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• W2274846837 endingPage "1551" @default.
• W2274846837 startingPage "1542" @default.
• W2274846837 abstract "Pichia (syn. Komagataella) pastoris is a widely used yeast platform for heterologous protein production. Expression cassettes are usually stably integrated into the genome of this host via homologous recombination. Although increasing gene dosage is a powerful strategy to improve recombinant protein production, an excess in the number of gene copies often leads to decreased product yields and increased metabolic burden, particularly for secreted proteins. We have constructed a series of strains harboring different copy numbers of a Rhizopus oryzae lipase gene (ROL), aiming to find the optimum gene dosage for secreted Rol production. In order to accurately determine ROL gene dosage, we implemented a novel protocol based on droplet digital PCR (ddPCR), and cross validated it with conventional real-time PCR. Gene copy number determination based on ddPCR allowed for an accurate ranking of transformants according to their ROL gene dosage. Results indicated that ddPCR was particularly superior at lower gene dosages (one to five copies) over quantitative real-time PCR (qPCR). This facilitated the determination of the optimal ROL gene dosage as low as two copies. The ranking of ROL gene dosage versus Rol yield was consistent at both small scale and bioreactor chemostat cultures, thereby easing clone characterization in terms of gene dosage dependent physiological effects, which could be discriminated even among strains differing by only one ROL copy. A selected two-copy strain showed twofold increase in Rol specific production in a chemostat culture over the single copy strain. Conversely, strains harboring more than two copies of the ROL gene showed decreased product and biomass yields, as well as altered substrate consumption specific rates, compared to the reference (one-copy) strain. Biotechnol. Bioeng. 2016;113: 1542–1551. © 2015 Wiley Periodicals, Inc." @default.
• W2274846837 created "2016-06-24" @default.
• W2274846837 creator A5028013763 @default.
• W2274846837 creator A5037504477 @default.
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• W2274846837 date "2016-03-16" @default.
• W2274846837 modified "2023-10-17" @default.
• W2274846837 title "Droplet digital PCR-aided screening and characterization of<i>Pichia pastoris</i>multiple gene copy strains" @default.
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• W2274846837 doi "https://doi.org/10.1002/bit.25916" @default.
• W2274846837 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/26704939" @default.
• W2274846837 hasPublicationYear "2016" @default.
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