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Applied and Environmental Microbiology, October 2002, p. 5034-5041, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.5034-5041.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Improvement of Recombinant Protein Yield by a Combination of Transcriptional Amplification and Stabilization of Gene Expression

A. Cebolla,1,2,3 J. L. Royo,1,3 V. de Lorenzo,4 and E. Santero1,2*

Laboratorio Andaluz de Biología, Universidad Pablo de Olavide, 41013 Seville,1 Departamento de Genética, Universidad de Sevilla, 41080 Seville,2 Biomedal S. L. Pabellón de Italia 3° NO, 41092 Seville,3 Centro Nacional de Biotecnología, CSIC, Madrid, Spain4

Received 18 March 2002/ Accepted 15 July 2002

We explored the use of a cascade circuit for heterologous gene expression that consists of a regulatory module with a salicylate-inducible system that controls the expression of a second regulator, xylS2, whose product is activated by common inducers. Activation and increasing the concentration of the second regulator synergistically induced heterologous genes downstream of the Pm promoter in the expression module. This module can be placed in multicopy vectors or in the chromosome of a host strain by means of minitransposons. Using reporter genes, we evaluated gene regulation capacity and gross production of the system with different configurations. The highest yield was obtained when the expression module was in a multicopy plasmid after a 6-h induction. However, expression modules in plasmids showed low stability after induction even with selective pressure. The chromosomal configuration had the lowest basal levels and induced levels comparable to those of plasmid configurations, resulting in accumulation of more than 10% of the total protein. Unlike the configurations in plasmids, the yield was maintained for at least 3 days even without selective pressure. In conclusion, the cascade system in the chromosome configuration is more efficient for long-term fermentation because of the great stability of the overexpressing phenotype in spite of the high levels of expression.

* Corresponding author. Mailing address: Laboratorio Andaluz de Biología (LAB), Universidad Pablo de Olavide, Ctra. de Utrera, Km 1, 41013 Seville, Spain. Phone: 34-954349386. Fax: 34-954349376. E-mail: esansan{at}dex.upo.es.

Applied and Environmental Microbiology, October 2002, p. 5034-5041, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.5034-5041.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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