| 1. |
- 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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| 2. |
- 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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| 3. |
- 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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