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 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.
Marx
Hans
H
Department of Biotechnology, BOKU, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Mecklenbräuker
Astrid
A
Gasser
Brigitte
B
Sauer
Michael
M
Mattanovich
Diethard
D
D004271:DNA, Fungal / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics*; D018628:Gene Dosage; D005800:Genes, Fungal; D005827:Genetics, Microbial / Q000379:methods*; D006801:Humans; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D012709:Serum Albumin / Q000096:biosynthesis / Q000235:genetics; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000235:genetics
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D011994:Recombinant Proteins; D012709:Serum Albumin; D013482:Superoxide Dismutase