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Applied and Environmental Microbiology, February 2007, p. 922-929, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01764-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Induction by Hypoxia of Heterologous-Protein Production with the KlPDC1 Promoter in Yeasts{triangledown}

Andrea Camattari,1 Michele M. Bianchi,2 Paola Branduardi,1 Danilo Porro,1 and Luca Brambilla1*

Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy,1 Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Roma "La Sapienza," P.le A. Moro 5, 00185 Rome, Italy2

Received 26 July 2006/ Accepted 24 November 2006

The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1ß was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get ß-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.

* Corresponding author. Mailing address: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy. Phone: 390264483451. Fax: 390264483565. E-mail: luca.brambilla{at}unimib.it.

{triangledown} Published ahead of print on 1 December 2006.

Applied and Environmental Microbiology, February 2007, p. 922-929, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01764-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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