Biochimie 70 (1988) 461-473 Soci6t6 de Chimie biologique/Eisevie ~, Paris 461 Molecular cloning of lactose genes in dairy lactic streptococci: the phospho-/3-galactosidase and/3-galactosidase genes and their expression products Willem M. DE VOS and Guus SIMONS Molecular Genetics Group, Department of Biophysical Chemistry, Research (NIZO), P.O. Box 20, 6710 BA Ede, The Netherlands (Received 2-12-1987, accepted with minor revision 15-12-1987) Netherlands Institute for Dairy Summary --- The mesophilic (S. iactis and S. cremoris) and thermophilic (S. thermophilus) dairy lactic streptococci, which are used in industrial dairy fermentations, contain two different lactose hydro!ysing enzymes, a phospho-/3-galactosidase and a/3-galactosidase. The central role of these enzymes m the pathways used for lactose transport and degradation is discussed along with their properties and distributions in lactic streptococci. In addition, recent results on the cloning, expression and sequence organization of the genes for the mesophilic phospho-B-galactosidase and thermophilic/3-galactosidase are reviewed. Original data are presented concerning heterologous gene expression in the study of lac- tose hydrolysis in lactic streptococci. These include 1) the purification of the S. lactis phospho-B- galactosidase from an overproducing Escherichia coli, and 2) the expression of the E. coli B-galacto- sidase (lacZ) gene in S. lactis employing a lactic streptococcal expression vector. recombinant DNA / heterologous gene expression / sequence organization / overproduction / regulation Introduction The lactic streptococci S. lactis, S. cremoris and S. thermophilus are employed as starter cultures [1-4f°ra] variety of industrial dairy fermentations . Their primary function is the rapid fer- mentative conversion of lactose into lactic acid both at mesophilic temperature (20-30°C; S. lactis and S. cremoris) and so called thermophilic temperature (37-45°C; S. thermophilus). In view of the biotechnological importance of this fermentation process, its relative simplicity and its high conversion efficiency, various molecular aspects of the lactose metabolism have been stu- died during the last few decades. The biochemical pathways involved in lactose transport and degradation have been established (Fig. 1) and subsequently analysed in more detail with specific attention for the energy transducing processes (see [5, 6] and [ 11-15] for reviews). Two different systems for lactose transport have been found in lactic streptococci: 1) the lactose phosphoenolpyruvate (PEP) dependent phosphotransferase system (PTS), in which lactose is phosphorylated during trans- location, and 2) the lactose permease system (PS), mediating the entry of unmodified lactose. These lactose transport systems differ with re- spect to their complexity and their bioenergetics. In addition, the mechanism by which lactose is transported determines the fermentative pathways (including the tagatose-6-P, Leloir and Embden-Mayerhof-Parnas pathways [5]) for the further conversion of the sugars into lactate (see Fig. 1). Key enzymes in these degradation routes are the lactose hydrolysing enzymes phospho-/3-galactosidase (P-/3-Gal, EC 3.2.1.85) and /3-galactosidase (/3-Gal, EC 3.2.1.23), which are specific for the phosphorylated and non-phosphorylated forms, respectively, of the internalized lactose. The observation that the ability to ferment