4.6 Article

Droplet digital PCR-aided screening and characterization of Pichia pastoris multiple gene copy strains

期刊

BIOTECHNOLOGY AND BIOENGINEERING
卷 113, 期 7, 页码 1542-1551

出版社

WILEY
DOI: 10.1002/bit.25916

关键词

ddPCR; gene dosage; Pichia pastoris; qPCR; Rhizopus oryzae lipase; recombinant protein

资金

  1. Generalitat de Catalunya [2014-SRG-452]
  2. Spanish Ministry of Economy and Competitivity [CTQ2013-42391-R]

向作者/读者索取更多资源

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 rate styled-content style=text-decoration:line-through styled-content, compared to the reference (one-copy) strain. Biotechnol. Bioeng. 2016;113: 1542-1551. (c) 2015 Wiley Periodicals, Inc.

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