Directed gene copy number amplification inPichia pastorisby vector integration into the ribosomal DNA locus

Abstract

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.

Genome‐scale metabolic model of methylotrophic yeast Pichia pastoris and its use for in silico analysis of heterologous protein production

“…As the model was based on the constitutive expression system using the GAP promoter and glucose as a substrate, 100 transcript copies were assumed per gene copy, as described for the Gapdh gene of S. cerevisiae. For both human serum albumin (HSA) and human superoxide dismutase (hSOD) approximately 10 5 protein copies per gene copy were calculated for steady state based on the experimental data described by Marx et al [33].The protein production reaction is summarized in equation (2) a∑dNTP(DNA) + b∑NTP(mRNA) + c∑(Amino acid) + xGTP(Energy for protein synthesis)…”

A color-based stable multi-copy integrant selection system forPichia pastorisusing the attenuatedADE1andADE2genes as auxotrophic markers

“…Using expression vectors that contain drug resistance genes like Kan R or Zeo R as selection markers, it is possible to increase the number of transformants harboring multiple copies of the expression vector by increasing the level of drug used for selection. 12,13 One significant disadvantage of the single-crossover type integration, however, lies in the fact that the multiple integrated copies can collapse back into a single copy by homologous recombination. This has recently been shown by Zhu and coworkers 14 and can be especially problematic during scale-up of the expression reaction during fermentation if the protein of interest is toxic to the cells or the eviction of several copies of expression plasmid results in other growth benefits for the cells.…”

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion

“…Although a linear correlation between gene copy number and cytoplasmic protein production has been demonstrated in yeast, this was not true for protein secretion (41). Recombinant protein secretion is often hampered along the secretory pathway or because of limitations in the central carbon metabolism (5).…”