Function of Corynebacterium glutamicum promoters in Escherichia coli , Streptomyces lividans , and Bacillus subtilis Miroslav Pa ´tek a, *, Gu ¨ nther Muth b , Wolfgang Wohlleben b a Institute of Microbiology, Academy of Sciences of the Czech Republic, Vı ´den ˇska ´ 1083, CZ-14220 Prague 4, Czech Republic b Microbiology/Biotechnology, Eberhard-Karls-University, Auf der Morgenstelle 28, D-72076 Tu ¨ bingen, Germany Received 19 November 2002; received in revised form 2 April 2003; accepted 7 April 2003 Abstract The function of seven promoters from Corynebacterium glutamicum , P-hom , P-leuA , P-per , P-aes 1, P-aes 2, P-45, and P-104, was analyzed in a heterologous background. DNA fragments carrying the promoters were cloned into shuttle promoter-probe vectors replicating in Escherichia coli and C . glutamicum (pET2), Streptomyces lividans (pGL7011) and Bacillus subtilis (pRB394). With the exception of P-hom , P-leuA and P-104 in B . subtilis , all promoters were found to be active in all species. Non-radioactive methods of primer-extension analysis and of S1-nuclease protection assay using automatic sequencer were developed to determine the respective transcriptional start points (TSPs). All TSPs were determined by primer extension and in two promoters (P-45 and P-hom ) the main TSPs were confirmed by S1-mapping. While the main TSPs were identical in all four species, utilization of multiple TSPs varied among the species and additional TSPs were detected in S . lividans . Knowledge of the efficiency of promoters and of exact respective TSPs may be of practical value for the construction of expression systems in a heterologous background. # 2003 Elsevier B.V. All rights reserved. Keywords: Corynebacterium glutamicum ; Escherichia coli ; Streptomyces lividans ; Bacillus subtilis ; Promoters; Heterologous expression 1. Introduction Introduction of heterologous genes into indus- trial microorganisms, where they constitute new metabolic pathways that may lead to the synthesis of novel products or creation of new degradative activities, belongs to the most popular strategies of strain improvement (Bailey, 1991). Well-defined regulated promoters coming from various bacteria serve also as efficient tools in rational metabolic engineering (Goldstein and Doi, 1995). The major barriers preventing general heterologous expres- sion of the genes in bacteria are the different structure of promoters and different recognition abilities of the respective RNA polymerases in individual species. The variations in core promoter sequence elements are connected especially with * Corresponding author. Tel.:  /420-241062398; fax:  /420- 241722257. E-mail address: patek@biomed.cas.cz (M. Pa ´tek). Journal of Biotechnology 104 (2003) 325  /334 www.elsevier.com/locate/jbiotec 0168-1656/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-1656(03)00159-7