| 1. |
- Chen, Xin, 1980, et al.
(författare)
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Dataset for suppressors of amyloid-beta toxicity and their functions in recombinant protein production in yeast
- 2022
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Ingår i: Data in Brief. - : Elsevier BV. - 2352-3409. ; 42
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Tidskriftsartikel (refereegranskat)abstract
- 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's 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] . (C) 2022 The Author(s). Published by Elsevier Inc.
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| 2. |
- Chen, Xin, 1980, et al.
(författare)
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Suppressors of amyloid-β toxicity improve recombinant protein production in yeast by reducing oxidative stress and tuning cellular metabolism
- 2022
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 72, s. 311-324
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Tidskriftsartikel (refereegranskat)abstract
- 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's 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.
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| 3. |
- Kreisel, Katrin, 1991, et al.
(författare)
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DNA polymerase η contributes to genome-wide lagging strand synthesis.
- 2019
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Ingår i: Nucleic acids research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 47:5, s. 2425-2435
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Tidskriftsartikel (refereegranskat)abstract
- DNA polymerase η (pol η) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol ηalso has other cellular roles. Here, we present evidence that pol η competes with DNA polymerases α and δfor the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol η,which contains a PCNA-Interacting Protein motif is required for pol ηto function in lagging strand synthesis. Finally, we provide evidence that a pol η dependent signature is also found to be lagging strand specific in patients with skin cancer. Taken together, these findings provide insight into the physiological role of DNA synthesis by pol η and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer.
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| 4. |
- Kroll, Alexander, et al.
(författare)
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A general model to predict small molecule substrates of enzymes based on machine and deep learning
- 2023
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Ingår i: Nature Communications. - 2041-1723 .- 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- For most proteins annotated as enzymes, it is unknown which primary and/or secondary reactions they catalyze. Experimental characterizations of potential substrates are time-consuming and costly. Machine learning predictions could provide an efficient alternative, but are hampered by a lack of information regarding enzyme non-substrates, as available training data comprises mainly positive examples. Here, we present ESP, a general machine-learning model for the prediction of enzyme-substrate pairs with an accuracy of over 91% on independent and diverse test data. ESP can be applied successfully across widely different enzymes and a broad range of metabolites included in the training data, outperforming models designed for individual, well-studied enzyme families. ESP represents enzymes through a modified transformer model, and is trained on data augmented with randomly sampled small molecules assigned as non-substrates. By facilitating easy in silico testing of potential substrates, the ESP web server may support both basic and applied science.
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| 5. |
- Kroll, Alexander, et al.
(författare)
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Deep learning allows genome-scale prediction of Michaelis constants from structural features
- 2021
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Ingår i: PLoS Biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 19:10
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Tidskriftsartikel (refereegranskat)abstract
- AU The:Michaelis Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly constant KM describes the affinity of an enzyme : for a specific substrate and is a central parameter in studies of enzyme kinetics and cellular physiology. As measurements of KM are often difficult and time-consuming, experimental estimates exist for only a minority of enzyme–substrate combinations even in model organisms. Here, we build and train an organism-independent model that successfully predicts KM values for natural enzyme–substrate combinations using machine and deep learning methods. Predictions are based on a task-specific molecular fingerprint of the substrate, generated using a graph neural network, and on a deep numerical representation of the enzyme’s amino acid sequence. We provide genome-scale KM predictions for 47 model organisms, which can be used to approximately relate metabolite concentrations to cellular physiology and to aid in the parameterization of kinetic models of cellular metabolism.
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| 6. |
- Li, Gang, 1991, et al.
(författare)
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Performance of Regression Models as a Function of Experiment Noise
- 2021
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Ingår i: Bioinformatics and Biology Insights. - : SAGE Publications. - 1177-9322. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Background: A challenge in developing machine learning regression models is that it is difficult to know whether maximal performance has been reached on the test dataset, or whether further model improvement is possible. In biology, this problem is particularly pronounced as sample labels (response variables) are typically obtained through experiments and therefore have experiment noise associated with them. Such label noise puts a fundamental limit to the metrics of performance attainable by regression models on the test dataset. Results: We address this challenge by deriving an expected upper bound for the coefficient of determination (R2) for regression models when tested on the holdout dataset. This upper bound depends only on the noise associated with the response variable in a dataset as well as its variance. The upper bound estimate was validated via Monte Carlo simulations and then used as a tool to bootstrap performance of regression models trained on biological datasets, including protein sequence data, transcriptomic data, and genomic data. Conclusions: The new method for estimating upper bounds for model performance on test data should aid researchers in developing ML regression models that reach their maximum potential. Although we study biological datasets in this work, the new upper bound estimates will hold true for regression models from any research field or application area where response variables have associated noise.
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| 7. |
- Peng, Martin, et al.
(författare)
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3D-Printed Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4-Hydroxystilbene
- 2019
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Ingår i: Chemistry - A European Journal. - : Wiley. - 1521-3765 .- 0947-6539. ; 25:70, s. 15998-16001
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Tidskriftsartikel (refereegranskat)abstract
- Continuous flow systems for chemical synthesis are becoming a major focus in organic chemistry and there is a growing interest in the integration of biocatalysts due to their high regio- and stereoselectivity. Methods established for 3D bioprinting enable the fast and simple production of agarose-based modules for biocatalytic reactors if thermally stable enzymes are available. We report here on the characterization of four different cofactor-free phenacrylate decarboxylase enzymes suitable for the production of 4-vinylphenol and test their applicability for the encapsulation and direct 3D printing of disk-shaped agarose-based modules that can be used for compartmentalized flow microreactors. Using the most active and stable phenacrylate decarboxylase from Enterobacter spec. in a setup with four parallel reactors and a subsequent palladium(II) acetate-catalysed Heck reaction, 4-hydroxystilbene was synthesized from p-coumaric acid with a total yield of 14.7 % on a milligram scale. We believe that, due to the convenient direct immobilization of any thermostable enzyme and straightforward tuning of the reaction sequence by stacking of modules with different catalytic activities, this simple process will facilitate the establishment and use of cascade reactions and will therefore be of great advantage for many research approaches.
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| 8. |
- Peng, Martin, et al.
(författare)
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Modeling-Assisted Design of Thermostable Benzaldehyde Lyases from Rhodococcus erythropolis for Continuous Production of α-Hydroxy Ketones
- 2022
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Ingår i: ChemBioChem. - : Wiley. - 1439-7633 .- 1439-4227. ; 23:7
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Tidskriftsartikel (refereegranskat)abstract
- Enantiopure α-hydroxy ketones are important building blocks of active pharmaceutical ingredients (APIs), which can be produced by thiamine-diphosphate-dependent lyases, such as benzaldehyde lyase. Here we report the discovery of a novel thermostable benzaldehyde lyase from Rhodococcus erythropolis R138 (ReBAL). While the overall sequence identity to the only experimentally confirmed benzaldehyde lyase from Pseudomonas fluorescens Biovar I (PfBAL) was only 65 %, comparison of a structural model of ReBAL with the crystal structure of PfBAL revealed only four divergent amino acids in the substrate binding cavity. Based on rational design, we generated two ReBAL variants, which were characterized along with the wild-type enzyme in terms of their substrate spectrum, thermostability and biocatalytic performance in the presence of different co-solvents. We found that the new enzyme variants have a significantly higher thermostability (up to 22 °C increase in T50) and a different co-solvent-dependent activity. Using the most stable variant immobilized in packed-bed reactors via the SpyCatcher/SpyTag system, (R)-benzoin was synthesized from benzaldehyde over a period of seven days with a stable space-time-yield of 9.3 mmol ⋅ L-1 ⋅ d−1. Our work expands the important class of benzaldehyde lyases and therefore contributes to the development of continuous biocatalytic processes for the production of α-hydroxy ketones and APIs.
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| 9. |
- Berglund, Anna-Karin, 1979, et al.
(författare)
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Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA. : Mapping ribonucleotides in mitochondrial DNA
- 2017
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Ingår i: PLoS genetics. - : Public Library of Science (PLoS). - 1553-7404 .- 1553-7390. ; 13:2
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Tidskriftsartikel (refereegranskat)abstract
- Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.
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| 10. |
- Börjesson, Johan, et al.
(författare)
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Effect of poly(ethylene glycol) on enzymatic hydrolysis and adsorption of cellulase enzymes to pretreated lignocellulose
- 2007
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Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 41:1-2, s. 186-195
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Tidskriftsartikel (refereegranskat)abstract
- There is a need to develop the enzymatic hydrolysis of cellulose for production of ethanol from biomass. In recent years the inhibitory effects of lignin in lignocellulosic substrates has been the focus of several studies. This points to the importance of understanding the interactions between cellulose degrading enzymes and lignin. Surface active substances have been shown to adsorb to lignin surfaces resulting in reduction of unproductive enzyme binding. It is essential to understand the surface properties of both enzymes and lignin to develop pretreatment methods, surface active additives and engineering of cellulose degrading enzyme systems. This study investigates the PEG-lignin interaction as well as interactions between lignin and the enzyme modules of the Hypocrea jecorina (Trichoderma reesei) enzymes Cel7A and Cel7B. Interactions were monitored with C-14 labelled PEG 4000 and by measuring the enzymatic activity in solution. It was found that the dominating driving force of PEG adsorption on lignin is hydrophobic interaction. The effect of PEG addition on enzyme conversion of lignocellulose increased with higher temperature due to increased adsorption of PEG on lignin, thus resulting in a higher surface density of PEG on the surface. The hydrophobic adsorption of enzymes to lignin induces denaturation of enzymes on lignin surfaces. The addition of PEG to the enzyme hydrolysis at a temperature of 50 degrees C is suggested to hinder deactivation of enzymes by exclusion of enzymes from lignin surfaces. The adsorption of full-length Cel7B to lignin was stronger than for Cel7A. A more hydrophobic surface on the flat face of the cellulose binding module (CBM) together with an additional exposed aromatic residue on the rough face of Cel7B CBM compared to Cel7A CBM gives a higher affinity to lignin for the Cel7B enzyme. (c) 2007 Elsevier Inc. All rights reserved.
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