Strain-dependent variance in short-term adaptation effects of two xylose-fermenting strains of Saccharomyces cerevisiae

Dijk, Marlous van; Erdei, Borbála; Galbe, Mats; Nygård, Yvonne; Olsson, Lisbeth

doi:10.1016/j.biortech.2019.121922

Cited by 29 publications

(19 citation statements)

References 33 publications

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“…Considering that the undetoxified hydrolysate was used in the fixed-bed bioreactor experiments, the results from the present work stand out even when compared to xylose fermentation in synthetic medium or in detoxified hydrolysates, which exhibited ethanol productivities below 0.8 g/L/h for recombinant S. cerevisiae strains [30,31]. Encapsulated T18 productivities were between 2.5 and 5.7 g/L/h.…”

Section: Discussionmentioning

confidence: 69%

Repeated batches as a strategy for high 2G ethanol production from undetoxified hemicellulose hydrolysate using immobilized cells of recombinant Saccharomyces cerevisiae in a fixed-bed reactor

Milessi

Perez

Zangirolami

et al. 2020

Biotechnol Biofuels

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Background: The search for sustainable energy sources has become a worldwide issue, making the development of efficient biofuel production processes a priority. Immobilization of second-generation (2G) xylose-fermenting Saccharomyces cerevisiae strains is a promising approach to achieve economic viability of 2G bioethanol production from undetoxified hydrolysates through operation at high cell load and mitigation of inhibitor toxicity. In addition, the use of a fixed-bed reactor can contribute to establish an efficient process because of its distinct advantages, such as high conversion rate per weight of biocatalyst and reuse of biocatalyst. Results: This work assessed the influence of alginate entrapment on the tolerance of recombinant S. cerevisiae to acetic acid. Encapsulated GSE16-T18SI.1 (T18) yeast showed an outstanding performance in repeated batch fermentations with cell recycling in YPX medium supplemented with 8 g/L acetic acid (pH 5.2), achieving 10 cycles without significant loss of productivity. In the fixed-bed bioreactor, a high xylose fermentation rate with ethanol yield and productivity values of 0.38 g ethanol /g sugars and 5.7 g/L/h, respectively were achieved in fermentations using undetoxified sugarcane bagasse hemicellulose hydrolysate, with and without medium recirculation. Conclusions: The performance of recombinant strains developed for 2G ethanol production can be boosted strongly by cell immobilization in alginate gels. Yeast encapsulation allows conducting fermentations in repeated batch mode in fixed-bed bioreactors with high xylose assimilation rate and high ethanol productivity using undetoxified hemicellulose hydrolysate.

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Section: Discussionmentioning

confidence: 69%

Repeated batches as a strategy for high 2G ethanol production from undetoxified hemicellulose hydrolysate using immobilized cells of recombinant Saccharomyces cerevisiae in a fixed-bed reactor

Milessi

Perez

Zangirolami

et al. 2020

Biotechnol Biofuels

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“…Despite designed to function as a transcriptional activator, dCas9-VPR has also previously in combination with certain sgRNAs been shown to confer transcriptional repression 32 . www.nature.com/scientificreports/ Our previous work has shown that robustness and tolerance can vary greatly between industrial strains and evolved mutants 41,48 , highlighting the need for a system that can be rapidly implemented in novel hosts. The benefit of plasmid-based expression of dCas9 and sgRNA in comparison with genomic integration is that the CRISPRa/i toolbox can rapidly be introduced to new strains and for performing novel genomic regulation events.…”

Section: Discussionmentioning

confidence: 99%

“…Our previous work has shown that robustness and tolerance can vary greatly between industrial strains and evolved mutants 41 , 48 , highlighting the need for a system that can be rapidly implemented in novel hosts. The benefit of plasmid-based expression of dCas9 and sgRNA in comparison with genomic integration is that the CRISPRa/i toolbox can rapidly be introduced to new strains and for performing novel genomic regulation events.…”

Section: Discussionmentioning

confidence: 99%

A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12

Cámara

Lenitz

Nygård

2020

Sci Rep

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A cRiSpR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12 elena cámara, ibai Lenitz & Yvonne nygård * Recent advances in CRISPR/Cas9 based genome editing have considerably advanced genetic engineering of industrial yeast strains. In this study, we report the construction and characterization of a toolkit for CRISPR activation and interference (CRISPRa/i) for a polyploid industrial yeast strain. In the CRISPRa/i plasmids that are available in high and low copy variants, dCas9 is expressed alone, or as a fusion with an activation or repression domain; VP64, VPR or Mxi1. The sgRNA is introduced to the CRISPRa/i plasmids from a double stranded oligonucleotide by in vivo homology-directed repair, allowing rapid transcriptional modulation of new target genes without cloning. The CRISPRa/i toolkit was characterized by alteration of expression of fluorescent protein-encoding genes under two different promoters allowing expression alterations up to ~ 2.5-fold. Furthermore, we demonstrated the usability of the CRISPRa/i toolkit by improving the tolerance towards wheat straw hydrolysate of our industrial production strain. We anticipate that our CRISPRa/i toolkit can be widely used to assess novel targets for strain improvement and thus accelerate the design-build-test cycle for developing various industrial production strains. The yeast Saccharomyces cerevisiae is one of the most commonly used microorganisms for industrial applications ranging from wine and beer fermentations to the production of biofuels and high-value metabolites 1,2. However, some of the current production processes are compromised by low yields and productivities, thus further optimization is required 3. In particular, the production of second-generation bioethanol and other biochemicals from lignocellulosic biomass, which provides an alternative to oil-based chemicals, suffers from sub-optimal productivity 4. During the hydrolysis of the raw material, inhibitory compounds (e.g. organic acids and aromatic aldehydes) are formed or released, compromising the microbial performance 5. While quite some work has been done on elucidating genes required for tolerance, much less work has been done on improving tolerance towards stress by altering expression of genes 6. Some previous studies demonstrated deletion 7-9 or overexpression 10-14 of endogenous genes to improve tolerance of S. cerevisiae towards inhibitors commonly found in lignocellulosic hydrolysates. However, most of the published work focuses on improving the tolerance of laboratory yeast strains that generally have weaker tolerance to stress 6 , while translation of beneficial modifications to more robust, industrial strains often is very challenging. The choice of yeast strain to be engineered is crucial for the successful implementation of the engineered phenotype in an industrial production process 15. Yeast strains used in industrial processes tend to be genetically diverse, since they usually arise from hybridization between different species 16. Hence...

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“…by a low spontaneous formation rate of heterogeneity) (pink-colored), case (3) partial decline to intermediate lower level due to rising subpopulation that overcame the production load through producing at lower level (e.g. [55]) (yellow-colored), case (4) homogenous increase in performance that increases the production load, in turn leading to higher enrichment rate of heterogeneity (purple-colored), case (5) enrichment of heterogeneity driven by a load, leading to complete production decline (e.g., [27]) (blue-colored) the propagated cells leading to an improved overall ethanol productivity [18], suggesting a positive role of adaptation to the environment when producing a primary metabolite, e.g. by increasing cell viability.…”

Section: Non-genetic and Genetic Heterogeneities Affect Bioprocess Pementioning

confidence: 99%

The future of self-selecting and stable fermentations

Rugbjerg

Olsson

2020

Journal of Industrial Microbiology and Biotechnology

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Unfavorable cell heterogeneity is a frequent risk during bioprocess scale-up and characterized by rising frequencies of low-producing cells. Low-producing cells emerge by both non-genetic and genetic variation and will enrich due to their higher specific growth rate during the extended number of cell divisions of large-scale bioproduction. Here, we discuss recent strategies for synthetic stabilization of fermentation populations and argue for their application to make cell factory designs that better suit industrial needs. Genotype-directed strategies leverage DNA-sequencing data to inform strain design. Self-selecting phenotype-directed strategies couple high production with cell proliferation, either by redirected metabolic pathways or synthetic product biosensing to enrich for high-performing cell variants. Evaluating production stability early in new cell factory projects will guide heterogeneity-reducing design choices. As good initial metrics, we propose production half-life from standardized serial-passage stability screens and production load, quantified as production-associated percent-wise growth rate reduction. Incorporating more stable genetic designs will greatly increase scalability of future cell factories through sustaining a high-production phenotype and enabling stable long-term production.

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Strain-dependent variance in short-term adaptation effects of two xylose-fermenting strains of Saccharomyces cerevisiae

Cited by 29 publications

References 33 publications

Repeated batches as a strategy for high 2G ethanol production from undetoxified hemicellulose hydrolysate using immobilized cells of recombinant Saccharomyces cerevisiae in a fixed-bed reactor

Repeated batches as a strategy for high 2G ethanol production from undetoxified hemicellulose hydrolysate using immobilized cells of recombinant Saccharomyces cerevisiae in a fixed-bed reactor

A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12

The future of self-selecting and stable fermentations

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