| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Maertens, Jeroen, 1990, et al.
(författare)
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Molecular-dynamics-simulation-guided membrane engineering allows the increase of membrane fatty acid chain length in Saccharomyces cerevisiae
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- The use of lignocellulosic-based fermentation media will be a necessary part of the transition to a circular bio-economy. These media contain many inhibitors to microbial growth, including acetic acid. Under industrially relevant conditions, acetic acid enters the cell predominantly through passive diffusion across the plasma membrane. The lipid composition of the membrane determines the rate of uptake of acetic acid, and thicker, more rigid membranes impede passive diffusion. We hypothesized that the elongation of glycerophospholipid fatty acids would lead to thicker and more rigid membranes, reducing the influx of acetic acid. Molecular dynamics simulations were used to predict the changes in membrane properties. Heterologous expression of Arabidopsis thaliana genes fatty acid elongase 1 (FAE1) and glycerol-3-phosphate acyltransferase 5 (GPAT5) increased the average fatty acid chain length. However, this did not lead to a reduction in the net uptake rate of acetic acid. Despite successful strain engineering, the net uptake rate of acetic acid did not decrease. We suggest that changes in the relative abundance of certain membrane lipid headgroups could mitigate the effect of longer fatty acid chains, resulting in a higher net uptake rate of acetic acid.
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| 3. |
- Olsson, Lisbeth, 1963, et al.
(författare)
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Microbial robustness in bioprocesses
- 2023
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Yeast is broadly exploited for industrial use, and strains are constantly improved to meet the requirements to produce the targeted product with high yield, productivity and titer. Successful strains have consistent performance also in presence of different perturbations, i.e. their performance is robust. The concept of microbial robustness will be discussed and contrasted to tolerance toward specific stresses. Furthermore, a method to quantitatively assess microbial robustness will be presented. This method allows a high throughput evaluation, in a perturbation space where different cellular function can form the basis for the evaluation. Another important tool box to examine intracellular status in face of pertubations are biosensors. Examples of applying these two methodologies towards microbial robustness will be discussed. We have used the tools to scale down bioprocesses and their perturbation, to follow adaptive laboratory evolution and to gain understanding of subpopulations.
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| 4. |
- Olsson, Lisbeth, 1963, et al.
(författare)
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Robustness: linking strain design to viable bioprocesses
- 2022
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Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799 .- 1879-3096. ; 40:8, s. 918-931
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Forskningsöversikt (refereegranskat)abstract
- Microbial cell factories are becoming increasingly popular for the sustainable production of various chemicals. Metabolic engineering has led to the design of advanced cell factories; however, their long-term yield, titer, and productivity falter when scaled up and subjected to industrial conditions. This limitation arises from a lack of robustness – the ability to maintain a constant phenotype despite the perturbations of such processes. This review describes predictable and stochastic industrial perturbations as well as state-of-the-art technologies to counter process variability. Moreover, we distinguish robustness from tolerance and discuss the potential of single-cell studies for improving system robustness. Finally, we highlight ways of achieving consistent and comparable quantification of robustness that can guide the selection of strains for industrial bioprocesses.
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| 5. |
- Torello Pianale, Luca, 1995, et al.
(författare)
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Exploring Microbial Robustness for a Sustainable and Efficient Bioproduction
- 2020
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Efficient microbial cell factories that produce valuable compounds are gaining increasing interest as one path towards a more sustainable economy. Therefore, there is an increasing need for robust microorganisms which can optimally perform even in harsh and challenging industrial conditions. The identification of robustness traits is crucial to improve the already-existing strains and develop new, better ones. Here, different approaches to study microbial robustness are presented. First, single-cell analysis in a cell population might give some insights on the development of more robust sub-populations. Physiological parameters (such as intracellular pH, fluxes, redox balance, etc.) and morphologic features were monitored with fluorescent biosensors and tagged proteins to study the single-cell status. Moreover, a barcoding technique will be used to discover and underline patterns in the development of population dynamics during the different industrial processes. Furthermore, an objective method to quantify robustness was developed for selection of useful strains and a large dataset was analysed to find predictive parameters for robustness. All together, these tools will give the possibility to identify robustness traits and understand robustness leading to improved industrial strains and processes.
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| 6. |
- Torello Pianale, Luca, 1995, et al.
(författare)
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Microbial robustness 101: tools and applications
- 2022
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Striving for a fossil-free society, bio-production is gaining increasing interest over time. Bioproduction applies microorganisms (bacteria, yeast, fungi) to produce valuable chemicals from different raw materials (plant biomass, waste materials, etc.) and offers sustainable use of side-streams and/or waste streams. Bioproduction suffers from challenges such as poor microbial performance and reproducibility. One key feature in this field is microbial robustness, i.e., the stability of a phenotype (cellular function) when a system is challenged by different perturbations. Microbial robustness, due to its abstract nature, has been poorly studied also due to the lack of tools available. Moreover, being able to include robustness evaluation in the early stages of bioprocess and strain design would facilitate their scaling up from the laboratory- to the industrial scales. Here two tools to explore microbial robustness with some applications and case studies in Saccharomyces cerevisiae are presented. First, a way to quantify the robustness of cellular functions was developed. The robustness coefficient proposed allows comparison between strains and cellular functions in a given perturbation space. This method, based on the Fano factor, is dimensionless, free from arbitrary control conditions and frequency-independent. Second, fluorescent biosensors sensing the intracellular environment were developed into a versatile and easy-to-use toolbox. Such toolbox was used in population studies to identify different physiological responses in different strains exposed to industrially-relevant media and conditions. In the future, it will be implemented in single-cell analysis in microfluidic devices and for studying the formation of subpopulations in large-scale fermentations. All together, these tools will give the possibility to identify robustness traits and mechanisms, allowing for physiological insights that are a foundation for improving industrial strains and process designs.
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| 7. |
- Trivellin, Cecilia, 1993, et al.
(författare)
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Performance and robustness analysis reveals phenotypic trade-offs in yeast
- 2024
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Ingår i: Life Science Alliance. - 2575-1077. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- To design strains that can function efficiently in complex industrial settings, it is crucial to consider their robustness, that is, the stability of their performance when faced with perturbations. In the present study, we cultivated 24 Saccharomyces cerevisiae strains under conditions that simulated perturbations encountered during lignocellulosic bioethanol production, and assessed the performance and robustness of multiple phenotypes simultaneously. The observed negative correlations confirmed a trade-off between performance and robustness of ethanol yield, biomass yield, and cell dry weight. Conversely, the specific growth rate performance positively correlated with the robustness, presumably because of evolutionary selection for robust, fast-growing cells. The Ethanol Red strain exhibited both high performance and robustness, making it a good candidate for bioproduction in the tested perturbation space. Our results experimentally map the robustness-performance trade-offs, previously demonstrated mainly by single-phenotype and computational studies.
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| 8. |
- Trivellin, Cecilia, 1993, et al.
(författare)
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Quantification of Microbial Robustness in Yeast
- 2022
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 11:4, s. 1686-1691
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Tidskriftsartikel (refereegranskat)abstract
- Stable cell performance in a fluctuating environment is essential for sustainable bioproduction and synthetic cell functionality; however, microbial robustness is rarely quantified. Here, we describe a high-throughput strategy for quantifying robustness of multiple cellular functions and strains in a perturbation space. We evaluated quantification theory on experimental data and concluded that the mean-normalized Fano factor allowed accurate, reliable, and standardized quantification. Our methodology applied to perturbations related to lignocellulosic bioethanol production showed that the industrial bioethanol producing strain Saccharomyces cerevisiae Ethanol Red exhibited both higher and more robust growth rates than the laboratory strain CEN.PK and industrial strain PE-2, while a more robust product yield traded off for lower mean levels. The methodology validated that robustness is function-specific and characterized by positive and negative function-specific trade-offs. Systematic quantification of robustness to end-use perturbations will be important to analyze and construct robust strains with more predictable functions.
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| 9. |
- Trivellin, Cecilia, 1993
(författare)
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Robustness quantification in yeast: A methodology to study phenotypic, evolutionary, and genomic aspects of microbial robustness.
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bioprocesses contribute to the shift towards a more sustainable economy. In bioprocesses, valuable chemicals can be generated from renewable resources while, at the same time, reducing carbon emissions. A major hurdle in bringing bio-based products to market is the time and cost involved in designing efficient cell factories. Cell factories developed in controlled laboratory settings achieve high yields and productivities, but often fail at a larger scale because of unforeseen perturbations. Microbial robustness, i.e., the ability to maintain functionality despite perturbations, is critical for designing cell factories but remains poorly studied, particularly with respect to quantification as well as evolutionary and genetic aspects. In this thesis work, mathematical evaluation, phenotypic characterization, evolution and genomics were applied to address the lack of quantification methods and explore robustness in yeast. A Fano factor-based approach for measuring robustness across multiple parameters and perturbations was created. Measurement of physiological data revealed trade-offs between robustness and performance in yeast. Moreover, when screening yeast deletion libraries, it pointed to the MET28 gene, which encodes a transcription factor regulating sulfur metabolism, as a mediator of robustness. Finally, evolution in fluctuating environments improved robustness in the industrial strain Ethanol Red, but not in two laboratory strains, contrasting with fitness trends. Altogether, applying robustness quantification to various experimental set-ups, enabled the identification of key genes and metabolic processes linked to enhanced robustness. This thesis thereby contributes to the field of physiology, particularly in the context of robustness. The developed techniques have the potential to advance design optimization and testing of robust strains in laboratory settings, thereby enabling a faster scale-up to industrial environments.
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