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- Blöbaum, Luisa, et al.
(author)
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Quantifying microbial robustness in dynamic environments using microfluidic single-cell cultivation
- 2024
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In: Microbial Cell Factories. - 1475-2859. ; 23:1, s. 44-
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Journal article (peer-reviewed)abstract
- BACKGROUND: Microorganisms must respond to changes in their environment. Analysing the robustness of functions (i.e. performance stability) to such dynamic perturbations is of great interest in both laboratory and industrial settings. Recently, a quantification method capable of assessing the robustness of various functions, such as specific growth rate or product yield, across different conditions, time frames, and populations has been developed for microorganisms grown in a 96-well plate. In micro-titer-plates, environmental change is slow and undefined. Dynamic microfluidic single-cell cultivation (dMSCC) enables the precise maintenance and manipulation of microenvironments, while tracking single cells over time using live-cell imaging. Here, we combined dMSCC and a robustness quantification method to a pipeline for assessing performance stability to changes occurring within seconds or minutes. RESULTS: Saccharomyces cerevisiae CEN.PK113-7D, harbouring a biosensor for intracellular ATP levels, was exposed to glucose feast-starvation cycles, with each condition lasting from 1.5 to 48 min over a 20 h period. A semi-automated image and data analysis pipeline was developed and applied to assess the performance and robustness of various functions at population, subpopulation, and single-cell resolution. We observed a decrease in specific growth rate but an increase in intracellular ATP levels with longer oscillation intervals. Cells subjected to 48 min oscillations exhibited the highest average ATP content, but the lowest stability over time and the highest heterogeneity within the population. C ONCLUSION: The proposed pipeline enabled the investigation of function stability in dynamic environments, both over time and within populations. The strategy allows for parallelisation and automation, and is easily adaptable to new organisms, biosensors, cultivation conditions, and oscillation frequencies. Insights on the microbial response to changing environments will guide strain development and bioprocess optimisation.
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| 2. |
- Laukkonen Ravn, Jonas, 1987, et al.
(author)
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Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
- 2024
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In: Microbial Cell Factories. - 1475-2859. ; 23:85
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Journal article (peer-reviewed)abstract
- Background The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. Results The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L− 1 after 48 h under oxygen limited condition in bioreactor fermentations. Conclusion This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast’s expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.
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| 3. |
- Torello Pianale, Luca, 1995
(author)
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Tools and applications to assess yeast physiology and robustness in bioprocesses: Lab-scale methods from single cells to populations
- 2024
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Doctoral thesis (other academic/artistic)abstract
- Bioprocesses enable the efficient production of valuable chemicals by microorganisms such as the yeast Saccharomyces cerevisiae . Predictable and stochastic perturbations affect microbial performance in an industrial-scale bioreactor. Because some of these complex and dynamic perturbations are difficult to mimic at a small scale, strains selected and developed in the lab might underperform in industrial settings, creating challenges during scale-up. Moreover, the ability of a system to maintain a stable performance, defined as microbial robustness, has been overlooked owing to a scarcity of suitable quantification methods. This thesis describes novel approaches for characterising industrially relevant microorganisms at laboratory scale. The developed methods and techniques were applied to one laboratory and two industrial yeast strains predominantly in the context of second-generation biofuel production. Yeast physiology was explored by both canonical methods and real-time monitoring of eight intracellular parameters using the Sc EnSor Kit. To complement physiology, the concept of robustness was explained and elaborated. A recently formulated method for quantifying robustness was applied to physiological data to determine the stability of cell performance and expand the concept of robustness itself. Lastly, the physiology and robustness of yeast cells exposed to rapid feast-starvation oscillations were investigated using dynamic microfluidics single-cell cultivation. This technique proved instrumental in mimicking, at a laboratory scale, the fast dynamics encountered within large-scale bioreactors. In summary, the tools presented in this thesis address some of the challenges associated with the scaling up of bioprocesses. Owing to the multilevel resolution, ranging from populations to single cells, the developed techniques have the potential to advance our understanding of microbial performance and robustness, ensuring more efficient and reliable industrial applications of engineered microorganisms.
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