Journal of Biotechnology 98 (2002) 199 – 213 Metabolic engineering of Lactococcus lactis : the impact of genomics and metabolic modelling Michiel Kleerebezem *, Ingeborg C. Boels, Masja Nierop Groot, Igor Mierau, Wilbert Sybesma, Jeroen Hugenholtz Department of Flaour, Nutrition and Natural Ingredients, Wageningen Centre for Food Sciences, NIZO Food Research, P.O. Box 20, 6710 BA Ede, The Netherlands Received 18 July 2001; received in revised form 11 February 2002; accepted 27 March 2002 Abstract Lactic acid bacteria display a relatively simple and well described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies that led to the efficient re-routing of the lactococcal pyruvate metabolism to end-products other than lactic acid, including diacetyl and alanine. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to exopolysaccharides and folic acid. Finally, the (future) impact of the developments in the area of genomics and corresponding high-throughput technologies will be discussed. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Lactic acid bacteria; Lactococcus lactis ; Metabolic engineering; Pyruvate metabolism; Genomics www.elsevier.com/locate/jbiotec 1. Introduction Lactic acid bacteria (LAB) are industrially im- portant microbes that are used all over the world in a large variety of industrial food fermentation. Their contribution in these fermentation processes primarily consists of the formation of lactic acid from the available carbon source resulting in a rapid acidification of the food raw material, which is a critical parameter in the preservation of these products. However, besides their lactic acid form- ing capacity, LAB also contribute to other product characteristics like flavour and texture. Next to their most important application, which is undoubtedly in the dairy industry, LAB are also applied at an industrial scale in the fermentation of other food-raw materials like meat, and vegetables. Lactococcus lactis is by far the most extensively studied lactic acid bacterium, and over the last decades elegant and efficient genetic tools have been developed. These tools are of critical impor- tance in metabolic engineering strategies that aim at inactivation of undesired genes and/or (con- trolled) overexpression of existing or novel ones. Especially, the nisin controlled expression (NICE) * Corresponding author. Tel.: +31-318-659629; fax: +31- 318-650400. E-mail address: kleerebe@nizo.nl (M. Kleerebezem). 0168-1656/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S0168-1656(02)00132-3