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Applied and Environmental Microbiology, September 2001, p. 4119-4127, Vol. 67, No. 9
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.9.4119-4127.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in Lactococcus lactis

Y. Le Loir, S. Nouaille, J. Commissaire, L. Brétigny, A. Gruss, and P. Langella*

Laboratoire de Génétique Appliquée, Unité de Recherches Laitières et de Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France

Received 8 June 2001/Accepted 22 June 2001

Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SPNuc) by that of L. lactis protein Usp45 (SPUsp) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SPUsp than when directed by SPNuc. This SPUsp effect on Nuc secretion is not due to a better antifolding activity, since SPUsp:Nuc precursor proteins display enzymatic activity in vitro, while SPNuc:Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SPUsp and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.

* Corresponding author. Mailing address: Laboratoire de Génétique Appliquée, Unité de Recherches Laitières et de Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France. Phone: 33 01 34 65 20 83. Fax: 33 01 34 65 20 65. E-mail: langella{at}jouy.inra.fr.

Applied and Environmental Microbiology, September 2001, p. 4119-4127, Vol. 67, No. 9
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.9.4119-4127.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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