| 1. |
- Kazemi Seresht, Ali, 1980, et al.
(författare)
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Long-Term Adaptation of Saccharomyces cerevisiae to the Burden of Recombinant Insulin Production
- 2013
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 110:10, s. 2749-2763
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Tidskriftsartikel (refereegranskat)abstract
- High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. A data-driven approach was applied to study the secretory production of a human insulin analog precursor (IAP) in Saccharomyces cerevisiae during prolonged cultivation (80 generations) in glucose-limited aerobic chemostat cultures. Physiological characterization of the recombinant cells involved a comparison with cultures of a congenic reference strain that did not produce IAP, and time-course analysis of both strains aimed at identifying the metabolic adaptation of the cells towards the burden of IAP production. All cultures were examined at high cell density conditions (30g/L dry weight) to increase the industrial relevance of the results. The burden of heterologous protein production in the recombinant strain was explored by global transcriptome analysis and targeted metabolome analysis, including the analysis of intracellular amino acid pools, glycolytic metabolites, and TCA intermediates. The cellular re-arrangements towards IAP production were categorized in direct responses, for example, enhanced metabolism of amino acids as precursors for the formation of IAP, as well as indirect responses, for example, changes in the central carbon metabolism. As part of the long-term adaptation, a metabolic re-modeling of the IAP-expressing strain was observed, indicating an augmented negative selection pressure on glycolytic overcapacity, and the emergence of mitochondrial dysfunction. The evoked metabolic re-modeling of the cells led to less optimal conditions with respect to the expression and processing of the target protein and thus decreased the cellular expression capacity for the secretory production of IAP during prolonged cultivation.
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| 2. |
- Kazemi Seresht, Ali, 1980, et al.
(författare)
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Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers
- 2013
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:9, s. 3939-3948
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Tidskriftsartikel (refereegranskat)abstract
- The use of auxotrophic Saccharomyces cerevisiae strains for improved production of a heterologous protein was examined. Two different marker genes were investigated, encoding key enzymes in the metabolic pathways for amino acid (LEU2) and pyrimidine (URA3) biosynthesis, respectively. Expression plasmids, carrying the partly defective selection markers LEU2d and URA3d, were constructed. Two CEN.PK-derived strains were chosen and insulin analogue precursor was selected as a model protein. Different truncations of the LEU2 and URA3 promoters were used as the mean to titrate the plasmid copy number and thus the recombinant gene dosage in order to improve insulin productivity. Experiments were initially carried out in batch mode to examine the stability of yeast transformants and to select high yielding mutants. Next, chemostat cultivations were run at high cell density to address industrial applicability and long-term expression stability of the transformants. We found that the choice of auxotrophic marker is crucial for developing a yeast expression system with stable heterologous protein production. The incremental truncation of the URA3 promoter led to higher plasmid copy numbers and IAP yields, whereas the truncation of the LEU2 promoter caused low plasmid stability. We show that the modification of the level of the recombinant gene dosage by varying the degree of promoter truncation can be a strong tool for optimization of productivity. The application of the URA3d-based expression systems showed a high potential for industrial protein production and for further academic studies.
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| 3. |
- Kazemi Seresht, Ali, 1980
(författare)
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Physiology of Saccharomyces cerevisiae Producing Recombinant Insulin in Continuous Culture
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The yeast Saccharomyces cerevisiae has widely been used as a host for the production of heterologous proteins. High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. Different target proteins often do not result in similar secretion yields, underlining the dependency of secretion efficiency on the physicochemical properties of the protein of interest. Two human insulin analogue precursors (IAPs) were used as model secretory proteins. The IAPs had minor differences in their amino acid sequences, yet pictured more than 7-fold difference in their secretion yields. Global transcriptome analysis carried out in aerobic glucose-limited chemostat experiments pinpointed distinct steps during the protein maturation pathway to be differentially regulated, and indicated an increased degradation of the IAP with the low secretion yield. The use of auxotrophic strains for improved IAP production was examined. The incremental truncation of the promoter of the auxotrophic marker gene URA3 led to higher plasmid copy numbers, and illustrated that the modification of the level of the recombinant gene dosage via the degree of promoter truncation can be a strong tool for optimizing the IAP productivity. The dynamic character of adaptive responses of S. cerevisiae towards a change in their nutrient access was studied based on global gene expression analysis, scrutinizing the impact of restricted supply of phosphate on the physiological state of IAP-expressing cells. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. A data-driven approach was applied to study the secretory IAP production in S. cerevisiae in prolonged chemostat cultures (80 generations), with the aim to explore the metabolic adaptation of the cells towards the burden of IAP production. Time-course analysis of global transcriptome and targeted metabolome analysis indicated at metabolic re-modeling of the recombinant cells based on augmented negative selection pressure on glycolytic overcapacity, changes in amino acid and central carbon metabolism, and mitochondrial dysfunction to account for decreased cellular expression efficiency in long-term chemostat cultures.
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| 4. |
- Kazemi Seresht, Ali, 1980, et al.
(författare)
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The Challenge of Improved Secretory Production of Active Pharmaceutical Ingredients in Saccharomyces cerevisiae: A Case Study on Human Insulin Analogs
- 2013
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 110:10, s. 2764-2774
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae has widely been used as a host for the production of heterologous proteins. Great attention has been put on improved secretory production of active pharmaceutical ingredients, and the secretory pathway of this eukaryotic host has been the playground of diverse strain engineering studies, aiming at enhanced cellular capacities for folding and trafficking of the target proteins. However, the cellular quality assessment for secretory proteins remains mostly unpredictable, and different target proteins often do not picture similar secretion yields, underlining the dependency of efficient secretion on the physicochemical properties of the protein of interest. In this study, two human insulin analog precursors (IAPs) with minor differences in their amino acid sequences were used as model secretory proteins. No differences between cells expressing these two proteins were found in the IAP transcript levels, gene copy numbers, or intra-cellularly accumulated proteins, yet a more than sevenfold difference in their secretion yields was found. Physiological characterization of cells expressing these proteins in batch processes revealed no significant difference in their specific growth rate, but an altered overflow metabolism. Global transcriptome analysis carried out in chemostat experiments pinpointed distinct steps during the protein maturation pathway to be differentially regulated and indicated an increased degradation of the IAP with the low secretion yield. In silico protein structure modeling of the IAPs suggested a difference in conformational stability, induced by the amino acid substitution, which most likely resulted in disparity in trafficking through the secretory pathway and thus a large difference in secretion yields.
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| 5. |
- Kazemi Seresht, Ali, 1980, et al.
(författare)
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The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses
- 2011
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background:The adaptation of unicellular organisms like Saccharomyces cerevisiae to alternating nutrient availability is of great fundamental and applied interest, as understanding how eukaryotic cells respond to variations in their nutrient supply has implications spanning from physiological insights to biotechnological applications.Results:The impact of a step-wise restricted supply of phosphate on the physiological state of S. cerevisiae cells producing human Insulin was studied. The focus was to determine the changes within the global gene expression of cells being cultured to an industrially relevant high cell density of 33 g/l cell dry weight and under six distinct phosphate concentrations, ranging from 33 mM (unlimited) to 2.6 mM (limited). An increased flux through the secretory pathway, being induced by the PHO circuit during low Pi supplementation, proved to enhance the secretory production of the heterologous protein. The re-distribution of the carbon flux from biomass formation towards increased glycerol production under low phosphate led to increased transcript levels of the insulin gene, which was under the regulation of the TPI1 promoter.Conclusions:Our study underlines the dynamic character of adaptive responses of cells towards a change in their nutrient access. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. Our work emphasizes the importance of reduced phosphate supply for improved secretory production of heterologous proteins.
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