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Enhancing the copy number of episomal plasmids in Saccharomyces cerevisiae for improved protein production

Yun Chen (Institutionen för kemi- och bioteknik, Systembiologi) ; Siavash Partow (Institutionen för kemi- och bioteknik, Systembiologi) ; Gionata Scalcinati (Institutionen för kemi- och bioteknik, Systembiologi) ; Verena Siewers (Institutionen för kemi- och bioteknik, Systembiologi) ; Jens B. Nielsen (Institutionen för kemi- och bioteknik, Systembiologi)
FEMS Yeast Research (1567-1356). Vol. 12 (2012), 5, p. 598-607.
[Artikel, refereegranskad vetenskaplig]

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.

Nyckelord: ubiquitin-tag, promoter, episomal plasmid, protein expression, copy number, ubiquitin/proteasome-dependent proteolysis, escherichia-coli, autoselection system, chemostat cultures, gene-expression, high-level, yeast, sesquiterpenes, maintenance, recognition

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Denna post skapades 2012-08-16. Senast ändrad 2015-12-17.
CPL Pubid: 161889


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Institutionen för kemi- och bioteknik, Systembiologi (2008-2014)



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Denna publikation ingår i:

Novel Synthetic Biology Tools for Metabolic Engineering of Saccharomyces cerevisiae



Denna publikation är ett resultat av följande projekt:

Industrial Systems Biology of Yeast and A. oryzae (INSYSBIO) (EC/FP7/247013)