| 1. |
- Laukkonen Ravn, Jonas, 1987, et al.
(författare)
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Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
- 2024
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Ingår i: Microbial Cell Factories. - 1475-2859. ; 23:85
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Tidskriftsartikel (refereegranskat)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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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- Torello Pianale, Luca, 1995, et al.
(författare)
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Fine-tuning the stress response of Saccharomyces cerevisiae using CRISPR interference technology
- 2019
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Efficient biochemical conversion of renewable carbon sources is crucial for the transition into an entirely renewable energy system and a resource-efficient society. However, the substitution of fossil-based chemicals with renewable biochemicals requires the production to be significantly more efficient and price competitive. Remediation of several technical bottlenecks is needed before this can be accomplished. Production of second-generation biochemicals (made from lignocellulosic biomass) is challenging due to presence of inhibitors in lignocellulosic hydrolysates. Weak acids, furans and phenolic compounds that are formed or released during hydrolysis of biomass are toxic for the producing cells and leads to suboptimal yield and productivity obtained during fermentation. In this project, we are trying to fine tune the expression of stress related genes to boost the stress tolerance in Saccharomyces cerevisiae using the CRISPR interference (CRISPRi) technology. CRISPRi is a genetic perturbation technique that allows sequence-specific repression or activation of gene expression, achieved by a catalytically inactive Cas9 protein fused to a repressor or activator, which can be targeted to any genetic loci using an sgRNA. Using a high-throughput yeast transformation method developed in our laboratory, we are generating a CRISPRi strain library. Each strain in this library has altered regulation for at-least one stress related gene. Next, high-throughput phenotypic evaluation of this library is performed by growing the strains under the exposure of inhibitors relevant to lignocellulosic hydrolysates. Here, we will demonstrate our primary CRISPRi library data. Further, we will explain the high-throughput methodologies for generating the CRISPRi mutants and to study their hydrolysate tolerance, adaptation and ethanol production capacity at microscale. In future, we will perform transcriptomics analysis of the most tolerant mutants to link superior phenotypes to the transcriptomic landscape. Subsequently, this novel information will be used as a resource to accelerate the design-build-test-learn cycle used for developing industrial yeast strains for efficient conversion of lignocellulosic hydrolysate.
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| 6. |
- Torello Pianale, Luca, 1995, et al.
(författare)
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Four ways of implementing robustness quantification in strain characterisation
- 2023
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Ingår i: Biotechnology for Biofuels and Bioproducts. - 2731-3654. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- Background. In industrial bioprocesses, microorganisms are generally selected based on performance, whereas robustness, i.e., the ability of a system to maintain a stable performance, has been overlooked due to the challenges in its quantification and implementation into routine experimental procedures. This work presents four ways of implementing robustness quantification during strain characterisation. One Saccharomyces cerevisiae laboratory strain (CEN.PK113-7D) and two industrial strains (Ethanol Red and PE2) grown in seven different lignocellulosic hydrolysates were assessed for growth-related functions (specific growth rate, product yields, etc.) and eight intracellular parameters (using fluorescent biosensors). Results. Using flasks and high-throughput experimental setups, robustness was quantified in relation to: (i) stability of growth functions in response to the seven hydrolysates; (ii) stability of growth functions across different strains to establish the impact of perturbations on yeast metabolism; (iii) stability of intracellular parameters over time; (iv) stability of intracellular parameters within a cell population to indirectly quantify population heterogeneity. Ethanol Red was the best-performing strain under all tested conditions, achieving the highest growth function robustness. PE2 displayed the highest population heterogeneity. Moreover, the intracellular environment varied in response to non-woody or woody lignocellulosic hydrolysates, manifesting increased oxidative stress and unfolded protein response, respectively. Conclusions. Robustness quantification is a powerful tool for strain characterisation as it offers novel information on physiological and biochemical parameters. Owing to the flexibility of the robustness quantification method, its implementation was successfully validated at single-cell as well as high-throughput levels, showcasing its versatility and potential for several applications.
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| 7. |
- 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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| 8. |
- Torello Pianale, Luca, 1995, et al.
(författare)
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Real-Time Monitoring of the Yeast Intracellular State During Bioprocesses With a Toolbox of Biosensors
- 2022
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Industrial fermentation processes strive for high robustness to ensure optimal and consistent performance. Medium components, fermentation products, and physical perturbations may cause stress and lower performance. Cellular stress elicits a range of responses, whose extracellular manifestations have been extensively studied; whereas intracellular aspects remain poorly known due to lack of tools for real-time monitoring. Genetically encoded biosensors have emerged as promising tools and have been used to improve microbial productivity and tolerance toward industrially relevant stresses. Here, fluorescent biosensors able to sense the yeast intracellular environment (pH, ATP levels, oxidative stress, glycolytic flux, and ribosome production) were implemented into a versatile and easy-to-use toolbox. Marker-free and efficient genome integration at a conserved site on chromosome X of Saccharomyces cerevisiae strains and a commercial Saccharomyces boulardii strain was developed. Moreover, multiple biosensors were used to simultaneously monitor different intracellular parameters in a single cell. Even when combined together, the biosensors did not significantly affect key physiological parameters, such as specific growth rate and product yields. Activation and response of each biosensor and their interconnection were assessed using an advanced micro-cultivation system. Finally, the toolbox was used to screen cell behavior in a synthetic lignocellulosic hydrolysate that mimicked harsh industrial substrates, revealing differences in the oxidative stress response between laboratory (CEN.PK113-7D) and industrial (Ethanol Red) S. cerevisiae strains. In summary, the toolbox will allow both the exploration of yeast diversity and physiological responses in natural and complex industrial conditions, as well as the possibility to monitor production processes.
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| 9. |
- Torello Pianale, Luca, 1995, et al.
(författare)
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ScEnSor Kit for Saccharomyces cerevisiae Engineering and Biosensor-Driven Investigation of the Intracellular Environment
- 2023
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Ingår i: ACS Synthetic Biology. - 2161-5063. ; 12:8, s. 2493-2497
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Tidskriftsartikel (refereegranskat)abstract
- In this study, the three-step build-transform-assess toolbox for real-time monitoring of the yeast intracellular environment has been expanded and upgraded to the two-module ScEnSor (S. cerevisiae Engineering + Biosensor) Kit. The Biosensor Module includes eight fluorescent reporters for the intracellular environment; three of them (unfolded protein response, pyruvate metabolism, and ethanol consumption) were newly implemented to complement the original five. The Genome-Integration Module comprises a set of backbone plasmids for the assembly of 1-6 transcriptional units (each consisting of promoter, coding sequence, and terminator) for efficient marker-free single-locus genome integration (in HO and/or X2 loci). Altogether, the ScEnSor Kit enables rapid and easy construction of strains with new transcriptional units as well as high-throughput investigation of the yeast intracellular environment.
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| 10. |
- Torello Pianale, Luca, 1995
(författare)
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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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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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