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      Hyperosmolarity adversely impacts recombinant protein synthesis by Yarrowia lipolytica—molecular background revealed by quantitative proteomics

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          Abstract

          Abstract

          In this research, we were interested in answering a question whether subjecting a Yarrowia lipolytica strain overproducing a recombinant secretory protein (rs-Prot) to pre-optimized stress factors may enhance synthesis of the rs-Prot. Increased osmolarity (3 Osm kg −1) was the primary stress factor implemented alone or in combination with decreased temperature (20 °C), known to promote synthesis of rs-Prots. The treatments were executed in batch bioreactor cultures, and the cellular response was studied in terms of culture progression, gene expression and global proteomics, to get insight into molecular bases underlying an awaken reaction. Primarily, we observed that hyperosmolarity executed by high sorbitol concentration does not enhance synthesis of the rs-Prot but increases its transcription. Expectedly, hyperosmolarity induced synthesis of polyols at the expense of citric acid synthesis and growth, which was severely limited. A number of stress-related proteins were upregulated, including heat-shock proteins (HSPs) and aldo–keto reductases, as observed at transcriptomics and proteomics levels. Concerted downregulation of central carbon metabolism, including glycolysis, tricarboxylic acid cycle and fatty acid synthesis, highlighted redirection of carbon fluxes. Elevated abundance of HSPs and osmolytes did not outbalance the severe limitation of protein synthesis, marked by orchestrated downregulation of translation (elongation factors, several aa-tRNA synthetases), amino acid biosynthesis and ribosome biogenesis in response to the hyperosmolarity. Altogether we settled that increased osmolarity is not beneficial for rs-Prots synthesis in Y. lipolytica, even though some elements of the response could assist this process. Insight into global changes in the yeast proteome under the treatments is provided.

          Key points

          • Temp enhances, but Osm decreases rs-Prots synthesis by Y. lipolytica.

          • Enhanced abundance of HSPs and osmolytes is overweighted by limited translation.

          • Global proteome under Osm, Temp and Osm Temp treatments was studied.

          Supplementary Information

          The online version contains supplementary material available at 10.1007/s00253-021-11731-y.

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4

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              The Perseus computational platform for comprehensive analysis of (prote)omics data.

              A main bottleneck in proteomics is the downstream biological analysis of highly multivariate quantitative protein abundance data generated using mass-spectrometry-based analysis. We developed the Perseus software platform (http://www.perseus-framework.org) to support biological and biomedical researchers in interpreting protein quantification, interaction and post-translational modification data. Perseus contains a comprehensive portfolio of statistical tools for high-dimensional omics data analysis covering normalization, pattern recognition, time-series analysis, cross-omics comparisons and multiple-hypothesis testing. A machine learning module supports the classification and validation of patient groups for diagnosis and prognosis, and it also detects predictive protein signatures. Central to Perseus is a user-friendly, interactive workflow environment that provides complete documentation of computational methods used in a publication. All activities in Perseus are realized as plugins, and users can extend the software by programming their own, which can be shared through a plugin store. We anticipate that Perseus's arsenal of algorithms and its intuitive usability will empower interdisciplinary analysis of complex large data sets.
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                Author and article information

                Contributors
                ewelina.celinska@up.poznan.pl
                Journal
                Appl Microbiol Biotechnol
                Appl Microbiol Biotechnol
                Applied Microbiology and Biotechnology
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                0175-7598
                1432-0614
                16 December 2021
                16 December 2021
                2022
                : 106
                : 1
                : 349-367
                Affiliations
                [1 ]GRID grid.410688.3, ISNI 0000 0001 2157 4669, Department of Biotechnology and Food Microbiology, , Poznan University of Life Sciences, ; Wojska Polskiego 48, 60-627 Poznan, Poland
                [2 ]GRID grid.5522.0, ISNI 0000 0001 2162 9631, Proteomics and Mass Spectrometry Core Facility, , Malopolska Centre of Biotechnology, Jagiellonian University, ; Gronostajowa 7a, 30-387 Krakow, Poland
                Article
                11731
                10.1007/s00253-021-11731-y
                8720085
                34913994
                b44c4c83-1e4a-497e-a5f1-73c068905de6
                © The Author(s) 2021

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                Categories
                Genomics, Transcriptomics, Proteomics
                Custom metadata
                © Springer-Verlag GmbH Germany, part of Springer Nature 2022

                Biotechnology
                yarrowia lipolytica,heterologous protein,proteomics of stress response
                Biotechnology
                yarrowia lipolytica, heterologous protein, proteomics of stress response

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