Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers
Artikel i vetenskaplig tidskrift, 2013

The use of auxotrophic Saccharomyces cerevisiae strains for improved production of a heterologous protein was examined. Two different marker genes were investigated, encoding key enzymes in the metabolic pathways for amino acid (LEU2) and pyrimidine (URA3) biosynthesis, respectively. Expression plasmids, carrying the partly defective selection markers LEU2d and URA3d, were constructed. Two CEN.PK-derived strains were chosen and insulin analogue precursor was selected as a model protein. Different truncations of the LEU2 and URA3 promoters were used as the mean to titrate the plasmid copy number and thus the recombinant gene dosage in order to improve insulin productivity. Experiments were initially carried out in batch mode to examine the stability of yeast transformants and to select high yielding mutants. Next, chemostat cultivations were run at high cell density to address industrial applicability and long-term expression stability of the transformants. We found that the choice of auxotrophic marker is crucial for developing a yeast expression system with stable heterologous protein production. The incremental truncation of the URA3 promoter led to higher plasmid copy numbers and IAP yields, whereas the truncation of the LEU2 promoter caused low plasmid stability. We show that the modification of the level of the recombinant gene dosage by varying the degree of promoter truncation can be a strong tool for optimization of productivity. The application of the URA3d-based expression systems showed a high potential for industrial protein production and for further academic studies.

Plasmid copy number

URA3d

High cell density cultivation

LEU2d

Human insulin

Promoter truncation

Författare

Ali Kazemi Seresht

Chalmers, Kemi- och bioteknik, Industriell Bioteknik

P. Norgaard

Novo Nordisk

E. A. Palmqvist

Novo Nordisk

A. S. Andersen

Novo Nordisk

Lisbeth Olsson

Chalmers, Kemi- och bioteknik, Industriell Bioteknik

Applied Microbiology and Biotechnology

0175-7598 (ISSN) 1432-0614 (eISSN)

Vol. 97 9 3939-3948

Ämneskategorier

Industriell bioteknik

Mikrobiologi

DOI

10.1007/s00253-012-4263-1