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Applied and Environmental Microbiology, March 1999, p. 1202-1206, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

A General Method for Selection of alpha -Acetolactate Decarboxylase-Deficient Lactococcus lactis Mutants To Improve Diacetyl Formation

Mirjana Curic,1 Birgitte Stuer-Lauridsen,1 Pierre Renault,2 and Dan Nilsson1,*

Research and Development, Chr. Hansen A/S, DK-2970 Hørsholm, Denmark,1 and Laboratorie de Génétique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy en Josas Cedex, France2

Received 19 August 1998/Accepted 10 December 1998

The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the alpha -acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp. lactis biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototrophic strains. Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC004 containing the ilv genes (encoding the enzymes involved in the biosynthesis of IV) of L. lactis NCDO2118 was constructed. Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained. These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC004. Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting. The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L. lactis strains.

* Corresponding author. Mailing address: Department of Physiology & Metabolism, Chr. Hansen A/S, Bøge Alle 10-12, DK-2970 Hørsholm, Denmark. Phone: 45 45 74 74 74. Fax: 45 45 74 89 94. E-mail: dn.dk{at}chr-hansen.com.

Applied and Environmental Microbiology, March 1999, p. 1202-1206, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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