| 1. |
- Liu, Zihe, 1984, et al.
(författare)
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Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering
- 2014
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Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 1098-5336 .- 0099-2240. ; 80:17, s. 5542-5550
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Tidskriftsartikel (refereegranskat)abstract
- The increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in the VTA1 gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.
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| 2. |
- Qin, J., et al.
(författare)
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Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of L-ornithine
- 2015
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 6:Sept., s. Art. no. 8224-
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Tidskriftsartikel (refereegranskat)abstract
- Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolites of industrial interest, and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products, and hence strains overproducing certain pathway intermediates can serve as platform cell factories for production of such products. Here we implement a modular pathway rewiring (MPR) strategy and demonstrate its use for pathway optimization resulting in high-level production of L-ornithine, an intermediate of L-arginine biosynthesis and a precursor metabolite for a range of different natural products. The MPR strategy involves rewiring of the urea cycle, subcellular trafficking engineering and pathway re-localization, and improving precursor supply either through attenuation of the Crabtree effect or through the use of controlled fed-batch fermentations, leading to an L-ornithine titre of 1,041±47 mg l-1 with a yield of 67 mg (g glucose)-1 in shake-flask cultures and a titre of 5.1 g l-1 in fed-batch cultivations. Our study represents the first comprehensive study on overproducing an amino-acid intermediate in yeast, and our results demonstrate the potential to use yeast more extensively for low-cost production of many high-value amino-acid-derived chemicals.
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| 3. |
- Huang, Mingtao, 1984, et al.
(författare)
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Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
- 2015
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 112:34, s. E4689-E4696
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Tidskriftsartikel (refereegranskat)abstract
- There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant a-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.
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| 4. |
- Sjöström, Staffan L., et al.
(författare)
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High-throughput screening for industrial enzyme production hosts by droplet microfluidics
- 2014
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 14:4, s. 806-813
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Tidskriftsartikel (refereegranskat)abstract
- A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its a-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in a-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains.
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| 5. |
- Yu, Tao, 1986, et al.
(författare)
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Reprogramming Yeast Metabolism from Alcoholic Fermentation to Lipogenesis
- 2018
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Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 174:6, s. 1549-1572
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Tidskriftsartikel (refereegranskat)abstract
- Engineering microorganisms for production of fuels and chemicals often requires major re-programming of metabolism to ensure high flux toward the product of interest. This is challenging, as millions of years of evolution have resulted in establishment of tight regulation of metabolism for optimal growth in the organism's natural habitat. Here, we show through metabolic engineering that it is possible to alter the metabolism of Saccharomyces cerevisiae from traditional ethanol fermentation to a pure lipogenesis metabolism, resulting in high-level production of free fatty acids. Through metabolic engineering and process design, we altered subcellular metabolic trafficking, fine tuned NADPH and ATP supply, and decreased carbon flux to biomass, enabling production of 33.4 g/L extracellular free fatty acids. We further demonstrate that lipogenesis metabolism can replace ethanol fermentation by deletion of pyruvate decarboxylase enzymes followed by adaptive laboratory evolution. Genome sequencing of evolved strains showed that pyruvate kinase mutations were essential for this phenotype.
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| 6. |
- Bao, Jichen, 1988, et al.
(författare)
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Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast
- 2018
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce recombinant proteins. However, S. cerevisiae naturally secretes only a few proteins, such as invertase and the mating alpha factor, and its secretory capacity is limited. It has been reported that engineering protein anterograde trafficking from the endoplasmic reticulum to the Golgi apparatus by the moderate overexpression of SEC16 could increase recombinant protein secretion in S. cerevisiae. In this study, the retrograde trafficking in a strain with moderate overexpression of SEC16 was engineered by overexpression of ADP-ribosylation factor GTP activating proteins, Gcs1p and Glo3p, which are involved in the process of COPI-coated vesicle formation. Engineering the retrograde trafficking increased the secretion of α-amylase but did not induce production of reactive oxygen species. An expanded ER membrane was detected in both the GCS1 and GLO3 overexpressio n strains. Physiological characterizations during batch fermentation showed that GLO3 overexpression had better effect on recombinant protein secretion than GCS1 overexpression. Additionally, the GLO3 overexpression strain had higher secretion of two other recombinant proteins, endoglucanase I from Trichoderma reesei and glucan-1,4-α-glucosidase from Rhizopus oryzae, indicating overexpression of GLO3 in a SEC16 moderate overexpression strain might be a general strategy for improving production of secreted proteins by yeast.
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| 7. |
- Bao, Jichen, 1988, et al.
(författare)
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Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae
- 2017
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Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 83:14, s. Article no. UNSP e03400-16
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae is widely used to produce biopharmaceutical proteins. However, the limited capacity of the secretory pathway may reduce its productivity. Here, we increased the secretion of a heterologous beta-amylase, a model protein used for studying the protein secretory pathway in yeast, by moderately overexpressing SEC16, which is involved in protein translocation from the endoplasmic reticulum to the Golgi apparatus. The moderate overexpression of SEC16 increased beta-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species resulting from the heterologous beta-amylase production was reduced. A genome-wide expression analysis indicated decreased endoplasmic reticulum stress in the strain that moderately overexpressed SEC16, which was consistent with a decreased volume of the endoplasmic reticulum. Additionally, fewer mitochondria were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-beta-glucosidase, indicating that this mechanism is of general relevance. IMPORTANCE There is an increasing demand for recombinant proteins to be used as enzymes and pharmaceuticals. The yeast Saccharomyces cerevisiae is a cell factory that is widely used to produce recombinant proteins. Our study revealed that moderate overexpression of SEC16 increased recombinant protein secretion in S. cerevisiae. This new strategy can be combined with other targets to engineer cell factories to efficiently produce protein in the future.
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| 8. |
- Dai, Zongijie, 1986, et al.
(författare)
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Global rewiring of cellular metabolism renders Saccharomyces cerevisiae Crabtree negative
- 2018
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae is a Crabtree-positive eukaryal model organism. It is believed that the Crabtree effect has evolved as a competition mechanism by allowing for rapid growth and production of ethanol at aerobic glucose excess conditions. This inherent property of yeast metabolism and the multiple mechanisms underlying it require a global rewiring of the entire metabolic network to abolish the Crabtree effect. Through rational engineering of pyruvate metabolism combined with adaptive laboratory evolution (ALE), we demonstrate that it is possible to obtain such a global rewiring and hereby turn S. cerevisiae into a Crabtree-negative yeast. Using integrated systems biology analysis, we identify that the global rewiring of cellular metabolism is accomplished through a mutation in the RNA polymerase II mediator complex, which is also observed in cancer cells expressing the Warburg effect.
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| 9. |
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| 10. |
- Huang, Mingtao, 1984, et al.
(författare)
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Efficient protein production by yeast requires global tuning of metabolism
- 2017
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- The biotech industry relies on cell factories for production of pharmaceutical proteins, of which several are among the top-selling medicines. There is, therefore, considerable interest in improving the efficiency of protein production by cell factories. Protein secretion involves numerous intracellular processes with many underlying mechanisms still remaining unclear. Here, we use RNA-seq to study the genome-wide transcriptional response to protein secretion in mutant yeast strains. We find that many cellular processes have to be attuned to support efficient protein secretion. In particular, altered energy metabolism resulting in reduced respiration and increased fermentation, as well as balancing of amino-acid biosynthesis and reduced thiamine biosynthesis seem to be particularly important. We confirm our findings by inverse engineering and physiological characterization and show that by tuning metabolism cells are able to efficiently secrete recombinant proteins. Our findings provide increased understanding of which cellular regulations and pathways are associated with efficient protein secretion.
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| 11. |
- Huang, Mingtao, 1984, et al.
(författare)
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Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production
- 2018
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:47, s. E11025-E11032
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Tidskriftsartikel (refereegranskat)abstract
- Baker’s yeast Saccharomyces cerevisiae is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. Here we identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications on the endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, we enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.
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| 12. |
- Huang, Mingtao, 1984, et al.
(författare)
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High-throughput microfluidics for the screening of yeast libraries
- 2018
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Ingår i: Synthetic Metabolic Pathways. - New York, NY : Humana Press. - 9781493972944 ; , s. 307-317, s. 307-317
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Bokkapitel (refereegranskat)abstract
- Cell factory development is critically important for efficient biological production of chemicals, biofuels, and pharmaceuticals. Many rounds of the Design–Build–Test–Learn cycles may be required before an engineered strain meeting specific metrics required for industrial application. The bioindustry prefer products in secreted form (secreted products or extracellular metabolites) as it can lower the cost of downstream processing, reduce metabolic burden to cell hosts, and allow necessary modification on the final products, such as biopharmaceuticals. Yet, products in secreted form result in the disconnection of phenotype from genotype, which may have limited throughput in the Test step for identification of desired variants from large libraries of mutant strains. In droplet microfluidic screening, single cells are encapsulated in individual droplet and enable high-throughput processing and sorting of single cells or clones. Encapsulation in droplets allows this technology to overcome the throughput limitations present in traditional methods for screening by extracellular phenotypes. In this chapter, we describe a protocol/guideline for high-throughput droplet microfluidics screening of yeast libraries for higher protein secretion. This protocol can be adapted to screening by a range of other extracellular products from yeast or other hosts.
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| 13. |
- Li, Feiran, 1993, et al.
(författare)
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Improving recombinant protein production by yeast through genome-scale modeling using proteome constraints
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Eukaryotic cells are used as cell factories to produce and secrete multitudes of recombinant pharmaceutical proteins, including several of the current top-selling drugs. Due to the essential role and complexity of the secretory pathway, improvement for recombinant protein production through metabolic engineering has traditionally been relatively ad-hoc; and a more systematic approach is required to generate novel design principles. Here, we present the proteome-constrained genome-scale protein secretory model of yeast Saccharomyces cerevisiae (pcSecYeast), which enables us to simulate and explain phenotypes caused by limited secretory capacity. We further apply the pcSecYeast model to predict overexpression targets for the production of several recombinant proteins. We experimentally validate many of the predicted targets for alpha-amylase production to demonstrate pcSecYeast application as a computational tool in guiding yeast engineering and improving recombinant protein production. Due to the complexity of the protein secretory pathway, strategy suitable for the production of a certain recombination protein cannot be generalized. Here, the authors construct a proteome-constrained genome-scale protein secretory model for yeast and show its application in the production of different misfolded or recombinant proteins.
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| 14. |
- Sjöström, Staffan, et al.
(författare)
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Droplet based directed evolution of yeast cell factories doubles production of industrial enzymes
- 2013
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Ingår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013. - : Chemical and Biological Microsystems Society. - 9781632666246 ; , s. 1270-1272
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Konferensbidrag (refereegranskat)abstract
- We present a high throughput micro-droplet based method for directed evolution of yeast cell factories for improved production of industrial enzymes. The workflow includes a fluorescently activated droplet sorter which was found to accurately sort droplets with a false positive rate of 0.0002 at 300 Hz. The workflow was used to screen a library of α-amylase expressing yeast mutants. A candidate yeast strain with a more than twofold increase in α-amylase production was isolated from a single round of directed evolution.
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| 15. |
- Sjöström, Staffan, et al.
(författare)
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Micro-droplet based directed evolution outperforms conventional laboratory evolution
- 2014
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Ingår i: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014. - : Chemical and Biological Microsystems Society. - 9780979806476 ; , s. 169-171
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Konferensbidrag (refereegranskat)abstract
- We present droplet adaptive laboratory evolution (DrALE), a directed evolution method used to improve industrial enzyme producing microorganisms for e.g. feedstock digestion. DrALE is based linking a desired phenotype to growth rate allowing only desired cells to proliferate. Single cells are confined in microfluidic droplets to prevent the phenotype, e.g. secreted enzymes, from leaking between cells. The method was benchmarked against and found to significantly outperform conventional adaptive laboratory evolution (ALE) in enriching enzyme producing cells. It was furthermore applied to enrich a whole-genome mutated library of yeast cells for α-amylase activity.
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| 16. |
- Wang, Guokun, 1988, et al.
(författare)
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Exploring the potential of Saccharomyces cerevisiae for biopharmaceutical protein production
- 2017
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Ingår i: Current Opinion in Biotechnology. - : Elsevier BV. - 0958-1669 .- 1879-0429. ; 48, s. 77-84
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Forskningsöversikt (refereegranskat)abstract
- Production of recombinant proteins by yeast plays a vital role in the biopharmaceutical industry. It is therefore desirable to develop yeast platform strains for over-production of various biopharmaceutical proteins, but this requires fundamental knowledge of the cellular machinery, especially the protein secretory pathway. Integrated analyses of multi-omics datasets can provide comprehensive understanding of cellular function, and can enable systems biology-driven and mathematical model-guided strain engineering. Rational engineering and introduction of trackable genetic modifications using synthetic biology tools, coupled with high-throughput screening are, however, also efficient approaches to relieve bottlenecks hindering high-level protein production. Here we review advances in systems biology and metabolic engineering of yeast for improving recombinant protein production.
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| 17. |
- Wang, Guokun, 1988, et al.
(författare)
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RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae
- 2019
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 116:19, s. 9324-9332
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Tidskriftsartikel (refereegranskat)abstract
- The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down-regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.
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