Enhancing the copy number of episomal plasmids in Saccharomyces cerevisiae for improved protein production
Artikel i vetenskaplig tidskrift, 2012

2 mu m-based episomal expression vectors are widely used in Saccharomyces cerevisiae for recombinant protein production and synthetic pathway optimization. In this study, we report a new approach to increase the plasmid copy number (PCN) and thus improve the expression of plasmid-encoded proteins. This was achieved by combining destabilization of the marker protein with decreasing the marker gene transcription level. Destabilization of the marker protein alone by fusing a ubiquitin/N-degron tag (ubi-tag) to the N-terminus of the Ura3 marker protein could increase the PCN and activity of LacZ expressed from the same vector. When arginine was exposed at the N-terminus of the marker protein after cleavage of ubiquitin, the PCN and LacZ activity were increased by 7080%. Replacement of the native URA3 promoter with the HXT1, KEX2 or URA3-d promoter resulted in an increase in the PCN and LacZ activity by about 30100%. Combining the ubi-tag and promoter modification of the marker gene, increased the PCN and LacZ activity by threefold. We also demonstrated that this new expression vectors can be used to increase enzyme activity by improving patchoulol production by threefold.

copy number

promoter

ubiquitin/proteasome-dependent proteolysis

yeast

maintenance

ubiquitin-tag

sesquiterpenes

gene-expression

autoselection system

chemostat cultures

recognition

high-level

protein expression

escherichia-coli

episomal plasmid

Författare

Yun Chen

Chalmers, Kemi- och bioteknik, Livsvetenskaper, Systembiologi

Siavash Partow

Chalmers, Kemi- och bioteknik, Livsvetenskaper, Systembiologi

Gionata Scalcinati

Chalmers, Kemi- och bioteknik, Livsvetenskaper, Systembiologi

Verena Siewers

Chalmers, Kemi- och bioteknik, Livsvetenskaper, Systembiologi

Jens B Nielsen

Chalmers, Kemi- och bioteknik, Livsvetenskaper, Systembiologi

FEMS Yeast Research

1567-1356 (ISSN) 1567-1364 (eISSN)

Vol. 12 5 598-607

Industrial Systems Biology of Yeast and A. oryzae (INSYSBIO)

Europeiska kommissionen (FP7), 2010-01-01 -- 2014-12-31.

Ämneskategorier

Kemiteknik

Styrkeområden

Livsvetenskaper och teknik

DOI

10.1111/j.1567-1364.2012.00809.x

Mer information

Skapat

2017-10-07