Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus

The yeast Pichia pastoris is a widely used host organism for heterologous protein production. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number integrants...

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Bibliographic Details
Published in:FEMS yeast research 2009-12, Vol.9 (8), p.1260-1270
Main Authors: Marx, Hans, Mecklenbräuker, Astrid, Gasser, Brigitte, Sauer, Michael, Mattanovich, Diethard
Format: Article
Language:English
Subjects:
Concatamers
Copy number
Cytoplasm
DNA, Fungal - genetics
DNA, Ribosomal - genetics
Gene Dosage
Genes, Fungal
Genetics, Microbial - methods
heterologous protein expression
Human serum albumin
human superoxide dismutase
Humans
Integration
Pichia - genetics
Pichia pastoris
post-transformational vector amplification
rDNA integration
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Recombination, Genetic
Ribosomal DNA
Serum Albumin - biosynthesis
Serum Albumin - genetics
Superoxide dismutase
Superoxide Dismutase - biosynthesis
Superoxide Dismutase - genetics
Transcription
Xenotransplantation
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Summary:The yeast Pichia pastoris is a widely used host organism for heterologous protein production. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number integrants of P. pastoris are achievable only by screening of random events or by cloning of gene concatemers. Methods for rapid and reliable multicopy integration of the expression cassette are therefore desirable. Here we present such a method based on vector integration into the rDNA locus and post-transformational vector amplification by repeated selection on increased antibiotic concentrations. Data are presented for two exemplary products: human serum albumin, which is secreted into the supernatant, and human superoxide dismutase, which is accumulated in the cytoplasm of the cells. The striking picture evolving is that intracellular protein production is tightly correlated with gene copy number, while use of the secretory pathway introduces a high clonal variability and the correlation with gene copy number is valid only for low gene copy numbers.
ISSN:1567-1356
1567-1364
DOI:10.1111/j.1567-1364.2009.00561.x