AfsR2 is a global regulatory protein involved in stimulating the biosynthesis of antibiotics in various Streptomyces species. Using 2-D gel electrophoresis and MALDI-TOF analyses, several putative AfsR2-target proteins were previously identified. In order to verify the biological significance of the proteomics-guided targets, the sixteen putative AfsR2 target genes were individually cloned under the strong-and-constitutive promoter (PermE(+)) of the Streptomyces expression vector, pSE34, followed by the transformation into both Streptomyces lividans and Streptomyces coelicolor. Among the putative AfsR2-target genes tested, the expression of a putative secreted solute binding protein, SCO6569 significantly repressed biosynthesis of actinorhodin in S. coelicolor, and the transcripts encoded by the pathway-specific activator, actll-ORF4 for actinorhodin were also reduced in SCO6569-overexpressing S. coelicolor. Moreover, SCO6569 gene-disruption in S. coelicolor further enhanced actinorhodin production, suggesting that the SCO6569 could be a possible AfsR2-dependent down-regulator acting in actinorhodin biosynthesis in S. coelicolor. (C) 2008 Elsevier Ltd. All rights reserved.