| 51. |
- Huang, Mingtao, 1984, et al.
(författare)
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Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
- 2015
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 112:34, s. E4689-E4696
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Tidskriftsartikel (refereegranskat)abstract
- There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant a-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.
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| 52. |
- Ishchuk, Olena, 1980, et al.
(författare)
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Genome-scale modeling drives 70-fold improvement of intracellular heme production in Saccharomyces cerevisiae
- 2022
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 119:30
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Tidskriftsartikel (refereegranskat)abstract
- Heme is an oxygen carrier and a cofactor of both industrial enzymes and food additives. The intracellular level of free heme is low, which limits the synthesis of heme proteins. Therefore, increasing heme synthesis allows an increased production of heme proteins. Using the genome-scale metabolic model (GEM) Yeast8 for the yeast Saccharomyces cerevisiae, we identified fluxes potentially important to heme synthesis. With this model, in silico simulations highlighted 84 gene targets for balancing biomass and increasing heme production. Of those identified, 76 genes were individually deleted or overexpressed in experiments. Empirically, 40 genes individually increased heme production (up to threefold). Heme was increased by modifying target genes, which not only included the genes involved in heme biosynthesis, but also those involved in glycolysis, pyruvate, Fe-S clusters, glycine, and succinyl-coenzyme A (CoA) metabolism. Next, we developed an algorithmic method for predicting an optimal combination of these genes by using the enzyme-constrained extension of the Yeast8 model, ecYeast8. The computationally identified combination for enhanced heme production was evaluated using the heme ligand-binding biosensor (Heme-LBB). The positive targets were combined using CRISPR-Cas9 in the yeast strain (IMX581-HEM15-HEM14-HEM3- δshm1-HEM2-δhmx1-FET4-δgcv2-HEM1-δgcv1-HEM13), which produces 70-foldhigher levels of intracellular heme.
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| 53. |
- Karlsson, Fredrik, 1984, et al.
(författare)
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Prospects for systems biology and modeling of the gut microbiome
- 2011
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Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799 .- 1879-3096. ; 29:6, s. 251-258
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Tidskriftsartikel (refereegranskat)abstract
- Abundant microorganisms that inhabit the human intestine are implicated in health and disease. The gut microbiome has been studied with metagenomic tools, and over 3 million genes have been discovered, constituting a 'parts list' of this ecosystem; further understanding requires studies of the interacting parts. Mouse models have provided a glimpse into the microbiota and host interactions at metabolic and immunologic levels; however, to provide more insight, there is a need to generate mathematical models that can reveal genotype-phenotype relationships and provide scaffolds for integrated analyses. To this end, we propose the use of genome-scale metabolic models that have successfully been used in studying interactions between human hosts and microbes, as well as microbes in isolation and in communities.
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| 54. |
- Karlsson, Fredrik, 1984, et al.
(författare)
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Symptomatic atherosclerosis is associated with an altered gut metagenome
- 2012
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Recent findings have implicated the gut microbiota as a contributor of metabolic diseases through the modulation of host metabolism and inflammation. Atherosclerosis is associated with lipid accumulation and inflammation in the arterial wall, and bacteria have been suggested as a causative agent of this disease. Here we use shotgun sequencing of the gut metagenome to demonstrate that the genus Collinsella was enriched in patients with symptomatic atherosclerosis, defined as stenotic atherosclerotic plaques in the carotid artery leading to cerebrovascular events, whereas Roseburia and Eubacterium were enriched in healthy controls. Further characterization of the functional capacity of the metagenomes revealed that patient gut metagenomes were enriched in genes encoding peptidoglycan synthesis and depleted in phytoene dehydrogenase; patients also had reduced serum levels of β-carotene. Our findings suggest that the gut metagenome is associated with the inflammatory status of the host and patients with symptomatic atherosclerosis harbor characteristic changes in the gut metagenome.
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| 55. |
- Kehoe, Laura, et al.
(författare)
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Make EU trade with Brazil sustainable
- 2019
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 364:6438, s. 341-
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 56. |
- Kjaer, Christina, et al.
(författare)
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Luminescence Spectroscopy of Rhodamine Homodimer Dications in Vacuo Reveals Strong Dye-Dye Interactions
- 2019
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Ingår i: ChemPhysChem. - : Wiley. - 1439-4235 .- 1439-7641. ; 20:4, s. 533-537
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Tidskriftsartikel (refereegranskat)abstract
- Being alone or together makes a difference for the photophysics of dyes but for ionic dyes it is difficult to quantify the interactions due to solvent screening and nearby counter ions. Gas-phase luminescence experiments are desirable and now possible based on recent developments in mass spectrometry. Here we present results on tailor-made rhodamine homodimers where two dye cations are separated by methylene linkers, (CH2)(n). In solution the fluorescence is almost identical to that from the monomer whereas the emission from bare cation dimers redshifts with decreasing n. In the absence of screening, the electric field from the charge on one dye is strong enough to polarize the other dye, both in the ground state and in the excited state. An electrostatic model based on symmetric dye responses (equal induced-dipole moments in ground state) captures the underlying physics and demonstrates interaction even at large distances. Our results have possible implications for gas-phase Forster Resonance Energy Transfer.
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| 57. |
- Kristensen, Frederik Pagh Bredahl, et al.
(författare)
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The Prevalence of Polyneuropathy in Type 2 Diabetes Subgroups Based on HOMA2 Indices of b-Cell Function and Insulin Sensitivity
- 2023
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Ingår i: Diabetes Care. - 0149-5992. ; 46:8, s. 1546-1555
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE Metabolic syndrome components may cumulatively increase the risk of diabetic polyneuropathy (DPN) in type 2 diabetes mellitus (T2DM) patients, driven by insulin resistance and hyperinsulinemia. We investigated the prevalence of DPN in three T2DM subgroups based on indices of b-cell function and insulin sensitivity. RESEARCH DESIGN AND METHODS We estimated b-cell function (HOMA2-B) and insulin sensitivity (HOMA2-S) in 4,388 Danish patients with newly diagnosed T2DM. Patients were categorized into subgroups of hyperinsulinemic (high HOMA2-B, low HOMA2-S), classical (low HOMA2-B, low HOMA2-S), and insulinopenic (low HOMA2-B, high HOMA2-S) T2DM. After a median follow-up of 3 years, patients filled the Michigan Neuropathy Screening Instrument questionnaire (MNSIq) to identify DPN (score ‡ 4). We used Poisson regression to calculate adjusted prevalence ratios (PRs) for DPN, and spline models to examine the association with HOMA2-B and HOMA2-S. RESULTS A total of 3,397 (77%) patients filled in the MNSIq. The prevalence of DPN was 23% among hyperinsulinemic, 16% among classical, and 14% among insulinopenic pa-tients. After adjusting for demographics, diabetes duration and therapy, lifestyle behaviors, and metabolic syndrome components (waist circumference, triglycer-ides, HDL cholesterol, hypertension, and HbA1c), the PR of DPN was 1.35 (95% CI 1.15–1.57) for the hyperinsulinemic compared with the classical patients. In spline analyses, we observed a linear relation of higher DPN prevalence with increasing HOMA2-B, independent of both metabolic syndrome components and HOMA2-S. CONCLUSIONS Hyperinsulinemia marked by high HOMA2-B is likely an important risk factor for DPN beyond metabolic syndrome components and insulin resistance. This should be considered when developing interventions to prevent DPN.
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| 58. |
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| 59. |
- Liu, Lifang, 1979, et al.
(författare)
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Balanced globin protein expression and heme biosynthesis improve production of human hemoglobin in Saccharomyces cerevisiae
- 2014
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 21, s. 9-16
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Tidskriftsartikel (refereegranskat)abstract
- Due to limitations associated with whole blood for transfusions (antigen compatibility, transmission of infections, supply and storage), the use of cell-free hemoglobin as an oxygen carrier substitute has been in the center of research interest for decades. Human hemoglobin has previously been synthesized in yeast, however the challenge is to balance the expression of the two different globin subunits, as well as the supply of the prosthetic heme required for obtaining the active hemoglobin (alpha(2)beta(2)). In this work we evaluated the expression of different combinations of alpha and beta peptides and combined this with metabolic engineering of the heme biosynthetic pathway. Through evaluation of several different strategies we showed that engineering the biosynthesis pathway can substantially increase the heme level in yeast cells, and this resulted in a significant enhancement of human hemoglobin production. Besides demonstration of improved hemoglobin production our work demonstrates a novel strategy for improving the production of complex proteins, especially multimers with a prosthetic group. Crown Copyright (C) 2013 Published by Elsevier Inc. on behalf of International Metabolic Engineering Society. All rights reserved.
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| 60. |
- Liu, Lifang, 1979, et al.
(författare)
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Improving heterologous protein secretion at aerobic conditions by activating hypoxia-induced genes in Saccharomyces cerevisiae
- 2015
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 15:7, s. 10-
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Tidskriftsartikel (refereegranskat)abstract
- Oxygen is important for normal aerobic metabolism, as well as for protein production where it is needed for oxidative protein folding. However, several studies have reported that anaerobic conditions seem to be more favorable in terms of recombinant protein production. We were interested in increasing recombinant protein production under aerobic conditions so we focused on Rox1p regulation. Rox1p is a transcriptional regulator, which in oxidative conditions represses genes induced in hypoxia. We deleted ROX1 and studied the effects on the production of recombinant proteins in Saccharomyces cerevisiae. Intriguingly, we found a 100% increase in the recombinant fungal alpha-amylase yield, as well as productivity. Varied levels of improvements were also observed for the productions of the human insulin precursor and the yeast endogenous enzyme invertase. Based on the genome-wide transcriptional response, we specifically focused on the effect of UPC2 upregulation on protein production and suggested a possible mechanistic explanation.
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