepsis, an increase of circulatingMDSCs is associated with adverse outcomes, but unique traits that can beused to identify increased activity of MDSCs are lacking. By using endotoxin toleranceas a model of sepsis-induced monocytic MDSCs (M-MDSC-like cells), this studyaims to identify the mediator and transcriptional regulator profile associated with MMDSCactivity. After analyzing 180 inflammation-associated proteins, a profile of differentiallyexpressed cytokines was found in M-MDSC-like cells versus normal monocytesstimulated with LPS. These cytokines were associated with 5 candidate transcriptionfactors,where particularly PU.1 showed differential expression on both transcriptionaland protein levels in M-MDSC-like cells. Furthermore, inhibition of PU.1led to increased production of CXCL5 and CCL8 in M-MDSC-like cells indicating itsrole in regulating the ability ofM-MDSC-like cells to recruit other immune cells. Takentogether, the study identifies a unique profile in the pattern of immune mediatorsdefining M-MDSC activity upon LPS stimulation, which offers a functional link to theircontribution to immunosuppression.Funding Agency:General Electric</p

Changes in the Proteome in the Development of Chronic Human Papillomavirus Infection&mdash;A Prospective Study in HIV Positive and HIV Negative Rwandan Women

Background: Effects on the proteome when a high risk (HR)-HPV infection occurs, when it is cleared and when it becomes chronic were investigated. Moreover, biomarker panels that could identify cervical risk lesions were assessed. Methods: Cytology, HPV screening and proteomics were performed on cervical samples from Rwandan HIV+ and HIV- women at baseline, at 9 months, at 18 months and at 24 months. Biological pathways were identified using the String database. Results: The most significantly affected pathway when an incident HR-HPV infection occurred was neutrophil degranulation, and vesicle-mediated transport was the most significantly affected pathway when an HR-HPV infection was cleared; protein insertion into membrane in chronic HR-HPV lesions and in lesions where HR-HPVs were cleared were compared; and cellular catabolic process in high-grade lesions was compared to that in negative lesions. A four-biomarker panel (EIF1; BLOC1S5; LIMCH1; SGTA) was identified, which was able to distinguish chronic HR-HPV lesions from cleared HR-HPV/negative lesions (sensitivity 100% and specificity 91%). Another four-biomarker panel (ERH; IGKV2-30; TMEM97; DNAJA4) was identified, which was able to distinguish high-grade lesions from low-grade/negative lesions (sensitivity 100% and specificity 81%). Conclusions: We have identified the biological pathways triggered in HR-HPV infection, when HR-HPV becomes chronic and when cervical risk lesions develop. Moreover, we have identified potential biomarkers that may help to identify women with cervical risk lesions