| 1. |
- Papapetridis, Ioannis, et al.
(författare)
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Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae
- 2017
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Ingår i: Biotechnology for Biofuels. - : BioMed Central. - 1754-6834. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD(+)-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining osmotolerance. Results: GPD2 encodes one of two S. cerevisiae isoenzymes of NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP(+)-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1 Delta gpd2 Delta strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L-1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway. Conclusions: Deletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP(+)-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes.
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| 2. |
- van Dijk, Marlous, 1990, et al.
(författare)
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Bottlenecks in lignocellulosic ethanol production: xylose fermentation and cell propagation
- 2017
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Ingår i: European biomass conference 2017, 25th edition, June 12-15; Stockholm, Sweden..
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- A remaining challenge for the development of economically feasible 2nd generation bio-ethanol is low xylose consumption rate and inhibitor tolerance of the utilized Saccharomyces cerevisiae strains. Yeast starter cultures produced for ethanol production in simultaneous saccharification and co-fermentation (SSCF) processes have to meet high, seemingly conflicting requirements. A high biomass yield during propagation is required to produce the high cell concentrations required for the harsh conditions in the proceeding fermentation. Inhibitor tolerance is essential for producing a highly viable starter culture as well as favorable fermentation kinetics. Short-term adaptation of yeast cultures during propagation has been shown to have a positive effect on pentose conversion as well as inhibitor tolerance. Here we propose a model propagation strategy for evaluating physiology of yeast cultures during propagation. This model propagation strategy will be implemented in a study comparing physiology of yeast cultures with and without exposure to lignocellulosic inhibitors during propagation to assess what molecular mechanisms underlie the short-term adaptation response phenotype. For industry, a better control of yeast properties during propagation will result in an improved and consistent performance of yeast starter cultures for SSCF purposes.
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| 3. |
- van Dijk, Marlous, 1990, et al.
(författare)
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Nutrient-supplemented propagation of Saccharomyces cerevisiae improves its lignocellulose fermentation ability
- 2020
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Propagation conditions have been shown to be of considerable importance for the fermentation ability of Saccharomyces cerevisiae. The limited tolerance of yeast to inhibitors present in lignocellulosic hydrolysates is a major challenge in second-generation bioethanol production. We have investigated the hypothesis that the addition of nutrients during propagation leads to yeast cultures with improved ability to subsequently ferment lignocellulosic materials. This hypothesis was tested with and without short-term adaptation to wheat straw or corn stover hydrolysates during propagation of the yeast. The study was performed using the industrial xylose-fermenting S. cerevisiae strain CR01. Adding a mixture of pyridoxine, thiamine, and biotin to unadapted propagation cultures improved cell growth and ethanol yields during fermentation in wheat straw hydrolysate from 0.04 g g−1 to 0.19 g g−1 and in corn stover hydrolysate from 0.02 g g−1 to 0.08 g g−1. The combination of short–term adaptation and supplementation with the vitamin mixture during propagation led to ethanol yields of 0.43 g g−1 in wheat straw hydrolysate fermentation and 0.41 g g−1 in corn stover hydrolysate fermentation. These ethanol yields were improved compared to ethanol yields from cultures that were solely short-term adapted (0.37 and 0.33 g g−1). Supplementing the propagation medium with nutrients in combination with short-term adaptation was thus demonstrated to be a promising strategy to improve the efficiency of industrial lignocellulosic fermentation.
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| 4. |
- van Dijk, Marlous, 1990, et al.
(författare)
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RNA sequencing reveals metabolic and regulatory changes leading to more robust fermentation performance during short-term adaptation of Saccharomyces cerevisiae to lignocellulosic inhibitors
- 2021
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: The limited tolerance of Saccharomyces cerevisiae to inhibitors is a major challenge in second-generation bioethanol production, and our understanding of the molecular mechanisms providing tolerance to inhibitor-rich lignocellulosic hydrolysates is incomplete. Short-term adaptation of the yeast in the presence of dilute hydrolysate can improve its robustness and productivity during subsequent fermentation. Results: We utilized RNA sequencing to investigate differential gene expression in the industrial yeast strain CR01 during short-term adaptation, mimicking industrial conditions for cell propagation. In this first transcriptomic study of short-term adaption of S. cerevisiae to lignocellulosic hydrolysate, we found that cultures respond by fine-tuned up- and down-regulation of a subset of general stress response genes. Furthermore, time-resolved RNA sequencing allowed for identification of genes that were differentially expressed at 2 or more sampling points, revealing the importance of oxidative stress response, thiamin and biotin biosynthesis. furan-aldehyde reductases and specific drug:H+ antiporters, as well as the down-regulation of certain transporter genes. Conclusions: These findings provide a better understanding of the molecular mechanisms governing short-term adaptation of S. cerevisiae to lignocellulosic hydrolysate, and suggest new genetic targets for improving fermentation robustness.
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| 5. |
- van Dijk, Marlous, 1990
(författare)
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Short-term adaptation of S. cerevisiae to lignocellulosic inhibitors: Underlying metabolic and physiological changes
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The limited tolerance of Saccharomyces cerevisiae (budding yeast) to inhibitors present in lignocellulosic hydrolysates is a major challenge in second-generation bioethanol production. Short-term adaptation of the yeast to lignocellulosic hydrolysates during cell propagation has been shown to improve its tolerance, and thus its performance in lignocellulose fermentation. The overall aim of this thesis was to identify molecular and physiological changes during short-term adaptation. In order to facilitate testing of S. cerevisiae physiology in lignocellulosic hydrolysate, a high-throughput methodology for the analysis of yeast strains in dark medium was developed. This methodology allows for monitoring of both aerobic and anaerobic growth of yeast in medium containing different hydrolysates at high reproducibility. The effect that individual nutrient components during propagation, rather than fermentation, has on lignocellulose fermentation performance is lacking. A high-throughput screening of certain vitamins, trace metals and nitrogen sources was performed. It was found that adding a mixture of pyridoxine, thiamine, and biotin to unadapted propagation cultures improved cell growth and ethanol yields during fermentation in wheat straw hydrolysate. Supplementing the propagation medium with nutrients in combination with short-term adaptation was thus demonstrated to be a promising strategy to improve the efficiency of industrial lignocellulosic fermentation. Different S. cerevisiae strain backgrounds are used in the production of a suitable second-generation bioethanol host. In order to facilitate application of results obtained in laboratory experiments it is important to know whether short-term adaptation affects different strains differently. The physiology of two industrial S. cerevisiae strains were investigated while being short-term adapted. During propagation, fed with a hydrolysate containing feed, ethanol accumulation was observed for strain CR01 but not for KE6-12. Additionally, a larger increase in specific ethanol productivity for CR01 was observed than for KE6-12. Thus, short-term adaptation was found to affect S. cerevisiae physiology differently depending on strain background. To gain a more complete insight into the metabolic changes that S. cerevisiae experiences during short‑term adaptation, RNA sequencing was performed on a time-series of samples taken from propagation cultures undergoing short-term adaptation. Expression data was compared to a non‑adapted control using differential gene expression analysis. Results demonstrate, among others, an interesting role for multidrug proton antiporters YHK8 and FLR1 in the process of short-term adaptation.
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| 6. |
- van Dijk, Marlous, 1990, et al.
(författare)
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Small scale screening of yeast strains enables high-throughput evaluation of performance in lignocellulose hydrolysates
- 2020
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Ingår i: Bioresource Technology Reports. - : Elsevier BV. - 2589-014X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Second generation biorefineries demand efficient lignocellulosic hydrolysate fermenting strains and recent advances in strain isolation and engineering have progressed the bottleneck in developing production hosts from generation of strains into testing these under relevant conditions. In this paper, we introduce a methodology for high-throughput analysis of yeast strains directly in lignocellulosic hydrolysates. The Biolector platform was used to assess aerobic and anaerobic growth of 12 Saccharomyces cerevisiae strains and their ΔPdr12 mutants in wheat straw hydrolysate. The strains evaluated included lab, industrial and wild type strains and the screening could capture significant differences in growth and ethanol production among the strains. The methodology was also demonstrated with corn stover hydrolysate and the results were in line with shake flask cultures. Our study demonstrates that growth in lignocellulosic hydrolysates could be rapidly monitored using 1 ml cultures and that measuring growth and product formation under relevant conditions are crucial for evaluating strain performance.
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| 7. |
- van Dijk, Marlous, 1990, et al.
(författare)
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Strain-dependent variance in short-term adaptation effects of two xylose-fermenting strains of Saccharomyces cerevisiae
- 2019
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Ingår i: Bioresource technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 292, s. 121922-
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Tidskriftsartikel (refereegranskat)abstract
- The limited tolerance of Saccharomyces cerevisiae to the inhibitors present in lignocellulosic hydrolysates is a major challenge in second-generation bioethanol production. Short-term adaptation of the yeast to lignocellulosic hydrolysates during cell propagation has been shown to improve its tolerance, and thus its performance in lignocellulose fermentation. The aim of this study was to investigate the short-term adaptation effects in yeast strains with different genetic backgrounds. Fed-batch propagation cultures were supplemented with 40% wheat straw hydrolysate during the feed phase to adapt two different pentose-fermenting strains, CR01 and KE6-12. The harvested cells were used to inoculate fermentation media containing 80% or 90% wheat straw hydrolysate. The specific ethanol productivity during fermentation was up to 3.6 times higher for CR01 and 1.6 times higher for KE6-12 following adaptation. The influence of physiological parameters such as viability, storage carbohydrate content, and metabolite yields following short-term adaptation demonstrated that short-term adaptation was strain dependent.
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