APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY High-level overproduction of Thermus enzymes in Streptomyces lividans Margarita Díaz & Eloy Ferreras & Renata Moreno & Ana Yepes & José Berenguer & Ramón Santamaría Received: 10 March 2008 / Revised: 3 April 2008 / Accepted: 5 April 2008 / Published online: 7 May 2008 # Springer-Verlag 2008 Abstract Biotechnology needs to explore the capacity of different organisms to overproduce proteins of interest at low cost. In this paper, we show that Streptomyces lividans is a suitable host for the expression of Thermus thermo- philus genes and report the overproduction of the corresponding proteins. This capacity was corroborated after cloning the genes corresponding to an alkaline phosphatase (a periplasmic enzyme in T. thermophilus) and that corresponding to a beta-glycosidase (an intracel- lular enzyme) in Escherichia coli and in S. lividans. Comparison of the production in both hosts revealed that the expression of active protein achieved in S. lividans was much higher than in E. coli, especially in the case of the periplasmic enzyme. In fact, the native signal peptide of the T. thermophilus phosphatase was functional in S. lividans, being processed at the same peptide bond in both organisms, allowing the overproduction and secretion of this protein to the S. lividans culture supernatant. As in E. coli, the thermostability of the expressed proteins allowed a huge purification factor upon thermal denatur- ation and precipitation of the host proteins. We conclude that S. lividans is a very efficient and industry-friendly host for the expression of thermophilic proteins from Thermus spp. Introduction Thermostable enzymes (thermozymes) from several sources are useful tools for different biotechnological process (Pantazaki et al. 2002). Frequent sources of these enzymes are strains of the genus Thermus, and the most common way to overproduce them involves use of a surrogate mesophilic host as cell factory, Escherichia coli being the one most widely used (Pessela et al. 2004). When production of the active form of a thermophilic enzyme in this mesophile is possible, the use of a purification step involving thermal denaturalization of the host proteins strongly facilitates its purification. Alternative methods for the overexpression of these proteins in engineered expression hosts of the same species have been successfully applied to the overproduction of different enzymes (Hidalgo et al. 2004; Kayser and Kilbane 2001; Moreno et al. 2003, 2005). However, the scaling up of these thermophilic expression systems is still poorly known in comparison to the long experience already available for classical industrial microorganisms such as Streptomyces. Appl Microbiol Biotechnol (2008) 79:1001–1008 DOI 10.1007/s00253-008-1495-1 M. Díaz : A. Yepes : R. Santamaría Instituto de Microbiología Bioquímica, Departamento de Microbiología y Genética, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain E. Ferreras : R. Moreno : J. Berenguer Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, 28049 Madrid, Spain M. Díaz (*) Instituto de Microbiología Bioquímica, CSIC /Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain e-mail: mardi@usal.es