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Applied and Environmental Microbiology, November 2005, p. 7092-7098, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7092-7098.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis

Paul A. Colussi and Christopher H. Taron*

New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938-2723

Received 18 April 2005/ Accepted 16 July 2005

The strong LAC4 promoter (PLAC4) from Kluyveromyces lactis has been extensively used to drive expression of heterologous proteins in this industrially important yeast. A drawback of this expression method is the serendipitous ability of PLAC4 to promote gene expression in Escherichia coli. This can interfere with the process of assembling expression constructs in E. coli cells prior to their introduction into yeast cells, especially if the cloned gene encodes a protein that is detrimental to bacteria. In this study, we created a series of PLAC4 variants by targeted mutagenesis of three DNA sequences (PBI, PBII, and PBIII) that resemble the E. coli Pribnow box element of bacterial promoters and that reside immediately upstream of two E. coli transcription initiation sites associated with PLAC4. Mutation of PBI reduced the bacterial expression of a reporter protein (green fluorescent protein [GFP]) by ~87%, whereas mutation of PBII and PBIII had little effect on GFP expression. Deletion of all three sequences completely eliminated GFP expression. Additionally, each promoter variant expressed human serum albumin in K. lactis cells to levels comparable to wild-type PLAC4. We created a novel integrative expression vector (pKLAC1) containing the PLAC4 variant lacking PBI and used it to successfully clone and express the catalytic subunit of bovine enterokinase, a protease that has historically been problematic in E. coli cells. The pKLAC1 vector should aid in the cloning of other potentially toxic genes in E. coli prior to their expression in K. lactis.

* Corresponding author. Mailing address: New England Biolabs, 240 County Road, Ipswich, MA 01938-2723. Phone: (978) 927-5054. Fax: (978) 921-1350. E-mail: taron{at}neb.com.

Applied and Environmental Microbiology, November 2005, p. 7092-7098, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7092-7098.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Read, J. D., Colussi, P. A., Ganatra, M. B., Taron, C. H. (2007). Acetamide Selection of Kluyveromyces lactis Cells Transformed with an Integrative Vector Leads to High-Frequency Formation of Multicopy Strains. Appl. Environ. Microbiol. 73: 5088-5096 [Abstract] [Full Text]  


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