| 1. |
- Perruca-Foncillas, Raquel, et al.
(författare)
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Assessment of fluorescent protein candidates for multi-color flow cytometry analysis of Saccharomyces cerevisiae
- 2022
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Ingår i: Biotechnology Reports. - : Elsevier BV. - 2215-017X. ; 34
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Tidskriftsartikel (refereegranskat)abstract
- Transcription factor-based biosensors represent promising tools in the construction and evaluation of efficient cell factories for the sustainable production of fuels, chemicals and pharmaceuticals. They can notably be designed to follow the production of a target compound or to monitor key cellular properties, such as stress or starvation. In most cases, the biosensors are built with fluorescent protein (FP) genes as reporter genes because of the direct correlation between promoter activity and fluorescence level that can be measured using, for instance, flow cytometry or fluorometry. The expansion of available FPs offers the possibility of using several FPs - and biosensors – in parallel in one host, with simultaneous detection using multicolor flow cytometry. However, the technique is currently limited by the unavailability of combinations of FP whose genes can be successfully expressed in the host and whose fluorescence can be efficiently distinguished from each other.In the present study, the broad collection of available FPs was explored and four different FPs were successfully expressed in the yeast Saccharomyces cerevisiae: yEGFP, mEGFP, CyOFP1opt and mBeRFPopt. After studying their fluorescence signals, population heterogeneity and possible interactions, we recommend two original combinations of FPs for bi-color flow cytometry: mEGFP together with either CyOFP1opt or mBeRFPopt, as well as the combination of all three FPs mEGFP, CyOFP1opt and mBeRFPopt for tri-color flow cytometry. These combinations will allow to perform different types of bi-color or possibly tri-color flow cytometry and FACS experiments with yeast, such as phenotype evaluation, screening or sorting, by single-laser excitation with a standard 488 nm blue laser.
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| 2. |
- Perruca Foncillas, Raquel, et al.
(författare)
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Assessment of the TRX2p-yEGFP Biosensor to Monitor the Redox Response of an Industrial Xylose-Fermenting Saccharomyces cerevisiae Strain during Propagation and Fermentation
- 2023
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Ingår i: Journal of Fungi. - 2309-608X. ; 9:6
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Tidskriftsartikel (refereegranskat)abstract
- The commercial production of bioethanol from lignocellulosic biomass such as wheat straw requires utilizing a microorganism that can withstand all the stressors encountered in the process while fermenting all the sugars in the biomass. Therefore, it is essential to develop tools for monitoring and controlling the cellular fitness during both cell propagation and sugar fermentation to ethanol. In the present study, on-line flow cytometry was adopted to assess the response of the biosensor TRX2p-yEGFP for redox imbalance in an industrial xylose-fermenting strain of Saccharomyces cerevisiae during cell propagation and the following fermentation of wheat-straw hydrolysate. Rapid and transient induction of the sensor was recorded upon exposure to furfural and wheat straw hydrolysate containing up to 3.8 g/L furfural. During the fermentation step, the induction rate of the sensor was also found to correlate to the initial ethanol production rate, highlighting the relevance of redox monitoring and the potential of the presented tool to assess the ethanol production rate in hydrolysates. Three different propagation strategies were also compared, and it was confirmed that pre-exposure to hydrolysate during propagation remains the most efficient method for high ethanol productivity in the following wheat-straw hydrolysate fermentations.
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| 3. |
- Perruca Foncillas, Raquel
(författare)
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Evaluation of biosensors and flow cytometry as monitoring tools in lignocellulosic bioethanol production
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The significant environmental impact of the current fossil fuel-based industry is a major concern for society. Consequently, various initiatives are being undertaken to establish a more sustainable industrial model. One example is via the transition from conventional fossil fuel refineries to biorefineries, where renewable raw materials are utilised. Amongst these raw materials, the use of lignocellulosic biomass from agricultural residues or wood has been favoured, as it does not compete with food or land resources. In particular, extensive research has been conducted to produce biofuels such as bioethanol from lignocellulosic biomass, referred to as second-generation (2G) bioethanol.In this thesis work, the goal was to develop and apply new tools to address challenges encountered in 2G bioethanol production. Specifically, the work focused on monitoring the impact of inhibitory compounds and mixed sugars on the fermentation performance of the yeast Saccharomyces cerevisiae.Inhibitory compounds are released during the pretreatment of the lignocellulosic biomass, a crucial step necessary to break down its complex structure and to enhance sugar accessibility This thesis work specifically focused on the redox imbalance induced in cells exposed to furaldehydes such as furfural or HMF. To study this effect, a biosensor for redox imbalance, TRX2p-yEGFP, was introduced into the cells and its fluorescence signal was monitored in real-time using flow cytometry. One potential strategy for enhancing the cells' tolerance to these inhibitors is to prepare them by introducing lignocellulosic hydrolysate in the feed during cell propagation. During this pre-exposure phase, a transient induction of the TRX2p-yEGFP biosensor signal for redox imbalance was observed, which gradually diminished. This indicated that, by the time of cell collection, the cells had adapted to the inhibitor concentration within the culture. To examine whether an increased induction level of the biosensor at the time of cell collection influenced the fermentation performance, an automated control system was devised. This system utilised data from the flow cytometry analysis to control the level of inhibitors in the cultivation feed. Consequently, when the biosensor signal began to decline, higher amounts of inhibitors were added, as long as the addition did not lead to an increase in the number of damaged cells.A second biosensor was used in this thesis work to investigate the sugar signalling response of S. cerevisiae to the presence of xylose. Xylose is the second most abundant sugar in lignocellulosic biomass; however, naturally, S. cerevisiae cannot metabolise it. Genetically modified S. cerevisiae strains have been generated by introducing heterologous pathways such as the XR/XDH or XI pathways to enable xylose consumption. Nevertheless, xylose consumption rates remain lower compared to glucose. Sugar signalling emerged as a potential bottleneck in the efficient utilisation of xylose. In the present work, the response of the SUC2p-yEGFP biosensor for sugar signalling was found to vary significantly depending on the pathway employed. A higher induction for the strains carrying the XI pathway was associated with poorer growth on xylose. Lastly, the effect of introducing a xylose epimerase capable of catalysing the conversion between the two anomers, α-D-xylopyranose and β-D-xylopyranose, as a strategy to improve xylose consumption was studied. The effect was enzyme-specific and proved to be particularly beneficial in strains utilising the xylose isomerase from Lachnoclostridium phytofermentans.In conclusion, the results presented in this thesis demonstrate how biosensors can facilitate the understanding and monitoring of intracellular processes that occur within the cell under stress conditions and be a key tool for improving production processes.
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| 4. |
- Persson, Viktor C., et al.
(författare)
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Impact of xylose epimerase on sugar assimilation and sensing in recombinant Saccharomyces cerevisiae carrying different xylose-utilization pathways
- 2023
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Ingår i: Biotechnology for Biofuels and Bioproducts. - 2731-3654. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundOver the last decades, many strategies to procure and improve xylose consumption in Saccharomyces cerevisiae have been reported. This includes the introduction of efficient xylose-assimilating enzymes, the improvement of xylose transport, or the alteration of the sugar signaling response. However, different strain backgrounds are often used, making it difficult to determine if the findings are transferrable both to other xylose-consuming strains and to other xylose-assimilation pathways. For example, the influence of anomerization rates between α- and β-xylopyranose in pathway optimization and sugar sensing is relatively unexplored.ResultsIn this study, we tested the effect of expressing a xylose epimerase in S. cerevisiae strains carrying different xylose-consuming routes. First, XIs originating from three different species in isogenic S. cerevisiae strains were tested and the XI from Lachnoclostridium phytofermentans was found to give the best performance. The benefit of increasing the anomerization rate of xylose by adding a xylose epimerase to the XI strains was confirmed, as higher biomass formation and faster xylose consumption were obtained. However, the impact of xylose epimerase was XI-dependent, indicating that anomer preference may differ from enzyme to enzyme. The addition of the xylose epimerase in xylose reductase/xylitol dehydrogenase (XR/XDH)-carrying strains gave no improvement in xylose assimilation, suggesting that the XR from Spathaspora passalidarum had no anomer preference, in contrast to other reported XRs. The reduction in accumulated xylitol that was observed when the xylose epimerase was added may be associated with the upregulation of genes encoding endogenous aldose reductases which could be affected by the anomerization rate. Finally, xylose epimerase addition did not affect the sugar signaling, whereas the type of xylose pathway (XI vs. XR/XDH) did.ConclusionsAlthough xylose anomer specificity is often overlooked, the addition of xylose epimerase should be considered as a key engineering step, especially when using the best-performing XI enzyme from L. phytofermentans. Additional research into the binding mechanism of xylose is needed to elucidate the enzyme-specific effect and decrease in xylitol accumulation. Finally, the differences in sugar signaling responses between XI and XR/XDH strains indicate that either the redox balance or the growth rate impacts the SNF1/Mig1p sensing pathway.
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