| 1. |
- Liu, Zihe, 1984, et al.
(author)
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Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering
- 2014
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In: Applied and Environmental Microbiology. - : American Society for Microbiology. - 1098-5336 .- 0099-2240. ; 80:17, s. 5542-5550
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Journal article (peer-reviewed)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. |
- Sjöström, Staffan L., et al.
(author)
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High-throughput screening for industrial enzyme production hosts by droplet microfluidics
- 2014
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In: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 14:4, s. 806-813
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Journal article (peer-reviewed)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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| 3. |
- Sjöström, Staffan, et al.
(author)
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Micro-droplet based directed evolution outperforms conventional laboratory evolution
- 2014
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In: 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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Conference paper (peer-reviewed)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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